(251e) A Scalable, High-Throughput Platform for Isolating Evs, Lipoproteins, and Rnps from Blood Plasma, Urine, and Saliva, Using Isoelectric Fractionation

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, and 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 stages of tumor progression and hence has the potential to serve as diagnostic and prognostic biomarkers. It is thus very important to separate these carriers and analyze them separately. However, the overlapping size and density of the nanocarriers have so far prevented their efficient physical fractionation, thus impeding independent downstream molecular assays. Moreover, affinity-based and liquid extraction technologies tend to exhibit significant bias and contamination, thus corrupting the quantification of expression level. Hence, there is a need for new technology to provide bias-free, high-throughput, and high-yield nanocarrier fractionation. To address these shortcomings, we take the advantage of their distinct isoelectric points and developed a novel free-flow isoelectric focusing (charge-based) technique to fractionate different exRNA-nanocarriers at high throughput, yield, and purity from various biofluids (plasma, urine, and saliva).

Our platform called the Continuous Isoelectric Fractionation (CIF) device consists of two modules of microfluidic chips: (i) pH gradient chip and (ii) separation chip. The pH gradient chip incorporates a pair of bipolar membranes, which splits water into H₃O⁺ and OH^– ions under optimized reverse bias voltage due to Wien effect. The separated H₃O⁺ and OH^– ions are separated and stabilized by flow (~12 ml/hour), zero-flux Boltzmann distributions of the inert buffer ions as well as rapid and reversible water dissociation reaction to form linear pH gradient (pH 3 to 11) without using commercial ampholytes. Depending on the application, the desired portion of the effluent from the pH gradient chip is then injected downstream into a separation chip(s) to produce a high-resolution pH gradient where the nanocarrier mixture is injected (~3ml/hour) and separated based on their charge (isoelectric point). To facilitate desired pH transfer from the pH gradient chip to the separation chip, a machine learning procedure is developed. For quantitative assessments, the effluents collected from all the outlets of the chip were qualitatively analyzed by various methods such as ELISA, gel electrophoresis, zeta potential, RT-qPCR, and TEM images.

We optimize the CIF technology by fractionating various combinations of binary mixtures of exRNA nanocarriers (EVs, LLPs, and RNPs) spiked in 1x PBS buffer (yield >80% and purity >90%). Its performance is then evaluated to fractionate RNPs from other nanocarriers in several biofluids, including plasma, urine, and saliva samples. Comprehensive, high-purity (plasma: >93%, urine: >95% and saliva: >97%), high-yield (plasma: >78%, urine: >87% and saliva: >96%), and probe-free isolation of ribonucleoproteins in 0.75 ml samples of various biofluids in 30 minutes is demonstrated, significantly outperforming affinity-based and highly biased gold standards having low yield and day-long protocols. The optimized technique has a resolution of 0.3 ΔpI, sufficient to separate many nanocarriers and even subclasses of nanocarriers. This device represents a significant advancement as it overcomes the various limitations described earlier of commonly utilized traditional isolation technologies including ultracentrifugation, immunocapture, and ultrafiltration techniques.

Collectively, this scalable device enables an exciting approach to early disease detection and may provide an effective technique to validate as well as identify new disease biomarkers in body fluids. Additionally, we envision CIF platform’s future application in exploring the heterogeneity of EVs such as fractionating different EVs types with cargoes derived from cancer cells (e.g. GPC-1, Active EGFR, AR-V7). It will require a fine pH gradient, which will likely require multiple separation devices to achieve sufficient pH resolution. We also anticipate that this technology can be used to purify other biological nanoparticles including virus vaccines, exosome drug carriers, amyloid-beta aggregates and peptide assemblies.