New immunoassay techniques using Nafion-modified electrodes and cationic redox labels or enzyme labels

Two new competitive immunoassay techniques with sensitive electrochemical detection at Nafion film modified electrodes are described and compared. Method A implies the use of an antigen labeled with a cationic redox group in homogeneous immunoassay. Method B involves an enzyme-labeled antigen, in connection with an anionic substrate + cationic electroactive 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 cobaltocenium and alkaline phosphatase to illustrate methods A and B, respectively. The enzymatic reaction was performed with N-ferrocenoyl)-6-amino-2,4-dimethylphosphate as substrate in method B. The dianionic substrate was repulsed from the Nafion film, whereas the corresponding enzymatically generated alcohol was selectively entrapped within the film as a ferricinium salt by applying a potential of 0.6 V vs. solAgAgCl during the accumulation step performed at pH 7.4. Both methods were tested for the determination of phenytoin in clinical serum samples.