An HNO3-Pretreated, Graphene Oxide-Modified Pencil Graphite Electrode for PSA Detection: Toward a Disposable Label-Free Electrochemical Immunosensor

Prostate cancer is a neoplastic disease that generally remains asymptomatic until its late-stage emergence. Among the techniques approved to date for prostate cancer diagnosis, prostate-specific antigen (PSA) screening is the most established method for its early detection. Currently, centralized clinical laboratories measure serum PSA using costly, time-consuming, multi-step immunoassays that rely heavily on lab technicians and highly sophisticated equipment. To address this issue, we developed a proof-of-concept, disposable, label-free electrochemical biosensor for rapid detection of PSA-α1-antichymotrypsin complex (PSA-ACT) and free PSA (fPSA) using a single antigen incubation step. The immunosensors were based on HNO3-pretreated pencil graphite electrodes (PGEs) modified with graphene oxide (GO). Antigen concentrations were determined by measuring signals from the [Fe(CN)6]3−/4− redox probe using cyclic voltammetry (CV); the presence of antigen molecules decreased anodic current response at the PGE surface. A log-linear calibration response over 1–1000 ng/mL and an estimated limit of detection (LOD) of 2.48 ng/mL were observed for PSA-ACT, indicating a detection range of 2.48–1000 ng/mL for the tPSA biosensor. The fPSA immunosensor showed a log-linear calibration response over 0.1–1000 ng/mL; however, an analytical LOD below the lowest experimentally evaluated calibrator (0.1 ng/mL) was not determined. Analyses of the biosensors’ performance, selectivity, repeatability, and short-term storage stability, along with a preliminary comparison with commercial enzyme-linked immunosorbent assay (ELISA) kits using real human samples, provided early evidence supporting further development and optimization of these platforms for point-of-care (POC) PSA testing.