Dean Flow-Assisted Microfluidic Aqueous Two-Phase Extraction for Purifying Extracellular Vesicles and Facilitating Single-Vesicle Analysis

Extracellular vesicles (EVs) play a crucial role in mediating and regulating biological processes, such as intercellular communication and signaling. The isolation and purification of EVs from biological samples are prerequisites for EV research. Herein, a Dean flow-assisted microfluidic aqueous two-phase extraction chip (DATPEC) was developed for isolating EVs from biological samples. In aqueous two-phase laminar flow, EVs with hydrophilic surfaces selectively migrate from the poly(ethylene glycol) (PEG) phase to the dextran (DEX) phase, which is accelerated by Dean flow. However, the proteins were still retained in the PEG phase due to the laminar effect and their surface properties. The separation performance of the DATPEC was investigated using breast cancer-derived EVs, and 91.1% EVs could be recovered from the cell culture medium supernatant with a protein removal rate of 95.6%. As a proof of concept, this technique was coupled with total internal reflection fluorescence (TIRF) microscopy to analyze surface proteins (EpCAM and EGFR) of EVs derived from eight cell lines. Fluorescent aptamers were specifically labeled on EVs, revealing the single-vesicle heterogeneity. In conclusion, DATPEC enables simple and efficient separation of high-purity EVs and exhibits the potential to be integrated with downstream techniques for EV analysis.