A new assay format for electrochemical immunosensors: polyelectrolyte-based separation on membrane carriers combined with detection of peroxidase activity by pH-sensitive field-effect transistor

A new rapid immunotechnique combining separation of reactants by filtration through a porous membrane and potentiometric detection of the bound enzyme label by a pH-sensitive field-effect transistor is proposed. The complexes to be detected are formed by the method described earlier in (Anal. Chem. 71 (1999) 3538), including a homogeneous binding of immunoreactants and a polyanion carrier (polymethacrylate) followed by heterogeneous separation on a membrane incorporating an immobilized polycation (poly- N-vinyl-4-ethylpyridinium). The proposed technique for a sensitive detection of peroxidase label is based on the measurement of pH changes in the optimised substrate solution containing o-phenylenediamine, hydrogen peroxide and ascorbic acid. The antigens studied were herbicide atrazine and hormone testosterone. Their specific detection is realised via competitive binding of free and peroxidase-labelled antigens by antibodies integrating with a (staphylococcal protein A–polyanion) conjugate. The total analysis time is 20–25 min. The range of quantitative detection is 0.2–100 ng ml −1 for atrazine and 5–300 ng ml −1 for testosterone. Data scatter of replicate tests varies from 3 to 10%. Application of protein A–polyanion conjugate allows to use the proposed protocol for different antigens without additional treatment of specific antisera.