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An Integrated Microfluidics Platform for Rapid and Sensitive Exosome RNA

用于快速、灵敏外泌体 RNA 的集成微流体平台

基本信息

批准号：
9352868
负责人：
Hsueh-Chia Chang
金额：
$ 19.31万
依托单位：
UNIVERSITY OF NOTRE DAME
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：
2016-09-14 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9352868
关键词：
Acoustics Affect Alzheimer&apos s Disease Animals Ascites Benchmarking Biocompatible Materials Biological Biological Markers Biological Models Biological Process Blood Cancerous Cardiac Cardiovascular Diseases Cell Communication Cell Culture System Cell Culture Techniques Cell Line Cells Charge Clinical Complex Coupling Detection Development Device or Instrument Development Devices Diagnosis Diagnostic Disease Early Diagnosis Engineering Epithelial Cells Evaluation Exclusion Genetic Transcription Goals Hour Human Human Resources Hybrids Immobilization Individual Infection Interdisciplinary Study Ion Exchange Liquid substance Malignant Neoplasms Malignant neoplasm of pancreas Measures Mechanics Membrane Messenger RNA Methods MicroRNAs Microfluidic Microchips Microfluidics Modeling Monitor Multiple Sclerosis Oligonucleotide Probes Outcome Pancreas Pancreatic Ductal Adenocarcinoma Patients Performance Physiological Play Polymerase Chain Reaction Production Proteins Quantitative Reverse Transcriptase PCR RNA RNA analysis Research Role Saliva Sampling Side Speed Stream Surface Techniques Therapeutic Time Travel Urine Work adaptive immune response biochip cancer biomarkers cost cost effective design diagnostic screening electric field exosome fight against improved instrument microvesicles mouse model novel operation outcome forecast pancreatic cancer cells pancreatic cell line particle portability prevent prognostic programs public health relevance rapid detection response screening sensor targeted biomarker tissue repair tumor initiation voltage

项目摘要

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.