Abstract: Extracellular vesicles (EVs) are bilayer lipid-structured nanoscale vesicles spontaneously secreted by almost all types of cells, which encapsulate a variety of bioactive substances including functional proteins, genomic DNA, and diverse RNA fragments. Serving as essential signaling mediators for intercellular material exchange and information transmission, EVs participate in the regulation of multiple physiological and pathological processes, drive the progression of numerous diseases, and have emerged as highly promising novel diagnostic biomarkers and ideal delivery carriers for precise targeted therapy in biomedical research and clinical practice. However, the rapid and highly sensitive detection of trace amounts of EVs in complex biological fluids remains a challenge. Complex endogenous components in biological fluids severely interfere with EVs detection and separation, and conventional detection methods usually require tedious purification pretreatment, suffer from low detection sensitivity, and fail to distinguish different EV subpopulations, which restricts the in-depth research and practical application of EVs. In this study, we established a capillary zone electrophoresis method based on field-enhanced sample stacking. By exploiting the difference in conductivity between the sample matrix solution and the background electrolyte solution, online enrichment of EVs was achieved through extending the sample injection time and modifying the composition of the injection buffer, enabling the rapid baseline separation and detection of different EV subpopulations. Using EVs from fetal bovine serum pre-treated by ultrafiltration as model samples, the separation of EV subpopulations was successfully achieved using a 75 µm inner diameter capillary with 50 mmol/L boric acid solution containing 100 mmol/L glucose (pH 8.0) as background electrolyte and an injection time of 30 s. Under the optimized conditions, the field-enhanced sample enrichment factor reached 92-fold, significantly improving the sensitivity of EVs detection. Using EVs purified from raw milk via an ion-exchange-based dual-nanomaterial method as the standard, a standard curve was plotted with good linearity (R²=0.990 1), thereby establishing a relative quantitative analysis method for EVs. This method was applied to the relative quantification of EVs in fetal bovine serum and raw milk, with relative errors of 5.79% and 2.34% compared to the EV concentrations measured by nanoparticle tracking analysis, respectively. The developed method was successfully applied to the separation and relative quantitative analysis of EV subpopulations in raw milk without complicated extraction and purification procedures. This simple, rapid, highly sensitive, and low-cost method provides a new technical approach for the separation and relative quantitative detection of different EV subpopulations in complex biological fluid samples, demonstrating considerable potential for widespread application.

Key words: extracellular vesicles (EVs), field-enhanced sample stacking (FESS), capillary zone electrophoresis (CZE), relative quantification