Electrochemical sandwich immunoassay for the prostate specific antigen using a polyclonal antibody conjugated to thionine and horseradish peroxidase

The authors present a voltammetric immunoassay for the quantification of the prostate specific antigen (PSA), a well-established tumor marker. Biotinylated anti-PSA antibodies were immobilized on the surface of a nano-TiO2-modified carbon paste electrode using specific interaction between streptavidin and biotin. Streptavidin itself was covalently grafted to the electrode surface via 4-aminobenzoic acid film. Signal transduction was then performed using polyclonal antibodies conjugated with horseradish peroxidase (HRP) and thionine. An amplified catalytic reduction current was observed in the presence of H2O2 because more than one polyclonal antibody linked to each antigen. Each step of the preparation of the immunosensor was monitored via electrochemical impedance spectroscopy. The electrode, if operated at a typical potential of 0.3 V vs. Ag/AgCl and using hexacyanoferrate as an electrochemical probe, exhibits linear responses in the 0.10 to 5.0 ng.mL(-1) and 5.0 to 100 ng.mL(-1) PSA concentration ranges, with a detection limit of 200 pg.mL(-1). The accuracy of the biosensor was confirmed by analyzing a certified human serum sample, and this indicated that the immunosensor is well suited for the quantification of PSA.