sensors Review Addressing the Selectivity of Enzyme Biosensors: Solutions and Perspectives Bogdan Bucur 1 , Cristina Purcarea 2 , Silvana Andreescu 3 and Alina Vasilescu 4,* 1 National Institute for Research and Development in Biological Sciences, 296 Splaiul Independentei, 060031 Bucharest, Romania; [email protected] 2 Institute of Biology, 296 Splaiul Independentei, 060031 Bucharest, Romania; [email protected] 3 Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13676, USA; [email protected] 4 International Centre of Biodynamics, 1B Intrarea Portocalelor, 060101 Bucharest, Romania * Correspondence: [email protected]; Tel.: +40-21-310-4354 Abstract: Enzymatic biosensors enjoy commercial success and are the subject of continued research efforts to widen their range of practical application. For these biosensors to reach their full potential, their selectivity challenges need to be addressed by comprehensive, solid approaches. This review discusses the status of enzymatic biosensors in achieving accurate and selective measurements via direct biocatalytic and inhibition-based detection, with a focus on electrochemical enzyme biosensors. Examples of practical solutions for tackling the activity and selectivity problems and preventing interferences from co-existing electroactive compounds in the samples are provided such as the use of permselective membranes, sentinel sensors and coupled multi-enzyme systems. The effect of activators, inhibitors or enzymatic substrates are also addressed by coupled enzymatic reactions and multi-sensor arrays combined with data interpretation via chemometrics. In addition to these more traditional approaches, the review discusses some ingenious recent approaches, detailing also on Citation: Bucur, B.; Purcarea, C.; Andreescu, S.; Vasilescu, A. possible solutions involving the use of nanomaterials to ensuring the biosensors’ selectivity. Overall, Addressing the Selectivity of Enzyme the examples presented illustrate the various tools available when developing enzyme biosensors for Biosensors: Solutions and new applications and stress the necessity to more comprehensively investigate their selectivity and Perspectives. Sensors 2021, 21, 3038. validate the biosensors versus standard analytical methods. https://doi.org/10.3390/s21093038 Keywords: selectivity; enzyme; electrochemical biosensor; enzymatic inhibition; biocatalytic sensor Academic Editor: Nicole Jaffreznic-Renault Received: 29 March 2021 1. Introduction Accepted: 15 April 2021 Selectivity represents the ability of an analytical method to detect the target analyte Published: 26 April 2021 without being influenced by other sample constituents. It represents one of the key advan- tages of biosensors, compared to other methods, as they allow to determine an analyte in a Publisher’s Note: MDPI stays neutral complex mixture without resorting to prior separation. with regard to jurisdictional claims in Enzyme based biosensors hold the largest market share of commercial biosensors published maps and institutional affil- iations. and continue to be widely investigated, along with devices based on antibodies, aptamers, cells and other biorecognition elements. Enzymes are biocatalysts, converting their target analyte at high rate. Enzymes are also activated or inhibited by various compounds including pollutants such as pesticides or heavy metals, which provide opportunities for the development of indirect inhibition-based measurements. The selectivity of enzymatic Copyright: © 2021 by the authors. biosensors, whether biocatalytic or inhibition based, is determined by the specificity of the Licensee MDPI, Basel, Switzerland. enzyme but the biosensor response is also influenced by design parameters such as: (i) the This article is an open access article biosensor type, e.g., first, second or third generation, (ii) the complexity of the sample and distributed under the terms and conditions of the Creative Commons (iii) the particularities of the detection method such as the electrode potential, electrode type Attribution (CC BY) license (https:// and surface modification, e.g., in the case of electrochemical measurements. The materials creativecommons.org/licenses/by/ used to create the sensing surface, modifiers, membranes, and the enzyme immobilization 4.0/). method are also contributing to the performance of the biosensors. Developing effective Sensors 2021, 21, 3038. https://doi.org/10.3390/s21093038 https://www.mdpi.com/journal/sensors Sensors 2021, 21, x FOR PEER REVIEW 2 of 38 Sensors 2021, 21, 3038 2 of 36 membranes, and the enzyme immobilization method are also contributing to the per- formance of the biosensors. Developing effective strategies for achieving high selectivity is strongly tiedstrategies on the