Drinks Reception and Poster Session 1

17:30 - 19:00 Tuesday, 20th May, 2025

Room Pavilion I

Presentation type Poster

P1.1 Bridge amplification for the detection of SNPs associated to cardiovascular diseases

Cansu Pinar Yenice, Vasoula Skouridou, Mohga Khater, Ciara Kathleen O'Sullivan

Rovira i Virgili University, Spain

Abstract

Single nucleotide polymorphisms(SNPs) are the most abundant genetic variations with occurring frequency of between 1 in 100 to 1 in 300 bases at defined genetic locations throughout the entire genome that are important genomic or physical markers with applications in many fields including predisposition to diseases.

We previously developed a technique for the multiplexed electrochemical detection of SNPs-associated with osteoporosis^[1] based on isothermal solid-phase primer extension exploiting recombinase polymerase amplification and ferrocene-labelled dNTPs. In this approach, 4 thiolated identical reverse primers with the exception of the terminal base are immobilized by chemisorption on individual electrodes of an array. The forward primer is in solution and upon addition of genomic DNA, primer extension only occurs where there is complementarity to the terminal base. To facilitate a clear differentiation between complementary and mismatch and to avoid primer-dimers, the concentration of the forward primer needs to be carefully optimized.

As an alternative we have applied bridge amplification where both forward and reverse primers of the target are immobilized on the surface of an electrode. Although the reaction kinetics are slower and steric hinderance is magnified, bridge amplification achieves spatial separation and solves the primer-dimer problem as well as inequivalent levels of amplification with surface tethered primers.

Here we report the use of bridge amplification for the detection of SNPs associated to cardiovascular diseases, the leading cause of death worldwide, to achieve multiplexed detection with high sensitivity and specificity. Our approach again exploits the use of ferrocene-modified dNTPs as electroactive markers for electrochemical detection, which are enzymatically incorporated during amplification and 5’-thiolated primers, which are immobilized on the surface of gold electrodes of an array and thus eliminating the primer-dimer problem. The approach is optimized for the surface chemistry of the electrochemical platform as well as bridge amplification reaction time and temperature.

Keywords

Williamson, P., Piskunen, P., Ijäs, H., Butterworth, A., Linko, V., & Corrigan, D. K. (2023). Signal amplification in electrochemical DNA biosensors using target-capturing DNA origami tiles. ACS Sensors, 8(4), 1471-1480. https://doi.org/10.1021/acssensors.2c02469

Keywords

Impedance

DNA origami

Self assembled monolayers (SAMs)

Nucleic acid amplification

P1.6 A sensitive and fast tetrodotoxin lateral flow biosensor by antibody-aptamer sandwich assay

Ulises Guillermo Díaz-Avello^1,2, Vasso Skouridou¹, Mònica Campàs², Ciara K. O'Sullivan^1,3

¹Interfibio Research Group. Chemical Engineering Department. Universitat Rovira i Virgili, Spain. ²IRTA, Institute of Agrifood Research and Technology, Spain. ³Institució Catalana de Recerca i Estudis Avancats (ICREA), Spain

Abstract

Tetrodotoxin (TTX) is a small and powerful marine toxin causing human intoxication upon consumption of contaminated fish worldwide. As a neurotoxin, TTX impairs nerve function by blocking the sodium voltage-gated channels leading to numbness, dyspnea, and even death. As cooking processes cannot destroy TTX and no antidotes are available, early detection is essential to prevent intoxications. However, detecting TTX is complicated since analytical techniques require extensive and costly processes to overcome matrix effects and achieve enough sensitivity thus limiting their field application. Lateral flow assays (LFAs) are rapid and robust analytical tools in a paper-based cost-efficient format that provide equipment-free signal read-out. Competitive LFAs are commonly used for small analytes like TTX due to the limited sites available for binding to more than one bioreceptor. These are more difficult to optimize, less robust and sensitive, and more difficult to evaluate than sandwich assays exploiting the dual binding of two bioreceptors. Aptamers are single stranded oligonucleotides with similar binding properties to antibodies and are extensively used for the detection of a variety of analytes, especially small molecules. In our previous work we established a microtiter plate-based hybrid sandwich assay based on a novel TTX aptamer and a commercial α-TTX monoclonal IgG antibody. In this work, we developed a colloidal gold-based LFA test for the rapid and equipment-free detection of TTX exploiting this antibody-aptamer sandwich format. The test achieved a visual limit of detection of 0.3 ng/mL TTX in 20 minutes, and was also shown to be highly specific, with long term storage stability. Its performance was validated with contaminated pufferfish samples detecting TTX far below the safety limits of 2 mg TTX/kg fish tissue established in Japan. Overall, this TTX LFA test is the first sandwich assay for TTX and significantly improves detection limits compared to tests commercially available.

Keywords

Neurotoxin

Marine Toxins

Pufferfish

Food safety

P1.7 A Pathway to Rational Aptamer-Based Biosensor Design for Continuous Biosensing - Decoding Aptamer-Protein Binding Kinetics Using Single-Molecule Techniques

Lena Fasching, Mike Filius, Raman van Wee, Chirlimin Joo, Alina Y. Rwei

Delft University of Technology, The Netherlands

Abstract

Real-time, continuous biosensing is essential to capture dynamic biological processes, necessitating highly sensitive, selective, and reusable biorecognition elements.¹Aptamers, short single-stranded nucleic acid sequences, are designed to bind specific targets, such as proteins, and have shown great promise as alternatives to antibodies and enzymes in biosensors due to their ease of production, high selectivity, and ability to regenerate.² Despite the development of numerous aptamers, their successful implementation in sensor systems remains limited, primarily due to difficulties in in-depth characterizing aptamer-target interactions.^3,4

To address this, we developed a single-molecule fluorescence platform to quantify aptamer-protein interactions with high temporal resolution. Using thrombin as a model, we measured the association (k_on) and dissociation (k_off) rates for three aptamers (HD1, NU172, and RE31), revealing distinct binding kinetics despite similar affinities (K_D). By integrating these single-molecule insights into a COMSOL simulation and validating the findings with quartz crystal microbalance (QCM) experiments, we demonstrated that sensor performance—specifically, sensitivity and detection limit—depends heavily on k_on and k_off rates. Despite similar K_D values among the tested aptamers, the NU172 aptamer achieved higher sensitivity and a lower limit of detection in our sensing example due to its slower k_off rate. This slower dissociation prolonged the lifetime of the thrombin-aptamer complex, enabling a more sensitive response in our flow-controlled QCM setup. These findings emphasize the importance of aptamer-protein kinetic analysis in selecting aptamers tailored for specific biosensor applications. This study provides a foundational framework for decoding aptamer-target kinetics at the single-molecule level, thereby facilitating the rational design of high-performance aptamer-based biosensors.

References

1. Kim, J., et al.; Nat. Biotechnol. 37, 389–406 (2019)

2. Ates, H. C. et al.; Nat. Rev. Mater. 7, 887–907 (2022)

3. Adachi, T. et al.; Molecules 24, 4229 (2019)

4. Ilgu, M. & Nilsen-Hamilton, M.; Analyst 141, 1551–1568 (2016)

Keywords

Single-molecule FRET

Continuous Biosensing

Aptamers

Aptamer-based Biosensors

P1.8 Prediction of an aptasensor recognition capacity by molecular docking

Danilo Ferreira, Anna Carolina Almeida de Paula, Bruna Dias, Antero Andrade, Estefânia Martins, Clascídia Furtado

Centro de Desenvolvimento da Tecnologia Nuclear, Brazil

Abstract

Docking is an in silico analysis capable of predicting the interaction between two molecules (in our case biomolecules) based on data such as spatial conformation, protonation at the chosen pH, flexibility of amino acid and nucleotide residues, and the definition of active and inactive groups. Its use allows predicting the spatial conformation of the complex formed by the recognition of a protein (CEA) by a specific aptamer (APTA3) and the intermolecular interactions that stabilize it. This makes it possible, through bioinformatics, to predict the ability of an aptasensor to recognize its target biomarker. Therefore, the aim of this in silico study was to predict the intermolecular interactions present in the APTA3-CEA complex and calculate its interaction energy under physiological conditions, assessing the viability of a biosensor. To this end, the methodology used was the selection of the most stable complex formed between APTA3 and CEA by the total system energy minimization method performed by the HADDOCK 2.4 platform, based on the active sites predicted by the PUResNet 2.0 platform, and the prediction of intermolecular interactions using ChimeraX software. With the predicted interactions, it was possible to calculate the Gibbs free energy of complex formation (-24.56 kJ mol⁻¹) and its chemical potential (-491.2 kJ) in the concentration range of 100 pmol L⁻¹ to 1 pmol L⁻¹. The values indicate the favorable formation and stability of the complex under physiological conditions (pH 7.4 and temperature of 310 K) and in the concentration range that includes values below and above the reference values for CEA in healthy individuals (2.5 pmol L⁻¹ to 5 ng mL⁻¹). It is concluded that APTA3 is an efficient candidate for the development of an aptasensor for CEA detection.

Keywords

Aptasensor

CEA

Docking molecular

Bioinformatics

P1.9 AuNP enhanced SPRI signal for picomolar detection of miRNAs

Coline Beltrami^1,2, Julien Moreau², Laurence Convert¹, Jean-François Bryche¹, Paul G. Charette¹

¹Université de Sherbrooke, Canada. ²Université Paris Saclay, France

Abstract

Organ donation, in the case of a brain-deceased organ donor requires in-depth monitoring of physiological conditions and adequate treatment to prevent the degradation of the tissues^[1]. One way of tracking this degradation is to follow the evolution of specific biomarkers, particularly miRNAs ^[2], of the inflammatory response (i.e. cytokine storm) occurring throughout the patient’s body. Here, we propose using an AuNP-enhanced Surface Plasmon Resonance Imaging (SPRI) biochip system to quantitatively detect those miRNAs.

SPRI allows multiplexing of targets, rapid measurements and quantitative measurements of target biomarkers without prior labelling. The limit of detection (LOD) for SPRI biosensors is typically in the nanomolar range for a small single-stranded DNA or RNA after a few minutes detection step^[3]. However, the miRNAs targeted in the context of organ detection are only present in the high femtomolar range, necessitating a reduction in the LOD by at least four orders of magnitude.

To achieve this goal, we employed several strategies. First, we optimized surface chemistry to maximize the affinity between our targets and the bioreceptors. We then worked on increasing the refractive index shift after the hybridization step with a sandwich-like assay using gold nanoparticles (AuNPs). These AuNPs were synthesized in a one-step process at ambient temperature and their functionalization was optimized to have a high solubility in saline solutions. A kinetic model was developed to understand the key parameters involved in this type of sandwich method. We achieved a LOD for the targeted miRNAs of less than 10 pM with an interaction time of less than 10 minutes.

[1] S. Weiss et al., Am J Transplant, vol. 7, n^o 6, p. 1584‑1593, juin 2007

[2] A.-A. Clément et al., Epigenetics, p. 1‑16, mai 2022

[3] M. Piliarik et J. Homola, Opt. Express, vol. 17, n^o 19, p. 16505, sept. 2009

Keywords

SPRI

miRNA

AuNPs

P1.10 Empowering on-site monitoring of fuel biocontamination using a smartphone-assisted colorimetric biosensor

Hui Jean Lim, Muhammad Harith Bin Mohammad Taufik, Shiuan Nee Goh, Zhi Yang Lian, Joy Pang, Susanna Su Jan Leong, Adison Wong

Singapore Institute of Technology, Singapore

Abstract

Microbial contamination in fuels degrades fuel quality and promotes microbiologically influenced corrosion, which can lead to fuel system failure if left unaddressed. Currently available test kits for fuel monitoring are limited by qualitative or semi-quantitative results and rely primarily on culture-based techniques that require several days to yield detectable outcomes. To address the urgent need for efficient fuel monitoring, we developed a rapid, affordable colorimetric biosensor that detects contamination through a red-to-purple colour change triggered by the salt-induced aggregation of aptamer-functionalised gold nanoparticles (AuNPs). Using Cell-SELEX, we identified a high-affinity DNA aptamer targeting Sphingomonas paucimobilis, a hydrocarbon-degrading bacterium isolated from an in-house fuel sample, marking the first aptamer developed specifically for this microbe. We subsequently optimised the aptamer by appending various functional groups and nucleotide sequences to the 5’ end, studying the effects of varying interaction strengths between the aptamer and AuNPs on the colloidal stability and colour profile of the resultant aptamer-AuNP conjugates. Notably, high sensitivity in colorimetric biosensing of the fuel microbe was achieved using a short nucleotide sequence of optimised length and base composition for the reversible immobilisation of aptamers onto AuNPs, yielding a broad detection range from 14.1 CFU/mL to 10⁸ CFU/mL, enabling early detection of fuel biocontamination. To facilitate on-site fuel monitoring, we developed a smartphone-based application as a portable alternative to ultraviolet-visible (UV-Vis) spectrophotometry for colorimetric readouts. The app analyses sample colour based on RGB channels to quantify nanoparticle aggregation, achieving a 98% correlation with UV-Vis measurements. Additionally, the biosensor performance was validated in spiked fuel samples, demonstrating its applicability in non-aqueous environments beyond typical environmental and food monitoring contexts. At a minimal assay cost of €0.11, our aptamer-based colorimetric biosensor offers a cost-effective solution for detecting microbial threats in fuel systems, with a plug-and-play protocol adaptable to diverse microbial detection applications.

Keywords

Colorimetric biosensor

Microbial contamination

Fuel monitoring

Smartphone readout

P1.11 Double-layer Film based on Schiff base-modified chitosan/PVA as an optical sensor for pH monitoring in smart food packaging

Sarah Ben Haj Fraj^1,2, Cristina Scolaro¹, Mohamed Hassen V Baouab², Annamaria Visco¹, Giovanni Neri¹

¹Department of Engineering, University of Messina, C. da Di Dio, I-98166 Messina, Italy. ²Research Unit Materials and Organic Synthesis (UR17ES31), Preparatory Institute for Engineering Studies of Monastir, University of Monastir, Tunisia

Abstract

In response to the critical need for rapid and accurate detection of food spoilage as well as the imperative to minimize food waste, recent scientific advancements have concentrated on the design of intelligent bio-films that utilize pH indicators for real-time monitoring. In this work, a double-layer Schiff base-modified chitosan/polyvinyl alcohol (PVA) film was prepared and characterized using different techniques such as FTIR, UV-vis, PL, SEM and NMR ¹H… This sensor leverages the sensitivity of Schiff base cross-linkage and the biocompatibility of chitosan and PVA matrix to create a robust, responsive material. Schiff base modification enhances the material’s optical properties, providing two distinct colorimetric and fluorescent responses that correlate with pH shifts. These dual optical signals enable precise detection across a broad pH range, making the sensor particularly suitable for real-time monitoring of pH changes that can indicate spoilage in packaged foods. Experimental analysis demonstrated the sensor’s stability, reusability, and high sensitivity to minor pH changes, which are critical for applications in the food packaging where early spoilage detection is essential. This novel sensor offers a promising approach to improving food safety and quality assurance in active packaging systems.

Keywords

Schiff base

pH sensor

Colorimetric-fluorescent

Food packaging

P1.12 Biosensors for the Diagnosis of Tuberculosis: MPT64 Aptazymes

Erkan Mozioğlu, Gizem Kılıç

Acibadem University, Turkey

Abstract

Tuberculosis is still one of the most important infectious diseases worldwide. According to WHO estimates, 36% of tuberculosis patients remain undiagnosed due to lack of facilities. Since the current diagnostic methods are based on traditional approaches with long-standing origins, their application is dependent on both well-equipped laboratories and specialists. Aptamers and aptazyme molecules are becoming important for biosensor designs in terms of both low cost and point-of-care diagnosis. In this study, aptazyme molecules specific for MPT64 proteins of M. tuberculosis were selected using in vitro methods as sensor molecules to develop a biosensor for the rapid diagnosis of tuberculosis. For the selection of aptazyme molecules specific for MPT64 proteins, a fluorometric DNA aptazyme library was first designed and selection cycles including binding-catalytic activity, extraction and amplification steps were completed. In this process, ligation for fluorescence labeling of the DNA aptazyme library, urea polyacrylamide gel electrophoresis for purification and demonstration of catalytic activity, DNA purification from the gel by ethanol precipitation, and amplification by polymerase chain reaction were used. As a result of the selection and negative-selection cycles using the designed aptazyme library, DNA molecules showing catalytic activity specific to MPT64 proteins were demonstrated using molecular techniques. Fluorometric aptazyme molecules specific for MPT64 proteins, which play an important role in the diagnosis of tuberculosis, have been selected.

Acknowledgments: We would like to thank TÜBİTAK for the support to Project titled “In-vitro Selection of DNA Aptazymes Specific for MPT64 Proteins” (Project No: 121Z220) and Acıbadem Mehmet Ali Aydınlar University for providing the infrastructure facilities for the conduct of this study.

Keywords

Tuberculosis

biosensor

aptazyme

MPT64 proteins

P1.13 Photoelectrochemical detection of dengue RNA: Tackling challenges related to secondary structures

Hannah Op de Beeck^1,2, Elise Daems^1,2, Anne Hauner³, Kevin K. Ariën^3,4, Karolien De Wael^1,2

¹Antwerp engineering, photoelectrochemistry and sensing (A-PECS), Department of Bioscience Engineering, University of Antwerp, Belgium. ²NANOlight Center of Excellence, University of Antwerp, Belgium. ³Institute of Tropical Medicine, Belgium. ⁴Department of Biomedical Sciences, University of Antwerp, Belgium

Abstract

Dengue is a rapidly emerging mosquito-borne virus, threatening half of the global population, particularly in the resource-limited settings where fast and accurate diagnosis is critical. Conventional methods such as reverse-transcription polymerase chain reaction are highly sensitive but time-consuming and expensive, while antigen-based tests often lack sensitivity. To overcome these limitations, we have developed an affordable, rapid point-of-care diagnostic biosensor based on singlet oxygen (¹O₂)-mediated photoelectrochemistry for the detection of dengue RNA.

This platform integrates: (i) photosensitizers that generate ¹O₂ upon illumination, (ii) a redox reporter to enhance the sensitivity, and (iii) magnetic beads for efficient target immobilisation onto the electrode surface and improved signal-to-noise ratios. The viral dengue RNA hybridises with both a capture probe, attached to a magnetic bead, and a detection probe, labelled with a photosensitizer. Upon illumination, the photosensitizer produces ¹O₂, which oxidises the redox reporter. This redox reporter is regenerated at the electrode surface, resulting in a photocurrent. As this photocurrent is only produced when the target-probe hybrid is formed, the matrix effects can be eliminated by switching the light on and off.

This work focuses on the critical factors affecting the biosensor performance, including challenges related to RNA instability and secondary structures, as well as optimizing magnetic bead handling. To disrupt secondary structures, we evaluated the impact of a thermal denaturation step and optimised probe design, such as the detection probe’s length and position. The optimised probe significantly improved probe-target hybridisation, achieving a limit of detection of 7 pM. Furthermore, the biosensor demonstrated high discrimination efficiency against Zika virus. Due to its potential for integration into a cost-effective, portable device and automated sample preparation, this platform shows great promise as a point-of-care tool for dengue diagnosis in resource-limited settings.

Keywords

Photoelectrochemical biosensing

Dengue RNA diagnostics

Secondary structures

P1.14 Engineering of single chain variable fragments (scFvs) to confer pH-dependent kinetics for use in biosensor applications

Ellie Wilson¹, David Probst¹, Madoka Nagata¹, Miho Oda^1,2, Mai Hamasaki^1,2, Qianming Xu², Ayumi Tanaka², Hirobumi Suzuki², Ryutaro Asano², Koji Sode¹

¹The University of North Carolina at Chapel Hill, USA. ²Tokyo University of Agriculture and Technology Department of Biotechnology and Life Science, Japan

Abstract

We engineered single chain variable fragments (scFvs) to confer pH-dependent kinetics for biosensor development to minimize the time lag in biosensor response, guided by predicted structures and interactions. As an example, we utilized an anti-insulin scFv. One of the constructed mutants, His-scFv, demonstrated favorable kinetic properties for insulin sensing at pH 6.0 and was predicted by our in-silico model to detect transient insulin concentrations more effectively than the wild type (WT) (Figure 1). This work provides a perspective on the requirements of utilizing antibodies as a biological recognition element (BRE) for developing biosensors for the in vivo continuous monitoring of proteins and peptides.

Selective modulation of receptor-ligand binding is a powerful tool in many areas of biotechnology and particularly in the development of biosensors. Biosensors for continuous monitoring require the use of a highly sensitive, specific, and reversible BRE. The required sensitivity and specificity of a BRE depend on the in vivo concentration of the target molecules of interest; however, regeneration of the molecular recognition site is crucial to transduce multiple measurements. Especially, the binding equilibrium of high affinity-type BREs, such as antibodies, strongly favors complex formation. The capacity to trigger a change in the kinetics of a high-affinity biological recognition element, such as an antibody, is necessary to facilitate reliable, continuous quantification of biomarkers present at low concentrations.

Followingly, we introduced histidine substitutions in the complementarity-determining regions (CDRs) of an anti-insulin scFv to confer pH-dependent binding. We designed four histidine mutants and characterized their binding affinity at pH 7.4 and pH 6.0 with Bio-layer interferometry. We define an in-silico pharmacokinetic and mass action model to understand how changes in kinetic parameters imparted by the histidine mutations allow for a reduction in time lag between the fraction of antibody bound versus the predicted in vivo concentration of insulin.

Uncaptioned visual

Keywords

scFv

Insulin

pH-dependent

Time Lag

P1.15 Bedside Aptamer-Based Biosensor for Monitoring Acute Chronic Liver Failure Therapy

Massimo Urban¹, Marianna Rossetti¹, Giulio Rosati¹, Gabriel Maroli¹, Stephanie Simon², Jonel Trebicka^3,4,5, Christophe Junot^2,6, Arben Merkoçi^1,7

¹Catalan Institute of Nanoscience and Nanotechnology, Spain. ²Université Paris-Saclay, CEA,Département Médicaments et Technologies pour la Santé (MTS), France. ³Medical Clinic 1, University Hospital, Goethe- University Frankfurt, Germany. ⁴Department of Internal Medicine B, Muenster University Clinic, University of Münster, Germany. ⁵European Foundation for the Study of Chronic Liver Failure, Spain. ⁶MetaboHUB, France. ⁷Catalan Institution for Research and Advanced Studies, Spain

Abstract

Acute-on-Chronic Liver Failure (ACLF) is a severe condition marked by rapid deterioration of liver function in patients with underlying chronic liver disease, leading to high mortality rates (Arroyo et al., 2020). Most patients with ACLF require terlipressin-therapy for renal failure due to hepato-renal syndrome (HRS), however is not always recommended by FDA, and terlipressin use may induce multi-drug resistance bacteria (Mücke et al., 2024). Therefore, early prediction of the therapeutic response is crucial for improving patient outcomes and avoid unnecessary administration. Urinary neutrophil gelatinase–associated lipocalin (uNGAL) has been shown to be a strong predictor of a patient’s response to terlipressin and albumin—standard treatments for HRS—and thus an important biomarker for disease management (Gambino et al., 2023). We present herein a bedside electrochemical aptamer-based biosensor for the detection of NGAL protein as a predictor of treatment response in ACLF.

We employed redox-tagged NGAL-aptamers as signaling element, interrogated by square wave voltammetry (SWV), leveraging the response-dependence on the applied frequency, to perform calibration-free measurements (Li et al., 2017). The sensor integration into a point-of-care platform for wireless data communication read by a smartphone, simplifies the sampling and measurement protocol.

As transducers, we used nanostructured gold inkjet-printed electrodes fabricated using simple consumer equipment given their robust fabrication protocol, low-cost, and enhanced electroanalytical properties and surface area (Rosati et al., 2022; Urban et al., 2023).

This platform offers a promising bedside tool for the monitoring of different ACLF-therapies, improving patient management.

References:

Arroyo, V., et al., 2020. N. Engl. J. Med. 382, 2137–2145.

Gambino, C., et al., 2023. Hepatology 77, 1630–1638.

Li, H., et al., 2017. 139, 11207–11213.

Mücke, M.M.,et al, 2024. Aliment. Pharmacol. Ther. 59, 877–888.

Rosati, G., et al., 2022. Biosens. Bioelectron. 196, 113737.

Urban, M., et al., 2023. Small 2306167, 1–13.

Keywords

Nanoporous gold electrode

Aptasensor

Inkjet printing

Clinical sensor

P1.16 Biphasic approach for pathogen detection in whole blood with single-molecule sensitivity

Enrique Valera, Jongwon Lim, Matthew Wester, Katherine Koprowski, An Van, Rashid Bashir

University of Illinois Urbana-Champaign Department of Bioengineering, USA

Abstract

Fast and accurate identiﬁcation of infection-causing microorganisms in blood remains a signiﬁcant diagnostic challenge. Blood stream infections (BSIs) are often associated with severe diseases and result in high morbidity and mortality, especially in critically ill patients. Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection, is the most important diagnostic and therapeutic challenge from BSIs. Timely and informed administration of antibiotics can signiﬁcantly improve patient outcomes. However, blood culture, performed for up to 5 days for a confirmed negative result or stopped as soon as a positive result is obtained, followed by PCR remains the gold standard for diagnosing BSI.

Inspired by our examination of the micro-nano-porous network in dried blood, we have developed a new approach for culture-free detection of bacteria from milliliters of whole blood that uses thermal heating/drying of blood resulting in a ‘biphasic’ reaction mix, as a method to improve the sensitivity, while avoiding the need for nucleic acid purification and the use of extraction kits. The term biphasic denotes the presence of a solid dried blood phase and a liquid solution phase. In the biphasic approach large blood volumes can be rapidly dried to create a physical microscale and nanoscale ﬂuidic network inside the dried blood matrix. Inhibitors are physically trapped in the solid-phase dried blood matrix, while amplification reagents can move into the solid porous network and initiate the amplification.

We demonstrate single-molecule sensitivity using LAMP ampliﬁcation reactions and detection of broad spectrum of pathogens, including gram-positive MRSA and MSSA bacteria, gram-negative E. coli bacteria, and Candida albicans (fungus) from whole blood with a limit of detection of 1.2 CFU/mL from 0.8-1 mL of starting blood volume in less than 2.5 hours. Our biphasic technique has been validated with dozens of spiked and clinical whole blood samples.

Keywords

Biphasic approach

Pathogen detection

Whole blood

single-molecule sensitivity

P1.17 Structure-Switching Electrochemical Aptasensor for Rapid, Reagentless, and Single-Step Nanomolar Detection of C-Reactive Protein

William Whitehouse^1,2, Julian Tanner^1,2, Simon Shiu¹, Andrew Kinghorn¹, Louisa Lo^1,2

¹The University of Hong Kong, Hong Kong. ²Advanced Biomedical Instrumentation Centre, Hong Kong

Abstract

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ArticleMarch 14, 2024

Structure-Switching Electrochemical Aptasensor for Rapid, Reagentless, and Single-Step Nanomolar Detection of C-Reactive Protein

William L. Whitehouse
Louisa H. Y. Lo
Andrew B. Kinghorn
Simon C. C. Shiu
Julian A. Tanner*

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ACS Applied Bio Materials

Cite this: ACS Appl. Bio Mater. 2024, 7, 6, 3721–3730

https://doi.org/10.1021/acsabm.4c00061

Published March 14, 2024

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Abstract

C-reactive protein (CRP) is an acute-phase reactant and sensitive indicator for sepsis and other life-threatening pathologies, including systemic inflammatory response syndrome. Currently, clinical turn-around times for established CRP detection methods take between 30 min to hours or even days from centralized laboratories. Here, we report the development of an electrochemical biosensor using redox probe-tagged DNA aptamers, functionalized onto inexpensive, commercially available screen-printed electrodes. Binding-induced conformational switching of the CRP-targeting aptamer induces a specific and selective signal-ON event, which enables single-step and reagentless detection of CRP in as little as 1 min. The aptasensor limit of detection spans approximately 20–60 nM in 50% human serum with dynamic response windows spanning 1–200 or 1–500 nM (R = 0.97/R = 0.98 respectively). The sensor is stable for at least 1 week and can be reused numerous times, as judged from repeated real-time dosing and dose–response assays. By decoupling binding events from the signal induction mechanism, structure-switching electrochemical aptamer-based sensors provide considerable advantages over their adsorption-based counterparts. Our work expands on the retinue of such sensors reported in the literature and is the first instance of structure-switching electrochemical aptamer-based sensors (SS-EABs) for reagentless, voltammetric CRP detection

Keywords

Reagentless

C-Reactive Protein

Electrochemical

Aptasensor

P1.18 A macromolecule regulation generated CMOS MEMS-based membrane-bridge type nanomechanical sensor for mercury ion concentration detection in serum.

Yu-Jie Tu, Hao-Yen Tang, Yi-Kuang Yen

National Taipei University of Technology, Taiwan

Abstract

Detection of heavy metal ions in human blood is critical, as excess mercury ions can cause harmful and potentially fatal effects on the nervous, digestive, immune systems, lungs, and kidneys. Although commonly used methods in hospitals and labs offer high sensitivity and precision, they are constrained by large equipment, high maintenance costs, and lengthy testing times. To overcome these challenges, we developed a membrane-bridge type nanomechanical biosensor using the CMOS MEMS standard process and integrated it with thymine-rich aptamers as the recognition element.

After binding to mercury ions, the thymine-rich aptamer undergoes conformational changes, resulting in variations in the chip's surface stress and quantifiable piezoresistive change rate. By optimizing the aptamer concentration to 1 µM, the sensor achieved maximum surface stress response. The sensor demonstrated strong linear responses to mercury ion concentrations from 0.001 to 100 ppm, with a linearity of= 0.989 in PBS solutions and= 0.981 in human serum samples. The detection of limit was determined as 0.55 ppb. The sensor was shown to have selectivity for mercury ions and with reusability verified as 1.5% coefficient of variation. Furthermore, the sensor showed high accuracy in complex media, with recovery rates in human serum ranging from 90.5% to 99.5%. The sensor costs approximately $0.07 per chip and requires only a 5 µL sample per test, with a rapid 10-minute detection time. This makes it suitable for both point-of-care and home real-time detection of heavy metal mercury ions in blood.

Uncaptioned visual

Keywords

membrane-bridge nanomechanical sensor

aptamer

cantilever beam

mercury ions

P1.19 Magnetic hybrid nanoparticle-based microfluidic aptasensor for rapid bacteria detection in complex food matrices

Sermet Can Beylikçi^1,2,3, İsmail Eren¹, Gökay Yıldız⁴, Barbaros Çetin⁵, Veli Cengiz Özalp⁶, Güneş Kibar^2,3,7

¹Dept. Food Eng., Manisa Celal Bayar Uni., Turkey. ²Micro Nano Particles (MNP) Research Group, Materials Science and Engineering Department, Adana Alparslan Turkes Science and Technology University, Turkey. ³UNAM - Institute of Materials Science and Nanotechnology Bilkent University, Turkey. ⁴TEKGEN Healthcare Services Inc., Turkey. ⁵Microfluidics & Lab-on-a-chip Res. Group, Mech. Eng. Dept. İ.D. Bilkent University, Turkey. ⁶Dept. Medical Biology, School of Medicine, Atılım Uni., Turkey. ⁷Atılım University Vocational School of Health Services, Turkey

Abstract

Here, we propose a novel and cost-effective Point-of-Care (PoC) system designed for early diagnosis of AD and monitoring of disease progression. Leveraging a low-cost green IR laser-assisted print/stamp technology, we fabricate reduced graphene oxide (rGO) electrodes directly integrated into lateral flow assay (LFA) strips [3]. These rGO electrodes can be functionalized with aptamers that specifically bind to key AD biomarkers, enabling real-time monitoring of the disease. Binding interactions between the aptamers and target biomarkers induce detectable changes in the electrochemical signal, enabling rapid, sensitive, and reliable detection.

This PoC system provides a promising, non-invasive approach to AD diagnostics, offering a streamlined, accessible solution for early detection and disease management.

Acknowledgements

This work is part of the 2D-BioPAD project that has received funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement No. 101120706. The ICN2 is funded by the CERCA programme/Generalitat de Catalunya, supported by the Severo Ochoa Centres of Excellence programme, Grant CEX2021-001214-S, funded by MCIN/AEI/10.13039.501100011033. We acknowledge Departament de Recerca i Universitats of Generalitat de Catalunya for the grant 2021 SGR 01464 and Grant PID2021-124795NB-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”.

References

Alzheimer’s Association. Alzheimer’s Disease Facts and Figures 2024
Arslan, B. et al. Clin Chem Lab Med. 2024; 62(6); 1063-1069
Calucho, E. et al. Biosens Bioelectron. 2024; 258, 116315

Keywords

graphene

electrochemical

lateral flow assay

alzheimer

P1.23 Development of an innovative assay for protein quantification based on loop-mediated isothermal amplification (LAMP): application to the cardiac biomarker troponin I

Mathilde Aubret¹, Patricia Laurent¹, Christine Saint-Pierre², Maud Savonnet^1,2, Arnaud Buhot², Myriam Cubizolles¹

¹Univ. Grenoble Alpes, CEA-Leti, France. ²Univ. Grenoble Alpes, CEA, CNRS, Grenoble INP, IRIG, SyMMES/CREAB, France

Abstract

Enzyme Linked Immuno Sorbent Assay (ELISA) is a gold standard method to perform quantification of proteins. Nevertheless, it could be challenging to develop and use ELISA when high sensitivity and large dynamic range are required in specific clinical situations. In case of myocardial infarction, levels of blood troponin I (cTnI), a cardiac biomarker, varies from low concentrations around 0.1ng/mL (4 pM) in the first hours of the infarction up to 100ng/mL (4 nM) after several hours. We therefore develop an innovative assay (1) (2) dedicated to the detection of troponin I on a large detection range.

Antibodies grafted on a solid support (constituted by magnetic beads) ensure troponin capture from the sample and a biomolecular complex enables the protein quantification. This detection complex comprises either aptamers or antibodies, coupled with a dumbbell, an oligonucleotide sequence needed for further isothermal amplification based on loop-mediated isothermal amplification (LAMP) using only two primers. With the aptameroLAMP method, we are able to detect troponin with a limit of detection of 1 nM in buffer medium. Consequently, this approach is not relevant for physio-pathological tests. We therefore implement another method, namely the immunoLAMP, to perform troponin I detection in both buffer and human plasma samples. Our results clearly demonstrate that the dynamic range covers from 5 nM down to 5 pM in buffer medium, with a limit of detection of 5pM. In addition, we validate the successful quantification troponin I in human plasma samples with similar dynamic range and limit of detection using this innovative assay combining antibody sandwich and dumbbell exponential amplification (3).

We are currently performing the integration of this validated protocol in a microfluidic chip, opening the way to the development of portable device allowing sensitive protein quantification.

Uncaptioned visual

Keywords

Loop-mediated isothermal amplification (LAMP)

Aptasensor

Immunosensor

Cardiac troponin I

P1.24 A novel rolling circle amplification-mediated photoelectrochemical detection methodology for Zika virus

Elise Daems¹, Sandra Tejerina Miranda², Mats Nilsson^3,4, Kevin Ariën⁵, Karolien De Wael¹

¹University of Antwerp, Belgium. ²Universidad Complutense de Madrid, Spain. ³SciLifeLab, Sweden. ⁴Stockholm University, Sweden. ⁵Institute of Tropical Medicine, Belgium

Abstract

The global incidence of arbovirus outbreaks, e.g. Zika, has been rising at an alarming rate. Such viruses are transmitted by mosquitos in tropical and subtropical regions and are responsible for a significant public health burden. Importantly, population growth, urbanisation and climate change led to an increase in the spread of arboviral diseases. Hence, diagnosing these viral infections is crucial to reduce the spread and disease burden. However, conventional diagnostic tools are expensive and time-consuming or lack accuracy and sensitivity.

To overcome these challenges, we developed a novel sensing methodology for viral Zika RNA that combines padlock probes and rolling circle amplification (PLP-RCA) with singlet oxygen-based photoelectrochemistry. Our technology integrates three key components: (i) photosensitisers that produce singlet oxygen upon light illumination, (ii) a redox reporter, and (iii) magnetic beads for immobilisation which allow easy sample handling and washing steps. Importantly, our strategy enables the direct detection of RNA targets without the need for conversion to DNA, simplifying the diagnostic process. First, the PLP-RCA protocol was optimised to achieve the highest amplification efficiency while maintaining specificity. Afterwards, the analytical performance of our methodology was evaluated and various PLP designs were compared aiding a rational design of PLPs in the future. In general, our findings indicate that our novel detection strategy offers enhanced sensitivity for viral Zika RNA thanks to the integration of PLP-RCA.

The underlying methodology can in principle be adapted to detect any nucleic acid sequence and, therefore, has the potential to be further extended for a wider range of applications (i.e. bacterial infections, antimicrobial resistance and cancer biomarkers).

Keywords

Photoelectrochemistry

Rolling circle amplification

Zika virus

P1.25 Combining aptamers and fluorescent silver nanoclusters on a DNA strand as a novel innovative biosensor for protein biomarker detection

Katharina Schieke¹, Daniel Martin¹, Doreen Lisicki¹, Dieter Frense¹, Peggy Reich², Doris Heinrich^1,2

¹Institute for Bioprocess and Analytical Measurement Technology e.V., 37308 Heilbad Heiligenstadt, Germany. ²TU Ilmenau, Faculty of Mathematics and Natural Sciences, 98693 Ilmenau, Germany

Abstract

Nucleic acids generally serve as versatile recognition elements in biosensors and show high efficiency in detecting various biomarkers with remarkable sensitivity and selectivity. Among them, aptamers and DNA-based silver nanoclusters as single-stranded oligonucleotides exhibit special properties such as fluorescence and electrochemical activity. In recent years, they were integrated into a variety of biosensors.

We investigated the combination of the classical thrombin aptamer [1] as a model biosensor with short-chain nucleotides that enable the formation of stable and fluorescent silver nanoclusters. These included the 12-base nucleotide used by Petty et al. to first report DNA-stabilized silver nanoclusters in 2004 [2], as well as the 28-base nucleotide that was extensively studied by the group of Gwinn et al. in 2013 [3]. We have continued this work by developing three additional Ag-DNA nanoagent designs that exhibit optical responses to the intracellular environment as well as ion sensing capabilities and are functional in living cells [4].

Here we show the optical and electrochemical properties of these fluorescent silver nanoclusters with a high affinity to the model biomarker thrombin. We investigated nucleotides dissolved in buffer as well as immobilized on gold surfaces by impedance spectroscopy and fluorescence. Our intention was to gain deeper insights into the biochemical processes involved in analyte binding to the receptor and thus increase the sensitivity of thrombin detection.

Further, we use these fluorescent silver nanoclusters for versatile applications in life sciences, food safety and environmental protection, ultimately focusing on their special optical properties and the generation of unique quantum effects.

1. Bock, L.C., et al., Nature, 1992. 355(6360): p. 564-566.

2. Petty, J.T., et al., Journal of the American Chemical Society, 2004. 126(16): p. 5207-5212.

3. Schultz, D., et al., Advanced Materials, 2013. 25(20): p. 2797-2803.

4. Bossert, N., et al., Sci Rep, 2016. 6: p. 37897.

Keywords

nanocluster

fluorescence

aptamer

biomarker

P1.26 Triplex DNA Nanoswitch-Driven SPR Platform for Enhanced Sensitivity in Cancer microRNA Detection

Ja-an Annie Ho¹, Pei-Ying Lin², Ying-Feng Chang^1,3, Li-Chen Su⁴, Itamar Willner⁵

¹National Taiwan University, Taiwan. ²National Taiwan University Hospital, Taiwan. ³Chang Gung University, Taiwan. ⁴Ming Chi University of Technology, Taiwan. ⁵Hebrew University of Jerusalem, Israel

Abstract

We present an innovative method for detecting bladder cancer-associated microRNAs utilizing pH-responsive triplex DNA switches. This approach integrates a stepwise surface plasmon resonance (SPR) biosensing platform, enabling the concurrent detection of two miRNAs. The detection process involves immobilizing two pH-responsive triplex probes, switch A and switch B, onto an SPR sensor surface. The probes bind to specific antibodies via SA/miR-183 and SB/miR-155, allowing attachment to the antibody-modified surface. Streptavidin-Au nanoparticles and biotinylated strands function as reporter units, marking the presence of the target miRNAs on the sensing interface. Sequential pH adjustments displace the reporter units, generating distinct SPR reflectivity changes: a shift at pH 5.0 for miR-183 and at pH 8.3 for miR-155, which correlate with miRNA concentrations. This dual-miRNA detection platform achieves detection limits as low as 0.6 pM for miR-183 and 0.8 pM for miR-155, highlighting its precision and sensitivity in biomarker analysis.

Keywords

DNA nanoswitch

Au nanoparticle

Multiplex analysis

microRNA

P1.27 An aptasensor utilizing electrochemical impedance spectroscopy for the simultaneous detection of Mycobacterium tuberculosis and SARS-CoV-2 biomarkers

Zhazira Zhumabekova, Damira Kanayeva

Nazarbayev University, Kazakhstan

Abstract

According to the WHO (2023), tuberculosis and coronavirus are the leading infectious diseases worldwide, identified by the key diagnostic proteins MPT64 and S-glycoprotein, respectively. This study aimed to develop an aptasensor for the simultaneous detection of both target proteins using single-stranded DNA aptamers attached to the surface of a dual screen-printed gold electrode (SPGE) and employing Electrochemical Impedance Spectroscopy (EIS). Our research group previously selected and characterized the aptamer sequence (17) against MPT64 (Sypabekova et al., 2017), while Liu et al. (2021) selected aptamer-1 for S-glycoprotein detection. In this study, these sequences were chemically modified at the 5' end by adding a thiol (-SH) C6 group, a spacer, and 5-T residues. The dual SPGE surface was functionalized with aptamers and then blocked with 6-mercapto-1-hexanol, followed by the simultaneous detection of antigens. To optimize the aptasensor’s performance, we evaluated the effects of buffer composition, aptamer concentration, and target incubation time: SELEX buffer was chosen as the working buffer, 0.5 µM was selected for aptamer concentration, and a 10-minute incubation time was used for the targets. The developed EIS-based aptasensor enabled the simultaneous and sensitive detection of MPT64 and S-glycoprotein, with limits of detection of 1.68 pg/ml and 3.39 pg/ml in buffer, respectively. Furthermore, the developed aptasensor is cost-effective, requiring only a 2.5 µl sample volume and a detection time of less than 30 minutes. In the future, clinical samples will be evaluated and an atomic force microscopy will also be performed to provide an overview of the events occurring on the dual SPGE surface during aptamer immobilization and the capture of target biomarkers.

References:

Liu X, Wang YL, Wu J, et al. Angew Chem Int Ed Engl. 2021;60(18):10273-10278.

Sypabekova M, Bekmurzayeva A, Wang R, Li Y, Nogues C, Kanayeva D. Tuberculosis. 2017;104:70-78.

WHO (2023). Tuberculosis and COVID-19.

Uncaptioned visual

Keywords

electrochemical impedance spectroscopy based aptasensor

Mycobacterium tuberculosis

SARS-CoV-2

simultaneous detection

P1.28 Microfluidic-Synthesized Fluorescent POSS Nanoparticles for Aptasensor-Based Selective Detection of Staphylococcus aureus

Fatma Okus¹, Efe Berk Altın^1,2, Sumeyye Altınok^1,3, Veli Cengiz Özalp⁴, Güneş Kibar^1,3,5

¹Micro Nano Particles (MNP) Research Group, Materials Science and Engineering Department, Adana Alparslan Turkes Science and Technology University Türkiye, Turkey. ²Department of Molecular Biology & Genetics İ.D. Bilkent University, Turkey. ³UNAM—National Nanotech. Research Center and Institute of Materials Science & Nanotech. İ.D. Bilkent University, Turkey. ⁴Department of Medical Biology, School of Medicine, Atilim University, Turkey. ⁵Atılım University Vocational School of Health Services, Turkey

Abstract

In this study, we introduce a novel fluorescent aptasensor for the selective detection of Staphylococcus aureus using microfluidic-synthesized fluorescent POSS (Polyhedral Oligomeric Silsesquioxane) nanoparticles. For the first time, green and red fluorescent-POSS nanoparticles were synthesized in a microfluidic reactor, enabling precise control over nanoparticle size and fluorescence properties. The green fluorescent-POSS nanoparticles were functionalized with a specific aptamer targeting S. aureus, while the red fluorescent-POSS nanoparticles remained unmodified to serve as a control for non-specific binding.

The designed nanoparticle system utilizes the selective binding of the aptamer to S. aureus, allowing the green-labeled particles to specifically target and bind to the bacteria, while the red-labeled particles exhibit minimal binding. S. aureus was cultured in tryptone soy broth (TSB) at 37°C, and bacterial concentration was determined spectrophotometrically by measuring optical density (OD). The selectivity of the aptasensor was evaluated by comparing the fluorescence intensity of green-labeled particles (aptamer-functionalized) and red-labeled particles (non-functionalized) in the presence of S. aureus. Non-target binding was also assessed with the red-labeled particles to confirm specificity. Fluorescence microscopy was employed to visualize and analyze the binding behavior of the particles, confirming the high selectivity of the aptasensor for S. aureus.

This work demonstrates the potential of microfluidic-synthesized fluorescent-POSS nanoparticles as a highly specific and sensitive platform for bacterial detection, providing a powerful tool for future diagnostic applications.

Funding: TUBITAK 2218 award (project number:122C228)

Keywords

Fluorescent POSS nanoparticles

Microfluidic nanoparticle synthesis

Aptasensor

Staphylococcus aureus

P1.29 Electrochemical Detection of Streptococcus pneumoniae from Blood Samples Based on Nuclease Activity on Oligonucleotide Probes-Decorated Magnetic POSS Nanoparticles

Güneş Kibar^1,2,3, Murat Kavruk⁴, Frank J. Hernandez⁵, Barış A. Borsa⁵, Veli Cengiz Özalp⁶

¹Micro Nano Particles (MNP) Research Group, Materials Science and Engineering Department, Adana Alparslan Turkes Science and Technology University, Turkey. ²UNAM—National Nanotech. Research Center and Institute of Materials Science & Nanotech. İ.D. Bilkent University, Turkey. ³Atılım University Vocational School of Health Services, Turkey. ⁴Department of Medical Biology, School of Medicine, Istanbul Aydın University, Turkey. ⁵Nucleic Acids Technologies Laboratory (NAT-Lab), Linköping University, Sweden. ⁶Department of Medical Biology, School of Medicine, Atilim University, Turkey

Abstract

The development of sensitive and specific diagnostic tools for Streptococcus pneumoniae (S. pneumoniae) detection is crucial for early diagnosis and treatment. In this study, we report a novel electrochemical biosensor based on oligonucleotide probe-functionalized magnetic POSS (Polyhedral Oligomeric Silsesquioxane) nanoparticles. The hybrid nanoparticles were synthesized by integrating superparamagnetic magnetic iron oxide (SPION) nanoparticles on POSS cores, followed by surface modification with polydopamine for enhanced stability and biofunctionalization. Nuclease-resistant oligonucleotide probes specifically degraded by S. pneumoniae nuclease were covalently immobilized on the nanoparticle surface. The magnetic-POSS nanohybrids were employed as a convenient tool for electrochemically quantifying S. pneumoniae-specific nuclease in a differential pulse voltammetry-based detection platform. The system demonstrated excellent specificity, discriminating S. pneumoniae from non-target bacteria. Reusability and stability tests further validated its potential for point-of-care applications. This study highlights the promise of hybrid nanomaterials for nucleic acid-based biosensors, paving the way for cost-effective, rapid, and reliable pathogen detection platforms. The electrochemical biosensor demonstrated a detection limit as low as 10² CFU mL⁻¹.

Funding: TUBITAK 2218 award (project number:122C228)

Keywords

Electrochemical DNA biosensors

HT self-assembled monolayer

electrocatalytic amplification

P1.33 A MXene ink-based biosensor with aptameric sequence for measuring Cystatin C in sweat

Giulia Matteoli¹, Elena De Gregorio^2,1, Lorena Tedeschi¹, Nicola Calisi³, Matteo Mannini⁴, Sofia Zanotti¹, Silvia Ghimenti², Denise Biagini², Tommaso Lo Monaco², Pietro Salvo¹

¹Institute of Clinical Physiology, Italian National Research Council (IFC-CNR), Italy. ²University of Pisa Department of Chemistry and Industrial Chemistry, Italy. ³University of Florence Department of Industrial Engineering, Italy. ⁴University of Florence Department of Chemistry 'Ugo Schiff ', Italy

Abstract

Cystatin C has recently gained attention as a critical biomarker for monitoring chronic and acute kidney diseases due to its elevated levels because of renal dysfunction. Current analytical techniques measure Cystatin C in plasma, saliva and urine but no reliable procedure has yet been developed in sweat. This study introduces a biosensor based on 2D nanomaterials and oligonucleotides capable of measuring Cystatin C levels in sweat. The biosensor uses aptamer immobilized onto a titanium carbide (Ti₃C₂) MXene-based ink for the non-invasive detection of Cystatin C. This biosensor is suitable for the integration in a wearable lab-on-chip for sweat analysis.

In our work, we synthesized and silanized Ti₃C₂ to elicit the bioconjugation of the aptamer onto the working electrode of an electrochemical cell. Bioconjugation was achieved by introducing maleimide groups onto the silanized surface, which bind to the thiolated aptamer, forming a disulphide bond. A control scrambled oligonucleotide sequence was designed to confirm the aptamer specificity for Cystatin C. The high electrical conductivity of Ti₃C₂ improved the exchange of electrical carriers during Electrochemical Impedance Spectroscopy. In artificial sweat, this biosensor had a detection range of 50-200 ng/mL, and achieved a low limit of detection of 8 ng/mL, a limit of quantification of 24 ng/mL, and a repeatability error of 7%.

This innovative biosensing approach is a significant advancement in renal health monitoring technology, paving the way for a wearable solution for continuous patient monitoring. By integrating non-invasive biomonitoring with lab-on-chip functionality, this device offers a promising method for patients to track renal biomarkers and improve personalized health monitoring solutions.

Keywords

aptamer

MXene

Cystatin C

Sweat

P1.34 Development of an extraction-free EC-LAMP sensor for the detection of Legionella spp. in water

Ane Rivas¹, Unai Eletxigerra², Ruth Diez², Santos Merino^2,3, Antton Sanjuan⁴, Mounir Bou-Ali⁴, Leire Ruiz^5,6, Jose Luis Vilas^5,6, Felipe Goñi¹, Garbiñe Olabarria¹

¹Gaiker Technology Centre, Spain. ²Tekniker Foundation, Spain. ³UPV/EHU Department of Electricity and Electronics, Spain. ⁴University of Mondragon, Spain. ⁵University of the Basque Country, Spain. ⁶BCMaterials, Spain

Abstract

Legionella spp. is a bacterium commonly present in both natural and man-made aquatic environments, often found at low concentrations in any public water system. Effective Legionella spp. monitoring is required along with the implementation of preventive interventions and predictive tools. Consequently, in-situ analysis of Legionella spp. in water systems is therefore crucial to ensures water safety.

Our group has previously developed a method that combines Loop-Mediated Isothermal Amplification (LAMP) with electrochemical transduction signalling (EC-LAMP) to provide a simple and cost-effective process, suitable for point-of-need solutions (1). This approach enables the detection of very low levels of Legionella spp. nucleic acid; specifically, 10 fg, which corresponds to 2.6 genomic copies. However, the main drawback for in-situ applications is the requirement for DNA extraction prior to EC-LAMP detection. To address this challenge, this work presents a low-cost, disposable, extraction free microfluidic EC-LAMP sensor capable of detecting as low as 500 cfu/L in tap water samples spiked with Legionella pneumophila NCTC 12821 (Fig 1). Detection was performed within the EC-LAMP sensor at the end-point of the reaction.

To achieve real-time EC-LAMP and therefore, to improve end-point detection, the formation of bubbles during the reaction at 66 ºC must be avoided to ensure accurate and reproducible electrochemical detection. To this end, a 3D-printed chamber has been specifically designed using numerical analysis to prevent bubble deposition over the sensor, which consists of 3 gold concentric electrodes placed at the bottom of the microfluidic chamber. In addition, the resins used for 3D printing were tested before manufacturing the EC-LAMP sensor to confirm their compatibility with the EC-LAMP reaction; both with the amplification reagents and the electroactive molecule.

