*2.1. Electrode Nanomaterials*

In DET-type and enzyme/mediator-immobilized MET-type bioelectrocatalysis, immobilization of as many amounts of enzymes and mediators as possible on the electrode surface is, in principle, able to increase the current density of bioelectrocatalytic systems. Therefore, nanostructured electrodes with a large ratio of the effective surface area against the projective surface area are useful and frequently utilized for bioelectrocatalytic systems. In addition, it is suggested that the heterogeneous electron transfer kinetics at the top edge of the microstructures of these electrodes is accelerated by the electric field strengthened by the expansion of the electric double layer [56] and the charge accumulation as expected by the Poisson equation [4]. This effect is very useful to decrease the overpotential due to the heterogeneous electron transfer in the DET-type reaction.

Electrode nanomaterials utilized in bioelectrocatalysis are roughly divided into two classes: carbon and metal. Carbon nanomaterials, such as carbon nanotubes [57–59], carbon blacks [60], carbon cryogels [61,62], and MgO-templated carbons [43,63,64], have properties to physically adsorb a large amount of enzymes and mediators at hydrophobic sites and are generally used as platforms favorable for bioelectrocatalysis. On the other hand, nanoporous gold constructed by anodization [27,65–67] or dealloying [68–70] and metallic nanoparticles of gold [57,71–78], silver [79–81], platinum [29–31], titanium oxide (TiO2) [80,81], iron oxide (Fe2O3) [82,83], and indium tin oxide (ITO) [84] are also widely used. Compared to carbon nanomaterials, the pore and particle sizes of metallic nanomaterials can be easily controlled according to several manufacturing methods. In addition, conductive supports, such as polymer hydrogels, act as nanostructured electrodes [85].

Furthermore, nanostructured electrodes exert another positive effect on DET-type bioelectrocatalysis from the viewpoint of the enzyme's orientation. The limited catalytic current density (*j*cat) in DET-type reactions is expressed by Equation (5):

$$j\_{\rm cat} = \pm \eta\_{\rm E} F k\_{\rm c,DET} \Gamma\_{\rm E,eff} \tag{5}$$

where *F* is the Faraday constant, *k*c,DET is the catalytic constant in DET-type bioelectrocatalysis (with *k*c,DET = (*n*S/*n*E)*k*c,DET(1) , *k*c,DET(1) being the catalytic constant for single turnover of the enzyme), and ΓE,eff is the surface concentration of the effective enzyme. Here, "effective" means being able to electrochemically communicate with electrodes. In other words, enzymes of which the electrode-active sites face away from the electrode are not included because the long-range electron transfer kinetic constant (k◦ ) exponentially decreases with an increase in the distance between the electrode surface and the electrode-active site in an enzyme (d) [86–88], as described by Equation (6):

$$\mathbf{k}^{\circ} = \mathbf{k}^{\circ}\_{\text{max}} \exp(-\beta \mathbf{d}),\tag{6}$$

where k◦ max is the rate constant at the closest approach (d = dmin) and β is the decay coefficient. Based on the random orientation model in which enzymes are assumed to be randomly oriented on electrodes [32,89,90], the enzymes can penetrate into nanostructured electrodes with mesopores, and the mesoporous electrodes can adsorb a large amount of enzymes suitable for DET-type reactions

compared with planar electrodes, as illustrated in Figure 2. This is called the curvature effect of mesoporous electrodes for DET-type bioelectrocatalysis and is also very effective in decreasing the overpotential of DET-type bioelectrocatalysis. In practical applications, it is necessary to select and optimize electrode nanomaterials based on the estimated size, shape, and hydrophobicity of enzymes.

**Figure 2.** Schematic of the enzyme orientations at the (**A**) planar and (**B**) mesoporous electrodes.

On the other hand, chemical modifications of electrode surfaces are very effective in controlling the enzyme orientation by electrostatic or other specific interactions between the electrode-active site and the electrode surface; therefore, they are useful to increase the population of the enzyme orientations with minimum distances between the electrode-active site and the electrode surface [91]. Such favorite orientations also contribute to decreasing the overpotential in the DET-type reaction. For example, [NiFe]-hydrogenase (H2ase) from *Desulfovibrio vulgaris* Miyazaki F and copper efflux oxidase (CueO) expressed in *Escherichia coli* showed strong DET-type bioelectrocatalysis activity at (positively charged) *p*-phenylenediamine-functionalized Ketjen-Black-modified glassy carbon electrodes compared with Ketjen-Black-modified electrode without any chemical functionalization and artificially introduced charged groups. The surfaces near the electrode-active sites of the enzymes are estimated to be negatively charged and the attractive electrostatic interaction with positively charged electrode surfaces increases the probability of enzyme orientations favorable for the interfacial electron transfer [92,93].

In contrast, bilirubin oxidase (BOD) from *Myrothecium verrucaria* showed strong DET-type bioelectrocatalytic activity at negatively charged electrodes. The surface near the electrode-active site of the enzyme is positively charged and the electrostatic interaction with negatively charged electrodes increases the probability of favorable orientations of the enzyme [94]. On the other hand, the DET-type bioelectrocatalysis of BOD was also improved by modifying an electrode with bilirubin (as the natural electron donor), which seemed to attractively interact with the electrode-active type I copper site as the electron-accepting site. The interaction increases the probability of favorable orientations of the enzyme [95]. In addition, membrane-bound d-fructose dehydrogenase (FDH) from *Gluconobacter japonicus* NBRC3260 showed strong DET-type bioelectrocatalytic activity at a 4-mercaptophenol-modified porous gold electrode, probably due to the stabilization of the enzyme layer by the hydroxy groups of 4-mercaptophenol [96]. FDH also showed the attractive and specific interaction with methoxy substituents on the electrode surface, which resulted in the favorable orientation [97].

In addition, gas-diffusion bioelectrodes are effective for enzymatic bioelectrocatalysis in which gaseous substrates such as dihydrogen, dioxygen, and carbon dioxide were used [98]. Since these gasses have low water solubility, the bioelectrocatalytic currents are often controlled and limited by diffusion processes of the substrates at usual electrodes. On the other hand, gas-diffusion bioelectrodes realize direct supplies of gaseous substrates from the gas phase to the reaction layer due to their suitable conductivity, hydrophobicity/hydrophilicity balance, and gas permeability, as illustrated in Figure 3.

**Figure 3.** Schematic of a bio-three-phase interface of a gas-diffusion bioelectrode.