type for of achieving enzyme highbiosensor, selectivity first, is second strongly and tied third on thegeneration, type of enzymeex- biosensor, emplified for superoxidefirst, second dismut and thirdase for generation, the detection exemplified of superoxide for superoxide anion, shown dismutase in Fig- for the detection ure 1. of superoxide anion, shown in Figure1. Figure 1. SchematicFigure 1. Schematic illustration illustration of first, second of first, and second third and generation third generation biosensors, biosensors, exemplified exemplified for biosensors for based on superoxidebiosensors dismutase based (SOD) on for superoxide the detection dismutase of superoxide (SOD) anion. for the Reproduced detection of fromsuperoxide [1] with anion. permission. Repro- duced from [1] with permission. In the first generation biosensors, the reactants or the reaction products are determined In the firstelectrochemically generation biosensors, at high the applied reactants potentials, or the being reaction prone products to interferences, are deter- which are elimi- mined electrochemicallynated by using at high membranes applied potentials, or sentinel being sensors, prone as detailedto interferences, below. The which second generation are eliminated biosensorsby using membranes make use of or mediators sentinel sensors, to reduce as the detailed overpotential below. requiredThe second for detection and generation biosensorsthus minimize make use interferences. of mediators For to some reduce redox the enzymes, overpotential direct required electron transferfor (DET), by detection and ‘wiring‘thus minimize or electrically interferences. connecting For the some enzyme redox with enzymes, the electrode direct is enabled,electron leading to third transfer (DET),generation by ‘wiring‘ biosensors or electrically with inherentlyconnecting higher the enzyme selectivity. with the electrode is enabled, leading to Thethird recent generation years biosensors witnessed awith tremendous inherently progress higher inselectivity. the development of enzymatic The recentbiosensors years witnessed as wearable a tremendous devices for progress the non-invasive in the development analysis of of biomarkers enzymatic in physiological biosensors as wearablefluids [2], devices for example for the for non-invasive the detection analysis of glucose, of lactate,biomarkers alcohol in physio- and uric acid analysis logical fluids [2],in sweatfor example [3]. The for inthe vivo detectionanalysis of isglucose, another lactate, area thatalcohol has and seen uric progress acid in designing analysis in sweatelectrochemical [3]. The in vivo enzyme analysis biosensors is another with area enhanced that has selectivityseen progress [4–6 in] bestde- illustrated by signing electrochemicalimplantable enzyme electrodes biosensors for the detectionwith enhanced of neurotransmitters selectivity [4–6] in best the brain illus- [7]. In addition trated by implantablevarious electrodes biosensors for were the developed detection of for neurotransmitters food analysis, targeting in the brain the detection [7]. In of pesticides, addition variousglucose, biosensors lactate, were glycerol developed (e.g., fo forr food monitoring analysis, fermentative targeting the processes), detection of biogenic amines pesticides, glucose,(for evaluating lactate, glycerol the freshness (e.g., for of fish monitoring and meat), fermentative bisphenol A, processes), etc. In the samebio- time, various genic amines (forenzyme evaluating biosensors the freshness were reported of fish and for meat), monitoring bisphenol the A, quality etc. In of the the same environment, for time, various enzymedetecting biosensors contaminants were such report as organophosphateed for monitoring and the carbamate quality of pesticides, the envi- [8] toxic metals ronment, for detectingsuch as arseniccontaminants [9] or chromiumsuch as organophosphate [10], etc. Applications and carbamate in agriculture pesticides, or livestock health [8] toxic metalsmonitoring such as arsenic have also[9] or been chromium reported, [10], with etc. some Applications advanced conceptsin agriculture to interface or biosensors livestock healthwith monitoring the Internet have of Thingsalso been [11 ].reported, with some advanced concepts to interface biosensorsWhile with the biosensors Internet haveof Things widespread [11]. applications, a critical question remains: how to While biosensorsachieve have accurate widespread and precise applications, measurements a critical and selectively question remains: detect analytes how to in such complex achieve accuratematrices? and precise For measurements example, for implantable and selectively biosensors, detect analytes the list ofin possiblesuch com- interfering