Ultrasensitive Immunoassay Based on Electrochemical Measurement of Enzymatically Produced Polyaniline

A novel ultrasensitive immunoassay method was developed based on the electrochemical measurement of polyaniline, which was catalytically produced by horseradish peroxidase-functionalized gold nanoparticle (HRP-Au NP) probe at an immunosensor. The immunosensor was prepared step-wise by first modifying the electrode with reduced graphene oxide (rGO)/Au NPs nanocomposite followed by the immobilization of capture antibodies on its surface. After performing a sandwich immunoreaction, the quantitatively captured HRP-Au NP nanoprobes could catalyze oxidation of aniline to produce electroactive polyaniline on the immunosensor surface. The electrochemical measurement of polyaniline enabled a novel detection strategy for HRP-based immunoassay. Both the signal amplification of the HRP-Au NP nanoprobe and the electron transfer acceleration of rGO/Au NPs on the immunosensor surface greatly improved the detection sensitivity of the immunoassay method. With the use of human IgG as a model analyte, this method showed a wide linear range over 4 orders of magnitude with a detection limit of 9.7 pg/mL. In addition, the immunosensor had low cost, satisfactory reproducibility and stability, and acceptable reliability. The relatively positive potential range for the polyaniline measurement completely excluded the conventional interference from dissolved oxygen. Thus, this method provides a promising potential for practical applications.