Non-invasive urine-based detection of PSMA-positive exosomes using a dual-aptamer electrochemical aptasensor for prostate cancer diagnosis.

A novel electrochemical aptamer-based biosensor is reported for label-free detection of prostate-specific membrane antigen (PSMA)-positive exosomes in human urine. The platform integrates magnetic nanoparticles functionalized with CD63-specific aptamers (mMNPs) for efficient exosome capture, and screen-printed electrodes (SPEs) modified with carbon nanofibers (CNFs) and immobilized PSMA-specific aptamers for targeted detection. Differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) confirmed the successful stepwise modification of the electrode surface and target recognition. The biosensor demonstrated excellent analytical performance for PSMA protein detection in exosome isolates, with a detection limit of 3 pg/mL and a linear response from 10 to 1400 pg/mL as quantified by ELISA. Specificity was confirmed using exosomes from PSMA-negative PC3 cell cultures and benign samples. Reproducibility and stability tests were conducted using exosomes isolated from the LNCaP cell culture, which confirmed operational robustness. Clinical urine testing showed high agreement with PSMA ELISA results, and urinary creatinine normalization improved data reliability across samples. This dual-aptamer biosensor offers a highly specific, reproducible, and non-invasive strategy for early prostate cancer diagnosis, with strong potential for point-of-care and clinical translation.