ELECTROCHEMICAL DEHYDROGENASE-BASED HOMOGENEOUS ASSAYS IN WHOLE-BLOOD

An electrochemical method has been developed for determining NADH in w hole blood for dehydrogenase-based assays by flow-injection analysis. NADH generated by dehydrogenase is oxidized by an electron-transfer co upling reagent, 2,6-dichloroindophenol (DCIP). The reduced form of DCI P (DCIPH2) is measured amperometrically by flow-injection analysis. En dogenous interferents were inhibited by p-hydroxymercuribenzoate. Elec trode fouling by proteins was not observed under assay conditions. The Emit(TM) theophylline enzyme immunoassay and the hexokinase glucose a ssay were used as models. For the glucose assay, the intraassay CVs we re 15% at 0.31 g/L and 3.5% at 1.82 g/L. Recoveries of glucose from wh ole 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 prec isions of 12%, 9.5%, and 8.8%. Both assays correlated well with result s by reference methods. These studies demonstrate that this method can measure NADH in whole blood without prior separation and that it is p otentially applicable to other dehydrogenase-based assays in whole blo od.