外泌体miRNA可以反映肿瘤进展和转移，是有效的癌症诊断生物标志物。然而，传统方法在外泌体miRNA基于癌症诊断中的准确性受到低灵敏度和复杂的RNA提取的限制。本文提出一种新型生物传感器，通过纳米荧光探针与催化发夹组装(CHA)技术的结合，实现了对外泌体miRNA的原位、无需提取和高灵敏度分析。纳米荧光探针能够直接进入外泌体与目标miRNA结合，并产生荧光信号，该信号可通过CHA反应放大以实现外泌体miRNA的原位和高灵敏度检测，而无需繁琐昂贵的miRNA提取或转染试剂。在最佳条件下，检测限为5 aM，对于三个外泌体miRNA，比定量实时聚合酶链式反应(qRT-PCR)低一个数量级。与线性判别分析算法相结合，可以100%准确区分出五种外泌体。通过检测临床血浆中的外泌体miRNA，可以以99%的准确度区分64名患者中的五种癌症，包括乳腺癌、肺癌、肝癌、宫颈癌和结直肠癌。这种简单、准确、敏感的生物传感器具有扩展成临床非侵入性癌症诊断测试的潜力。 版权所有 © 2023 Elsevier B.V.。保留所有权利。

Exosomal miRNAs can reflect tumor progression and metastasis, and are effective biomarkers for cancer diagnosis. However, the accuracy of exosomal miRNA-based cancer diagnosis is limited by the low sensitivity and complicated RNA extraction of traditional approaches. Herein, a novel biosensor is developed for in situ, extraction-free, and highly sensitive analysis of exosomal miRNAs via nanoflare combined with catalyzed hairpin assembly (CHA) amplification. Without cumbersome and costly miRNA extraction or transfection agents, nanoflare can directly enter the exosomes to bind target miRNAs and generate a fluorescence signal that can be amplified by the CHA reaction to achieve the in situ and highly sensitive detection of exosomal miRNAs. Under the optimal conditions, the detection limit of 5 aM is obtained for three exosomal miRNAs, which is an order of magnitude lower than quantitative real time polymerase chain reaction (qRT-PCR). In combination with the linear discriminant analysis algorithm, five exosomes are distinguished with 100% accuracy. Importantly, five cancers including breast, lung, liver, cervical, and colon cancer from 64 patients are distinguished with 99% accuracy by testing exosomal miRNAs in clinical plasma. This simple, accurate, and sensitive biosensor holds the potential to be expanded into clinical non-invasive cancer diagnostic tests.Copyright © 2023 Elsevier B.V. All rights reserved.