(1) https://doi.org/10.1016/j.talanta.2020.121061

Uncaptioned visual

Fig.1 Schematic workflow for Legionella spp. detection in tap water with the presented EC-LAMP sensor. a) End-point detection and b) Real-time-detection approaches

Keywords

EC-LAMP

Legionella spp.

Electrochemical sensor

3D-printing chamber

P1.35 Apta-MIP Hybrid Sensor Approach for Detection of Food Allergen Gliadin at Femtogram Level

Selenay Sadak^1,2, Gözde Aydoğdu Tığ³, Bengi Uslu¹

¹Ankara University Faculty of Pharmacy, Turkey. ²Ankara University Graduate School of Health Science, Turkey. ³Ankara University Faculty of Science, Turkey

Abstract

Gliadins are allergenic proteins that can pose various risks to human health and are found in high amounts in the most commonly consumed foods. Gliadin, a component of gluten, triggers oxidative stress in celiac disease. The use of sensitive analytical methods, especially in foods containing high amounts of these proteins, is very important for food safety. Biosensors have been developed as an alternative to laborious, expensive methods used in the food industry. Biosensors are selective, sensitive, simple, fast and low-cost systems. Aptamer-based biosensors are widely preferred in electrochemical studies today. One of the promising approaches regarding biosensors is the studies in which aptamer-based sensors are combined with molecularly imprinted polymers. The antibody-like binding and ability of MIP to distinguish between molecules increases the selectivity of the method.

In this study, the determination of gliadin was aimed by combining aptamer-based biosensor with MIP method. For this purpose, screen printed gold electrode was used. The electrode surface was electrochemically coated with gold solution in order to bind the aptamer to the surface, then the aptamer-gliadin complex was dripped onto the surface and adhered to it Orthophenylene diamine monomer was used to create a MIP surface. Gliadin was determined by usin differential pulse voltammetry (DPV) in a wide range such as 0.25 fg/mL -1 ng/mL with the developed method. LOD was calculated as 1.1 x 10^-8 ng/mL and LOQ as 3.4 x10^-8 ng/mL with the developed method. Interference studies were performed with similar molecules such as bovine serum albumin (BSA), casein, and ara-H1 to evaluate the selectivity. With the developed method, determination of gliadin was carried out in the recovery range of 98.1-104.6% from real samples gluten-free bread, popcorn and biscuits. It is believed the method will be promising for the successful determination of food allergens in the future.

Keywords

Aptamer

Gliadin

Food allergen

MIP

P1.36 Towards multiplex electrochemical detection on universally modified carbon electrodes for integrated molecular diagnostics

Dominic Kleinknecht¹, Martin Trotter¹, Cornelia Keß^1,2, Judith Sum¹, Felix von Stetten^1,2, Nadine Borst^1,2, Andreas Schreiber^1,2

¹Hahn-Schickard, Freiburg, Germany. ²University of Freiburg, Germany

Abstract

At the point of care, compact and versatile molecular diagnostic solutions are needed, as existing devices often rely on expensive, proprietary consumables and complex systems that are impractical in resource-limited or field settings. A flexible system with easily adaptable consumables is needed to rapidly respond to emerging health threats, such as the recent SARS-CoV-2 pandemic or Mpox outbreaks.

Here we demonstrate electrochemical signal-on, multiplex loop-mediated isothermal amplification (LAMP) detection in real-time. Within 30 minutes pathogens are detected on low-cost screen-printed carbon electrodes, covalently functionalized with generic solid-phase probes. The complementary redox-labelled probe is specifically released during LAMP, making the detection principle 'universal' and easily adaptable to different targets. We show that the electrochemical LAMP assay is faster and has twice the signal-to-noise ratio compared to the corresponding fluorescent LAMP. Electrochemical duplex detection is demonstrated by using two probes with different electrochemical labels, which are detected upon release on one working electrode with two co-grafted universal surface probes. The presented duplex LAMP assay detects 100 copies of the antibiotic resistance gene CTX-M1 in the crude lysate of 10,000 E. coli cells, tracing the uidA-housekeeping gene. These results show that electrochemical detection on functionalized carbon electrodes is a strong and adaptable alternative to optical nucleic acid detection methods. Importantly, the use of universal surface modifications allows for the simple transfer of existing LAMP assays, for different target pathogens, to our platform.

We are convinced, that the described detection system can be incorporated into miniaturized diagnostic devices. By using the same generic solid-phase probes, sensitive multiplex detection of a multitude of DNA and RNA targets at the point-of-care become electrochemically detectable on the same platform with minimal assay adaption.The presented platform could become a valuable tool to make molecular diagnostic testing more accessible, affordable and flexible in low-resource settings.

Keywords

Probe based LAMP

Carbon electrodes

Multiplex, signal on

Electrochemical detection

P1.37 Kinetic inversion in hybridization-based biosensors with multi-step reaction mechanisms

Yannick Stulens¹, Brittany Mueller², Dmitry Kolpashchikov², Jef Hooyberghs¹

¹Hasselt University, Belgium. ²University of Central Florida, USA

Abstract

DNA plays a major role in many biological processes and technological applications. The recent increased interest in minimally invasive techniques in medicine shows the need to improve mutant detection in an excess of single nucleotide variant background. We explore new detection and quantification concepts by improving our understanding of the thermodynamic and kinetic properties of DNA.

Typically, hybridization-based biosensors make use of the difference in binding free energy between the matching target and mismatched target. In a single-step reaction, i.e. the formation of a probe + target duplex, this leads to optimal discrimination at equilibrium. Earlier work on a multistranded sensor attributed the improved selectivity to the sensor operating under non-equilibrium conditions. In contrast to single-step reactions, it was also observed that matching target equilibrated faster compared to mismatched target, a novel phenomenon dubbed ‘kinetic inversion.' It is our understanding that this behavior is unique to sensors with multi-step reaction mechanisms. In such a system, the optimal time scale to discriminate between matching and mismatched target is not at equilibrium but at a specified time during the reaction kinetics (see figure).

We studied the presence and properties of kinetic inversion in a multi-stranded probe with a two-step reaction mechanism. A theoretical analysis shows that it is sensitively dependent on the reaction rates and the thermodynamics of the interacting strands. Selective binding should occur in the first of the two steps, and the second step should be indiscriminate. In addition to this, the first step should be noticeably faster compared to the second step. Experimental data is currently being collected using a multi-stranded probe designed to operate around room temperature. Kinetic inversion could increase the biosensor performance by extending the dynamic range and shortening the readout time.

Uncaptioned visual

Keywords

Kinetics

SNV detection

Multi-step reaction

Multi-stranded probe

P1.38 Rapid resolution of KRAS gene mismatches in colorectal cancer using LAMP-amplified photoelectrochemistry

Johana Strmisková¹, Alejandro Valverde², Ludmila Moráňová¹, Martin Bartošík¹, Karolien De Wael²

¹Masaryk Memorial Cancer Institute, Czech Republic. ²University of Antwerp, Belgium

Abstract

Despite significant advances, colorectal cancer (CRC) remains the second leading cause of cancer-related deaths worldwide, although the recent decline in incidence reflects the effectiveness of screening programs. Timely diagnosis and targeted treatments are essential to improve survival rates, highlighting the continuing need to optimize current clinical technologies, such as digital polymerase chain reaction (dPCR) or next-generation sequencing (NGS). Their high costs and complex protocols have positioned biosensor technology as a great alternative in terms of simplicity, speed and cost. However, challenges related to the high specificity and sensitivity required in the analysis of complex biological samples hinder their large-scale adoption in clinical environments.

To address these current limitations, this communication presents an innovative biosensing strategy using photosensitizers (PS) to generate singlet oxygen (¹O₂) upon illumination in photoelectrochemical (PEC) detection. The main advantage of PS, pioneered by our research group, is their light-activated measurement, which allows a clear distinction between signal and background by controlling the light source using enzyme-free labels^[1]. Ongoing efforts to exploit the unique capabilities of ¹O₂-based PEC bioplatforms will be presented, in particular the incorporation of locked nucleic acids (LNA) to improve mismatch recognition^[2] and loop-mediated isothermal amplification (LAMP) to increase sensitivity using DNA isolated from cell lines containing KRAS mutations, clinical biomarkers for assessing predisposition risk and predicting therapeutic response in CRC^[3]. This synergistic combination (Figure 1) holds great promise for overcoming gaps in the state-of-the-art and contributing to further progress in CRC diagnostics and personalized medicine using biosensing technologies.

Uncaptioned visual

^[1] S. Trashin et al., Nat. Commun, 8 (2017) 16108. ^[2] R. Sebuyoya et al., Sens. Actuators B Chem., 394 (2023) 134375. ^[3] K. Ondraskova et al., Anal. Bioanal. Chem., 415 (2023), 1065-1085.

The financial support of SOCan consortium (iBOF/23/030) and FWO Junior postdoctoral fellowship (1251525N) are gratefully acknowledged.

Keywords

Photoelectrochemical biosensor

LAMP amplification

Colorectal cancer

KRAS mutations

P1.39 Neutrophil Extracellular Trap Quantification in Microarray

Shan Wang, Jia Qiu, Xianwen Tan, Yizhe Zeng, Xiaoyu Zhou, Mengsu Yang

City University of Hong Kong, Hong Kong

Abstract

Neutrophil extracellular traps (NET), composed of decondensed chromatin and antimicrobial proteins, are crucial in the innate immune response against pathogens. However, current NET visualization techniques, such as morphological assessment and ELISA, are often limited by subjectivity, time intensity, and specificity. To address these limitations, there is an emerging demand for high-throughput, objective NET detection methods that ensure reproducible data through careful sample preparation. In this study, we introduce a novel, high-throughput aptamer-based microarray assay for the objective detection and quantification of NETs, overcoming the limitations of traditional methods. The microarray confined the NETosis restricted spatial domain to minimize the quantification difficulties due to DNA structure entanglement in NETs. The assay leverages a fluorescent aptamer targeting neutrophil elastase (NE) and nuclei acid dyes to measure NETs, optimizing sensitivity by minimizing background fluorescence. Validated against NE-antibody with time-lapse microscopy, the assay demonstrated a strong correlation with PMA-induced NET formation. It was successfully applied to compare NETosis rates in cancer patients and healthy controls, offering a rapid, scalable alternative to existing techniques.

Keywords

Neutrophil extracellular trap

aptamer

microarray

circulating tumor cells

P1.40 A Two-Pot, Quadruple-Signal Amplified Nanozymatic Biosensor for Ultra-Sensitive Detection of Salmonella Typhimurium in Foods

Lizhou Xu^1,2, Xingkai Hao^1,2, Yuhao Wen², Danyang Li³

¹Zhejiang University, China. ²ZJU-Hangzhou Global Scientific and Technological Innovation Center, China. ³Sun Yat-Sen University, China

Abstract

In-field detection of foodborne pathegens is of high importance. We developed a nanozymatic colorimetric biosensor for the rapid and ultra-sensitive detection of Salmonella Typhimurium (S.T.) in food samples, featuring simplified two-pot sample preparations and quadruple-signal amplifications.

A novel nanozyme is synthesized through rolling circle amplification (RCA) to produce repeated S.T. aptamers and gold nanoparticle (AuNP) hybridization sites on the surface of generation 6.5 dendrimers (G6.5), which is followed by hybridization with ssDNA-AuNPs probes to generate RCA-G6.5-AuNPs complexes. The morphology and structure of nanocomplexes were thoroughly characterized using TEM, FTIR, fluorescent microscopy. For food samples detection, the RCA-G6.5-AuNPs complexes specifically capture S.T. in a sandwich format with S.T. antibody-labeled magnetic nanoparticles, followed by a magnetic separation to remove food matrixes. Subsequently, resulting sandwich complexes react with glucose and MnO₂ nanosheets to initiate cascade reactions, where H₂O₂ and gluconic acid are generated in positive samples to trigger MnO₂ decomposition, inhibiting color formation with the TMB substrate.

Results showed that the RCA-G6.5-AuNPs complexes significantly enhance detection signals owning to their dual-amplification mechanisms: 1) G6.5 dendrimers offer multiple binding sites, facilitating multi-copy RCA product conjugation, and 2) RCA products create abundant AuNP binding sites, greatly increasing AuNPs loading. Additionally, H₂O₂ and gluconic acid synergistically reacting with MnO₂ nanosheets provide a third amplification stage. Results also showed that the sensor exhibits a linear detection range of 10–10⁶ CFU/mL and a detection limit of 5 CFU/mL. Tests on real samples (milk and beef) yielded recoveries of 93.3%–107.3% and high accuracy (RSD < 10%), highlighting the excellent detection efficacy of the biosensor.

The advantages of our biosensor, including simplicity, sensitivity, and robustness, suggest a practical approach for the rapid and sensitive detection of foodborne bacteria.

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Scheme 1. Schematic illustration of the biosensor.

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Figure 1. Detection performance of the biosensors for bacteria detection.

Keywords

rolling circle amplification

dendrimers

cascade enzymatic reactions

foodborne pathogens

P1.41 Aptamer Functionalized Sodium Alginate-based Conducting Hydrogel Scaffold for Electrochemical Sensing of Virulence Factor of H. pylori: CagA

Amit K. Yadav¹, Damini Verma², Dhiraj Bhatia¹

¹Department of Biological Sciences and Engineering, Indian Institute of Technology Gandhinagar, Gujarat-382355, India. ²Centre for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India

Abstract

Colonization of the human stomach by Helicobacter pylori (H. pylori) significantly raises the risk of gastritis, ulcers, and gastric cancer. The primary virulence factor facilitating its persistence in gastric cells is the protein cytotoxin-associated gene A (CagA). Consequently, there is considerable interest in creating a scalable, affordable, and adaptable biosensor platform for the rapid and sensitive detection of CagA, which could be highly valuable for healthcare and medical research. We developed a flexible aptasensing platform utilizing a hierarchically structured sodium alginate (SA)-based conductive polymer hydrogel to detect the H. pylori virulence factor, CagA. The synthesized SA hydrogel was thoroughly characterized using various techniques, including X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), elemental mapping, atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FT-IR) and Raman spectroscopy. To achieve covalent attachment of the CagA aptamer to the SA hydrogel-modified ITO electrode, we employed 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) coupling chemistry. Electrochemical surface characterization of the aptasensor at each fabrication stage was performed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). Thanks to the distinctive properties of conductive SA hydrogels, such as high permeability to biological substrates and fast electron transfer, our aptasensor exhibits outstanding sensing capabilities. It offers a broad linear detection range (0.1–80 ng mL⁻¹), high sensitivity [22.64 μA log₁₀(mL ng⁻¹) cm⁻²], a low LOD (0.17 ng mL⁻¹), LOQ (0.54 ng mL⁻¹), and a rapid response time (~14 minutes). With the ease and scalability of hydrogel processing, this conductive SA hydrogel-based aptasensor platform holds significant potential as a cost-effective sensor kit to enhance patient prognosis and quality of life. This work presents an important advancement toward creating a non-invasive, highly sensitive diagnostic platform for the rapid detection of pathogenic bacteria, specifically for CagA.

Keywords

Conducting Hydrogel

Aptasensor

Sodium alginate

Cancer

P1.42 Plasmonic fiber optic absorbance biosensor for amplification-free nucleic acid detection

Udiptya Saha, Tanushree Mana, Narayanan Madaboosi, V. V. Raghavendra Sai

Indian Institute of Technology Madras, India

Abstract

Detection of nucleic acid biomarkers offers critical potential for clinical applications, enabling early, rapid, and specific diagnoses of diseases, ranging from infections to cancers. The widely used PCR technique, while reliable, has been largely confined to centralized laboratories due to its limitations, such as the need for thermal cycling and specialized reagents. The development of an amplification-free quantification of DNA/RNA targets, as proposed in this work, has the potential to revolutionize diagnosis by making this technology simple and accessible to resource-limited settings.

Here, we present a plasmonic fiber optic absorbance biosensor (P-FAB) designed to detect nucleic acid targets down to ultralow concentrations. This assay uses a U-bent fiber optic sensor (U-FOS) with capture oligonucleotides (cONs) immobilized on the sensor surface and gold nanoparticles (AuNPs) conjugated with detection oligonucleotides (dONs) as labels. The cONs and dONs are designed such that they are partially complementary to the target ON, facilitating a sandwich hybridization on the sensor surface. In this assay, the cON-coated U-FOS probe is first incubated in the target solution for 1 hour, followed by immersion in AuNP-dON conjugate solution. The target recognition is realized and quantified in real-time by monitoring the absorption of light propagating through the U-FOS probe, which is coupled to a green LED and photodetector. Our findings emphasize the significance of optimizing several parameters, including cON concentration (100 nM), choice of blocking agent and its concentrations for effective surface blocking (aminated hexaethylene glycol, 50 mM for 30 min), and AuNP label size (30 nm), all of which contribute to achieving desirable dynamic range, high sensitivity and lower detection limits. The optimised parameters will be employed for specific detection of respiratory tract pathogens in clinically relevant ranges. This P-FAB approach offers a promising pathway for sensitive and cost-effective nucleic acid detection, potentially advancing point-of-care diagnostics.

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Keywords

Carbon dots

Aptasensor

Vancomycin

Therapeutic drug monitoring

P1.47 Development of aptamer-based hydrogel with gold nanoparticles for sensitive neurotransmitter detection by enhanced holding ability of target molecules

Chang-yun Lee, Seungsoo Kim, Minjeong Kim, Hyeon-Yeol Cho

Kookmin University, Republic of Korea

Abstract

Aptamers are short, single-stranded DNA or RNA molecules that bind selectively to their target molecules with high affinity and specificity. Because of these properties, aptamers have been widely used in disease-specific diagnostic kits and sensors to detect specific substances. However, the binding of aptamers to their target molecules is not permanent, and sensors using monolayer aptamers suffer from dissociation of the target molecules. This phenomenon is a limitation when the product needs to be continuously detected while changing the culture medium, such as in cell monitoring. To overcome this, we developed a hydrogel with aptamers synthesized in three dimensions and evaluated its sensing performance using dopamine, a biomarker for Parkinson's disease, as a model material. The hydrogel was fabricated by the rolling circle amplification method and incorporated RGD-bound gold nanoparticles (AptaGo hydrogel) to enhance conductivity and cell binding. Confocal microscopy confirmed that the hydrogel exhibited a higher retention property for dopamine compared to the aptamer. The hydrogel also selectively captured dopamine by discriminating against L-dopa, a structurally similar molecule. Electrochemical measurements showed that the AptaGo hydrogel exhibited higher sensitivity for dopamine compared to aptamer monolayers and bare aptamer hydrogel, with a linear increase with concentration, indicating its potential as a sensor. Furthermore, electron microscopy and immunofluorescence analysis confirmed that stem cells cultured on AptaGo hydrogel not only stably adhered but also promoted differentiation into neurons. In conclusion, the developed AptaGo hydrogel was not only capable of highly sensitive detection of target substances, but also applicable to cell differentiation monitoring, demonstrating its potential as a multifunctional platform for biomedical applications.

Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00211360) and Biomaterials Specialized Graduate Program through the Korea Environmental Industry & Technology Institute (KEITI) funded by the Ministry of Environment (MOE).

Keywords

Apta-hydrogel

Biosensor

Electrochemical analysis

Biomedical application

P1.48 Towards the point of care detection of Phytophthora palmivora, the coconut bud rot pathogen

Remya Eapen, Jose Joseph

Kerala University of Digital Sciences, Innovation and Technology, India

Abstract

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Coconut industry is the bread and butter for millions of lives engaged in producing, processing, and marketing kernel, inflorescence, shell, and coconut water-based products. In 2022, 466,941 million tons of desiccated coconut were imported worldwide. Apart from this, more than 15 value added products are being made from coconut and coconut trees. Coconut trees typically take about 6 to 10 years to start producing fruit after planting. Phytophthora palmivora, a species of oomycete, can severely affect coconut trees, leading to disease and potentially death, particularly through bud rot and stem bleeding diseases. The inter-tree and intra-tree spread of disease by these oomycetes can happen in a few weeks time, leading to huge economic loss, considering the long time from seed to fruit.

A quick, simple, and accurate method to detect the disease at a low cost can help develop point-of-care devices to detect Phytophthora palmivora. In this paper, we report a biosensor for detecting Phytophthora palmivora through DNA hybridization. This biosensor wields differential pulse voltammetry (DPV) to quantify the presence of the analyte. Gold nanostructures electrodeposited onto a stainless steel substrate from a precursor solution is used as the working electrode of the sensor. It facilitates the formation of a self-assembled layer of thiol-modified single-stranded probe sequences on the surface. The target sequence is a 30-mer oligonucleotide of Phytophthora palmivora isolate obtained from GenBank. The change in peak current of differential pulse voltammetry before and after the hybridization of the single-stranded target oligonucleotides with their complementary probe sequences immobilized on the surface of the working electrode is normalized and recorded as ΔI_p %. The fabricated sensor yielded a sensitivity of 1.7 ΔI_p % per decade change in the molarity of the target when the target concentration varied in the range of 1 µM to 1pM.

Keywords

Biosensors

Electrochemical

Plant disease

DNA

P1.49 Osmium-based organometallic complexes as highly stable redox reporters for electrochemical aptamer-based sensors

Andrea Montón-Vicente^1,2, Lucía Morillo-Victorero^1,3, Eduardo Buxaderas^2,4, David Díaz Díaz^2,5, María Alba¹

¹ARQUIMEA Research Center, Spain. ²University of Laguna University Institute of Bio-Organic Antonio González, Spain. ³Institute of Chemical Research of Catalonia, Spain. ⁴South Institute of Chemistry, Argentina. ⁵University of La Laguna Department of Organic Chemistry, Spain

Abstract

Electrochemical aptamer-based (EAB) sensors enable rapid, cost-effective, and continuous monitoring of biomarkers, with their performance being highly reliant on the redox reporter attached to the aptamer. To date, only a limited number of redox reporters have been employed for aptamer labeling in EAB sensors, with methylene blue serving as the gold standard. This redox marker has limitations such as pH sensitivity and a redox potential that overlaps with secondary and non-specific biological redox processes. This is an enormous constraint for achieving EAB sensors with the highly demanding features required for proper signaling and long operational stability in biological fluids. Thus, there is a critical need to develop new, pH insensitive, electrochemically reversible and chemically stable reporters for EAB sensing. Here, we present a comprehensive study of various osmium-based organometallic complexes as candidates for redox labeling of aptamers. On the basis of an Os(II/III) complex previously reported in the literature, we designed and synthesized new organometallic structures and evaluated them electrochemically both in solution and covalently attached to aptamers in different chemical environments. We explored alternative ligands with different electronic properties to study the electronic effects on the redox potential of the whole complex, laying the foundation for understanding the structure‒redox relationship. Furthermore, we tested different DNA linkage chemistry through amide coupling and click chemistry and its influence on the stability of the resulting electrochemical signal. We also compared the performance of these Os(II/III) complexes attached to aptamers with different sequences, lengths and linkers, suggesting appropriate combinations for use in EAB sensing. With our results, we aim to contribute to the advancement of novel redox reporters with outstanding properties for aptamer labeling and thus to the development of EAB sensors with longer lifetimes and superior operational stability.

Keywords

Electrochemical aptamer-based sensors

Osmium-based complexes

Redox reporters

Organic synthesis

P1.50 A novel aptasensing interface based on the electrodeposition Au-nanodendirites on microfabricated gold band array electrodes for DMD diagnosis

Gözde Aydoğdu Tığ¹, Serap Evran², Vuslat B. Juska^3,4

¹Ankara University, Turkey. ²Ege University, Turkey. ³Tyndall National Institute, Ireland. ⁴Northwestern University - Chicago Campus, USA

Abstract

Duchenne Muscular Dystrophy (DMD) is a rare genetic muscle disease in boys that causes progressive muscle wasting and weakness. DMD is the most common muscle disease of childhood, and its incidence is reported to be approximately one in 3500-4000 boys worldwide [1].

Early diagnosis of DMD by applying rapid and sensitive techniques that allow quantitative determinations of the specific biomarkers is highly important to slow down the disease's progress and determine the appropriate treatment. Recent studies have shown the available DMD-specific biomarkers in bodily fluids, such as urine. Therefore, a non-invasive diagnostic method specific to urinary biomarkers is urgently needed to diagnose diseases early and monitor their progression. Recent studies support the high potential of titin N-terminal fragments detected in urine samples of DMD patients as a quantitative biomarker in diagnosing the disease [2-3]. Ultramicron-scaled microfabricated electrodes/chips have been gaining attention in particularly in the field of biosensors, due to the excellent adaptation of the electroactive surface area with varying size and dimensions. The aptamer sequence for the titin N-terminal fragment was identified by the SELEX (systematic evolution of ligands by exponential enrichment) method. Then, this specific and novel aptamer was applied as a biorecognition element on the ultra-micro Au electrode, which is used to detect the titin N-terminal fragment with high selectivity and provide quantitative data on the protein concentration. Various experimental parameters of the realized DNA aptamer-based ultra-micro structured interface were studied and optimized using electrochemical techniques.

Magdolna Casian^1,2,3, Oana Hosu-Stancioiu¹, Ioana Manea¹, Dimas Suárez², Natalia Díaz², María Jesús Lobo Castañón^2,3, Noemí de-los-Santos-Álvarez^2,3, Cecilia Cristea¹

¹Department of Analytical Chemistry, Faculty of Pharmacy, Iuliu Hațieganu University of Medicine and Pharmacy Cluj-Napoca, 4 Pasteur Street, 400349, Cluj-Napoca, Romania. ²Departamento de Química Física y Analítica, Universidad de Oviedo, c/Julián Clavería 8, 33006 Oviedo, Spain. ³Instituto de Investigación Sanitaria del Principado de Asturias, Av. de Roma s/n, 33011, Oviedo, Spain

Abstract

In recent years, the emergence of antibiotic-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), has become a significant challenge in the treatment of bacterial infections, particularly in patients in the intensive care unit. Glycopeptide antibiotics, such as vancomycin, are the first-line treatment for severe infections caused by MRSA, thus monitoring is crucial for proper dosing and for optimizing the patient’s clinical outcome. Due to its narrow therapeutic window and concentration-related toxicity, vancomycin should be prescribed and monitored in a highly personalized manner [1].

In the field of precision medicine, aptamers have emerged as an innovative class of biorecognition elements due to their enhanced stability and specificity, holding great promise for the development of point-of-care monitoring systems [2].

In this study, magnetic bead-assisted SELEX technology was applied for the screening of novel vancomycin-specific aptamers. The designed strategy and the introduction of negative and counter-selection steps allowed the selection of specific aptamers after only seven rounds of selection, capable of discriminating vancomycin from other structural analogs. The affinity of the most promising candidates was evaluated by surface plasmon resonance, obtaining dissociation constants in the nanomolar range. The structure of the aptamer with the highest affinity was predicted computationally, and its complex with vancomycin was characterized by docking and molecular dynamics calculations. Studies regarding the development of an electrochemical aptasensor for monitoring vancomycin in the serum of patients in the intensive care unit will be presented.

References:
[1] Swartling M, et al., Int J Antimicrob Agents. 2024 Jan 1;63(1):107032.

[2] Liu Y, et al., ACS Sens. 2024 Jan 26;9(1):228-35.

Acknowledgements:

This work was supported by a grant of Romanian Ministry of Education and Research PN-IV-P8-8.1-PRE-HE-ORG-2023-0076 contract no. 26 PHE/2022, Spanish Ministerio de Ciencia e Innovación Project PID2021-123183OB-100 MICIN/AEI/10.13039/501100011033/FEDER UE and Iuliu Hațieganu UMF internal grant no. 779/1/13.01.2025.

Keywords

antibiotic resistance

aptamer

therapeutic drug monitoring

electrochemical aptasensor

P1.54 A high sensitive fluorescent aptasensor for detecting β-amyloid oligomers as Alzheimer’s disease biomarker based on catalytic hairpin assembly and magnetic separation

Chun-Hsien Chen¹, Chun-Chi Wang², Yen-Ling Chen¹

¹National Chung Cheng University Department of Chemistry and Biochemistry, Taiwan. ²Kaohsiung Medical University School of Pharmacy, Taiwan

Abstract

The abnormal accumulation of beta amyloid peptides (Aβ) in brain tissues was deduced as one of determining causes of Alzheimer’s disease (AD). Besides, Aβ was also found neurotoxic to brain tissues in its oligomeric form (AβO) which was regarded as a biomarker of AD. Therein, a fluorescent aptasensor coupled with the catalytic hairpin assembly (CHA) and the magnetic separation was developed for the detection of AβO. The technique of CHA is a non-enzymatic circular reaction based on the migration and the displacement among two metastable hairpins and an initiating fragment (Ini). The Ini can facilitate the hybridization between two hairpins. To satisfy strategies of recognizing AβO and triggering the CHA reaction, magnetic beads modified with duplexes of AβO-specifically binding aptamer (Apt) and CHA initiating fragment (Apt-Ini@MBs) were constructed. AβO can bind with Apt and induces the detachment of Apt out of Ini@MBs. The free Ini on MBs can further initiate the CHA reaction achieved by hairpin 1 (H1), hairpin (H2) and DNA duplex labeled with FAM and BHQ (F-Q). The production of H1-H2 duplex can further competitively hybridize with FAM-labeled fragment (F) and form to be the complex of H1-H2-F which possesses a turn-on of fluorescence and can serve as the signal indicator corresponding to the level of AβO. The linear range of this sensor can be obtained to be from 10 pg/mL to 10 ng/mL with a limit detection as 4 pg/mL (r = 0.9821). Besides, this technique was also applied to plasma samples from healthy individuals and levels of AβO were ranged from 87 pg/mL to 290 pg/mL. The outcome demonstrated that the proposed method possessed the potential to be a tool for the early diagnosis of AD.

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This study presents a novel nonessentials-guided in situ aptamer truncation strategy to enhance the analytical performance of aptamer-based biosensors by optimizing the sequence and structure of aptamers for improved target binding affinity and specificity. Aptamers, widely used as bio-recognition elements, often contain nonessential nucleotides that hinder effective target binding due to steric hindrance or self-hybridization, thereby limiting sensor performance. Our strategy utilizes bidirectional exonuclease digestion to selectively remove these unbound regions in situ, while preserving the critical binding domains.

Applying this strategy to vaspin-binding aptamers, we successfully developed a pair of engineered aptamers, V1(3′-5′)–26 and V49(3′-5′)–30. These aptamers showed distinct binding sites on the vaspin protein, as confirmed through molecular docking simulations and mutation studies. In addition, surface plasmon resonance (SPR) analysis demonstrated significantly enhanced binding specificity and affinity compared to the original sequences.

Incorporating this engineered aptamer pair into a sandwich-type electrochemical biosensor significantly improved analytical performance. Chronoamperometric (CA) measurements showed a 16-fold improvement in sensitivity, achieving a limit of detection (LOD) of 57 pM in buffer and 84 pM in serum. Importantly, this level of sensitivity enables reliable detection of vaspin within its physiological concentration (0.2–2.5 ng/mL) in human serum, facilitating its application in the diagnosis and monitoring of metabolic disorders such as obesity and type 2 diabetes.

This study highlights the potential of our novel aptamer engineering strategy to enhance the performance of biosensors targeting various analytes. This advancement opens new pathways for developing cost-effective, highly sensitive, and point-of-care diagnostic tools.

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Keywords

Exonuclease

Engineered aptamer

Electrochemical biosensor

Vaspin

P1.60 Novel amperometric aptasensor for creatinine detection

Daniela Flamino, Katherine Bettencourt

University of Lisbon, Portugal

Abstract

Chronic kidney disease (CKD) impacts over 850 million people worldwide and caused over 3.1 million deaths in 2019, making it the 8th leading cause of death [1]. This non-communicable disease can lead to renal failure, requiring dialysis or a transplant; thus, early diagnosis and monitoring are crucial to slow progression and improve outcomes. Creatinine, a muscle metabolism byproduct, serves as a primary biomarker of kidney function, rising as impairment worsens. This works presents a novel surface-modified aptamer biosensor for continuous creatinine monitoring [2].

In the methodology, a gold screen-printed electrode was modified with electrodeposited gold nanoparticles to improve signal sensitivity. Polydopamine - a biocompatible polymer known for its exceptional adhesive properties - and ethanolamine film was deposited over the gold nanoparticles, establishing a robust binding with the recognition element [3]. This surface allows binding aptamers via 1,2-Michael addition or thiol-gold interactions while ethanolamine minimizes nonspecific adsorption. To refine specificity, 11-mercapto-1-undecanol was introduced as a blocking agent. Experiments were performed in diluted human serum (Figure 1) after surface optimization in phosphate-buffered saline (PBS) medium.

Uncaptioned visual

Figure 1 - Step-by-step modification of electrodes for creatinine detection.

The sensor detects current changes through interactions between creatinine and the aptamer with a methylene blue redox probe. Upon binding, electron transfer is hindered by large molecules in human serum, producing an amperometric response inversely proportional to creatinine concentration. Calibration demonstrated a strong linear correlation between current response and creatinine concentration, with a stabilization time of around four minutes, validating our work’s potential for CKD monitoring (Figure 2).

Uma imagem com texto, captura de ecrã, file, GráficoDescrição gerada automaticamente

Figure 2 - Creatinine concentration calibration curve in human serum.

References:

[1] Institute for Health Metrics and Evaluation. GBD results. Available from: https://vizhub.healthdata.org/gbd-results/

[2] Ferguson MA, Waikar SS. Clinical Chemistry. 2012;58(4):680–689. doi: 10.1373/clinchem.2011.167494.

[3] Almeida LC, et al. Scientific Reports. 2021;11(1). doi: 10.1038/s41598-021-81816-1.

Keywords

creatinine

aptamer-based biosensor

electrochemical detection

continuous monitoring

P1.61 A TF-based estrogen biosensor optimized using machine learning

Scott Gaines, Patrick Lally, Omri Yosfan, James Galagan

Boston University, USA

Abstract

Allosteric transcription factors (aTFs) are a class of regulatory proteins that modulate their DNA-binding affinity in response to small molecule binding. While traditionally used in in vivo biosensors, aTFs are now being adapted for real-time, ex vivo applications. Previously, our lab created the first biosensor of this class with our optical progesterone sensor. We further demonstrated that selecting different binding sequences for the progesterone aTF can be used to tailor sensor performance. Here we apply the same strategy to develop an ex vivo aTF-based biosensor for estrogen.

An estrogen-responsive aTF was first identified from a steroid-degrading microbe. We have experimentally confirmed the aTF’s ability to bind to a putative operator DNA sequence and to modulate this binding in response to estrogen. We then employ a machine learning approach to optimize sensor performance by quantitatively designing DNA binding sequences. This approach utilizes our tool BoltzNet: a biophysically motivated and interpretable neural network that accurately predicts aTF binding energy from DNA sequence. Once kinetically calibrated for a given aTF, the model can be used to create sensors with desired quantitative performance specifications (e.g. limit of detection (LOD), dynamic range). In this presentation, we will describe the results of this optimization and the performance of our estrogen aTF-based sensor.

Keywords

Transcription Factors

Estrogen

Machine Learning

P1.62 CRISPR-Cas13a-mediated Digital Profiling of Single Cancer Exosomes: Simultaneous Detection of Surface Proteins and miRNAs through Selective Fusion in Droplet Microfluidics

Shu Xiao, Jingyu Shi, Mo Yang

The Hong Kong Polytechnic University, Hong Kong

Abstract

The heterogeneity of breast cancer results in suboptimal performance of traditional and emerging technologies in detecting single exosomal biomarkers, thereby limiting their potential for clinical application. Therefore, simultaneous detection of two different types of biomarkers, i.e., protein and microRNAs (miRNAs) combined biomarkers can aid in more accurately identifying specific cancer types and subtypes, thereby guiding personalized treatment. We developed an innovative exosome-selected protein miRNAs one-stop biosensor based on a digital microfluidic chip, which contains an array of one million droplets capable of in situ simultaneous detection of various exosomal biomarkers, such as surface protein HER2 and miR-21. These droplets self-assemble into high-density three-dimensional spheroid structures within a large observation chamber, enabling visualization and analysis of individual exosomes. We developed an aptamer-functionalized liposome that specifically recognizes exosomal surface protein receptors through a surface fluorescence-labeled nucleic acid aptamer probe, which restores fluorescence signal via conformational change, and subsequently selectively fuses with target exosomes. This strategy employs liposome-mediated membrane fusion to transfect the CRISPR/Cas13a system into exosomes, achieving precise and sensitive detection of tumor-associated miRNAs. This approach allows ultrasensitive detection of exosomal miRNAs and membrane proteins without RNA extraction. This method significantly reduces false positive rates by providing a more comprehensive cancer biomarker profile. It increases the accuracy of cancer diagnosis and staging monitoring to nearly 100%, which is crucial for effective cancer treatment and favorable prognosis.

Keywords

Droplet Digital Exo

one-stop biosensor

selective membrane fusion

CRISPR-Cas13a

P1.63 A Novel Aptameric Biosensor Based on Germanene Nanosheet for Rapid and Sensitive BRCA1 Mutation Detection

Yingying HUANG¹, Shu Xiao¹, Siu Hong Dexter WONG², Mo YANG¹

¹The Hong Kong Polytechnic University, Hong Kong. ²Ocean University of China, China

Abstract

The breast cancer 1 (BRCA1) gene is a well-known tumor suppressor, yet its role in T cells suggests a potential to enhance anti-tumor immunity. Consequently, the sensitive detection of mutated or defective BRCA1 is crucial for optimizing T cell-mediated breast cancer therapy. In this study, we introduce a novel biosensor utilizing methylgermane (GeCH3), a two-dimensional nanomaterial that remains underexplored in biosensing applications. We investigated the correlation between the size of GeCH3 nanosheets and their photophysical properties. We demonstrated that GeCH3 nanosheets exhibit a broad absorption spectrum and strong fluorescence emission at 640 nm. Building on these properties, we developed an aptamer-based GeCH3 nanosheet platform to detect the BRCA1 gene. Our results indicate that this platform enables rapid detection of BRCA1 at picomolar (pM) concentrations within 30 minutes through ratiometric fluorescence sensing. Furthermore, we extracted and amplified the mature BRCA1 gene from both activated and non-activated CD8+ T cells, employing our platform for nucleic acid detection. This work not only pioneers the application of GeCH3 in nucleic acid biosensing but also elucidates the impact of BRCA1 mutations on T cells, thereby laying the groundwork for future biomedical sensing applications using GeCH3 nanosheets

Keywords

methylgermane nanosheet

BRCA1

CD8+ T cells

nucleic acid biosensing

P1.64 Simultaneous detection of inflammatory cytokines in biological fluids with an innovative aptasensor

Maria-Bianca Irimes¹, Mihaela Tertis¹, Alexandra Pusta^1,2, Radu Oprean¹, Cecilia Cristea¹

¹Analytical Chemistry Department, Faculty of Pharmacy, Iuliu Haţieganu University of Medicine and Pharmacy, 4 Louis Pasteur St., Cluj-Napoca, Romania, Romania. ²Medical Devices Department, Faculty of Pharmacy, Iuliu Haţieganu University of Medicine and Pharmacy, 4 Louis Pasteur St., Cluj-Napoca, Romania, Romania

Abstract

Cytokines are signaling biomolecules involved in cell growth, immune response, inflammation, and cancer-related mechanisms. Due to their critical roles, cytokines are important biomarkers for diagnosing specific medical conditions and assessing responses to pharmacological treatments ¹^,². This study aimed to develop a customized platform for the simultaneous electrochemical detection of Interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α) in biological fluids.

Customized electrochemical cells were printed in-lab. The working electrodes were modified with Au and Pt nanoparticles to increase sensitivity, and two specific aptamers labeled with two different redox labels to ensure specificity. All modification steps were confirmed using cyclic voltammetry and electrochemical impedance spectroscopy and the detection was performed by cyclic voltammetry. The optimized platform was applied for real sample analysis represented by saliva and sweat collected from patients and healthy subjects. The developed platform was characterized through electrochemical and morphological techniques and subsequently applied for the simultaneous detection of IL-6 and TNF-α. Key analytical parameters, including limit of detection, limit of quantification, and sensitivity for cytokines were assessed. The aptasensor was able to determine the analytes on a linear domain between 5- 5000 pg/mL with an LOD of 1.7 pg/mL. Additionally, the platform was tested on real samples, highlighting its applicability in clinical practice. The developed sensor enables the specific simultaneous electrochemical detection of IL-6 and TNF-α, highlighting its suitability for medical applications.

Acknowledgment. This work was supported by the Romanian Ministry of Education and Research, CNCS-UEFISCDI, project number PN-IV-P8–8.1-PRE-HE-ORG-2023-0076. M-B. Irimeș thanks the Iuliu Hațieganu UMF internal grant no.779 /2.13.01.2025.

References

1. Irimes MB et al. Multiplexed electrochemical sensing devices for chronic diseases diagnosis and monitoring. TrAC Trends Anal Chem. 2024 Mar 1;172:117560.

2. Arévalo B, et al. Simultaneous electrochemical immunosensing of relevant cytokines to diagnose and track cancer and autoimmune diseases. Bioelectrochemistry. 2022 Aug 1;146:108157.

Keywords

simultaneous detection

aptasensor

cytokines

real samples

P1.65 Aptamer – based biosensors: towards electrochemical monitoring of wounds healing using printed electronics

Marta Jarczewska¹, Julia Czopinska¹, Jakub Moszczynski¹, Andrzej Peplowski²

¹Warsaw University of Technology, Faculty of Chemistry, The Chair of Medical Biotechnology, Poland. ²Warsaw University of Technology, Centre for Advanced Materials and Technologies CEZAMAT, Department of Printed Electronics, Textronics and Assembly, Poland

Abstract

As the wound care market costs estimate up to 96.8 billion annually [1], there is a need for development of methods allowing for precise determination of the stage of healing on skin surface. Contemporary, wound stage can be judged upon visual inspection, cell cultivation taken from wound exudate or pH measurement [2]. Unfortunately, those methods require frequent removal of wound dressing and might not deliver an immediate information on actual condition of inflamed area.

The alternative approach could be determination of concentration of certain cytokines which elevated level might indicate the progression of inflammation both in acute and chronic wound. This can be achieved by application bioreceptor elements of high specificity towards cytokines such as DNA aptamers. Such measurements could be executed by placing a biosensing layer on printed electronics that would be formed directly on wound dressing.

In this presentation we will focus on characterization of various carbon-based surfaces including glassy carbon, edge-plane pyrolytic graphite as well as screen printed electrodes fabricated using graphene nanoplatelets’ paste for immobilization of aptamers specific to interleukin-6 and tumor necrosis factor alpha. The studies referred to selection of aptamer tethering method as well as experimental conditions to carry out voltammetry and impedimetric measurements. The possibility of aptamer layers formation on carbon materials was also studied using imaging methods such as atomic force microscopy. Further research was aimed on determination of aptasensor working parameters as well as its testing using artificial exudate samples.

[1] C. K. Sen, Adv Wound Care (New Rochelle), 2019, 8(2), 39–48.

[2] G. Kallstrom, Journal of Clinical Microbiology, 2014, 52 (8), 2753–2756

This work was financially supported by the Warsaw University of Technology: IDUB program Young PW 2 grant no. 504/04496/1020/45.010032 “Studies on the aptamer-based layers properties combined with printed electronics for electrochemical monitoring of wounds healing”.

Keywords

Aptamers

Electrochemistry

Wearable sensors

Protein biomarkers

P1.66 Fabrication and characterization of a novel highly porous graphene-gold microelectrode based on laser-induced graphene for molecular biosensing of uropathogenic E. coli

genosensor

cancer biomarkers

P1.69 Pyrococcus furiosus argonaute and rolling circle amplification integrated multiplex platform for circRNA detection

Xinxin Ke, Tao Hu

Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, China

Abstract

Accurate detection of circular RNA (circRNA) is essential for their clinical use. However, circRNA detection is hampered by the low abundance of circRNA and interference from their linear isomers. Here, we have integrated reverse transcription-rolling circle amplification (RT-RCA) with pfAgo cleavage to achieve one-pot, sensitive and multiplexed detection of circRNA. The RT-RCA process of circRNA generated repeat units of the back-splicing junction (BSJ) serve as substrate for pfAgo cleavage and the cleaved products act as guide DNA to further activate the second cleavage of pfAgo for fluorescence detection. The method is capable of simultaneously detecting circRNAs as low as 1 fM and has shown high selectivity. In particular, the proposed method can identify specific circRNAs in real samples without the use of RNase R. Furthermore, universal non-coding RNA (ncRNA) detection such as miRNA can be achieved by the introduction of padlock probes for the RCA process. Thus, the proposed method using RCA and pfAgo cleavage holds great potential as a universal platform for diagnosing ncRNA-associated diseases.

Keywords

Circular RNA

Argonaute

RT-RCA

P1.70 Split Prism-assisted Optical Aptasensor for Ultrasensitive and Reliable Detection of SARS-CoV-2

Hyun Mo Cho^1,2, Jung Hyun Choi^1,3, Jiyoon Bu³, Dong Hyung Kim¹

¹Korea Research Institute of Standards and Science, Republic of Korea. ²SIS Sensor corporation, Republic of Korea. ³Inha University, Republic of Korea

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has caused a significant number of deaths due to infections by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Ensuring the early detection of the virus with accuracy and reliability is of great significance to the public health for preventing its spread. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard for detecting SARS-CoV-2, but is expensive and time-consuming methodology. In this study, a real-time split prism-assisted solution-immersed silicon (SP-SIS) aptasensor was designed to achieve ultrasensitive detection of SARS-CoV-2 spike proteins with high accuracy. The sensor assembly, which includes a newly designed split prism, effectively eliminates background noise, primarily caused by strong reflection from the inner prism surface. This is achieved by reflecting and absorbing the noise light with a thin metallic plate bonded between the two optical components of the split prism. To improve biosensing accuracy, the incident angle of the laser beam was precisely aligned with a low variation of ± 0.005 degrees for each newly attached sensor assembly. Surface modification of the sensor, by controlling its hydrodynamic properties, enhanced aptamer immobilization, resulting in improved detection efficiency. The SP-SIS aptasensor showed exceptional sensitivity, with detection limits of 76.8 fg/mL for spike proteins and 22.1 particles/mL for virus-like particles. The proposed system demonstrated reliable detection of SARS-CoV-2 variants and high specificity in distinguishing SARS-CoV-2 from other coronaviruses. These results suggest that this aptasensor system can provide accurate and sensitive detection of SARS-CoV-2 at early stages and monitor the rapid transmission of other infectious viruses.

Keywords

Optical aptasensor

Split prism

SARS-CoV-2

Ultra-sensitivity

P1.71 Optoelectronics properties of Polyhexylthiophene-chitosan Nanoparticles for Ochratoxin A biosensing

Fatma Besbes^1,2, Rym Mlika², Hafsa Korri-Youssoufi¹

¹Paris-Saclay University, France. ²University of Monastir, Tunisia

Abstract

Ochratoxin A (OTA), a potent mycotoxin produced by Aspergillus and Penicillium species, poses a significant threat to public health and food safety. Given the carcinogenic and nephrotoxic properties of OTA, its rapid and accurate detection in food samples has become a critical goal. This study aims to develop a dual electrochemical and fluorescence-based biosensor using poly(3-hexylthiophene) (P3HT)-chitosan nanoparticles for the detection of OTA.

The synthesis of these nanoparticles involved the integration of P3HT, a semiconducting polymer known for its exceptional photoluminescence properties and electron mobility, with chitosan, a bio-sourced biopolymer, to form stable nanoparticles with optoelectronics properties. Aptamer binding was achieved via electrostatic interactions between the negatively charged carboxyl groups of the aptamer and positively charged amine groups of chitosan. The formation of nanoparticles was verified through dynamic light scattering (DLS) and zeta potential measurements, which demonstrated particle stability, optimal size distribution, and colloidal stability in an aqueous solution.

We demonstrate that P3HT-chitosan nanoparticles exhibited distinct fluorescence emission peaks and a strong response to OTA binding, enabling sensitive detection based on fluorescence quenching properties and electrochemical sensing through the variation of the optoelectronic properties of the nanocomposite.

The nanoparticles P3HT/Chitosan based aptasensors demonstrates high sensitivity for detection of mycotoxins’ and represent an innovative platform for detection of various contaminant by tailoring the aptamer. The nanoparticles P3HT/Chitosan presents also a printing compatibility enabling sensor fabrication via low cost techniques and a potential adaptability in wearable sensors that can hold potential application in food safety and environmental monitoring.

Keywords

aptasenor

optoelectronics

ochratoxin

biopolymer

P1.72 Design of an innovative aptamer-enhanced electrochemical biosensor for the detection of Listeria monocytogenes

Giuseppe Lamberti¹, Laura Martina², Daniela Chirizzi³, Livia Giotta¹, Paola Semeraro¹, Anna Rita De bartolomeo¹, Roberta Catanzariti³, Maria Rachele Guascito¹

¹Università del Salento, Italy. ²Unviersità del Salento, Italy. ³Istituto Zooprofilattico Sperimentale di Puglia e Basilicata, Italy

Abstract

Healthcare and food industry have always been in collaboration in order to grant clean and safe food. Listeria monocytogenes is a peculiar bacterium found in various consumed foods, such as raw meat and vegetables, milk and its derivatives. Even not being lethal, people with weak immune system and other pathologies might suffer much worse problems. In order to prevent it, various methods were developed to detect this bacterium. Biosensors are obtaining an increasing interest from the scientific community, especially for their extreme versatility and efficiency. Within this vast group, Aptasensors are extremely interesting: these sensors exploit aptamers, custom-made oligonucleotides that can specifically recognized various proteins. The aptasensor was characterized through electrochemical techniques such as Cyclic Voltamemtry (CV) and Electrochemical Impedance Spectroscopy (EIS). The biosensor working process is based on the aptamer, which is custom-made to target Internalin A: this protein is responsible of the entrance of Listeria into cells, through the interaction between Internalin A and E-Cadherin. The biosensing platform is characterized by various layers. The first one is an electrodeposited Polydopamine (PDA) layer on a gold electrode, necessary in order to bind the aptamer to the electrode. The polymer is then functionalized with the aptamer, and exposed to the bacterium.

Each step of the design was characterized through electrochemical techniques such as CV and EIS, spectroscopical techniques such as FT-IR and Raman Spectroscopy, and microscopical techniques such as Scansion electronic microscopy (SEM).

Aknowledgments: The authors thank the project IZSPB 02/21 RC “Sviluppo di un sensore innovative per la ricerca quali – quantitiativa di microrganismi patogeni in matrici alimentary (BIMPA).

[1] L. Hosseinzadeh, M. Mazloum-Ardakani, Advances in aptasensor technology, Advances in Clinical Chemistry, 2020, 99, 237-279. https://doi.org/10.1016/bs.acc.2020.02.010

[2] F. Di Pietrantonio, D. Cannatà, M. Benetti, Biosensor technologies based on nanomaterials, Func. Nano. Interfaces for Env. And Biom. Applications, 2019, 181-242. 10.1016/B978-0-12-814401-5.00008-6

Keywords

Biosensor

Aptasensor

Aptamer

Electrochemistry

P1.73 Development of a Visual Biosensor for Colorectal Cancer Using Functionalized Metal-Organic Frameworks and Rolling Circle Amplification

Siang-Ren Yu, Yu-Ju Teng, Yu-Fen Huang, Wen-Chuan Ku, Hsin-Yu Yeh, Cai-Yu Pao, Cheng-Yu Lee

Chung Yuan Christian University Department of Chemistry, Taiwan

Abstract

Abstract

Surface-Enhanced Raman Spectroscopy (SERS) is emerging as an essential analytical technique due to its efficiency, simplicity, and rapidity. It enables real-time, non-destructive, and ultrasensitive detection of target analytes with exceptionally low detection limits. The primary challenge lies in developing substrates that are reproducible, cost-effective, and efficient. In this context, we have designed and optimized a novel plasmonic paper-based nanoplatform for rapid and portable SERS detection.

Two different types of gold nanoparticles were used: gold nanospheres (AuNPs) were first deposited via an immersion approach onto Whatman paper, and then gold nanorods (AuNRs) were added dropwise. Various nanoparticle concentrations were systematically tested and optimized, establishing a correlation between detection intensity, nanoparticle concentration, and their plasmonic resonance properties.

