Visualized separation of exosomes in urine using carrier ampholyte-free free-flow isoelectric focusing...

Free flow electrophoresis (FFE) is a fully liquid-phase electrophoretic separation technique without supporting media, featuring mild separation conditions, continuous separation capability, and compatibility with multiple electrophoretic modes compared with other electrophoretic methods. In this work, a carrier ampholyte-free free-flow isoelectric focusing (CA-free FFIEF) device was constructed via anion/cation exchange membranes paired with strong acid/alkali electrode solutions (500 mmol·L⁻¹ phosphoric acid/1000 mmol·L⁻¹ sodium hydroxide). A stable linear pH gradient (4.07-9.35) was formed along the electric field direction in the separation chamber under an applied electric field of 300 V, enabling isoelectric focusing separation of exosomes. Based on the differences in particle size and surface charge of exosomes, a separation and preparation workflow for exosomes via CA-free FFIEF was established by optimizing electric field strength, buffer pH, flow rate and other parameters, achieving efficient separation of urinary exosomes. Carbon quantum dots (CQDs) were utilized to label exosomes to realize visualized separation throughout the process. Afterwards, metal oxide affinity chromatography (MOAC) with TiO₂ microspheres was applied to purify FFIEF-fractionated exosomes to eliminate protein contaminants and enrich target vesicles. The CA-free FFIEF system separated two subtypes of urinary exosomes with distinct isoelectric points, with a total exosome recovery of 95.91%. After sequential FFIEF and TiO₂ purification, the overall protein residual ratios in fraction 7 was as low as 1.77%. A total of 1802 proteins were identified in high-abundance fractions, among which 84.2% were exosome marker proteins recorded in the ExoCarta database. Distinct protein expression profiles were observed across different fractions, providing resources for screening differential biomarkers of urinary system diseases. This study offers a novel strategy for rapid and high-efficiency separation of urinary exosomes and lays a foundation for fundamental exosome research and translational clinical applications.