An integrated human organ-on-chip ultrasensitive miRNA detection platform for novel biomarker discovery

用于新型生物标志物发现的集成人体器官芯片超灵敏 miRNA 检测平台

• 批准号: 10226151
• 负责人: Pinar Zorlutuna
• 金额: $ 38.57万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10226151
• 关键词: Address; Animal Model; Benchmarking; Biological Assay; Biological Markers; Biosensing Techniques; Biosensor; Blood Circulation; Blood specimen; Cardiovascular Diseases; Cell Culture Techniques; Cells; Clinical; Coronary Arteriosclerosis; Culture Media; Detection; Development; Devices; Diagnostic; Disease; Disease model; Distress; Event; Fingerprint; Glucose; Goals; Health; Heart; Hematological Disease; Histologic; Human; Human Engineering; Hypoxia; Individual; Ischemia; Life; Ligation; Link; Liquid substance; Malignant Neoplasms; Methods; MicroRNAs; Microfluidic Microchips; Microfluidics; Modeling; Monitor; Myocardial; Myocardial Infarction; Myocardial tissue; Myocardium; Oxidative Stress; Patients; Performance; Physiological; Preparation; Property; Protocols documentation; RNA; Reperfusion Injury; Reperfusion Therapy; Role; Sampling; System; Techniques; Technology; Testing; Time; Tissue Engineering; Tissue Model; Tissue Sample; Tissues; Validation; Ventricular; base; biomarker development; biomarker discovery; biomarker signature; circulating microRNA; clinical application; clinical translation; clinically relevant; conditioning; deep sequencing; detection method; detection platform; disease phenotype; drug discovery; exosome; experimental study; human model; human subject; human tissue; induced pluripotent stem cell; innovation; ischemic conditioning; miRNA expression profiling; microRNA biomarkers; normoxia; novel; novel marker; novel strategies; organ on a chip; percutaneous coronary intervention; personalized medicine; potential biomarker; predicting response; prognostic; real time monitoring; sensor; specific biomarkers; tool

Abstract Circulating miRNAs have proposed as specific biomarkers of disease states, including some of the most prevailing ones such as cardiovascular diseases and cancer. However using miRNAs as biomarkers is very challenging despite recent advances in high-throughput miRNA profiling. Various detection technologies, protocols, ligation and extraction/purification methods have led to varying miRNA profiling results of cells and biofluids under different conditions. Most importantly, all require days long sample-to-answer assay times, thus ruling them out for detection and monitoring of urgent, life threatening conditions such as myocardial infarction (MI). A rapid real-time, PCR-free miRNA-profiling device would be exceedingly valuable for precision, personalized medicine in years to come. However, it is very difficult to start even developing such a platform because of the limitations in testing models. Animal models often fail to predict responses in humans; and studies of human subjects do not readily allow for precise control over the disease events or temporal correlation of the disease state and biomarker expression dynamics. To address this challenge, in this study, we will develop an organ-on-a-chip device with an integrated attomolar (aM)-level miRNA sensing capability, which we will use for optimizing real-time monitoring of fluctuations in multiple miRNAs for novel biomarker discovery. As an immediate application, we will start with a human myocardium-on-chip (MoC) as a clinically relevant model and imitate the course of a heart attack. We hypothesize that using the MoC with ultrasensitive miRNA detection, we will discover a unique signature that indicates the onset of reperfusion injury during MI treatment. Finally, we will test the sensor device and the miRNA signature using clinical blood samples. Our microfluidic organ-on-a-chip platform will consist of four basic components: 1) the tissue engineered human MoC from human induced pluripotent stem cells (hiPSCs), 2) the exosome lysing unit, 3) the concentration unit for the lysed RNAs and 4) the detection unit for the miRNAs. In Aim 1, we will couple these components into a fully integrated microfluidic platform. First we will validate the clinical relevance of the MoC model by comparing with human tissue and blood samples. Then we will characterize and optimize the performance of a novel miRNA detection biosensor using MoC and benchmark it against established miRNA analysis techniques. In Aim 2 we will focus on multiplexing the sensing approach for the real-time detection of a panel of miRNAs, and 1) use the MoC to discover a miRNA signature to be used as a novel biomarker that captures the RI onset, as well as 2) to optimize the multiplexed sensor for faster clinical translation. In Aim 3 we will determine the diagnostic and prognostic capabilities of the novel biosensor and miRNA biomarker signature we developed in Aims 1 and 2 using the MoC model, with clinical samples from MI patients. Our long-term goal is to utilize this integrated platform to study exosomes and their RNA content to advance current understanding of their role in human health and to determine their potential as biomarkers for disease states.

抽象的 循环 miRNA 已被提议作为疾病状态的特定生物标志物，包括一些最常见的疾病状态。 心血管疾病、癌症等常见疾病。然而，使用 miRNA 作为生物标志物是非常困难的。 尽管高通量 miRNA 分析最近取得了进展，但仍具有挑战性。各种检测技术， 方案、连接和提取/纯化方法导致细胞和细胞的 miRNA 分析结果不同 不同条件下的生物体液。最重要的是，所有这些都需要数天的样本到结果分析时间，因此 排除它们来检测和监测紧急的、危及生命的疾病，例如心肌梗死 （MI）。快速实时、免 PCR 的 miRNA 分析设备对于精确、 未来几年的个性化医疗。然而，即使是开发这样的平台，也很难开始 由于测试模型的限制。动物模型通常无法预测人类的反应；和研究 人类受试者的数据不容易精确控制疾病事件或疾病的时间相关性 疾病状态和生物标志物表达动态。为了应对这一挑战，在本研究中，我们将开发一种 具有集成阿摩尔 (aM) 级 miRNA 传感功能的器官芯片设备，我们将使用它 优化多个 miRNA 波动的实时监测，以发现新的生物标志物。作为一个 立即应用，我们将从人类芯片上心肌（MoC）作为临床相关模型开始， 模仿心脏病发作的过程。我们假设使用具有超灵敏 miRNA 检测功能的 MoC，我们 将发现一个独特的特征，表明心肌梗死治疗期间再灌注损伤的发生。最后，我们将 使用临床血液样本测试传感器设备和 miRNA 签名。我们的微流控芯片器官 该平台将由四个基本组成部分组成：1）来自人类诱导的组织工程人类MoC 多能干细胞 (hiPSC)，2) 外泌体裂解单位，3) 裂解 RNA 的浓度单位，4) miRNA 的检测单元。在目标 1 中，我们将把这些组件耦合到一个完全集成的微流体中 平台。首先，我们将通过与人体组织和血液进行比较来验证 MoC 模型的临床相关性 样品。然后我们将使用以下方法表征和优化新型 miRNA 检测生物传感器的性能 MoC 并将其与已建立的 miRNA 分析技术进行基准测试。在目标 2 中，我们将重点关注多路复用 用于实时检测一组 miRNA 的传感方法，以及 1) 使用 MoC 发现 miRNA 签名用作捕获 RI 起始的新型生物标志物，以及 2) 优化多重检测 传感器可实现更快的临床转化。在目标 3 中，我们将确定诊断和预后能力 我们使用 MoC 模型在目标 1 和 2 中开发了新型生物传感器和 miRNA 生物标志物特征，其中 来自 MI 患者的临床样本。我们的长期目标是利用这个集成平台来研究外泌体和 它们的 RNA 含量可促进目前对其在人类健康中的作用的了解并确定其潜力 作为疾病状态的生物标志物。