Real-time bioluminescent detection of circulating biomarkers

循环生物标志物的实时生物发光检测

基本信息

批准号：
9330155
负责人：
CHRISTOPHER C. W. HUGHES
金额：
$ 19.31万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-15 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9330155
关键词：
Address Affect Alzheimer&apos s Disease Antibodies Binding Biological Assay Biological Markers Biology Biomedical Research Biomimetics Blood Glucose Blood Vessels C-terminal Cell Culture Techniques Cell physiology Cells Chimeric Proteins Clinical Coupled Detection Development Devices Diabetes Mellitus Dimerization Disease Environment Enzyme-Linked Immunosorbent Assay Enzymes Feedback Fostering Funding Funding Mechanisms Glucose Goals Grant Growth Health Heart Diseases Hour Human Human Biology Imagery Imaging technology Individual Insulin Insulin Receptor Islets of Langerhans Laboratories Light Liquid substance Luciferases Malignant Neoplasms Measurement Measures Mediating Metabolic Metabolic Diseases Methods Microfluidic Microchips Microfluidics Molecular Conformation Organ Output Pancreas Phase Process Proteins Reporter Research Sensitivity and Specificity Source Specimen Staining method Stains Structure System Technology Testing Time Tissues U-Series Cooperative Agreements United States National Institutes of Health Validation base blood glucose regulation circulating biomarkers design detector diabetic dimer human disease imaging approach in vivo infancy innovation islet metabolic abnormality assessment monomer noninvasive diagnosis novel personalized medicine protein biomarkers receptor receptor binding sample fixation small molecule tool

项目摘要

Project Summary / Abstract Organ-on-a-chip technology has recently presented new tools for studying cellular and micro-organ function within a 3D environment mimicking native organ structures. However, in these enclosed systems, it remains difficult to evaluate the health and function of live cells contained within these devices without retroactive fixation and antibody staining. Secreted protein biomarkers may provide an opportunity to non- invasively evaluate the health and function of these cells, although the tools to reliably sense these biomarkers in real-time have yet to be developed. We have recently applied an organ-on-a-chip approach to study the metabolic disorder diabetes. In healthy individuals, pancreatic islet structures secrete the small molecule insulin to negatively regulate blood glucose levels, a process that is disrupted in diabetic individuals resulting in elevated blood glucose levels. In order to study the mechanisms underlying defective islet function ex vivo, we have recently incorporated human pancreatic islets within a 3D, vascularized organ-on-a-chip platform which mimics the pancreas in vivo. To fully exploit this platform, a simple, reliable, and real-time method for sensing the secreted biomarker insulin is necessary to evaluate islet function within the chip. Here, we propose to design, develop, and validate a receptor-based, bioluminescent imaging approach to optimize the real-time detection of small protein biomarkers. Specifically, we will utilize split luciferase constructs coupled to the human insulin receptor and its substrate, IRS-1, to detect insulin binding, using light output as a read-out of insulin concentration. The sensitivity and specificity of these tools will be extensively validated in two developmental phases: static 2D culture; followed by application in a 3D, biomimetic pancreas-on-a-chip platform. While this proposal focuses on developing these tools for insulin detection, our approach has broad implications in the detection of other secreted biomarkers in both static culture and dynamic microfluidic platforms.

项目摘要 /摘要 Organ-A-A-Chip技术最近提出了研究细胞和微孔器的新工具在模仿天然器官结构的3D环境中起作用。但是，在这些封闭的系统中，在没有这些设备中包含的活细胞的健康和功能的情况下仍然难以评估这些设备的健康和功能追溯固定和抗体染色。分泌的蛋白质生物标志物可能为非 - 尽管可以可靠地感知这些生物标志物的工具，但可以侵入地评估这些细胞的健康和功能实时尚未开发。我们最近采用了一种器官芯片方法来研究代谢疾病糖尿病。在健康个体中，胰岛结构分泌小分子胰岛素对负调节血糖水平进行负调节，这一过程在糖尿病患者中被破坏，导致血糖水平升高。为了研究有缺陷胰岛功能的机制，我们最近已将人类胰岛融合在一个3D的血管化器官片平台中模仿体内胰腺。为了充分利用此平台，一种简单，可靠和实时的传感方法分泌的生物标志物胰岛素对于评估芯片中的胰岛功能是必要的。在这里，我们建议设计，开发和验证一种基于受体的生物发光成像方法，以优化实时检测小蛋白质生物标志物。具体而言，我们将利用分裂的荧光素酶构造与人类胰岛素受体及其底物IRS-1，以光输出为读出胰岛素结合胰岛素浓度。这些工具的敏感性和特异性将在两个中得到广泛验证发展阶段：静态2D文化；然后在3D植物胰腺上施用平台。尽管该建议着重于开发这些胰岛素检测工具，但我们的方法已经在静态培养和动态中检测其他分泌生物标志物的广泛含义微流体平台。