*2.2. E*ff*ects of Surface Chemistry*

Carbon nanomaterials are frequently formed with hydrophobic and chemically inactivate surfaces, whereas functional groups, such as hydroxyl, carboxyl, and carbonyl, can be formed at the surfaces of carbon nanomaterials, depending on the preparation and treatment approaches. For example, carboxyl groups are frequently found in oxidized CNTs, which can be produced by treatment with acid or peroxides. By contrast, redox enzyme surfaces usually consist of polar amino acids, some of which can even be charged in a solution. Intricate interactions between carbon nanomaterials and redox enzymes have been proposed to play important roles in the orientation and interfacial electron transfer rate of redox enzymes [40]. For example, an improved electron transfer has been reported in BOD on UV-ozone treated carbon black [41] and carbon nanofiber [42] surfaces. UV-ozone treatment was proposed to produce hydrophobically and negatively oxygenated functional groups in carbon nanomaterials. In addition, the O2-reduction current produced from the DET-type bioelectrocatalyst of *Tr*Lac at the CNT decreases with increasing N dopant ratio [43]. Strong electrostatic interactions between N-doped CNTs, which have a positive surface potential, and laccase has been proposed to cause the denaturation of redox enzymes and/or decrease the DET reaction rate. Furthermore, the zeta potential (i.e., surface charge density) of graphene oxide has been reported to affect the enzyme adsorption, direct electron transfer, and catalytic current [44]. In a recent study, three types of carbon nanotubes with different lengths were utilized as platforms for the DET-type bioelectrocatalysis of three redox enzymes, viz. BOD from *Myrothecium verrucaria* (*Mv*BOD), CueO from *Escherichia coli*, and H2ase from *Desulfovibrio vulgaris* Miyazaki F (*Dv*H2ase) [45]. As a result, diffusion-controlled O<sup>2</sup>

reduction reaction with catalytic current density of ~8 mA cm−<sup>2</sup> in an O2-saturated neutral buffer was realized by BOD in CNTs of a length of 1 µm, but the catalytic current densities decreased as the length of CNTs increased [45] (Figure 3). However, in the cases of CueO and H2ase, the catalytic current (O<sup>2</sup> reduction for CueO and H<sup>2</sup> oxidation for *Dv*H2ase) increases as the length of CNTs increased. This tendency can be weakened by the addition of Ca(NO3)<sup>2</sup> to the electrolyte. The CNT surface is negatively charged in neutral solutions owing to the dissociation of the carboxyl group on the CNT surface, and the number of carboxy groups increases with a decreasing CNT length. It was concluded through this research that the electrostatic interaction between the region close to the active site of the enzymes and CNTs is one of the most important factors controlling the enzyme adsorption for DET-type bioelectrocatalysis. <sup>−</sup> μ <sup>−</sup> μ

ω υ <sup>−</sup> **Figure 3.** (**A**) Rotating disk linear scan voltammograms (LSVs) of O<sup>2</sup> reduction for *Mv*BOD/CNT1 (**solid line**), *Mv*BOD/CNT2 (**dashed–dotted line**), and *Mv*BOD/CNT3 (**broken line**). The dotted line represents an LSV on a CNT1 without enzyme. All measurements were carried out in O<sup>2</sup> -saturated phosphate buffer (0.1 M, pH 7.0, and 25 ◦C) at ω = 4000 rpm and υ = 5 mV s−<sup>1</sup> . (**B**) Relationships between the steady state current density and the relative amount of carboxy groups at different CNT surfaces (*N*-COOH). Reprint from [45]. Copyright (2017), with permission from Elsevier.

ω υ <sup>−</sup>
