Aptamer-based CRISPR/Cas12a assay for the ultrasensitive detection of extracellular vesicle proteins

Tumor-derived extracellular vesicles (TEVs) have emerged as promising sources of diagnostic and prognostic biomarkers for nasopharyngeal carcinoma (NPC). However, the lack of high-sensitivity analytic methods for ultratrace membrane proteins on TEVs hamper their clinical application of TEVs. Herein, by combining aptamers that specifically bind to protein targets on TEVs, PCR-based exponential amplification and CRISPR/Cas12a real-time DNA detection, we developed a novel technique, termed the aptamer-CRISPR/Cas12a assay, to detect CD109⁺ and EGFR⁺ TEVs from cell lines and complex biofluids. The platform enables highly sensitive detection of CD109⁺ and EGFR⁺ TEVs at as low as 100 particles/mL with a linear range spanning 6 orders of magnitude (10²-10⁸ particles/mL), which was found to be sufficient to effectively detect TEV proteins directly in low-volume (50 μl) samples. Furthermore, clinical serum sample analysis verified that the combination of serum CD109⁺ and EGFR⁺ TEV levels yielded high diagnostic accuracy, with an AUC of 0.934 (95% CI: 0.868-1.000), a sensitivity of 84.1% and a specificity of 85.0%, in discriminating NPC from healthy controls. Moreover, the dramatic decrease in both biomarkers in responders after radiotherapy indicated their potential roles in radiotherapy surveillance. Given that the aptamer-CRISPR/Cas12a assay rapidly and conveniently detects ultralow concentrations of CD109⁺ and EGFR⁺ TEVs directly in serum, it could be useful in NPC diagnosis and prognosis.