Enabling exosome biomarker development via digitized single vesicle analysis

项目总结/摘要 外泌体代表了诊断生物标志物的下一个“组学”前沿。因此，许多技术 已经开发了利用外来体蛋白或微小RNA（miRNA）货物的检测作为潜在的生物标志物 发展一般来说，这些方法是基于这样的假设，即检测到特定的 外泌体蛋白或miRNA谱将与特定疾病相关。然而，这一假设被以下因素所破坏： 从生物流体中收集的外来体群体的异质性导致的固有可变性。 此外，先前开发蛋白质，特别是基于miRNA的生物标志物的尝试已经揭示， 一个小RNA序列可以与许多疾病相关，即使没有干扰， 外来体的异质性。因此，我们假设，为了获得适当的特异性， 外泌体生物标志物，外泌体蛋白和miRNA的相关性-同时检测和可分辨 在单个外泌体水平上-是必要的。在这里，我们提出了一个高密度的多路复用微系统 实施用于数字外泌体分析的新的等温核酸扩增方法， 在单个外泌体水平上，膜蛋白标记物和miRNA序列的特异性相关性。一个低- 具有集成的扩增试剂的成本和易于使用的热塑性芯片使得能够自离散化 用于空间多路复用分析的外泌体，可扩展到一百万个反应。用于特异性检测 miRNA序列和膜蛋白，我们设计了一个等温反应优化的， 扩增短DNA和RNA序列，其鉴定miRNA序列以及DNA 与标记外泌体表面蛋白的抗体缀合的寡核苷酸。该反应称为 转录循环扩增（TCA），利用具有核酸外切酶活性的DNA聚合酶降解 供体/猝灭剂标记的探针（即，FRET探针）用于特异性和实时定量检测。一前一后， RNA聚合酶作用于聚合的寡核苷酸，用于循环产生RNA，导致指数生长。 扩增，提供高度灵敏的检测。通过整合这两种工具，我们将开发一种生物标志物， 发现平台，该平台首先通过密集的反应阵列数字化外泌体，并将miRNA 在单个外泌体中具有膜蛋白的序列。

PROJECT SUMMARY/ABSTRACT Exosomes represent the next “omic” frontier in diagnostic biomarkers. As such, numerous technologies have been developed to exploit detection of exosomal proteins or microRNA (miRNA) cargo for potential biomarker development. In general, these approaches are based on the assumption that detection of a particular exosomal protein or miRNA profile will correlate with a specific disease. Yet, this assumption is undermined by the inherent variability resulting from heterogeneity of exosome populations collected from biological fluids. Further, previous attempts to develop protein and, especially, miRNA-based biomarkers have revealed that a single miRNA sequence can be correlated with numerous diseases even without the confounding heterogeneity of exosomes. Thus, we hypothesize that to achieve appropriate specificity for the discovery of exosomal biomarkers, correlation of exosomal proteins and miRNAs – simultaneously detected and resolvable at the single-exosome level – is necessary. Here we propose a highly dense multiplexed microsystem implementing a new isothermal nucleic acid amplification method for digital exosome analysis, enabling the specific correlation of membrane protein markers and miRNA sequences at the single exosome level. A low- cost and easy-to-use thermoplastic chip with integrated amplification reagents enables self-discretization of exosomes for spatially multiplexed analysis, scalable up to one million reactions. For specific detection of miRNA sequences and membrane proteins, we have designed an isothermal reaction optimized for the amplification of short DNA and RNA sequences that identifies miRNA sequences as well as DNA oligonucleotides conjugated to antibodies that label the exosome surface proteins. The reaction, referred to as transcription cycling amplification (TCA), utilizes DNA polymerase with exonuclease activity to degrade donor/quencher-labeled probes (i.e., FRET probes) for specific and real-time quantitative detection. In tandem, RNA polymerase acts on the polymerized oligo for the cyclical generation of RNA that leads to exponential amplification, providing highly sensitive detection. By integrating these two tools, we will develop a biomarker discovery platform that first digitizes the exosomes across a dense reaction array and correlates miRNA sequences with membrane proteins in single exosomes.