An electrochemical NADH biosensor based on a nanoporous gold electrode modified with diaphorase and an osmium polymer

• Use of nanoporous gold electrode as support for diaphorase. • Sensor for the determination of NADH utilising an osmium redox polymer and diaphorase. • Limit of detection of 0.8 µM and a linear range from 5 to 100 µM. NADH is a cofactor used by a wide range of dehydrogenases. Measurement of the concentration of NADH is widely used to measure the activity of enzymes. Extensive efforts have been made to develop electrochemical sensors for NADH determination at low overpotentials to avoid interferences. The development of an enzymatic electrochemical biosensor for the detection of NADH is described. Nanoporous gold electrodes were utilised for the immobilization of diaphorase and osmium-based polymer Os(bpy) 2 (PVI). Nanoporous gold electrodes of different pore sizes were manufactured by varying the dealloying temperature. To optimise the enzymatic response, the concentrations of polymer and enzyme, average pore size and the operating temperature were examined with the optimal performance observed using 10 µL of Os(bpy) 2 (PVI) and 5 µL of diaphorase at concentrations of 6 and 10 mg/mL, respectively, on nanoporous gold electrodes with an average pore size of 5.9 nm. The biosensor showed a high sensitivity of 89.6 µA/cm 2 mM, a low LOD of 0.8 µM and a linear range from 5 to 100 µM at a potential of 0.35 V vs Ag/AgCl at a temperature of 40 °C.