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. .