Photoactivated liquid-liquid phase separation-based cascade CRISPR/Cas12a electrochemical platform for sensitive small extracellular vesicles detection.

Small extracellular vesicles (sEVs) have emerged as promising liquid biopsy biomarker for gastric cancer (GC) diagnosis. However, profiling sEVs surface proteins in complex clinical matrices remains challenging by limited sensitivity and high background interference. In this study, we developed an electrochemical platform based on the photoactivated liquid-liquid phase separation cascaded Cas12a amplification (PL-cCas12a) for ultrasensitive and specific detection of sEVs surface proteins. The rationally designed system enables precise spatiotemporal control over the reaction. Photoactivation ensures precise reaction initiation. The spatially confined microenvironment accelerate reaction efficiency by local substrates enrichment and suppresses non-specific trans-cleavage leakage via steric hindrance. The PL-cCas12a platform achieved the limit of detection (LOD) as low as 3.95 × 103 particles/μL with negligible background leakage. By integrating the aptamers targeting GC-associated sEV surface proteins, this platform accurately discriminated GC patients from healthy donors (AUC = 0.912). Therefore, this platform represents a promising tool for non-invasive early cancer diagnosis.