细胞外囊泡（EV）表面蛋白是原发性肿瘤表达的重要早期癌症诊断生物标志物。然而，由于EV蛋白的稀有性和临床样本中非EV成分的干扰，这些EV蛋白的检测变得复杂。本文介绍了一种直接检测EV表面蛋白的MEmbrane-Specific Separation and two-step Cascade AmpLificatioN（MESS2CAN）策略，处理时间仅为4小时。MESS2CAN利用新型脂质探针（长链与PEG2K连接，一端带有生物素，另一端带有DSPE）和带有链霉亲和素涂层的磁珠，使得一小时内EV的回收率可达到49.6%。随后通过引物交换反应（PER）与Cas12a系统级联的双重扩增策略，实现对目标蛋白在每微升10个EV颗粒的高敏感检测。通过使用4株细胞系和90个临床检测样本，我们证明MESS2CAN可用于分析从细胞和患者血浆中获得的EV上的HER2、EpCAM和EGFR的表达。MESS2CAN显示出EGFR的所需特异性与敏感性（AUC=0.98）以及HER2的所需特异性与敏感性（AUC=1），可用于区分HER2阳性乳腺癌、三阴性乳腺癌和健康捐助者。MESS2CAN是一种非常敏感的体外EV诊断方法，适用于样本中含有微量EV的临床样本。© 2023 The Authors. Journal of Extracellular Vesicles由Wiley Periodicals, LLC代表国际细胞外囊泡学会出版。

Extracellular vesicle (EV) surface proteins, expressed by primary tumours, are important biomarkers for early cancer diagnosis. However, the detection of these EV proteins is complicated by their low abundance and interference from non-EV components in clinical samples. Herein, we present a MEmbrane-Specific Separation and two-step Cascade AmpLificatioN (MESS2CAN) strategy for direct detection of EV surface proteins within 4 h. MESS2CAN utilises novel lipid probes (long chains linked by PEG2K with biotin at one end, and DSPE at the other end) and streptavidin-coated magnetic beads, permitting a 49.6% EV recovery rate within 1 h. A dual amplification strategy with a primer exchange reaction (PER) cascaded by the Cas12a system then allows sensitive detection of the target protein at 10 EV particles per microliter. Using 4 cell lines and 90 clinical test samples, we demonstrate MESS2CAN for analysing HER2, EpCAM and EGFR expression on EVs derived from cells and patient plasma. MESS2CAN reports the desired specificity and sensitivity of EGFR (AUC = 0.98) and of HER2 (AUC = 1) for discriminating between HER2-positive breast cancer, triple-negative breast cancer and healthy donors. MESS2CAN is a pioneering method for highly sensitive in vitro EV diagnostics, applicable to clinical samples with trace amounts of EVs.© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.