Electrochemical enzyme immunoassay for phenytoin by flow-injection analysis incorporating a redox coupling agent

Using phenytoin as a model analyte, we demonstrate an electrochemical enzyme immunoassay based on flow-injection analysis and incorporating 2,6-dichloroindophenol (DCIP) as a redox coupling agent. DCIP reacts with NADH to form NAD+ and DCIPH2, the reduced form of the coupling agent. The production of DCIPH2 is monitored at +250 mV vs Ag/AgCl. This low applied potential improves selectivity in the biological matrix, differentiating against components that are oxidizable at the more-positive potentials required for direct electrochemical detection of NADH. The kinetics-based assay also eliminates other common interferences, mainly from ascorbic acid and glutathione. This system does not require precolumns or analytical columns for isolation of the NADH response. Good agreement with a routine clinical laboratory procedure for phenytoin is obtained for clinical samples (r = 0.95), illustrating the feasibility of such an approach.