Enzymes are natures´ highly active biocatalysts for a manifold of conversions of biologically relevant substances and the activation of small molecules. Thus, they are important elements in the metabolism of organisms. Redox proteins are a specific sub-class of enzymes that catalyze redox reactions, a process in which an electron transfer step is involved.
The electrical coupling of such biocatalysts with electrode surfaces either in a direct electron transfer regime, where the enzyme is directly wired to the electrode surface, or in a mediated electron transfer regime, where an electron relay matrix is used for electron transfer between electrode and protein, allows for the direct control over the catalytic reaction by applying appropriate potentials to the electrochemical transducer. Hence, redox enzymes can be used to drive the production/conversion of a certain product/substrate like the energy vector H2 for energy storage/conversion systems by wiring of so-called hydrogenases or the oxidation/reduction of important analytes like glucose by wiring sugar converting oxidases (e.g. glucose oxidase (GOx) and others).
The high activity of enzymes makes them highly suitable to act as biocatalysts or biorecognition elements in efficient energy conversion systems and sensitive sensor devices. However, their high turnover rates are often coupled to a rather high intrinsic instability against inhibitors/interferences. Moreover, the selectivity of the enzymes is often related to a specific compound class (e.g. sugars) rather than to an individual molecule, which might be the substance of interest. Evidently, these features limit the use of such redox proteins in technological applications and intelligent electrode design with multifunctional properties is highly desired, including an active layer (Figure 1a, blue shaded area) and a protection shield (red shaded area) able to remove detrimental compounds and interfering co-substrates.
Polymer Multilayer Systems For the Full Protection of Hydrogenases
Recently, the authors of this article demonstrated that by incorporation into a low potential viologen modified polymer (an example is shown in Figure 1b, blue) that is capable of reducing O2
, highly air-sensitive hydrogenases (H2
ases) like [NiFe]-  or [NiFeSe]-H2ases  which are promising alternatives for noble metal catalysts in fuel cells, can be efficiently protected from harmful O2
under turnover conditions .
While this single layer system (polymer/H2ase layer) prevents a deactivation of the catalyst in the active layer, the current output of the electrode and thus also the power output of a corresponding biofuel cell (BFC) is reduced because electrons generated from H2 oxidation (i.e. the conversion of the fuel) are used for the reduction of O2 at the reduced viologen mediator species [1,2]. Hence, protection systems that are decoupled from the H2 oxidation process are highly desired.
Stimulated by these results, the possibility to introduce alternative protection strategies to fully protect the hydrogenase and decouple the protection mechanism from the catalytically active layer to prevent a decrease in current in the presence of O2 were exploited. An efficient way of O2 removal is provided by the bienzymatic reaction cascade based on an oxidase and catalase (CAT) . The oxidase catalyzes the oxidation of a substrate (eq. 1), e.g. glucose in case of GOx, with the concomitant consumption of O2 that is the natural electron acceptor in this system. The formed H2O2 is then further disproportioned by catalase to harmless H2O and ½ O2 again (eq. 2). Hence, in each catalytic cycle, ½ O2 molecule is removed (eq. 3).
substrate + O2 → product + H2O2 (1)
H2O2 → H2O + ½ O2 (2)
substrate + ½ O2 → product + H2O (3)
Evidently, the use of such an O2 removal system in a BFC requires the immobilization of the individual components to avoid side reactions at the opposite electrode. Thus, a redox silent but rather hydrophilic polymer (P(SS-GMA-BA), Figure 1b) has been used for immobilization of the oxidase and catalase on the electrode surface, ensuring a solvated environment for the enzymes . For the fully protected H2 oxidizing H2ase-based bioanode a polymer multilayer approach with an underlying active H2ase ([NiFe]- or [NiFeSe]-H2ase)/redox polymer layer and an O2 removal layer bearing a sugar converting oxidase GOx and catalase were selected (Figure 1a). In contrast to the electrodes equipped only with the H2ase/polymer layer the developed polymer multilayer-based bioanodes show a constant current output even in the presence of up to 5 % O2 in the gas feed (Figure 2a) .
The lifetime of the bioanodes could be considerably enhanced by using pyranose oxidase as sugar converting oxidase instead of GOx for the O2 removal layer since this enzyme produces a stable product that is not prone to undergo side reactions which may influence the activity of the electrode. This nicely demonstrates that not only the electrode architecture but also the type and nature of the used proteins in the protection layer is crucial to contribute to the stability of the electrode.
