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Amperometric Biosensors Based on Direct Electron Transfer Enzymes

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Amperometric Biosensors Based on Direct Electron Transfer Enzymes molecules Review Amperometric Biosensors Based on Direct Electron Transfer Enzymes Franziska Schachinger †, Hucheng Chang † , Stefan Scheiblbrandner and Roland Ludwig * Biocatalysis and Biosensing Laboratory, Department of Food Sciences and Technology, BOKU–University of Natural Resources and Life Sciences, 1190 Vienna, Austria; [email protected] (F.S.); [email protected] (H.C.); [email protected] (S.S.) * Correspondence: [email protected] † These authors contribute equally to this work. Abstract: The accurate determination of analyte concentrations with selective, fast, and robust methods is the key for process control, product analysis, environmental compliance, and medical applications. Enzyme-based biosensors meet these requirements to a high degree and can be operated with simple, cost efficient, and easy to use devices. This review focuses on enzymes capable of direct electron transfer (DET) to electrodes and also the electrode materials which can enable or enhance the DET type bioelectrocatalysis. It presents amperometric biosensors for the quantification of important medical, technical, and environmental analytes and it carves out the requirements for enzymes and electrode materials in DET-based third generation biosensors. This review critically surveys enzymes and biosensors for which DET has been reported. Single- or multi-cofactor enzymes featuring copper centers, hemes, FAD, FMN, or PQQ as prosthetic groups as well as fusion enzymes are presented. Nanomaterials, nanostructured electrodes, chemical surface modifications, and protein immobilization strategies are reviewed for their ability to support direct electrochemistry of enzymes. Citation: Schachinger, F.; Chang, H.; Scheiblbrandner, S.; Ludwig, R. The combination of both biosensor elements—enzymes and electrodes—is evaluated by comparison Amperometric Biosensors Based on of substrate specificity, current density, sensitivity, and the range of detection. Direct Electron Transfer Enzymes. Molecules 2021, 26, 4525. https:// Keywords: biosensor; current density; direct electron transfer; enzyme; selectivity; sensitivity; doi.org/10.3390/molecules26154525 nanomaterial; self-assembled monolayers; nanocomposite; nanoparticle Academic Editors: Carlo Augusto Bortolotti and Gianantonio Battistuzzi 1. Introduction 1.1. Background Received: 29 June 2021 Chemical sensors consist of a chemical recognition system, a physicochemical trans- Accepted: 23 July 2021 Published: 27 July 2021 ducer and a signal processing unit that transforms the concentration of the analyte into a quantifiable signal. A biosensor employs a biological recognition (biorecognition) element working on the principle of bioaffinity or biocatalysis. Bioaffinity sensors use receptor Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in proteins, antibodies, or polynucleotides as biorecognition elements, whereas enzymes are published maps and institutional affil- used as bioelectrocatalysts. Several types of signal transducers can be distinguished, for iations. example: mass-sensitive, optical, and electrochemical. Electrochemical biosensors can be based on the detection of a measurable current (amperometric), the accumulation of charge (potentiometric), changes in current as a result of a potentiometric effect at a gate electrode (field effect), changes in the conductive properties of a medium between elec- trodes (conductometric), or the changes of resistance and reactance close to an electrode Copyright: © 2021 by the authors. (impedimetric). Licensee MDPI, Basel, Switzerland. This article is an open access article 1.2. Amperometric Biosensors distributed under the terms and conditions of the Creative Commons Amperometric biosensors utilizing oxidoreductases were classified into three genera- Attribution (CC BY) license (https:// tions (Figure1): 1st generation biosensors employing oxidases based on the electrocatalytic creativecommons.org/licenses/by/ monitoring of substrate consumption or product formation, 2nd generation biosensors 4.0/). employing oxidases or dehydrogenases based on the electrocatalytic recycling of suitable Molecules 2021, 26, 4525. https://doi.org/10.3390/molecules26154525 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x 2 of 32 1.2. Amperometric Biosensors Amperometric biosensors utilizing oxidoreductases were classified into three Molecules 2021, 26, 4525 generations (Figure 1): 1st generation biosensors employing oxidases based on 2the of 32 electrocatalytic monitoring of substrate consumption or product formation, 2nd generation biosensors employing oxidases or dehydrogenases based on the redoxelectrocatalytic mediators, recycling and 