Low potential detection of NADH with Prussian Blue bulk modified screen-printed electrodes and recombinant NADH oxidase from Thermus thermophilus

A biosensor for the determination of the reduced coenzyme nicotinamide adenine dinucleotide (NADH) has been assembled using a recombinant enzyme NADH oxidase from Thermus thermophilus covalently immobilized on Prussian Blue bulk-modified screen-printed electrodes. Flow injection analysis (FIA) coupled with amperometric detection was used to detect NADH. Various parameters such as cofactor (FMN, flavin mononucleotide) concentration (2 mM), flow rate (0.35 mL/min), buffers (citrate–phosphate, phosphate and glycine–KOH), pH dependence (range 3.0–10.5), response time (12 s) and operational stability (120 injections) were evaluated and optimised. At pH 5.0, for which the biosensor showed the highest response, the detection and quantification limits were 1.1 × 10−7 and 3.6 × 10−7 M, respectively, and the linear working range was comprised between 1 and 400 μM. The proposed biosensor was stable for 2 months (preserved in 50 mM phosphate buffer, pH 6.8, at 4 °C). The possibility to co-immobilize glycerol dehydrogenase (GDH) and the NADH oxidase in order to measure glycerol, a key target analyte during the alcoholic fermentation of grapes, was also investigated. Different dilutions of a complex matrix such as wine were tested to assess the interferences, the probe recovery and stability.