The performance of the sensor was first evaluated using 4-mercaptobenzoic acid (4-MBA), a well-known SERS probe. In a second step, we detected a simple DNA strand composed of 20 adenines (PolyA20) to determine if our sensor can be used to observe biomolecules. The limits of detection of 4-MBA and PolyA20 were determined, demonstrating the platform’s high sensitivity and versatility for studying nucleic acid interactions.

The final goal was to detect the interaction between the DNA strand, used as a bioreceptor, and RNA. In this case, RNA of 20 uracils (PolyU20) was deposited as a complementary strand to PolyA20 to study the influence of the interaction on the SERS signal.

This paper-based plasmonic nanoplatform offers a cost-effective and easily producible solution for portable SERS applications, paving the way for broader implementation in analytical and diagnostic fields.

Keywords

Nanobiosensor

Fast detection

DNA

P1.80 Cost-Effective and Highly Sensitive Inkjet-printedAptasensor for Real-Time Monitoring of EnvironmentalAntibiotic Contamination

Davi Farias^1,2, Marianna Rossetti², Andy Bruno², Ruslán Alvarez-Diduk², Giulio Rosati², Thiago Paixão¹, Arben Merkoçi^2,3

¹University of São Paulo - USP, Brazil. ²Nanobioelectronic and Biosensors Group, Catalan Institute of Nanoscience and Nanotechnology - ICN2, Spain. ³ICREA, Institució catalana de Recerca i Estudis Avançats, Spain

Abstract

The widespread presence of antibiotics in the environment represents a critical threat to ecosystems and public health, highlighting the urgent need for real-time monitoring solutions. This study introduces an aptamer-based, inkjet-printed nanostructured biosensor designed for the sensitive and selective detection of antibiotics, specifically ampicillin, in environmental matrices. Inkjet printing allows for precise deposition of nanomaterial inks, providing a cost-effective, scalable, and reproducible method for fabricating electroanalytical platforms [1]. The biosensor achieves enhanced selectivity and sensitivity by using a nanostructured gold electrode, functionalized with biorecognition elements through a robust thiol-gold chemistry bond [2]. The biorecognition element, an aptamer — short single-stranded DNA or RNA molecules — binds specifically to the target antibiotic, ampicillin, offering high specificity and stability. The aptamer is conjugated with a redox probe, methylene blue, enabling to detect the presence of the antibiotic analyte through the difference in the electron transfer observable in the absence and the presence of the target. The proposed inkjet-printed aptasensor exhibits high sensitivity, surpassing the performance of previously reported electrochemical aptasensors (E-AB)[3] through the use of nanostructured electrodes that achieve heightened sensitivity at a lower cost. This aptasensor platform represents a rapid, cost-effective alternative for in-field monitoring of antibiotic contamination, with potential applicability for clinical samples as well.

Reference:

1. Rossetti, M.; Srisomwat, C.; Urban, M.; Rosati, G.; Maroli, G.; Yaman Akbay, H. G.; et al. Biosensors and Bioelectronics 2024, 250.

2. Rossetti, M.; Brannetti, S.; Mocenigo, M.; Marini, B.; Ippodrino, R.; Porchetta, A. Angewandte Chemie International Edition 2020, 59(35), 14973–14978.

3. Yu, ZG; Lai, RY. Talanta 2018, 176, 619-624

Keywords

Inkjet-Printing

Nanostructured- Gold

Apatasensor

Environmental analysis

P1.81 Two-Dimensional Spacing optimization of PEG-Silane Monolayers for Enhanced miRNA Detection in Silicon Photonic Biosensors.

Paul Michel-Lara^1,2, Cesar Sanchez-Huertas^1,2, Apriliana Ellya Ratna Kartikasari^3,4, Guanghui Ren^1,2, Tetsuya Shimogaki^1,2, Crispin Szydzik^1,2, Magdalena Plebanski^3,4, Arnan Mitchell^1,2

¹RMIT University STEM College, Australia. ²RMIT University Integrated Photonics and Applications Centre, Australia. ³RMIT University - Bundoora Campus, Australia. ⁴CAVAL Ltd, Australia

Abstract

References

[1] D. Ozkan-Ariksoysal, Biosensors, 12 (2022) 607.

[2] H. Subak, F. F. Yilmaz, D. Ozkan-Ariksoysal, Microchemical Journal, 204 (2024) 110976.

[3] H. Subak, G. Selvolini, M. Macchiagodena, D. Ozkan-Ariksoysal, M. Pagliai, P. Procacci, G. Marrazza, Bioelectrochemistry 138 (2021) 107691.

Keywords

Aptasensor

Mycotoxin

Electrochemical DNA biosensor

Antibiotic resistance

P1.85 A Portable, Power-Free Platform for Real-Time Colorimetric Nucleic Acid Amplification Detection

Jiho Park, Min-Gon Kim

Gwangju Institute of Science and Technology, Republic of Korea

Abstract

Isothermal amplification has gained attention as an alternative method to Polymerase Chain Reaction (PCR) for nucleic acid amplification testing (NAAT) in point-of-care testing (POCT). In particular, real-time monitoring of isothermal amplification results offers notable advantages in rapid results and quantitative analysis. However, detecting fluorescence signal increases during real-time nucleic acid amplification requires expensive and sophisticated equipment and detectors. In this study, we developed a portable, power-free POCT platform that enables real-time colorimetric detection of nucleic acid amplification by integrating Reverse Transcription Recombinase Polymerase Amplification (RT-RPA) reactions with lateral flow assay on a paper chip. The developed device has the following advantages: 1) sample purification using a small, portable, power-free tip column, 2) a water-triggered, power-free heater that satisfies the temperature requirements for RT-RPA reactions, 3) a paper strip that enables simultaneous lateral flow assay and real-time colorimetric detection of the RT-RPA amplification process and 4) results can be obtained from sample preparation to answer within 30 minutes. This study is expected to be widely applicable in situations where rapid nucleic acid amplification-based diagnostics are needed, especially in resource-limited settings.

Keywords

Isothermal amplification

Real-time detection

Point of care testing

Power-free platfrom

P1.86 Application of a Selected Aptamer in an Electrochemical Magneto-Assay for Sensitive Malaria Diagnosis in Whole Blood

Briza Pérez-López¹, Liga Kunrade², Judit Prat-Trunas¹, Una Riekstina², Eva Baldrich¹

¹Vall d’Hebron Hospital Institut de Recerca, Spain. ²University of Latvia, Latvia

Abstract

Malaria is a poverty-related disease that affects especially low- and middle-income countries. Eradication of this life-threatening disease, which is caused by several species of Plasmodium parasite, will require population mass screening and treatment. Extremely cheap, fast, sensitive and easy-to-use analytical tools will be needed for the early diagnosis of symptomatic infections, including Plasmodium species identification and quantitation, but also the identification of asymptomatic carriers. Certainly, rapid tests (RDT) exist for malaria diagnosis that employ antibodies for detection of Plasmodium antigens in patient blood. However, antibodies are an expensive biocomponent of limited shelf-life. As an alternative, aptamers are short oligonucleotide sequences, selected and produced in vitro, that selectively bind to specific targets as monoclonal antibodies do.

We selected Plasmodium falciparum lactate dehydrogenase (Pf-LDH) specific ssDNA aptamers using a protein-SELEX assay. The best-performing aptamers showed nanomolar binding affinity towards Pf-LDH, with different levels of cross-reactivity to P. ovale and P. malariae LDHs. They were stable in 100% human serum for up to 3 h and in 10% human serum for up to 24 h.

One of the selected aptamers was successfully implemented as the detection receptor in a rapid electrochemical magneto-biosensor. This detected Pf-LDH between 0.5 and 15.6 ng/mL with a limit of detection below 1,0 ngꞏmL^-1and provided Pf-LDH quantitation in less than 15 min.

The results show that customized aptamers could serve as sensitive and selective synthetic receptors to develop cost-effective point-of-care biosensors for malaria diagnosis.

Keywords

Malaria

Fast Diagnostics

Plasmodium-LDH

Magnetic Beads

P1.87 A Capacitance Biosensor for Prostate Cancer Detection via Normalised Urinary Extracellular Vesicles

Khageephun Permpoka¹, Phuritat Kaewarsa¹, Wattanai Paekoh¹, Wanida Laiwattanapaisal¹, Pedro Estrela²

¹Chulalongkorn University, Thailand. ²University of Bath, UK

Abstract

Extracellular vesicles (EVs), especially exosomes, play a part in intercellular communication and can be found in biological fluids. Inevitably, EVs have emerged as a promising biomarker for cancer diagnosis. Detecting EVs in urine is inherently less invasive than blood. However, reliable EVs quantitation in urine is still challenging due to low EVs concentration, high sample variation, and lack of standardization. Herein, we introduce a capacitance-based electrochemical detection of dual markers including CD63 and prostate-specific membrane antigen (PSMA) – the former being a generic marker present in all exosomes and the latter a prostate cancer marker. With this strategy, urinary EVs detection is more reliable owing to incorporating CD63 as a normalisation marker. Integrating a capacitance technique (non-Faradaic impedance) provides highly sensitive and reagent-free technique capabilities ideal for point-of-care diagnosis.

For the sensor fabrication, both thiolated CD63 and PSMA aptamers were self-assembled on reduced graphene oxide and molybdenum disulfide (rGO/MOS₂) screen-printed electrodes. The immobilisation of the aptamers was optimised, with a 1:10 mercaptohexanol: thiolated aptamer ratio providing the best results for both CD63 and PSMA; results can be obtained in 10 minutes using 10 µL from EVs isolated samples. Under these conditions, the associated CD63 and PSMA detection limits are 1.6×10⁶and 5.2×10⁶ particles/mL, respectively. The linear range of CD63 extended from 1.83×10⁶ to 2.28×10⁸ particles/mL and for PSMA ranging from 3.66×10⁶ to 4.57×10⁷ particles/mL. We are currently evaluating the sensor’s performance with clinical samples (10 urine samples from healthy individuals and prostate cancer patients).

The capacitance sensor provides a simple, fast, reagent-free, and more reliable detection technique for urinary EVs detection to improve prostate cancer diagnosis.

Keywords

Extracellular vesicles detection

Capacitance-based biosensor

Prostate cancer

Aptasensor

P1.88 Hybridization chain reaction coupled to photoelectrochemical biosensors to advance isothermal, enzyme-free clinical diagnostics

Anouk Peymen^1,2, Alejandro Valverde^1,2, Ludmila Moráňová³, Martin Bartošík³, Karolien De Wael^1,2

¹Antwerp engineering, photoelectrochemistry and sensing (A-PECS), Department of Bioscience Engineering, University of Antwerp, Belgium. ²NANOlight Center of Excellence, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium. ³Research Center for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic

Abstract

The detection of DNA mutations is critical for cancer diagnosis, prognosis, and treatment, as these genetic alterations often drive tumor progression. PCR-based techniques remain the gold standard for DNA biomarker detection thanks to their exceptional sensitivity and specificity. However, their application in decentralized settings and point-of-care diagnostics is limited by high costs, time-intensive protocols, and reliance on enzymes and thermal cycling. Over the years, electrochemical nucleic acid biosensors have attracted significant attention as a cost-effective and user-friendly alternative, offering rapid and reliable results [1, 2]. Nonetheless, their clinical translation is hampered by their inability to achieve the high sensitivity required for identifying genetic cancer mutations.

To overcome this limitation, novel amplification approaches are being explored. Many strategies, however, remain reliant on thermal cycling or enzymes, making them susceptible to specific storage, complex sample purification, matrix inhibition and irreproducible results. Thus, a trend emerged towards enzyme-free isothermal amplification techniques, such as hybridization chain reaction (HCR) [3]. Through a cascade of repeated hybridization events involving metastable hairpin structures, HCR produces a long double-stranded DNA labeled with multiple signal molecules for each target. In that manner, it is a straightforward and powerful amplification tool, that served to be a reliable technique to enhance the sensitivity across various applications. Therefore, in this study, HCR is integrated into a photoelectrochemical biosensor for the detection of KRAS G12C, a clinically significant mutation associated with tumor progression [4]. By combining the simplicity and cost-effectiveness of photoelectrochemical biosensors with the high sensitivity and specificity required for DNA biomarker detection, this research aims to bridge the gap between experimental development and clinical implementation of a promising nucleic acid biosensor.

1. Trotter et al. Biosens Bioelectron, 2020.

2. Mariani et al. Mikrochim Acta, 2022.

3. Chai et al. ACS Appl Mater Interfaces, 2021.

4. Daems et al. Biosens Bioelectron, 2023.

Keywords

Cancer

DNA biomarker

Photoelectrochemical biosensor

Hybridization chain reaction

P1.89 A lab-on-paper device for monitoring aquatic environments on-site using environmental DNA (eDNA).

Chau Ha Pham¹, Birgitte Kasin Hønsvall², Erik Andrew Johannessen¹, Bao Quoc Ta¹

¹University of South-Eastern Norway - Campus Vestfold, Norway. ²Zimmer & Peacock AS, Norway

Abstract

Biodiversity monitoring is critical for evaluating ecosystem health, detecting environmental stress, and conserving endangered species, particularly in aquatic environments such as rivers, lakes, and oceans. It underpins sustainable resource management, public health, and economic stability by ensuring ecosystems deliver essential services like clean air and water. Environmental DNA (eDNA) analysis has emerged as a non-invasive tool for biodiversity assessment, leveraging DNA shed by organisms through biological materials (e.g., hair, feces, saliva, skin cells) found in water, soil, air, and snow. eDNA analysis provides rapid insights into species presence and ecosystem health, avoiding the limitations of traditional, labor-intensive monitoring techniques. Here, we present a lab-on-paper device that integrates sample input and multiple results outputs, offering on-site, rapid tests that can be operated by minimally trained personnel. The device consists of two main components: an eDNA concentration and extraction unit and an amplification and detection unit. The extraction unit includes multiple layers, such as dual filtration layers for debris removal and a nucleic-acid capture pad, which is modified with high-molecular-weight chitosan and silica dioxide to enhance DNA binding. Once the nucleic acids are captured, the user simply slides the capture pad into alignment with the amplification chamber, pre-loaded with a dry master mix and primers for isothermal amplification. Results are visualized by a color change: blue indicates a positive detection, while purple indicates a negative result. Our device aims to analyze and detect three targets at the same time: Esox lucius (Northern Pike), Anguilla anguilla (European Eel), and Salmo salar (Atlantic Salmon). These species are categorized as regionally invasive, endangered, and of least concern, respectively. This approach holds promise for accessible and efficient biodiversity monitoring in aquatic ecosystems.

The work was supported by the Ministry of Church and Education and the Research Council of Norway (Norfab project no. 245963).

Keywords

eDNA

Lab-on-a-paper

isothermal amplification

aquatic environments

P1.90 Graphene-based aptasensor for the detection of matrix metalloproteinases in complex samples

Annabel Pohl, Felix Hempel, Chandan Singh, Irini Petrou, Alexey Tarasov

University of Applied Sciences Kaiserslautern, Germany

Abstract

Matrix metalloproteinase-9 (MMP-9) is implicated in a range of pathological conditions, including cancers, cardiovascular diseases, and neurodegenerative disorders. Recent studies also link elevated MMP-9 levels to major depressive disorder (MDD), a highly prevalent psychiatric disorder affecting over 300 million people worldwide making it a promising biomarker for both disease progression and treatment response (https://doi.org/10.3389/fneur.2022.861843). MMP-9 plays a critical role in maintaining blood-brain barrier integrity and synaptic plasticity which are two factors central to the neurobiology of depression. Elevated MMP9 levels in patients with MDD have been associated with neural inflammation and degradation, which may contribute to the onset and persistence of depressive symptoms.

This project aims to establish a graphene field-effect transistor (GFET)-based aptasensor for MMP-9 detection in complex biological media. Using MMP9-specific aptamers, the sensor platform enables the sensitive, selective and label-free electronic measurements of MMP-9 concentrations in a variety of biological media. Initial results show a low limit of detection in the pM range with a high reproducibility. Polyethylene glycol is implemented into the surface functionalization to achieve anti-fouling properties (https://doi.org/10.1002/admt.201800186) creating a novel tool to detect proteases as MDD biomarkers.

By enabling continuous, non-invasive tracking of MMP-9 levels, this biosensor has potential as a tool for monitoring therapeutic efficacy in depression. Tracking MMP-9 responses to medication could provide clinicians with real-time insight into treatment effects, ultimately helping to personalize care for patients with MDD. In addition, this project explores the feasibility of integrating aptamer-functionalized GFETs into a multi-sensing platform, potentially advancing biosensing systems for broader psychiatric and neurological applications.

Keywords

GFET

Aptasensor

Enzyme-based biosensing

Major Depressive Disorder

P1.91 Rationally designed peptide recognition elements for miRNA: Biosensor development for cancer diagnostics

Soumya Rajpal¹, Muhammed Abdel-Hamied^1,2, Christine Kranz¹, Boris Mizaikoff^1,3

¹Ulm University, Germany. ²Cairo University, Egypt. ³Hahn-Schickard Ulm, Germany

Abstract

miRNAs have emerged as critical cancer biomarkers due to their cancer-specific expression patterns and their potential for early detection. Additionally, their stability in biofluids highlights their suitability for clinical applications using minimally invasive sampling techniques. However, certain factors –including their short and highly homologous sequences (e.g., single base mismatch), large variation in base composition (i.e., G/C or A/T rich), and the presence of secondary structures such as primary and precursor forms –limit their adaptability to diagnostic approaches, making specific detection more challenging. Biomimicking natural recognition processes involving microRNA-binding proteins can help to identify key peptide motifs that can serve as specific recognition elements for microRNA biosensors.

Highly specific and sensitive biosensing relies on high-affinity candidates, which can be identified through in silico screening of rationally designed peptide libraries tailored for target recognition. This study focuses on the computational selection of peptide-based recognition elements for the detection of miR-21, a key miRNA present in cancer cells. Four distinct peptide pools were curated based on their structural and functional properties: (1) peptides derived from RNA-binding proteins, designed to mimic key motifs involved in RNA recognition; (2) peptides enriched in basic and aromatic amino acids, aiming to enhance electrostatic and hydrophobic interactions; (3) peptides featuring both basic and acidic residues, intended to promote charge-based interactions; and (4) peptides with coplanar amino acid arrangements, selected to maximize structural complementarity. We used molecular docking and molecular dynamics simulations to analyze peptide-miRNA interactions, assessing binding affinities, stability and conformational dynamics. The findings highlight promising binding profiles for the design of peptide-based biosensors targeting miR-21 and thus contribute to the development of improved sensors for miRNA detection and quantification with potential for early cancer diagnosis.

Keywords

Cancer diagnostics

RNA sensors

in silico screening

peptide simulations

P1.92 Construction of aptamer based colorimetric assay for whole blood glycated haemoglobin (HbA1c) estimation using gold nanoparticles

Dr. Rooma Devi¹, Dr. Aman Chauhan¹, Prabhat Kumar², Ajay Singh³, Neeru Bhaskar⁴

¹Maharishi Markandeshwar Institute of Medical Science and Research, Maharishi Markandeshwar (Deemed to Be University), Mullana, Haryana, India. ²Singhania University, Pacheri Bari, Distt. Jhunjhunu, Rajasthan, India. ³Indian Institute of Technology, Jammu, Jammu and Kashmir, India. ⁴Adesh Medical College And Hospital, Shahabad, Kurukshetra, haryana, India

Abstract

Thiol-modified aptamer oligonucleotides containing gold nanoparticles (G@NPs) bound to glycated hemoglobin (HbA1c) with high affinity in whole blood samples. Aptamer thiol groups enhanced the stability of aptamers adsorbed on the surface of G@NPs. A detection strategy was used to visually confirm the presence of HbA1c and the absorbance of the coloured compounds at selected wavelengths. Modified G@NPs are used in many colorimetric detection strategies due to their high extinction coefficient and strongly distance-dependent optical properties. In the present study, as the target analyte reports a method to prepare gold nanoparticle-bound purple aptamer aggregates that rapidly collapse into bluish red dispersed nanoparticles upon binding of HbA1c. HbA1c was detected in unpretreated whole blood after dilution at pH 7.4 and with UV-VIS double beam spectrophotometer with a lower limit of detection (LOD) (0.1 μM) and a much broader linear detection range (0.1 μM–100 μM). This current colorimetric method provides a rapid (7 min), accurate and high determination of whole blood HbA1c with minimal interference from uric acid, bovine serum albumin, hemoglobin, and glucose. The lower detection limit and broader linearity of aptamer-bound G@NPs hybrids improve the accuracy and precision of HbA1c measurements.

Keywords

Aptamer

Whole Blood

Glycated hemoglobin

Gold nanoparticles

P1.93 BIOSENSORS FOR THE MONITORING OF COLORECTAL CANCER METASTASIS

Katarzyna Ratajczak, Magdalena Stobiecka

Warsaw University of Life Sciences, Poland

Abstract

Cancer is one of the main cause of death worldwide. Thus, early diagnosis become crucial in the fight against cancer. Currently research studies efforts are focused on finding new strategy for the rapid detection of two or more cancer biomarkers. It has been shown that biosensors can be used for these purposes and offer the short reading time, portability, low cost, rapidness and high sensitivity of the measurements [1-2].

In our work, we have designed and tested fluorescence and electrochemical biosensors for the detection of cancer biomarkers such as adenosine triphosphate (ATP) and survivin – a member of the inhibitor of apoptosis protein (IAP) family. The developed probes consisted of a single-stranded oligonucleotides with attached fluorescence dye and also antibody attached to gold electrode.

In the absence of ATP or survivin mRNA a strong or weak signal from fluorecence dye was recorded, respectively. In the presence of the molecules the conformation of the oligonucleotides probes was changed which also resulted in a change of the obtained signal. We have also registered change in electrochemical signal when survivne protein was added to developed immunosensor. In our study, we have performed the sensitivity and selectivity of the proposed biosensors. The application in real samples obtained from SW480 cell lysate was also investigated.

We would like to thank for financial support from the National Science Centre (NCN), Poland: Program OPUS Grant No. 2023/49/B/ST4/04280.

References:

[1] İnce B., et.al.; (2024), Essential Chem, 1(1), 1-23

[2] Bhardwaj H., et. al.; (2024), Mater. Adv., 5, 475-50

Keywords

biosensors

cancer biomarkers

cancer cells

fluorescence and electrochemical techniques

P1.94 Bispecific-aptamer based electrochemical sensor for direct quantification of glycated albumin

Andrea Rescalli^1,2, Carlotta Gastaldi³, David Probst¹, Mika Hatada¹, Francesco Cellesi², Pietro Cerveri^3,2, Koji Sode¹

¹The University of North Carolina at Chapel Hill, USA. ²Polytechnic University of Milan, Italy. ³University of Pavia, Italy

Abstract

We report an innovative electrochemical aptamer sensor for monitoring glycated albumin values (GA) using a bispecific-aptamer to recognize both human serum albumin (HSA) and glycated human serum albumin (GHSA), eliminating the need for multiple assays. This sensor functions without pre-digestion steps and directly quantifies GA exploiting the different binding dynamics between the aptamer and the target proteins.

GA is a biomarker for diabetic patients and for pre-diabetic conditions, that reflects average blood glucose levels over the previous 2–3 weeks. GA is defined as the percentage ratio of GHSA to total HSA concentrations, and its quantification is currently based on assay kits that rely on two separate measurements—a proteolytic digestion of GHSA and a second assay for total HSA—making integration into a single point-of-care testing (POCT) platform challenging. The requirement for proteolytic processing further limits its clinical utility outside of centralized laboratories [1, 2].

In response, we developed a novel electrochemical aptamer sensor for GA detection that leverages a bispecific-aptamer as biological recognition element to interact with HSA and/or GHSA, eliminating the need for multiple assays. Using square wave voltammetry (SWV), we characterized the sensor response over time and across frequencies, detecting changes in electron transfer kinetics to correlate dynamic response patterns with specific glycation ratios representative of healthy and diabetic conditions (10%, 20%, 40%). By tracking signal evolution within 20 minutes, our sensor demonstrated consistent quantification of GA across physiological albumin concentrations.

This approach marks an additional step toward accessible, rapid GA monitoring not only for POCT settings, but also for the personal use with non-invasive samples, such as tear, saliva and sweat.

References

[1] Rescalli et al., Biosensors, 2022, 12(9), 687

[2] Hatada et al., Sensors and Acutators:B, Chemical, 2022, 130914

Keywords

glycated albumin

bispecific-aptamer

aptasensor

diabetes

P1.95 A Flexible Paper-Based Biosensor for Rapid miRNA Detection for High-Throughput and On-Site Applications

Marianna Rossetti¹, Liming Hu¹, Ruslan Álvarez-Diduk¹, Chiara Capolungo^1,2, Gabriel Maroli¹, Enric Calucho¹, Arben Merkoçi1¹

¹Catalan Institute of Nanoscience and Nanotechnology, Spain. ²University of Bologna, Italy

Abstract

The detection of oligonucleotide biomarkers in biological fluids is crucial for medical research and diagnostics, particularly in the context of liquid biopsies. Among these biomarkers, microRNAs (miRNAs) have emerged as pivotal biomarkers due to their stability in various bodily fluids and their associations with diseases, including cancer. Current detection methods, such as microarrays and qRT-PCR, are often hindered by complexity and the need for sophisticated instrumentation, making them unsuitable for point-of-care applications in resource-limited settings. To overcome these challenges, we propose a dry-reagent paper-based biosensor that utilizes fluorescent molecular beacon -modified gold nanoparticles embedded in nitrocellulose strips for the rapid and quantitative detection of miRNAs. This system leverages the high sensitivity and specificity of molecular beacons, which undergo a conformational change upon target binding, resulting in a strong fluorescent signal. Our biosensor can be employed with a microplate reader for high-throughput analysis or adapted to a portable platform using a low-power LED and smartphone camera for on-site diagnostics. This approach holds promise for accessible and cost-effective miRNA detection in clinical settings, advancing liquid biopsy technologies, and improving patient outcomes through early diagnosis and monitoring.

Acknowledgements

This project is partly funded from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 101029884. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them. The ICN2 is funded by the CERCA programme/Generalitat de Catalunya, supported by the Severo Ochoa Centres of Excellence programme, Grant CEX2021-001214-S, funded by MCIN/AEI/10.13039.501100011033. We acknowledge Departament de Recerca i Universitats of Generalitat de Catalunya for the grant 2021 SGR 01464 and Grant PID2021-124795NB-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”

Keywords

fluorescence

miRNA detection

paper-based platform

P1.96 Development of next-generation portable magnetic device for detection of antimicrobial resistance genes

Maria Leonor Santos¹, Débora Albuquerque^2,3, Cristina Bárbara^1,4, Miguel Teixeira^3,5, Aida Duarte^6,7, Rui Pinto^6,8, Diogo Caetano^2,3, Cátia Caneiras^1,7,9

¹Institute of Environmental Health (ISAMB), Associate Laboratory TERRA, Faculdade de Medicina, Universidade de Lisboa, Portugal. ²Institute for Systems and Computer Engineering - Microsystems and Nanotechnologies (INESC-MN), Portugal. ³Instituto Superior Técnico (IST), Universidade de Lisboa, Portugal. ⁴Santa Maria Local Health Unit, Portugal. ⁵Institute for Bioengineering and Biosciences (2iBB), The Associate Laboratory Institute for Health and Bioeconomy (i4HB), Portugal. ⁶Faculdade de Farmácia, Universidade de Lisboa, Portugal. ⁷Egas Moniz Interdisciplinary Research Center, Egas Moniz School of Health and Science, Portugal. ⁸Clinical Analysis Laboratory Dr. Joaquim Chaves (JCS), Portugal. ⁹Institute of Preventive Medicine and Public Health (IMP&SP), Faculdade de Medicina, Universidade de Lisboa, Portugal

Abstract

A major clinical and public health concern is the presence of antibiotic resistance genes in bacteria. When pathogenic bacteria carry these genes, antibiotics become ineffective, leading to infections with increased morbidity and mortality. There is therefore a clear need for methods that can detect antibiotic resistance genes at the point of care. The objective was to develop an innovative technology that could detect these genes at the point of care.

Bactometer has been developed as a next-generation portable point-of-care device that employs magnetic and machine learning techniques to detect cells and analyse DNA. Using microfluidics, this lab-on-a-chip device provides a rapid response without the need for additional equipment or dedicated technicians, offering a convenient and efficient solution for identifying bacterial resistance genes in a sample. Magnetic applications have distinct advantages in robustness, stability, sensitivity and specificity, with reduced background interference and time-consuming sample preparation steps.

A total of 102 biochips were used to test 25 bacterial isolates from two Portuguese hospitals to see if Bactometer could detect one of the most important resistance genes, Klebsiella pneumoniae carbapenemase (KPC). Primers were selected that were both sensitive and specific for the KPC gene, and probes complementary to the PCR product were designed. As the biochip is magnetoresistive, the magnetic field changes in the presence of magnetically labelled targets (the KPC gene). Bactometer is 97% accurate.

Bactometer is an innovative device that can identify the clinically relevant strains in a sample and their resistance genes, particularly the KPC gene. This technology is a reliable and accurate diagnostic tool with the potential to revolutionise healthcare, particularly in early diagnosis and guiding prescribing decisions. In addition, Bactometer can facilitate patient triage and isolation, outbreak detection and may prove vital in the fight against the emerging global antibiotic pandemic.

Keywords

Antimicrobial resistance genes (ARGs)

Point-of-care testing (PoCT)

Magnetic sensors

Deoxyribonucleic acid (DNA) analysis

P1.97 A novel, enzyme-free combination of MNAzyme and Aptamer technology for sensitive, fast and affordable decentralized diagnostic assay development

Jörg Schönfelder, Lars Verschuren, Frank Simons, Bart Keijser

TNO Location Leiden Sylviusweg, The Netherlands

Abstract

The market for decentralized in vitro diagnostic testing is steadily growing, urged by the increasing shortage and need in healthcare personal and infrastructure and the increasing availability of meaningful decentral tests.

Currently, enzyme immunoassays (EIA) either in the form of lateral flow assays (LFA) or on small user-friendly devices are still the mainstay for decentral testing. However, many of them lack specificity and sensitivity compared to laboratory based, centralized testing. In recent years more types of decentralized molecular tests were introduced with improved assay performance, but these are limited to nucleic acid biomarkers. The only accepted and widely used POC technology for non-nucleic acid biomarkers is the well-known test for blood glucose levels (Glucometer).

We develop a potential point of care (POC) method applicable to non-nucleic acid biomarkers by combining MNAzyme (Multicomponent Nucleic Acid enzyme) with Aptamer technology. MNAzyme technology involves an enzyme-like reaction in the presence of metal ions triggered by the assembly of its components. In our approach we leverage the biomarker affinity of the aptamer to assemble and trigger the MNAzyme reaction, translating the biomarker presence into a readable output signal. Aptamer technology is cost-effective, sensitive and applicable to a wide range of biomarker types. Both technologies are solely based on nucleic acids and thus completely enzyme free, offering significant advantages such as low reagent cost, extremely temperature insensitivity, and broad test application possibilities.

In this study, we present the initial results of our Aptamer-MNAzyme test approach, combining a well-known MNAzyme with an adenosine binding aptamer. We demonstrate that the MNAzyme is activated in the presence of adenosine, with a response time of 10-15 min and a limit of detection in the low µM range. Other types of biomolecules also showed promising results, suggesting that this approach could lead to a new strategy for POC test design.

Keywords

MNAzyme

Aptamer

POC

Adenosine

P1.98 CRISPR AMR DX: an optimised CRISPR/Cas12a-based multiplex biosensor for rapid diagnosis of enteric fever and antimicrobial resistance

Mujar Minette Shalo, Jennifer Molloy

University of Cambridge, UK

Abstract

Enteric fever is a common cause of morbidity and mortality in underdeveloped countries where it is endemic. Clinical management and control of the disease rely on prompt diagnosis and effective treatment. Nevertheless, current diagnostic methods are not sufficiently specific and sensitive while antimicrobial resistance is becoming widespread, limiting treatment efficacy. Significant delays to treatment are due to the widely-used blood culture diagnostics which take several days to return a result and have limited sensitivity of only 40–60% ¹. The emergence of novel molecular techniques such as Clustered regularly interspaced short palindromic repeats (CRISPR-Cas12a)-based diagnostics has improved the potential of point-of-care molecular diagnostics. Cas12a is an RNA-guided endonuclease that detects specific DNA sequences. Hybridisation to the target strand activates indiscriminate cleavage of a single-stranded reporter DNA in the reaction mix, releasing a fluorescent read-out².

CRISPR AMR Dx seeks to engineer an affordable, sensitive multiplex Cas12a-based biosensor to diagnose enteric fever caused by typhoidal Salmonella and identify related antibiotic-resistant genes. A fast-folding fusion domain was added to Cas12a to improve its binding affinity and sensitivity to target DNA. Computational tools and molecular dynamic simulations will further inform the engineering of a thermostable Cas12a to improve its processivity and sensitivity. RNA guides specific to multiple bacteria and AMR targets will be predicted and optimised using machine learning algorithms. The best-performing guides will be incorporated into the diagnostic assay. The improved assay will then be integrated into a multichannel microfluidic device. This will enhance specificity and sensitivity while reducing complexity and the need for instrumentation, in line with the REASSURED criteria for point-of-care diagnostics.

Keywords

Enteric fever

CRISPR-Cas12a

Antimicrobial resistance

molrcular diagnostics

P1.99 Picomolar detection of sars-cov-2 spike protein enabled by liquid-liquid phase separation on an electrochemical aptamer biosensor

HoPing Siu¹, Yage Zhang², Sihan Liu³, HoCheung Shum^4,5, Wei Guo^3,5, Julian Tanner^6,5

¹The University of Hong Kong, Hong Kong. ²Shenzhen University, China. ³The University of Hong Kong Department of Mechanical Engineering, Hong Kong. ⁴Department of Chemistry & Department of Biomedical Engineering, City University of Hong Kong, Hong Kong. ⁵Advanced Biomedical Instrumentation Centre, Hong Kong. ⁶The University of Hong Kong School of Biomedical Sciences, Hong Kong

Abstract

Liquid-liquid phase separation (LLPS) has been proposed to dissect intracellular pathological events. A typical example is the colocalization of SARS-CoV-2 nucleocapsid protein and RNA to form a membrane-less condensate crucial for viral replication. However, this phenomenon has not been utilized in translational research such as molecular diagnosis. In this study, we first formulated a LLPS system to allow the condensation of SARS-CoV-2 spike (S) protein within a dextran-rich aqueous layer, verified by microscopic imaging and biochemical assays. Then we designed an electrochemical aptamer biosensor (E-AB) to selectively capture the S protein and transduce it into electrical signal via a peroxidase redox reaction. The integration of LLPS enabled an over 100-fold increase in detection sensitivity compared to the E-AB without LLPS. While the primary rationale is the local enrichment of protein via LLPS condensation, the macromolecular crowding environment from the dextran-rich solution is believed to facilitate the enzymatic performance and possibly aptamer capturing efficiency. Overall, LLPS is a single-step approach to improve E-AB sensitivity, and the protein condensation can be achieved in synthetic human biofluid, further suggesting potential applications in medical diagnostic research.

Keywords

P1.100 Biofunctionalized fiber optic sensors for viral RNA detection

Patryk Sokołowski¹, Paweł Wityk², Adam Władziński¹, Joanna Raczak-Gutknecht², Wiktoria Brzezińska², Małgorzata Szczerska¹

¹Gdańsk University of Technology, Poland. ²Medical University of Gdańsk, Poland

Abstract

Rapid and accurate viral diagnostics are critical for responding effectively to epidemiological threats. Fiber optic sensors provide near real-time measurement capabilities, making them particularly useful for epidemic monitoring and early pathogen detection. The ability to establish sensor networks enables large-scale monitoring, which is essential for widespread diagnostic applications.

In this study, we present the application of fiber optic sensors based on biofunctionalized microspheres for viral RNA detection. The microsphere fabricated on the end of fiber is 280μm. By employing specific surface modifications, such as the immobilization of nucleic acid probes on gold layer, we achieved high selectivity and sensitivity. The developed method enabled the detection of viral RNA at a concentration as low as 1pM in a time of 10mins, highlighting its potential for viral diagnostics. The sensor was fabricated using a single-mode telecommunication fiber, making integration into existing fiber optic networks possible, which enhances applicability in real-time epidemiological monitoring systems.

Uncaptioned visual

Figure 1. Principle of operation of the microsphere-based fiber-optic sensor, a) clear fiber, b) with functionalized layer and RNA, c) sensor’s head under microscope, d) obtained spectra

Acknowlegment: This work was supported by DS programs of Faculty of Electronics, Telecommunications and Informatics of Gdańsk Tech, by the 14/1/2024/IDUB/III.4.1/Tc grant under the TECHNETIUM Talent Management Grants and by COST Action [CA21159].

References:

K. Cierpiak, P. Wityk, M. Kosowska, et al. C-reactive protein (CRP) evaluation in human urine using optical sensor supported by machine learning. Sci Rep (2024). https://doi.org/10.1038/s41598-024-67821-0
P. Sokołowski, K. Cierpiak, M. Szczerska, et al/ Optical method supported by machine learning for dynamics of C-reactive protein concentrations changes detection in biological matrix samples, Journal of Biophotonics (2024). https://doi.org/10.1002/jbio.202300523
Wityk, P.; Terebieniec, A.; Nowak, R.; Łubiński, J.; Mroczyńska-Szeląg, M.; Wityk, T.; Kostrzewa-Nowak, D. Reusable Biosensor for Easy RNA Detection from Unfiltered Saliva. Sensors 2025. https://doi.org/10.3390/s25020360

This study aimed to develop a competitive electrochemical aptasensor for the fast and specific detection of S. aureus through its surface component, protein A (PrA). The biosensor was designed on gold screen-printed electrodes (AuSPE) to enable the testing of real samples and the incorporation in a point-of-care device. An aptamer for the specific recognition of PrA was immobilised on the gold surface using multi-pulse amperometry, followed by the deposition of titanium carbide nanosheets (MXene) to enhance the electrochemical signal and complementary DNA sequences labelled with ferrocene (cDNA-Fc) to improve the selectivity². The unbound sites of the surface were blocked using a thiolic compound and the target molecule was detected after incubation on the modified electrode, indirectly, measuring the Fc-electrochemical signal using cyclic voltammetry. Multiple PrA concentrations were incubated and tested and a linear correlation between the electrochemical signal and the levels of the target molecule was obtained. The sensor was also successfully tested in haemoculture samples, from patients with systemic S. aureus infections, proving it can be used as a tool for rapid diagnostic.

(1) Canciu, A. et all. TrAC Trends in Analytical Chemistry 2023, 161, 116983.

(2) Wang, H. et all . Anal Chim Acta 2020, 1094, 18–25.

This study was founded by the UMF internal grant 778/4/13.01.2025 and the HORIZON FishEUTrust project.

Keywords

competitive aptasensor

Staphylococcus aureus

Protein A

MXene

P1.104 Label-free Aptamer Based Biosensors for Ultrasensitive Determination of an Amino Acid L-Citrulline

Selenay Sadak^1,2, Gözde Aydoğdu Tığ³, Bengi Uslu¹

¹Ankara University Faculty of Pharmacy, Turkey. ²Ankara University Graduate School of Health Science, Turkey. ³Ankara University Faculty of Science, Turkey

Abstract

Abstract

Mpox is a zoonotic disease caused by the Monkeypox virus (MPXV), a double-stranded DNA virus belonging to the Orthopoxvirus genus. In 2024, the WHO reclassified mpox as a Public Health Emergency of International Concern (PHEIC) due to the emergence of Clade I, a virulent strain associated with severe symptoms and higher mortality. Current WHO data show that nearly half of mpox cases involve co-infection with Human Immunodeficiency Virus (HIV), underscoring the urgent need for efficient diagnostic tools to contain potential epidemics. While PCR methods are reliable, they require complex laboratory infrastructure and lengthy processing. Addressing these challenges, a dual electrochemical DNA (deDNA) biosensor has been developed for ultrasensitive, simultaneous detection of mpox and HIV co-infection. The biosensor employs cytosine–cytosine (C–C) mismatch-driven nanocomplexes, stabilized by Ag⁺ ions, that mimic natural base mutations, enabling selective hybridization with target MPXV DNA and HIV cDNA. The excellent redox properties of these nanocomplexes allow direct, sensitive electrochemical detection without amplification, achieving a linear range from 10¹ to 10⁵ pM and a detection limit of 2 pM. Successful detection of viral particles in urine and saliva highlights this as the first electrochemical sensor for MPXV and HIV co-infection. With portable readout technology, the deDNA biosensor supports remote, real-time analysis, providing a practical tool for monitoring and containing disease spread.

Keywords

Mpox

HIV

Electrochemical detection

P1.108 Carbon-based aptasensor platforms for vancomycin electrochemical detection

Izabela Zaraś¹, Marta Jarczewska¹, Andrzej Pepłowski²

¹Warsaw University of Technology Faculty of Chemistry, Poland. ²CEZAMAT PW sp. z o. o., Poland

Abstract

Due to adverse side effects, the concentration of drugs with a narrow therapeutic window must be monitored in a patient's blood. One such drug is vancomycin, an overdose of which can result in hearing loss or kidney damage. Currently, methods such as immunoassays or HPLC-MS are used to determine blood levels of this antibiotic. The limitation of these methods is that they are time-consuming and often require the use of sophisticated apparatus [1, 2]. An alternative could be the application aptamer-based biosensors. In the project, electrodes based on different carbon materials were used including glassy carbon, edge plane pyrolytic graphite and miniaturized electrodes made of pastes containing graphene nanoplatelets. To immobilize the receptor layer on the carbon surfaces, the aptamers modified with pyrene or anthracene were introduced, which allowed tethering through π-π interactions between carbon surface and aromatic anchor groups conjugated to aptamer 5’ end. Measurements were performed using electrochemical techniques such as cyclic voltammetry and electrochemical impedance spectroscopy. The studies were focused on choice of the carbon material allowing for the most efficient aptamer immobilization as well as type of aromatic anchor attached to aptamer probe. This was proceeded by characterization of aptamer-based biosensor working parameters.

[1] Dauphin-Ducharme P, Yang K, Arroyo-Currás N, et al. Electrochemical Aptamer-Based Sensors for Improved Therapeutic Drug Monitoring and High-Precision, Feedback-Controlled Drug Delivery. ACS Sens. 2019;4(10):2832-2837.

[2] Mu F, Zhou X, Fan F, Chen Z, Shi G. A fluorescence biosensor for therapeutic drug monitoring of vancomycin using in vivo microdialysis. Anal Chim Acta. 2021;151:338250.

This work was financially supported by the Warsaw University of Technology: IDUB programme Materials for Young-2 grant no. 504/04496/1020/45.010523 “Application of abiological (carbon and carbon/gold) – biological (nucleic acids) hybrid materials for noninvasive skin-wearable electrochemical biosensing platforms for therapeutical drug monitoring (TMD) and control of drug delivery system”

Keywords

aptasensor

vancomycin

electrochemical biosensor

therapeutic drug monitoring

P1.109 Development of electrochemical aptasensor for detection of fertilization period of cattle

SHUNSUKE YOSHIMURA

University of Fukui, Japan

Abstract

Currently, artificial insemination is the primary method used to increase cattle production in the livestock industry. However, determining the estrus period is challenging, and mistimed insemination can reduce conception rates. To address this, we focused on luteinizing hormone (LH) as an indicator for predicting ovulation timing. LH surges during the estrus cycle, leading to ovulation 27–30 h later. In this study, we developed an electrochemical aptasensor that utilizes the structural change of nucleic acid aptamers upon binding to the target. As shown in Fig. 1, the aptamer used is modified with a mediator at its terminal and immobilized on the electrode surface. When the aptamer binds to bovine LH (bLH), it undergoes a structural change that brings the mediator closer to the electrode, promoting an electrochemical reaction enabling LH detection.

The bLH sensor was prepared as follows. Since no bLH aptamer has been reported, a human LH (hLH) aptamer was used. The hLH aptamer was dropped onto the working electrode of a screen-printed electrode with electrodeposited gold nanoparticles, immobilizing the aptamer onto the working electrode. Next, amine-reactive PES was dropped onto the electrode to modify the amino group at the aptamer terminal with the mediator. Then, 6-mercapto-1-hexanol (6MCH) was used to modify the electrode surface. The prepared electrode was used as the bLH sensor.

The bLH sensor was evaluated by differential pulse voltammetry. The addition of bLH led to an increase in response current from the oxidation of PES. This suggests the sensor can detect bLH. However, a larger increase in response current was observed when hLH was added. In the future, we aim to improve the sensitivity of the sensor by optimizing the aptamer sequence

Uncaptioned visual

Fig. 1 Reaction scheme of the bLH sensor

Keywords

aptasensor

luteinizing hormone

electrochemistry

P1.110 Rapid colorimetric detection of D-dimer based on unmodified aptamer-gold nanoparticle conjugates

Hai-Jo Lin, Lin-Chi Chen

National Taiwan University, Taiwan

Abstract

D-dimer is a biomarker of blood coagulation, and elevated levels are associated with an increased risk of cardiovascular diseases. Therefore, a cheap, rapid, and visible diagnostic method for D-dimer is essential for point-of-care diagnostics. Unmodified aptamer-gold nanoparticle (Apt-AuNP) conjugates were assembled and studied for colorimetric D-dimer assay in this study. The D-dimer aptamer, named B5R10-4, was identified by the homemade systematic evolution of ligands by exponential enrichment (SELEX) process and utilized as the sensing probe. The AuNPs were synthesized with a diameter of 19.2 nm and high batch-to-batch stability. AuNPs aggregated in a high-salt solution, causing a color change from wine-red (A₅₂₀) to purple-blue (A₆₀₀). However, B5R10-4 Apt-AuNP conjugates did not aggregate in a high-salt solution, displaying a wine-red color. When D-dimer specifically interacted with B5R10-4, B5R10-4 would no longer attach to the AuNP surface, leading to AuNP aggregation and a color change from wine-red to purple-blue. This dose-dependent colorimetric assay showed a linear calibration range of A₆₀₀/A₅₂₀from 0.25 nM to 10 nM. The sensitivity was 0.055 nM^-1, with a limit of detection as low as 22.7 pM. Furthermore, the color change of the AuNP solution could be clearly distinguished at a D-dimer concentration above 2 nM. Specificity tests were performed using human serum albumin (HSA), and no significant changes in the A₆₀₀/A₅₂₀ ratio were observed. Interference tests were also tested using 1% fetal bovine serum (FBS) and the A₆₀₀/A₅₂₀ ratio still showed linearity with D-dimer concentration. This method utilizes aptamer and AuNPs without modifications, making it cost-effective. Moreover, the AuNP aggregation occurs within a few seconds, enabling rapid detection. Finally, the color change can be observed with the naked eye, providing a visible diagnosis. This Apt-AuNP conjugates colorimetric method for D-dimer demonstrates its niche potential in point-of-care applications.

Uncaptioned visual

Keywords

D-dimer

Aptamer

Mridula Bhadra, Manisha Sachan, Seema Nara

Motilal Nehru National Institute of Technology Allahabad, India

Abstract

The detection of miRNAs by liquid biopsy techniques has enormous potential for early metastatic cancer diagnosis. miRNA-145-5p have shown to repress epithelial ovarian cancer (EOC) metastasis by inhibiting cell proliferation and promoting cell death. In our initial study, its downregulated expression has been validated in tissue and serum samples of EOC patients. Among all screened microRNAs, namely miR-200c-3p, miR-200b-3p, miRNA-145-5p and miR-148a-3p, miRNA-145-5p has the best diagnostic potential with the area under the ROC curve (AUC) of 0.868 (p<0.05) in EOC tissue and 0.916 in serum cohort. Also, miR-145-5p showed 80% sensitivity and 100% specificity, establishing that, among the others, miR-145-5p could prove to be a potent biomarker for non-invasive early detection of EOC. Therefore, suggesting its high diagnostic potency, here we attempt to standardize the electrochemical sensing of miRNA-145-5p on a fluorine-doped tin oxide (FTO) working electrode. The underlying sensing principle is a strand displacement reaction in which the target microRNA first displaces a DNA probe hybridized to the immobilized capture probe. The miRNA is subsequently replaced by a gold nanorod (AuNR) conjugated reporter DNA. The intrinsic peroxidase activity of AuNRs will generate the signal and would correlate with the concentration of miRNA on the electrode. For this, AuNRs were successfully synthesized, characterized using UV-Vis, and transmission electron microscopy and used for exploring their intrinsic peroxidase. These were then conjugated with reported DNA (H0) and the conjugation was confirmed colorimetrically and electrochemically. The working FTO electrode was prepared using streptavidin chemistry by immobilizing the biotinylated capture probe (H1). All fabrication steps were monitored using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The sensor generated a distinctive voltammetric peak on FTO electrodes modified with AuNRs and streptavidin upon successful hybridization. The working electrode was found promising to detect miRNA in the initial stages and is being validated for performance.

Keywords

Biosensor

Epithelial Ovarian Cancer

Nanozyme

MicroRNA

P1.115 Development of a sensitive biosensor for detection of amyloid beta 42 (Aβ42) in SH-SY5Y neuroblastoma cells using methylene blue- modified DNA aptamer and zinc oxide (ZnO) nanoparticles.

Dr. Aman Chauhan¹, Dr. Rooma Devi¹, Sukhpal Singh¹, Ajay Singh², Neeru Bhaskar³, Adesh K Saini⁴, Sasanka Chakrabarti¹, Karanpreet Bhutani¹

¹Maharishi Markandeshwar Institute of Medical Science and Research, Maharishi Markandeshwar (Deemed to Be) University, Mullana, Ambala, Haryana, India. ²Indian Institute of Technology, Jammu, India. ³Adesh Medical College and Hospital, Mohri, Shahbad, Ambala, Haryana, India. ⁴Maharishi Markandeshwar Engineering College, Maharishi Markandeshwar (Deemed to Be) University, Mullana, Ambala, Haryana, India

Abstract

Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder resulting in loss of memory and cognition. The characteristic feature of AD is accumulation of Aβ42 in the extracellular space of the brain, forming amyloid plaques. We utilized experimental AD model using commercially available Aβ 1-42 peptide to induce amyloid plaque formation in SHSH-5Y cells and by using DRB 18 (5,5′-(((4-Chloro-1,2-phenylene)bis(azanediyl))bis(methylene))bis(2-methylphenol). DRB 18, inhibits glucose transporter (GLUT), responsible for hypometabolic stress in SHSY-5Y cells that mimics the metabolic dysfunction as observed in AD (Model of AD).

SH-SY5Y neuroblastoma cells were cultured in 6-well plates using a 1:1 mixture of DMEM (Dulbecco's Modified Eagle Medium), HAM's F-12 medium, 10% fetal bovine serum and 1% penicillin-streptomycin. Upon reaching 60% confluency, the cells were divided into four experimental groups: A control group with no treatment, A dimethyl sulfoxide (DMSO) treatment group, An amyloid-beta 42 (Aβ42) peptide treatment group, and A GLUT inhibitor group treated with DRB 18. After 48 hours, Aβ42 levels were quantified using a methylene blue- modified aptamer immobilized on a zinc oxide (ZnO) coated glassy carbon electrode in each group. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) analyses revealed distinct electrochemical responses. In the Aβ42 peptide treatment group and DRB 18 treatment group, decrease in current were observed, reflecting elevated levels of Aβ42 in the cell supernatant in these groups. In the DMSO and control groups, negligible electrochemical responses were recorded, demonstrating low or undetectable Aβ42 levels. In conclusion, this method is highly sensitive and specific as compared to traditional methods and a simpler and more efficient approach for the early detection of AD with minimal processing time.

Keywords

ZnO nanoparticles

DNA Aptamer

Alzheimer's Diseases

DRB 18

P1.116 Fluorescence-based detection of amyloid-beta 42 (Aβ42) peptide employing FITC (fluorescein isothiocyanate)- labelled aptamers immobilized on ZnO nanoparticles and electrodes in a cell-based model of Alzheimer's disease (AD) induced by GLUT hypometabolic stress.

