The simultaneous detection of proteins and microRNA (miRNA) at the single extracellular vesicle (EV) level shows great promise for precise disease profiling, owing to the heterogeneity and scarcity of tumor-derived EVs. However, a highly reliable method for multiple-target analysis of single EVs remains to be developed. In this study, a digital dual CRISPR-Cas-powered Single EV Evaluation (ddSEE) system was proposed to enable the concurrent detection of surface protein and inner miRNA of EVs at the single-molecule level. By optimizing simultaneous reaction conditions of CRISPR-Cas12a and CRISPR-Cas13a, the surface protein of EVs was detected by Cas12a using antibody-DNA conjugates to transfer the signal of the protein to DNA, while the inner miRNA was analyzed by Cas13a through EV-liposome fusion. A microfluidic chip containing 188,000 microwells was used to convert the CRISPR-Cas system into a digital assay format to enable the absolute quantification of miRNA/protein-positive EVs without bias through fluorescence imaging, which can detect as few as 214 EVs/μL. Finally, a total of 31 blood samples, 21 from breast cancer patients and 10 from healthy donors, were collected and tested, achieving a diagnostic accuracy of 92% in distinguishing patients with breast cancer from healthy donors. With its absolute quantification, ease of use, and multiplexed detection capability, the ddSEE system demonstrates its great potential for both EV research and clinical applications.

中文翻译：

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使用数字双 CRISPR-Cas 检测在单个细胞外囊泡水平同时检测蛋白质和 miRNA


由于肿瘤来源的 EV 的异质性和稀缺性，在单个细胞外囊泡 （EV） 水平同时检测蛋白质和 microRNA （miRNA） 显示出精确疾病分析的巨大前景。然而，一种高度可靠的单 EV 多靶点分析方法仍有待开发。在这项研究中，提出了一种数字CRISPR-Cas 驱动的 Single E E 评估（ddSEE） 系统，能够在单分子水平同时检测 EV 的表面蛋白和内部 miRNA。通过优化 CRISPR-Cas12a 和 CRISPR-Cas13a 的同时反应条件，Cas12a 使用抗体-DNA 偶联物检测 EVs 的表面蛋白，将蛋白质的信号传递给 DNA，同时通过 EV-脂质体融合通过 Cas13a 分析内部 miRNA。使用包含 188,000 个微孔的微流控芯片将 CRISPR-Cas 系统转换为数字检测形式，以便通过荧光成像对 miRNA/蛋白质阳性 EV 进行无偏差的绝对定量，荧光成像可检测低至 214 个 EVs/μL。最后，共收集和检测了 31 份血液样本，其中 21 份来自乳腺癌患者，10 份来自健康供体，在区分乳腺癌患者和健康供体方面达到了 92% 的诊断准确率。凭借其绝对定量、易用性和多重检测能力，ddSEE 系统展示了其在 EV 研究和临床应用方面的巨大潜力。