Autocatalytic Signal Amplification via Orthogonal Recognition of Dual Proteins on the Surface of Serum Extracellular Vesicles for Precise Diagnosis of Prostate Cancer

Prostate-specific antigen (PSA) testing is now commonly used for the clinical screening of early prostate cancer (PCa). However, its low specificity, especially in the gray area, usually leads to overtreatments or missed diagnoses. Serum extracellular vesicles (EVs) derived from PCa cells are considered to be novel biomarkers for diagnosing PCa, offering promising clinical application prospects. Due to the high heterogeneity and complexity of serum EVs, specific capture and sensitive detection are essential for EV analysis. Here, through targeted recognition of the EV surface membrane protein cluster of differentiation 63 (CD63) and the prostate-specific membrane antigen (PSMA), we developed a dual-protein orthogonal triggered autocatalytic signal amplification (DOTASA) strategy to specifically recognize PCa-derived EVs and trigger allosteric unwinding of DNA duplexes, thereby inducing catalytic hairpin assembly (CHA) and generating fluorescence resonance energy transfer (FRET) for ultrasensitive detection of PCa-derived EVs in clinical serum samples. Our protocol is able to differentiate between early PCa and benign prostatic hyperplasia with a specificity of 100%, which is higher than the specificity of 73.3% for conventional PSA testing. Characterization of subjects' work in clinical trials showed significant PCa discrimination with an area under the curve of 0.9956. Our work provides a rapid and powerful method for accurate diagnosis of early PCa.