Aptamer-based CRISPR/Cas12a assay for the ultrasensitive detection of extracellular vesicle proteins
Huilan Li, Shan Xing, Jianhua Xu, Yi He, Yanzhen Lai, Yu Wang, Ge Zhang, Songhe Guo, Min Deng, Musheng Zeng, Wanli Liu
Abstract
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.