Aulia Ayuning Tyas, Setyawan Purnomo Sakti, Duangjai Nacapricha, Kanchana Uraisin, Hani Susianti, Akhmad Sabarudin, Phoonthawee Saetear
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. © 2025 The Author(s)
Department of Chemistry, Faculty of Science, Mahidol University, 272 Rama VI Road, Bangkok, Ratchathewi, 10400, Thailand; Flow-Innovation Research for Science and Technology Laboratories (FIRST Labs), Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, 272 Rama VI Road, Bangkok, Ratchathewi, 10400, Thailand; Department of Chemistry, Faculty of Science, Brawijaya University, Jl. Veteran, Malang, 65145, Indonesia; Department of Physics, Faculty of Science, Brawijaya University, Jl. Veteran, Malang, 65145, Indonesia; Research Center for Advanced System and Material Technology, Brawijaya University, Jl. Veteran, Malang, 65145, Indonesia; Department of Clinical Pathology, Faculty of Medicine, Brawijaya University, Jl. Veteran, Malang, 65145, Indonesia
