Exosomes that carry multiple proteins from the originating cells are known as emerging biomarkers for tumor diagnostics. However, it is still technically challenging to accurately evaluate subtle differences of exosomal membrane proteins. Here, we developed a rolling circle amplification (RCA)-assisted flow cytometry approach (FCA) to simultaneously profile surface proteins and quantify exosomes. In this work, specific anti-CD63 antibody-conjugated magnetic beads were first utilized to capture exosomes. Then, the captured exosomes were bound with DNA primers, which comprise exosomal surface protein-specific recognition aptamers. The RCA reaction generates repeat DNA sequences for fluorescent probe hybridization. Finally, a conventional flow cytometer was introduced to phenotype exosomal protein markers. Such a sensitive RCA-assisted FCA displays an excellent detection limit of 1.3 × 10⁵ exosome/mL. The variable composition of four protein markers on different cell-derived exosomes was sensitively detected through changing the protein-recognition sequence of the DNA primer, which reveals a heterogeneous pattern. Exosomes from different cell sources could be distinguished by the abundance difference of multiple surface proteins. Furthermore, the developed RCA-assisted FCA enabled quantitative analysis of blood samples from lung cancer patients, indicating its potential for early clinical diagnosis and prognosis of cancer.

中文翻译：

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滚环扩增辅助流式细胞术方法同时分析外泌体表面蛋白

携带来自原始细胞的多种蛋白质的外泌体被称为肿瘤诊断的新兴生物标志物。然而，准确评估外泌体膜蛋白的细微差异在技术上仍然具有挑战性。在这里，我们开发了一种滚环扩增 (RCA) 辅助流式细胞术方法 (FCA)，以同时分析表面蛋白和量化外泌体。在这项工作中，首先使用特异性抗 CD63 抗体偶联的磁珠来捕获外泌体。然后，捕获的外泌体与包含外泌体表面蛋白特异性识别适体的 DNA 引物结合。RCA 反应生成用于荧光探针杂交的重复 DNA 序列。最后，将常规流式细胞仪引入表型外泌体蛋白标记物。⁵外泌体/毫升。通过改变 DNA 引物的蛋白质识别序列，灵敏地检测到不同细胞来源的外泌体上四种蛋白质标志物的可变组成，揭示了一种异质性模式。来自不同细胞来源的外泌体可以通过多种表面蛋白的丰度差异来区分。此外，开发的 RCA 辅助 FCA 能够对肺癌患者的血液样本进行定量分析，表明其在癌症早期临床诊断和预后方面的潜力。