CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2020 Dec 1;92(23):15497-15505. doi: 10.1021/acs.analchem.0c03374. Epub 2020 Nov 11.
Exosomes, which are phospholipid bilayer nanovesicles, can transfer their content to recipient cells, playing a crucial role in intercellular communication. Exosomes have emerged as promising cancer biomarkers. However, a convenient, efficient, and economical approach for their isolation and comprehensive analysis is still technically challenging. In this study, aptamer-based immunoaffinitive magnetic composites, MagG@PEI@DSP@aptamer, were prepared to achieve the convenient capture, efficient enrichment, and mild release of exosomes. The constructed composites contain three segments: a PEI-modified magnetic graphene scaffold, an aptamer CD63 sequence, and a cleavable cross-linker in between. Notably, the binding capacity of MagG@PEI@DSP for an aptamer is 93 nmol/mg, and per milligram MagG@PEI@DSP@aptamer could capture 450 μg exosomes. Moreover, the released exosomes from MagG@PEI@DSP@aptamer composites were intact and well-dispersed. The prepared composites were then applied to profile the metabolite composition of exosomes secreted by breast cancer cells MCF-7, and the number of detected features was obviously increased when compared to that obtained by the traditional ultracentrifugation method (4528 vs 3710 and 3967 vs 3785 in the positive and negative ionization modes). Besides, the exosomes secreted by MCF-7 and normal breast cells MCF-10A were isolated from cell culture medium with MagG@PEI@DSP@aptamer, and their metabolic profiles were then comprehensively analyzed; in total, 119 metabolites in MCF-7 and MCF-10A were identified. Compared with exosomes from MCF-10A, 43 and 42 metabolites were upregulated and downregulated, respectively, in those from MCF-7. These data showed that the prepared MagG@PEI@DSP@aptamer composites can be used to effectively capture exosomes and further for metabolomics analysis.
外泌体是一种磷脂双层纳米囊泡，可以将其内容物转移到受体细胞中，在细胞间通讯中发挥着至关重要的作用。外泌体已成为有前途的癌症生物标志物。然而，一种方便、高效和经济的方法来分离和全面分析它们在技术上仍然具有挑战性。在这项研究中，基于适配体的免疫亲和磁性复合材料 MagG@PEI@DSP@aptamer 被制备出来，以实现外泌体的方便捕获、高效富集和温和释放。所构建的复合材料包含三个部分：PEI 修饰的磁性石墨烯支架、适配体 CD63 序列和中间的可切割交联剂。值得注意的是，MagG@PEI@DSP 对适配体的结合能力为 93 nmol/mg，每毫克 MagG@PEI@DSP@aptamer 可以捕获 450 μg 的外泌体。此外，从 MagG@PEI@DSP@aptamer 复合材料中释放的外泌体完整且分散良好。然后将制备的复合材料应用于分析乳腺癌细胞 MCF-7 分泌的外泌体的代谢物组成，与传统的超速离心法相比，检测到的特征数量明显增加（正、负离子模式下分别为 4528 对 3710 和 3967 对 3785）。此外，用 MagG@PEI@DSP@aptamer 从细胞培养液中分离 MCF-7 和正常乳腺细胞 MCF-10A 分泌的外泌体，然后对其代谢谱进行全面分析；总共鉴定出 MCF-7 和 MCF-10A 中的 119 种代谢物。与 MCF-10A 来源的外泌体相比，MCF-7 来源的外泌体中分别有 43 种和 42 种代谢物上调和下调。这些数据表明，所制备的 MagG@PEI@DSP@aptamer 复合材料可有效捕获外泌体，并进一步用于代谢组学分析。
