High-Throughput Electrokinetic Fractionation and Analysis of Extracellular RNA Nano-Carriers

项目摘要 细胞外RNA（exRNA），包括信使RNA（mRNA）和微小RNA（miRNA），起着重要的作用 在内分泌信号传导中，因此，对细胞与细胞的通讯至关重要。越来越多的证据表明 exRNA对疾病的发展至关重要，因此，对这些exRNA的分析将在以下方面发挥重要作用： 继续进行生物发现、诊断、治疗和药物开发。有三种重要的exRNA 保护exRNA不受大多数生理生物流体中存在的RNA酶影响的载体- 细胞外囊泡（EV）、脂蛋白（LLP）和核糖核蛋白（RNP）。但目前的 这些技术在区分载体特异性exRNA来源的能力方面受到限制， 无法准确建立exRNA图谱。除了缓慢和耗时之外，大多数 用于exRNA载体分离的技术本质上是低效和有损的，限制了 绝对或相对量化。在这项研究计划中，我们将开发一套高通量的 微流控技术将整合整个分析过程-exRNA的分离和隔离 载体，裂解和解离载体以释放它们的exRNA货物，以及敏感性和选择性的 靶exRNA、蛋白质和脂质的检测。通过利用微流体平台， 我们的成熟技术，我们预计，我们将能够完成整个分析小组的5 在3小时内从仅100 μL人血浆样品中检测靶向exRNA。这套工具将有一个 对我们理解exRNA生物学和检测exRNA产生了深远的变革性影响 表达作为生物标志物用于广泛的疾病。 我们的载体分离策略的基础将是基于连续等电分馏（CIF）的 我们的新型膜基自由流动电泳微流控装置。我们将使用离子交换 膜（IEM）作为pH致动器以建立自由流动的pH梯度，通过以下方式分离exRNA载体： 它们的等电点并将分离的载体洗脱成单独的等分试样。这个过程需要~30 分钟然后将EV和LLP等分试样注入表面声波（SAW）微流体装置中， 机械裂解它们以释放它们的exRNA货物，而RNP将在整合的盐上加工， 该方法包括分离、蛋白质/exRNA分离和纯化芯片，其利用IEM的离子耗尽特征。 最后，将开发四种检测方法：基于IEM的高丰度exRNA（> 106）传感器 拷贝），使用浸入式AC电喷雾（iACE）液滴产生的液滴PCR装置，用于低丰度 exRNA（102-106个拷贝），以及芯片上2D聚丙烯酰胺凝胶电泳（2D PAGE）和胶束电泳（2D PAGE）。 分别用于蛋白质和脂质的电动色谱（MEKC）。我们将优化整个过程， 包括样品转移、体积和时间，以满足我们的总体吞吐量和样品体积目标。

PROJECT ABSTRACT Extracellular RNA (exRNA), including messenger RNA (mRNA) and microRNA (miRNA), play an important role in endocrine signaling and as such, are critical to cell-to-cell communication. There is increasing evidence that exRNA are critical to disease development, and analysis of these exRNA, therefore, will play a vital role in continued biodiscovery, diagnostics, therapeutics, and drug development. There are three essential exRNA carriers that protect the exRNA from ever-present RNAses present in most physiological biofluids – extracellular vesicles (EVs), lipoproteins (LLPs), and ribonucleoproteins (RNPs). However, current technologies are limited in their ability to discriminate from which carrier specific exRNA originate and thus unable to accurately establish exRNA profiles. In addition to being slow and time consuming, most technologies for exRNA carrier isolation are inherently inefficient and lossy, limiting the effectiveness of absolute or even relative quantification. In this research program, we will develop a suite of high-throughput microfluidic technologies that will integrate the entire analysis process – separation and isolation of exRNA carriers, lysing and dissociation of the carriers to release their exRNA cargo, and the sensitive and selective detection of target exRNA, proteins, and lipids. By utilizing microfluidic platforms that build upon and expand our proven technologies, we anticipate that we will be able to complete the entire analysis for a panel of 5 target exRNA in 3 hours from only a 100 μL human blood plasma sample. This suite of tools will have a profound and transformative impact on advancing our understanding of exRNA biology and detecting exRNA expression as biomarkers for a wide range of diseases. The foundation for our carrier isolation strategy will be continuous isoelectric fractionation (CIF) based on our novel membrane-based free-flow electrophoresis microfluidic device. We will use ion exchange membranes (IEMs) as pH actuators to establish a free flowing pH gradient, separating the exRNA carrier by their isoelectric point and eluting the separated carriers into individual aliquots. This process should take ~30 min. The EV and LLP aliquots will then be injected into a surface acoustic wave (SAW) microfluidic device that mechanically lyses them to release their exRNA cargo, while RNPs will be processed on an integrated salt dissociation, protein/exRNA separation, and purification chip that utilizes the ion depletion feature of IEMs. Finally, four assays will be developed for detection: an IEM-based sensor for high abundance exRNA (> 106 copies), a droplet PCR device using immersed AC electrospray (iACE) droplet generation for low abundance exRNA (102-106 copies), and on-chip 2D polyacrylamide gel electrophoresis (2D PAGE) and micellar electrokinetic chromatography (MEKC) for proteins and lipids, respectively. We will optimize the entire process, including sample transfer, volume, and timing, to meet our overall throughput and sample volume targets.