Electrochemical investigations of the reaction mechanism and kinetics between NADH and redox-active (NC)2C6H3–NHOH/(NC)2C6H3–NO from 4-nitrophthalonitrile–(NC)2C6H3–NO2-modified electrode

A simple and sensitive method for the electrocatalytic detection of NADH on a carbon paste electrode modified with a redox-active (NC)2C6H3–NO/(NC)2C6H3–NHOH (NOPH/NHOHPH) electrogenerated in situ from 4-nitrophthalonitrile (4-NPHN) is presented. The electrode modified with 4-NPHN showed an efficient electrocatalytic activity towards the oxidation of NADH with activation overpotential of 0.12 V vs. Ag/AgCl. The formation of an intermediate charge transfer complex is proposed for the charge transfer reaction between NADH and the 4-NPHN-resulting system. The second-order rate constant for electrocatalytic oxidation of NADH, κobs, and the apparent Michaelis–Menten constant KM, at pH 7.0 were evaluated with rotating disk electrode (RDE) experiments, giving 1.0 × 104 mol−1 L s−1 and 2.7 × 10−5 mol L−1, respectively. Employing the Koutecky–Levich approach indicated that the NADH oxidation reaction involves two electrons. The sensor provided a linear response range for NADH from 0.8 up to 8.5 μmol L−1 with sensitivity, detection, quantification limits and time response of 0.50 μA L μmol−1, 0.25 μmol L−1, 0.82 μmol L−1 and 0.1 s, respectively. The repeatability of the measurements with the same sensor and different sensors, evaluated in terms of relative standard deviation, were 4.1 and 5.0%, respectively, for n = 10.