(505-M) Continuous Isoelectric focusing microfluidics device for bias-free and high-throughput fractionation of nanocarriers (EVs, lipoproteins, and RNPs) from blood plasma, urine, and saliva

Introduction:: Extracellular nanocarriers (exosomes, lipoproteins, and ribonucleoproteins) of protein and nucleic acids mediate intercellular communication and are clinically adaptable as circulating biomarkers. Both the nanocarrier identity and their molecular cargo are relevant for detecting and characterizing various diseases. 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. We have 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).

Materials and Methods:: 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 H 3 O + and OH – ions under optimized reverse bias voltage due to Wien effect. The separated H 3 O + 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, and TEM images.

Results, Conclusions, and Discussions:: 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 with 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. 

Additionally, we envision that CIF platform’s future application in exploring the heterogeneity of EVs such as fractionating different EV types with cargoes derived from cancer cells (e.g., GPC-1, Active EGFR, AR-V7) 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.

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