Single Extracellular Vesicle Sorting and Analysis

项目摘要 胞外囊泡是一种由多种细胞分泌的膜包裹颗粒。 进入细胞外空间。电动汽车是高度异质的，并包括一组不同的表面 蛋白质标记物以及囊泡内的货物，如RNA。当前的隔离方法 电动汽车的研究缺乏必要的敏感性和精确度，无法充分描述和理解 可能存在的各种EV亚群的构成和分布。事实上，最新的 EV分离方法，包括差速离心法、亲和/免疫磁法、 基于聚合物的沉淀，基于大小的排除，可能容易受到污染；例如， 亲和/免疫磁性方法可能会受到非特异性相互作用的不利影响，这些相互作用可能 造成污染物与电动汽车的共沉淀。 此外，这些批量技术只能报告平均超过数百万 EVS，因此不能用来理解存在于 电动汽车。然而，了解电动汽车及其运载的货物的多样性是迈向 更好地了解电动汽车在生理和心理方面所扮演的确切角色 病理生理过程。反过来，这种理解对于实现潜力是重要的。 电动汽车在诊断(例如，作为一类新的生物标志物)和治疗(例如，作为药物载体)方面的应用。 为了解决当前方法的局限性，我们建议开发和应用三套 在单一电动汽车水平上研究高通量电动汽车的新的但相关的技术。这些新的 技术包括纳米级流动分析仪和流动分类器，用于分析和分类个体 具有高灵敏度和高吞吐量的电动汽车。此外，我们还计划演示一种成像 用于量化单个电动汽车和小群电动汽车的RNA含量的数字聚合酶链式反应平台。

Project Summary Extracellular vesicles (EVs) are membrane-enclosed particles, which are secreted from various cell types into the extracellular space. EVs are highly heterogeneous and comprise a diverse set of surface protein markers as well as intra-vesicular cargoes, such as RNAs. Current approaches to the isolation and study of EVs lack the necessary sensitivity and precision to fully characterize and understand the make-up and the distribution of various EV subpopulations that may be present. In fact, most current EV isolation methodologies, including differential centrifugation, affinity/immuno-magnetic isolation, polymer-based precipitation, size-based exclusion, can be prone to contaminations; for example, affinity/immuno-magnetic methods can be adversely affected by non-specific interactions that can cause co-precipitation of contaminants with EVs. Additionally, these bulk techniques can only report information that is averaged over many millions of EVs, and thus cannot be used to understand the high degree of heterogeneity that exists among the EVs. Yet, understanding the diversity of EVs and the cargos they carry is an essential step towards gaining a better understanding of the precise roles EVs play in both physiological and pathophysiological processes. This understanding, in turn, is important towards realizing the potentials of EVs in diagnostics (e.g. as a new class of biomarkers) and therapeutics (e.g. as drug carriers). To address the limitations of the current methodologies, we propose to develop and apply three sets of new but related technologies for studying EVs with high-throughput at the single-EV level. These new techniques include a nanoscale flow analyzer and flow sorter for the analysis and sorting of individual EVs with high sensitivity and high throughput. Furthermore, we also plan to demonstrate an imaging digital PCR platform for quantifying the RNA contents of single EVs as well as small groups of EVs.