3rd generation of suitable biosensors redox mediators, employing and preferentially 3rd generation dehydrogenases biosensors capableemploying of direct preferentially electron transferdehydrogenases to bare or capable modified of electrodes.direct electron Direct transfer electron to bare transfer or (DET)modified forms electrodes. the basis ofDirect 3rd generation electron transfer biosensors (DET) [1]. forms DET is the the basis ability of of 3rd a solubilized generation or immobilizedbiosensors [1]. redox DET active is the proteinability of to a exchange solubilized electrons or immobilized with an redox electrode active upon protein contact; to forexchange example, electrons oxidoreductases with an electrode directly upon donating contact; electrons for example, gained oxidoreductases from substrate oxidation directly todonating the electrode. electrons Therefore, gained 3rd from generation substrate biosensors oxidation can to bethe operated electrode. at lowerTherefore, potentials 3rd (closegeneration to the biosensors midpoint potentialcan be operated of the enzyme’sat lower potentials prosthetic (close group) to the than midpoint those needed potential for of the enzyme’s prosthetic group) than those needed for the detection of enzymatic the detection of enzymatic reaction products, e.g., H2O2, in 1st generation biosensors, or evenreaction the redoxproducts, potential e.g., H needed2O2, in to1stapply generation redox biosensors, mediators or to shuttleeven the electrons redox potential between theneeded enzyme to apply cofactor redox and mediators the electrode to shuttle in 2nd el generationectrons between biosensors. the enzyme The specificity cofactor ofand 3rd generationthe electrode biosensors in 2nd generation is thus less biosensors. affected byThe electroactive specificity of interferents 3rd generation like biosensors ascorbic acid is andthus also less by affected the absence by electroactive of the redox interferents active mediator. like ascorbic acid and also by the absence of the redox active mediator. Figure 1. Schematic representation of the working principle of the three generations of enzyme- based amperometric biosensors. 1st generation: based on the electrocatalytic detection of enzymatic Figure 1. Schematic representation of the working principle of the three generations of enzyme- substrate conversion or product formation. 2nd generation: based on the electrocatalytic recycling of based amperometric biosensors. 1st generation: based on the electrocatalytic detection of enzymatic asubstrate redox mediator. conversion 3rd generation:or product formation. based on direct 2nd ge electronneration: transfer based fromon the enzyme electrocatalytic to electrode. recycling Yellow moleculesof a redox depict mediator. the FAD 3rd cofactorgeneration: offlavoenzymes based on direct and electron the red transfer molecule from depicts enzyme the hemeto electrode. cofactor inYellow the mobile molecules cytochrome depict the domain FAD ofcofactor a multi-cofactor of flavoenzymes enzyme. and the red molecule depicts the heme cofactor in the mobile cytochrome domain of a multi-cofactor enzyme. 1.3. Applications for DET-Based Biosensors 1.3. PotentialApplications applications for DET-Based of amperometric Biosensors biosensors range from the monitoring of envi- ronmentalPotential markers applications like pesticides, of amperometric medical analytes biosensors in blood range or urinefrom likethe glucose,monitoring lactate, of andenvironmental cholesterol, markers biomarkers like forpesticides, cancer or medical diabetes, analytes to various in blood industrially or urine important like glucose, sub- stanceslactate, as and saccharides, cholesterol, alcohols, biomarkers and phenols for ca forncer process or diabetes, monitoring. to various Moreover, industrially biosensors forimportant the monitoring substances of cellulose-degrading as saccharides, alcohols, enzymes and and phenols lignocellulosic for process biomass monitoring. hydrolysis haveMoreover, been proposed biosensors [2,3 ].for Interestingly, the monitoring enzymes of for applicationcellulose-degrading in 3rd generation enzymes ampero- and metriclignocellulosic biosensors biomass are mostly hydrolysis heme, flavo-,have been or quino-enzymes, proposed [2,3].but Interestingly, copper enzymes enzymes are for also knownapplication to interact in 3rd withgeneration electrodes amperometric through DET biosensors (Figure are2). mostly heme, flavo-, or quino- enzymes, but copper enzymes are also known to interact with electrodes through DET (Figure 2). Molecules 2021, 26, x 3 of 32 Molecules 2021, 26, 4525 3 of 32 Figure 2. Schematic overview on application areas, analytes, enzymes, and the architecture of Figure 2. Schematic overview on application areas, analytes, enzymes, and the architecture of 3rd 3rdgeneration
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