Graphene and Nanogold-Functionalized Immunosensing Interface with Enhanced Sensitivity for One-Step Electrochemical Immunoassay of Alpha-Fetoprotein in Human Serum

A new electrochemical immunosensing protocol for sensitive detection of alpha-fetoprotein (AFP, as a model) in human serum was developed by means of immobilization of horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFT) onto graphene and nanogold-functionalized biomimetic interfaces. The low-toxic and high-conductive graphene complex provided a large capacity for nanoparticulate immobilization and a facile pathway for electron transfer. With a one-step immunoassay format, the antigen-antibody complex was formed between the immobilized HRP-anti-AFP on the electrode and AFP in the sample. The formed immunocomplex was coated on the electrode surface, inhibited partly the active center of HRP, and decreased the catalytic reduction of HRP toward the enzyme substrate of H2O2. Under optimal conditions, the decrease of reduction currents was proportional to AFP concentration, and the dynamic range was 1.0-10 ng/mL with a relative-low detection limit (LOD) of 0.7 ng/mL AFP. Intra- and inter-assay coefficients of variation (CVs) were less than 10%. The assay was evaluated for clinical human serum samples, including 8 (possible) patients with hepatocarcinoma and 3 normal human sera. Correct identification of negative/positive samples and perfect accordance with results from Elecsys 2010 Electrochemiluminescent Automatic Analyzer as a reference was obtained. Importantly, the graphene and nanogold-based sensor provided a promising platform for the detection of other biocompounds, and could be further applied for development of other potential electrochemical bio/chemosensors.