特定细胞外囊泡 (EV) 亚群的分离和富集对于精准医学至关重要。然而，目前的方法主要依赖于单一阳性标记，并且容易受到可溶性蛋白质或杂质的干扰。这一限制是电动汽车在生物研究中广泛应用的重大障碍。在此，我们提出了一种新方法，利用邻近连接测定（PLA）和 DNA-RNA 杂交提前促进两种蛋白质在 EV 膜上的结合，从而能够分离和富集具有双阳性膜蛋白的完整 EV，然后使用用于捕获和酶裂解以释放分离的 EV 的功能化磁珠。分离的 EV 亚群可进一步用于细胞摄取研究、高通量小 RNA 测序和乳腺癌诊断。因此，开发和实施用于分离和富集特定细胞外囊泡亚群的专门系统可以增强该领域的基础和临床研究。本文受版权保护。保留所有权利。本文受版权保护。版权所有。

The isolation and enrichment of specific extracellular vesicle (EV) subpopulations are essential in the context of precision medicine. However, the current methods predominantly rely on a single-positive marker and are susceptible to interference from soluble proteins or impurities. This limitation represents a significant obstacle to the widespread application of EVs in biological research. Herein, we propose a novel approach that utilizes proximity ligation assay (PLA) and DNA-RNA hybridization to facilitate the binding of two proteins on the EV membrane in advance enabling the isolation and enrichment of intact EVs with double-positive membrane proteins followed by using functionalized magnetic beads for capture and enzymatic cleavage for isolated EVs release. The isolated subpopulations of EVs can be further utilized for cellular uptake studies, high-throughput small RNA sequencing, and breast cancer diagnosis. Hence, developing and implementing a specialized system for isolating and enriching a specific subpopulation of extracellular vesicles can enhance basic and clinical research in this field. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.