Amperometric NADH determination via both direct and mediated electron transfer by NADH oxidase from Thermus aquaticus YT-1

Using NADH oxidase extracted from Thermus aquaticus YT-1, the determination of NADH by the direct oxidation of labile FADH2 and by mediated electron transfer using the Ru(III/IV) redox couple is described. A scheme for enzyme amplification is outlined using alcohol dehydrogenase (ADH) and NADH oxidase in solution, and enables 1–10 μM NADH to be determined. Detection is performed at + 150 mV using the RuCl62−/RuCl63− couple. Additionally, an electrode is modified by an ADH/NADH oxidase bilayer, enabling ethanol determination (20–200 μ M) at 0 mV via the oxidation of ruthenium red.