Ionic liquid and nanogold-modified immunosensing interface for electrochemical immunoassay of hepatitis B surface antigen in human serum

A mediator-free electrochemical immunoassay protocol based on a disposable immunosensor for the detection of hepatitis B surface antigen (HBsAg) in human serum was developed. To fabricate such an immunosensor, a layer of sol–gel composite film containing room temperature ionic liquid and chitosan was initially formed on a glassy carbon electrode. Nanogold particles were then adsorbed onto the membrane via the amine groups of chitosan molecules, and then horseradish peroxidase (HRP)-labeled hepatitis B surface antibodies (HRP-anti-HBs) were immobilized onto the nanogold surface. With a non-competitive immunoassay format, the antibody–antigen complex could be formed by a simple one-step immunoreaction between the immobilized HRP-anti-HBs and HBsAg in sample solution. The formed immunocomplex inhibited partly the active center of the HRP, which decreased the immobilized HRP toward the reduction of H₂O₂. The performance and factors influencing the performance of the immunosensor were evaluated. Under optimal conditions, the current change obtained from the carried HRP relative to H₂O₂ system was proportional to HBsAg concentration in the range of 1.5–400 ng/mL with a detection limit of 0.5 ng/mL (at 3δ). The reproducibility, selectivity, and stability of the proposed immunosensor were acceptable. Moreover, the proposed immunosensors were used to analyze HBsAg in human serum specimens. Analytical results of clinical samples suggested that the developed immunosensor has a promising alternative approach for detecting HBsAg in the clinical diagnosis.