SYNTHESIS OF ELECTROACTIVE PROTEIN HYBRID, FEC-BSA-DIG, AND ITS APPLICATION TO A NOVEL HOMOGENEOUS ELECTROCHEMICAL IMMUNOASSAY

A novel homogeneous electrochemical immunoassay using ferrocene- (Fec-), digoxin- (Dig-) doubleconjugated protein, has been developed. The system is based on the principle that electrochemical activity of the conjugate is suppressed in the presence of an antibody, and restored in the presence of free digoxin. Hence, a new homogeneous immunoassay can be carried out when the electroactivity of the conjugate is determined. Here, we synthesize the conjugate by controlling the numbers of ferrocene and digoxin on individual carrier proteins simultaneously Bovine serum albumin (BSA) is selected as a carrier protein to which the electrochemical labels and the several hapten molecule are bound by Schiff's base formation. Since the conjugate synthesized here behaves as a multivalent antigen, and is capable of binding to antidigoxin antibody, therefore the antigen having electrochemical activity loses its activity depending on antibody concentration. When the conjugate, in which 39 molecules of ferrocene and 8 molecules of digoxin are bound, is used for the inhibition experiment of electrochemical reaction before and after the immunoreaction, peak height currents, responsible for the redox reactions of the bound ferrocene, decreased depending on the added antibody in a dose-dependent fashion. This system is feasible as a homogeneous electrochemical immunoassay, since the method may provide a simple and a rapid approach toward a homogeneous assay of bioactive substances.