Dr. Aman Chauhan¹, Dr. Rooma Devi¹, Ajay Singh², Asha Kumari¹, Rohini Sharma¹, Adesh K. Saini³, Karanpreet Bhutani¹

¹Maharishi Markandeshwar Institute of Medical Science and Research, Maharishi Markandeshwar (Deemed to Be) University, Mullana, Ambala, Haryana, India. ²Indian Institute of Technology, Jammu, Jammu and Kashmir,, India. ³Department of Biotechnology, Maharishi Markandeshwar Engineering College, Maharishi Markandeshwar (Deemed to Be) University, Mullana, Ambala, Haryana, India

Abstract

¹Nalecz Institute of Biocybernetics and Biomedical Engineering Polish Academy of Sciences, Poland. ²Institute of Organic Chemistry Polish Academy of Sciences, Poland. ³National Medicines Institute, Poland

Abstract

Monitoring of drug concentrations in biological fluids is a critical aspect of pharmacological research and clinical practice, helping to ensure both, efficacy and safety in drug therapy. For 6-mercaptopurine (6-MP), the active metabolite of thiopurines, precise concentration assessment is particularly important due to its narrow therapeutic index, where the difference between an effective and toxic dose is minimal. Thiopurines, including 6-MP, are commonly used in managing inflammatory bowel disease (IBD), autoimmune disorders, and some types of cancer. However, their potential adverse effects necessitate careful monitoring during therapy. However, there is a lack of analytical tools enabling physicians to optimize therapy quickly and easily.

This research introduces the first-reported electrochemical biosensor based on drug-DNA interaction to detection of 6-mercaptopurine. It utilizes a dsDNA sequence (5’-GGCAGGACGGAG-3’) that selectively interacts with 6-MP. The interaction of the DNA sequence with 6-MP was also confirmed through molecular modeling methods, incorporating empirical data from NMR and HPLC experiments. The biosensor is designed as a simple system, utilizing screen-printed graphite electrodes with an adsorbed dsDNA layer. It provides a linear response within the nanomolar concentration range, aligning well with the concentrations typically found in biological samples. This compatibility makes the biosensor a valuable tool for precise and reliable 6-MP detection in pharmacological and clinical research.

There is a significant gap in the development of an electrochemical measurement system that utilizes a bioreceptor ensuring the selectivity towards 6-MP needed for analyzing complex samples. The proposed bioanalytical tool offers a promising solution, combining simplicity, and affordability, making it applicable for rapid and efficient 6-MP determination.

Acknowledgment: The study was supported by Polish National Science Center, Project 2019/35/O/ST5/01886

Keywords

6-mercaptopurine

Short double-stranded DNA

Electrochemical biosensor

DNA-drug interaction

P1.121 Smart diagnosis based on image analysis of the rotational diffusion of oligonucleotide probe-modified Janus particles.

AIRI YANAGIHARA¹, YUI WATANABE¹, SATOSHI AMAYA², Han-Sheng Chuang³, HIROAKI SAKAMOTO¹

¹University of Fukui, Japan. ²The University of Tokyo, Japan. ³National Cheng Kung University, Taiwan

Abstract

To diagnose cancer and elucidate pathology using AI, samples must be obtained from specimens, analyzed, and analyzed. Because AI accuracy improves with the amount of data analyzed, high-throughput analysis of a large number of samples is required; however, this is difficult with current cancer testing technology.

In this study, we developed a novel biosensor that can specifically, quickly, and easily detect the target Ribonucleic acid (RNA) by fabricating Janus particles with half the surface of fluorescent particles coated with gold, supplementing miRNA produced from laryngeal cancer on the gold surface and measuring its rotational Brownian motion changes by image analysis. According to the Stokes-Einstein-Debye (SED) equation, the rotational diffusivity of a particle is inversely proportional to the cube of the particle diameter when the solution temperature and liquid viscosity are controlled. Therefore, particles with smaller diameters have larger diffusivities, whereas particles with larger diameters have smaller diffusivities. For the Janus particles, the diffusivity was calculated from the correlation intensity of the flashing signal obtained from the rotational motion of the particles at each elapsed time, and the correlation time was calculated from the correlation intensity of the flashing signal obtained from the rotational motion of the particles. We also introduced rolling circle amplification (RCA) to increase the size of Janus particles, further increasing the correlation time for image analysis. Experiments based on this principle revealed that in the presence of the target RNA, the Janus particles captured the RNA and formed a Janus particle-RNA complex, which increased the particle volume and correlation time.

The correlation time can be measured using only a microscope. Because blood or saliva is collected from patients with laryngeal cancer to detect and quantify the target miRNA, a simple and minimally invasive testing technique is expected.

Uncaptioned visual

Keywords

Janus particle

miRNA

Image Anarist

Rolling Circle Amplification (RCA)

P1.122 The effect of CpG methylation on the structure and stability of the i-motifs located in the CpG islands

Shintaro Inaba¹, Daiki Oshikawa², Yudai Kitagawa¹, Kaori Tsukakoshi¹, Kazunori Ikebukuro¹

¹Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Japan. ²Department of Industrial Technology and Innovation, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Japan

Abstract

Introduction:

CpG methylation plays an important role in gene regulation. Our previous study identified G-quadruplex (G4)-forming regions within CpG island, where methylation rates varied significantly. We showed that CpG methylation affected the structural topology of G4-forming oligonucleotides [1]. The complementary cytosine-rich strands of these oligonucleotides can form i-motifs via cytosine-cytosine hydrogen bonding. However, little is known about the effect of CpG methylation on i-motif. In this study, we investigated the effect of CpG methylation on the structure and thermal stability of several i-motif-forming DNAs.

Methods:

We used five i-motif-forming DNAs from four cancer-related genes (unmethylated VEGF, C-KIT, BCL2, HRAS1, and HRAS2), and DNA oligonucleotides methylated at all CpG sites (methylated VEGF, C-KIT, BCL2, HRAS1, and HRAS2). CD spectra of the DNAs were measured at pH 4.4 ~ 8.0 to analyze the effect of the methylation on i-motif structure and thermal stability. Native PAGE was performed to investigate the effect of CpG methylation on the structure.

Results and Discussion:

CD spectra indicated that all oligonucleotides formed i-motif under acidic conditions. CpG methylation altered the molar ellipticities of these DNAs at each pH, suggesting that the modification caused structural differences. In certain sequences, the transitional pH, recognized as an index of i-motif stability and calculated by fitting the molar ellipticities at 290 nm of each pH, increased with CpG methylation, indicating that the methylation increased pH stability. And the melting temperature (Tm) at transitional pH of each unmethylated DNA was consistently higher in methylated sequences, indicating improved thermal stability.

Native PAGE revealed the methylation altered the DNA mobility at the transitional pH, indicating that it stabilized the structure and formed a stable monomer-type i-motif. We would like to develop the biosensor to detect CpG methylation focusing on the structural change by the modification.

Reference:

[1] Tsukakoshi, K. et.al., Molecules, 2018 (23), 1-12.

Keywords

CpG methylation

I-motif

pH dependence

structural change

P1.123 Functionalized Aerogel-Based Disposable Electrochemical Biosensor for Hormone Detection

Shan-Rong Wu, Yu-Ju Teng, Chuan-Chun Lu, Jen-Chieh Yeh, Jia-Ying Weng, Chia-Ling Tsai, Cheng-Yu Lee

Chung Yuan Christian University Department of Chemistry, Taiwan

Abstract

This study presents a disposable electrochemical biosensor for detecting cortisol, a hormone associated with stress, designed to be rapid, cost-effective, and highly sensitive biosensing platform.

The sensor utilizes a functionalized silica aerogel as a nanocarrier, where the aerogel surface is modified with Linker DNA to secure the electrochemical signal molecule, methylene blue (MB), within the porous structure of silica aerogel. To control the release of MB, a cortisol-specific aptamer, conjugated with bovine serum albumin (BSA), forms a blocking layer by binding to the Linker DNA. When cortisol is present, it selectively binds to the aptamer, disrupting the blocking layer and releasing MB, thus producing an electrochemical signal that indicates cortisol levels.

This sensor design offers notable advantages, including high sensitivity and fast detection, without complex operational steps. Additionally, by adjusting the nucleic acid sequences of the Linker DNA and aptamer, the platform can be adapted to detect various analytes relate to replaced aptamer, enhancing its versatility for a range of diagnostic applications. This approach marks a significant advancement in real-time hormone detection, offering a practical solution for rapid and accessible diagnostics with the potential of developing an universal diagnostic platform.

Keywords

Cortisol

Silica aerogel

Electrochemical biosensor

Nanocarrier

P1.124 Development of a novel method for neural activity assessment using microelectrode array technology

SunHyun Park, Kyungmin Kang

Division of Advanced Predictive Research, center for Bio-Signal Research, Korea Institute of Toxicology (KIT), Republic of Korea

Abstract

Neurological disorders are related to exposure to various environmental toxicants. However, the toxicity evaluation of environmental chemicals through animal testing has challenges due to time delay and ethical problem. There are needs for advanced neurotoxicity assessments. The development of alternative toxicity testing methods is crucial for improving the time efficiency and accuracy of chemical safety evaluations while addressing ethical concerns regarding animal use. In this study, we introduce an innovative in vitro neurotoxicity assessment platform that integrates microelectrode array (MEA) technology to evaluate neuronal activity and network dynamics. Unlike conventional methods, MEA analysis provides real-time, high-resolution insights into functional changes in neuronal networks, enabling a deeper understanding of neurotoxic effects. Primary cortical neurons derived from fetal rats were cultured on MEA plates. Neuronal activity was monitored across six parameters: the number of active electrodes, mean firing rate, burst rate, spike-in-burst ratio, network burst, and synchrony index. This robust analytical framework facilitated the detection of subtle changes in neuronal function in neurotoxicity assessments. By focusing on functional neurophysiology, this method paves the way for more ethical, efficient, and precise toxicity testing. These findings suggested developing standardized alternative neuronal toxicity testing protocols. This platform not only enhances our ability to evaluate neurotoxic effects but also represents a significant step toward reducing animal use in toxicity research while improving safety evaluations for chemicals and pharmaceuticals.

Keywords

Keywords

bio-impedance

endothelial tissue

tight junction formation

organ-on-chip

P1.128 Application of a ready-to-use cell sensor for dioxins and dioxin-like compounds screening in meat samples

Yangsheng Chen¹, Songyan Zhang², Bin Zhao¹

¹Hangzhou Institute for Advanced Study, UCAS, China. ²Tox Sense (Shenzhen) Technology Innovation Co. Ltd., China

Abstract

Dioxins and dioxin-like compounds (DLCs) in foodstuffs are closely related to human health. As China is the largest food-consuming country, there is a potentially large demand for screening bioassays that are rapid, cost-effective and capable of determining dioxins and DLCs in foodstuffs. CBG2.8D is a reporter gene-based recombinant cell sensor that was recently developed for determining dioxin and DLCs in ambient and seafood samples. In this study, we established a bioanalytical method with this ready-to-use cell sensor for the bioanalysis of dioxins and DLCs in different types of meat samples. Twenty-nine samples from three typical types of meat (beef, pork and fish) were collected and subjected to both instrumental analysis and a CBG2.8Dbioassay. The intra- and inter-lab reproducibility of the bioassay was evaluated, and the coefficients of variation (CVs) were lower than 25%, suggesting that the cell sensor had a good reproducibility for the meat samples. Based on the correlation equation and coefficient obtained by comparing the data from the instrumental analysis and CBG2.8D bioassay, we found that this method had better performance with pork and fish than with beef. The compliance rate was also determined by comparing the results from the instrumental analysis and there were no false results for the pork and fish samples. Lastly, a complete operation procedure was summarized as a guideline for practical application. In conclusion, the CBG2.8D cell sensor exhibits excellent stability and is capable of screening dioxins and DLCs in meat samples.

Keywords

aryl hydrocarbon receptor

cell sensor

dioxin

food

P1.129 Development of bacteriophage plate for rapid detection of carbapenem-resistant Escherichia coli using MALDI-TOF MS

Youngkeun Yoo, Jaewoong Kim, Taekyu Park, Kanghyeon Kim, Jong-Min Park

Hallym University, Republic of Korea

Abstract

Antibiotic-resistant bacteria are a serious threat to global public health because they make antibiotic treatment of diseases difficult. In particular, carbapenem-resistant enterobacteriaceae have been listed as an urgent threat of antibiotic resistance by the U.S. Centers for Disease Control and Prevention in 2019. However, traditional antibiotic susceptibility tests based on molecular diagnostics and biochemical diagnostics, including disk diffusion tests and PCR, are time-consuming and labor-intensive. Therefore, development of a rapid antibiotic resistance testing method is urgently needed.

Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was widely used for rapid bacterial identification based on a database of mass peak patterns of various microorganisms. In this study, a bacteriophage plate was developed for rapid detection of carbapenem-resistant Escherichia coli using MALDI-TOF MS and the bacterial identification system. A bacteriophage plate was prepared using two unmodified bacteriophages that can be attached specifically with carbapenem-resistant E. coli. To immobilize bacteriophages with appropriate orientation, functionalized parylene (poly-p-xylylene) thin film with different surface charges was coated onto the MALDI-TOF plate. Orientation of bacteriophages and affinity to the carbapenem-resistant E. coli on the bacteriophage plate were evaluated by fluorescence spectroscopy analysis and confocal microscope images. To evaluate detection performance and specificity of the bacteriophage plate, carbapenem-resistant E. coli, carbapenem-susceptible E. coli, and other bacterial species, including Klebsiella pneumonia and Pseudomonas aeruginosa, were treated on the bacteriophage plate and analyzed by the bacterial identification system in the MALDI-TOF MS. Finally, quantitative analysis was carried out to demonstrate rapid and sensitive detection within 10 minutes of carbapenem-resistant E. coli using bacteriophage plate-based MALDI-TOF MS.

Keywords

MALDI-TOF MS

bacteriophage

antibiotic susceptibility testing

carbapenem

P1.130 Pain-on-a-Chip: A Microfluidic Device for Neuron Differentiation and Functional Discrimination in Animal Models of Chronic Pain

Douer Zhu¹, Dusan Matusica², Nicholas Veldhuis³, Azadeh Nilghaz¹, Ziqiu Tong¹, Rainer Haberberger⁴, Daniel Poole³, Kelly O’Sullivan⁵, Wendy Imlach⁵, Nicolas Voelcker^1,6,7

¹Monash Institute of Pharmaceutical Sciences Drug Delivery Disposition and Dynamics, Australia. ²Flinders Health and Medical Research Institute, Australia. ³Monash Institute of Pharmaceutical Sciences Drug Discovery Biology, Australia. ⁴The University of Adelaide School of Biomedicine, Australia. ⁵Monash University Monash Biomedicine Discovery Institute, Australia. ⁶Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, Clayton, Australia. ⁷Monash University Department of Materials Science and Engineering, Australia

Abstract

Chronic pain is a global health issue that is poorly understood and challenging to treat. Improving pain classification and treatment requires new strategies that objectively discriminate between pain conditions and minimise subjectivity associated with the perception of pain. To address this, we have developed a microfluidic biosensor - termed ‘pain-on-a-chip’ - that leverages recent advancements in biocompatible microfluidic technology with on-chip differentiation of nociceptor-like cells, enabling small sample volumes to be used. Following neuronal differentiation, we employed on-chip live-cell imaging to validate the chip’s functionality. For the first time, we confirmed that neuronal sensory cells can be robustly differentiated in a microfluidic system. Our results demonstrate that this model can discriminate between biological fluids from distinct chronic pain models. This system has potential as an objective, rapid, cost-effective and minimally invasive method for distinguishing between different subtypes of chronic pain.

Keywords

Chronic pain

Microfluidics

nociceptor

Biosensor

P1.131 Evaluating the Vibrio harveyi whole-cell biosensor for assessing the effects of Lavender preparations on Campylobacter jejuni intercellular signalling

Dina Jug, Blaž Jug, Sonja Smole Možina, Anja Klančnik

University of Ljubljana Biotechnical faculty, Slovenia

Abstract

Intercellular bacterial communication is essential for biofilm formation and represents a promising target for novel antimicrobial strategies. In Campylobacter jejuni, the signalling molecule autoinducer-2 (AI-2), produced by the enzyme LuxS, plays a central role in communication, motility, virulence, and biofilm development. Although the specific AI-2 receptor in C. jejuni remains unidentified, understanding how to modulate this signalling pathway could provide new preventative measures. In this study we used the Vibrio harveyi MM30 whole-cell biosensor to evaluate the effects of lavender (Lavandula angustifolia) preparations (essential oil and ethanol extracts) and pure compounds (linalool and linalyl acetate) on C. jejuni AI-2 signalling. Vibrio harveyi, which produces bioluminescence in response to AI-2, showed a significant decrease in bioluminescent response when exposed to spent media of C. jejuni treated with lavender preparations, indicating disruption of AI-2 signalling. Interestingly, while lavender treatments affected the biosensor's response, they did not change AI-2 concentrations or luxS expression in C. jejuni. These findings indicated that either lavender preparations are interacting with AI-2 in the media or they are preventing their detection by V. harveyi. To investigate further, we performed an additional test by adding lavender preparations directly to the spent media of untreated C. jejuni, which resulted in the same reduction in bioluminescent response as spent media of C. jejuni treated with lavender preparations.

These findings suggest that lavender preparations can modulate bacterial communication pathways by inhibiting AI-2 receptor activity, positioning them as promising natural antimicrobial agents. The V. harveyi biosensor proves to be a powerful tool for investigating bacterial signalling and its disruption, paving the way for future research into novel strategies for preventing biofilm formation in C. jejuni and other pathogens.

Acknowledgment: Funding from the Slovenian Research Agency was provided research for PhD 51861, projects no. J4-2542, no. J4-3088, no. J4-4548, and program P4-0116.

Keywords

Vibrio harveyi

Campylobacter jejuni

whole cell biosensor

intercellular communication

P1.132 Microfluidic approach as a new platform for the assessment of Campylobacter jejuni biofilms on microplastics

Blaž Jug¹, Andreja Slejko¹, Andreja Rajković², Tadej Kokalj³, Anja Klančnik¹

¹University of Ljubljana Biotechnical faculty, Slovenia. ²Ghent University Faculty of Bioscience Engineering, Belgium. ³Institute of Metals and Technology, Slovenia

Abstract

Pathogenic bacteria associated with food poisoning originate from persistent contamination of food-related environments. Campylobacter are highly prevalent in poultry production and processing systems, yet the bacterial transmission through these systems remains unexplored. These bacteria persist largely due to biofilms, which can endure and cause cross-contamination. We introduced a microfluidic approach as a new platform for the assessment of Campylobacter jejuni biofilms on microplastics and compared the detection of C. jejuni in biofilms on microplastics under both static (without flow) and dynamic (with flow) conditions.

We fabricated the microfluidic chip in the foil lamination technology (Kokalj et al., 2014). A base for chip was fabricated by milling in polycarbonate (PC) including the in/outlets and main chamber, the pressure sensitive adhesive (PSA) foil with cut channels was laminated on top of the chamber. After manual adding of microparticles the MF system was closed with PC foil. Tubes connected to the chip allowed a steady flow of liquid via a syringe pump. In addition to our standard setup, we incorporated standardized microplastics (Polypropylene-PP) of 150-250 µm into some of the chips to study its influence on biofilm dynamics. We inoculated C. jejuni and allowed them to adhere for 4 hours. After that we started the flow rate of 5 µl/min with different time points of 24, 48 and 72 hours at 37 °C in a microaerophilic atmosphere to form the biofilm. We then analysed passive and active detachment (by increasing the flow rate to 50 µl/min). After detachment, the biofilm cells inside the chip were collected by ultrasound and analysed by cultivation methods.

This microfluidic platform offers precise environmental control and real-time monitoring, providing novel insights into the formation, stability, and shedding of C. jejuni biofilms.

Acknowledgment: Research supported by the Slovenian Research Agency (projects J4-3088, J4-4548, and program P4-0116).

Keywords

Campylobacter jejuni

microfluidics

biofilm

microplastics

P1.133 Label-Free and Non-Destructive Monitoring of Differentiation and Aging in Mesenchymal Stem Cells Based on Cellular Metabolism

Kyeong-Mo Koo¹, Chang-Dae Kim¹, Tae-Hyung Kim^1,2

¹Department of Intelligent Precision Healthcare Convergence, Sungkyunkwan University (SKKU), Seobu-ro 2066, Jangan-gu, Suwon, Gyeonggi-do, 16419, Republic of Korea. ²Department of Biomedical Engineering, SKKU, Seobu-ro 2066, Jangan-gu, Suwon, Gyeonggi-do, 16419, Republic of Korea

Abstract

Cellular aging and passage-associated changes can diminish the therapeutic efficacy of mesenchymal stem cells (MSCs), despite their pivotal role in regenerative medicine. Recent analytical tools often require invasive processes, limiting their applicability for real-time and long-term monitoring. In this study, we developed a label-free and non-destructive electrochemical (EC) platform to monitor MSC aging and osteogenic differentiation by focusing on mitochondrial metabolism. We dynamically detected these distinct metabolic shifts, serving as biomarkers for cellular aging and differentiation, through EC signals. This platform enabled the detection of mitochondrial metabolic changes during osteogenic differentiation across multiple passages (from passage 4 to 10), highlighting functional changes with increasing passage number. Furthermore, we investigated the role of culture media in modulating osteogenic differentiation. Interestingly, in high-glucose media, MSCs exhibited rapidly proliferation during the initial culture phase; however, from day 3 after that, their proliferation decreased, resulting in cellular senescence. The electrical signals diminished by approximately 15%, and cellular multipotency was substantially affected. The differentiation rate into the osteogenic lineage has been delayed by approximately 7 days compared to that on normal medium. EC signals accurately reflected changes in mitochondrial metabolism and differentiation efficiency affected by medium composition, illustrating their sensitivity to biological variables.

This study provides an accurate, non-invasive approach for the real-time assessment of MSC quality, providing crucial information into cellular senescence and differentiation capability. Our methodology offers a thorough approach to improving quality control in stem cell therapies, supporting customized methods for optimal regeneration prospects.

Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (Grant Nos. NRF-2022R1A4A2000776, NRF-2022R1A2C4002217) and the Korean Fund for Regenerative Medicine funded by Ministry of Science and ICT and the Ministry of Health and Welfare. (Grant numbers RS-2022-00070316, RS-2024-00331844, and RS-2024-00410437).

Keywords

Cellular Aging

Osteogenesis

Mitochondrial Metabolism

Electrochemical Biosensor

P1.134 Application of insect olfactory receptor-expressing sensor cells for screening olfactory inhibitors

Hidefumi Mitsuno, Yuji Sukekawa, Sawako Niki, Eri Kuroda, Takuya Nakajo, Eiji Ohya, Stephan Shuichi Haupt, Ryohei Kanzaki

The University of Tokyo Research Center for Advanced Science and Technology, Japan

Abstract

Abstract

Microbial infection, as the most frequent complication of chronic wounds, has become a global healthcare challenge because of the impairment of the wound healing process and the risks for organ failure, sepsis, amputations, and death. Chronic wounds are estimated to affect 8.2 million individuals in the US alone and cost from $28.1 to $96.8 billion annually. These numbers are expected to increase significantly in the next few decades with an ageing population and the recent epidemic of obesity and diabetes.

When mature biofilms form on wounds, treatment becomes challenging. Their resistance to antibiotics, disinfectants, and the host immune system can be up to 1000 times higher than that of treating infections caused by planktonic bacteria. Therefore, the fast and early-stage detection of microbial biofilms or their planktonic forms is urgently needed.

The broad spectra of Gram-positive and Gram-negative bacteria, frequently found in wounds, were used to investigate their biofilm detection by potentiometric and current measurements. The results confirmed that the development of negative open circuit potential of bioanode is mainly caused by mediated electron transfer (MET). The control measurements with polymicrobial culture prove that different bacteria species are more electroactive and can possibly share electron transfer mediators synergistically. This phenomenon results in higher (more negative) OCP and current/power densities. This synergy might benefit potentiometric or/and “amperometric” detection of multi-species wound infections.

Additionally, we introduce a novel proof-of-concept of wireless and battery-less biofilm detection. The design uses medically relevant bacteria-based biofilms as electron suppliers to reduce a non-conducting antenna material to an excellent conductor. It combines modern knowledge of nanobiotechnology, a current understanding of microbial fuel cells, and wireless signal registration using radio frequency (RF) antenna tags.

Keywords

Wireless

Biofilm

Biosensor

Electrogenicity

P1.138 Discrimination of subtle structural variations in alcohol compounds using insect odorant receptor-expressing cells

Rui Zhou, Yuji Sukekawa, Sawako Niki, Eri Kuroda, Ryohei Kanzaki, Shigehiro Namiki, Hidefumi Mitsuno

The University of Tokyo Research Center for Advanced Science and Technology, Japan

Abstract

Subtle differences in the chemical structure, such as modifications in functional groups or carbon chain length, generally have minimal effects on a molecule's overall physicochemical properties, including electronic characteristics, boiling point, and polarity. This similarity makes it challenging for artificial odorant sensors to differentiate minor variations owing to the significant overlap in their response signals. In contrast, insect odorant receptors are naturally sensitive to these subtle structural differences due to their complex living environments and chemical communication systems. By expressing odorant receptor proteins in heterologous cells, it is possible to mimic the specific responses of these natural odorant receptors, aiding in the development of biosensors for screening structurally similar chemicals.

To explore this, we first assessed the ligand responses of various alcohol compounds that elicit distinct response values according to a public database cataloging Drosophila melanogaster odorant receptors and their ligands. We utilized three odorant sensor cell lines we previously generated, each expressing D. melanogaster odorant receptors, the odorant receptor co-receptor, and a calcium indicator in the Sf21 cell line. Subsequently, ligand-screening assay with structurally similar alcohols that varied in functional group position or carbon chain length was performed.

Notably, sensor cells expressing D. melanogaster receptors Or13a, Or47a, and Or98a produced ligand-evoked responses consistent with the alcohol response profiles in the database. Moreover, the cells exhibited distinct fluorescence changes in response to functional group position or carbon chain length variations, suggesting that our sensor cells can differentiate between various chemicals and detect subtle structural variations akin to natural odorant receptors expressed in living insects. This advancement supports the development of insect odorant receptor-based biosensors for precise odorant screening and discrimination.

Keywords

insect odorant receptors

in vitro expression system

odorant sensor

structural discrimination

P1.139 Bacteria as Sensor Elements – Shewanella oneidensis in Electrochemical Environmental Biosensors

Kaja Fołta¹, Abdullah Abdullah¹, Divine Yufetar Shyntum², Katarzyna Krukiewicz¹

¹Department of Physical Chemistry and Technology of Polymers, Silesian University of Technology, Poland. ²Biotechnology Centre, Silesian University of Technology, Gliwice, Poland of Technology, Poland

Abstract

The development of electrochemical biosensors opens new possibilities for the rapid and selective detection of microorganisms. Here, we present an innovative approach to constructing biosensors based on electroactive bacteria, with a particular focus on Shewanella oneidensis MR-1. This microorganism, capable of extracellular electron transfer (EET), serves as a bioactive sensing element, enabling the detection of electrochemical changes in response to selected analytes. The study employed various electrochemical techniques, including cyclic voltammetry, impedance spectroscopy, and chronopotentiometry, to assess the activity of S. oneidensis biofilms deposited on indium tin oxide (ITO) electrodes, both unmodified and modified with a conductive coating (PEDOT:PSS) and a glucose layer. The results demonstrated that the presence of the biofilm significantly influences the electrochemical response of the electrode, confirming the feasibility of using S. oneidensis as a biocomponent in electrochemical biosensors. The findings presented in our study highlight the potential applications of the developed detection platform in environmental diagnostics, quality control in the food industry, and biotechnology.

This research was funded by the Polish Ministry of Science and Higher Education – Wsparcie studentów w zakresie podniesienia ich kompetencji i umiejętności [W125].

P1.140 Portable device for in vivo ecotoxicological studies of Polyethylene terephthalate (PET) nanoplastics impact on Artemia franciscana

Antonella Giacovelli¹, Eleonora Giannotta¹, Silvia Rizzato¹, Francesca Lionetto², Anna Grazia Monteduro¹, Carola Esposito Corcione², Alfonso Maffezzoli², Gayatri Udayan³, Giuseppe Maruccio¹, Maria Giulia Lionetto³

¹Omnics Research Group, Department of Mathematics and Physics “Ennio De Giorgi”, University of Salento, Italy. ²Department of Engineering for Innovation, University of Salento, Italy. ³Department of Biological and Environmental Sciences and Technologies (DISTEBA), University of Salento, Italy

Abstract

Nanoplastics (NPs) are increasingly recognized as a significant environmental and health concern. Studying the responses of living organisms to nanoplastic exposure is crucial for understanding the environmental and health impacts of NP pollution. To address this challenge, sensitive and user- friendly assays are required for rapid, cost-effective and simple detection of these emerging pollutants for the preservation of all living beings.

With this aim, we propose an innovative portable smartphone-based platform for behavioral and ecotoxicological in vivo studies of the model microcrustacean organism Artemia franciscana. Herein, a common smartphone with a camera replaces a traditional microscope enabling real-time observation of the effect of the exposure of this organism to increasing concentrations of polyethylene terephthalate (PET) nanoparticles (from 0.5 to 50 µg/L) in 15,6 mm wells. This device allows the operator to perform accurate and quick field analyses without the use of complex instrumentation. In particular, movement velocities and trajectories and the viability of A. franciscana are evaluated at two different life stages, newborn and adults, in comparison with stand and methods.

Results contribute to elucidating how nanoplastics affect neurological and physiological functions in invertebrates, offering a valuable tool for environmental monitoring and pollution assessment.

The proposed smartphone platform enables real-time behavioral and ecotoxicological analysis of microscrustaceans during nanoplastics exposure, enhancing traditional toxicity tests with a cost- effective and field-applicable tool.

Keywords

Nanoplastics ecotoxicology

Smartphone-based biosensing

Artemia franciscana behavior

Polyethylene terephthalate (PET) toxicity

P1.141 Measuring the transendothelial electrical resistance in a blood-brain barrier-on-chip exposed to physiological shear stress levels

Muriel A. Holzreuter¹, Quinty Halmingh², Loes I. Segerink¹

¹BIOS Lab on a Chip group, University of Twente, The Netherlands. ²University of Twente, The Netherlands

Abstract

Drugs targeted to the brain for treatment of various neurological diseases have an especially high failure rate for clinical approval. One reason for this high failure rate is the blood-brain barrier (BBB) which controls the passage of substances and restricts the uptake of drugs into the brain. [1] Therefore, better models with integrated sensors are necessary to increase our understanding of the BBB and to improve the drug development process. Here, we present a BBB-on-chip in the physiological size range of brain microvasculature. The 60 µm x 80 µm cross-section of the microfluidic channels allows for the application of physiological shear stress during the entire culture period while keeping medium consumption low. COMSOL simulations of this chip design showed that a 0.01 µL/s flow rate reaches shear stress values in the physiological range of 0.1 – 10 Pa [2]. For monitoring of the barrier function, electrodes are integrated into the channels to measure the transendothelial electrical resistance (TEER). The electrode design was optimized using COMSOL to achieve uniform sensitivity. Platinum electrodes were patterned on glass using lithography, and showed a linear response to changing conductivity of KCl solutions. We showed that hCMEC/D3 cells can be cultured for 5 days inside the channels. In a next step, the TEER will be quantified daily for the entire culture period. The simple chip design allows easy upscaling of production. Therefore, we believe that this chip will be useful for investigating the BBB in health and disease once fully optimized.

Uncaptioned visual

[1] W.M. Pardridge, “Drug targeting to the brain,” Pharmaceutical Research, vol. 24, no. 9, pp. 1733–1744, Sep. 2007, doi: 10.1007/s11095-007-9324-2.

[2] A.G. Koutsiaris et al., “Volume flow and wall shear stress quantification in the human conjunctival capillaries and post-capillary venules in vivo,” Biorheology, vol. 44, no. 5–6, pp. 375–386, 2007.

Keywords

Organs-on-chips

Trans-endothelial electrical resistance

Electrodes

Microfluidics

P1.142 Development of a mobile odorant sensing device using small insect electroantennography-based sensor and its application towards robotic sensing

Yuji Sukekawa, Jeongmin Lee, Takuya Nakajo, Stephan Shuichi Haupt, Ryohei Kanzaki, Hidefumi Mitsuno

The University of Tokyo Research Center for Advanced Science and Technology, Japan

Abstract

Male silkmoths use their antennae as sensors to detect sex pheromones emitted from female silkmoths and exhibit searching behavior once they detect pheromones. Their antennae have olfactory sensory neurons in which chemo-electrical transduction is performed by odorant receptor proteins to generate signals processed by the brain. Electroantennogram (EAG) is a routine method to acquire odor evoked electrical antennal responses that have potential to be used as an odorant / pheromone sensor.

In this study, we developed a mobile odorant / pheromone sensing device based on a sensor with a small-sized EAG amplifier and evaluated its pheromone detection capability. We used pheromones as a target odorant in the test. The developed sensor showed dose dependent responses and repeatable responses with high reproducibility. Moreover, the sensor was integrated with a battery and DC fan, comprising in a mobile sensing device. The DC fans generated airflow to allow target gas to be taken into the device through suction holes. To demonstrate the devices sensing capability, we prepared two dishes which contained a piece of filter paper, and aliquots of pheromone solution were put in one of the dishes. The device successfully discriminated the presence of pheromones when the device sucked in air near the dishes. Moreover, the device also responded to a real female silkmoth placed near the device. Furthermore, a developed sensor was used for mobile odorant / pheromone sensing with a robot and a drone. A small-sized drone equipped with EAG amplifier sensors was able to respond to pheromones when the drone hovered in a room.

Therefore, we demonstrated the feasibility of using the device for mobile sensing of odorants. Although limitations in continuous device operation time still have to be addressed, the developed sensor has great potential in signaling the presence of otherwise hard to detect odorants.

Keywords

electroantennogram

insect olfaction

odor sensor

mobile sensing

P1.143 An intelligent organ-on-chip for ECM remodeling and alveolar dynamics simulation: Revolutionizing idiopathic pulmonary fibrosis research

Chuan-Yi Yang¹, Jia-Wei Yang², Chong-You Chen¹, Guan-Yu Chen^3,2

¹Department of Electrical Engineering, College of Electrical and Computer Engineering, National Yang Ming Chiao Tung University, Hsinchu, Taiwan. ²Anivance AI, Engineering bldg.6, 1001 Ta-Hsueh Road, Hsinchu, Taiwan 300, R.O.C., Taiwan. ³Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Taiwan

Abstract

With the rising incidence of respiratory diseases and post-infectious complications, pulmonary fibrosis has emerged as a significant concern within the global medical community. We center on idiopathic pulmonary fibrosis (IPF), a condition marked by the progressive proliferation of fibroblasts and pathological accumulation of extracellular matrix (ECM). Due to the limited understanding of disease progression in humans, we designed an in vitro model of pulmonary fibrosis to address this challenge and explore potential therapeutic strategies. This model simulates respiration based on the physiological parameters of clinical IPF patients. This tunable hydrogel replicates physiological ECM stiffness of 7-25 kPa and simulates alveolar expansion and contraction through laser-induced cyclic deformation at 0.3 Hz. It enables investigation of ECM remodeling and fibroblast - ECM interactions, marking a significant advancement over previous in vitro models. Then, through photothermal energy conversion, we controlled the autologous environment to study dynamic interactions between fibroblasts and the ECM. This approach confirmed the crucial role of ECM stiffness and mechanical deformation in IPF progression. Finally, within a biomimetic lung microenvironment, clinical drug therapy reduced fibrosis fluorescence intensity from 0.055 a.u. to 0.02 a.u., marking an approximate 63.6% decrease. Integrating this model into drug development could accelerate its translation to clinical applications. This progress relies on collaboration among biomedical researchers, clinicians, and pharmaceutical manufacturers.

Keywords

Idiopathic Pulmonary Fibrosis

Organ-on-Chip

Alveolar dynamics simulation

Drug development

P1.144 A novel optical sensor for quercetin monitoring in wine by a vapor-phase synthesized molecularly imprinted polymer (MIP)

Tiziano Di Giulio¹, Muhammad Ibrar Asif¹, Cosimino Malitesta¹, Martina Corsi², Giuseppe Barillaro², Elisabetta Mazzotta¹

¹University of Salento Department of Biological and Environmental Sciences and Technologies, Italy. ²University of Pisa Department of Information Engineering, Italy

Abstract

Molecularly imprinted polymers (MIPs) are synthetic materials that exhibit unique molecular recognition abilities, directly resulting from their tailored synthesis process[1]. Due to these features, MIPs have been applied in various fields, including sensor development. In this study, we propose to use a room-temperature vapor-phase synthesis approach[2] to obtain a robust, highly sensitive, and selective MIP-based optical sensor for quercetin (QU), combining the selective capabilities of MIPs with the optical properties of nanostructured porous silicon (PSiO₂) used as an interferometer. The MIP synthesis was guided by specific design criteria supported by computational simulations[3]. Prior to synthesis, QU was anchored onto the PSiO₂ surface, by a naïve carbonyldiimidazole linker, capitalizing on the benefits of target pre-immobilization during the imprinting process[4]. Pyrrole (Py) was chosen as the functional monomer due to its room temperature volatility and strong affinity for QU. Molecular mechanics and quantum chemistry calculations demonstrated an energetically favorable interaction between the monomer and target, facilitated by π-π stacking interactions and hydrogen bonding. Polypyrrole (PPy) films embedding the target molecules were directly obtained on the PSiO₂ surface following vapor-phase polymerization. The MIP-coated PSiO₂ substrates resulted in sensors with high sensitivity and selectivity for QU detection. Using UV-VIS reflectance spectroscopy, the sensor demonstrated effective detection of QU in aqueous and ethanolic solutions within a concentration range of 2.5 to 200 μM. Selectivity was confirmed through tests involving mixture with potential interfering substances and by detecting QU in real samples of red and white wines. The results underscore the practical applicability of the developed sensor for its real use and highlight the efficacy of the imprinting process.

Acknowledgements

This work was funded by the European Union Horizon Europe programme under grant agreement No 101046946 (RESORB).

References

[1] https://doi.org/10.1021/acs.biomac.5b01454

[2] https://doi.org/10.1002/SMLL.202302274

[3] https://doi.org/10.3390/IJMS24076785.

[4] https://doi.org/10.3390/bios11010003

Keywords

molecularly imprinted polymers (MIPs)

computational simulations

quercetin

optical sensor

P1.145 Rational Design of Electrosynthesized MIP-Based Sensors Using In Silico Models

Tiziano Di Giulio¹, Muhammad Ibrar Asif¹, Khadijia Eddahaoui¹, Cosimino Malitesta¹, Elisabetta Mazzotta¹, Boris Mizaikoff², Soumya Rajpal²

¹University of Salento Department of Biological and Environmental Sciences and Technologies, Italy. ²Ulm University Institute of Analytical and Bioanalytical Chemistry, Germany

Abstract

Electrochemical sensors are pivotal for rapid and sensitive analyte detection in environmental and biomedical applications [1]. However, they may face challenges in achieving specificity due to potential interference from background signals or other electroactive species in the sample. Additionally, stability issues over time, such as electrode fouling, surface degradation, or drift, can pose further challenges, particularly when natural recognition elements are utilized as receptors. Molecular imprinting introduces synthetic receptors to enhance specificity and stability of the recognition element, and complementarily electrochemical synthesis of MIPs provides better control over the fabrication process. This combined with computational pre-selection of the optimal combination of polymerization components, such as monomers and template molecules, can accelerate the development of next-generation sensor design processes [2]. This research focuses on the strategic screening of monomers from a unique electropolymerizable monomer library, with the aim of developing a generic process applicable to both small and large molecules; advancing from the conventional electro-synthesized MIPs (e-MIPs) based on arbitrary selection of polymerization mixtures. Quantum mechanical calculations are employed for estimating interaction energies for small targets, while molecular mechanics to predict interactions with larger complexes like proteins. Further on this versatility, we employ two model targets, first, a pesticide imidacloprid whose detection as a water pollutant is crucial for environmental sustainability; second, a vital heart disease biomarker, troponin I, to underscore the potential of MIPs in biomedical applications, particularly in diagnostics. The monomer library comprises electrochemically significant compounds such as scopoletin, pyrrole, phenol, o-aminophenol, o-phenylendiamine, m-phenylendiamine, p-phenylendiamine and aminophenylboronic acid. Spatial distribution, non-covalent bonds and binding energies are utilized to characterize the impact of monomer type and ratio within the complex. The integration of theoretical and experimental approaches aims to establish a comprehensive protocol for the rational design of eMIPs, advancing the field of sensor technology.

References

1. doi:10.1007/S00216-022-03981-0

2. doi:10.3390/IJMS24076785

Keywords

Rational Design

In silico models

molecularly imprinted polymers

proteins detection

P1.146 Design of peptide probes for volatile organic compound GFET sensing

Mina Okochi, Tharatorn Rungreungthanapol, Kae Senoo

Tokyo Institute of Science, Japan

Abstract

The development of ultra-sensitive volatile organic compounds sensing technology is expected to have a wide range of applications, including environmental monitoring, hazardous materials detection and quarantine, manufacturing flow and quality control, and health and disease monitoring. Existing large-scale analyzers, such as gas chromatography, are capable of high-precision detection, but they have some issues in complicated operation, real-time and on-site detection. Therefore, miniaturized sensor development such as graphene field-effect transistors (GFETs) has been widely and vigorously pursued. However, the area that FETs can sense is limited to the Debye length range in the extreme vicinity of the channel surface. Consequently, for even higher sensitivity detection, the size of the recognition molecule to be modified on the electrode surface must be smaller than the Debye length. Antibodies and receptors used as recognition molecules in biosensors have excellent sensitivity and specificity in their interactions with target molecules however there is a problem of reduced sensitivity due to the effect of Debye shielding. In addition, the cost of production and storage stability by expression purification using cells were also issues. To address these issues, this study aimed to design peptide probes for sensing volatile organic compounds and to develop ultra-sensitive sensing technology by constructing GFET peptide interfaces. For example, peptide probes for targeting skatole were explored based on the amino acid sequence of CquiOR10, an olfactory receptor of mosquitoes, using binding and selectivity to the target compounds as indices. The selected binding peptides were linked to a graphene self-assembling peptide to create bifunctional peptides and enable a one-step functionalization on GFET for label-free, ultrasensitive, and selective detection. The skatole binding peptide-modified GFET sensor exhibited high sensitivity with a detection limit of 2 pM and high selectivity for skatole.

Keywords

peptides

volatile organic compound

graphene

FET

P1.147 Biomimetic polymer receptor-based electrochemical sensor for direct detection of clinically relevant analytes

Jekaterina Reut, Roman Boroznjak, Akinrinade George Ayankojo, Vitali Syritski

Department of Materials and Environmental Technology, Tallinn University of Technology, Estonia

Abstract

The use of Molecularly Imprinted Polymers (MIPs) as biomimetic receptors in sensing devices is a promising strategy to address limitations of biological recognition elements, such as instability under thermal and pH variations and limited shelf-life. Molecular imprinting creates molecular cavities within a polymeric network that mimic the size, conformation, and chemical properties of target molecules, ensuring high specificity and efficient binding. Combining MIPs with electrochemical transducers offers a powerful approach for developing portable sensors with real-time monitoring, user-friendliness, low cost, and high sensitivity. A direct sensing approach that eliminates the need for external redox probes significantly improves sensor performance by better mimicking complex sample environments and reducing result interpretation errors. Ruthenium oxide (RuO2) electrodes, known for their excellent electrochemical properties, have received limited attention in molecular imprinting.

This study demonstrates the synergy between a MIP layer and RuO₂ electrode in an electrochemical sensor for the direct detection of disease biomarkers in biological samples. Protein biomarkers, including brain-derived neurotrophic factor (BDNF) and growth factor X, were selected as target analytes. In this sensor configuration, RuO₂ serves as both an electrode transducer and an integrated redox probe, enabling signal measurement directly in the analyte solution. Following the optimization of the template elution, rebinding time, and MIP layer thickness, the sensors demonstrated an effective functionality within a relevant analytical ranges in serum samples. Furthermore, the sensors demonstrated high reusability, maintaining performance across multiple rebinding-regeneration cycles.

This work was supported by the Estonian Research Council (grant PRG2113).

Keywords

molecularly imprinted polymer

electrochemical sensor

ruthenium oxide

protein detection

P1.148 Synthesis and Application of Polynorepinephrine-Based Biomimetic Receptors: Nanoparticles and Films for Fiber-Optic Biosensing and Beyond

Simone Ventisette¹, Pasquale Palladino¹, Maria Minunni², Simona Scarano¹

¹University of Florence Department of Chemistry 'Ugo Schiff ', Italy. ²University of Pisa Department of Pharmacy, Italy

Abstract

A new generation of molecularly imprinted biopolymers derived from the endogenous neurotransmitter polynorepinephrine (MIPNEs) has been developed, highlighting their potential as versatile and cost-effective materials for protein recognition. A straightforward synthesis method produces films and nanoparticles imprinted with specific peptide sequences from target proteins through alkali-induced autoxidation of the monomer at low temperatures in aqueous media, resulting in three-dimensional recognition cavities capable of reversible and selective binding to the target protein. MIPNEs demonstrate significant promise as receptor mimics in bioanalytical affinity-based assays. These bioreceptors have been effectively integrated with various analytical techniques, including Surface Plasmon Resonance (SPR)¹ and Fiber-Optic Bio-Layer Interferometry (BLI)², achieving performance in human serum comparable to standard detection methods, using immunoglobulin G1 as the target. Similar outcomes are anticipated for MIPNE-based sensors targeting proteins such as human serum albumin (HSA) or myoglobin across diverse matrices.

The versatility of MIPNEs has also enabled the development of innovative therapeutic tools. By harnessing their molecular recognition properties and biocompatibility, MIPNE NPs are being explored in biomedical applications as effective molecular catchers or drug delivery systems.³

These results confirm the efficacy and flexibility of MIPNEs as biomimetic receptors, paving the way for their potential use as substitutes for antibodies.

Acknowledgments:

Project funded under the National Recovery and Resilience Plan (NRRP), European Union -NextGenerationEU, as part of the Tuscany Health Ecosystem THE (ECS_00000017) spoke 4 -Nanotechnologies for diagnosis and therapy. Authors acknowledge MUR-Dipartimenti di Eccellenza 2018 - 2022 and 2023 - 2027 (DICUS 2.0) to the Department of Chemistry “Ugo Schiff”, University of Florence.

References:

1. Torrini, F. et al., Biosens. Bioelectron. 217, 114706 (2022)

2. Ventisette, S. et al., Biosens. Bioelectron. under revision

3. S. Xu et al., Angew. Chem. International Edition. 60, (2021)

Keywords

Molecularly Imprinted Polymers

Biomimetic receptors

Bio-Layer Interferometry

Optical detection

P1.149 Nanozymatic MOF-on-MOF hybrid material for colorimetric ammonium ion detection from soil water

Eda Akin, Mattis Neubauer, Sruthi Prasood Usha, Zeynep Altintas

Kiel University, Germany

Abstract

The development of technologies for continuous measurement of nitrogen forms in the soil is essential for optimizing the application of fertilizers in agriculture and preventing water-resource pollution [1]. Ammonium ion determination in soil water is crucial for sustainable agriculture and environmental monitoring. However, there is no effective commercial technology available for continuous monitoring of ammonium species in soil water [2]. Herein, a colorimetric nanoreactor integrating nanozyme as a transducer component and metal organic framework (MOF) as receptor was combined with another MOF for the selective detection and removal of ammonium ion in soil water. The synthesis of nanozyme involves post-synthetic modification of gold nanoparticles (AuNPs) with zeolitic imidazolate frameworks-8 (ZIF-8) in an inorganic solvent. The resulting composite, AuNPs@ZIF-8, exhibits tuneable peroxidase-mimicking activity with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate, and H₂O₂, generating a blue-color compound (TMB_ox). An enhanced catalytic activity is observed due to the functional synergy between AuNPs and ZIF-8 [3]. Subsequently, the nanozyme is mixed with pre-synthesized host MOF consisting of zirconium and mellitic acid via post-functionalization strategy. The novel MOF-on-MOF hybrid structure was comprehensively characterized using XPS, XRD, SEM, and AFM. In the absence of ammonium ion, nanozyme efficiently catalyses the H₂O₂ oxidation of TMB, developing a blue-color reaction mixture. The presence of NH₄⁺ results in their adsorption to MOF, which inhibits TMB oxidation, and reaction mixture color changes to light blue, corresponding to NH₄⁺ concentration. This approach combining two distinct MOFs offers a robust material for in situ quantification of NH₄⁺ in soil water.

[1] Bouraoui, F., & Grizzetti, B. (2014). Science of the Total Environment, 468, 1267-1277.

[2] Yupiter, R., Arnon, S., Yeshno, E., Visoly-Fisher, I., & Dahan, O. (2023). npj Clean Water, 6(1), 25.

[3] Kang, N., Wei, X., Shen, R., Li, B.,... & Astruc, D. (2023). Applied Catalysis B: Environmental, 320, 121957.

Uncaptioned visual

Keywords

Nanozyme

Metal-organic frameworks

Ammonia ion detection

Soil water

P1.150 A baiting strategy for the development of aptamer as a bioreceptor for tuberculosis heterodimer antigen detection

Shaira Jane Acosta¹, Vasso Skouridou¹, Ciara K. O'Sullivan^1,2

¹Interfibio Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, 43007, Spain. ²Institució Catalana de Recerca i Estudis Avancats (ICREA), 08010 Barcelona, Spain

Abstract

A substantial proportion of individuals infected with tuberculosis (TB) still go undiagnosed. Given the limitations of sputum-based TB diagnostics, developing methods based on non-sputum sample analysis, such as serum and urine, enables TB detection at peripheral settings. The antigens CFP-10 and ESAT-6, which form a heterodimeric complex upon secretion, have demonstrated a good diagnostic potential. However, TB antigens are typically present at low concentrations, and the lack of robust bioreceptors capable of selectively binding TB antigens hinders antigen-based detection.

We developed aptamers that recognize the ESAT-6/CFP-10 heterodimer using a combination of capture SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and a novel antibody-pulldown SELEX, in which the target-bound single-stranded DNA (ssDNA) sequences were baited with an α-CFP-10 IgG antibody immobilized on magnetic beads. Specifically, we performed two parallel selections using two different ssDNA libraries, where each was immobilized to magnetic beads through hybridization with a complementary docking probe. Upon addition of the target in solution, high-binding affinity ssDNA sequences were released from the beads and bound to the target. These eluted target-bound ssDNA sequences were subsequently pulled-down with α-CFP-10 IgG antibody-conjugated magnetic beads, thereby isolating the sequences that can effectively bind the ESAT-6/CFP-10 heterodimer forming a sandwich with the antibody.

A total of six rounds were performed and candidate aptamers were identified using next-generation sequencing. The binding properties of the selected aptamers were evaluated using enzyme-linked aptamer assays, demonstrating affinity dissociation constants in the nanomolar range. Overall, the selected aptamers exhibited selective binding to the heterodimer as bioreceptors, making them adaptable for biosensing applications.

Keywords

tuberculosis

ESAT-6/CFP-10 heterodimer

aptamer

pulldown capture SELEX

P1.151 Development of an electrochemical sensor for detecting melamine

Ernestas Brazys¹, Vilma Ratautaite², Arunas Ramanavicius^1,2

¹Vilnius University, Lithuania. ²State Research Institute Center for Physical Sciences and Technology, Lithuania

Abstract

Melamine is a compound commonly used in the plastics industry to produce resins. The misuse of melamine by unethical manufacturers to inflate protein levels in dairy products is well-documented. Notably, in 2008, melamine-contaminated milk caused urinary stones in children under three in China [1]. Melamine is nephrotoxic to humans, and its consumption can lead to renal diseases. This study demonstrates melamine detection using molecularly imprinted polypyrrole [2].

The molecular imprinting procedure involves three steps: 1) self-assembly of monomer, cross-linker, and template molecules complexes; 2) polymerization; and 3) removal of the template to create specific binding sites in the polymer structure [3]. This enables the development of sensors for specific detection.

In this study [2], a melamine-imprinted polypyrrole-based (MIP) sensor was developed. The pre-polymeric mixture contained 50 mM of pyrrole and 5 mM of melamine in a 10 mM PBS solution (pH 7.4). The polymeric layer was electrochemically deposited on a graphite electrode using potential pulses, and the template was extracted with 1 M H₂SO₄ solution for 60 minutes. In addition, MIP layers were modified with gold nanoparticles (AuNPs) of 3.5 nm, 6 nm, and 13 nm diameters, along with gold(I) complexes during polymer preparation.

