(28d) Continuous Free-Flow Isoelectric Separation of Extracellular RNA Nanocarriers from Plasma

Extracellular RNA (exRNA) nanocarriers secreted in biological fluids play a crucial role in a variety of complex cellular functions by trafficking signaling molecules from cell to cell. exRNAs abundant in physiological fluid (such as blood, urine, lymph fluids) are encased and protected by three classes of nanocarriers: Extracellular vesicle (EVs), Lipoprotein (LLPs) and Ribonucleic protein (RNPs). Several recent studies show that exRNA nanocarriers from cancer tumor cells contain a unique set of molecular cargos that can indicate the different stage of tumor progression and hence has a potential to serve as diagnostic and prognostic biomarkers. It is thus very important to separate these carriers and analyze them separately. The currently available separation techniques use density-gradient multiple stage ultracentrifugation, immunocapture and ultrafiltration techniques, which are cumbersome and personnel intensive. Moreover, owing to overlapping nanocarrier size distributions and densities, these isolation technologies often suffer from low yield, poor purity, and quantification inaccuracy. To address these shortcomings, we take the advantage of their distinct isoelectric points and developed a high-throughput membrane-based free-flow isoelectric separation technology for rapid isolation of exRNA carriers (exosome, LLPs and RNPs) with high yield and purity within minutes from a plasma sample.

The device is configured by a single upstream channel, wedged between two bipolar membranes, further connected to the downstream channel (Fig. 1). Under reverse bias, bipolar membrane depletes the ions along their field lines to reduce the ionic current and increase the field strength due to the ion depletion zone at the junction. The excessively high field at the junction of the bipolar membrane allows splitting of water into H+ and OH-. The generated H+ and OH-, then precisely driven out from their respective ion exchange membrane. Further, their diffusion and continuous injection in the downstream channel allows the establishment of the pH gradient from 2 to 11, with distinct pH bands. A constant transverse electric field up to ±150 V/cm is then applied in the downstream channel for the separation of three carriers in three isolated chambers.

The activated device is successfully used for the generation of a constant pH gradient for isoelectric focusing and monitoring of separation (Fig. 2). Free-Flow isoelectric focusing is demonstrated with the fluorescently tagged high-density lipoprotein, low-density lipoprotein and ribonucleoprotein protein, covering a range of isoelectric point from 4 to 8. Separation of carriers was accomplished within a residence time of 10 seconds at a flow rate of 3ml/hr. Furthermore, the characterization of outlet sample by UV spectrometry and Gel-Electrophoresis suggests the purity of the isolated particles more than 90%.

The defined microfluidic device has shown the potential and benefits of providing a high-throughput and high-precision separation of disease cargos with different range of pIs, within a few seconds. The continuous free flow allows to process a large volume of sample in a short time. Collectively, this device enables the sophisticated approach for early disease detection and may provide an effective technique to validate and identify new disease biomarkers in body fluids. With a proper extension, it can also be used to purify nanoparticles like virus vaccines, exosome drug carriers, amyloid-beta aggregates, peptide assemblies etc.