Multi-enzyme anode composed of FAD-dependent and NAD-dependent enzymes with a single ruthenium polymer mediator for biofuel cells

•A Ru–quinone polymer worked as a mediator for FAD-dependent glucose dehydrogenase.•Enzymes with different coenzymes were fixed and electrically wired by a redox polymer.•A redox polymer mediates the electron transfer of both FAD- and NAD-enzymes.

A multi-enzyme electrode composed of FAD-dependent and NAD-dependent enzymes was fabricated using a poly-ruthenium complex (PAHA–Ru), which has two 1,10-phenanthroline-5,6-dione molecules as ligands. PAHA–Ru was used to immobilize FAD-dependent glucose dehydrogenase (FAD–GDH) onto an electrode and to examine PAHA–Ru containing the quinone moieties as an electron mediator. In cyclic voltammetry measurements of the FAD–GDH modified electrode in the presence of D-glucose, a catalytic current was obtained, which indicated electron transfer from FAD–GDH to PAHA–Ru. Our previous study has reported that PAHA–Ru with the quinone ligands also works as a mediator for NADH oxidation on an NAD-dependent alcohol dehydrogenase (NAD–ADH) modified electrode. Hence, FAD–GDH and NAD–ADH were co-immobilized with PAHA–Ru to make a multi-enzyme electrode. Using this multi-enzyme electrode as an anode, catalytic currents were observed in D-glucose solution, ethanol solution, and a mixed D-glucose and ethanol solution. The catalytic current in the mixed solution was greater than the currents obtained in the single substrate solutions, indicating bioelectrocatalysis reactions by the two enzymes and the single mediator in the mixed solution. Thus, we demonstrated that PAHA–Ru modified electrode enables selection of enzymes and their substrates from a wider range for enzymatic biofuel cells.

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