Reduced graphene oxide-gold/methylene blue composite on miniaturized electrochemical immunosensor for the label-free voltammetric determination of hepatitis B surface antigen

• A SPCE immunosensor with rGO-Au/MB composite for HBsAg detection is developed. • In-situ electroreduction and In-situ electropolymerization is used in fabrication. • HBsAg antibody (HBsAb) is employed for specific HBsAg detection. • Methylene blue is utilized as a redox mediator to monitor current signals. • Current signal changes are inversely proportional to HBsAg amounts. This study introduces a miniaturized, label-free electrochemical immunosensor incorporating a reduced graphene oxide-gold/methylene blue (rGO-Au/MB) composite-modified electrode for the detection of hepatitis B surface antigen (HBsAg). The rGO-Au/MB composite was developed on a screen-printed carbon electrode (SPCE) through a two-step process involving in-situ electrochemical reduction and in-situ electropolymerization. Initially, cyclic voltammetry (CV) was used to create rGO-Au composite films from an aqueous mixture of graphene oxide (GO) and Au(III). Subsequently, in-situ electropolymerization of methylene blue (MB) was performed using CV to form the rGO-Au/MB composite. The composite-modified electrode’s physical and electrochemical properties were characterized using CV, electrochemical impedance spectroscopy, field emission scanning electron microscopy, and energy dispersive X-ray analysis. For HBsAg detection, the electrode was functionalized with HBsAg antibodies (HBsAb) and bovine serum albumin (BSA) as a blocking agent to prevent non-specific binding, resulting in the rGO-Au/MB/HBsAb/BSA immunosensor. Differential pulse voltammetry was employed to quantify HBsAg levels by measuring the current signals of polymerized MB, which served as a redox mediator. The decrease in polymerized MB signal correlated with the amount of HBsAg due to the binding of non-electrochemically active HBsAg to HBsAb. Under optimal conditions, the immunosensor demonstrated a linear detection range of 60–1060 ng/mL and a detection limit of 25.7 ng/mL (3SD of regression/slope). Recovery studies in diluted serum samples showed satisfactory results, ranging from 80.4 % to 117.6 %. The proposed immunosensor offers potential as a point-of-care device due to its miniaturization and performance comparable to the chemiluminescence immunoassay method.