5.3.3. Protection against Reactive Oxygen Species (ROS) Production

Two electron reduction of O<sup>2</sup> yields the formation of unstable superoxide O<sup>2</sup> •− which is the precursor for other reactive species including H2O<sup>2</sup> and •OH. Incomplete oxygen reduction can be encountered in the course of certain enzymatic reactions, or when O<sup>2</sup> is directly reduced at an electrochemical interface. A relevant example of ROS produced by enzymatic reactions is the oxidation of sugars by the flavin-based GOx. Here, O<sup>2</sup> is a co-substrate of the enzyme and is reduced to H2O<sup>2</sup> after the release of D-glucono-δ-lactone within glucose oxidation [290]. Identically, oxidation of lactate-by-lactate oxidase produces H2O2. To avoid any detrimental effect of H2O2, one solution is to electroenzymatically reduce this latter by peroxidases [291]. Alternatively, catalase has been used to decompose H2O<sup>2</sup> [292]. Engineering of flavoproteins is also largely reported to decrease O<sup>2</sup> activity [293,294]. Such strategy is particularly useful when the flavoprotein is embedded in redox hydrogels, where the competition between reaction of the enzyme with the redox mediator or O<sup>2</sup> occurs within the polymer, decreasing the efficiency of the bioelectrocatalysis. An additional issue comes from the reduction of O<sup>2</sup> directly by the redox entities of the polymer.

Reactions other than the oxidation of sugar by GOx embedded in Os-based polymers can expose enzymes to ROS. Actually, low potential viologen-based redox polymers developed to protect HASEs against high potential inactivation also serve as shields against O<sup>2</sup> [224,237,288]. However, the viologen-based matrix itself may generate ROS in the course of the HASE protection mechanism, inducing its progressive decomposition [295]. To tackle this issue, a top polymer layer containing both GOx and catalase was used to ensure a complete HASE protection [143]. Alternatively, iodide was added in the whole system to induce dismutation of H2O2. An improvement of the half-life of the HASE in the presence of O<sup>2</sup> was demonstrated [295]. In the case of O2-tolerant HASEs, their specific sensitivity/tolerance to O<sup>2</sup> has been shown to lead to an even greater sensitivity to ROS. Hence, it was reported that ROS formed by applying low potentials to a HASE-based carbon felt electrode in the presence of O<sup>2</sup> irreversibly deactivated the enzyme contrary to reversible O<sup>2</sup> inactivation [111,198] (Figure 14). In this case, a protective upper layer composed of a 3D porous carbon matrix increased the stability by 4–6 times against ROS compared to a 2D electrode. It was proposed that the 3D matrix could scavenge ROS before reaching the enzymes inside the pores.

#### **6. Conclusions and Future Directions**

Electrocatalysis involving redox enzymes takes advantage of the high selectivity and affinity of the biomolecules, as well as the large diversity of identified enzymes, hence a large variety of substrates. Possible applications range from implantable medical devices (insulin pump, neurological stimulator, artificial organs, etc.), wearable sensors, and environmental diagnostic to energy converting devices or CO<sup>2</sup> capture [16,17,296–299]. Many strategies are available to enhance enzyme stability in general. However, the specificity of redox enzymes that sense an electric field and require a controlled orientation to operate on electrochemical interfaces makes the stability of redox enzymes one of the most important issues to resolve before a large-scale commercial development of bioelectrocatalysis. The search for a good balance between stability and activity of enzymes is a prerequisite, even complexified by the search for efficient ET in the case of redox enzymes. Recent examples in the literature concerning MCOs revealed the difficulty of getting improved interfacial ET parameters or improved redox properties while maintaining both the stability and the activity of the enzyme [300,301]. No doubt that protein engineering, which is increasingly carried out with the aid of innovative solutions based on emerging genetic tools, will offer new opportunities of producing stable enzymes. There is also no doubt that the screening of the biodiversity will allow the discovery of redox enzymes with outstanding properties, which can be applied either directly in bioelectrochemical devices, or as model enzymes for protein engineering for high stability.

Alternative strategies can be envisioned in case enzyme stability is limiting the targeted device. The first one would be to refresh the bioelectrodes regularly, so as to renew inactivated enzymes by new active ones. Increasing salt concentration in the medium was reported to allow LAC desorption, rendering the electrode available for fresh enzyme adsorption [209]. Refreshment of the bioelectrode can also be done by adsorbing the enzyme on magnetic nanoparticles [302]. Applying a suitable magnetic field may allow assembling or release of the biocatalyst on the electrode. Cosnier et al. proposed to use enzymes and redox mediators encapsulated in glyconoparticles, both diffusing in solution [303]. This strategy was supposed to allow protein and mediators to freely rotate for an efficient ET and to offer in addition the possibility of easy refreshing of the biocatalyst by a simple exchange of the solutions. Although suitable to overcome enzyme instability, these procedures based on enzyme renewal, however, do not appear to be sustainable or cost effective. Nanozymes, or bioinspired catalysts, could be desirable alternatives to enzymes in the future [304–307]. Taking benefit of the fundamental understanding of bioelectrocatalysis, these synthetic catalysts may provide higher stability while operating the closest to the enzymatic catalysis.

Should redox enzymes or biosynthetic catalysts be used, novel methodologies must be developed in order to get new insights in the parameters that limit the stability of bioelectrocatalysis. In situ and *in operando* methods are certainly the most appealing within the objective of understanding and enhancing bioelectrode stability [308,309]. Recent reviews by Reisner et al. and by Crespilho et al. discussed various methods, and especially coupled methods, currently available to study bioelectrode interfaces [309,310]. Methods allowing mass or layer thickness quantification, i.e., SPR, QCM-D and ellipsometry, coupled to electrochemistry will give access to the relationship between enzyme loading or release and catalytic response [21,116,271,272,311]. Electrochemistry coupled to spectroscopies (IR (including SEIRA, PMIRRAS), XAS, fluorescence, Raman (including SERRS), EPR, CD, mass spectrometry) will help in the determination of conformational change once enzyme is immobilized or as a function of applied potential or current [312–317]. Microscopy coupled to electrochemistry will allow the mapping of the electrocatalysis [318–320]. Finally, getting more and more accurate experimental data on enzyme behavior on electrodes may be implemented in theoretical models that in return will help in bioelectrode rationalization [21,117,321–323].

**Author Contributions:** Writing, C.B.; review and editing, H.-M.M., A.d.P. and I.M.; writing, editing, and supervision, E.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by ANR (ENZYMOR-ANR-16-CE05- 0024). H-M Man grant was funded by Région PACA, France, Centre National de la Recherche Scientifique, CNRS, France and Hyseas company, Cannes, France. C. Beaufils grant was funded by Aix-Marseille University and by "Agence Innovation Defense" of the French army ministry.

**Conflicts of Interest:** The authors declare no conflict of interest.
