Substrate-Modification Method for Enhancing Performance of a Direct Electron Transfer-Type Biocathode and a Biofuel Cell
Authors: So Keisei , Kawai Shota , Kitazumi Yuki , Shirai Osamu , Kano Kenji 
Published: February 2020

Introduction
Biofuel cells, which are electric generators that utilize redox enzymes as catalysts, are very secure devices due to the enzyme reactions that can operate under mild conditions (room temperature, atmospheric pressure and neutral pH). Generally, enzymes in the biofuel cells oxidize several biologically related reductants such as alcohols, sugar, amines, organic acids, or hydrogen at the anode and reduce dioxygen (O2) at the cathode. There are two types of electric connection between enzymes and electrodes to realize bioelectrocatalysis. One is mediated electron transfer (MET)-type, in which the mediator shuttle the electron between the enzyme and the electrode. The other is direct electron transfer (DET)-type, in which the enzyme can directly transfer the electron to the electrode.
Although the number of redox enzymes capable of direct communication with electrodes is limited, DET-type biofuel cell has several advantages over the MET-type one. Mediator-less configuration does not require any separator, which leads to simplifications, and DET-type cell can also reduce the possible health hazard problems due to artificial mediators and thermodynamic losses arising from the difference in the redox potentials between the active site. For enhancing the performance of DET-type biofuel cells, controlling the enzyme orientation on the electrode surface is one of the major concerns, since the electron transfer rate constant decreases exponentially with an increase of the distance between redox sites and electrode.
In this study, we focus on bilirubin oxidase (BOD), the promising DET-type enzyme for O2 reduction at a biocathode. Biologically, BOD oxidizes bilirubin at T1 copper site and reduces O2 at T2-3 coppers site. We have a hypothesis that when the electrode is modified with the substrate of BOD, BOD will attractively interact with the modified compound in such an orientation that the electron-accepting T1 site faces the electrode surfaces. The orientation seems to be convenient in the DET-type bioelectrocatalysis.
Experimental
Ketjien Black (KB) powder, one of the carbon materials, was mixed with PTFE and homogenized. The KB slurry was applied to glassy carbon electrode (GCE). Subsequently, bilirubin solution was applied on the KB-modified GCE (KB-GCE) and dried. BOD solution was then applied to the electrode. Rotating disk voltammograms were recorded in an O2-saturated phosphate buffer (pH 7) at room temperature.
Air-breathing gas-diffusion-type electrode was prepared by applying KB slurry to the carbon paper electrode (CPE). Similar modification with bilirubin was carried out on KB-modified CPE (KB-CPE). Cyclic voltammograms were recorded and one compartment fructose/O2 biofuel cell was constructed by combining with a bioanode, which fructose dehydrogenase was adsorbed on the mesoporous carbon electrode.
Results and Discussions
The effect of the bilirubin-modification on the catalytic reduction of O2 was examined using BOD-adsorbed bilirubin-KB-GCE and BOD-adsorbed KB-GCE. Under O2-saturated conditions, the cathodic waves are observed with both BOD-adsorbed electrodes. The catalytic wave at the BOD-adsorbed bilirubin-KB-GCE is much larger than that at the BOD-adsorbed KB-GCE and shows sigmoidal characteristics, while the BOD-adsorbed KB-GCE shows rather straight characteristics in the voltammograms. The sigmoidal and straight characteristics of the catalytic waves can be ascribed to the controlled and random orientation of the adsorbed enzyme, respectively. Therefore, we may assume that bilirubin adsorbed on the electrode can assist BOD to face the electrode surface in an orientation convenient for the DET catalysis.
In the case of air-breathing gas-diffusion-type electrode, the cathodic wave of BOD-adsorbed bilirubin-KB-CPE also shows sigmoidal characteristics, therefore we succeeded in applying bilirubin-modification method to the practical electrode for biofuel cells. Finally, we constructed a DET-type fructose/O2 biofuel cell, and the maximum power density was 2.6 mW cm-2 under quiescent and air atmospheric conditions at room temperature.