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提出是疾病状态的特定生物标志物，包括最多的疾病状态 诸如心血管疾病和癌症之类的流行患者。但是使用miRNA作为生物标志物非常 尽管高通量miRNA分析取得了进步，但仍具有挑战性。各种检测技术， 方案，连接和提取/纯化方法已导致细胞和 在不同条件下的生物流体。最重要的是，所有这些都需要长时间的样品到答案时间，因此 排除它们以检测和监测紧急，威胁生命的状况，例如心肌梗塞 （Mi）。快速实时的无PCR miRNA促进装置对于精确的精确度非常有价值， 未来几年的个性化药物。但是，即使开发这样的平台也很难 由于测试模型的局限性。动物模型通常无法预测人类的反应。和研究 人类受试者不容易允许精确控制疾病事件或时间相关性 疾病状态和生物标志物表达动力学。为了应对这一挑战，在这项研究中，我们将开发一个 具有集成attomolor（AM）级miRNA感应能力的器官芯片设备，我们将用于 优化对新生物标志物发现的多个miRNA中波动的实时监测。作为 立即应用，我们将从片上的人类心肌（MOC）作为临床相关模型开始 模仿心脏病的过程。我们假设将MOC与超敏感miRNA检测到，我们 将发现一个独特的特征，表明在MI治疗期间再灌注损伤发作。最后，我们会的 使用临床血液样本测试传感器设备和miRNA特征。我们的微流体器官片 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的人类MOC 多能干细胞（HIPSC），2）外泌体裂口单元，3）裂解RNA的浓度单元和4） miRNA的检测单元。在AIM 1中，我们将将这些组件融入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，我们将使用并优化使用新型miRNA检测生物传感器的性能 MOC和基准对建立的miRNA分析技术进行基准测试。在AIM 2中，我们将专注于多重 实时检测一个miRNA的传感方法，1）使用MOC发现miRNA 签名用作捕获RI发作的新型生物标志物，以及2）以优化多路复用 传感器用于更快的临床翻译。在AIM 3中，我们将确定该诊断和预后能力 新型生物传感器和miRNA生物标志物签名我们使用MOC模型在AIMS 1和2中开发 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 他们的RNA含量以提高当前对人类健康作用的了解并确定其潜力 作为疾病状态的生物标志物。