Bioanalytical method for NAD detection in blood plasma utilizing solution-phase formate dehydrogenase and electrochemical detection.

Nicotinamide adenine dinucleotide is a crucial coenzyme in cellular metabolism and is implicated in various diseases. This work introduces an electrochemical bioanalytical method utilizing solution-phase formate dehydrogenase (CbFDH) for detecting its oxidized form (NAD) in human blood plasma samples. The detection mechanism involves the catalytic conversion of NAD to NADH, facilitated by CbFDH in the presence of formate. This NADH is then quantified by electrochemical measurements at disposable carbon screen-printed electrodes. The reaction is completed within one minute. The assay exhibits a linear response range from 3.74 μM to 2.00 mM, a sensitivity of 8.98 ± 0.18 μA mM, and a limit of detection (3/) of 1.12 μM. It demonstrates selectivity against common interferences found in plasma samples, including glucose, urea, creatinine, guanosine 5'-monophosphate, cytidine 5'-monophosphate, flavin adenine dinucleotide, adenosine 5'-triphosphate, and lactate, with interference levels below 5% relative to the unperturbed NAD signal. Recovery studies showed 98.0-104.4% recoveries, with further validation against a colorimetric alcohol dehydrogenase assay confirming accuracy in plasma samples.