ENZYME IMMUNOASSAYS WITH AN ELECTROCHEMICAL DETECTION METHOD USING ALKALINE-PHOSPHATASE AND A PERFLUOROSULFONATED IONOMER-MODIFIED ELECTRODE - APPLICATION TO PHENYTOIN ASSAYS

Enzyme immunoassays were performed with a Nafion-modified glassy carbo n electrode as sensor, alkaline phosphatase as the enzyme label, and 6 -(N-ferrocenoylamino)-2,4-dimethylphenyl phosphate (S-) as the enzyme substrate. The three-step procedure required successive pH changes and was set up as a homogeneous or heterogeneous competitive immunoassay; phenytoin was selected as a model antigen. In step I, the competitive immunoreaction was performed in physiological neutral medium. In step II, the enzyme generation of 6-(N-ferrocenoylamino)-2,4-dimethylpheno l (P) took place after addition of S- in alkaline buffered solution (p H 10.2) containing a Mg2+ salt. In step III, the accumulation of P int o the modified electrode proceeded by applying a potential of 0.6 V vs Ag/AgCl for 5 min in neutral medium (pH 7.5) and was followed by a sq uare wave voltammetric scan, Substrate S- was dianionic and therefore was repelled from the anionic Nafion film, whereas P was entrapped wit hin the Nafion film as a ferrocenium salt, and so the sensitive electr ochemical detection of P was possible (detection limit, 10(-8) mol . L (-1)). Both homogeneous and heterogeneous immunoassay techniques (meth ods A and B, respectively) were applied for the determination of pheny toin in clinical seric samples. Phenytoin at therapeutic concentration s could be sensitively determined in clinical samples with method B, b ut not so with method A, which can be envisioned only for semiquantita tive assays (positive/negative tests).