Electrochemical dehydrogenase-based homogeneous assays in whole blood

An electrochemical method has been developed for determining NADH in whole blood for dehydrogenase-based assays by flow-injection analysis. NADH generated by dehydrogenase is oxidized by an electron-transfer coupling reagent, 2,6-dichloroindophenol (DCIP). The reduced form of DCIP (DCIPH2) is measured amperometrically by flow-injection analysis. Endogenous interferents were inhibited by p-hydroxymercuribenzoate. Electrode fouling by proteins was not observed under assay conditions. The Emit theophylline enzyme immunoassay and the hexokinase glucose assay were used as models. For the glucose assay, the intraassay CVs were 15% at 0.31 g/L and 3.5% at 1.82 g/L. Recoveries of glucose from whole blood (compared with that for aqueous standards) were 109%, 97.9%, and 101% at 0.050, 2.00, and 5.00 g/L glucose, respectively, and 104%, 101%, and 102% for theophylline at concentrations of 5.0 (low), 16.4 (medium), and 30.2 (high) mg/L, respectively, with corresponding precisions of 12%, 9.5%, and 8.8%. Both assays correlated well with results by reference methods. These studies demonstrate that this method can measure NADH in whole blood without prior separation and that it is potentially applicable to other dehydrogenase-based assays in whole blood.