Dual Marker Co-Expressed Exosome-Based Liquid Biopsy Electrochemical Assay for Enhanced-Accuracy Diagnosis of Prostate Cancer.

Tumor-derived exosomes (TDEs) have emerged as clinically significant liquid biopsies for noninvasive early cancer diagnosis. Although serum prostate-specific antigens (PSA) are widely used for prostate cancer (PCa) screening, their diagnostic accuracy is compromised by elevated levels in nonmalignant conditions. B7H3, a protein overexpressed on cancer cell membranes, shows promise as a tumor diagnostic marker but lacks PCa specificity. To address these limitations, we developed an analytical platform for high-accuracy PCa screening via the co-recognition of dual exosomal markers (PSA and B7H3) using a paper-based electrochemical immunosensor. Magnetic Fe3O4 nanoparticles functionalized with multivalent PSA aptamers captured PSA+ TDEs, while anti-B7H3-conjugated iron-manganese silicate nanozymes (IMSNs) amplified the electrochemical signals. A specific electrochemical response occurred only when both markers were highly expressed on TDEs, eliminating false positives from inflammation-induced PSA elevation. Coupled with high sensitivity (detection limit: 81 particles/mL) and specificity for TDEs detection, the assay successfully screened 50 PCa patients, 33 benign prostatic hyperplasia (BPH) patients, and 20 healthy controls, enabling enhanced-accuracy diagnosis of PCa.