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.