An Integrated Microfluidics Platform for Rapid and Sensitive Exosome RNA

 描述（由申请人提供）：最近的发现表明，称为外泌体的微囊泡通常在细胞到细胞的信息传递中发挥关键作用，作为携带信使RNA（mRNA）和微小RNA（miRNA）的运输平台。至关重要的是，这种细胞与细胞之间的交流会影响基因转录，并最终影响受体细胞的蛋白质生产，通常会将它们转化为疾病状态。出于这个原因，外泌体RNA被认为在各种各样的生物学功能中发挥重要作用，例如适应性免疫反应和心脏组织修复，以及在从阿尔茨海默病和多发性硬化到心血管疾病和癌症的疾病的形成和发展中发挥重要作用。此外，外泌体存在于大多数生物流体中，包括血液、唾液和尿液，因此是作为早期疾病检测的生物标志物的首选。然而，提取外来体并分析它们携带的RNA是一项漫长、复杂且昂贵的实验任务，需要分离、分离和操作的多个步骤。在这项研究计划中，我们将开发一种低成本、高通量的微流控平台，可以快速从生物样品中分离外来体，并灵敏地检测它们携带的mRNA和miRNA。我们设想，这种通过检测和定量外泌体RNA的新型仪器将对临床医生如何能够诊断早期疾病并为疾病发展建立诊断标准产生重大影响，成为一种改变游戏规则的诊断和治疗仪器。我们将设计集成设备，使其快速，灵敏，具有成本效益，需要很少的生物材料，并利用一次性组件。我们将通过在健康和癌性胰腺细胞系中筛选靶生物标志物外泌体miRNA来证明微流控生物芯片的分析能力，并建立该设备的诊断和预后能力。所提出的微流体平台将从其背景生物材料中分离和分离外泌体，然后随后裂解它们并直接检测释放的靶miRNA。我们将利用尺寸排阻膜将外泌体（~30-100 nm）与复杂样品（细胞培养基、血液、腹水）中的其他生物分子分离。在压电微流体基底的表面上产生的表面声波（SAW）将产生高剪切力（经由声流）和高电场（经由机电耦合）以裂解分离的外泌体并释放它们的RNA。我们将用离子交换膜检测miRNA，其中固定的寡核苷酸探针将与靶miRNA杂交，并从根本上改变膜的电流-电压关系。我们多元化的跨学科研究团队将优化和整合这些组件中的每一个，表征来自健康患者和胰腺导管腺癌患者的上皮细胞系分泌的外泌体模型系统的性能，以标准分析技术为基准。我们的目标是将分析时间从使用当前方法的数小时/天减少到60分钟以下，检测限低于pM。

 DESCRIPTION (provided by applicant): Recent discoveries have shown that microvesicles, called exosomes, often take on a pivotal role in cell-to-cell messaging, serving as the transport platform that carries messenger RNA (mRNA) and microRNA (miRNA). Crucially, this cell-to-cell communication can affect gene transcription and ultimately, the protein production of recipient cells, often converting them to a disease state. For this reason, exosomal RNA is believed to play an important role in a wide variety of biological functions, such as adaptive immune response and cardiac tissue repair, as well as in the formation and development of diseases ranging from Alzheimer's disease and multiple sclerosis to cardiovascular disease and cancer. Further, exosomes are found in most biological fluids, including blood, saliva, and urine, and therefore are prime to serve as biomarkers for early disease detection. However, extracting exosomes and analyzing the RNA they carry, is a long, involved, and expensive experimental task, requiring multiple steps of separation, isolation, and manipulation. In this research program, we will develop a low-cost, high-throughput microfluidic platform that rapidly isolates exosomes from biological samples and sensitively detects the mRNA and miRNA they carry. We envision that this novel instrument for by detecting and quantifying exosomal RNA will have a major impact on how clinicians are able to diagnose early-stage disease and establish prognoses for disease development, becoming a game-changing diagnostic and therapeutic instrument. We will engineer the integrated device to be rapid, sensitive, and cost effective, requiring little biological material and utilizing disposable components. We will demonstrate the analytical proficiency of the microfluidic biochip by screening for a target biomarker exosomal miRNA in both healthy and cancerous pancreatic cell lines and establish the device's diagnostic and prognostic capability. The proposed microfluidic platform will separate and isolate exosomes from their background biological material and then subsequently lyse them and directly detect released target miRNA. We will utilize a size-exclusion membrane to separate the exosomes (~30-100 nm) from other biomolecules in complex samples (cell media, blood, ascites fluid). Surface acoustic waves (SAWs) generated on the surface of a piezoelectric microfluidic substrate will produce both high shear forces (via acoustic streaming) and high electric fields (via electro-mechanical coupling) to lyse the separated exosomes and release their RNA. We will detect miRNA with an ion-exchange membrane where immobilized oligonucleotide probes will hybridize with target miRNA and fundamentally alter the current-voltage relationship of the membrane. Our diverse, interdisciplinary research team will optimize and integrate each of these components, characterizing performance on a model system of exosomes secreted by epithelial cell lines from both healthy patients and those with pancreatic ductal adenocarcinoma, benchmarking against standard analysis techniques. Our goal is to reduce the analysis time from many hours/days using current methods to under 60 min with sub-pM limits of detection.