To create an enzyme-based biological fuel cell generating electricity from glucose and O2, we modified a glassy carbon electrode with a bi-layer polymer membrane, the inner layer containing diaphorase (Dp) and the outer, glucose dehydrogenase (GDH, an NAD+-dependent enzyme). The Dp membrane was formed from a newly synthesized 2-methyl-1,4-naphthoquinone (Vitamin K3; VK3)-based polymer. This polymer showed reversible redox activity at a potential close to that of free VK3 (-0.25 V vs. Ag/AgCl sat. KCl), and served as an electron mediator of Dp for the electrocatalytic oxidation of NADH to NAD+. The addition of Ketjenblack into the Dp/VK3 film enhanced the generation of NAD +. The outer GDH membrane oxidized glucose continuously using NAD+ generated at the inner Dp film. To construct the glucose/O 2 biological fuel cell, we coupled the enzyme-modified anode with a polydimethylsiloxane-coated Pt cathode. The cell's open circuit voltage was 0.62 V and its maximum power density was 14.5 μW/cm2 at 0.36 V in an air-saturated phosphate buffered saline solution (pH 7.0) at 37 °C containing 0.5 mM NADH and 10 mM glucose. Although its performance deteriorated to ca. 4 μW/cm2 over 4 days, the cell subsequently maintained this power density for more than 2 weeks.
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