Multiplexed 3D-rGO/AuNP electrochemical aptasensor for ultrasensitive detection of urinary PSMA in prostate cancer diagnosis.

Prostate cancer (PCa) is one of the most common cancers in men worldwide and remains a major cause of cancer-related deaths. The current blood test for prostate-specific antigen (PSA) lacks specificity, often producing false positives and leading to unnecessary biopsies. Prostate-specific membrane antigen (PSMA), a cell-surface glycoprotein highly overexpressed in prostate cancer, can also be detected in urine and provides a promising non-invasive biomarker for accurate diagnosis. In this work, we developed a multiplexed electrochemical aptasensor based on a three-working-electrode screen-printed platform (TWSPE) for ultrasensitive detection of urinary PSMA. Each electrode was sequentially modified with three-dimensional reduced graphene oxide (3D-rGO) and gold nanoparticles (AuNPs) to form a high-surface-area, conductive interface for covalent immobilization of a PSMA-specific DNA aptamer. This nanostructured interface enhanced electron transfer, improved probe stability, and amplified electrochemical signals. The three-electrode configuration enabled simultaneous triplicate measurements from a single sample, improving analytical precision and reducing variability. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) confirmed stepwise electrode modification and selective PSMA recognition. Under optimized conditions, the aptasensor achieved a detection limit of 5 pg/mL, a wide linear range of 15-2000 pg/mL, and excellent reproducibility (RSD <4 %). Preliminary validation with human urine samples successfully discriminated prostate cancer patients from healthy donors, underscoring the biosensor's potential for accurate and non-invasive prostate cancer diagnosis. These results establish the 3D-rGO/AuNP multiplexed aptasensor as a sensitive, reliable, and practical platform for urinary PSMA detection, with strong promise for clinical translation.