The properties of all polypyrrole films were evaluated using differential pulse voltammetry (DPV) in PBS with a 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] redox probe. The interaction between melamine and the polymer layers was assessed by comparing oxidation peak currents and calculating the apparent imprinting factor. The optimal results were obtained with the MIP modified with 0.05 nM AuNPs of 3.5 nm diameter.

References:

1. A. K. -C. Hau, et al., 2009, Journal of the American Society of Nephrology, 20.

2. E. Brazys, et al., 2024, Microchemical Journal, 199.

3. V. Ratautaite, et al., 2022, Journal of Electroanalytical Chemistry, 917.

Keywords

molecularly imprinted polymers

melamine

polypyrrole

sensors

P1.152 An AI assisted biosensing device for early diagnosis of Alzheimer’s disease

Sudhaunsh Deshpande, Arjun Ajith Mohan, Guoyi Liu, Sanjiv Sharma

University of Liverpool, UK

Abstract

Introduction: Accurate and early diagnosis of Alzheimer's disease is crucial for timely intervention. Currently, confirmation of AD often relies on invasive lumbar punctures to extract cerebrospinal fluid for analysis, a procedure that is uncomfortable and not readily available in many settings. This work presents a patient-friendly alternative: a cost-effective electrochemical biosensor for detecting p-tau 181, a key AD biomarker, directly in blood (plasma and serum). This point-of-care device overcomes the limitations of conventional methods and the need for invasive CSF testing by integrating a novel porous gold electrode, a highly specific molecularly imprinted polymer (MIP), and automated data processing via machine learning.

Methods: A MIP was electropolymerized on a porous gold electrode to selectively capture p-tau 181. This porous surface enhances sensitivity and reduces biofouling common in biological samples. A custom handheld potentiostat with integrated wireless communication enables decentralized measurements. Fabrication involved PCB manufacturing with gold electroplating, porous surface modification, optimized monomer preparation, and precise MIP fabrication. Sensor characterization included cyclic voltammetry and scanning electron microscopy with energy-dispersive X-ray spectroscopy (EDS). The device wirelessly transmits data to a remote server for analysis by a machine learning algorithm trained to identify p-tau 181 concentrations from electrochemical signals.

Results and Conclusion: SEM imaging confirmed the porous morphology of the gold electrode and uniform polymer coating. EDS analysis validated the MIP's elemental composition. Electrochemical characterization demonstrated successful sensor function, detecting p-tau 181 across a clinically relevant range of 0-50 pg/mL with a remarkable limit of detection (LOD) of 40 fg/mL and a sensitivity of 4.23 μA/pg/mL in PBS, human serum, and plasma with different anticoagulants. This AI-assisted p-tau 181 biosensor offers a sensitive, specific, and cost-effective solution for AD diagnosis, paving the way for accessible early detection and improved patient care by eliminating the need for invasive lumbar punctures.

Uncaptioned visual

Keywords

Alzheimer's Disease

point of care

MIPs

Machine intelligence

P1.153 Enzymatic electrochemical biosensing for point of care detection of total sugar

Sudhaunsh Deshpande, Arjun Mohan, Sanjiv Sharma

University of Liverpool, UK

Abstract

Introduction: Rapid and accurate total sugar detection is crucial for managing metabolic disorders like fructosemia and galactosemia. Current point-of-care diagnostics often focus on individual sugars like glucose, leaving a critical gap in comprehensive metabolite monitoring, particularly for disorders involving multiple sugar imbalances. This study presents an enzymatic electrochemical biosensor for efficient total sugar measurement in human serum.

Methods: A glucose oxidase (GOx) based sensor was developed, leveraging its high specificity and stability. To broaden its substrate range for total sugar analysis, GOx was encapsulated within gold nanoparticles (AuNPs) and co-entrapped in CTAB, a unique approach that enhances enzyme stability and promotes signal amplification. Fabrication involved PCB electrode production with porous gold modification to increase surface area and sensitivity. AuNPs were synthesized using a thermal reduction method. Gox was then encapsulated within AuNPs along with CTAB and phenol red, which serves as a redox mediator and facilitates immobilization. This complex was immobilized onto the porous gold working electrode by electrochemical polymerization. Sensor characterization included cyclic voltammetry and surface analysis using SEM and EDS.

Results and Conclusion: Electrochemical characterization confirmed sensor functionality with a distinct oxidative peak at 0.15V . The sensor demonstrated high sensitivity towards a range of sugars, including glucose, fructose, lactose, and galactose, with minimal interference from other serum components. While further investigation into long-term stability is ongoing, this cost-effective and user-friendly biosensor offers a promising solution for point-of-care total sugar detection. Its rapid detection capabilities and potential for miniaturization and integration with portable potentiostats pave the way for revolutionizing the diagnosis and management of metabolic disorders.

Keywords

total sugar

modified enzyme

point of care

low cost

P1.154 Decentralised electroanalysis of emerging contaminants: molecularly imprinted polymer sensor for the selective detection of the antidepressant venlafaxine in water

María Cerrato-Álvarez^1,2, Javier Menéndez Menéndez¹, Patricia Rebelo^1,3, Pablo Rioboó-Legaspi¹, Henri P. A. Nouws³, Cristina Delerue-Matos³, Estefanía Costa-Rama¹, M. Teresa Fernández-Abedul¹

¹Department of Physical and Analytical Chemistry, University of Oviedo, Spain. ²Department of Analytical Chemistry, University of Extremadura, Spain. ³REQUIMTE/LAQV, ISEP, Polytechnic of Porto, Portugal

Abstract

Decentralised analysis has become of paramount relevance for obtaining accurate information and making rapid decisions. Artificial intelligence will be soon applied to measurement procedures, requiring large datasets that could be obtained from analytical platforms. In this context, electrochemical detection systems could be the basis of field-deployable devices for real-time continuous environmental measurements.

Environmental analysis, commonly approached with centralised and robust high-tech equipment, is moving towards on-site strategies [1]. The presence of emerging contaminants in inland waters and wastewater treatment pools is among the main environmental concerns. Although low-cost electrochemical cells have been designed for pharmaceutical analysis [2,3], more work is required to attain adequate selectivity for real decentralization.

In this work, a methodology for monitoring electroactive contaminants in real time is presented. A discharge of redox pollutants in a water reservoir is simulated to study the effect of different conditions. Since a very large group of substances could be present, selectivity is required. This could be approached using molecularly imprinted polymers [4]. A sensor was carefully designed and optimised for venlafaxine determination. Experimental parameters related to the different steps: electropolymerization, extraction and incubation, were optimised. The analytical features of the methodology were evaluated and analysis of water samples of Febros (Portugal) wastewater treatment plant was performed.

This work was supported by PCI2022-133007 (Biodivrestore Cofund 2020, funded by MCIN/AEI/10.13039/501100011033 and EU (NextGenerationEU/PRTR funds) and AYUD/2021/51289 (Government of the Principality of Asturias and EU FEDER Program). M.C.-A. was supported by Margarita Salas grant (UNI/551/2021-Complementaria 2022-MS18), P.R.-L. by Severo Ochoa BP-21/029 grant and E.C.-R. by Beatriz Galindo BG20/00027 grant.

Najib Messaoud, Paula Lopes, João Barbosa, Raquel Queirós

International Iberian Nanotechnology Laboratory, Portugal

Abstract

Accurate monitoring of lactate levels in amniotic fluid during labour is essential for assessing fetal well-being and guiding timely clinical interventions. Elevated lactate concentrations (above 11 mM) are indicative of fetal distress [1], demanding rapid decision-making to improve neonatal outcomes. In this study, we developed an electrochemical sensor based on a molecularly imprinted polymers (MIPs) for the selective and sensitive detection of lactate in amniotic fluid. We explored two different monomers, 3-aminophenylboronic acid (APBA) and dopamine, to create highly specific MIP layers. This approach offers a reusable alternative to traditional bio-affinity sensors, such as antibodies, which are generally limited to single use applications.

Optimization studies focused on the electropolymerization of monomers, sensor stability and template removal using chemical and chemical-free strategies. The morphology and composition of the MIP sensors were characterized using Scanning Electron Microscopy and Raman spectroscopy. To validate the MIP layer's selectivity, a non-imprinted polymer (NIP) was settled as a control.

To ensure applicability in complex biological matrices, specifically in amniotic fluid, the sensor was designed for integration into a catheter-based system, enabling real-time, intrapartum monitoring of lactate within the clinically relevant range (2-12 mM). This innovative sensor platform aims to provide obstetricians with a reliable tool for assessing fetal condition during labour, potentially reducing the need for emergency interventions and improving perinatal outcomes.

[1] The Journal of Maternal-Fetal & Neonatal Medicine, 35(25), 7306–7311

Keywords

Sensor

Lactate

Intrapartum Monitoring

Amniotic fluid

P1.159 Electroactive NanoMIPs for Early Detection of Acute-on-Chronic Liver Failure (ACLF)-Associated Small-MoleculeBiomarkers

Jose Marrugo-Ramírez^1,2,3, Giulio Rosati^1,2,3, Andrew Piper^1,2,3, Jonel Trebicka^4,5,6, Christophe Junot⁷, Cristopher Zaleski⁸, Alvaro Garcia-Cruz⁸, Sergey A. Piletsky⁸, Arben Merkoçi^1,2,3,9

¹Catalan Institute of Nanoscience and Nanotechnology (ICN2), Spain. ²CSIC, Spain. ³BIST (ICN2), Spain. ⁴Department of Internal Medicine I, University of Frankfurt, Germany. ⁵Medizinische Klinik B, Universitätsklinikum Münster, Münster University, Germany. ⁶European Foundation of Chronic Liver Failure, EFCLIF, Spain. ⁷French alternative Energies and Atomic Energy commission (CEA), CEA Saclay, France. ⁸Department of Chemistry, University of Leicester, UK. ⁹ICREA, Institució Catalana de Recerca i Estudis Avançats, Spain

Abstract

Cirrhosis can result from a variety of factors, including chronic alcohol consumption, hepatitis C, bile duct disease, and diabetes, among others. An advanced stage of this condition, known as Acute-on-Chronic Liver Failure (ACLF), presents significant challenges, including an increased risk of mortality and organ failure. This study aims to aid the understanding of kynurenic acid (KA) and N-acetylneuraminic acid (NANA), recently identified as promising urinary biomarkers for ACLF. Detecting these metabolites and other small-molecule targets is complex, underscoring the necessity for innovative molecular tools to navigate the challenges associated with the lack of suitable bioreceptors. In this context, this work proposes the development and use of electroactive molecularly imprinted polymer nanoparticles (e-nanoMIPs) as an effective and stable alternative to traditional antibodies and enzymes for detecting KA and NANA. Our findings indicate that the developed sensors successfully achieve subfM limits of detection for both biomarkers in buffer solutions and diluted synthetic urine, demonstrating good selectivity towards structurally similar and co-occurring metabolites.

Keywords

ACLF

(Bio)sensor

nanoMIPs

Electrochemistry

P1.160 Biomimetic materials for the diagnosis and follow-up of age- related diseases

Felismina Moreira

Polytechnic Institute of Porto School of Engineering, Portugal

Abstract

The most common age-related diseases (ArD) include dementia (especially Alzheimer's), cardiovascular disease and diabetes. Despite their social and economic impact, there is currently no test that can accurately and simultaneously diagnose multiple ArDs, which is crucial for appropriate therapy and follow-up care. Existing detection methods rely mainly on immune reactions, which are expensive, unsustainable and time-consuming. Alternatively, sophisticated chromatographic techniques are also expensive and therefore impractical for point-of-care (PoC) analysis. Synthetic materials with high affinity for specific molecules can overcome these challenges. The aim is to demonstrate that an affordable electrochemical sensor platform can be created to screen ArD biomarkers at the PoC in patients by developing a low-cost, portable, cross-platform biosensor material that is specific for each biomarker.

Here we present a novel sensing platform based on advanced sensory biomaterials to monitor biomarkers related to ArD in a PoC. The design of the biosensing materials involves the synthesis of plastic antibodies (PA) using innovative molecular imprinting (MI) technology. The nanostructured sensor units were fabricated by modifying conductive electrodes with nanomaterials such as carbon, metals or semiconductors compounds for subsequent assembly of the PAs.

Overall, these PoC tests enable early diagnosis, monitoring of disease progression and evaluation of new drugs during clinical trials or treatment response.

Acknowledge: This study was financed by project IBEROS+ (0072_IBEROS_MAIS_1_E, Interreg-POCTEP 2021-2027); This work has been partially supported by the Portuguese Foundation for Science and Technology (FCT), through grants UIDB/04730/2020 and UIDP/04730/2020.

Keywords

Ageing diseases

biomarkers

Plastic antibodies

Point-of-Care

P1.161 Surface-Imprinted Polymer Photonic Sensors for Improved Point-of-Care Detection of Bacterial Urinary Tract Infections

Valerii Myndrul¹, Rocio Arreguin-Campos¹, Igor Iatsunskyi², Flavia Di Scala¹, Kasper Eersels¹, Bart van Grinsven¹

¹Sensor Engineering Department, Faculty of Science and Engineering, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands. ²NanoBioMedical Centre, Adam Mickiewicz University, 3, Wszechnicy Piastowskiej Str., 61 614 Poznan, Poland

Abstract

Bacteria play a critical role in human health, aiding in essential processes like digestion and immunity while also causing diseases, such as urinary tract infections, which are predominantly caused by Escherichia coli (E. coli) [1]. Rapid detection of E. coli is vital, as traditional diagnostic methods like urine culture and polymerase chain reaction (PCR) can take 1–2 days, delaying treatment and increasing the risk of inappropriate antibiotic use [2].

This study introduces a novel biosensor platform combining Surface Imprinted Polymers (SIPs) with porous silicon (PSi) substrates for the (photoluminescence-) PL-based detection of E. coli. SIPs provide an artificial recognition layer, ensuring high affinity for bacterial targets through non-covalent interactions with outer membrane proteins, carbohydrate patterns, and phospholipids [3]. Utilizing polydimethylsiloxane (PDMS) as the imprinting polymer enhances transmittance, ensuring efficient PL signal generation within the underlying PSi surficial layer.

Uncaptioned visual

Figure 1. Schematic Representation of the PSi/SIP Biosensor for E. coli Detection. The upper pathway shows PSi/NIP fabrication, lacking E. coli specificity, causing no PL change. The lower pathway demonstrates PSi/SIP with imprinted cavities, enabling selective bacterial binding and noticeable PL intensity change.

The resulting PSi/SIP biosensors demonstrate exceptional selectivity, reproducibility, and rapid detection capabilities, with a detection time of approximately 20 minutes. These biosensors require no pre-treatment, simplifying the workflow and minimizing resource requirements. PL readout methods address the sensitivity limitations of artificial receptors, enabling detection at low concentrations [4]. This platform presents a scalable and efficient solution for UPEC detection in real-world samples, facilitating reliable PoC diagnostics while reducing reliance on laboratory methods.

Reference.

[1]. A.W. Walker, L. Hoyles, Nat. Microbiol. 8 (2023) 1392–1396.

[2]. B. Foxman, Dis. Mon. 49 (2003) 53–70.

[3]. T.M. Iakimova, et al., Cell Reports Phys. Sci. (2024) 101853.

[4]. V. Myndrul et al., Biosens. Bioelectron. (2024) 116916, https://doi.org/10.1016/j.bios.2024.116916

Keywords

Porous Silicon biosensor

Surface Imprinted Polymers

Photoluminescence

Urinary Tract Infections

P1.162 MIP-based Electrochemical Tuberculosis Biosensor for HIV Positive Patients

Diego Nigoa¹, Thomas Lee¹, Maria Barbosa², Azrin Jamison¹, Megan Brobst¹, Lauren Murphy¹, Elizabeth Hause¹, Delphine Dean¹

¹Clemson University Department of Bioengineering, USA. ²Medical University of South Carolina, USA

Abstract

Tuberculosis (TB) and HIV/AIDS stand at number 1 and 2 top causes of death in Tanzania, as reported by the World Health Organization1. The WHO has also categorized Tanzania as high-burden for TB and high-burden for HIV-associated TB2. Furthermore, coinfection of TB and HIV allows latent TB infections to more quickly progress to active TB disease. Therefore, early diagnosis of TB in these patients is of utmost importance. However, some of these coinfected patients may receive false negative test results for TB due to their weakened immune system, which common tests for TB rely on. By creating an electrochemical biosensor based on antigens found in mycobacterium tuberculosis, the team plans to more effectively and quickly diagnose tuberculosis in HIV positive individuals.

A study3 published in 2023 showed high concentrations of three antigens in the urine of patients positive for both TB and HIV/AIDS: ESAT-6, CFP-10, and MPT-64. This study aims to test the efficacy of a single MIP biosensor for all three antigens, compared to using three separate sensors for each antigen. The research group has successfully created MIPs using this method targeting creatinine4.

This biosensor approach hopes to greatly reduce the turnaround time of TB testing, by allowing testing to be done at the point-of-care using a handheld cyclic voltammetry device after the synthesis has been performed in a lab.

References:

United Republic of Tanzania Health Data Overview, World Health Organization <https://data.who.int/countries/834>
“WHO releases new global lists of high-burden countries for TB, HIV-associated TB and drug-resistant TB,” World Health Organization. <https://www.who.int/news/item/17-06-2021-who-releases-new-global-lists-of-high-burden-countries-for-tb-hiv-associated-tb-and-drug-resistant-tb>
DK Turbawaty, et al. <https://doi.org/10.1177/2632010X231198831>
AM Jamison, et al. “Detection of Antiretroviral Drugs in Urine (The Kugundua)” Biomedical Engineering Society Conference, 2019. Abstract Database <https://www.bmes.org/archive>

(2) Mohsenzadeh, E.; Ratautaite, V.; Brazys, E.; Ramanavicius, S.; Zukauskas, S.; Plausinaitis, D.; Ramanavicius, TrAC Trends in Analytical Chemistry 2024, 171, 117480.

(3) Quadrini, L.; Laschi, S.; Ciccone, C.; Catelani, F.; Palchetti, I. TrAC Trends in Analytical Chemistry 2023, 168, 117345.

Keywords

Molecularly imprinted polymers (MIPs)

Electrochemical urea detection

Selective sensing

Molecular recognition

P1.166 Integration of Artificial Intelligence and Molecularly Imprinted Polymer-Based Electrochemical Multi-Sensor for Aging Clock

Youngjun Seo

Korea University, Republic of Korea

Abstract

The aging of populations worldwide represents a major demographic trend with far-reaching implications for public health and socioeconomic systems [1]. This shift has precipitated a surge in research focusing on age-related disorders and their potential interventions. Of particular concern are geriatric disease conditions, notably cancer and dementia, which not only impact individual health outcomes but also pose substantial burdens on healthcare systems and social structures. In response to these challenges, there is an increasing societal imperative for developing and implementing innovative technologies aimed at early identification and proactive management of the aging process. Here, we developed and evaluated the performance of an electrochemical multi-sensor-based aging clock that can simultaneously measure various inflammatory cytokine markers. The aging clock incorporates molecularly imprinted polymers (MIPs), selected for their high reliability and reusability in point-of-care (POC) [2]. MIPs demonstrate biomimetic properties analogous to metabolite-specific antibodies and exhibit superior biocompatibility in complex biological matrices. To validate the efficacy of the aging clock, we conducted analyses on samples derived from lipopolysaccharide (LPS)-induced senescence cell models and blood specimens from age-stratified cohorts. The resultant data were subsequently applied to a machine learning algorithm for the quantification and pattern recognition of age levels. This process facilitates the quantification and longitudinal tracking of aging processes, enables early detection of physiological anomalies, and supports the analysis of age-related phenotypic patterns. Integrating MIP-based sensors, multi-analyte detection capabilities, and machine-learning algorithms in this aging clock offers a comprehensive analytical tool for both research and clinical applications in age-related disorders, promising new insights and potential solutions.

Keywords

Aging clock

MIP

Artificial Intelligence

Aging monitoring

P1.167 Machine learning-guided (L)SPR analysis of epitope imprinting efficiency in polynorepinephrine biopolymers

Davide Sestaioni¹, Giulia Ciacci², Andrea Barucci², Pasquale Palladino¹, Simona Scarano¹

¹University of Florence Department of Chemistry 'Ugo Schiff ', Italy. ²Nello Carrara Institute of Applied Physics National Research Council, Italy

Abstract

The dream of having innovative materials capable of functioning as affinity “binding partners” instead of antibodies is a groundbreaking research field in bioanalytics and biosensing, aligned with EU directive on animal protection in research (2010/63/EU). Molecularly Imprinted Polynorepinephrine (MIPNE), synthesized by co-polymerizing Norepinephrine (NE) with a peptide following the "Epitope Imprinting Approach" (EIA), constitutes a promising alternative[1]. EIA improves receptor performance by selecting and imprinting a specific peptide sequence from a target protein[2], rather than the whole protein itself. A key challenge, however, remains in defining optimal selection criteria for epitopes, which significantly impacts the sensitivity and stability of the receptor and, consequently, the biosensor’s performance.

Here is introduced an innovative, Machine Learning-assisted method for epitope selection. Developed starting from an LSPR assay, this method overcomes the unreliability of the traditional "trial and error" approach. A training library of 50 peptides was analyzed by measuring the maximum plasmonic absorption wavelength of AuNPs grown directly on each MIPNE[3]. Results clustered into distinct output classes, used as experimental labels. From this data, 101 peptide descriptors from Phyton environment were extracted and correlated with the experimental classes, leading to find reliable descriptors capable of predicting optimal peptide templates based on the protein’s sequence. The method was finally validated through affinity and kinetic SPR assays, confirming its effectiveness.

Acknowledgments:

Project funded under National Recovery and Resilience Plan (NRRP) – Discovering the SEcret woRld of pOlyseroTONin for green molecular ImprINting and its application in bioanalytics (SEROTONIN), CUP: B53D23025250001 – Authors acknowledge MUR - Dipartimenti di Eccellenza 2018 - 2022 and 2023 - 2027 (DICUS 2.0) to the Department of Chemistry “Ugo Schiff”, University of Florence.

References:

[1] Torrini F. et al., Sens Actuators B Chem. (2023)

[2] Torrini F. et al., Biosens Bioelectron. (2022)

[3] Scarano et al., Microchimica Acta. (2019)

Keywords

Optical Biosensors

Mimetic Receptors (MIPs)

Machine Learning

Epitope Selection

P1.168 Recombinant Saxiphilin as a Tool for the Detection of Paralytic Shellfish Toxins: Towards the Development of a Biosensor

Ambbar Aballay-González^1,2, Mònica Campàs³, Jorge Diogène³, Maximiliano Figueroa⁴, Allisson Astuya^1,2

¹Biotoxins laboratory, Faculty of Natural and Oceanographic Sciences, University of Concepción, Chile. ²Center for Oceanographic Research COPAS COASTAL, Universidad de Concepción, Chile. ³Marine and Continental Waters Program, Institute of Agrifood Research and Technology (IRTA) La Ràpita, Spain. ⁴Molecular biophysics laboratory, Faculty of Biological Sciences, University of Concepción, Chile

Abstract

Paralytic shellfish poisoning (PSP) toxins comprise a group of compounds, with over 50 congeners described, all of which are structural analogs of saxitoxin (STX). These toxins are responsible for approximately 35% of human intoxications associated with marine toxins worldwide. To ensure food safety, continuous monitoring of shellfish intended for human consumption is implemented. Ethical and economic concerns have driven the development of alternative methods that can complement official detection protocols. However, most of these methods rely on antibodies, which, due to their high specificity, exhibit limited cross-reactivity with different PSP toxin congeners. Therefore, the use of a natural receptor for paralytic toxins, such as the Saxiphilin protein, is proposed for the development of a novel detection method with potential application in biosensor technology.

The C-lobe of Saxiphilin from Lithobates catesbeianus was recombinantly produced in insect cells using the MultiBac baculoviral system (Genova Biotech), yielding 6 mg of protein per liter of culture medium. The developed system consists of immobilized Saxiphilin, a labeled saxitoxin tracer, and a competitive assay to evaluate the detection of paralytic toxins. The determined IC50 for saxitoxin was 60 ng of STX·mL⁻¹, with demonstrated cross-reactivity for PSP toxin congeners such as GTX2&3 and dcSTX.

These results highlight the potential of the Saxiphilin-based system as an alternative tool for PSP toxin detection in shellfish. Its ability to recognize various PSP toxin congeners positions it as a promising approach for the development of biosensors applicable in monitoring programs, contributing to food safety and reducing the reliance on animal-based assays

Keywords

Saxitoxin

protein

detection

paralytic shellfish poisoning

P1.169 Advancing mpox detection: the potential of electrochemical sensors

Dmitrij Gritsok¹, Maria Montenegro¹, Martin Hedström², Célia Amorim¹

¹University of Porto, Portugal. ²Lund University, Sweden

Abstract

Mpox is a zoonotic viral disease currently declared a Public Health Emergency of International Concern by the WHO¹. The 2022–2023 multi-country outbreak resulted in approximately 100,000 cumulative cases². The virus’s dissemination beyond endemic regions in Africa underscores the urgent need for effective diagnostic tools. Sporadic cases persist, viral mutations lead to asymptomatic or severe presentations, complicating detection and management of disease. Well-established diagnostic methods such as PCR and ELISA are highly sensitive and specific but rely on specialized labs, expensive equipment, and trained personnel. These requirements limit the suitability for point-of-care testing and rapid management of outbreaks. For instance, many mpox cases have been only classified as suspected due to the lack of appropriate diagnostic tools³.

Electrochemical sensors offer a promising alternative due to rapid analysis, portability, miniaturization, cost-effectiveness, and ease of use. They enable direct detection of mpox biomarkers (i.e., viral DNA, proteins, antibodies) through electrochemical transduction. While mpox-specific electrochemical sensing is emerging, progress has been made. Current studies focus on detecting A29 surface protein, a key immunosensor target, while genosensors and CRISPR-based biosensors gain traction for their selectivity and detection limits^4,5. Future efforts should explore additional electrochemical designs adaptig them for mpox detection including alternative targets, diverse recognition elements, dual-mode and multiplex detection strategies.

Acknowledgements

This work received financial support from the PT national funds (FCT/MECI, Fundação para a Ciência e Tecnologia and Ministério da Educação, Ciência e Inovação) through the project UID/50006 – Laboratorio Associado para a Química verde – Tecnologias e Processos Limpos.

References

1. C. Wenham et al., Lancet, 2022, 400, 2169-2171.

2. B. Cabanillas et al., Allergy, 2024, 79, 3285-3309.

3. J. Garrigues et al., J. Clin. Microbiol., 2022, 60, e01655-22.

4. L. de Lima et al., ACS Appl. Mater. Interfaces, 2023, 15, 58079-58091.

5. J. Lee et al., Nanoscale, 2024, 16, 11318-11326.

Keywords

Monkeypox

Electrochemical sensors

P1.170 Advancements of a molecularly imprinted polymer biosensor for detection of ARV drugs in HIV-positive patients in low-resource settings

Azrin Jamison¹, Maria Eduarda Barbosa de Camargo², Taya Lee³, Diego Nigoa¹, Thomas Lee¹, Megan Brobst¹, Ella Hause¹, Aaron Spearman¹, Jeremiah Carpenter¹, Melissa McCullough¹, Melinda Harman¹, John DesJardins¹, Delphine Dean¹, Lauren Murphy⁴

¹Clemson University Department of Bioengineering, USA. ²Medical University of South Carolina, USA. ³University of Maryland, USA. ⁴Clemson University, USA

Abstract

Adherence to antiretroviral (ARV) therapy is crucial for managing HIV, particularly in resource-limited, low-and middle-income countries (LMICs). Molecularly imprinted polymers (MIPs) offer a promising, cost-effective option for drug monitoring in these settings, due to their durability and reusability compared to other biosensors, such as synthetic and natural proteins and aptamers. These other sensors have limited shelf life and are more expensive, making MIPs the ideal sensor type. However, MIPs present fabrication challenges, such as weak adherence to the gold-screen printed electrode (Au-SPE) surface and inconsistencies in the calibration curves. One of the goals has been to establish a consistent pre-treatment process for Au-SPE to promote a more consistent monolayer of carboxylic groups.

There have been several identified critical factors in the fabrication process that influence the consistency. Several pre-treatment methods were established and tested. The control procedure consisted of washing Au-SPE in ethanol (EtOH) for 5 minutes and then drying with an air hose while method A consisted of 5-minute EtOH and Acetone wash respectively followed by drying with an air hose. Method B utilized plasma treatment for 1 minute followed by nitrogen exposure and method A washing steps. Contact angle measurements were taken before and after each wash step and after 11-mercaptoundecanoic acid deposition to evaluate surface hydrophilicity. Acetone was eventually excluded from all methods due to sensor well degradation. Contact angle measurements were then retaken to confirm the Au-SPE was still hydrophilic in nature. 

The group found that utilizing a combination of plasma treatment and nitrogen exposure followed by an EtOH wash presented the most hydrophilic surface. Future work will aim to fine-tune treatment durations and steps to achieve greater surface hydrophilicity, further improving sensor performance for ARV adherence monitoring. This work highlights the critical need to optimize Au-SPE pre-treatment to improve MIP sensor reproducibility.

Keywords

Antiretroviral (ARV) Monitoring

Molecularly Imprinted Polymers

Point-of-Care

Urine

P1.171 Electrochemical Techniques for Investigating Bio interfaces

Sunil Luhar^1,2, Kamila Sadowska¹

¹aNalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Ks. Trojdena 4, 02-109 Warsaw, Poland, Poland. ²RMIT University, Australia

Abstract

Electrochemical techniques play a crucial role in the study of bio interfaces, offering valuable insights into biomolecular interactions, surface properties, and sensor performance. This literature review explores the application of fundamental electrochemical methods—such as cyclic voltammetry, electrochemical impedance spectroscopy, and chronoamperometry—in characterizing material stability, surface functionalization, and signal transduction for biosensing and diagnostic applications. Additionally, emerging advancements, including wearable electrochemical sensors and 3D-printed interfaces, are revolutionizing real-time monitoring and point-of-care diagnostics. By integrating electrochemical approaches with modern fabrication techniques, researchers are developing highly sensitive and reliable biosensors tailored for diverse healthcare and analytical applications. This review highlights recent progress in the field and discusses future directions for optimizing electrochemical methodologies in bio interface studies

Acknowledgments: Kamila Sadowska would like to acknowledge financial support from National Science Centre, project number 2023/50/E/ST5/00347.

Keywords

Electrochemical techniques

Bio interfaces

Biosensing,

Diagnostics

P1.172 Combined electrochemical and synthetic recognition sensing technology for the real time detection of adenosine: a crucial biomarker in bowel health

Molly Wild¹, Perrine Lasserre², Bhavik Patel³, Nicholas Turner¹

¹The University of Sheffield, UK. ²University of Strathclyde, UK. ³University of Brighton, UK

Abstract

Adenosine is a clinically important biomarker for the detection of irritable bowel diseases, and research has shown its importance in the modulation of homeostatic systems within the bowel¹. Current methods of detection require sampling from the patient and subsequent preparation before chromatographic quantification can be performed^2,3.

However, the half life of adenosine stands at approximately 10 seconds⁴ and therefore, despite excellent sensitivity through methods such as HPLC, the delay from sample collection to quantification can “mask” the accurate concentration of the marker in blood due to decay.

With this in mind, this work presents the effort to produce an adenosine sensing device that can provide real-time monitoring of the marker through a combinatorial approach of electrochemical quantification methods (differential pulse voltammetry (DPV)) and synthetic recognition technology (Molecular Imprinting).

This approach demonstrated excellent affinity/selectivity towards the desired target adenosine (K_D = 692 nM), a 10-fold increase compared to other nucleosides, whilst the use of DPV in an electrochemical MIP device allowed for a limit of detection in the pM range, comparable to current methods, without the drawbacks of sample prep or run time.

The sensor was then tested with simulated “real-world” samples, with spiked serum and no significant loss in efficacy was observed. It is envisaged this sensor can be used in point of care scenarios where timely and accurate monitoring of adenosine is of critical and clinical importance.

1 L. Antonioli, M. Fornai, C. Pellegrini, L. Bertani, Z. H. Nemeth and C. Blandizzi, Front. Immunol., DOI:10.3389/fimmu.2020.01310.

2 G. Haink and A. Deussen, Journal of Chromatography B, 2003, 784, 189–193.

3 L. Löfgren, S. Pehrsson, G. Hägglund, H. Tjellström and S. Nylander, PLOS ONE, 2018, 13, e0205707.

4 D. Faulds, P. Chrisp and M. M.-T. Buckley, Drugs, 1991, 41, 596–624.

Keywords

Electrochemical sensor

Biomarkers

Synthetic sensing technology

Electrochemical quantification

P1.173 Automated platform for the sample preparation of sequencing: from sample to library preparation

Mélissa BAQUE, Nicolas SARRUT-RIO, YVES FOUILLET, François BOIZOT, Mahfod Benessalah, Jean-Maxime ROUX

CEA-Leti, France

Abstract

Field detection of biothreat agents are usually based on immunologic Lateral Flow Assays and nucleic acid amplification tests that are both biased detection method because of their agents specific reagents. Nowadays the portable sequencing is becoming a promising field unbiased detection method that allows the simultaneous identification of multiple biothreat microorganisms in suspicious samples. New sequencing technologies are more and more accessible and affordable with high performances as the MinION, a real time device for DNA or RNA sequencing. However, this technology requires multistep process before the sequencing as the extraction and the purification of DNA and the library preparation. All these steps involve pipetting and handling which are hardly compatibles with on-field operations. Here we present the development of an automated platform from the sample to the Oxford Nanopore Technologies library preparation.

The overall protocol is performed on two microfluidics cartridges made of COC, managed by a unique instrument thanks to pneumatic actuations. The first cartridge is dedicated to DNA recovery (figure 1): concentration and mechanical lysis with the integration of a polycarbonate membrane and purification with the integration of a silica membrane. The cartridge allow to simply drop off 1mL of raw sample (as puddle, powder or soil matrix) and to obtain 50µL of concentrated purified DNA (60% of efficiency) ready for the library preparation. A second cartridge (figure 2A) is dedicated for the library preparation specific to the MinION device which include embedded lyophilized reagents and thermal incubations. This microfluidic protocol is successfully performed using an elastic membrane pneumatically actuated (figure 2B) and allows the successive resuspension of lyophilized reagents in volume from 10 to 70µL (figure 2C).

Our automated platform allows non-experts operators such as firefighters to prepare samples before sequencing with few manipulations and only one instrument from raw samples.

Uncaptioned visual

Figure 1

Uncaptioned visual

Figure 2

Keywords

sequencing

microfluidic

sample preparation

pathogens

P1.174 Development of an electromagnetic micropump for liquids handling in flexible biomedical microfluidic devices

Adrien Geffrelot, Mehdi Ammar, Elisabeth Dufour-Gergam

Centre for Nanosciences and Nanotechnology, France

Abstract

Biomedical microfluidic devices (BMD) perform specific functions (biomarker extraction, cell sorting, biochemical reactions, etc.) for disease diagnosis, medical monitoring, personalized medicine, etc. Integration of microfluidic functions in these devices is a major challenge to achieve. For this purpose, solutions are under development [1],[2] and marketed products [3],[4] already exist. There main technological issues concern the integrability of the components for use in BMD.

Our work proposes the development of a fully integrable micropump for liquids handling in BMD. The main advantages are that the design can be easily customized (micropump dimensions and performances) for a specific applied BMD. In addition, our microfabrication steps for the micropump can be easily combined with standard BMD manufacturing process.

Our micropump operates on an electromagnetic principle. It consists of a microcoil and a deformable magnetic membrane underneath a pumping chamber (fig.1), embedded in a flexible biocompatible material. By generating a magnetic field with microcoil, the deformable magnetic membrane is subjected to a force and deforms. This induces volume and pressure changes in the pumping chamber, causing liquid to flow.

We first modelled the micropump to investigate microfluidic behaviour resulting from membrane deformation. A membrane deformation of 10 µm should be the minimum to achieve pumping for a specific design. Then, we developed a microfabrication process under clean room conditions. The electrodeposition and transfer of the magnetic material onto a deformable material and its assembly with a microfluidic chip and microcoil were the main microfabrication steps to be worked on. Finally, a setup based on laser displacement sensing was mounted to characterize the micropumps (fig.2a). Membrane deformation of 15 µm was measured, which corroborates with modelling, and the behaviour as function of actuation current and frequency was studied (fig.3). After the pumping achievement, the micropump is now ready for use in BMD (fig.2b).

Uncaptioned visual

Keywords

Microfabrication

Integration

Membrane deformation

Pumping

P1.175 A rapid one-pot ATP detection strategy based on self-priming extension of phosphorothioated aptamer hairpin

Yuri Jo¹, Junhyeok Yoon², Hyun Gyu Park¹

¹Korea Advanced Institute of Science and Technology Department of Chemical and Biomolecular Engineering, Republic of Korea. ²Korea University College of Life Sciences and Biotechnology Department of Biotechnology, Republic of Korea

Abstract

Herein, we investigated a method for ATP detection based on aptamer-mediated phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) technology. In the absence of ATP, the stem region of the aptamer hairpin (AptHP) adopts a double-stranded conformation, which is subsequently cleaved by exonuclease III, a double-strand-specific exonuclease. The cleaved AptHP probe cannot bind to the trigger, thereby inhibiting the subsequent THSP reaction. In contrast, in the presence of ATP, the aptamer region of the AptHP binds to the target, inducing a conformational change in the hairpin probe. The conformational shift inhibits the stem from developing a double-stranded configuration, so preventing it against exonuclease III. The intact AptHP then binds to the trigger, facilitating probe extension mediated by DNA polymerase. This extension reaction initiates the self-folding of the terminal hairpin, triggering subsequent extension cycles. As a result, an elongated DNA concatemer is generated, which can be monitored in real-time using LAMP dye, a fluorescent agent that intercalates into double-stranded DNA. This isothermal detection method allows for the specific identification of target ATP. By utilizing this approach, we successfully demonstrated the feasibility of the ATP detection method within 25 minutes, with a broad linear range spanning from 0.25 to 250 µM.

Keywords

PS-THSP

Fluorescence-based biosensor

Aptamer

ATP detection

P1.176 Numerical modelling of complex structures in paper-based microfluidic channels to predict flow and transport phenomena of analytes

Ramy Moumneh, Grégoire Le Brun, Sami Yunus, Jean-Pierre Raskin

Catholic University of Louvain, Belgium

Abstract

Paper-based analytical devices (PADs) are well-known rapid and portable biosensors for testing a wide range of analytes (from medical to environmental applications) at the point-of-need. While the most popular form of PADs is the lateral flow assay, paper-based microfluidic devices (µPAD) are gaining increasing interest. Their more elaborate structures can achieve new functionalities for sample processing and detection, such as flow at constant rate or multiplexing. However, their increased complexity makes it more difficult to control sample flow and species transport due to the lack of understanding, hampering the development of functional tools in practice.

In this work, we propose a new simulation-based approach to compensate for the lack of a paper-based microfluidic model predicting flow behavior in complex structures (e.g., curvature, multiple inlets, channel division, etc.). The uniqueness of our approach lies in the consideration of the paper matrix as a network of interconnected capillaries (or pores). For this purpose, a simulation of paper-based microfluidic channels is developed based on three variables: contact angles of the membrane types, fluid viscosities and rate factors of the species in the fluid, and is calibrated on experimental results for benchmark geometries. First, we present the simulation validation for existing models (Lucas-Washburn, Mendez and Hong-Kim’s models, among others). Secondly, the simulation is applied to improve understanding and prediction of flows in complex structures (curvatures, combination of constraints, etc.) and compared with experimental results. Finally, the transport of species is investigated to complete the model.

The developed model demonstrates its utility by enhancing comprehension and predictive capabilities regarding transport phenomena in µPADs. It enables analysis from ionic flows for electrochemical detection to the selection of parameters for the flow, immobilization and detection of (lysed-)bacteria within membranes (including membrane type, solvent choice, and geometry), which posed significant challenges in previous studies.

Keywords

paper-based microfluidic

modelling

transport phenomena

sample processing

P1.177 Targeted Molecular Engineering for the Development of Advanced Biosensing Technologies

Miguel António Dias Neves^1,2, Ines MendesPinto^1,2

¹University of Porto Institute for Research and Innovation in Health Sciences, Portugal. ²University of Porto Faculty of Medicine, Portugal

Abstract

Biosensors are analytical devices that convert a biomolecular recognition event into a measurable signal proportional to the concentration of the analyte recognized. This is achieved through three interconnected components: 1) the biomolecular recognition element, which is a biomolecule that binds to a specific analyte; 2) the transducer which converts the biomolecular binding event into a measurable physical/chemical signal; and 3) the signal processor which captures and processes the physical/chemical signal to an output readable by the user. Often the molecular recognition element and the transducer must be interconnected (e.g. an aptamer or protein immobilized onto an electrode or plasmonic sensing surface) to ensure efficient signal transduction. Furthermore, in some cases, signal transduction can be dependent on physical interaction, dissociation or conformation change between the molecular recognition element and transducer after analyte binding. In recent years, our team has focused on optimizing the interface between the molecular recognition element and transducer by combining surface chemistry with molecular engineering of proteins and aptamers. This approach enhances the sensitivity and reproducibility of electrochemical, optical, and piezoelectric biosensors. From these studies, we have developed molecular engineering strategies adaptable across various biosensor configurations. Within this framework, we highlight: 1) the effects of surface chemistry on biomolecular probe immobilization and biosensor performance; 2) a combined platform of protein engineering and surface chemistry that enables functional immobilization of structure-dependent protein heterodimers, like integrins, on hydroxyl-terminated surfaces for autoimmune antibody detection; and 3) the engineering of a solution-based aptamer-antibody FRET biosensor adaptable to any antibody produced in rabbit.

Keywords

Surface Chemistry

Aptasensors

Immunosensors

P1.178 Advances in Real-Time Endotoxin Detection Using Functionalized Silicon Photonic Sensors in Biomanufacturing

Guillaume Nonglaton¹, Benoit Gilquin¹, Hippolyte Durand¹, Malika Amdaoud¹, Darzhan Sadvokassova¹, Ali Kheir Aldine¹, Mahfod Benessalah¹, Caroline Fontelaye¹, Loïc Laplatine¹, Sandy Mathew¹, Audrey Guilain², Charlotte Parent¹, Frédéric Revol-Cavalier¹, Nicolas Sarrut-Rio¹, Eric Calvosa², Stanislas Lhomme¹

¹French Alternative Energies and Atomic Energy Commission Electronics and Information Technology Laboratory, France. ²Sanofi Pasteur Research and Development Centre Marcy-l'Etoile, France

Abstract

Biomanufacturing utilizes living organisms such as Escherichia coli or mammalian cells to produce complex biomolecules like vaccines, monoclonal antibodies (mAbs), and recombinant proteins. A critical step in these processes is downstream processing (DSP), which removes impurities, including endotoxins, host cell proteins (HCP), and DNA, to comply with stringent regulatory standards.

Endotoxins, derived from Gram-negative bacteria, are toxic molecules that can trigger severe inflammatory responses. Rapid and precise detection is essential to ensure the safety of injectable products. While conventional methods like LAL and rFC tests are widely used, they have limitations in terms of response time and reliance on animal-derived resources.

In this study, we present an innovative solution based on functionalized silicon photonic sensors integrated with a microfluidic set-up. Using an advanced biofunctionalization protocol, these sensors enable near-real-time detection of endotoxins in matrices resembling real biomanufacturing conditions. Our results demonstrate the ability of these sensors to detect endotoxin concentrations as low as a few hundred ng/ml within minutes. This technology represents a significant advancement in real-time impurity monitoring during biomanufacturing processes.

Silicon photonic sensors pave the way for continuous and reliable monitoring of critical impurities, enhancing safety, efficiency, and regulatory compliance in industrial processes. This presentation will highlight the progress achieved in sensor surface functionalization and the potential impact of these innovations on the biopharmaceutical industry.

Keywords

Biomanufacturing

Endotoxins

Photonics sensors

Real-time detection

P1.179 Protein detection of extracellular vesicles to monitor and predict the therapeutic response in ovarian cancer using a micropillar patch-integrated microfluidic chip

Yu-sian Huang, Hung-Wei Yang

National Cheng Kung University, Taiwan

Abstract

Ovarian cancer, although less common than other cancers, ranks first in mortality among gynecological cancers. Currently, there is no highly effective screening method for early diagnosis. The main clinical screening methods include pelvic examinations and the CA-125 tumor marker test. However, none of these methods have proven effective in monitoring the therapeutic response in ovarian cancer. Now, molecular profiling of circulating extracellular vesicles (EVs) provides a promising noninvasive means to diagnose, monitor, and predict the course of cancers. Therefore, we have developed a micropillar patch-integrated microfluidic chip (MPMC) to detect the protein markers (HSP70, CD147, and EpCAM) of EVs for monitoring and predicting the therapeutic response in ovarian cancer (Figure 1). CD147 is used to determine drug resistance, HSP70 to assess metastatic potential, and EpCAM to identify tumor-derived EVs. We constructed amine-modified micropillar patches using amine-terminated PLA, followed by antibody conjugation on the micropillar surfaces. The results indicated that the antibody conjugation efficiency on the micropillar patches could reach up to 80%. Our findings demonstrate that the MPMC provides high accuracy in distinguishing between ovarian cancer, non-metastatic ovarian cancer, and healthy individuals. For ovarian cancer patients undergoing treatment, the MPMC can accurately monitor therapeutic response and serve as an independent prognostic factor for progression-free survival. Collectively, this work highlights the potential clinical utility of MPMC in detecting protein markers of EVs for managing ovarian cancer.

Uncaptioned visual

Figure 1. The schematic illustrates the protein detection of EVs for monitoring and predicting the therapeutic response in ovarian cancer using a micropillar patch-integrated microfluidic chip.

Keywords

Microfluidic

Ovarian cancer

EVs

biosensor

P1.180 Development of core-shell molecularly imprinted polymers: A comparative study of oriented imprinting, via covalent and semi-covalent strategies, and non-oriented imprinting

Rahil Radfar, Zeynep Altintas

Kiel University, Germany

Abstract

Molecularly imprinted polymers (MIPs), also known as antibody mimics, are tailor-made polymer matrices integrating recognition cavities that are complementary to a target of interest in terms of size, shape, and functional groups. MIPs are at the centre of attention for decades due to their outstanding features making them suitable in a wide range of applications from extraction, purification, and sensing to biomedical applications [1]. Among different formats of MIPs, core-shell structures combine the unique characteristics of an inorganic core with binding capabilities of the MIP shell, thereby, strengthening their utilization in targeted multimodal imaging, and theranostics. However, the process with which the shell is imprinted on the core significantly effects the binding capacity of the MIP particles [2].

In this study, three different imprinting approaches, namely, non-oriented imprinting, oriented covalent imprinting, and oriented semi-covalent imprinting, were employed to synthesize IL-6 specific core-shell MIP nanostructures by imprinting an epitope of IL-6 on a magnetic core with a size of about 50 nm (Fig. 1). The as-synthesized nanocomposites have been characterized and compared using light scattering methods, Fourier Transform infrared spectroscopy, fluorescent spectroscopy, contact angle measurements, transmission, and fluorescence microscopies [3]. Furthermore, the effect of imprinting approach on the binding performance of the magnetic MIPs, in terms of affinity, sensitivity, and specificity, was studied using voltametric measurement methods with an investigation range of 0.01 pg mL^-1 to 100,000 pg mL^-1. The results emphasized how synthesis approach can impact the physiochemical and binding properties of core-shell MIPs. The study provides valuable insights into the selection of imprinting strategies best suited for synthesizing core-shell MIPs, allowing researchers to adapt their approach based on specific applications and target requirements.

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[1] Haupt et al., Chemical reviews, 2020.

|2] Bhogal et al., TrAC Trends in Analytical Chemistry, 2020.

|3] Radfar et al., Analytical and Bioanalytical Chemistry, 2024.

Keywords

Core-shell imprinting

Non-oriented imprinting

Oriented immobilization

IL-6-specific epitope imprints

P1.181 Towards surface-independent protein immobilization for biosensing applications enabled by atmospheric cold plasma technology

Lieze Dankers¹, Karen Leirs¹, Bernard Nisol², Jeroen Lammertyn¹

¹Department of Biosystems, Biosensors Group, KU Leuven, Leuven, Belgium. ²Molecular Plasma Group, Belgium

Abstract

Biosensors have shown their great potential across various fields (e.g., diagnostics, drug development, environmental monitoring). However, the wide variety of readout systems and/or applications has made it challenging to establish a universal surface for biorecognition element attachment. Additionally, traditional immobilization techniques are complex or can compromise the biofunctionality. This highlights the need for a standardized biomolecule immobilization process, enabling straightforward target detection on a multitude of surfaces. The Molecular Plasma Group's cold atmospheric plasma technology (Figure 1A) offers a scalable solution, combining surface activation with the ability to graft molecules in a single step.

We employed this technology to create stable surface-independent carboxylic acid linker-layer coatings on polymethylmethacrylate (PMMA), cyclic olefin copolymer (COC), polyvinylchloride (PVC), and glass. The linker-layer enables covalent protein immobilization via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry, creating a robust and specific biointerface, serving as a foundation for bioassay integration.

Coating composition, thickness, and topography was analyzed using FT-IR and SEM, showing a stable carboxylic acid-rich layer. Next, the biofunctionalization capacity of the linker-layer was evaluated by the immobilization of three different proteins on PMMA, i.e., IgG, streptavidin, and protein G. Fluorescent microscopy was used to proof the presence of Cy5-streptavidin and its stability when stored in buffer for 5 days (Figure 1B). ELISA-based model bioassays (Figure 1C) demonstrated protein-independent functionalization capacity (Figure 1D) and a linker-layer stability of at least one month (stored in air) (Figure 1E). Finally, a calibration curve was composed for IgG detection on PMMA, showing high signal-to-noise ratio (Figure 1F) and consistent performance was obtained across all four (PMMA, COC, PVC, glass) substrates tested (Figure 1G), proving the method's universal applicability, independent of the surface type.

Hence, this technology offers versatile biosensor fabrication and can revolutionize the biosensors field by enabling surface-independent (bio)functionalization in a scalable and cost-effective manner.

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Keywords

Cold atmospheric plasma

Standardized protein immobilization

Surface-independent

Bio-assay and Biosensor applications

P1.182 ^{Development of a whole-cell based bacterial biosensor assay for quantification of AI-2 signalling molecules in complex media}

Dina Jug¹, Anja Klančnik¹, Iztok Dogša², Sonja Smole Možina¹

¹University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, 1000 Ljubljana, Slovenia. ²University of Ljubljana, Biotechnical Faculty, Department of Microbiology, 1000 Ljubljana, Slovenia

Abstract

Bacteria produce different signalling molecules (SM) for communication and complex social behaviours. Detection and quantification of bacterial SM can be performed with classical analytical methods (colorimetric, liquid and gas chromatography techniques), or by biosensor-based methods using engineered fluorescent and luminescent bacterial sensors. In order to use biosensor-based methods we must define a whole-cell biosensor organism which can detect and quantify SM of studied bacteria.

We used Campylobacter jejuni as a model organism, known as food borne pathogen which produces AI-2, a ‘universal signal’ for interspecies communication and we tested different Vibrio harveyi biosensor strains (BB170, MM30, BB152 and MM32) to select the best luminescent response to C. jejuni AI-2. After V. harveyi biosensor strain selection, various modelling and experimental approaches were used to quantify the concentration of AI-2 in defined growth medium and in a model food system (5% chicken juice in MH broth). The strongest response to C. jejuni AI-2 had V. harveyi MM30, which can amplify the effects of AI-2 on the bioluminescent response. This self-amplification of the response to AI-2 by V. harveyi MM30 enabled the measurement of extremely low AI-2 concentrations produced by C. jejuni (< 0.001 µM). The Hill equation was used as mathematical model to calculate the concentration of C. jejuni AI-2. The concentration of AI-2 in the C. jejuni defined growth medium after 20 h of incubation was (1.21 ±0.05) μM and in the model food system was 5-fold higher [(6.1 ±2.3) μM].

