Enzyme immunoassay with flow-amperometric detection of NADH

Using phenytoin as a model analyte, we demonstrate that an enzyme-coupled immunoassay based on flow-injection analysis and amperometric detection of NADH is both feasible and practical. Good agreement with a routine clinical laboratory procedure for phenytoin was obtained for patients' serum samples. The electrode must be protected to prevent fouling by proteins in the analytical sample. The optimum detection range for NADH was at 0.01 of the concentrations of NADH generated during the several minutes required for each analysis. This suggests that the electrochemical technique should be extendable to the determination of species at concentrations in the microgram per liter range.