Allosteric Probe-Initiated Wash-Free Method for Sensitive Extracellular Vesicle Detection through Dual Cycle-Assisted CRISPR-Cas12a.

Extracellular vesicles (EVs) are emerging as promising biomarkers for cancer diagnosis and therapy. Recognizing low-abundance EVs from clinical samples in an easy-to-operate way is highly desired but remains a challenge. Herein, we established an allosteric probe-initiated dual cycle amplification-assisted CRISPR-Cas12a (AID-Cas) platform for sensitive detection of EVs in a wash-free way. In AID-Cas, the allosteric probe can specifically recognize and bind with target EVs and thus initiate the following dual-cycle amplification. Subsequently, the amplified products were transcribed to generate numerous single-stranded RNAs, which could work as crRNA to trigger the trans-cleavage of CRISPR-Cas12a. Consequently, the proposed approach achieved a good linear response to extracted EVs in a concentration range from 102 to 106 particles/μL. Because of its high sensitivity, together with its wash-free convenience, the proposed strategy could have promising clinical potentials for early diagnosis of cancers.