NEW IMMUNOASSAY TECHNIQUES USING NAFION-MODIFIED ELECTRODES AND CATIONIC REDOX LABELS OR ENZYME LABELS

Two new competitive immunoassay techniques with sensitive electrochemi cal detection at Nafion film modified electrodes are described and com pared. Method A implies the use of an antigen labeled with a cationic redox group in homogeneous immunoassay. Method B involves an enzyme-la beled antigen, in connection with an anionic substrate + cationic elec troactive product of the enzymatic reaction in heterogeneous assay. A 5 min accumulation step precedes the electrochemical detection in both methods. Method B can be applied to the determination of any kind of antigen, whereas method A is restricted to haptens. Phenytoin, an anti -epileptic drug, was chosen as a model antigen and labeled by cobaltoc enium and alkaline phosphatase to illustrate methods A and B, respecti vely. The enzymatic reaction was performed with (N-ferrocenoyl)-6-amin o-2,4-dimethylphosphate as substrate in method B. The dianionic substr ate was repulsed from the Nafion film, whereas the corresponding enzym atically generated alcohol was selectively entrapped within the film a s a ferricinium salt by applying a potential of 0.6 V vs. Ag/AgCl duri ng the accumulation step performed at pH 7.4. Both methods were tested for the determination of phenytoin in clinical serum samples.