Anodized gold surface enables mediator-free and low-overpotential electrochemical oxidation of NADH: A facile method for the development of an NAD+-dependent enzyme biosensor

Abstract

An efficient electrochemical oxidation strategy for the reduced form of nicotinamide adenine dinucleotide (NADH) is required to develop NAD+-dependent enzyme-based biosensors. However, oxidation of NADH at conventional electrodes requires high overpotentials. Here, we report that a nanoporous gold (NPG) surface prepared by a facile method via anodization of a conventional polycrystalline gold electrode enables “direct” electrochemical oxidation of NADH at an extremely low overpotential. In the voltammogram, the oxidation peak current was observed at −0.075 V vs. Ag|AgCl at pH 7.5 without the use of mediators. The potential was substantially more negative than that obtained at a conventional gold electrode (0.6 V). The activity could be explained in terms of the crystal planes at the NPG surface. Because of the low detection potential, interference by ascorbic acid was negligible. We evaluated d-glucose sensing using NAD+-dependent d-glucose 1-dehydrogenase from Bacillus megaterium (BmGDH-IV) at the NPG. Wild-type and a G259A mutant of BmGDH-IV exhibited clear bioelectrocatalytic signals in the presence of d-glucose, in accordance with their inherent properties. The mutant did not exhibit detectable signals for saccharides other than d-glucose, confirming the highly specific detection. Thus, the excellent NADH oxidation activity of NPG allows the development of NAD+-dependent enzyme-based biosensing.