Zhao Xianxian, Zeng Leili, Mei Qiang, Luo Yang
Department of Clinical Laboratory, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing 400038, China.
Emergency Department of PLA 922 Hospital, Hengyang, Hunan 421002, China.
ACS Sens. 2020 Jul 24;5(7):2239-2246. doi: 10.1021/acssensors.0c00944. Epub 2020 Jul 14.
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 10 to 10 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.
细胞外囊泡（EVs）正成为癌症诊断和治疗中颇具前景的生物标志物。以易于操作的方式识别临床样本中低丰度的细胞外囊泡是非常必要的，但仍然是一项挑战。在此，我们建立了一种变构探针引发的双循环扩增辅助CRISPR-Cas12a（AID-Cas）平台，用于以无需洗涤的方式灵敏检测细胞外囊泡。在AID-Cas中，变构探针可以特异性识别并与目标细胞外囊泡结合，从而启动后续的双循环扩增。随后，扩增产物被转录以产生大量单链RNA，这些单链RNA可作为crRNA触发CRISPR-Cas12a的反式切割。因此，所提出的方法在10至10颗粒/微升的浓度范围内对提取的细胞外囊泡实现了良好的线性响应。由于其高灵敏度以及无需洗涤的便利性，所提出的策略在癌症早期诊断方面可能具有广阔的临床应用潜力。
