使用原子力显微镜、扫描电子显微镜和拉曼光谱法对细胞外囊泡进行免疫捕获，以实现个体的多模式特征分析。
Immuno-capture of extracellular vesicles for individual multi-modal characterization using AFM, SEM and Raman spectroscopy.
机构信息
Laboratory of Organic Chemistry, Wageningen University, The Netherlands and Applied Microfluidics for BioEngineering Research, MESA+ Institute for Nanotechnology and TechMed Center, University of Twente, The Netherlands.
Medical Cell BioPhysics, TechMed Center, University of Twente, The Netherlands.
出版信息
Lab Chip. 2019 Jul 23;19(15):2526-2536. doi: 10.1039/c9lc00081j.
Tumor-derived extracellular vesicles (tdEVs) are promising blood biomarkers for cancer disease management. However, blood is a highly complex fluid that contains multiple objects in the same size range as tdEVs (30 nm-1 μm), which obscures an unimpeded analysis of tdEVs. Here, we report a multi-modal analysis platform for the specific capture of tdEVs on antibody-functionalized stainless steel substrates, followed by their analysis using SEM, Raman spectroscopy and AFM, at the single EV level in terms of size and size distribution, and chemical fingerprint. After covalent attachment of anti-EpCAM (epithelial cell adhesion molecule) antibodies on stainless steel substrates, EV samples derived from a prostate cancer cell line (LnCAP) were flushed into a microfluidic device assembled with this stainless steel substrate for capture. To track the captured objects between the different analytical instruments and subsequent correlative analysis, navigation markers were fabricated onto the substrate from a cyanoacrylate glue. Specific capture of tdEVs on the antibody-functionalized surface was demonstrated using SEM, AFM and Raman imaging, with excellent correlation between the data acquired by the individual techniques. The particle distribution was visualized with SEM. Furthermore, a characteristic lipid-protein band at 2850-2950 cm-1 was observed with Raman spectroscopy, and with AFM the size distribution and surface density of the captured EVs was assessed. Finally, correlation of SEM and Raman images enabled discrimination of tdEVs from cyanoacrylate glue particles, highlighting the capability of this multi-modal analysis platform for distinguishing tdEVs from contamination. The trans-instrumental compatibility of the stainless steel substrate and the possibility to spatially correlate the images of the different modalities with the help of the navigation markers open new avenues to a wide spectrum of combinations of different analytical and imaging techniques for the study of more complex EV samples.
肿瘤衍生的细胞外囊泡（tdEVs）是癌症疾病管理有前途的血液生物标志物。然而，血液是一种高度复杂的流体，其中包含与 tdEVs（30nm-1μm）相同大小范围内的多个物体，这阻碍了对 tdEVs 的无障碍分析。在这里，我们报告了一种多模式分析平台，用于在抗体功能化不锈钢基底上特异性捕获 tdEVs，然后使用 SEM、拉曼光谱和 AFM 在单个 EV 水平上对其进行分析，从大小和尺寸分布以及化学指纹的角度分析。在不锈钢基底上共价附着抗-EpCAM（上皮细胞黏附分子）抗体后，将源自前列腺癌细胞系（LnCAP）的 EV 样品冲洗到装有该不锈钢基底的微流控装置中进行捕获。为了在不同分析仪器之间跟踪捕获的物体并进行后续相关分析，导航标记是用氰基丙烯酸酯胶在基底上制造的。使用 SEM、AFM 和拉曼成像证明了 tdEVs 在抗体功能化表面上的特异性捕获，并且各个技术获得的数据之间具有极好的相关性。使用 SEM 可视化了颗粒分布。此外，在拉曼光谱中观察到 2850-2950cm-1 处的特征脂质-蛋白质带，并且通过 AFM 评估了捕获的 EV 的大小分布和表面密度。最后，SEM 和拉曼图像的相关性使得能够将 tdEVs 与氰基丙烯酸酯胶颗粒区分开来，突出了这种多模式分析平台用于区分 tdEVs 与污染的能力。不锈钢基底的跨仪器兼容性以及借助导航标记将不同模式的图像进行空间相关的可能性为研究更复杂的 EV 样品的不同分析和成像技术的广泛组合开辟了新途径。
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