Amperometric determination of NADH and dehydrogenase enzymes at a redox-active nanocomposite

Aminated carbon nanotubes, CNT_NH2, were covalently modified with glutardialdehyde (GDI) and the redox dye Azure to form a new electrode material CNT_NH2-GDI-Azure (CGA). The nanocomposite of CGA and polysaccharide chitosan was used for the anodic determination of NADH. Compared to conventional carbon and metal electrodes, the CGA electrode drastically lowered the overpotential for NADH oxidation (by > 0.40 V), which minimized the interferences from the matrix of real-life samples (human serum). At -0.10 and 0.00 V, the CGA detected NADH and two NADH-dependent enzymes (lactate and malate dehydrogenase, LDH and MDH) down to 0.6 μM, 1.5 U L^-1, and 0.5 U L^-1, respectively. The activity of enzymes was quantified in 200-μL samples of human serum by the rapid (5 min) internally calibrated electrochemical continuous enzyme assay (ICECEA) at CGA within the clinically relevant linear ranges of 1.5-30 U L^-1 (R² = 0.994) for LDH and 0.5-8.1 U L^-1 (R² = 0.996) for MDH. Another attractive feature of CGA was that it maintained a stable NADH current (∼103 %) during a continuous 10-h long oxidation of 0.10 mM NADH, which sharply contrasted with a decaying NADH current observed at conventional electrodes. The CGA is a stable material that can be used in the form of a film or renewable paste electrode for the determination of NADH and hundreds of NADH-dependent dehydrogenases as biomarkers of human diseases.