The functional membrane proteins on tumor-cell-derived EVs contain a large amount of biomolecular information, and can serve as a comprehensive marker to delineate the molecular nature of cancer. However, due to low secretion rates, it is difficult to perform accurate quantification and biomolecular analysis with conventional EV analysis technologies such as the Western blots. Here, we introduce a multifunctional EV analysis technology based on an automated microfluidic chemiluminescent ELISA (Enzyme-Linked ImmunoSorbent Assay) platform. With this system, we were able to achieve rapid EV quantification (<1 h) with relatively small sample volume (~8 μL) and high sensitivity (optimal LOD = $8.7\times {10}^7$ EV/mL). In addition to the EV quantification, we evaluated the expression levels for a panel of four cancer-related EV membrane proteins (EGFR, HER2, MHC-I, and EpCAM) using a newly developed immunoprofiling assay that combines immunoprecipitation and sandwich ELISA. Due to high sensitivity, this immunoprofiling assay only requires a very small amount of input protein (<40 ng/marker). Our studies show that the expression level of functional EV membrane proteins is stable under external stimulation, which suggests that the expression profile of the EV membrane proteins may serve as a robust and unique “molecular fingerprint” for the immunophenotyping of cancer cell lines.

肿瘤细胞衍生的电动汽车上的功能性膜蛋白包含大量的生物分子信息，可以用作描述癌症分子性质的综合标记。但是，由于分泌率低，因此难以通过传统的EV分析技术（例如Western印迹）进行准确的定量和生物分子分析。在这里，我们介绍基于自动微流化学发光ELISA（酶联免疫吸附测定）平台的多功能EV分析技术。使用此系统，我们能够以相对较小的样品量（〜8μL）和高灵敏度（最佳LOD = $8.7\times {10}^7$EV / mL）。除了EV定量分析外，我们还采用了新开发的结合免疫沉淀和三明治ELISA的免疫谱分析方法，评估了一组四种与癌症相关的EV膜蛋白（EGFR，HER2，MHC-1和EpCAM）的表达水平。由于灵敏度高，这种免疫谱分析仅需要非常少量的输入蛋白（<40 ng / marker）。我们的研究表明，功能性EV膜蛋白的表达水平在外部刺激下是稳定的，这表明EV膜蛋白的表达谱可作为癌细胞系免疫表型的强大而独特的“分子指纹”。