A Glucose/O2 BFC Based On an O2 Tolerant GOx/Polymer Bioanode
Since the active layer and the protection layer are decoupled from each other, this protection strategy can be transposed to other O2 sensitive systems. GOx is a highly active, cheap, and robust enzyme that is used as catalyst for the fabrication of bioanodes in many glucose/O2-based BFCs. However, the fact that O2 acts as the natural electron acceptor for GOx hampers the fabrication of a simple membrane-free BFC. A common approach to circumvent the O2 dilemma is based on the use of O2 insensitive but usually more complex sugar converting enzymes like dehydrogenases. Moreover, for reasonable performance of a BFC, low potential mediators must still be used which may act as catalyst for the reduction of O2 and thus lower the power output of the assembled device. By combining the oxidase/catalase-based O2 removal layer with a redox polymer/GOx active layer, it was possible to build an O2 tolerant bioanode that could be operated under ambient conditions in combination with a bilirubin oxidase-based biocathode in a glucose/O2 powered membrane-free BFC. As oxidase for the O2 shield, lactate oxidase was used that converts lactate into pyruvate in the presence of O2. This makes this bioanode highly attractive for implantable sensors (e.g. for glucose) since lactate is present in blood in sufficiently high concentrations.
Interference-free Detection of Lactose
The protection shield comprised of the bienzymatic system does not only remove O2 but also the corresponding substrate (eq. 3) i.e. glucose in case of GOx. Consequently, a protection layer was integrated into a cellobiose dehydrogenase (CDH)-based biosensor for the detection of lactose in the presence of high concentrations of glucose in dairy products . The enzyme catalyzed splitting of lactose into its monosaccharides galactose and glucose is conventionally used during the production of lactose-free products thus leading to high concentrations of glucose. Since, standard CDHs show high turnover rates for the conversion of lactose and glucose, the removal of the latter is a prerequisite for the quantification of the target analyte in lactose-free products. The sensor was able to detect lactose in the µM level in the presence of up to 140 mM glucose which was removed at the polymer/electrolyte interface .
Polymer multilayer structured bioelectrodes equipped with an active layer and a carefully designed interference removal layer represent a powerful strategy for enhanced power outputs in energy conversion systems (e.g. H2 or glucose oxidation in BFCs) and interference-free detection with amperometric biosensors. It can be anticipated that this protection concept can be transposed to any sensitive catalyst since the active layer and the protection layer are decoupled from each other. Moreover, specific enzyme cascade reactions by incorporation of additional biocatalysts in the hydrophilic polymer layers might become accessible that can even further enhance the performance of the active or the protection layer.
Julian Szczesny1, Felipe Conzuelo1, Adrian Ruff1
1Analytical Chemistry - Center for Electrochemical Sciences (CES), Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany
Dr. Adrian Ruff
Center for Electrochemical Sciences (CES), Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany
 N. Plumeré, O. Rüdiger, A. A. Oughli, R. Williams, J. Vivekananthan, S. Pöller, W. Schuhmann, W. Lubitz, A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage, Nat. Chem. 2014, 6, 822, DOI 10.1038/NCHEM.2022.
 A. Ruff, J. Szczesny, S. Zacarias, I. A. C. Pereira, N. Plumeré, W. Schuhmann, Protection and Reactivation of the [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hidenborough under Oxidative Conditions, ACS Energy Lett. 2017, 2, 964, DOI 10.1021/acsenergylett.7b00167.
 N. Plumeré, Anal. Bioanal. Interferences from oxygen reduction reactions in bioelectroanalytical measurements: the case study of nitrate and nitrite biosensors, Chem. 2013, 405, 3731, DOI 10.1007/s00216-013-6827-z.
 A. Ruff, J. Szczesny, N. Marković, F. Conzuelo, S. Zacarias, I. A. C. Pereira, W. Lubitz, W. Schuhmann, A fully protected hydrogenase/polymer-based bionode for high-performance hydrogen/glucose biofuel cells, Nat. Commun. 2018, 9, 3675, DOI 10.1038/s41467-018-06106-3.
 F. Lopez, S. Zerria, A. Ruff, W. Schuhmann, An O2 Tolerant Polymer/Glucose Oxidase Based Bioanode as Basis for a Self-powered Glucose Sensor, Electroanalysis 2018, 30, 1311, DOI 10.1002/elan.201700785.
 F. Lopez, S. Ma, R. Ludwig, W. Schuhmann, A. Ruff, A Polymer Multilayer Based Amperometric Biosensor for the Detection of Lactose in the Presence of High Concentrations of Glucose, Electroanalysis 2017, 29, 154, DOI 10.1002/elan.201600575.