In this study we developed a whole-cell based biosensor assay for simple, safe, rapid, sensitive and accurate AI-2 quantification in complex matrices. This biological approach enabled the quantification of AI-2 in complex media of C. jejuni, which can help to improve the quality and safety of food production.

Funded by ARIS for PhD 51861 and projects J4-2542, J4-3088, J4-4548, P4-0116.

Keywords

Biosensor method

Vibrio harveyi

Campylobacter jejuni

AI-2 quantification

P1.183 Development of oral mucosa-on-achip for anticancer drug toxicity evaluation and drug screening

Younggyun Kim^1,2, Sunghan Lee^1,2, Hyeop Oh³, Sangeun Lee⁴, Bongseop Kwak⁵, Bo Hae Kim³

¹Yonsei University, Republic of Korea. ²Dongguk University College of Medicine, Republic of Korea. ³Dongguk University Ilsan Hospital, Republic of Korea. ⁴Dongguk University Ilsan Hospital,, Republic of Korea. ⁵Dongguk University College of Medicine,, Republic of Korea

Abstract

Oral mucositis is a adverse effect of cytotoxic anticancer drug treatment, leading to significant clinical issues, including severe pain, malnutrition, and increased morbidity and mortality in cancer patients. Despite its danger, effective prevention and therapy are insufficient, underscoring the urgent need for novel drug development. Conventional cell culture and animal models are widely employed for drug screening and testing. However, conventional cell culture models have limits in mimicking complex biological features, and animal models have ethical concerns and cannot observe specific disease processes. Organ-on-a-chip technology offers a promising alternative by providing a more physiologically relevant model that bridges the gap between conventional cell culture models and animal models. In this context, Organ-on-a-chip has gained increasing research interest. However, the application of organ-on-a-chip to the oral mucosa remains underexplored, with no existing studies focusing on evaluating therapeutic interventions for oral mucositis. In this study, we developed an oral mucosa-on-a-chip capable of assessing mucosal damage from cytotoxic anticancer drugs and the efficacy of therapeutic agents. This chip consists of three channels for mimicking the blood vessel, the lamina propria, and the epithelium. The blood vessel channel is fabricated by making a lumen structure with HUVECs (human umbilical vein endothelial cells), simulating in vivo drug delivery environment. The lamina propria channel consists of fibroblasts and extracellular matrix, which play a key role in the wound-healing process. Finally, the epithelium channel mimics the epithelium including basal cells by dispersing keratinocyte spheroids. This chip holds great potential as a drug discovery model for evaluating the healing efficacy of candidate therapeutics for oral mucositis. By providing a more accurate representation of the biological complexity of the oral mucosa, our oral mucosa-on-a-chip model could pave the way for new treatments, accelerating the development of effective therapies for oral mucositis.

Keywords

Microfluidic Devices

Mucositis

Organ-on-a-Chip

Antineoplastic Agents

P1.184 Mixing of LAMP reagents for screening applications on a Lab-on-a-Disc

Donal Duignan¹, Eimantas Davalis¹, Nicklas Rondot¹, Kayla Bruno², Darren McAuley¹, James Landers¹, Eadaoin Carthy¹, David Kinahan¹

¹Dublin City University, Ireland. ²Dublin City University, RAPID, Ireland

Abstract

The development of centrifugal microfluidic platforms has revolutionised molecular diagnostics by enabling rapid, automated, and cost-effective analysis. This study presents the design and development of a low-cost instrument for DNA (Deoxyribonucleic acid) LAMP (Loop-Mediated Isothermal Amplification) amplification integrated with a microfluidic “Lab-on-a-Disc” (LoaD) system. The system automates nucleic acid amplification and detection through isothermal conditions (65°C) and employs a touchscreen and webcam for colorimetric detection, identifying specific wells during rotation.

The instrument incorporates a precision-controlled rotational mechanism, a microfluidic disc with optimised reagent flow paths, and an integrated heating system. The heating mechanism utilises an enclosure-based air convection system, ensuring uniform distribution of heat for reliable amplification. The control system is implemented within LabVIEW, consisting of an Arduino Unos based controller network to regulate rotation speed, detection signal and perform temperature readings.

Extensive thermal modelling and validation experiments demonstrate the efficiency of the instrument in maintaining stable isothermal conditions and detecting amplified nucleic acid sequences with high sensitivity.

The design optimises reagent mixing and downstream processing through centrifugal actuation, reducing complexity and costs associated with traditional molecular diagnostic workflows. Furthermore, we aim to demonstrate multiplex screening on a single disc and minimise liquid handling provided by centrifugal forces. The developed system presents a viable alternative to PCR-based diagnostics, offering rapid and portable nucleic acid detection suitable for point-of-care applications.

Keywords

Lab on a disc

Colorimetric LAMP assay

DNA amplification

Cell lysis

P1.185 On-Chip Micromixer Synthesis of Extracellular Vesicle-Imprinted Nanoparticles for Biosensing Approaches

Kadriye Ölmez^1,2, Özgecan Erdem¹, Beyza Nur Küçük^1,2, Eylul Gulsen Yilmaz^1,2, Fatih Inci^1,2

¹Bilkent University, UNAM-National Nanotechnology Research Center, Ankara, Turkey. ²Bilkent University, Institute of Materials Science and Nanotechnology, Ankara, Turkey

Abstract

Molecularly imprinted polymer nanoparticles are a cost-effective antibody alternative, making monodisperse synthesis essential. Micromixer-based microfluidics offers MIP synthesis by controlling the flow rate and mixing, enhancing reaction kinetics and particle uniformity. Optimized shear and flow rates improve shape, size, and binding specificity control. As biomarkers for cancer, extracellular vesicles (EVs) carry molecular information valuable for early detection and personalized treatments.

This study presents a micromixer based on convergence-divergence principles for the controlled synthesis of extracellular vesicle-imprinted nanoparticles (EV-MIPs). We developed a nanoplasmonic sensor with these EV-MIPs for real-time, kidney-specific extracellular vesicle detection with high specificity and affinity. EVs were isolated from HEK 293 cell cultures using on-chip ultrafiltration and characterized by Nanoparticle Tracking Analysis (NTA) and Scanning Electron Microscopy (SEM) before EV-MIP synthesis. COMSOL Multiphysics simulations optimized mixing efficiency across flow rates for successful nanoparticle synthesis. EV-MIPs were characterized using NTA, Dynamic Light Scattering (DLS), SEM, and Fourier-transform infrared spectroscopy (FTIR). A plasmonic metasurface modified with EV-MIPs was validated through X-ray photoelectron spectroscopy (XPS) to confirm surface chemistry. The microfluidic plasmonic sensor detected EVs by recording wavelength shifts, indicating binding to EV-MIPs on the surface.The isolated EVs measured approximately 180 nm with a concentration of 7 x 10⁹ particles/mL using NTA, confirming their spherical morphology via SEM. Optimization through COMSOL simulations identified 150 µL/min as optimal for achieving EV-MIP nanoparticles with a narrower size distribution, measuring around 80 nm with a concentration of 1 x 10⁹ particles/mL. The introduction of EVs shifted the baseline signal to 1.34 nm, confirming binding to EV-MIPs on the sensor surface. This study demonstrates a micromixer-based approach for continuous flow synthesis of EV-MIPs, enabling accurate detection of HEK-293 cell-derived EVs using nanoplasmonic sensors. This novel approach shows considerable promise for advancing real-time disease diagnostics by integrating on-chip MIP technology with nanoplasmonic sensing.

Keywords

Microfluidic

Micromixers

Extracellular Vesicle-Imprinted Nanoparticles

Biosensor

P1.186 Fiber optic nanogold-linked immunosorbent assay for ultrasensitive detection of beta-amyloid aggregates

Chun-Ping Chang, Yi-Ting Yang, Yen-Ling Chen, Lai-Kwan Chau

National Chung Cheng University Department of Chemistry and Biochemistry, Taiwan

Abstract

Fiber Optic Particle Plasmon Resonance (FOPPR) is a biosensing technology that utilizes the surface plasmon resonance effect of gold nanoparticles (AuNPs). When specific wavelengths of light are directed at the AuNPs, they resonate with the collective oscillation of free electrons on the nanoparticles’ surface. This resonance causes changes in the intensity of reflected light, which can be measured to detect targets that bind to the fiber. In this study, we developed a fiber optic nanogold-linked sorbent assay (FONLISA) based on FOPPR technology to detect amyloid aggregates in body fluids. Beta-amyloid oligomer (AβO) is a protein complex formed by the aggregation of beta-amyloid monomers. The accumulation of Aβ in the brain is the primary factor initiating the pathogenesis of Alzheimer's disease (AD).In this method, antibodies and aptamers (Apt) specific to AβO are modified on the surface of optical fibers and AuNPs respectively. When the Apt- AuNP are mixed with AβO and injected into the fiber sensing zone, they form a sandwich structure consisting of AuNP-Apt-AβO-antibody. This structure leads to significant changes in optical signals, allowing for quantitative detection. The research results demonstrate the several advantages of this method: (1) it offers high detection sensitivity, reaching pM levels for detecting AβO; (2) the detection process is rapid, taking less than 20 min; (3) it demonstrate excellent selectivity, effectively identifying the oligomeric form of beta-amyloid. This technological development provides a new tool for the early diagnosis of Alzheimer's disease, showing significant potential for clinical application.

Keywords

Beta amyloid oligomer

Alzheimer's disease

Fiber optic nanogold-linked immunosorbent assay

Fiber optic particle plasmon resonance

P1.187 Towards clinical translation of a singlet oxygen-based photoelectrochemical platform for KRAS point mutation detection

Jorine Arnouts¹, Senada Koljenovic^1,2, Marc Peeters^1,2, Greetje Vanhoutte², Timon Vandamme^1,2, Karen Zwaenepoel^1,2, Karolien De Wael¹

¹University of Antwerp, Belgium. ²University Hospital Antwerp, Belgium

Abstract

The rapid and accurate detection of cancer biomarkers, such as KRAS point mutations, is crucial for timely diagnosis and personalized treatment. KRAS mutations play a pivotal role in tumorigenesis and serve as important prognostic and predictive markers in several cancers, including colorectal, pancreatic, and lung cancer. Our research group previously developed a singlet oxygen-based photoelectrochemical platform capable of detecting single-point DNA mutations, as demonstrated in a proof-of-concept study targeting KRAS mutations.

To bridge the gap between laboratory research and clinical application, we have optimized the assay protocol for high-throughput analysis, transitioning from a single-Eppendorf workflow to a 96-well plate format, using the KRAS Q61H mutation. During this optimization, we scaled down the working volume from 1 mL to 100 µL, decreased the streptavidin bead volume from 10 µL to 2.5 µL, and fine-tuned the concentrations of the capture and detection probes. This updated protocol maintains equivalent analytical performance compared to the original method while significantly improving throughput and clinical applicability.

To further enhance the platform’s clinical utility, we are investigating various amplification strategies, such as Loop-Mediated Isothermal Amplification (LAMP), Hybridization Chain Reaction (HCR), and Rolling Circle Amplification (RCA). Planned studies will assess the feasibility of integrating one of these methods into the 96-well plate workflow to improve assay sensitivity and enable the reliable detection of low-abundance mutations directly from clinical samples. Additionally, the platform will be tested for the first time on patient samples to evaluate its performance in a clinical context.

This work represents a significant first step toward developing a robust, sensitive, and scalable diagnostic platform for cancer biomarker detection, laying the foundation for translating innovative biosensor technology into clinical practice.

Keywords

Photoelectrochemical biosensing

Cancer biomarker detection

High-throughput assay

Clinical biosensor development

P1.188 Capillary-Driven Microfluidic Approaches for Metal-Enhanced Fluorescent Immunodetection of Proteins

Keywords

Microfluidic chip

Exosome capture

3D nanostructure

HER2-positive cancer

P1.192 Integrable PET-Based Microfluidic Platform for Small-Scale Preanalytical Separation of human IgG

Karolina Porycka, Sylwia Pniewska, Marcin Drozd, Katarzyna Tokarska, Kamil Żukowski, Elżbieta Malinowska

Warsaw University of Technology, Poland

Abstract

Disease detection and immune response monitoring through serological methods offers wide possibilities for accurate diagnostics. These methods often rely on qualitative antibody tests for viral infections (e.g., COVID-19, HIV), bacterial infections, and autoimmune diseases. Immunoenzymatic assays using whole blood or serum samples can be prone to errors due to sample complexity. While IgG separation can reduce these issues, the process is generally time-consuming. Current small-scale IgG purification methods use microplates coated with bacterial proteins, requiring complex manual procedures. To simplify this, researchers are developing cost-effective microfluidic devices enabling analytical system miniaturization. These innovations aim to improve serological diagnostics by simplifying IgG separation and enabling more efficient health monitoring systems.

The presented study focuses on developing flexible, PET foil-based disposable platforms for rapid antibody separation from whole serum. The method relies on reversible IgG antibody capture using protein A/G immobilized on polyester foil. pH-controlled antibody capture and elution occur entirely within the microfluidic system through a sequence of injections. This process is driven by an external pumping system automated with an Arduino microcontroller. To preserve antibody activity, a microfluidic mixer module neutralizes the eluate. It has been demonstrated that the system enables pre-analytical, preliminary IgG separation for qualitative analyses, including serological testing for COVID-19. The selectivity of IgG separation for endogenous proteins other than IgG (e.g., CRP and hemoglobin) has also been confirmed. This system offers a straightforward approach for pre-treating small-scale samples, enabling their use in bioanalytical assays such as ELISA, lateral flow tests, or serological immunosensors. The module could be integrated into advanced micro total analysis systems (μTAS) for thorough serological profiling or antibody-antigen complex capture.

This work has been financially supported by Young PW projects granted by Warsaw University of Technology under the Excellence Initiative: Research University (IDUB) program.

Keywords

antibodies separation

serological assay

protein A/G affinity

IgG antibodies

P1.193 Stenosis CFD simulations and evaluation of their interaction with drug delivery microparticles

Yago Radziunas-Salinas¹, Santiago Paramés-Estévez², Alberto Otero-Cacho², María Teresa Flores-Arias¹, Ana Isabel Gómez-Varela¹, Mª Carmen Bao-Varela¹, Alberto P. Muñuzuri²

¹Photonics4Life Research Group, Applied Physics Department, Facultade de Física and Instituto de Materiales (iMATUS), Universidade de Santiago de Compostela, Campus Vida, E15782 Santiago de Compostela, Spain. ²GFNL Non linear physics group, Dept. of Physics, and Galician Centre for Mathematical Research and Technology (CITMAga). Universidade de Santiago de Compostela, E15782 Santiago de Compostela, Spain

Abstract

Cardiovascular diseases are the leading cause of death globally, recognizing the necessity to create new treatment methods. In many of them, such as stenosis or atherosclerosis, inadequate blood flow plays a pivotal role in the physiological origin of the disease. Consequently, new therapeutic strategies based on blood flow characteristics arise as a potential solution to guide drug carriers to overcome these problems.

Computer fluid dynamics (CFD) simulations give important insight on the physical variables characterising the blood flows in the vicinity of the pathology. Among them, the velocity of the flow, the wall shear stress (WSS) on the surface of the affected area, and the vorticity within the system are key parameters to this purpose.

To study this phenomenon, an idealised stenosis was simulated. Blood flow analysis suggests an increase in the WSS at the surface surrounding the stenosis. On the other hand, notorious changes in the velocity profiles are obtained in its vicinity. This effect in addition with the sudden change in the vessel morphology yields to the appearance of vortexes next to the stenosis, highlighting the nonlinear nature of the flow.

This framework suggested to study the interaction between microparticles and the fluid dynamics to demonstrate whether a difference within the fluid flow arises according to the geometry and size of the particle. Simulations suggest that the residence times of the particles near the stenosis is dependent on the geometry. Finally, their spatial distribution after piercing through the affected area shows nuances associated with the particle shape and volume.

This approach emphasizes the importance of selecting the most adequate drug carrier geometry to interact within the blood flow, highlighting the need to develop personalized medical strategies to treat these conditions adequately.

Keywords

Stenosis

CFD

microparticles

drug delivery

P1.194 Automated Rapid Blood Typing using Lab-on-a-Disc Technology

Anna Roy, David Kinahan, Eadaoin Carthy

Dublin City University, Ireland

Abstract

With blood supplies at critically low levels in several European countries, including Ireland, the urgency for precise blood compatibility testing has never been more apparent. Traditional blood typing methods require large sample volumes and can be time-consuming, limiting their use in urgent or resource-constrained settings. This work presents a microfluidic lab-on-a-disc (LoaD) platform that provides rapid, comprehensive blood typing using just 400 μL of blood. Designed for point-of-care applications, this platform meets the critical need for efficient, patient-specific blood matching to address current shortages.

The LoaD device automates blood compatibility testing using centrifugation to channel blood samples precisely into multiple reaction chambers. Each chamber contains immobilised antibodies that bind specifically with target antigens, achieving high sensitivity and stability. This targeted approach allows the platform to determine a patient’s exact blood type and compatibility with donor blood, ensuring a comprehensive analysis with minimal sample volume.

Incorporating dissolvable film tabs, the device controls the timing of sample movement to maximise the assay’s precision and reliability. Image analysis software further enhances accuracy by assessing blood reactivity in real time, reducing potential human error and ensuring reproducibility. The workflow is streamlined to a single pipetting step followed by centrifugation, making the process highly efficient, user-friendly, and suitable for a variety of clinical settings.

This microfluidic approach represents a significant advancement in addressing blood shortages and transfusion safety. By consolidating multiple laboratory processes into one integrated platform, the LoaD device provides a robust, point-of-care solution for rapid blood typing. Its reliable and reproducible results offer a safer, more efficient alternative to traditional methods, especially valuable in emergency and critical care. Ultimately, this innovation promises to enhance patient care by supporting safe and efficient blood transfusions in today’s challenging healthcare landscape.

Keywords

Microfluidics

Point of Care

Blood Matching

Lab-On-a-Disc

P1.195 Controlled Synthesis of PLGA Nanoparticles in Microfluidic Chips: Investigating the Influence of Mixing on Particle Size

Muhammad Mubashar Saeed, Eadaoin Carthy, David Kinahan, Nicholas Dunne

Dublin City University, Ireland

Abstract

Nanoparticles (NPs) have gained pivotal importance in modern biomedical research, due to their unique nanoscale properties that enhance therapeutic outcomes. Poly(lactic-co-glycolic acid) (PLGA) NPs stand out for their biodegradability and biocompatibility, making them highly suitable for different applications. However, traditional synthesis methods such as emulsification, and solvent evaporation, often lack precise control over NPs physical properties like size, polydispersity index (PDI), and surface charge. Microfluidic synthesis provides a promising alternative to conventional approaches as it can enable an enhanced control over particle characteristics.

In this study, we investigated how varying mixing parameters in a microfluidic chip affects PLGA NPs properties. Utilizing a continuous hydrodynamic flow-focusing (HFF) setup, we introduce 10mg/ml PLGA-acetonitrile solution through the central channel and 0.3% polyvinyl alcohol (PVA) solution in deionized (DI) water through side channels. Initially flow visualization done by 5% solution of potassium cyanide and 5% solution of iron (III) chloride to evaluate mixing dynamics based on their visible color change upon mixing, Following flow visualization and python based image analysis we tested 25 different flow conditions (different flow rates and flow ratios) and measured time for liquids to be fully mixed. Later we corelated mixing time with NPs size. We also identified different flow conditions where mixing time, and thereby NPs size, were identical. For example at flow rates 50:5 and 20:1 mixing time was identical. we synthesized PLGA NPs at these optimized conditions and assessed their properties using dynamic light scattering (DLS) and electron microscopy (SEM/TEM).

Our results demonstrate that tuning flow rates and flow rate ratios in the microfluidic system allows precise control over the mixing of reagents, which directly influences the size, PDI, and surface properties of the resulting PLGA NPs. This study highlights the potential of microfluidic platforms for fine-tuning NP synthesis.

Keywords

Lab on a chip

PLGA Nanoparticles Synthesis

hydrodynamic flow focusing

Mixing

P1.196 Optimization of the biofunctionalization process for FET-based sensors: a 2² factorial experimental design and validation by SPR

Beatriz Sequeira-Antunes^1,2,3, Ana S. Viana⁴, Ana Francisca Martins³, Nuno Marujo², Susana Cardoso^3,5, Hugo Alexandre Ferreira^1,2

¹University of Lisbon Institute of Biophysics and Biomedical Engineering, Portugal. ²Exotictarget Lda, Portugal. ³INESC Microsystems and Nanotechnologies, Portugal. ⁴Centro de Química Estrutural, Institute of Molecular Sciences, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Portugal. ⁵University of Lisbon Higher Technical Institute, Portugal

Abstract

Biofunctionalization is critical for biosensor development, ensuring reliable biomolecule immobilization on surfaces, such as those in field-effect transistor (FET)-based sensors. However, optimizing this process is challenging due to various variables. In this study, we optimized the biofunctionalization process using a 2² factorial experimental design (Table 1), which evaluates two factors at two levels: (1) the concentration ratio of mercaptoundecanoic acid (MUA) to 11-mercapto-1-undecanol (MUD) to form a mixed self-assembled monolayer (SAM), and (2) the use of Protein G (PrG) for antibody orientation.

Uncaptioned visual

The process included cleaning gold electrodes, forming a mixed-SAM, activating it with N-ethyl-N′-(3-(dimethylamino)propyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS), immobilizing PrG (where applicable), and sequential incubations with anti-immunoglobulin G (IgG), bovine serum albumin (BSA), and IgG. Each step was characterized by ellipsometry to measure the optical thickness of the resulting nearly transparent layers.

Figure 1 shows that the E4 protocol was the optimal condition, given the highest IgG thicknesses. This was anticipated due to the 1:1 ratio in the mixed-SAM, which increased covalent bonding between the COOH and NH₂ groups of the antibody. Furthermore, PrG inclusion enhanced antibody orientation, positioning them for efficient IgG detection.

Uncaptioned visual

Surface plasmon resonance provided further validation of the E4 biofunctionalization surface’s process. After immobilizing PrG onto the gold electrodes, subsequent steps, including Anti-IgG immobilization, BSA blocking, IgG binding, PBS washes, and glycine-HCl washes, to regenerate the sensor’s surface, were monitored in real-time. The results support the viability of the biofunctionalization approach.

This work was conducted with the aim of applying this optimized functionalization method to FET-based biosensors, which would represent a much more significant contribution by enhancing robustness, selectivity and sensitivity in biosensing applications.

Reference

1. Almeida, L.C., et al. Electrosynthesis of polydopamine-ethanolamine films for the development of immunosensing interfaces. Sci Rep 11, 2237 (2021)

Project partially funded by FCT: UID/00645/2025.

Keywords

Biofunctionalization

Self-assembly monolayer

Protein G

Surface plasmon resonance

P1.197 Revealing the matrix mechanical regulation on cancer stem cells by integrated microfluidic platform

JIALEI SONG, Mo YANG, Yi LIU

Hongkong Polytechnic University, Hong Kong

Abstract

Cancer recurrence is a major factor in the failure of treatments. Since cancer stem cells (CSCs) are closely linked to tumor formation, metastasis, and drug resistance, understanding their heterogeneity is crucial for effective therapy. Current analysis techniques often average data across many molecules at the whole-cell level, overlooking differences between individual cells. To address this, we designed an integrated microfluidic platform for high-throughput single-cell analysis, aimed at uncovering the mechanical regulation of CSCs.

Keywords

microfluidic

cancer stem cells

mechanical regulation

P1.198 Classical to clinical: a new computational approach to measure the variation of binding kinetics in affinity biosensors

Brandon Tipper, Lisa Miller, Mohammad Nasr Esfahani, Steven Johnson

University of York, UK

Abstract

Classical binding kinetic approximations assume idealised conditions, with multiple aspects of the Langmuir approach being overlooked in most computational models. These assumptions/omissions lead to invalid estimates of the association/dissociation rates with errors of as much as 40-60% being commonplace, with inaccuracies in response curves from even the most sensitive and specific molecular interactions.

To illustrate this, we have analysed a series of experimental immunoassay measurements (obtained using a commercial quartz crystal microbalance with dissipation monitoring (QCMD) machine) using a range of analytical approaches, where limitations associated with the theoretical assumptions were assessed and evaluated.

Following from this critique, we have developed a new approach to modelling biosensor binding curves that more accurately reflects interactions between a surface-bound probe molecule (anti-IgG) and a target antigen (IgG) diffusing in solution. Specifically, our new model introduces both an increased localised surface concentration and a normalised distribution of binding kinetics for the immobilised layer of probe molecules. We show this model has the capability to accurately describe experimental data (providing effective association/dissociation rates that are variable throughout an experiment to within 20% accuracy) and allow for a precise analysis of the anti-IgG-IgG interaction under a range of flowrates and IgG concentrations.

This data not only reveals the significant errors associated with traditional analysis of binding affinity in immunoassays, but also allows evaluation of the ‘quality’ and uniformity of a layer of surface immobilised probe molecules. For example, our new analytical approach may provide insight into how experimental non-idealities, such as surface roughness, aggregation and non-specific binding impact the observed rate of association. It is our expectation that this new computational approach will have significant impact on the development of more reliable/robust affinity biosensors, changing the way in which molecular detection is understood and evaluated while opening further opportunities for examination.

Keywords

Computational Modelling

Localized Concentration

Variable Binding Kinetics

Microfluidics

P1.199 Self-powered microfluidic analysis for simultaneous isolation, capture, and detection

Hyunjun Kim¹, June Soo Kim¹, Noah Jang¹, Da Ye Kim¹, Yujin Nam¹, Jinkyung Kim¹, Jin Park¹, Kihyun Kim¹, Maeum Han², Seong Ho Kong¹

¹Kyungpook National University, Republic of Korea. ²Institute of Semiconductor Fusion Technology, Kyungpook National University, Republic of Korea

Abstract

¹Warsaw University of Technology, Centre for Advanced Materials and Technologies CEZAMAT, Poland. ²Faculty of Mechanical and Industrial Engineering, Poland

Abstract

Microfluidic devices are increasingly essential in applications such as point-of-care diagnostics, wearable health monitors, and lab-on-a-chip platforms. However, conventional fabrication methods often involve rigid substrates and potentially toxic materials that limit their use in flexible, biocompatible devices. This work demonstrates the fabrication of flexible microfluidic channels, ranging from 100 to 300 µm in width, utilizing the aerosol jet printing (AJP) technique.

AJP is an additive manufacturing process that employs a finely focused aerosolized ink stream, enabling precise deposition of functional materials with high resolution on diverse substrates. Here, biocompatible polymer inks are printed onto flexible films under controlled conditions, creating sealed microchannels in a single manufacturing step. This approach eliminates the need for extensive photolithography, chemical etching, or time-consuming layering steps, thereby reducing production costs and complexity.

The printed microfluidic channels exhibit robust mechanical flexibility and chemical stability. Their small footprint and high aspect ratio allow for efficient fluid transport in real-time monitoring and diagnostic assays. Moreover, the nontoxic nature of the materials ensures compatibility with physiological samples, making these channels suitable for wearable devices that come into direct contact with skin or biological fluids. Comprehensive characterization, including dimensional accuracy, channel integrity, and flow performance, confirms the reliability and reproducibility of the printed structures.

By integrating the microfluidic channels with other additively manufactured components (e.g., electrodes, sensors), this platform paves the way for fully printed wearable devices such as smart patches for continuous health monitoring or single-use diagnostic cartridges for rapid disease screening. The simplicity, versatility, and scalability of the AJP approach position it as a compelling manufacturing method for next-generation microfluidics. This research explores the characterization and integration of those printed channels with real-time sensing elements, advancing the development of cost-effective, user-friendly, and personalized healthcare solutions.

Keywords

microfluidics

aerosol jet printing

microprinting

wearables

P1.204 Fabrication and Characterization of a 3D Printed LOC Device for in-line Monitoring of Biofilm Growth using Integrated Raman Spectroscopy

Casper Kunstmann¹, Arkadiusz Goszczak¹, Zuzanna Parnicka¹, Jacek Fiutowski¹, Jonathan Brewer^2,3

¹University of Southern Denmark NanoSYD, Denmark. ²University of Southern Denmark, epartment of Biochemistry and Molecular Biology, Denmark. ³Danish Molecular Biomedical Imaging Center (DaMBIC), Denmark

Abstract

The development of advanced in vitro skin models is essential for biomedical applications such as drug testing and tissue engineering. We present a novel 3D printed skin-on-chip device designed to support pressure-adaptive human cell growth. Fabricated using high-resolution micro-precision 3D printing, the device incorporates a PETE membrane for skin cell growth and features flexible, biocompatible resin with pressure chambers. These chambers exert lateral pressure on the membrane, simulating dynamic skin conditions.

Structural integrity, surface morphology, and fluid dynamics were assessed, alongside finite-element simulations to analyze heat distribution and material properties. Device testing focused on optimizing laminar flow and membrane integrity using syringe-driven flows and optical microscopy. The goal was to maximize nutrient delivery without exceeding membrane capillary pressure.

To evaluate performance, bacterial biofilms (Staphylococcus carnosus) were grown as a model system. The device integrates a mini-Raman spectrometer capable of surface-enhanced Raman scattering (SERS) for real-time biofilm monitoring. This enables detailed analysis of bacterial growth, biofilm behavior, and response to treatments.

In conclusion, this 3D printed skin-on-chip platform effectively replicates the natural microenvironment of human skin under dynamic conditions. Its adaptability to pressure changes makes it a valuable tool for dermatology, pharmacology, and tissue engineering research. This work contributes to the advancement of biosensor technologies for health and well-being.

Keywords

3D printed Microfluidics

Biofilm growth

LOC Incubator

Raman spectroscopy

P1.205 Mass-manufacturable microfluidic devices for power-free, high-performance enzymatic immunoassays

Naveen Kumar Mehto^1,2, Chris Bowen³, Nuno M. Reis^1,2

¹University of Bath Department of Chemical Engineering, UK. ²University of Bath Center for Bioengineering & Biomedical Technologies, UK. ³University of Bath Department of Mechanical Engineering, UK

Abstract

Precise sequential fluid flow and efficient washings are crucial in microfluidics-based biomedical diagnostics and lab-on-a-chip devices, especially bioassays involving enzymatic amplification and complex sample matrices. Control of multiple fluid streams within microfluidic channels has been achieved with both active and passive manipulation techniques. Active control methods such as electric pumps, acoustic valves, and syringe pumps, are often complex and involve expensive microfabrication and present limited bio-compatibility and scalability. In contrast, passive microfluidic devices require no external forces, with fluid flow driven exclusively by gravity and/or capillary forces. We report for the first time sequential fluid flow and efficient washings in multiplexed microfluidic channels fabricated from flat sheets of transparent fluorinated ethylene propylene (FEP) material. Coating of FEP surface with polyvinyl alcohol (PVOH) rendered the surface of the microfluidic devices hydrophilic, combining controlled capillary-driven flow with effective adsorption of diagnostics antibodies in FEP and fluorescence detection in the devices. Extensive fluidic characterization using colorimetric (1.0 mg/ml indigo carmine) and fluorescence (0.5 mg/ml fluorescein) confirmed effective and controlled sequential fluid flow of immunoassay reagents, including remarkable washouts, without crosstalk between sequential reagents, for a range of shapes and geometries. The reagent's superficial flow velocity inside the microfluidic channels was found to range from 20 to 40 mm/s and captured by a pressure balance between resistance forces and Laplace pressure, following closely Washburn’s equation. This offers to fine-tune fluid flow by controlling dimensions of the channel but also the type of absorbent pad at the channel's outlet. Washings operated at ~5 mm/s ensured controlled and efficient separation of bound from unbound molecules, required for immunoassays with minimal background noise and high signal-to-noise. We are currently applying and characterising analytical performance of these mass-manufacturable devices for smartphone quantitation of cardiac biomarkers through heterogeneous enzymatic immunoassays, which will be shared.

Keywords

Passive microfluidic

Sequential fluid flow

Fluorinated ethylene propylene

Enzymatic immunoassays

P1.206 Miniaturised DNA extraction: entwining microfluidics and nanoarchitectonics

Pujari Ramya Priya, Satish Kumar Dubey, Sanket Goel

BITS Pilani - Hyderabad Campus, India

Abstract

DNA extraction is one of the critical steps in nucleic acid testing. It is a complex process involving reagent mixing, centrifugal separation, and substantial manual intervention. Conventional DNA extraction methods are unsuitable for point-of-care diagnostics because they are labour-intensive. Microfluidic devices are highly promising for point-of-care applications, but sample preparation, especially DNA isolation from blood samples, is often challenging. Nevertheless, microfluidics for DNA extraction has been limited by its potential to decrease the dependency on bulky fluidic actuation components and simplify the process. This work describes a microfluidics-based platform for efficient and rapid DNA extraction from blood samples, overcoming challenges with current techniques. The continuous microchannel of the proposed device integrates passive mixing, thereby streamlining the process. Our protocol used blood samples, magnetic beads, and various liquids, allowing for automated and precise extraction. DNA with a purity of 1.8 (A260/280) and a yield of 83 ng/µL from 10 µL was achieved with the system, showing its efficiency. This method provides a simplified and reliable workflow for field diagnostics by minimising manual handling, reducing reagent consumption, and significantly shortening processing time. It is especially advantageous in emergency and resource-limited settings where rapid and accurate results are crucial. Integrating passive mixing and continuous flow enhances the device's efficiency, making it a promising solution for point-of-care diagnostics and advancing the applicability of microfluidics in biomedical and clinical diagnostics. Uncaptioned visual Figure: Representation of DNA extraction on microfluidic chips A, B, C, and D is the inlets where buffers are sent into the microchannel. I and II represent the magnetic bead position during binding and elution.

Keywords

Microfluidics

Point of care Diagnostics

Nanoarchitectonics

DNA extraction

P1.207 Analytical performance of an in-house automated microfluidic DNA extraction device for gastrointestinal pathogens

Rukmini Sarma¹, Ada Zwetlana¹, Kabir Khan¹, Rishi Kant Thakur¹, Tushar Jeet¹, Prabhu Balasubramanian¹, Suneeta Meena², Vivekanandan Perumal¹, Ravikrishnan Elangovan¹

¹Indian Institute of Technology Delhi, India. ²All India Institute of Medical Sciences New Delhi, India

Abstract

Background: Diarrheal infections are one of the leading causes of unnecessary antibiotic usage in developing countries, leading to antimicrobial resistance (AMR). Therefore, rapid detection of pathogens is crucial to providing suitable and timely treatment to prevent AMR. To bridge this gap, this study aimed to evaluate the analytical performance of an in-house automated microfluidic device for rapid bacterial DNA extraction from diarrheal stool samples. The device consists of a microfluidic chip containing pre-loaded reagents for silica-based nucleic acid extraction, an inbuilt optical reader, and an IOT system for data analysis and reporting. The automated device is an electromechanical system programmed to sequentially transport the necessary reagents within the microfluidic chip using pneumatic control.

Methodology: Using a commercial DNA extraction kit, we extracted DNA from Shigella flexneri and Salmonella Typhi. The extracted DNA was used to optimize the qPCR reaction conditions using literature primers. Next, we optimized the in-house stool DNA extraction protocol using diarrheal stool samples (which tested negative for S. flexneri and S. Typhi) spiked with cultured bacteria in the spin column format. We used a commercial stool DNA extraction kit as a gold standard reference. Next, we tested the in-house developed protocol with the automated device and evaluated its performance using qPCR.

Results: Our in-house protocol for stool DNA extraction showed similar performance as the commercial kit in terms of DNA yield, purity, and qPCR amplification. The in-house protocol was replicated in the microfluidic device, which showed high repeatability and reproducibility of PCR amplification of the extracted DNA. The limit of detection was found to be ~100 CFUs.

Conclusions: The microfluidic device is cost-effective, has reduced hands-on time because of minimal sample pre-processing steps, is rapid (15-20 min per sample) and sensitive, and is, therefore, a suitable alternative for low-resource settings.

Keywords

Nucleic acid extraction

Microfluidics

Diarrheal pathogens

Antimicrobial resistance

P1.208 Advances in microfluidic approach for rapid on-site magnesium analysis for heart failure patients

Miguel Vidal^1,2,3, Jorien Berendsen², Sónia O. Pereira¹, Loes Segerink², Aoife Morrin³, Cátia Leitão¹

¹i3N, Department of Physics, University of Aveiro, Portugal. ²BIOS Lab on a Chip group, University of Twente, The Netherlands. ³School of Chemical Sciences, Dublin City University, Ireland

Abstract

Point-of-care biosensors have attracted significant interest and investment throughout the years, broadening in scope and application, as a way to improve patient care and relieve strained health systems. These devices can target a range of cardiovascular diseases and conditions including heart failure. Patients suffering from heart failure often exhibit electrolyte imbalances such as magnesium (Mg²⁺) and potassium (K⁺) depletions that can lead to life-threatening arrhythmias. Magnesium is the second most abundant cation in intracellular tissues and it is crucial in several physiological processes, therefore it is important to manage Mg²⁺levels in the organism.

This research involves the development of a microfluidic chip containing a colorimetric assay of calmagite (1-(1-hydroxy-4-methyl-2-phenylazo)-2-naphthol-4-sulfonic acid) to measure Mg²⁺concentration in a liquid sample. The calmagite assay was prepared in 2-Amino-2-methyl-1-propanol buffer and the response to Mg²⁺ was first studied resorting to both a smartphone camera and a UV-VIS spectrophotometer in a cuvette system. Subsequently, microfluidic PDMS (Polydimethylsiloxane) chips were fabricated incorporating the assay, and the chips’ features were optimized, including the channels’ pattern and height, to enhance the optical density and the fluid flow. Image acquisition was performed inside a white light box and a linear increase in Euclidean distance was obtained with Mg²⁺ concentration for both the cuvette system (volume ~ 3 mL) and the microfluidic chips (volume < 30 µL).

Preliminary results demonstrate the feasibility of the calmagite chip for Mg²⁺ measurement in the range of clinical interest in serum and highlight its potential to be used for analysis of very small plasmatic sample volumes, targeting heart failure management at the point of interest.

Keywords

Calmagite

Biosensor

Colorimetric

Electrolytes

P1.209 Going small to study stress: using microscale geometries for delivering rapid cortisol quantitation

Visesh Vignesh¹, Bernardo Castro-Dominguez¹, Tony James¹, Julie Gamble-Turner², Stafford Lightman³, Nuno Reis¹

¹University of Bath, UK. ²Bournemouth University, UK. ³University of Bristol, UK

Abstract

Keywords

Microfluidic

Long-lasting

Biopolymer

sensing materials

P1.213 Wax-Sealing Method for Integrating Microfluidic Devices with Screen-Printed Electrodes

Yen-Wei Chang, Yen-Wen Lu

National Taiwan University Department of Biomechatronics Engineering, Taiwan

Abstract

Microfluidic electrochemical sensors have gained popularity due to their precision and low reagent consumption. However, one of the key challenges in their development is effectively integrating microfluidic devices with screen-printed electrodes (SPEs) while preventing leakage.

Conventional integration methods, such as plasma bonding, Van der Waals force, or screw assembly, present several limitations. Plasma bonding works only with glass-substrate SPEs, Van der Waals force often results in leakage, and screw assembly can cause deformation of the microfluidic device or electrode due to the pressure applied. Additionally, the common materials used for these sensors such as polydimethylsiloxane (PDMS) for microfluidic devices and plastic for commercial SPEs are challenging to integrate using these conventional methods.

To integrate microfluidic devices with commercial SPEs, a wax sealing method is demonstrated. Figure 1(a) shows the microfluidic device designed for wax sealing, featuring three components: a wax channel, a liquid channel, and a slot for the SPE. The integration process involves inserting the SPE into the slot, placing the device on a hot plate, and adding 20 mg of solid paraffin wax to the wax channel inlet. As the wax melts, the surface properties of PDMS and the surface tension of the molten wax guide it to fill the junction between the microfluidic device and the SPE, as shown in Figure 1(b). Once solidified, the wax forms a seal that prevents leakage during liquid flow through the channel.

This wax-sealing method offers a simple and effective solution for integrating microfluidic devices with plastic SPEs. The entire process can be completed in 5 minutes, eliminates the risk of deformation, and prevents leakage from both aqueous solutions and organic solvents like ethanol, as shown in Figure 1(c). This makes the method versatile for a wide range of applications.

Uncaptioned visual

Keywords

Microfluidic electrochemical sensor

Wax-sealing

sensor integration

leakage-free

P1.214 Accelerating drug discovery with focal molography: Precision label-free kinetics in complex biological environments

Simona Notova, Sophia Yajie Zhai, Volker Gatterdam, Roman Popov, Andreas Frutiger

lino Biotech AG, Switzerland

Abstract

In drug discovery, the need for efficient technologies to rapidly screen and characterize potential therapeutic candidates is critical. Traditional methods, such as surface plasmon resonance (SPR), offer valuable insights into biomolecular interactions but often struggle with nonspecific binding and environmental noise, especially in complex biological samples. Such limitations underscore the demand for innovative, robust solutions capable of interaction analysis in biological relevant sample conditions. This is especially important during a certain development phase of the drug candidate when it will be applied to patients and the desired protein interaction must reliable work in the presence of the patients' proteome.

Focal molography is a novel optical diffraction-based technology that addresses these challenges with a unique label-free detection method. By utilizing engineered “molograms” – nanostructures on a sensor chip designed for specific molecular binding – focal molography produces high-sensitivity while minimizing interference from nonspecific binding. This approach is particularly effective for crude samples, such as tissue extracts and human serum, where it achieves precise kinetic measurements despite the complexities of the sample matrix.

In various study, we applied focal molography to challenging condition for biosensors, like detection of pM protein concentration in 50% human serum, detection of cell secreted proteins in cell culture medium or for hit validation of DNA-encoded library (DEL) hits. Our method enabled multiplexed kinetic analysis across 54 individual sensing spots, allowing for the simultaneous characterization of multiple interactions. Key binding parameters, including association (k_on) and dissociation rates (k_off), and the dissociation constant (K_D), were reliably measured.

Focal molography’s robustness in the face of environmental fluctuations and biological interference provides a quicker, more dependable approach to drug discovery workflows, enabling the precise identification of promising drug candidates. This technology serves as a valuable asset for enhancing biosensing applications in both research and therapeutic development.

Keywords

drug discovery

label-free

multiplexed

complex media

P1.215 Gii based fully integrated handheld electrochemical dx platform for diabetic ketoacidosis monitoring

Yousillya Bunga, Pablo Lozano-Sanchez, Marco Caffio, Prosper Kanyong, Joanne Holmes

iGii, UK

Abstract

Ketoacidosis, especially diabetic ketoacidosis (DKA), is a life-threatening condition resulting

from the accumulation of ketone bodies, such as β-hydroxybutyrate (β-HB) and acetoacetate,

due to disrupted glucose metabolism. Early detection is critical, but often delayed with

conventional methods like urine dipsticks.

This study introduces an electrochemical sensor designed for the rapid detection of ketone

bodies in urine. Using our 3D carbon nanomaterial, Gii, the sensor was optimised for detecting

acetoacetate via square wave voltammetry (SWV).

Testing with standard solutions and artificial urine samples demonstrated high sensitivity and

selectivity, with detection limits as low as 1.53 mM; thus, demonstrating its potential utility

analysis of ketones clinical samples. The sensor offers an accurate, reliable, and faster

alternative to traditional methods, making it highly suitable for point-of-care applications in

managing ketoacidosis and diabetes. Crucially, the use of Gii allows us to deliver superior

sensitivity, sustainably.

Keywords

Biosensors

Diagnostics

Electrochemistry

Ketone

P1.216 Commercial large-scale production of electrochemical sensors for environmental monitoring

Miaosi Li, Chathurika Abeyrathne, Luke Cossins

Universal Biosensors Pty Ltd, Australia

Abstract

Universal Biosensors (UBI) is specializing in electrochemical point-of-use diagnostics. Our patented technology focuses on utilizing a portable handheld device that analyzes a small blood sample from a finger prick. UBI manufactures roll-to-roll consumable test strips in-house at our ISO 13485 certified facility, with a capacity of 30 million test strips per year.

The presence of heavy metals, such as lead (Pb) in tap water—often stemming from aging plumbing materials—poses a significant health risk, as Pb can leach into the water over time. Even at low concentration levels, Pb can adversely affect health. The World Health Organization (WHO) drinking water guideline for Pb is 10 µg/L. The gold standard method for Pb detection in tap water, ICP-MS, offers exceptional sensitivity but is impractical for on-site testing due to its complexity. To address this issue, UBI has recently commercialized a low-cost, disposable electrochemical sensor for precise Pb detection in drinking water, ensuring timely identification and mitigation of potential health hazards.

Utilizing patented strip technology and innovative buffer formulations, UBI has effectively scaled the manufacturing of a sensor system for the detection of Pb in drinking water. It achieves LoD of 5 µg/L and a quantification range of 5-100 µg/L. The buffer can be efficiently dried using freeze dryer technology, enhancing manufacturing capabilities. Notably, the detection process can be completed within 5 minutes. Unlike many comparable systems, this sensor does not necessitate pH or temperature corrections. Additionally, UBI has developed a sensor for copper (Cu) detection, achieving a LoD of 50 µg/L and a quantification range of 50-3000 µg/L, with detection time < 3 minutes.

These advancements underscore the feasibility of large-scale production of heavy metal sensors for drinking water monitoring. Furthermore, UBI is actively developing other assays, including chromium, arsenic, and per- and polyfluoroalkyl substances (PFAS).

P1.217 An ultrasensitive bio-chip sensor for detection of aflatoxin M1 in milk

NEELAMMA KUTTAIAH MANDEPANDA¹, LIZY KANUNGO¹, SUNIL BHAND²

¹Birla Institute of Technology and Science Pilani - K K Birla Goa Campus, India. ²Birla Institute of Technology & Science Pilani - K K Birla Goa Campus, India

Abstract

Aflatoxin M1 is a potential human carcinogen, teratogen, hepatoxic, and nephrotoxic that contaminates milk. Due to its hazardous nature, its detection before consumption is important as per the stringent regularity standards such as 50 ng/L for European Union. Herein, we present an ultrasensitive, label-free biochip that can detect AFM1 spiked in European reference materials (ERM-BD 282) and commercial milk samples. The biochip could detect AFM1 in milk with a linear range of detection 25- 1000 ng/L, with a limit of detection (LOD) of 25 ng/L with an analysis time of 15 minutes. The developed bio-chip comprised a gold-coated interdigitated electrode functionalized with monoclonal antibodies of AFM1, crosslinked via a self-assembled monolayer of 11-MUA. Under the optimized condition, the non-faradic mass change due to antigen-antibody binding was investigated by Impedimetric and Capacitive analysis. The bio-chip was characterized using FE-SEM and ATR- FTIR analysis. EIS characterization of the biochip was carried out in the presence and absence of AFM1. The standard operating protocol for the development of bio-chips was optimized. Under the optimized conditions, intra- and inter-day reproducibility performance was studied. The intra-day and inter-day relative standard deviation (RSD %) values for the developed biochip obtained were found to be satisfactory [ 1.2 % (n=5) and 4.9 % (n=5) respectively]. The developed biochip is field deployable and has an advantage over other available commercial kits. Real samples available off the shelf were analyzed and cross-validated using ELISA kits. The sensitivity of the biochip is studied, and recovery analysis are carried out.

Uncaptioned visual

Keywords

AFM1 bio-chip

label-free detection

ultra-sensitive

capacitance

P1.218 Nanochannels for biosensing: a travel from the stochastic sensing to the use of nanoporous membranes for diagnostics

Alfredo de la Escosura-Muñiz

University of Oviedo, Spain

Abstract

The stochastic sensing based on biomimetic single nanopores is considered as one of the more relevant breakthroughs in bioanalysis, being the DNA sequencing tool an outstanding milestone in modern research [1,2]. Inspired by it, the use of solid-state nanoporous membranes has paved the way to novel and versatile biosensing systems ranging from electrical to optical detection devices, bringing new advantages for biosensor development and application. The great potential of these systems for electrochemical clinical diagnostics deserves a special focus [3].

In this context, the purpose of this communication is to give a travel from the research in this field along the last two decads, from the basis of the stochastic sensing to the recent trends in the use of nanoporous membranes for biosensing applications. An overview on the appplications for DNA sequencing and the detection of proteins, virus and other analytes will be given. Special focus will be put in recent approaches for the in situ monitoring of biomarkers for chronic wound infection diagnosis [4-7] and for the screening of antibiotics [4,8] a hot topic for facing antimicrobial resistance (AMR).

References

[1] A. de la Escosura-Muñiz, A. Merkoçi, ACS Nano 6(9) (2012) 7556.

[2] A. de la Escosura-Muñiz, A. Merkoçi, TrAC - Trends Anal. Chem. 79 (2016) 134.

[3] D. Valero-Calvo, A. de la Escosura-Muñiz, TrAC - Trends Anal. Chem. 172 (2024) 117568.

[4] A. de la Escosura-Muñiz, et al., ACS Appl. Mater. Interfaces, 11 (2019) 13140

[5] A. Iglesias-Mayor, A. de la Escosura-Muñiz, et al., Biosens. Bioelectron. 209 (2022) 114243.

[6] D. Valero-Calvo, A. de la Escosura-Muñiz, et al., Microchim. Acta 190 (2023) 257

[7] C. Toyos-Rodríguez A. Escosura-Muñiz et al., Front. Bioeng. Biotechnol. 12 (2024) 1310084.

[8] C. Toyos-Rodríguez, A. de la Escosura-Muñiz, et al., Anal. Bioanal. Chem. 415 (2023) 1107.

Acknowledgements

PID2020-115204RB-I00 and PID2023-149004OB-I000 projects from MICINN.

Keywords

Nanopores

Nanochannels

Nanoporous membranes

Diagnostics

P1.219 Effect of substrate type on the surface properties and functionalization of Au-electrodes in pathogen detection for aquaculture

Marcin Paweł Prządka^1,2, Agata Obstarczyk¹, Paweł Chodasewicz¹, Paulina Kapuścik¹, Sylwia Baluta³, Piotr Poręba^2,4, Patrycja Pokora¹, Ewa Mańkowska¹, Jarosław Domaradzki¹, Michał Mazur¹, Katarzyna Pala², Małgorzata Kot⁵, Jan Ingo Flege⁵, Damian Wojcieszak¹

¹Faculty of Electronics, Photonics and Microsystems, Wroclaw University of Science and Technology, Janiszewskiego 11/17, Wroclaw 50-372, Poland. ²Food4Future Technologies Sp. z o.o., Tarasa Szewczenki 24, Wrocław 51-351, Poland. ³Faculty of Chemistry, Wrocław University of Science and Technology, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland. ⁴Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Bellaterra, 08193 Barcelona, Spain. ⁵Applied Physics and Semiconductor Spectroscopy, Brandenburg University of Technology Cottbus-Senftenberg, Konrad-Zuse-Strasse 1, Cottbus D-03046, Germany

Abstract

Biosensors offer a promising alternative for a wide range of diagnostic tests due to their real-time and continuous analysis capabilities, automation, and miniaturization potential. A key application for biosensors is in environmental protection, particularly for monitoring water quality, as they enable rapid in situ detection of contaminants such as pathogens.

This study focused on evaluating how different substrates for depositing a Au electrode influence the stable formation of a bioreceptor layer. Substrates tested included ceramic, glass, and epoxy laminate, onto which thin gold coatings were applied using the electron beam evaporation (EBE) method. EBE allows producing extremely smooth layers, and the surface properties of the produced sensor structures depend on the type of substrate. The layers characteristics were examined using optical profilometry, scanning electron microscopy (SEM), and atomic force microscopy (AFM), to analyze surface topography before and after bioreceptor layer formation. Reflectance and wettability measurements provided additional details, facilitating comparisons of how Au surface morphology affects bio-functional layer formation.

The essential stage of the study involved analyzing surface oxidation levels via photoelectron spectroscopy, specifically through the Au4f, O1s, and C1s states. This helped determine oxidation levels post-deposition and estimated the coverage area of the bioreceptor layer.

The resulting biosensing systems show potential in aquaculture for detecting pathogens such as Salmonid alphavirus (SAV) using Electrochemical Impedance Spectroscopy (EIS). Complex analysis identified substrate factors affecting the bioreceptor layer’s formation, which is essential for precise pathogen detection in aquaculture. The results have great commercial impact, as they relate to performance of sensors that have been commercially available for a short time, and understanding the mechanism that determines their reliability and reproducibility of the production process.

