癌细胞源小型细胞外囊泡（csEVs）是反映多种恶性肿瘤存在和进展的重要液体活检指标。然而，由于正常sEVs（nsEVs）的干扰和早期癌症低丰度的存在，可靠地鉴别csEVs仍然是一个巨大的挑战。在这项工作中，我们开发了一种双元素选择性触发csEVs识别（TESTER）策略，用于选择性地从复杂的临床体液样品中鉴定csEVs。该方法基于MNAzyme控制的EpCAM和CD63蛋白在csEVs膜上的同步识别。通过EpCAM适配体和CD63适配体对csEVs的高效识别，促使Partzyme A和Partzyme B探针的释放，诱导MNAzyme结构形成，从而循环裂解底物链产生级联荧光信号放大。所开发的TESTER方法在复杂生物样品中对csEVs的检测阈值为72个颗粒μL-1，实现了对csEVs的高灵敏度和选择性定量。与此同时，我们成功构建了一种新的双分子同时识别平台，为未来构建双分子激活的检测开关提供了良好的思路。版权所有 © 2023 Elsevier B.V. 保留所有权利。

Cancer-derived small extracellular vesicles (csEVs) are crucial liquid biopsy indicators that reflect the presence and progression of many malignancies. However, reliable discrimination of csEVs remains a great challenge owing to the interference from normal sEVs (nsEVs) and low abundance in the early stages of cancer. In this work, we developed a Two-Elements Selectively Triggered csEVs Recognization (TESTER) strategy for selective identification of csEVs from the complex clinical body fluid samples. This method was based on the MNAzyme-controlled synchronous recognition to EpCAM and CD63 proteins on the membrane of csEVs. Efficient recognition to csEVs via EpCAM aptamer and CD63 aptamer prompted the release of Partzyme A and Partzyme B probes to induce a MNAzyme structure formation, resulting in the cyclic cleavage of substrate chain to produce cascade fluorescence signal amplification. The detection threshold of the developed TESTER approach for csEVs in complicated biological samples was 72 particles μL-1, accomplishing the highly sensitive and selective quantification of csEVs. At the same time, we successfully constructed a new platform for bimolecular simultaneous recognition, which provides a good idea for the construction of bimolecular-activated detection switch in the future.Copyright © 2023 Elsevier B.V. All rights reserved.