The development of NAD⁺-dependent dehydrogenase screen-printed biosensor based on enzyme and nanoporous gold...

Given the significance of dihydronicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide (NAD⁺) in numerous biochemical fields such as clinical diagnostics and fermentation monitoring, a synergistic strategy was proposed based on the co-catalysis of NAD⁺-dependent dehydrogenases and nanoporous gold (NPG) towards the oxidation of substrates and NADH, respectively. An NAD⁺-dependent dehydrogenase/NPG/SPE biosensing platform was developed by modifying screen-printed electrode (SPE) with NPG and NAD⁺-dependent dehydrogenase for the electrochemical detections of NADH, ethanol, and glucose. Owing to the exceptional oxidation activity of NPG towards NADH, the amperometric detection of NADH exhibited good linearity from 50 μM to 2.0 mM with a low detection limit (LOD) of 15.18-16.39 μM and a satisfactory sensitivity of 1.58-1.72 μA mM^-1 within a wide pH range. With alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) as model enzymes, ADH/NPG/SPE and GDH/NPG/SPE exhibited excellent analytic characteristics (sensitivity of 0.66 μA mM^-1 and 2.04 μA mM^-1, LOD of 40.72 μM and 14.83 μM) of ethanol and glucose detection in buffer solution as well as in human serum and fermentation broth. The sensitive micro-sample detections of ethanol and glucose in both real samples were achieved using the proposed biosensors with comparable accuracy (deviation rates of 0.85-7.92%) as automatic analyzers. The proposed biosensing platform elicited many advantageous properties in practical applications, including micro-sample analysis, cost-efficient, easy fabrication, and flexible adaptability, which made it a promising candidate for the clinical blood test, urinalysis, and fermentation monitoring.