Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles

Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples.