Quantitative Analysis of Bioactive NAD+Regenerated by NADH Electro-oxidation

The bioactivity of NAD+ electrogenerated at a high-surface-area composite anode was verified spectroscopically. The anode was composed of poly(methylene green) electropolymerized on carbon nanotubes (PMG-CNT) which was in turn immobilized on carbon paper. A mathematical model calibrated by measurements of NADH oxidation at PMG-CNT-modified glassy carbon electrodes was applied to predict transient NADH consumption. The model showed good agreement with the experimental data, and 80% conversion of NADH was observed after 1 h of electrochemical oxidation. Using a spectroscopic enzyme cycling assay, the yield of enzymatically active NAD+ was verified at 93% and 87% for applied potentials of 500 and 150 mV vs Ag|AgCl, respectively. This suggests that roughly 10% of oxidized NADH may be lost due to dimerization or some other side reaction after accounting for self-decay. These results prove that bioactive NAD+ can be efficiently produced using electrochemical techniques, enabling application in bioconversion, biosensor, and bioenergy processes.