One-step electrochemical immunoassay of biomarker based on nanogold-functionalized graphene sensing platform

Abstract

A rapid and sensitive one-step electrochemical immunoassay for the determination of α-fetoprotein (AFP, as a model biomarker) was developed by immobilizing anti-AFP antibody on a nanogold-functionalized graphene interface. Initially, thionine molecules as electron mediators were conjugated onto the surface of graphene nanosheets, then gold nanoparticles were sandwiched onto the graphene, and then horseradish peroxidase-anti-AFP (HRP-anti-AFP) conjugates were immobilized on the nanogold surface. The presence of two-dimensional graphene nanosheets with high conductivity not only increased the surface coverage of the electrode, but also enhanced the immobilized amount of biomolecules. With the AFP analyte, the formed antigen–antibody complex was coated on the electrode surface, which partially hindered the immobilized HRP toward the catalytic reduction of H₂O₂ in the solution. Under optimal conditions, the electrochemical immunosensor displayed a wide dynamic range of 0.1–200 ng mL⁻¹ with a detection limit (LOD) of 0.05 ng mL⁻¹ AFP at 3s_B. The reproducibility, selectivity and stability of the immunosensor were investigated with the acceptable results. In addition, the one-step immunoassay was evaluated and validated by assaying 5 positive and 5 negative serum specimens, and the obtained results were compared with those by the referenced electrochemiluminescent, receiving a good correlation between the two methods.