P1.220 Portable high-performance magnetoelectric sensor for fetal magnetocardiography measurement

Enzhong Song¹, Yinan Wang¹, Dongshan Su¹, Tao Wang¹, Yuanhang Wang¹, Xiaoding Guo², Zhibo Ma¹

¹Northwestern Polytechnical University, China. ²College of Equipment Management and Engineering University of PAP, China

Abstract

Uncaptioned visual

Introduction:

Vernix caseosa, a highly insulating substance that surrounds the fetus, presents significant challenges for fetal electrocardiogram (ECG) measurement. However, magnetic field lines are not impeded by vernix, making fetal magnetocardiogram (MCG) a feasible and non-invasive alternative for cardiac monitoring. To address the demand for precise detection of weak magnetic signals, this study proposes a high-sensitivity, broadband cylindrical magnetoelectric (ME) sensor that leverages magnetic frequency conversion (MFC) to enhance its performance for detecting cardiac magnetic signals.

Methods:

The cylindrical ME sensor consists of two primary components: axially polarized piezoelectric ceramic PZT-5H and axially magnetized magnetostrictive material Fe₈₁Ga₁₉ tube. The inherent self-bound effect of the cylindrical structure enables the sensor to exhibit two distinct vibration modes—axial vibration (d₃₃) and radial vibration (d₃₁). The coupled multi-mode vibrational response significantly enhances the magnetoelectric coupling effect. To further suppress 1/f noise, an MFC technique was applied, utilizing the sensor’s mechanical resonance properties to transfer low-frequency signals to its resonance frequency, thereby improving its capability to detect low-frequency magnetic fields. Details of the sensor's performance testing system and packaging design are depicted in Figures 2 (a) and (b).

Results:

The testing results demonstrate that the cylindrical ME sensor exhibits excellent performance in detecting magnetic fields below 500 Hz under MFC. Specifically, the sensor achieves a LOD of 50 pT@10 Hz. Notably, the inherent high floor noise level allows operation without shielding, overcoming limitations in harsh shielding environments. In addition, the zero-biasing magnetic field of the sensor brings the advantage of size and weight. This device provides a means for low-cost, portable detection of magnetic fields at the pT level, aiding in the prevention of brain and heart diseases in fetuses. Finally, Figure 3 shows the typical placement of the sensor on a patient’s body is provided, offering a visual representation of the testing process.

Keywords

Cylindrical magnetoelectric (ME) sensor

Fetal magnetocardiograms

Magnetic frequency conversion (MFC)

P1.221 Development of a Portable Non-Contact Magnetoelectric Sensor Array for Non-Invasive Cardiac Monitoring

Yinan Wang¹, Enzhong Song¹, Yuanhang Wang¹, Dongshan Su¹, Zhibo Ma¹, Xiaoding Guo²

¹Northwestern Polytechnical University, China. ²College of Equipment Management and Engineering University of PAP, China

Abstract

In this study, we introduce a novel array-based magnetoelectric (ME) material sensor designed to advance magnetocardiography (MCG) for cardiac monitoring. Traditional MCG systems have been limited by large size and the requirement for superconducting environments, making them impractical for widespread use. Our innovative sensor system overcomes these limitations with an advanced ME material that achieves a magnetic field detection limit of 8.12 picotesla at 20 Hz. This high sensitivity is essential for accurately capturing the subtle magnetic fields generated by cardiac electrical activity, positioning our sensor as a compelling alternative to conventional electrocardiography (ECG).

The array configuration of our sensor provides detailed spatial mapping of cardiac magnetic fields, offering a non-invasive, contactless method for cardiac monitoring. This approach enhances patient comfort and is ideal for long-term and ambulatory assessments, overcoming the limitations of ECG related to skin contact and impedance variations.

Compact and cost-effective, our sensor is well-suited for integration into wearable devices, enabling continuous health monitoring in everyday settings. With Bluetooth connectivity, users can track their cardiac health in real-time and efficiently share data with healthcare providers. This advancement democratizes access to advanced cardiac diagnostics and supports personalized healthcare and remote management.

In summary, our ME-based MCG sensor represents a significant advancement in cardiac diagnostics by offering a sensitive, non-invasive, and user-friendly solution. It addresses the demand for portable, affordable health monitoring tools, underscoring its potential to revolutionize cardiac care and inspire future innovations in biomedical sensing technologies.

Uncaptioned visual

Keywords

Magnetoelectric Sensor

Non-Invasive Monitoring

Cardiac Magnetic Fields

Non-Contact Technology

P1.222 A Paper-based Dual Functional Biosensor for Safe and User-Friendly Point-of-Care Urine Analysis

Yujia Li^1,2, Yingqi Kong^1,2, Yixuan Li^1,2, Bing Li¹

¹Institute for Materials Discovery, University College London, London, WC1E 7JE, UK. ²Department of Chemistry, University College London, London, WC1E 7JE, UK

Abstract

Safe, accurate, and reliable analysis of urinary biomarkers is clinically important for the early detection and monitoring the progression of chronic kidney disease (CKD), as it becomes one of the world’s most prevalent non-communicable diseases. However, current technologies for measuring urinary biomarker are either time-consuming and limited to well-equipped hospitals; or lack the necessary sensitivity for the quantitative analysis and post a health risk to the frontline practitioners. Here we report a robust paper-based dual functional biosensor, which is integrated with the clinical urine sampling vial, for the simultaneous and quantitative analysis of pH and glucose in urine. The pH sensor was fabricated by electrochemically depositing IrOx onto the paper substrate using optimised parameters, which enabled an ultrahigh sensitivity of 71.58 mV·pH^-1. Glucose oxidase (GOx) was used in combination with an electrochemically deposited Prussian blue layer for the detection of glucose, and its performance was enhanced by the gold nanoparticles (AuNPs), chitosan, and graphite composites, achieving a sensitivity of 1.5 μA·mM^-1. This dual functional biosensor was validated using clinical urine samples, where the correlation coefficient as 0.96 for pH and 0.98 for glucose detection were achieved with the commercial methods as references. More importantly, the urine sampling vial was kept sealed throughout the sample-to-result process, which minimised the health risk to the frontline practitioners and simplified the diagnostics procedures. This diagnostic platform, therefore, holds high promise as a rapid, accurate, safe, and user-friendly point-of-care (POC) technology for the analysis of urinary biomarkers in frontline clinical settings.

Keywords

Chronic kidney disease (CKD)

Urinary biomarkers

Paper-based dual functional biosensor

Frontline clinical settings

P1.223 UriMoni™: A Microfluidic-Based (Bio)sensing Platform for Clinically Relevant Biomonitoring and Beyond

S1 protein

Bicelle

P1.226 Advanced Software Analysis Platform for Multi-Technology Biosensor Data Integration and Interpretation

Daniel Arismendi-Arrieta¹, John Strangård¹, Sina Bondza^1,2, Jos Buijs^1,2

¹Ridgeview Instruments AB, Sweden. ²Department of Immunology, Genetics and Pathology, Uppsala University, Sweden

Abstract

TraceDrawer addresses the analytical challenges in processing complex real-time interaction data from diverse biosensor platforms. This comprehensive analysis software facilitates the interpretation of molecular recognition events and binding kinetics through both label-free [1] and label-based [2] approaches, supporting research across biological, chemical, and materials science domains.

The TraceDrawer platform enables systematic analysis of data from multiple biosensor technologies, including label-free techniques such as Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance (QCM), as well as LigandTracer that works with label-based methods utilizing fluorescence and radioactive detection. The software implements advanced mathematical models for data integration across different sensing platforms, providing a unified approach to real-time interaction analysis. A notable feature is the InteractionMap algorithm [3], which enables the deconvolution of binding heterogeneity in real-time measurements without a priori assumptions, facilitating the elucidation of complex molecular interaction mechanisms.

The analytical framework incorporates sophisticated preprocessing and evaluation tools for the characterization of interaction rates, binding affinities, and mechanistic parameters. These capabilities extend across various experimental systems, from protein-protein interactions to materials surface characterization and ligand-target interactions on live cells, enabling quantitative analysis of both rapid and slow interaction kinetics. Additionally, the platform enables concentration estimation of analytes, providing flexibility in experimental design and quantification strategies.

References:

[1] Biochemistry 63, 2816 (2024)

[2] Sci Rep 13, 10031 (2023)

[3] Biochem Biophys Res Commun 428, 74 (2012)

Keywords

Real-Time Interactions Analysis

Binding Kinetics

TraceDrawer

InteractionMap

P1.227 Fast-track diagnosis of urinary tract infections

Fatemeh Arvaneh¹, Susan Ibi Preus¹, Lea Holritz¹, Beate Ramshøj Knudsen¹, Line Vigga Kristensen², Kim Langaas², Kristian Stærk³, Karen Andersen Ranberg³, Winnie Edith Svendsen¹

¹Technical University of Denmark, Denmark. ²UVigga diagnostics, Denmark. ³University of Southern Denmark, Denmark

Abstract

Urinary tract infections (UTIs) represent a significant global health challenge, accounting for over 404 million cases and nearly 237,000 deaths annually. They are especially prevalent among the elderly and immune-compromised, where untreated UTIs can escalate to severe complications, including kidney infections and sepsis, which carry a mortality rate of up to 40%. Current diagnostics rely on symptom assessment and urine cultures, which can take up to 72 hours to yield results. This delay often leads to antibiotic treatment based on dipstick tests alone which are often inaccurate and contribute to antibiotic overuse. Consequently, Danish health authorities have advocated for phasing out dipstick tests in clinical setting due to their low diagnostic reliability.

Traditional biomarkers including C-reactive protein (CRP), urine nitrite, and leukocyte esterase have low sensitivity and specificity, often resulting in both over- and undertreatment. A pediatric study of 1,186 patients revealed that 43% were overtreated, and 13% were undertreated, leading to recurrent infections, risk of antibiotic resistance, and kidney damage.

To address these critical diagnostic limitations, we have developed a lateral flow assay (LFA) that detects Interleukin-6 (IL-6), a key infection marker that rises significantly in UTIs. Measuring IL-6 in urine enhances diagnostic accuracy and enables differentiation between UTIs and asymptomatic bacteriuria (ABU), providing a valuable complement to standard urine cultures. Additionally, we are investigating the potential of incorporating a second biomarker to enhance specificity, which we look forward to discussing in detail at the conference. This rapid point-of-care solution leverages LFA technology to deliver results within minutes, offering a transformative alternative to costly and time-consuming laboratory tests.

Uncaptioned visual

Keywords

UTI

LFA

Biomarker

POC-testing

P1.228 Science Meets Society: The Value of SSH in EU Biosensor R&I and Market Research

Kristen Connor, Rita Campos, Filipe Santos, Joana Sousa, Bernardo Valente

University of Coimbra Centre for Social Studies, Portugal

Abstract

What is the role of public dialogue, the social sciences, humanities, and arts in biosensor market research, innovation and development in the European Union? In this poster presentation, a team of social scientists from the Centre for Social Studies (CES) at the University of Coimbra, Portugal, will present their findings from the BioAssembler.eu consortium (Horizon Europe No 101070589). This project is developing a bio-inspired assembly technology for scalable manufacturing of silicon-based label free multiplex biosensors in semiconductor fabrication platforms.

European Commission R&I (research and innovation) funding increasingly requires and prioritizes projects with deep SSH (social sciences and humanities) integration and SSH mainstreaming. This presentation will discuss how the BioAssembler consortium integrated SSH into the project, and the centrality of SSH for field-based market research based on two case studies: marine monitoring and forensics. Audience members will gain an understanding of the role of SSH in biosensor R&I in the EU; ideas for how to integrate SSH into their projects; and have the chance to engage in dialogues about the value artists, humanists, and social scientists bring to the field both at the R&I and user adoption phase. Given the expansiveness of “SSH” practitioners and experts in the EU, audience members will have the chance to engage with seasoned social scientists about what SSH approach might work best for their project, research, or product.

BioAssembler engaged artistic performers, illustrators, and comic book artists as part of its science communication strategy. Images, examples, and drafts of these materials will be available to the audience. SSH practitioners with experience engaging artists will be able to discuss the relationship between biosensor R&I, SSH mainstreaming, and the role of the arts in effective science communication.

Keywords

Market research

SSH integration

multiplexed biosensors

science communication

P1.229 Puremilk: decentralized human milk analysis ensuring donor milk in hospital settings

Lea Holritz, Susan Ibi Preus, Beate Knudsen, Pulkit Saluja, Maria Dimaki, WinnieE. Svendsen

Technical University of Denmark, Denmark

Abstract

Human milk is incredibly important for premature babies, providing essential nutrients and antibodies that are crucial for their development and immune systems. It helps protect against infections, supports healthy growth, and can significantly improve health outcomes for these vulnerable infants. However, once expressed, human milk can become a medium for bacterial growth if not handled and stored properly, thereby greatly posing risks, especially for premature and neonatal infants. Currently, testing donor milk for bacterial contamination can be challenging due to the need for specialized equipment, high costs, and time-consuming procedures, which may not be feasible in all hospital settings and add to time and resources for testing in milk kitchens. The PureMilk test kit addresses this by offering a simple and fast method for ensuring the safety of donor milk in hospitals or at milk kitchens. The kit includes a patented pretreatment protocol, that standardizes the milk sample, ensuring consistency and accuracy in testing. The kit also features a paper-based device that detects bacteria directly in the milk, leveraging the advantages of paper-based analytical systems, such as point-of-use, ease-to-use and low cost. Finally, a reader interprets the signals from the paper-based device, translating them into quantifiable data that calculates the initial concentration of bacteria in the milk sample. This innovative approach ensures high sensitivity and quality assurance, with a testing time of less than 20 minutes and no special training or equipment needed, making donor milk safe for neonatal units and protecting infants from potential infections while preserving the nutritional benefits of human milk.

Keywords

Human milk

Bacteria

Decentral analysis

premature infants

P1.230 Novel electrospun materials for the advancement of lateral flow diagnostics

Nicola Kelly, Jenny Aveyard

University of Liverpool, UK

Abstract

First developed in the 1980s, lateral flow tests (LFTSs) are membrane-based diagnostics devices that are simple to use, cheap, robust, and accessible. However, their true potential was not recognised until more recently during the COVID-19 pandemic which saw LFTs used on an unprecedented scale globally with over 20 million tests used within the UK in 12 months, allowing diagnosis and monitoring to occur beyond healthcare settings. Despite this, LFTs are fraught with issues relating to sensitivity and selectivity.

Currently, casted nitrocellulose membranes are the industry standard for LFT membranes due to their low cost, mechanical strength, and high protein binding. However, nitrocellulose membranes also have shortfalls such as sensitivity to environmental factors which affect their mechanical strength, highly flammable behaviour, and batch to batch variation due to the manufacturing method. By reconsidering the materials and production method, the membrane design could significantly change LFT performance.

Manufacturing techniques such as electrospinning allow the fabrication of nanofibrous polymer materials with high porosity, interconnected porous networks, and a high surface-to-volume ratio. The porosity of the membrane governs the capillary flow of a sample/reagent through the LFT. This in turn governs the sensitivity of the LFT.

Electrospun polymeric membranes where characterised and compared to commercial nitrocellulose membranes using methods such as microCT and BET analysis and displayed superior properties relating to porosity, pore interconnectivity and reproducibility. Protein binding analysis also displayed an increase in binding capacity compared to the commercial standard.

This project facilitates a step change in materials and manufacturing techniques in LFT production by investigating the use of electrospinning to fabricate polymer membranes with superior properties that will improve the sensitivity and selectivity of membrane-based diagnostics. This approach has the potential to address existing challenges and further expand the application of LFTs in point-of-care testing.

Keywords

Electrospinning

bioconjugation

antibodies

detection format

P1.231 An affordable, field-deployable fluorometer for detection of pollutants using engineered protein biosensors

Nathaniel Smith, Adrian Rizk, Owen Rizk, Shahir Rizk

Indiana University South Bend, USA

Abstract

Fluorescence is a widely used technique for a vast number of biochemical and biomedical applications. Many biosensors rely on a change in fluorescence or bioluminescence to detect the presence of a substance of interest. However, fluorometers are expensive and require expert users, constraining their implementation in field detection. Here, we describe the development and implementation of a portable fluorometer device that can detect the presence of glyphosate in different water and soil samples treated with commercial herbicides. Glyphosate is the most commonly used herbicide in the United State, often contaminating groundwater and posing potential health risks to humans. Our device is 3D-printed and uses small, affordable electronic components for excitation and detection of emission. The device utilizes an engineered biosensor protein modified to detect glyphosate through fluorescence changes. Our results show that the device successfully distinguishes between glyphosate-contaminated and uncontaminated samples in water and soil samples, including those treated with commercial herbicides. The low cost of the device (under $40) and the ease of use open the potential for applications in remote areas without the need for expert operators. Our work is expending the use of the device for the detection of several analytes with a focus on neglected tropical infections.

Keywords

Fluorescence

Glyphosate

Pollution detection

Diagnostics

P1.232 Lab-made flexible electrode based on PVP-CuO film for dopamine detection

Duygu Zabitler, Esra Ülker, Gözde Aydoğdu Tığ

Ankara University, Turkey

Abstract

The scientific community has recently noted a growing focus on developing cost-effective and disposable electrochemical devices for analyte detection [1]. This study aims to develop environmentally friendly technologies that use waste materials for screen-printed electrodes (SPEs), with a particular focus on carbon-based conductive inks and colorless nail polish to create disposable electrodes on polyethylene terephthalate (PET) plastic, which is derived from recyclable bottles. Preparation of lab-made electrodes, carbon-based conductive inks, and silver inks were utilized, along with surface modifications for the fabrication of the working electrode. Lab-made electrodes advance environmental impact reduction and embody green chemistry principles, promoting sustainability and resource responsibility. Lab-made SPEs were modified with PVP-based (polyvinylpyrrolidone) CuO (copper oxide) nanoparticles via drop-casting to improve analytical performance. PVP forms a uniform film, boosting stability, while CuO's conductivity and catalytic properties increase electrode sensitivity and accelerate electrochemical reactions. This combination enhances analytical capabilities, lowering detection limits and more reliable sensor responses for detecting dopamine (DA). This detection is critically necessary because it is found in biological samples, and abnormal level is associated with different severe conditions - DA with neurological disorders such as Parkinson's [2]. Therefore, diagnosing and effectively managing health risks associated with this condition is important for developing low-cost, eco-friendly, and practical electrodes for biological molecule measurement.

References:

[1] Moro, G., Bottari, F., Van Loon, J., Du Bois, E., De Wael, K., Moretto, L.M. 2019. "Disposable electrodes from waste materials and renewable sources for (bio) electroanalytical applications," Biosensors and Bioelectronics, 146, 111758.

[2] Krishnamoorthy, K., Sudha, V., Kumar, S.M.S., Thangamuthu, R. 2018. "Simultaneous determination of dopamine and uric acid using copper oxide nano-rice modified electrode," Journal of Alloys and Compounds, 748, pp. 338-347.

Keywords

Flexible electrode

lab-made electrode

nanoparticle

dopamine

P1.233 DEVELOPMENT OF INNOVATIVE (BIO)SENSOR FOR THE DETECTION OF BIOCIDE DISINFECTANTS IN DAIRY INDUSTRIES

Youssef ALI^1,2, Valerie GAUDIN², Christophe Soumet², Michel Laurentie², Pascal Mailley³

¹French National Interprofessional Centre for Dairy Economics, France. ²Anses Fougeres Laboratory, France. ³CEA, LETI, Departement of Microtechnologies for Health and Biology , Directorate of CEA Technological Research Division, France

Abstract

Quaternary ammonium compounds (QAs) are used in the dairy industry in cleaning and disinfection protocols. Chlorinated products are widely used for water treatment, leads to the birth of toxic by-products such as chlorates (ClO₃^-). Studies reported the contamination of dairy products by excessive concentrations of biocide, much higher than the European maximum residue limit (MRL). MRL is 100 µg/kg for QAs and ClO₃^- in milk and 10 µg/kg for ClO₃^- in baby food. To date, no commercial self-monitoring method is capable of achieving the MRL.

The objective of the SensoMilk project is to develop portable, easy to use, sensitive (10-100 µg/kg), rapid , and inexpensive (bio)sensor for the detection and quantification of QAs & ClO₃^- residues in milk and rinsing water, in the dairy industry.

Electrochemical and colorimetric enzymatic biosensors are in development for the detection of QAs, based on the principle of inhibition of an enzyme acetylcholinesterase (AchE) by QAs. In the presence of QAs, the intensity of the oxidation thiocholine peak decreases (the product of the enzymatic reaction). Colorimetric biosensor is based on the classical Ellmann's method [1] (λ=405 nm). When a QA is present, the intensity of yellow color decreases. The results showed the detection of QAs as low as 100 µg/L and 5000 µg/L by electrochemical and colorimetric biosensors respectively.

For chlorates, a colorimetric sensor based on the oxidation of a dye, indigo carmine, by ClO₃^- , in strong acidic conditions, is in development [2]. The absorbance (λ= 630 nm) decreases when the concentration of ClO₃^- increases. At that time a preliminary detection limit of 500 µg/l of ClO₃^- in water was obtained in a microplate format. Some work remains to be performed on optimization of assay parameters and sample preparation, especially for milk.

[1]http://dx.doi.org/10.1155/2013/932946

[2]https://doi.org/10.1016/j.talanta.2009.04.065

Keywords

Bio(sensor)

Biocide

Acetylcholinesterase

Milk & rinsing water

P1.234 NanoAmp: towards sensitive protein PCR

Saurabh Buchude^1,2, Denise Di Lena^3,1, Edoardo Sisti^4,1, Alessandro Bertucci³, Eleonora Dapozzo⁴, Rudy Ippodrino¹, Bruna Marini¹

¹Ulisse Biomed SRL, Italy. ²University of Rome Tor Vergata, Italy. ³University of Parma, Italy. ⁴University of Pisa, Italy

Abstract

Quantitative detection and monitoring of antigens and therapeutic antibodies is the fundamental cornerstone of in vitro diagnostics. Accurate quantification of these biomolecules is essential for disease diagnosis or theragnostic application. NanoAmp, a novel quantitative polymerase chain reaction (qPCR)-based diagnostic platform developed by Ulisse Biomed S.p.A., is a homogeneous assay, leverages the principles of local concentration increase induced by the target analyte, combined with PCR amplification, to achieve rapid, sensitive, and quantitative detection of target analytes in a single step reaction. In contrast to heterogenous assays like ELISA, CLIA and lateral flow assays, which require multiple steps, NanoAmp reaction takes place in a single tube without compartmentalization of steps, giving a streamlined approach avoiding the laborious, expensive, and potentially contaminating protocols often associated with traditional methods. Technology also exhibits capability of multiplexing detection through high resolution melting curve analysis or using target specific probes. Characterization and standardization of the technology was done using a Digoxigenin model system to detect as low as 50 picomolar of anti-digoxigenin antibody. The assay has a rapid turnaround time of approximately 45 minutes inclusive of hands-on time, making it an ideal diagnostic technology to be deployed in emergency cases like Sepsis. Furthermore, the platform is robust enough to work on all existing qPCR machines. Future efforts will focus on targeting therapeutic monoclonal antibodies in theragnostic and optimizing the platform for Sepsis infection directly from complex matrices.

Keywords

Quantitative PCR

In Vitro Diagnostics

Single-Step Assay

Theragnostic Applications

P1.235 Technology challenges review for in-vivo aptamer based biosensors

Jörg Fochtmann¹, Md Rafsunjani²

¹Harz University of Applied Sciences, Germany. ²TU Bergakademie Freiberg University, Germany

Abstract

In-vivo sensor integrated with aptamer technology have been emerged in biomedical industry as a tool for real-time biomedical monitoring which offers great sensitivity, accuracy and specificity to detect biomolecules. The results of several investigations on sensor technology are combined in this review and merged with the aptamer-functionalized platform to demonstrate the advancement of aptamer-based hybrid electrochemical platforms.

Concrete diagnosis and therapeutic mediations have been made possible by transduction principles like electrochemical and optical signal conversion. Microfabrication, soft-lithography, carbon modification, and hybrid material integration are examples of new and creative techniques that have aided in miniaturization and improved stability and sensitivity in physiological settings. The electrochemical, optical, and piezoelectric mechanisms that underpin signal conversion processes have made significant strides in enhancing signal stability and reducing interference in vivo. On the other hand, aptamer-based interfaces for improved bio-recognition and the significance of metabolic and nanostructured material changes in improving device efficiency are highlighted in brain neurochemical monitoring. Aptamer functionalized cell membranes for brain applications validate improved target specificity. Some significant studies outperform the traditional external powered sensor by offering enhanced integration in living organisms which includes methods like micro plating, subtract affect and logic gate functions.

Future directions indicate how to adopt personal healthcare in a seamless manner. Advanced sensor design combined with aptamer-based technology has revolutionary promise for therapeutic monitoring, diagnostics, and precision medicine. This review highlights ongoing research and offers a thorough assessment of the state of advancement in aptamer-based in-vivo sensors technologies.

Keywords

Aptamer

Semiconductor

P1.236 Direct electron transfer type spermine sensor with engineered spermidine dehydrogenase with enhanced electron transfer ability

Mao Fukushi¹, Sheng Tong¹, Yuki Yaegashi¹, Takumi Yanase¹, Junko Okuda-Shimazaki¹, Ryutaro Asano¹, Kazunori Ikebukuro¹, Koji Sode², Wakako Tsugawa¹

¹Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Japan. ²Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, USA

Abstract

In this study, we present a novel direct electron transfer (DET) type electrochemical spermine sensor using engineered spermidine dehydrogenase (SpDH), which shows 3 times higher current density than the wild type (WT) one.

Polyamines are recognized as the biomarkers for variety of diseases including cancer. Among them, salivary spermine was reported to be utilized for the early diagnosis of pancreatic cancer (PC) ^[1]. Therefore, we have been engaged in the development of handheld, rapid and sensitive spermine sensor suitable for salivary samples, by utilizing oxidoreductases with the combination of electrochemical sensing principle. We focused on a unique enzyme, spermidine dehydrogenase (SpDH), which is composed of a catalytic domain with redox cofactor, flavin adenine dinucleotide (FAD) and a heme b domain. SpDH was recombinantly produced using Escherichia coli. An enzymatic sensor was constructed by immobilizing SpDH on gold electrodes. The cyclic voltammograms of the electrode with SpDH showed that catalytic current toward spermine was observed with onset potential of -150 mV vs Ag/AgCl in the absence of any additional electron acceptor in the solution. These results demonstrated that SpDH is capable of DET with electrode. The chronoamperometry under applied potential of 0 mV vs Ag/AgCl showed linear current responses toward spermine with LOD of 0.72 µM.

To further improve the DET-properties of SpDH, we introduced mutations to the heme b axial ligands in SpDH. One mutant showed the spermine-concentration dependent catalytic current higher than WT. The spermine concentration dependent currents was also observed by chronoamperometry, showing 3 times higher response current than those with WT with LOD of 0.42 µM. The current achievements promise the future realization of a simple detection method of spermine for PC diagnosis.

[1] Asai et al., Cancers 2018, 10, 43

Keywords

Spermine

Spermidine dehydrogenase

Direct electron transfer

Protein engineering

P1.237 A flexible biosensing platform for the simple detection of metabolites

Jacopo Giaretta^1,2, Ronil Rath^1,2, Riccardo Zulli³, Theja Prabhakar¹, Suvan Shrestha¹, Sina Naficy^1,2, Syamak Farajikhah^1,2, Fariba Dehghani^1,2

¹The University of Sydney, Australia. ²The University of Sydney Nano Institute, Australia. ³University of Padova Department Industrial Engineering, Italy

Abstract

Point-of-care testing and patient-centred care are the new paradigm in an evolving healthcare system moving away from hospital-centred approaches. This has driven the development of portable and wearable sensing devices, ideally cost effective and simple to use. Nevertheless, electrochemical sensors often result quite expensive, bulky, and invasive, while also requiring large sample volumes.

We designed a sensing platform to detect biomarkers commonly present in biofluids employing conductive polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) combined with enzyme horseradish peroxidase (HRP). The PEDOT:PSS/HRP system exhibited a linear decrease in resistivity when exposed to just 40 µL of hydrogen peroxide down to a micromolar level. This was true both when using a cellulosic substrate and when embedding the system in a hydrogel matrix.

Using the PEDOT:PSS/HRP system as a building block, we added a second enzyme to allow the detection of more complex molecules. For instance, the addition of enzyme lipoxygenase allowed the detection of linoleic acid, which concentration is related to metabolic disorders, cardiovascular diseases, and cancer. The sensor could detect linoleic acid down to a nanomolar level and function also at different pHs. Glucose oxidase, instead, was added to PEDOT:PSS/HRP to allow glucose detection, crucial for diabetes management and cardiovascular diseases. In this case, the sensor could detect glucose down to 10 micromolar and exhibited selectivity among interferents commonly found in saliva. In this case, the effect of enzyme immobilization on sensor stability was also investigated, with sensors remaining stable for up to 10 days.

In conclusion, the PEDOT:PSS/HRP system proved as a promising flexible platform for the simple detection of complex molecules, in the range of interest in human biofluids and requiring only a few drops of analyte for analysis. This approach may guide the development of cost-effective point-of-care sensing devices.

Keywords

Chemiresistive

Biosensor

Metabolites

PEDOT:PSS

P1.238 Combination of Raman spectroscopy and electrochemistry for the easy detection and characterization of enzymes

David Ibáñez, Paula Caldevilla-Collado, Laura García-Alcalde, David Hernández-Santos, Pablo Fanjul-Bolado

Metrohm DropSens, Spain

Abstract

Raman spectroscopy is one of the most promising chemical analysis techniques due to its inherent fingerprint properties which allow the identification of different species present in a studied system. Although low sensitivity has limited its use as a detection method, the surface-enhanced Raman scattering (SERS) effect has improved its effectivity for a huge variety of applications. In particular, the energy provided by the 638 nm laser ensures a balance between risking damage to the sample and the generation of fluorescence, making this laser popular for most biological applications.

As proof of concept, Raman spectroelectrochemistry was employed for the detection of aldehyde dehydrogenase (ALDH) and cytochrome c. Electrochemical SERS (EC-SERS) protocol requires two steps in a single experiment: the electrochemical generation of silver nanoparticles with SERS features, and then, the spectroscopic detection of the enzyme present in the sample.

The proposed EC-SERS procedure with a Raman spectroelectrochemical instrument offers an interesting alternative for the characterization of ALDH in solution not previously reported in the literature (Figure 1a). The developed protocol also allows the easy detection and the characterization of the oxidation state of cytochrome c. Considering that cytochrome c exists as interconvertible reduced and oxidized forms, the oxidation state of the Fe ion can be determined according to the position of their characteristic Raman bands. Reduced cytochrome c shows one Raman band centered at 1604 cm-1, while the oxidized form displays an upshifted band centered at 1636 cm-1. According to the Raman spectrum (Figure 1b), the reduced form of the analyte is detected during the EC-SERS experiment. This experiment demonstrates the possibility to detect cytochrome c as well as characterize its oxidation state.

a)	b)

Figure 1. SERS spectrum of (a) ALDH and (b) cytochrome c obtained in KCl aquous solution with silver electrodes.

Keywords

Electrochemistry

Raman spectroscopy

SERS

Screen-printed electrodes

P1.239 Determination of lactic acid in beer with Screen-Printed Electrodes

Daniel Antuña-Jiménez, Laura García-Alcalde, Paula Caldevilla-Collado, David Hernández-Santos, Pablo Fanjul-Bolado, María Begoña González-García

Metrohm DropSens SL, Spain

Abstract

The presence of lactic acid in beer is essential in a certain amount to afford acidity and lactic aromas so it is usually introduced to the wort on purpose. Although lactic acid usually appears as the result of controlled bacteria fermentation, its amount in excess it is considered a defect because it can also appear as result of an infection. Controlling the level of lactic acid is essential not only for the purpose to offer a certain organoleptic properties but to monitor fermentation processes and control food quality/safety.

In this work, a commercial enzymatic sensor (DRP-LACT10) is used to measure this analyte in four commercial beers (from local brewery Cotoya) as a proof of concept to its potential application in fermentation monitoring. Determination is based on the detection via mediator of hydrogen peroxide produced by the enzymatic reaction onto the working electrode. With a sample of 50 µL, this analyte is measured in a range of 0 to 0.4 mM in less than 75 s with an amperometric detection at -0.1 V.

To avoid matrix effects, a dilution of 1:10 were made with each beer in Tris-NO3 0,1M pH 7,2 aqueous solution before measuring. To validate data obtained, a commercial L-Lactate Assay kit was use, demonstrating the viability to detect lactate in beer with a portable device without the requirements of complex pretreatments or the use of non-portable optical devices.

In combination to the electrochemical sensing platform, a novel portable user-friendly potentiostat reader DRP-DROPSTATPLUS was used, where lactic acid value of the sample can be easily viewed on its LCD display. Thanks to its small size and its portability, its combination with the lactate biosensor offers a winner combo to acquire in-situ measurement in industrial or household breweries with a simple low-cost setup easy to operate.

Keywords

Lactic Acid

Commercial beers

Screen-printed electrodes

Portable reader

P1.240 Development of a wash-free bacterial detection method based on the proximity-unlocked luminescence by sequential enzymatic reactions

Shogo Izumi¹, Daimei Miura^2,3, Kaori Tsukakoshi², Koji Sode⁴, Wakako Tsugawa², Kazunori Ikebukuro², Ryutaro Asano²

¹Department of Industrial Technology and Innovation, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Japan. ²Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Japan. ³Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Japan. ⁴Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill & North Carolina State University, USA

Abstract

We report a convenient luminescent bacterial detection method based on the sequential antibody-conjugated enzymatic reactions, which can provide the signal, only when two labeled enzymes localize in proximity on bacterial surface. This method achieved homogeneous detection by simply mixing reagents.

Detection of pathogenic bacteria such as Escherichia coli (E. coli) O157 strain is critical for public health. As conventional detection methods, including colony counting and PCR, are time-consuming and require well-trained technicians, a convenient detection method for on-site testing is desired. Recently, we developed a wash-free virus detection system, designated as proximity-unlocked luminescence by sequential enzymatic reactions from antibody and antibody/aptamer (PULSERAA)^[1]. PULSERAA enables convenient detection and visualization of infectious spots. Thanks to the simple design of the sensing components, PULSERAA can be easily customized for various targets by employing specific antibodies for each target.

In this study, we adapted PULSERAA for bacterial detection, targeting lipopolysaccharide (LPS) and outer membrane protein C (OmpC). ScFv-glucose oxidase (GOx) complex was prepared using anti-LPS scFv and the SpyCatcher (SC)/SpyTag (ST) system. SC-fused GOx and ST-fused anti-LPS scFv were recombinantly prepared and mixed to form scFv-GOx. For the detection of bacteria, commercially available HRP-labeled anti-OmpC antibody was combined with the anti-LPS scFv-GOx. As a model case, the detection of E. coli DH5α strain was attempted using customized PULSERAA system. The chemiluminescence from HRP reaction was measured after adding glucose and luminol to detect H₂O₂ produced by GOx reaction, which occurs only when the anti-LPS scFv-GOx and HRP-labeled anti-OmpC antibody localize in close proximity on the bacterial surface. As a result, E. coli concentration-dependent chemiluminescence change was observed, and high linearity was obtained in the range of 10²-10⁵ cells/mL, suggesting this system has a potential as a convenient E. coli detection method.

[1] Miura et al., Adv. Sci., 2024, 240387 Uncaptioned visual

Keywords

Immunosensor

Escherichia coli

Antibody-enzyme complex

Homogeneous detection

P1.241 Development of a Nanomaterial and Conductive Polymer Based Amperometric Biosensor for Levodopa Determination

Sinem Boyraz, Ceren Kaçar Selvi

Diabetes mellitus, a chronic metabolic disease, is projected to increase globally by 59.7% by 2050, highlighting the urgent need for rapid, accurate, non-invasive, and cost-effective diagnostic tools. Current glucose monitoring devices, such as finger-prick blood tests, continuous interstitial fluid monitors, and colorimetric urine strips, have drawbacks like discomfort, high costs, and limited sensitivity. Consequently, demand is rising for minimally invasive point-of-care solutions to monitor glucose in biofluids like saliva, sweat, and urine.[1]

Nanotechnology is revolutionizing biosensing by harnessing nanomaterials with advanced properties, such as higher sensitivities, lower detection limits, and compact platforms. Laser-induced graphene (LIG) stands out due to its outstanding electrochemical properties, simplicity, scalability, rapid production, and ease of patterning. These attributes have positioned LIG as a key enabler for high-performance biosensors, particularly for the development of enzymatic and non-enzymatic glucose sensors.[2-5]

This work compares the performance of LIG-based enzymatic and non-enzymatic glucose biosensors. Non-enzymatic sensors incorporate composites of LIG with gold nanoparticles (LIG/AuNPs), operating in alkaline pH conditions. In contrast, enzymatic sensors utilize mediators to transfer electrons from glucose oxidase (GOx) enzymes to LIG-based transducers, functioning at neutral pH.

The biosensors were evaluated in a glucose concentration range from 1 to 1400 mg/dL in buffered solutions. Additionally, synthetic and real urine samples were tested, and compounds that interfere with glucose detection were identified. The developed sensors demonstrated promising performance within the clinical glucose detection range, highlighting their potential for practical, non-invasive glucose monitoring in biofluids such as urine.

References

[1] Sehit, E., Altintas, Z., 2020, Biosensors and Bioelectronics, 159, 112165.

[2] Liu, J., et al., 2022, Microchimica Acta, 189 (54).

[3] Pereira S. O., et al., 2021, Nanomaterials, 11 (8), 1893.

[4] Santos, N. F., et al., 2021, Advanced Materials Technologies, 6, 2100007.

[5] Kulyk, B., et al., 2022, Carbon 197, 253–263.

Keywords

Laser-induced graphene

Nanoparticles

Electron mediators

Glucose

P1.245 Bioreference electrode; a reference electrode based on biological recognition elements for stable, versatile biosensing

Joseph A. Kerrigan Jr., David Probst, Koji Sode

Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC, USA

Abstract

We present a unique reference electrode design, designated as a Bioreference electrode, which maintains stable potential based on reduction/oxidation state of an inactive enzyme. To create the Bioreference electrode, we engineered a direct electron transfer (DET)-type enzyme which is “inactive” or unable to transfer electrons to the electrode, keeping a stable redox-potential, while immobilized on the electrode. This Bioreference electrode was revealed to improve the stability of open circuit potential (OCP) based enzymatic sensors.

OCP based enzymatic sensors are comprised of a recognition electrode used to measure a change in potential over the working electrode due to the oxidoreductase reaction with substrates, resulting in the enzymes with reduced status. Commonly used reference electrode is Ag/AgCl, which can drift by as much as 100 mV due to fluctuations in chloride ion, or due to oxidation. This has limited the translation of OCP type sensors form in vitro, to in vivo use cases. To overcome this limitation, we designed a Bioreference electrode, engineered inactivated redox enzyme harboring a redox group—allowing for a stable redox potential.

As a representative example, we prepared a Bioreference electrode with inactivated DET-type glucose dehydrogenase, and investigated its feasibility using both amperometry and OCP glucose sensors. Our developed OCP glucose sensor with Bioreference electrodes, revealed less than 50 mV drift, maintaining an error rate below 10% across pH shifts (6–8) and NaCl concentration changes (50–500 mM). Furthermore, the Bioreference electrodes allow for greater reference electrode miniaturization, providing stable potential even with 100 µm electrodes.

We also confirmed the advantage of using the Bioreference electrode in an amperometric based enzymatic sensor for the detection of glucose. This study revealed that the Bioreference electrode will be used for creating stable biosensors and demonstrates their application for long-term, in vivo continuous monitoring.

Keywords

Bioreference electrode

Direct electron transfer

reference elecrtode

continuous monitoring

P1.246 Non-invasive ethanol detection via skin gas using an ear-mounted wearable biosensor

Takahiro Arakawa¹, Riki Ishikawa², Kenta Iitani², Koji Toma³, Kohji Mitsubayashi²

¹Tokyo University of Technology, Japan. ²Institute of Science Tokyo, Japan. ³Shibaura Institute of Technology, Japan

Abstract

Volatile organic compounds in human exhalations and skin emissions are associated with metabolic processes and diseases, making their measurement valuable for non-invasive, straightforward metabolic assessments and disease diagnosis. Skin gas, in particular, offers a promising avenue for continuous, constraint-free collection, which is ideal for the next generation of wearable diagnostic devices. In this study, we developed a wearable biochemical gas sensor (biosniffer) for ethanol detection by constructing a headset-type system that can perform unrestrained measurements of ethanol emissions from the ear canal, integrated with a complementary metal-oxide semiconductor (CMOS) camera. This system detects ethanol gas from skin emissions by measuring the increase in fluorescence of NADH, a product of the enzymatic oxidation of ethanol by alcohol dehydrogenase (ADH). The system utilizes a UV-LED for excitation, passing through a band-pass filter to illuminate an ADH enzyme-immobilized membrane, while a compact CMOS camera with near-ultraviolet (N-UV) functionality detects the resulting NADH fluorescence. Incorporated into an earmuff, this wearable setup facilitates continuous ethanol monitoring. Fluorescence imaging reveals an increase in output correlated with ethanol gas concentration, ranging from 11 ppb to 444 ppm, which spans typical skin ethanol levels following alcohol consumption. Moreover, tests on human participants who consumed alcohol demonstrated an increase and subsequent decrease in fluorescence output corresponding to ethanol absorption and metabolism, respectively. This device shows promise for real-time, direct ethanol monitoring from the ear canal with minimal interference from perspiration.

Keywords

Enzyme

Gas sensor

Imaging

Ear

P1.247 Nanoparticle polymer-brush hybrid systems for enzyme-based biosensor

Anila Antony¹, Hongtao Cai¹, Anne Linhardt¹, Alla Synytska¹, Milkin Pavel¹, Petr Formanek²

¹University of Bayreuth, Germany. ²Leibniz Institute of Polymer Research Dresden, Germany

Abstract

Enzymes have been explored for their potential use in biosensing applications due to specificity. However, the limitations of enzyme-based biosensors, such as poor long-term stability, loss of enzyme and proper electron transfer during the sensor application, remain significant challenges. To address these issues, we are developing a suitable matrix, based on organic-inorganic hybrid nanoparticles for enzyme immobilization. We do surface modification on Multi-Walled Carbon Nanotubes (MWCNTs) using Polydimethylamino-ethyl methacrylate (PDMAEMA) brushes via Surface Initiated-Atom Transfer Radical Polymerization (SI-ATRP). The highly positively charged PDMAEMA brushes enable the covalent immobilization of enzymes with negative surface charge, preventing enzyme denaturation and improving stability over time. Additionally, we can control enzyme immobilization efficiency by varying the polymer brush thickness and grafting density, allowing the system to be tailored to different biosensing applications. Moreover, these organic-inorganic systems can be used for immobilization of different enzymes according to the application. These particles are readily dispersible in suitable buffers, and our work shows how they are successfully used as ink for electrode functionalization, demonstrating their potential for scalable production of enzyme-based biosensors. This versatile platform is applicable to various enzymes, making it suitable for a wide range of applications, including medical diagnostics, environmental monitoring, and food safety. Our approach represents a promising solution to enhance the performance of enzyme-based sensors.

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Reference:

1. Antony, A; Cai, H; Linhardt, A; Synytska; A. Polymer modified MWCNTs; suitable matrix for enzyme immobilization (manuscript in preparation)

2. Cai, H; Antony, A; Linhardt, A; Formánek, P; Ionov, L; Synytska; A. Can conductivity be introduced into polymer-brush functionalized core-shell particles with high enzyme loading capacity? (manuscript in preparation)

Acknowledgements:

The authors would like to acknowledge the project funding from BMBF (Bundesministerium für Bildung und Forschung, (FKZ: 031B1118B) and DFG Grant SY125/15-1 for financial support.

Keywords

MWCNT

SI-ATRP

Enzyme immobilization

Polymer brush

P1.248 Paper-Based Sensing Strip for On-Skin Sweat Analysis

Jinze Chen, Toshihiro Kasama, Madoka Takai, Ryo Miyake

The University of Tokyo, Japan

Abstract

Wearable sensors play crucial roles in self-health monitoring, sports tracking, and other applications, but the utilization of metal and polymer as ingredients brings the problem of high cost and environment impact. To address these issues, we proposed a minimized paper-based sensing strip for human sweat analysis and selected lactate as detection target. In the characterization, our strip exhibits a good conductivity and an expanded detection range up to 10 mM, which corresponds to the lactate concentration range of sweat.

The initial fabrication step is forming the graphene electrode and absorbent channel on paper. We innovatively integrated the electrode and absorbent channel together as graphene is an ideal electrode material and paper has innate capillary force and could realize the sampling and liquid transportation automatically. The designed pattern was processed by CO2 laser cutter under certain parameters (Fig. 1.2). After engraving, electrodes were connected to conductive tape and Ag/AgCl ink was used to form reference electrode.

Afterwards, a modification was applied to graphene electrode to make the lactate sensor (Fig. 1.4). The electrode surface was coated with three sequential layers: first, a mediator layer of 20 mM/mL tetrathiafulvalene in an acetone/ethanol mixture. Next, an enzyme layer was added, containing lactate oxidase mixed with chitosan. Finally, a PVC coating dissolved in tetrahydrofuran was applied to enhance detection range. Calibration plot was determined using a three-electrode lactate sensor (Fig.2). It showed that this sensor exhibited an expanded detection range up to 10 mM and a good linearity with acceptable deviation. To test the sensing performance of this strip, chronoamperometry was conducted with working potential of 0.15 V, and the concentration of lactate increased continuously (Fig. 3). The result indicated that the absorbent channel enabled the successive liquid transportation and this strip had an effective response to the change of concentration.

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Keywords

Paper-based Sensor

Disposable

On-Skin Analysis

P1.249 Simple and Effective: The portable and low-cost biosensor for quick pathogens detection in foods

Evgeni Eltzov

Agricultural Research Organization, Israel

Abstract

This study introduces an innovative biosensor system designed for the rapid and simultaneous detection of multiple foodborne pathogens, including Bacillus cereus, Staphylococcus aureus, Bacillus subtilis, and Escherichia coli, enhancing critical food safety measures. Given the widespread impact of foodborne illnesses and the diverse nature of causative agents, this research developed a biosensor capable of functioning effectively within complex food matrices. The biosensor operates through a layered structure with a special water-proof reactive layer sensitive to specific extracellular enzymatic activities of targeted pathogens. When pathogens in a food sample release enzymes, these interact with the layer, causing it to degrade. This degradation allows the sample to contact a subsequent layer containing a dry, color-changing dye. The movement through the degraded gelatin rehydrates and activates this dye, leading to a color shift that accumulates in an absorbent layer, producing a visible signal indicative of pathogen presence. Employing such a specially modified layer, the biosensor demonstrated high specificity and sensitivity, effectively distinguishing between the targeted pathogens even at low bacterial concentrations, such as 1 cell/mL. Extensive testing across various food types highlighted the sensor’s adaptability, showing how food composition influences detection efficacy. The results confirmed the biosensor's robustness in maintaining accuracy and reliability across different food environments. This advancement in food safety technology offers a practical, rapid detection tool that holds promise for on-site testing, potentially transforming food safety protocols and ensuring higher standards of public health and food quality.

Keywords

Food safety

point of care device

Foodborne pathogens

Public health

P1.250 Microparticle-based biosensor for the detection of endocrine-disrupting chemicals

Rosa Louisa Gehring, Veronika Riedl, Andreas Müller, Tilo Pompe

Leipzig University, Germany

Abstract

The presence of endocrine-disrupting chemicals (EDC) in our environment is steadily increasing, e.g. through pathways such as wastewater disposal. Estrogen and its derivatives interfere and affect the health of humans and ecosystems through their endocrine-disrupting properties. Given their low environmental concentrations, the conventional methods for the detection are high-performance liquid chromatography coupled to mass spectrometry. These methods are highly sensitive but are associated with lab-based expensive instrumentation and the requirement of trained personnel.

We recently introduced a hydrogel microparticle-based biosensor designed for cost-effective, point-of-use monitoring of low molecular weight analytes. This competitive biosensing assay is based on estrogen-derivative functionalized polyethylene glycol (PEG) hydrogel microparticles, which interact with a glass chip surface functionalized with the specific binding partner: the human estrogen sulfotransferase (SULT1E1). The free analyte (estrogen-derivatives) in solution and the estrogen-functionalized microparticles compete for the available binding sites on the chip surface. Following that, the adhesion and deformation of the microparticles on the chip surface allow for a straightforward optical read-out of the interaction, e.g. by microscopes or mobile optical devices.

In a proof-of-concept study, we demonstrated the detection of estrogen with high selectivity using this method. Currently, new experiments to optimize the biosensor improved the limit of detection towards nanomolar range and the steadiness of immobilization strategies for mobile, point-of-use applications. Specifically, we studied covalent random coupling via maleic anhydride copolymer coatings and oriented immobilization using the SpyCatcher/SpyTag system with the recombinant expressed proteins (SULT1E1-His₆, SpyCatcher-SULT1E1). Ongoing analysis focuses on robust handling and storage conditions to facilitate an application as fast and easy monitoring kit in environmental applications, e.g. for the detection of estrogen-derivates or bisphenol A in waste and drinking water samples.

Keywords

biosensor

hydrogel microparticles

enzyme immobilization

environmental monitoring

P1.251 Thermodynamic requirements for specific oligonucleotide probes for magnetic biosensors.

Songeun Kim^1,2, Jisoo Im^1,2, Shan Wang^3,4, Jung-Rok Lee^1,2

¹Department of Mechanical and Biomedical Engineering, Ewha Womans University, Seoul 03760, Republic of Korea. ²Graduate Program in Smart Factory, Ewha Womans University, Seoul 03760, Republic of Korea. ³Department of Materials Science and Engineering, Stanford University, Stanford, California 94305, USA. ⁴Department of Electrical Engineering, Stanford University, Stanford, California 94305, USA

Abstract

Heterogeneous hybridization is a key mechanism for obtaining genomic information, such as genotyping, gene profiling, and single-nucleotide polymorphism (SNP) analysis, through the utilization of solid-phase biosensors, including planar and bead-based types. In these sensors, short oligonucleotide probes are immobilized on the surface, enabling them to bind with target DNA in solution, leading to heterogeneous hybridization. By employing solid-phase biosensors in nucleic acid-based assays and utilizing a DNA structural prediction method based on Gibbs free energy and DNA melting temperatures, it is possible to enhance the binding affinity between probes and on-target sequences while minimizing off-target interactions. However, predicting the affinity of specific hybridization and the degree of cross-hybridization between surface-tethered probes and target DNAs in solutions remains challenging due to the complexity of heterogeneous hybridization. This challenge is particularly caused by the unpredictable thermodynamic penalties resulting from the constraints of the solid surface. The majority of the data on DNA thermodynamic parameters that has been previously examined is derived from solution-phase predictions, leading to a restricted comprehension of the consequences imposed by solid surfaces. In this study, we propose practical guidelines for designing oligonucleotide probes, based on thermodynamic properties calculated from solution-phase predictions and data obtained from planar magnetic biosensors. We analyze the thermodynamic conditions in which short oligonucleotide probes achieve optimal hybridization to a target on the planar biosensor. The proposed criteria include a Gibbs free energy of at least -7.5 kcal·mol^-1 and a melting temperature at least 10 ºC below the hybridization temperature. Furthermore, we assessed the impact of secondary structures, such as hairpins and homodimers, to enhance the optimization of oligonucleotide probe design. We suggest that these guidelines will help researchers in creating planar biosensors that have a significant level of sensitivity and specificity for detecting clinical samples, while also anticipating the ability to multiplex targets.

Keywords

Magnetic biosensors

Heterogeneous hybridization

Nucleic acid structure

Specificity

P1.252 Enzymatic biosensor system for gas-imaging of ethanol and acetaldehyde vapor using bicolor fluorometry

Kenta Iitani¹, Rintaro Miura¹, Kenta Ichikawa¹, Koji Toma², Takahiro Arakawa³, Kohji Mitsubayashi¹

¹Institute of Science Tokyo, Japan. ²Shibaura Institute of Technology, Japan. ³Tokyo University of Technology, Japan

Abstract

Humans emit volatile organic compounds (VOCs) that reflect their biochemical state. While gas imaging can reveal where and how VOCs are emitted, current systems lack the selectivity and sensitivity to image multiple VOCs simultaneously. Here, we have developed a gas imaging system that uses two-wavelength fluorometry to selectively and simultaneously image VOCs. As a proof of concept, ethanol (EtOH) and acetaldehyde (AcH) - key molecules in alcohol metabolism - were selected as model VOCs.

