High-Throughput Electrokinetic Fractionation and Analysis of Extracellular RNA Nano-Carriers

细胞外 RNA 纳米载体的高通量电动分离和分析

• 批准号: 9811910
• 负责人: Hsueh-Chia Chang
• 金额: $ 46.35万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9811910
• 关键词: Acoustics; Aliquot; Biological Assay; Biological Markers; Biology; Blood; Cell Communication; Cells; Centrifugation; Chromatography; Communication; Consumption; DNA; Detection; Development; Devices; Diagnostic; Disease; Dissociation; Effectiveness; Electrophoresis; Endocrine; Extracellular Protein; Filtration; Foundations; Fractionation; Generations; Genetic; Genetic Transcription; Genomics; Goals; Health; Heterogeneity; Hour; Human; Imaging Techniques; Immune Cell Suppression; Immune response; Individual; Intake; Ion Exchange; Ions; Isoelectric Point; Lead; Lipids; Lipoproteins; Liquid substance; Lymph; Malignant - descriptor; Mechanics; Membrane; Messenger RNA; Methods; MicroRNAs; Microfluidic Microchips; Microfluidics; Molecular; Nanoporous; Neoplasm Metastasis; Neurons; Pathway interactions; Phase; Physiological; Plasma; Play; Precipitation; Process; Proteins; Proteomics; Protocols documentation; RNA; Reporting; Research; Research Personnel; Ribonucleases; Ribonucleoproteins; Role; Sampling; Signal Transduction; Sodium Chloride; Surface; Surface Antigens; Techniques; Technology; Time; Two-Dimensional Polyacrylamide Gel Electrophoresis; Ultracentrifugation; Ultrafiltration; Urine; base; digital; drug development; drug discovery; extracellular; extracellular vesicles; magnetic beads; microfluidic technology; nanocarrier; nanoparticle; nanoscale; novel; pH gradient; programs; rate of change; response; screening; sensor; therapeutic development; tool; trafficking

PROJECT ABSTRACT Extracellular RNA (exRNA), including messenger RNA (mRNA) and microRNA (miRNA), play an important role in endocrine signaling and as such, are critical to cell-to-cell communication. There is increasing evidence that exRNA are critical to disease development, and analysis of these exRNA, therefore, will play a vital role in continued biodiscovery, diagnostics, therapeutics, and drug development. There are three essential exRNA carriers that protect the exRNA from ever-present RNAses present in most physiological biofluids – extracellular vesicles (EVs), lipoproteins (LLPs), and ribonucleoproteins (RNPs). However, current technologies are limited in their ability to discriminate from which carrier specific exRNA originate and thus unable to accurately establish exRNA profiles. In addition to being slow and time consuming, most technologies for exRNA carrier isolation are inherently inefficient and lossy, limiting the effectiveness of absolute or even relative quantification. In this research program, we will develop a suite of high-throughput microfluidic technologies that will integrate the entire analysis process – separation and isolation of exRNA carriers, lysing and dissociation of the carriers to release their exRNA cargo, and the sensitive and selective detection of target exRNA, proteins, and lipids. By utilizing microfluidic platforms that build upon and expand our proven technologies, we anticipate that we will be able to complete the entire analysis for a panel of 5 target exRNA in 3 hours from only a 100 μL human blood plasma sample. This suite of tools will have a profound and transformative impact on advancing our understanding of exRNA biology and detecting exRNA expression as biomarkers for a wide range of diseases. The foundation for our carrier isolation strategy will be continuous isoelectric fractionation (CIF) based on our novel membrane-based free-flow electrophoresis microfluidic device. We will use ion exchange membranes (IEMs) as pH actuators to establish a free flowing pH gradient, separating the exRNA carrier by their isoelectric point and eluting the separated carriers into individual aliquots. This process should take ~30 min. The EV and LLP aliquots will then be injected into a surface acoustic wave (SAW) microfluidic device that mechanically lyses them to release their exRNA cargo, while RNPs will be processed on an integrated salt dissociation, protein/exRNA separation, and purification chip that utilizes the ion depletion feature of IEMs. Finally, four assays will be developed for detection: an IEM-based sensor for high abundance exRNA (> 106 copies), a droplet PCR device using immersed AC electrospray (iACE) droplet generation for low abundance exRNA (102-106 copies), and on-chip 2D polyacrylamide gel electrophoresis (2D PAGE) and micellar electrokinetic chromatography (MEKC) for proteins and lipids, respectively. We will optimize the entire process, including sample transfer, volume, and timing, to meet our overall throughput and sample volume targets.

项目摘要