Jonsson Comprehensive Cancer Center , University of California Los Angeles , Los Angeles , California 90024 , United States.
ACS Sens. 2019 Feb 22;4(2):488-497. doi: 10.1021/acssensors.8b01564. Epub 2019 Jan 31.
Exosomes contain cell- and cell-state-specific cargos of proteins, lipids, and nucleic acids and play significant roles in cell signaling and cell-cell communication. Current research into exosome-based biomarkers has relied largely on analyzing candidate biomarkers, i.e., specific proteins or nucleic acids. However, this approach may miss important biomarkers that are yet to be identified. Alternative approaches are to analyze the entire exosome system, either by "omics" methods or by techniques that provide "fingerprints" of the system without identifying each individual biomolecule component. Here, we describe a platform of the latter type, which is based on surface-enhanced Raman spectroscopy (SERS) in combination with multivariate analysis, and demonstrate the utility of this platform for analyzing exosomes derived from different biological sources. First, we examined whether this analysis could use exosomes isolated from fetal bovine serum using a simple, commercially available isolation kit or necessitates the higher purity achieved by the "gold standard" ultracentrifugation/filtration procedure. Our data demonstrate that the latter method is required for this type of analysis. Having established this requirement, we rigorously analyzed the Raman spectral signature of individual exosomes using a unique, hybrid SERS substrate made of a graphene-covered Au surface containing a quasi-periodic array of pyramids. To examine the source of the Raman signal, we used Raman mapping of low and high spatial resolution combined with morphological identification of exosomes by scanning electron microscopy. Both approaches suggested that the spectra were collected from single exosomes. Finally, we demonstrate for the first time that our platform can distinguish among exosomes from different biological sources based on their Raman signature, a promising approach for developing exosome-based fingerprinting. Our study serves as a solid technological foundation for future exploration of the roles of exosomes in various biological processes and their use as biomarkers for disease diagnosis and treatment monitoring.
外泌体包含细胞和细胞状态特异性的蛋白质、脂质和核酸货物，并在细胞信号转导和细胞间通讯中发挥重要作用。目前基于外泌体的生物标志物的研究主要依赖于分析候选生物标志物，即特定的蛋白质或核酸。然而，这种方法可能会错过尚未被识别的重要生物标志物。替代方法是分析整个外泌体系统，要么通过“组学”方法，要么通过提供系统“指纹”而不识别每个单个生物分子成分的技术。在这里，我们描述了一种后一种类型的平台，该平台基于表面增强拉曼光谱（SERS）与多元分析相结合，并展示了该平台用于分析来自不同生物来源的外泌体的实用性。首先，我们检查了这种分析是否可以使用简单的、市售的分离试剂盒从胎牛血清中分离的外泌体进行，或者是否需要“金标准”超速离心/过滤程序所达到的更高纯度。我们的数据表明，对于这种类型的分析，后一种方法是必需的。在建立了这一要求之后，我们使用独特的混合 SERS 基底对单个外泌体的拉曼光谱特征进行了严格分析，该基底由含有准周期性金字塔阵列的石墨烯覆盖的 Au 表面制成。为了检查拉曼信号的来源，我们使用低和高空间分辨率的拉曼映射结合扫描电子显微镜对外泌体的形态识别进行了分析。这两种方法都表明，光谱是从单个外泌体中收集的。最后，我们首次证明，我们的平台可以根据其拉曼特征区分来自不同生物来源的外泌体，这是开发基于外泌体的指纹识别的一种很有前途的方法。我们的研究为未来探索外泌体在各种生物过程中的作用以及将其用作疾病诊断和治疗监测的生物标志物提供了坚实的技术基础。
