5.2.3. Conductive Porous Material as Redox Enzyme Host Matrices Favoring Fast ET

We discussed in the previous sections how porous materials can be beneficial for the stabilization of embedded enzymes. The further requirements for their use as host matrices of redox enzymes are that either the porous material is conductive to ensure DET, or redox mediators can be added either diffusing or co-immobilized within the matrix. We emphasized that MOFs are nowadays considered as very attractive materials. To ensure electron conduction within MOFs, multicomponent systems can be synthetized composed of MOFs, enzymes and conductive nanomaterials such as graphene nanosheets or CNTs [147]. A nice example is provided by Li et al. who encapsulated a LAC in ZIF-8 in the presence of CNTs (LAC@ZIF-8) [247]. Fluorescence measurements highlighted a negligible enzyme leaching. When compared to free enzyme or to enzyme embedded in MOF with no CNT, the LAC@ZIF-8 hybrid displayed improved thermal stability, enhanced resistance to solvents as well improved long-term storage stability. However, although DET was demonstrated, the catalytic efficiency was greatly enhanced by redox mediator addition, suggesting that a large enzyme population was not directly wired in the hybrid material. Mesoporous silica nanotubes are another example of a suitable matrix when coated with a graphene sheet layer to enhance its electrical conductivity. BOD entrapped in the material was able to reduce O<sup>2</sup> in the absence of redox mediators, and the bioelectrode stored in water at 4 ◦C for 15 days retained around 80% of initial current [248]. The stability of the bioelectrode was suggested to be linked to the size of the nano-channels (12 nm) being close to the size of the enzyme, limiting its mobility. Fructose dehydrogenase and HASE were immobilized in MgOC with pore size in the range 10–150 nm [249,250]. DET occurred between enzymes and the material regardless of the pore size, but a pore diameter close to the enzyme size enhanced the thermal and long-term stabilities.

The relationship between pore size and stability is shared by non-redox and redox enzymes. However, for redox ones, the orientation of the enzyme toward the material wall must also be considered for a direct wiring. In pores presenting a diameter close to the enzyme size, the distance between electrical relays on the surface of enzymes and the material will be statistically minimized. Multi-point contacts are expected to occur at the same time. This means that both stability and ET rates should be enhanced simultaneously [238,251]. Porous gold obtained either by dealloying processes or anodization is increasingly used for immobilization of redox enzymes. Their pore sizes are compatible with efficient enzyme entrapment [144]. Using electrochemistry, a narrow distribution of orientation of BOD favorable for enhanced DET was measured [252]. A model based on close packing spheres explained why the curvature of pores was beneficial for DET. Unfortunately, no examination of the enzyme conformation in the porous gold material was provided. Effect of nanomaterial curvature was evocated in another work to explain LAC properties once immobilized in a CNT network in the presence of ethanol [253]. An improved direct catalytic current for O<sup>2</sup> reduction as well as reduced chloride inhibition was measured. CD and ATR-IR demonstrated a net difference in the secondary structure when the enzyme/CNT/ethanol system was immobilized at the electrode as compared with enzyme in solution. The structure stabilization suggested that ethanol favors contact between CNT and LAC in its native form with an orientation favorable for DET. Although not deeply investigated, CNT curvature was suggested to help in the stabilization of the immobilized dehydrated enzyme.
