Enzyme-Polymer stickers for assembling single and multi-analyte biosensors
Abstract
Electrochemical biosensors have achieved widespread commercial success due to their high selectivity, ease of use, and low cost.However, the fabrication of many such systems, especially when targeting multianalyte sensing, is often constrained by the preparation of the sensing film, which typically relies on drop-casting polymer-enzyme solutions followed by drying to form the active layer.Depending on the enzyme-polymer formulation, this step can be difficult to integrate into large-scale, roll-to-roll production processes and lacks flexibility for constructing multianalyte sensors.Here, we propose a new assembly concept in which the sensing film containing enzymes and redox polymers is pre-fabricated as sensor stickers that can be transferred in a single step either onto individual electrodes to generate singleanalyte sensors or combined on electrode arrays for multianalyte sensing.We demonstrate the sensor sticker concept using four oxidoreductases as biorecognition elements that catalyze the reaction of four different analytes (hydrogen, formate, nitrate and NADPH).Accurate sensing was achieved on both standard glassy carbon electrodes and on specifically designed microelectrode arrays modified with the sensor stickers.The stickers exhibited high selectivity with minimal interference, even in mixed samples containing all four analytes.This modular approach to assembling multianalyte sensors on microarrays is broadly applicable to other analytes and holds strong promise for flexible, customizable, and scalable fabrication of multianalyte biosensors.Enzyme-based electrochemical biosensors are widely used for quantitative analysis in the food industry and healthcare 1 .Their high affinity and specificity toward their natural substrates ensure excellent sensitivity and selectivity, even in complex media 2 , thereby eliminating the need for sample pretreatment, making them highly compatible with point-of-use sensing.Redox-active enzymes are often used as biorecognition elements, as they catalyze the oxidation or reduction of specific analytes, therefore generating or consuming electrons.Redox-active polymers (e.g.Os complex-or viologen-modified polymers) are commonly employed as a conducting matrix, within which redox-active enzymes are immobilized and deposited onto electrodes as thin films, and to mediate electron transfer to or from the electrode 3,4 , generating an electrical current that serves as the sensing signal.The production of several polymer-enzyme-based electrodes is well established, most notably the glucose biosensor, which is manufactured at a scale exceeding 10¹⁰ units annually.5 However, similar large-scale success has not yet been achieved for many other analytes, and even less so for multianalyte sensors.At the research and development stage, the most widely used method for sensor fabrication involves mixing the enzyme with the polymer to form an aqueous formulation, which is drop-cast onto the electrode surface.Upon drying, a hydrogel film containing the enzyme forms the sensing layer.This approach has enabled proof-of-concept demonstration of amperometric biosensors for a wide range of analytes, including lactose 6 , galactose 7 , fructose 8 , creatinine 9 , lactate 9 , nitrate 10 , hydrogen 11 , and phosphate 12 .This diversity in biosensing systems has additionally served as a basis for the development of multianalyte sensing platforms, such as wearable, multiplexed sensors for personalized medicine and fitness, integrating quantitative measurements of physiological parameters and biomarkers 13,14 .However, drop-casting methods are often incompatible with large-scale, continuous roll-to-roll manufacturing of such sensors which includes multiple steps, such as electrode fabrication, sensor layer deposition, capillary channel assembly, and so on, ideally performed in one continuous process.Achieving homogeneous, reproducible films by drop-casting, often requires controlled and lengthy drying conditions, which are difficult to integrate into fast, continuous assembly processes.Moreover, multianalyte sensors typically require multiple sensing layer depositions involving different enzyme polymer formulations, with different requirements for drying steps and thus necessitate multistep assembly processes 15 .Here, we introduce a "sensor sticker" approach in which electrodes are modified with a preformed hydrogel film (Fig. 1) to separate the dropcasting and drying steps of sensing-layer preparation from the main sensor assembly process, making it compatible with mass production.In this approach, the sensor stickers are formed by first drop-casting a liquid formulation of enzyme and polymer on a hydrophobic Teflon tape.After drying, transferable enzyme-polymer stickers are obtained, which are transferred directly onto electrodes using a controllable force gauge, forming functional sensing layers.This strategy provides a flexible approach for tailoring multianalyte sensors to different target analytes on demand.To demonstrate this concept, we fabricated several enzyme-polymer stickers using viologen-modified polymers as the immobilization matrix and four different enzymes as biorecognition elements: nitrate reductase (NaR), ferredoxin-NADP + reductase (FNR), formate dehydrogenase (FdH), and hydrogenase (DdH) for the sensing of nitrate, NADPH, formate, and hydrogen, respectively.Proof-of-concept for the sensing stickers was first realized by using standard glassy carbon electrodes (GCEs) for single analyte sensing.Ultimately, the four sensor stickers were assembled as multianalyte sensors on a single gold microelectrode array to evaluate their selectivity towards a mixed-substrate analyte.This sensor fabrication principle is expected to be universally applicable to other sensing elements, allowing for the production of customizable multianalyte sensors that can selectively profile targeted sample components.The self-adhesive property of the sensor stickers renders them potentially applicable to a variety of surfaces and simplifies the mass production of diverse sensing systems.
Authors 22
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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Fraunhofer Institute for Interfacial Engineering and Biotechnology
Affiliation as printed
Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Schulgasse 11a, Straubing, Bayern, Germany, 94315
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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Affiliation as printed
Center for Electrochemical Sciences (CES), Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Universitätsstr. 150, Bochum, North Rhine-Westphalia, Germany, D-44780
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Fraunhofer Institute for Interfacial Engineering and Biotechnology
Affiliation as printed
Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Schulgasse 11a, Straubing, Bayern, Germany, 94315
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Fraunhofer Institute for Interfacial Engineering and Biotechnology
Affiliation as printed
Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Schulgasse 11a, Straubing, Bayern, Germany, 94315
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Instituto de Tecnologia Química e Biológica António Xavier · Universidade Nova de Lisboa
Affiliation as printed
Instituto de Tecnologia Química e Biológica António Xavier (ITQB NOVA), Universidade NOVA de Lisboa, Av. da República, Oeiras, Portugal, 2780-157
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Instituto de Tecnologia Química e Biológica António Xavier · Universidade Nova de Lisboa
Affiliation as printed
Instituto de Tecnologia Química e Biológica António Xavier (ITQB NOVA), Universidade NOVA de Lisboa, Av. da República, Oeiras, Portugal, 2780-157
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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Affiliation as printed
Technologie Campus Parsberg-Lupburg, OTH Ostbayerische Technische Hochschule Regensburg, Am Campus 1, Parsberg, Germany, 92331
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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Consejo Superior de Investigaciones Científicas · Instituto de Catálisis y Petroleoquímica
Affiliation as printed
Department of Biocatalysis, ICP-CSIC, Institute of Catalysis, C/ Marie Curie 2, Cantoblanco, Madrid, Spain, 28049
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Consejo Superior de Investigaciones Científicas · Instituto de Catálisis y Petroleoquímica
Affiliation as printed
Department of Biocatalysis, ICP-CSIC, Institute of Catalysis, C/ Marie Curie 2, Cantoblanco, Madrid, Spain, 28049
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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Affiliation as printed
Catalysis and Surface Chemistry, Leiden University, P.O. Box 9502, Leiden, Netherlands, 2300 RA
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Technical University of Munich
Affiliation as printed
Electrobiotechnology, Technical University of Munich, Uferstraße 53, Straubing, Bayern, Germany, 94315
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