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.

PROJECT NARRATIVE This research will develop a suite of microfluidics tools that will be able to separate the three exRNA carriers (extracellular vesicles, lipoproteins (LLPs), and ribonucleoproteins), release their cargo, and detect the resulting exRNA, proteins, and lipids. The suite of tools is projected to enable the detection of a panel of microRNA and messenger RNA over a 6 order of magnitude in copy number in under three hours and using only 100 microliters of blood plasma. this suite of microfluidic chips and operating protocol will be enable researchers to assess the role exRNA in human health and disease, advance our understanding of exRNA biology, and lead to the detection of exRNA biomarkers for a wide range of diseases.