5.2.4. Redox Enzyme Encapsulation in Hydrogels for Bioelectrocatalysis

Entrapping redox enzymes into hydrogels is expected to provide a suitable hydrated environment able to maintain the enzyme in a functional active state. Some examples of use of a sole polymer for direct electrocatalysis involving redox enzymes were recently reported [254]. Conductive polyaniline (PANi) forms a nanostructured hydrogel once deposited on an electrode [255]. The PANi hydrogel exhibits a continuously connected hierarchical 3D network with pore diameter of 60 nm, allowing a short electron diffusion length. The PANi-formate dehydrogenase electrode was efficient for catalytic reduction of CO<sup>2</sup> into formate. Although no exhaustive study of stability was made, conversion of CO<sup>2</sup> was efficient for 12 h. However, hydrogel matrices are very often poorly conductive, and conductive nanostructures or redox mediators must be co-immobilized in the gel to ensure electron conduction. Chitosan is a natural polyamino-saccharide that forms thin hydrogel films at electrodes through controlled electrodeposition process. The amine groups are available for attachment to a functionalized electrode or for anchorage of the enzyme. CNTs mixed with chitosan render the material conductive. Hosting a LAC, the so-built bioelectrode retained 70% initial current after 60 days of continuous measurement [209,256]. The same authors reported an improved bioelectrode using genipin as a cross linker. The LAC-based electrode was implanted in rats and remained operational for 167 days in vivo [257].

Alternatively, redox mediators can act as electron relays between the electrode and the enzyme inside the hydrogel. In the search for enzyme stabilization, Matsumoto et al. prevented leaching of a biotinylated GDH by immobilizing it into a streptavidine-hydrogel prepared by click chemistry between sortase A and PEG [258]. In the presence of a ferrocene derivative as a redox mediator, the so-designed electrode showed improved stability when compared to the electrode with no hydrogel. One issue was the weak stability of the hydrogel itself on the electrode. This last result points out one more requirement for redox enzyme-based electrode stabilization: Not only the enzyme must be stable but the interaction between the electrode and the hybrid enzyme-host matrix must also be strong enough to avoid leaching of the whole biomaterial. To ensure stability of the bioelectrode, hydrogel can be combined within porous conductive material. As an illustration, a hydrogel formed by cross-linking bovine serum albumin, GOx and arginine was interlocked within the pores of a carbon cloth [259]. Leaching of enzyme was less than 0.5% of the total amount of embedded protein within 48 h. UV-Visible spectrometry and CD demonstrated that 90% of the enzyme secondary structure was preserved in the network. In the presence of ferricyanide as a redox mediator, the current density for glucose oxidation was maintained for 25 days.

Diffusing redox mediators are however undesirable in many cases, either because they limit the rate of reaction or because they can be toxic when leaching to the environment in which the enzyme operates. Redox hydrogels, introduced 30 years ago by Adam Heller, are now widely exploited for immobilization of a variety of enzymes [260]. These hydrogels combine the advantages of a hydrated matrix ensuring protection of the enzyme and rapid diffusion of the substrate with the presence of tethered redox moieties (they can be based on osmium, ruthenium, ferrocene, colbaltocene, quinone, or viologen derivatives) that can electrically wire the enzyme by electron hopping inside the gel [261–263]. The redox potential of the hydrogel matrix can be adapted to the desired reaction to be catalyzed by tuning the ligands of the redox moieties. Ferrocene entity was tethered to linear PEI and used to cross-link GOx [264]. Of initial current, 70% and 36% was retained after 21 days of storage and 6 h of continuous operation, respectively. This marked difference underlines the effect of applied potential on the stability of the bioelectrode, an issue that we will further discuss in Section 5.3.2. Os-tethered hydrogel embedding GDH was loaded in porous MgOC deposited by ink-drop-casting on an electrode [265]. The high catalytic currents for glucose oxidation were linked to the controlled pore diameter of the MgOC matrix that allows high loading of redox polymer and rapid substrate diffusion. Moreover, the combined advantage of hydrated redox polymer and porous structure

allowed improved stability. Of initial catalytic current, 44% was recovered after 10 days of operation against 3% for the hydrogel-enzyme hybrid deposited on a glassy carbon electrode. Direct oxidation of glucose by *Corynascus thermophilus* cellobiose dehydrogenase was compared to the mediated one obtained when the CNT-based electrode was further modified by an Os-based redox hydrogel [200]. MET process generated higher catalytic currents than DET one proceeding at a lower potential through the FAD cofactor instead of the heme domain in the case of the DET process. Of the mediated electrocatalytic current, 30% remained after 9 days of CV cycling. In such configuration, it will be interesting in the future to get the comparative stability through the DET process. Last but not least, redox hydrogels have been shown in recent years to act as shields against O<sup>2</sup> for sensitive enzymes. This topic will be further developed below in Section 5.3.3.
