Improvement of NADH detection using Prussian blue modified screen-printed electrodes and different strategies of immobilisation

Amperometric biosensors based on NADHox/FMN and Prussian blue modified screen-printed electrodes (SPE) have been developed in order to improve the NADH detection. The catalytic effect, the sensitivity and the stability of PB-modified SPEs were studied for different pH buffer solutions and different voltage range, by cyclic voltammetry and amperometric measurements. NADH oxidase was immobilised on Prussian blue modified SPEs by entrapment in sol–gel, PVA-AWP polymer matrices or using glutaraldehyde. The amperometric detection of NADH was performed at +0.25 V versus Ag/AgCl. FMN was added in reaction medium or either immobilised. Better results were obtained when FMN was entrapped with NADHox in the biocatalytic layer using sol–gel matrix. The sensitivity was 4.57 mA M−1 cm−2 (R.S.D. = 9.2%, n = 4), the linear range up to 1.61 mM and the detection limits about 1.17 μM. These biosensors present a good long term and operational stability, the response decreases from 3.4% after 2 days, to 50% of the analytical signal after 6 months.