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Microfluidic filtration and electrophoretic separation system to isolate extracellular vesicles from blood

Microfluidic filtration and electrophoretic separation system to isolate extracellular vesicles from blood
Ryan T Davies
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Extracellular vesicles are released by various cell types, particularly tumor cells, and may be potential targets for blood-based cancer diagnosis. However, studies performed on blood-borne vesicles to date have been limited by lack of effective, standardized purification strategies. Using in-situ prepared nanoporous membranes, we present a simple strategy employing a microfluidic filtration system to isolate vesicles from raw blood samples. This method can be applied to purify nano-sized particles from small volumes of blood allowing isolation of intact extracellular vesicles, and avoiding the need for laborious and potentially damaging centrifugation steps or overly specific antibody-based affinity capture. Porous polymer monoliths were integrated as membranes into PMMA microfluidic chips by benchtop UV photopolymerization through a mask, allowing precise positioning while preserving simplicity of device preparation. Pore size could be manipulated by changing the ratio of porogenic solvent to prepolymer solution, and was tuned to a size proper for extraction of vesicles. Using the membrane as a size exclusion filter, we separated vesicles from cells and large debris by injecting whole blood under pressure through the microfluidic device. Electrophoretic injection was employed as an alternative driving force to propel particles across the filter and increase the separation efficiency of vesicles from proteins in a parallel technique. We found purity of the vesicle isolation could be improved by exploiting the higher electrophoretic mobility of vesicles compared to proteins. From the whole blood of melanoma-grown mice, we isolated extracellular vesicles and performed RT-PCR to verify their contents of RNA. Melan A mRNA present in melanoma-derived vesicles were found enriched in filtered samples, allowing sensitive detection of tumor markers compared to original blood. This filtration system can be incorporated into other on-chip processes enabling integrated sample preparation for the downstream analysis of blood-based extracellular vesicles.
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