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

Oct 20, 2020Talanta

Highly sensitive detection of proteins on cell-released vesicles using aptamer-guided CRISPR technology

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Abstract

The aptamer-CRISPR/Cas12a assay detects CD109 and EGFR tumor-derived extracellular vesicles at concentrations as low as 100 particles/mL.

This technique can measure TEV proteins in low-volume samples of 50 μl.
The assay demonstrated a high diagnostic accuracy for nasopharyngeal carcinoma with an area under the curve (AUC) of 0.934.
Sensitivity and specificity rates of the assay were 84.1% and 85.0%, respectively, for distinguishing NPC from healthy controls.
A significant decrease in CD109 and EGFR levels was observed in patients responding to radiotherapy, suggesting their potential as biomarkers for treatment response.

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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 CD109and EGFRTEVs from cell lines and complex biofluids. The platform enables highly sensitive detection of CD109and EGFRTEVs at as low as 100 particles/mL with a linear range spanning 6 orders of magnitude (10-10particles/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 CD109and EGFRTEV 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 CD109and EGFRTEVs directly in serum, it could be useful in NPC diagnosis and prognosis. + + + + 2 8 + + + +

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

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