The system utilizes the reversible reaction of nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase (ADH), as shown in Figure 1. ADH catalyzes the oxidation of EtOH with NAD⁺ under alkaline conditions (ADH_OX) and the reduction of AcH with NADH under acidic conditions (ADH_RD). NADH produced during EtOH oxidation and consumed during AcH reduction fluoresces at 490 nm when excited at 340 nm. However, because both reactions emit at the same wavelength, simultaneous imaging is not possible. To overcome this, diaphorase (DP) was combined with ADH_OX to convert NADH to resorufin (RN), which fluoresces at 590 nm (excitation at 560 nm), allowing EtOH and AcH to be distinguished by wavelength.

For the experiment, ADH_OX-DP and ADH_RD were immobilized on separate meshes, stacked without contact, and exposed to a mixture of EtOH and AcH gases at different concentrations. The system quantitatively imaged red fluorescence for EtOH (dynamic range 1-300 ppm) and blue fluorescence for AcH (0.2-5 ppm). By changing the NADH-dependent enzyme, the target VOCs can be adjusted. This system shows potential for future applications in monitoring metabolic disorders and pharmacokinetics because we can change the target VOCs by changing the enzymes.

Uncaptioned visual

Figure 1. Overview of the system and typical results of the simultaneous imaging of EtOH and AcH

Keywords

Gas-imaging

Fluorescence

Enzyme

Volatile organic compounds

P1.253 Micro fluidic paper based analytical devices for point-of-care biochemical assays of chronic diseases biomarkers

Raquel Mesquita, Francisca Ferreira, António Rangel

Universidade Católica Portuguesa, CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Portugal

Abstract

The recent interest in on-hand, easy-to-use analytical methods has grown exponentially, and if used correctly, they can be an effective aid in healthcare, such as point-of-care testing (POCT). To attain an immediate, on-hand response, the use of low-cost, easy-to-use devices like microfluidic paper-based analytical devices (µPADs) is a powerful tool. However, if a faster analysis results in a faster decision on the course of action, then it is crucial to have an accurate and reliable tool. The downscaling of the analytical procedure raises sample handling challenges, namely the reduced sample volume and matrix complexity, particularly when using biological fluids. The use of µPADs as innovative diagnostic tools aims to combine microfluidic principles with paper substrates to offer simple, portable, and cost-effective solutions. The µPADs do not require external pumps or power sources, and the colorimetric reactions performed directly on paper enable immediate visual detection. For accurate quantification of the biochemical parameters, the image of the coloured product can be scanned, and the colour intensity converted into absorbance units to attain precise quantification. These measurements then correlate with the concentration of the individual biomarkers, like glucose and urea, enabling real-time, quantitative analysis without complex equipment. These features make µPADs ideal for point-of-care applications as easy-to-use and disposable devices, especially when developed targeting biological samples of non-invasive collection, namely saliva. The use of saliva as a biological sample for diagnosis and monitorization of health conditions is more convenient as it reduces patient discomfort compared to traditional blood sampling and it is also easier to handle in field settings, making it highly valuable for low-resource environments.

Ackowlegments

F. T. S. M. Ferreira thanks FCT- Fundação para a Ciência e a Tecnologia for the grant SFRH/BD/144962/2019. This work was also supported by National Funds from FCT through project UIDB/50016/2020.

Keywords

Microfluidic

Enzymatic reactions

Urea and glucose determination

Saliva samples

P1.254 Electrocatalytic Nanobiosensor for Simultaneous Measurement of Glucose and Ethanol in Sweat Using Palladium Nanoclusters

Alejandro Rodríguez-Penedo, Estefanía Costa-Rama, Beatriz Fernández, Rosario Pereiro, M. Teresa Fernández-Abedul

University of Oviedo Department of Physical and Analytical Chemistry, Spain

Abstract

The design of analytical devices that enable decentralized diagnostics has gained enormous importance in analytical and clinical chemistry. A crucial aspect of performing these analyses effectively is obtaining information from samples that are easy to extract, with minimally- invasive procedures. In this context, both glucose [1] and ethanol [2] could be effective biomarkers, being especially interesting for minimally invasive samples.

Palladium nanoclusters (PdNCs), composed of a few hundred of palladium atoms, have demonstrated excellent catalytic activity over the oxygen reduction reaction (ORR). Additionally, when coated with dihydrolipoic acid, these are stable, exhibit low polydispersity, and can be conjugated with biomolecules [3], such as glucose oxidase (GOx) and alcohol oxidase (AOx). Since the voltametric signal obtained for ORR is significantly influenced by the oxygen concentration, the determination of substrates of oxidases could be made following its decrease. Then, electrocatalysis by PdNCs can be used to measure the decrease in dissolved oxygen near the electrode, which is proportional to the amount of ethanol and glucose in the solution.

Therefore, this study focuses on the development and validation of a bianalyte biosensor platform that uses, apart from biocatalysts (GOx and AOx), PdNCs as electrocatalylsts. Both are deposited on screen-printed carbon electrodes for the simultaneous determination of glucose and ethanol in sweat. Furthermore, the integration of this biosensor into wearable devices could allow rapid and cheap measurements on a minimally invasive sample such as sweat.

Acknowledgements: This work was supported by the projects PID2022-137319OB-C21 and PID2023-148375OB-I00 (MCIU/AEI/10.13039/501100011033/ FEDER, EU), and IDE/2024/000694 (Government of the Principality of Asturias, FEDER, EU through SEKUENS). A.R.P. was supported by the “Severo Ochoa” grant (BP21-029)

References:

[1] O. Amor-Gutiérrez et al., Biosens. Bioelectron. 2019, 135, 64-70.

[2] E. Costa-Rama et al., Anal. Chim. Acta 2012, 728, 69-76.

[3] A. Rodríguez-Penedo et al., Microchim. Acta 2023, 190, 493.

Keywords

Palladium nanoclusters

Enzymes

Electrocatalysis

Sweat

P1.255 Peptide-Functionalized Nanoprobes for Rapid and Affordable Colorimetric Serodiagnosis of Typhoidal Salmonellosis

Jai Kumar Saini, Vijayender Bhalla

CSIR-Institute of Microbial Technology (CSIR-IMTECH), India

Abstract

Typhoidal salmonellosis, a systemic febrile infection caused by Salmonella enterica serovar Typhi, remains a significant public health concern in endemic regions, associated with severe morbidity and high mortality rates. The current gold standard for diagnosis, blood culture, is labor-intensive and time-consuming, while the widely used Widal test often yields false-positive results, limiting its reliability.

The present study introduces an innovative, rapid, and low-cost colorimetric assay designed for the serodiagnosis of typhoidal Salmonellosis based on dual (antigen+ urease inhibitor) coated probes. The presence of specific antibodies in the patient sera creates a shielding layer that render the enzyme tracer free in solution and consequently its activity gives a red color endpoint/alarm indicating infection. For antigen coating we selected flagellin protein as a biomarker hit after FliC emerged as a key target in our immunoproteomics LC-MS data, enabling a focused approach for detection. Using in silico studies, we have identified flagellin peptides from Salmonella Typhi that can be used to interact with antibodies of sera-positive patients. Silver nanoparticles bio-conjugated with these peptides showed a unique color shift response after antibody binding leading to a visually observable pink color change within 15 minutes, signifying a positive result.

The proposed assay is highly user-friendly, requiring minimal resources, no complex instrumentation, and minimal technical expertise. Its affordability and portability make it an ideal point-of-care diagnostic tool for low-resource settings, addressing a critical need in endemic regions. This innovative biosensing platform offers a promising alternative to existing methods, enabling rapid, accurate, and accessible detection of typhoidal salmonellosis.

Keywords

Point-of-Care

Silver-Urease Interaction

Serodiagnosis

Nanoprobes

P1.256 An ultrasensitive, portable colorimetric biosensing array using functionalized nanozyme based on metal-organic framework for simultaneous detection of multiple pathogenic bacteria in foods

Lizhou Xu^1,2, Yuhao Wen², Xinghao Hao^1,2, Danyang Li³

¹Zhejiang University, China. ²ZJU-Hangzhou Global Scientific and Technological Innovation Center, China. ³Sun Yat-Sen University, China

Abstract

There is a great need for rapid, simple, and in-field detection of multiple pathogenic bacteria for various applications. In this work, we developed an ultrasensitive and convenient colorimetric immunosensor based on nanozyme compatible with portable optical analysis for rapid and sensitive detection of multiple pathogenic bacteria.

Firstly, a nanocomposite {PDA@(ZIF-8@Pt)³} was synthesized based on nanozyme (platinum nanoparticles) and sequentially coated with multiple layers of metal-organic frameworks (ZIF-8). The nanocomposite conjugated with specific antibodies was used for detecting target bacterial cells, which were initially captured on the sensing array via primary antibodies. Absorbance intensities were measured after the addition of the substrate solution. A portable fluorescent imaging system was fabricated to capture images within a dark chamber, and the average grayscale value was determined using a smartphone equipped with a color analysis application to quantify the concentration of target. Additionally, leveraging the catalytic properties of Pt nanoparticles with different substrates, a multicolor sensor array was developed for the detection of multiple pathogenic bacteria.

The synthesized nanocomposite was found with high antibody loading efficiency and catalytic efficiency. This method exhibits a favorable linear relationship within the range of 10−10⁶ CFU/mL, with a detection limit as low as 1.67 CFU/mL. When applied to the detection of milk and beef samples, the method demonstrated satisfactory recovery rates (97.3%−104.3%) and accuracy (RSD < 6.11%). Furthermore, the PDA@(ZIF-8@Pt)³ exhibited excellent selectivity and stability.

A colorimetric biosensor was developed based on PDA@(ZIF-8@Pt)³-based immunoassay compatible with portable optical analysis for rapid and sensitive detection of multiple pathogenic bacteria including Salmonella Typhimurium, E. coli, V. parahaemolyticus, S. aureus. The proposed biosensing array is highly potential for fast screening and visual identification of infectious diseases across a broader range of fields.

Uncaptioned visual

Scheme 1.

Uncaptioned visual

Figure 1. Detection of four bacteria simultaneously.

Keywords

Nanozyme

Metal-organic framework

Naked-eye detection

Sensor array

P1.257 High-entropy prussian blue analogue-based glucose detection under physiological pH

Huan-Yu Liang, Wei-Li Shih, Lin-Chi Chen

National Taiwan University Department of Biomechatronics Engineering, Taiwan

Abstract

With rising diabetes rates, glucose monitoring is critical for metabolic health. Electrochemical sensors offer a promising solution for real-time, minimally invasive tracking and supporting personalized healthcare. Prussian Blue (PB) is a widely used electrocatalyst in redox reactions involving hydrogen peroxide, which is essential in glucose sensing via glucose oxidase (GOx) activity. However, PB deteriorates in neutral and alkaline pH environments due to the decomposition of Fe–(CN) bonds, making glucose sensing at neutral pH unachievable. This study aims to enhance the stability of glucose sensors in physiological conditions (pH 7.4) using a high-entropy Prussian blue analogue (HEPBA). High-entropy materials represent an emerging concept that employs the "cocktail effect," combining multiple elements in equal proportions. This creates a highly disordered structure, where complex interactions enhance stability and resilience across thermal, mechanical, and chemical environments. Our experiments show that HEPBA-modified electrodes maintain 87% of their initial current density after 20 cycles at pH 7.4, while traditional PB retains only 26% after five cycles, demonstrating superior stability. Glucose sensing was conducted in 0.1 M NaClO₄ and 5 mM phosphate buffer (pH 7.4) with GOx/HEPBA-modified electrode, fabricated by directly drop-casting HEPBA sub-micron particle ink and glucose solution onto GCE. GOx/HEPBA/GCE shows a sensitivity of 5.5 μA/mM·cm² across a linear range of 0.5–5 mM and 92% current retention after 5 cycles. In conclusion, these findings highlight the enhanced stability through a high-entropy strategy and the potential of HEPBA-modified electrodes for reliable biosensing in physiological conditions. Future research can further explore the applicability of high-entropy materials across diverse biosensing platforms, potentially transforming healthcare diagnostics and management through highly stable, efficient, and long-lasting sensors.

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Keywords

Glucose sensing

High-entropy material

Electrochemical sensor

Metabolic health

P1.258 Electrochemical enzyme-based biosensor with a microfluidic multichannel cell

Amirmansoor Ashrafi, Tong Liu, Karel Chadt, Jiří Homola

Institute of Photonics and Electronics Czech Academy of Sciences, Czech Republic

Abstract

National Institute of Research and Development for Biological Sciences, Romania

Abstract

The dopamine is a relevant biomarker usually determined electrochemically, but for a thorough bioclinical status investigation the dopamine 4-sulfate should be simultaneously analysed. Dopamine 4-sulfate is a major metabolite from both exogenous and endogenous dopamine found in plasma, urine and other biological samples. Unlike dopamine, dopamine 4-sulfate is not electrochemically active and it is detected after its hydrolysis onto an arylsulfatase biosensor. The simultaneous detection of both dopamine and dopamine 4-sulfate is achieved with a bielectrode system one for free dopamine electrochemical detection and the second for both free dopamine and dopamine 4-sulfate quantification after hydrolysis by immobilized arylsulfatase. For optimum analytical perfomances, i.e. low interferences and good limit of detection, the electrodes were modified with carbon-based nanoparticles, organic monolayer containing acid moieties (carboxylic and sulphate) using diazonium electrodeposition in presence of radical quenchers to avoid surface passivation. Due to the complex nature of the biological samples, extensive studies of antifouling layers and reactivation of the surface were carried out. It was observed that several step potentials in both reduction and oxidation range between the measurements allowed a surface regeneration of the (bio)electrodes ant achieve a good operational stability.

Acknowledgements: This work was performed with the support of MRID through the Core Program within the National Research, Development and Innovation Plan 2022–2027 project no. 23020101(SIA-PRO), contract no 7N/2022 and project PNRR-III-C9-2022 – I5 cod 18/16.11.2022 (RESPONSE).

Keywords

dopamine sulfate

dopamine

arylsulfatase

functionalised electrode

P1.262 Acetylcholinesterase and butyrylcholinesterase bienzymatic biosensors for electrochemical screening of neurotoxic compounds

Madalina-Petruta Bucur¹, Maria-Cristina Bucur¹, Bogdan Bucur¹, Adela Teban-Man^2,3, Maria Daniela Nicoară^2,3, Horia Leonard Banciu³, Gabriel Lucian Radu¹

¹National Institute of Research and Development for Biological Sciences, Romania. ²Institute of Biological Research Cluj, Romania. ³Babeș-Bolyai University, Romania

Abstract

Neurotoxins of natural (cyanotoxins) or anthropic (insecticides) origins have dramatic impact on human and environmental health. Fast environmental screening with cholinesterases-based biosensors is a useful approach to monitor and prevent potential harmful effects on resident fauna and/or humans. Monoenzymatic biosensors usually based on acetylcholinesterase or to a lesser extent on butyrylcholinesterase fail to provide information over entire spectrum of the inhibitors since there are compounds that specifically inhibit one of the two enzymes, e.g. methomyl inhibits only acetylcholinesterase while pirimicarb inhibits the butyrylcholinesterase at much lower concentrations than the acetylcholinesterase. A bienzymatic biosensor with both acetylcholinesterase and butyrylcholinesterase co-immobilized on a single electrode has the advantage that each neurotoxic compound is detected by the enzyme with the highest affinity in just one measurement. Here, we aim to test the detection limits of organophosphorus/carbamate insecticides (pirimicarb methomyl) and cyanobacterial guanitoxin by using monoenzymatic and bienzymatic biosensors. We found that the detection limits for pirimicarb were 50 ng/mL with the bienzymatic biosensor and 400 ng/mL with the acetylcholinesterase biosensor. Similarly, the detection limits for methomyl were 6 ng/mL with the bienzymatic biosensor and 700 ng/mL with the butyrylcholinesterase biosensor. Furthermore, out of 24 cyanobacterial strains that were screened for guanitoxin-like cholinesterase inhibition, only one strain related to Aphanizomenon sp. demonstrated the potential for significant inhibition of both acetylcholinesterase and butyrylcholinesterase. Noteworthy, the fresh cell lysates led to a higher enzymatic inhibition compared to lyophilized biomass thus underscoring the importance of sample preparation in environmental screening.

Acknowledgements: This work was performed with the support of MRID through the Core Program within the National Research, Development and Innovation Plan 2022–2027 project no. 23020101(SIA-PRO), contract no 7N/2022 and project PNRR-III-C9-2022 – I5 cod 18/16.11.2022 (RESPONSE).

Keywords

acetylcholinesterase

butyrylcholinesterase

cyanotoxins

insecticides

P1.263 Enzymatic microbiosensors for the local characterization of electrode materials used in energy conversion

Felipe Conzuelo

NOVA University Lisbon Institute of Chemical and Biological Technology António Xavier, Portugal

Abstract

The possibility of performing electrochemical characterizations at the microscale allows direct local analysis of chemical reactions with higher spatial and temporal resolution. Therefore, the use of miniaturized electrochemical probes offers important advantages for analytical purposes. Since the probe has a substantially smaller size compared to the investigated sample, the analysis is carried out with a rather low substrate consumption, which ensures minimal invasiveness and avoids possible interferences during a measurement. In addition, the small size of the sensor helps to increase mass transport rates and allows the measurement of catalytic reactions in operando conditions. On the other hand, and as is well known in electrochemical analysis, enzyme-modified electrodes provide relevant advantages, as they offer highly sensitive and selective conversions. Such enzyme-modified electrodes can also be applied to local analysis. It is in this context that we present the development of enzyme-modified microelectrodes, for the research and characterization of electrode materials for energy conversion. Specifically developed microbiosensors allow the local analysis and detection of reaction products during the operation of investigated materials. Examples will be shown using different microbiosensors for the characterization of electrodes applied for light energy conversion and the fabrication of biofuel cells. The presented strategy opens new possibilities for the optimization of the investigated materials by a deeper analysis at the micrometer scale.

Acknowledgements:

This work was supported by FCT - Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020). F.C. acknowledges FCT for the researcher contract [2022.05842.CEECIND/CP1725/CT0001] under the Scientific Employment Stimulus – Individual Call 2022.

Keywords

Microelectrodes

Enzymes

Redox reactions

SECM

P1.264 Cytochromes p450 as bio-tool for a multitargeted screening of chemicals in food matrices – A case study for the analysis of pesticides in mushroom

Hydrogel microparticles

Antibiotics

P1.267 Investigation of ACE2 coupled with antibody Fc Fragment as a biorecognition element for the detection of SARS-CoV-2 spike protein omicron variant

Silvija Juciute¹, Asta Luciunaite², Vincentas Maciulis³, Ieva Plikusiene¹

¹Vilnius University, Lithuania. ²Vilnius University Life Sciences Center, Lithuania. ³State Research Institute Center for Physical Sciences and Technology, Lithuania

Abstract

Sensitive and fast detection of target analyte is essential for the development of biosensing devices. Rapid tests for identification of SARS-CoV-2 virus mainly rely on specific monoclonal antibodies against this virus’ proteins. Due to mutations of SARS-CoV-2, affinity of antibodies to virus becomes lower. To overcome evasion caused by mutations, we investigated ACE2 coupled with antibody Fc fragment as a biorecognition element for the detection of SARS-CoV-2 spike protein Omicron (SCoV2-oS) variant.

In our investigation, we performed ACE2 receptor coupled with antibody Fc fragment immobilization on the gold-covered sensing surface. We immobilized ACE2 via self-assembling monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) or via protein G through antibody Fc fragment attached to ACE2. Utilization of 11-MUA SAM resulted in random orientation of ACE2, while protein G gave site-directed immobilization. In addition, affinity interactions between ACE2 and SARS-CoV-2 spike protein’s Omicron variant were investigated.

In our study, we applied spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation (QCM-D) methods combination. Both of these techniques allow us to measure biomolecules interactions in real time. Moreover, SE and QCM-D do not require any specific preparation of sample and proteins do not need any label to be detected. Combination of SE and QCM-D can provide profound information about optical and mechanical properties of protein layers from single measurement.

From obtained data, we calculated dry and wet surface mass densities, hydration for randomly and site-direct immobilized ACE2 and SCoV2-oS layers. In addition, we applied two step binding model for kinetic parameters computations. We calculated association and dissociation rate and affinity constants for ACE2 and SCoV2-oS interactions.

This research was funded by a grant (No. S-MIP-23-82) from the Research Council of Lithuania.

Keywords

References

[1] A. Popov, et al., Sensors 2021, 21(3), 948.

[2] S. Adomaviciute-Grabusove, et al., Chemosensors 2021, 9, 223.

Keywords

Biosensor

MXenes

Glucose

P1.271 Reagentless electrochemical glucose biosensor based on dendritic gold nanostructures

Almira Ramanaviciene, Katazyna Blazevic, Anton Popov, Asta Kausaite-Minkstimiene

NanoTechnas – Center of Nanotechnology and Materials Science, Faculty of Chemistry and Geosciences, Vilnius University, Lithuania

Abstract

Electrochemical enzyme-based biosensors have applications across diverse sectors, including the food industry, agriculture, veterinary medicine, and clinical diagnostics. Nanostructure-based electrochemical biosensors present innovative solutions to complex bioanalytical challenges and significantly impact glucose biosensors' sensitivity, limit of detection, selectivity, stability, and duration of analysis [1-3].

In this research branched, dendritic gold nanostructures (DGNs) were synthesized on the graphite rod electrode surface (DGNs/GR) by one-step procedure, in the absence of any template, surfactant and stabilizer and modified by redox mediator – tetrathiofulvalene (TTF) and glucose oxidase (GOx) to development of reagentless amperometric glucose biosensor. Optimal conditions for DGN synthesis were identified, and the electrode surfaces were characterized through electrochemical techniques and scanning electron microscopy. GOx was adsorbed on the modified electrode surface and cross-linked (GOx/TTF/DGNs/GR). The main advantage of this second generation amperometric glucose biosensors is that all required reagents are immobilized on the electrode surface. The biosensor performance was investigated in the buffer and blood serum samples. Finally, the developed biosensor was tested for non-invasive glucose monitoring in model systems.

Acknowledgement:

This research has received funding from the Research Council of Lithuania (LMTLT), agreement No. S-MIP-24-7.

References:

1. M-J. Lee, J-H. Choi, J-H. Shin, J. Yun, T. Kim, Y-J. Kim, B-K. Oh. Gold nanoclusters with two sets of embedded enzyme nanoparticles for applications as electrochemical sensors for glucose. ACS Appl. Nano Mater. 2023, 6, 13, 12567–12577.

2. L. Sakalauskiene, B. Brasiunas, A. Popov, A. Kausaite-Minkstimiene, A. Ramanaviciene. The development of reagentless amperometric glucose biosensor based on gold nanostructures, Prussian blue and glucose oxidase. Biosensors 2023, 13, 942.

3. N. German, A. Popov, A. Ramanavicius, A. Ramanaviciene. Development and practical application of glucose biosensor based on dendritic gold nanostructures modified by conducting polymers. Biosensors 2022, 12, 641.

Keywords

glucose oxidase

glucose biosensor

gold nanostructures

electrochemical methods

P1.272 Large-scale fabrication of silk-based enzymatic biosensors through microelectronic technologies.

Pablo Rodríguez-Núñez¹, Carla Blanes¹, Silvia Mena¹, Sebastian Gavira¹, S.D. Aznar-Cervantes², Carlos Dominguez¹, Gonzalo Guirado³, Sara Santiago⁴, Xavier Muñoz Berbel¹

¹Microelectronics Institute of Barcelona, Spain. ²Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, Spain. ³Autonomous University of Barcelona, Spain. ⁴Complutense University of Madrid Department of Analytical Chemistry, Spain

Abstract

Uncaptioned visual

Silk fibroin, a protein derived from the silk of Bombyx mori silkworms, is gaining attention as a sustainable and biocompatible biomaterial, especially for enzyme-based biosensors. Previous studies have demonstrated the compatibility of silk fibroin with microelectronic technologies and the long-term stability of enzymes within the silk matrix, lasting over 8 months. In this study, we explore the potential of producing enzymatic biosensors through photolithographic techniques employed in the semiconductor industry.

A novel silk-crystallization protocol base on the use of green chemical compounds is here optimized, enabling silk reorganization and crystallization in under 5 hours and without the need for water annealing or the use of organic solvents. This new protocol simplifies silk crystallization to a single step, reduces crystallization time compared to the conventional 48-hour water annealing process, and does not compromise enzyme activity (>90%). By contrast, in methanol or ethanol, over 65% of enzymatic activity is lost in less than 15 minutes of incubation.

Crystalline domains (beta-sheets) in the silk matrix highly enhance the stability of enzymes such as glucose oxidase, even under harsh conditions. Enzymes encapsulated within these crystalline structures retain their activity after exposure to organic solvents such as methanol, which typically inactivate most enzymes (as confirmed in the case of nitrocellulose matrices).

Utilizing spin-coating, we produced thin silk fibroin films (50-80 nm) on silicon wafers, ideal for microelectronic integration. By employing precision electron beam lithography (EBL) for nanoscale patterning, we maintain the structural integrity and biosensing capabilities of the fibroin films, supporting scalable biosensor production.

Our biosensor fabrication marks a significant leap toward eco-friendly cleanroom processes that minimized environmental impact. By utilizing biodegradable silk fibroin and a solvent-free approach, we align with green manufacturing principles. This innovative strategy facilitates the integration of biosensors into microelectronic platforms for diverse applications, including diagnostics, environmental monitoring, and industrial processes.

Keywords

Silk fibroin

Enzyme-based biosensors

Microelectronic

Crystallization

P1.273 Development of an electrochemical label-free biosensor to assess the peroxidase activity of hemoglobin

Alfonso Sequeda-Juárez¹, Diego Ortega-Picazo¹, Flor del Carmen Cortés-Ortegón¹, Ana M. Espinosa-García², José A. García-García², Sánchez-Pérez Celia¹

¹National Autonomous University of Mexico, Mexico. ²General Hospital of Mexico Dr Eduardo Liceaga, Mexico

Abstract

Hemoglobin (Hb) is a biologically significant protein that transports oxygen from the lungs to tissues via the bloodstream. In addition to its oxygen transport function, Hb can undergo redox reactions, generating radicals such as O₂⁻ and, indirectly, hydrogen peroxide (H₂O₂). This peroxidase activity of Hb plays a key role in degrading toxic substances that produce free radicals, oxidative stress, cellular damage, and is also associated with the development of inflammatory diseases and cancer.

This study reports the analysis of the catalytic activity of Hb using cyclic voltammetry (CV) and chronoamperometry (CA) with H₂O₂ as the substrate. A platinum microarray electrode was functionalized with gold nanoparticles (20 nm in diameter) was used as a sensor of methemoglobin solution prepared at concentrations of 250 ng/mL (3.88 nM) in PBS at a pH 7.4 exposed to H₂O₂. As a negative control, a methemoglobin solution was exposed to 15 mM sodium azide (NaN₃) for 10 minutes to inhibit its peroxidase activity. Following this, hydrogen peroxide (25 and 50 ng/ml) was added to assess its electrochemical activity through CV and CA.

We present cyclic voltammograms and chronoamperometry plots recorded upon exposing hemoglobin to H₂O₂, revealing changes in the electrical response due to variations in its Hb peroxidase redox activity and a significant change in electric current over time under different conditions. These findings highlight the potential of this study for assessing hemoglobin activity in response to environmental pollutants, tobacco smoke exposure, and other radical-generating substances linked to adverse health effects and disease progression.

Keywords

Hemoglobin

Peroxidase

Electrochemistry

Biosensor

P1.274 Electrochemical biosensor based on replaceable immobilized enzyme reactor and silver amalgam screen-printed electrode for determination of pyruvate in flow injection analysis

Sofiia Tvorynska, Bohdan Josypcuk

J. Heyrovsky Institute of Physical Chemistry, Czech Republic

Abstract

The sensitive, rapid, and cost-effective determination of pyruvate is of growing interest in clinical analysis as well as food industry. Diabetes, liver cirrhosis, cardiovascular diseases, and severe brain abnormalities are several disorders caused by pyruvate deficiency in the human body. It has also been shown to increase pyruvate concentrations in blood serum and saliva by 2.0 to 2.8 times in patients with oral cancer. In addition, elevated pyruvate levels are known to play a prominent role in autism. In the food industry, pyruvate serves as an indicator of bacterial contamination of dairy products, and is also monitored during wine production.

In order to determine pyruvate, a flow injection analysis (FIA) system has been developed by coupling an immobilized enzyme reactor (IMER) connected upstream to a silver amalgam screen-printed electrode acting as a transducer. The preparation of the high-performance IMER included the evaluation of two enzymatic systems: (i) pyruvate oxidase and (ii) lactate dehydrogenase and lactate oxidase. In addition, pyruvate oxidase was subjected to further experiments to compare two different immobilization protocols: (i) covalent enzyme attachment via glutaraldehyde on the previously prepared aminated mesoporous silica powder aminopropyl-SBA-15 and (ii) physical adsorption on bare mesoporous carbon powder Starbon^®300. Both protocols provide a relatively high load of pyruvate oxidase, which was found by determining the amount of immobilized enzyme by the Bradford method. The principle of pyruvate determination, based on amperometric monitoring of oxygen consumption via its four-electron reduction at a negative detection potential using the silver amalgam screen-printed electrode (−0.85 V vs. a pseudo-reference Ag electrode), avoids interference from commonly interfering oxidising compounds. The newly developed FIA biosensor platform has been successfully used for the quantitative determination of pyruvate in human biological fluids such as urine and saliva, as well as in food stuffs such as wine and dairy products.

Keywords

enzymatic biosensor

flow injection analysis

amperometric detection

pyruvate determination

P1.275 Advancing glucose biosensors: Role of gold nanorods morphology for electrochemical performance

Aleksej Zarkov, Marina Sapauskiene, Anton Popov

Vilnius University, Lithuania

Abstract

In recent years, gold nanorods (AuNRs) have gained considerable attention due to their distinctive physical, optical, and chemical properties, which make them well-suited for various biomedical applications, including drug and gene delivery, photothermal and photodynamic therapies, and theranostic approaches [1]. In addition, their exceptional electrochemical properties position AuNRs as promising candidates for biosensor development, such as in enzymatic biosensors [2]. These advantageous properties are strongly influenced by the morphology of AuNRs, particularly the length-to-width ratio [3].

This study aimed to investigate the effect of AuNRs morphology on the analytical performance of electrochemical glucose biosensors. Three different types of AuNRs were synthesized and characterized through scanning electron microscopy (SEM), UV-VIS spectroscopy, and electrochemical methods. These AuNRs were subsequently employed in the fabrication of electrochemical glucose biosensors, whose analytical performance was evaluated and compared.

References

[1] X. Hao, et al., Chem. Commun., 2024, 60, 469-481

[2] Y. Xie, et al., JEAC, 2021, 893, 115328

[3] I. Ahmad, Colloids Surf. A: Physicochem. Eng. Asp., 2024, 703, 135410

P1.276 Development of an on-site organophosphorus pesticide detection system based on gold nanoparticle aggregation-LFA platform

A-in Seo, Na Hun Kim, Kyeong Ho Lee, Hye Jin Lim, Hyo Young Mun

Apteasy MJ Inc., Republic of Korea

Abstract

Uncaptioned visual

In this study, we developed an organophosphorus pesticide detection system that is economical and easy to use by non-experts using the gold nanoparticle aggregation-based lateral flow assay (LFA) in the field. Organophosphorus pesticides primarily used in insecticides, herbicides, and chemical weapons, disrupt neurotransmitters, leading to various toxicities such as respiratory distress, headache, and death. This emphasizes the critical need for rapid detection in field. Current detection methods, such as HPLC and GC-MS, are restricted to trained personnel and laboratories, limiting on-site handling of organophosphorus pesticides. To address this, our research team developed a method based on the gold nanoparticle aggregation-based LFA, enabling detection within 3 minutes. This method utilizes acetylcholinesterase (AChE) which breakdown neurotransmitters and a substrate, acetylthiocholine (ATC), where the presence of organophosphorus pesticides inhibits AChE, preventing ATC breakdown and gold nanoparticle aggregation. Conversely, in the absence of pesticides, AChE activity is maintained, leading to ATC breakdown into thiocholine (TC) and acetic acid. Through the aggregation of gold nanoparticles induced by the presence of TC, which is generated during this process, the development of the system for distinction through the LFA has been completed. As a result, it had an accuracy of 94%, a correlation of 98% in a comparative evaluation with HPLC, and detection of up to 1ppb, which is lower than the EPA standard daily intake amount. Furthermore, when evaluated based on over 100 types of pesticides available on the market, detection was confirmed for 73 types pesticides. Through this study, we have developed a rapid and user-friendly system for on-site detection of organophosphorus pesticides, which can be applied to ensure food safety, prevent pesticide poisoning among agricultural workers, and manage risks related to chemical weapons.

Keywords

pesticide detection

LFA

P1.277 Enzymatic urea flexible biosensor based on CuS/Bioglass 45S5.TiO₂ Nps thin films with digital output

Manuel Alejandro Chairez Ortega¹, Rafael Gonzalez-Landaeta¹, Marcos Pita², Antonio L. De Lacey², Adriana A. Siller-Ceniceros³, Amanda Carrillo Castillo¹

¹Institute of Engineering and Technology, Autonomous University of Ciudad Juarez. Cd. Juarez, Chihuahua. ZC: 32310, Mexico. ²Instituto de Catálisis y Petroleoquímica, CSIC, c/Marie Curie 2, 28049 Madrid, Spain. ³Faculty of Chemical Sciences, Autonomous University of Nuevo León. San Nicolás de los Garza, Nuevo León. ZC: 66455, Mexico

Abstract

In this work we report the fabrication of an enzymatic urea biosensor based on CuS/Bioglass 45S5.TiO₂ nanoparticles (Nps) thin films deposited over PET ITO substrates. The materials were synthesized by soft chemistry methods, such as chemical bath for CuS and sol-gel for bioglass, and then deposited by spin coating. The urease was immobilized on the bioglass surface by crosslinking with glutaraldehyde. The chemical characterization by FTIR of the enzymatic detection process showed the vibrations of the characteristic groups of urea hydrolysis, where at 1145 and 1615 cm^-1 the CO₂ bonds were observed, and at 1589 and 1685 cm^-1 the ammonia ions bonds were observed. UV-Vis characterization showed hypochromic behavior in thin films, attributed to the interaction of CuS with ammonium ions and CO₂. The interaction of the thin film materials and the reaction products of the urease activity led to an increase of electrical resistance of the device. A sensitivity of 10.523 kΩ/mM (urea) was measured directly with a multimeter and observed in impedance spectroscopy by the increment of impedance mid circles in Cole-Cole plots. The biosensor was connected to a Wheatstone bridge, obtaining a voltage output proportional to resistance change with a sensitivity of 2.2 mV/mmol/l, obtaining a decrease in the voltage after the connection to an LT1167 amplifier. Finally, the amplifier output was digitalized with an Arduino esp32, indicating the urea concentration in an LCD display.

Keywords

urea

enzymatic biosensor

flexible electronics

digital output

P1.278 The Ultrafast Ligand (S)-To-Metal (Cu II) Charge Transfer in Laccase Copper Site T1: Correlation Between Redox Potential and Timing

Antonella Cartoni^1,2, Alessandra Paladini², Daniele Catone³, Patrick O'Keeffe², Patrizia Gentili¹, Federica Palmeri^1,2, Mattea Carmen Castrovilli²

¹Department of Chemistry, Sapienza University, P. le Aldo Moro 5, 00185 Rome, Italy. ²Istituto di Struttura della Materia-CNR, (ISM-CNR), Area della Ricerca di Roma 1, 00015, Monterotondo, Italy. ³Istituto di Struttura della Materia—CNR (ISM-CNR), Area di Ricerca di Tor Vergata via del Fosso del Cavaliere, 100, Rome, Italy

Abstract

Laccases are blue multicopper oxidases that catalyze the reduction of oxygen to water while oxidizing various substrates, included several pollutants. They are used in applications such as biofuel cells and biosensors [1-5]. Laccases contain four cupric ions at three distinct sites: type 1 (T1), type 2 (T2) and binuclear type 3 (T3). Electrons from the substrate transfer to the T1 site and then to the T2/T3 trinuclear copper cluster (TNC), where oxygen is reduced to water. Laccases are classified based on the redox potential at the T1 site, ranging from 0.4–0.8 V vs. NHE. The T1 site has a unique trigonal geometry coordinated by one cysteine and two histidines residues. In high and medium redox potential laccases, the T1 site has also an axial residue, Phe or Leu, whereas in low redox potential laccases, it has a trigonal elongated tetrahedral structure with a coordinating Met. The T1 site also exhibits strong absorption at ~600 nm due to charge transfer from the cysteine sulphur ligand to T1. In recent decades, significant efforts have been devoted to uncovering the factors behind the notable variations in ET1 among laccases [6]. This work is focused on the relaxation dynamics of excitation at 600 nm studied with pump-probe in a fs time scale of two laccases with different redox potentials (Trametes villosa, 780 mV, and Myceliophthora thermophila, 470 mV) to find possible correlation between redox potentials and timing. The results seem indicate such a correlation discussed in the presentation.

References:

[1] M.C. Castrovilli et al. Biosens. Bioelectron. 2025, 267,116758

[2] M.C. Castrovilli et al. ACS Sustainable Chem. Eng. 2022, 10, 1888–1898

[3] M.C. Castrovilli et al. Biosens. Bioelectron. 2020, 163, 112299

[4] ESILARANTE 202283YHXY Project PRIN 2022

[5] Ateneo Project 2021 RP12117A5D2DEE6B

[6] I. Mateljak et al. ACS Catal. 2019, 9, 4561−4572

Keywords

laccase

biosensor

dynamics

redox potential

P1.279 Open circuit potential based continuous ketone monitoring using a quasi-direct electron transfer type engineered β-hydroxybutyrate dehydrogenase

Kurea Ikegai¹, Junko Okuda-Shimazaki¹, Mika Hatada², David Probst², Ryutaro Asano¹, Kazunori Ikebukuro¹, Koji Sode², Wakako Tsugawa¹

Abstract

We demonstrate an open circuit potential (OCP) principle based enzymatic sensor for continuous ketone monitoring, using a quasi-direct electron transfer type engineered β-hydroxybutyrate (BHB) dehydrogenase (BHBDh).

Diabetic ketoacidosis (DKA) is a serious and fatal condition of diabetes, and is caused by excess accumulation of ketone bodies, especially BHB, in the blood. While blood fingerstick test strips are mostly used for BHB detection, the continuously ketone monitoring (CKM) system will be enable the early detection of rising ketone level, thereby to prevent DKA. Considering the advantage of the OCP based sensing principle, where the signal is independent on the size of the electrode^[1,2], this study aims to develop OCP based enzymatic sensor for CKM by applying engineered quasi-direct electron transfer type BHBDh^[3], which will be suitable for minimally invasive in vivo monitoring.

Phenazine ethosulfate (PES) modified BHBDhs were prepared by incubating amine- or thiol-reactive PES with engineered BHBDhs. PES modified BHBDhs were immobilized on gold electrodes via a self-assembled monolayer to construct enzyme electrodes. In electrochemical measurement, OCP value is continuously measured in phosphate buffer or artificial interstitial fluid (ISF) containing NAD⁺.

The electrodes with engineered BHBDh modified with PES showed OCP change upon the addition of BHB in the sample solution. With the increase of BHB concentration, the changes in the OCP were observed within the range from 0.1mM – 4mM BHB, which covers the physiological concentration range for both normal and DKA condition. These results revealed that the construction of OCP based sensor using quasi-DET-type enzyme was constructed which is suitable for continuous ketone monitoring system.

Carina Vieira¹, Sara Cravo^2,3, Emilia Sousa^2,3, Marcela Segundo¹, Alberto Araújo¹

¹REQUIMTE LAQV Porto, Portugal. ²University of Porto Interdisciplinary Centre of Marine and Environmental Research, Portugal. ³Laboratory of Organic and Pharmaceutical Chemistry (LQOF), Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Portugal

Abstract

Cytochrome P450 (CYP450) enzymes, namely CYP3A4, metabolize 50–70% of pharmaceuticals in phase I drug metabolism, with their catalytic cycle relying on electron transfer from redox partners. Electrochemical techniques represent an alternative approach by enabling direct electron transfer between the enzyme and the electrode, bypassing the need for natural electron donors. Nevertheless, unlocking the full potential of CYP450 biosensors hinges on overcoming challenges related to enzyme orientation, immobilization, and substrate accessibility.

In this study, we introduce a novel CYP3A4-based biosensor designed to detect testosterone constructed on a carbon screen-printed electrode. The biosensor was improved using a tailored modification mixture comprising synthesized pyrene-linear poly(ethyleneimine), multi-walled carbon nanotubes, and CYP3A4 enzyme. This modification improved the electroactive surface area by 150% and produced striking structural changes in the polymer network, as revealed by SEM imaging, with the formation of interconnected spherical patterns.

Oxygen levels are a pivotal factor for enzymatic activity, while the sigmoidal current response to testosterone concentration (up to 15 μM), highlighted CYP3A4's homotropic positive cooperativity, characterized by a Hill coefficient of 2.4 ± 0.2, an I_max of 2.6 ± 0.1 μA, and a K_M of 7 ± 4 μmol L⁻¹.

This work underscores the critical role of innovative enzyme immobilization strategies in optimizing biosensor efficiency and sheds light on the interplay between enzyme activity, substrate levels, and oxygen availability. Our findings lay the groundwork for advanced CYP450 biosensors, propelling the field of drug metabolism monitoring and precision diagnostics.

Funding

This work received financial support from the PT national funds (FCT/MECI, Fundação para a Ciência e Tecnologia and Ministério da Educação, Ciência e Inovação) through the project UID/50006 - Laboratório Associado para a Química Verde - Tecnologias e Processos Limpos.

Acknowledgments

Carina S. P. Vieira thanks FCT and National Funds and European Social Fund for financial support, for her Ph.D. Grant Ref. 2022.11691.BD

Keywords

Metabolism

Cytochrome P450 3A4

Biosensor

Enzymatic Kinetics

P1.283 Application of SECM-Based Sensors using low-concentration GOx for surface modifications

Beatričė Kulikauskaitė¹, Antanas Zinovičius^2,3, Tomas Mockaitis², Inga Morkvėnaitė-Vilkončienė², Arūnas Ramanavičius^1,2

¹Vilnius University, Lithuania. ²State Research Institute Center for Physical Sciences and Technology, Lithuania. ³Vilnius Gediminas Technical University Faculty of Mechanics, Lithuania

Abstract

Analysis and diagnostic devices are essential in modern medicine, particularly with the rise of fast and ultra-sensitive electrochemical biosensors. However, these biosensors face persistent challenges, including signal amplification and reusability. While electrochemical impedance spectroscopy (EIS) has shown promise in biosensor applications, the direct immobilization of recognition elements onto electrodes often hinders the diffusion of reaction products, resulting in diminished signals and typically limiting these sensors to single use.

To address these limitations novel method scanning electrochemical impedance microscopy (SEIM) (combined EIS and scanning electrochemical microscopy) was used. This approach allows for non-destructive experiments and enables the immobilization of recognition elements onto desired surface. The sample can then be scanned with an ultramicroelectrode in close proximity to the sample surface. facilitating improved diffusion of reaction products towards the electrode. Additionally, ultramicroelectrode could be reused and only inexpensive surface with recognition element has to be replaced.

In this study 100 ng/mL of glucose oxidase enzyme was immobilized on a dielectric surface, and SEIM was utilized in redox competition mode. Experimental data demonstrated that charge transfer resistance is the most effective parameter for evaluating glucose oxidase-catalysed reactions at varying glucose concentrations.

In conclusion, the SEIM method represents a promising approach for in situ evaluation of glucose biosensors.

Keywords

SECM

EIS

GOx

low concentration

P1.284 New approaches in biosensor technology for assessing the vitality of trees after forest fires by analyzing the sugar metabolism in the cambium

Raffaella Margherita Zampieri^1,2, Eleftherios Touloupakis¹, Isabela Calegari Moia¹, Alessio Giovannelli^1,3, Claudia Cocozza⁴

¹Research Institute on Terrestrial Ecosystems National Research Council Florence Branch, Italy. ²University of Florence Department of Agricultural Food Environment and Forestry Sciences and Technologies, Italy. ³National Biodiversity Future Centre, Italy. ⁴University of Florence Department of Agricultural Food Environment and Forestry, Italy

Abstract

In Mediterranean countries, forest fires are a recurrent event that must be considered as a key component of regional and global forest management strategies and biodiversity restoration programs. The development of tools to quickly determine the fate of damaged trees after a stressful event such as wildfire is of great importance. In this context, an innovative approach to assess irreversible physiological damage in trees could help to support the planning of management decisions for disturbed sites to restore biodiversity, protect the environment, and understand the adaptation of ecosystem functionality. The vitality of trees can be estimated by various physiological indicators, such as cambium activity and the amount of starch and soluble sugars, while the accumulation of ethanol in cambial cells and phloem is considered an alarm signal for cell death. However, their determination requires time-consuming laboratory protocols, making the approach impractical in the field. The aim of our project is to develop biosensors capable of determining the concentration of soluble sugars and ethanol in the stem tissue of injured trees as indicators of tree vitality to directly distinguish between damaged and recovering trees in the forest. To achieve this goal, we select sites where prescribed fires or recent forest fires have occurred as experimental setups.

Keywords

sugar

tree vitality

forest fire

P1.285 The Genotoxic Effect Of Morpholinum-Based Ionic Liquids On Pseudomonas aeruginosa Les B58: In Vitro And In Silico Mechanistic Study

Jakub Michalski, Oskar Szczepaniak, Dorota Narożna

Poznan University of Life Sciences, Poland

Abstract

Pseudomonas aeruginosa is a multi-drug resistant pathogen, which pose a serious health threat being a major cause of nosocomial infections. Therefore, there is an urgent need for the development of non-traditional agents limiting the viability of this bacterium. Ionic liquids may be such potent compounds. This work aimed to determine the bacteriostatic effect against P. aeruginosa by interaction with DNA and promoting damages to genetic material. We used the well-characterized P. aeruginosa LES B58) as model strain and morpholinium-based cations combined with herbicidal anions. We noticed a decrease in the bacterium viability along with the decline in the amount of DNA isolated from the treated cells. Electrochemical analysis with carbon paste electrode as a working electrode showed the presence of oxidation changes in genome of P. aeruginosa treated with 1024 mg/Ldose of [DecEtMor][2,4-D]. The potential interaction between the tested cations and the model DNA fragment was confirmed using Hartree-Fock method. Our results confirmed the previous findings that morpholinum-based ionic liquids may be applied to fight P. aeruginosa infections.

Keywords

Pseudomonas sp.

DNA oxidation

ionic liquids

P1.286 Electrochemical determination of three factor problem: SWV study of interaction between diadenosine polyphosphates (Ap₃A and Ap₄A), allicin and dsDNA.

Oskar Szczepaniak, Małgorzata Pietrowska-Borek

Poznan University of Life Sciences, Poland

Abstract

Thiopolysulfides (TPS) are a group of organic sulfur compounds and are a part of secondary metabolites produced by the plants of the Alliaceae family. A key representing compound of TPS is allicin, which has documented bacteriostatic properties by damaging bacterial DNA. However, in eukaryotic organisms it exhibits no genotoxic effects, except for cancer cells for which the IC₅₀ is significantly higher than that of non-cancer ones.

The cellular regulators of the cell cycle Ap₄A and Ap₃A play an essential role in the eukaryotic response to inflammation. As TPS also exhibit documented anti-inflammatory effects by inducing kinases and inhibiting cytokine production, the question arises whether they may interact with Ap₄A and Ap₃A regulators. Such interaction could result in synergistic stronger anti-microbial and anti-tumour effect or conversely decrease the DNA-intercalation and genotoxic effect in eucaryotic cells.

To determine whether cell cycle regulators may limit the degradation of nitrogen bases in DNA induced by the presence of allicin, we applied dsDNA-covered the carbon paste electrode (CPE) in three electrode set. After electrostatic deposition of dsDNA, the CPE was transferred to well containing different concentration of Ap₄A or Ap₃A aliquots for kinematic association with bonded DNA strands. Then we moved CPE to next well filled with different allicin concentrations, and finally we performed SWV measurement. Changes in signals of purine bases (guanine at potential E = 1.0V and adenine E = 1.2V) showed that the tested cell cycle regulators may affect allicin ability to induce DNA susceptibility to oxidative damage.

Funding source

The research has been funded with sources of the National Science Centre of Poland (2024/08/X/NZ3/00519). The attendance at the conference was co-funded with Young Researcher and Innovator Conference Grant, as a part of COST Action "SENESCENCE2030: Targeting Cell Senescence to Prevent Age-Related Diseases" (E-COST-GRANT-CA23119-8e194595).

Keywords

Ap3A

Ap4A

carbon paste electrodes

DNA oxidation

P1.287 Real-time, in vivo monitoring of plasma urea concentrations

Shaylee Larson, Chelsea Brown, Lisa Fetter, Tod Kippin, Kevin Plaxco

University of California Santa Barbara, USA

Abstract

The current gold-standard approach for assessing the efficacy of hemodialysis is the intermittent measurement of plasma urea concentrations in blood samples from which estimates of urea clearance are derived. A significant limitation of this approach, however, is that it fails to deliver immediate feedback that could be used to adjust and optimize treatment in real-time. By reducing the risk of under-treatment, minimizing the costs and inconvenience associated with over-treatment, and ultimately improving patient outcomes through a more responsive and precise approach to dialysis management, the clinical value of such personalized treatment could be substantial.

Motivated by its potential clinical impact, numerous researchers have pursued technologies for real-time measurement of plasma urea. The most widely explored of these involves electrochemical sensors that use an enzyme that converts urea into a more easily detectable product, producing a potentiometric signal monotonically related to urea concentration. To date, however, this class of urea sensors has been restricted solely to ex vivo applications, presumably because both pH and ammonia ion concentrations are tightly regulated in the body, rendering their levels dependent on factors other than just urea concentration.

Here we explore an alternative approach to the molecule’s real-time, in vivo measurement. Specifically, we describe the design of a urea-detecting electrochemical aptamer-based (EAB) sensor and its successful deployment in the veins of live rats. Signal generation in an EAB sensor offers distinct advantages for in vivo applications. Most notably, due to its biomimetic nature, this signal transduction mechanism is selective and specific enough to work in situ in the living body. Motivated by these advantages, we demonstrate the sensitivity and responsiveness of our sensor in vivo across healthy, uremic, and kidney-diseased conditions in living rats.

Keywords

Aptamers

Electrochemisty

in vivo

Kidney Disease

P1.288 A rational optimisation approach for the development of a multiplexed lateral flow immunoassay detecting ovarian germ cell tumour markers in serum

Aida Abdelwahed¹, Luca Panariello^2,3, Andre Shamsabadi^2,4, Carl E. Eskildsen^2,4, Edmund H. Wilkes⁵, Federico Galvanin⁶, Srdjan Saso^7,8, Molly M. Stevens^2,3,4

¹Department of Materials, Department of Bioengineering and Institute of Biomedical Engineering, Imperial College London, UK. ²Department of Materials, Department of Bioengineering and Institute of Biomedical Engineering, Imperial College London, London, UK. ³Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Sweden. ⁴Department of Physiology, Anatomy and Genetics, Department of Engineering Science, and Kavli Institute for Nanoscience Discovery, University of Oxford, UK. ⁵Department of Clinical Biochemistry, North West London Pathology, Imperial College Healthcare NHS Trust, Charing Cross Hospital, UK. ⁶Department of Chemical Engineering, University College London, UK. ⁷West London Gynaecological Cancer Centre, Hammersmith Hospital, Imperial College NHS Trust, UK. ⁸Department of Metabolism, Digestion and Reproduction, Imperial College London, UK

Abstract

We will discuss the gathering of the raw data, and the processing to remove interferences otherwise present in real world samples.

Keywords

Screen printed electrodes

Potentiostats

Electrochemical sensor

Commercialisation