Bioanalytical method for NAD+ detection in blood plasma utilizing solution-phase Candida boidinii 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 Candida boidinii 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⁻¹, and a limit of detection (3s_b/m) 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.