An ultrasensitive hybridization chain reaction-amplified CRISPR-Cas12a aptasensor for extracellular vesicle surface protein quantification

Tumor-derived extracellular vesicle (TEV) protein biomarkers facilitate cancer diagnosis and prognostic evaluations. However, the lack of reliable and convenient quantitative methods for evaluating TEV proteins prevents their clinical application. Methods: Here, based on dual amplification of hybridization chain reaction (HCR) and CRISPR-Cas12a, we developed the apta-HCR-CRISPR assay for direct high-sensitivity detection of TEV proteins. The TEV protein-targeted aptamer was amplified by HCR to produce a long-repeated sequence comprising multiple CRISPR RNA (crRNA) targetable barcodes, and the signals were further amplified by CRISPR-Cas12a collateral cleavage activities, resulting in a fluorescence signal. Results: The established strategy was verified by detecting the TEV protein markers nucleolin and programmed death ligand 1 (PD-L1). Both achieved limit of detection (LOD) values as low as 10² particles/µL, which is at least 10⁴-fold more sensitive than aptamer-ELISA and 10²-fold more sensitive than apta-HCR-ELISA. We directly applied our assay to a clinical analysis of circulating TEVs from 50 µL of serum, revealing potential applications of nucleolin⁺ TEVs for nasopharyngeal carcinoma cancer (NPC) diagnosis and PD-L1⁺ TEVs for therapeutic monitoring. Conclusion: The platform was simple and easy to operate, and this approach should be useful for the highly sensitive and versatile quantification of TEV proteins in clinical samples.