DSPE-Ti-Chip: A Dual-Functionalized Microfluidic Device for High-Efficiency Isolation of Extracellular Vesicle from Microvolume Biofluids

Isolating extracellular vesicles (EVs) from microvolume samples remains a major technical challenge, as conventional methods are often hampered by low yield, poor purity, and impracticality when processing trace volumes. Herein, we developed a dual-functionalized microfluidic platform (DSPE-Ti-Chip) that synergistically combines Ti⁴⁺ coordination for phosphate binding and 1,2-Distearoyl-<i>sn</i>-glycero-3-phosphorylethanolamine (DSPE) phospholipid probes for membrane insertion, enabling efficient capture of EVs from biofluids with volumes as low as 20 μL. The dual-affinity strategy proved superior, capturing twice as many EVs as chips with either ligand alone. Compared to ultracentrifugation (UC), the DSPE-Ti-Chip demonstrated significantly enhanced performance, improving the EV purity by 2.2 times and increasing EV yield by 2.5 times. Furthermore, the platform achieved an 86.9% recovery rate, representing a 5.2-fold improvement over UC, while reducing the processing time from 4 to 1 h. We applied this platform to isolate EVs from the culture medium of a human alveolar-capillary lung-on-a-chip model exposed to combustible cigarette smoke (CS) or electronic cigarette aerosols (ECA). Subsequent label-free quantitative proteomics identified 393 ± 13 proteins per sample group (n = 3), and comparative analysis revealed distinct proteomic signatures. CS-EVs were enriched in inflammatory and immune-related proteins like S100A8/A9, whereas ECA-EVs were more associated with extracellular matrix remodeling and cell adhesion. Our results demonstrate that the DSPE-Ti-Chip is a robust and efficient tool for high-quality EV isolation from limited samples, enabling downstream omics analysis and revealing divergent pathophysiological pathways induced by different aerosol exposures.