Microfluidic enrichment of urinary small extracellular vesicle-associated proteins for proteomic biomarker discovery

Isolation of small extracellular vesicles (sEVs) typically <150 nm from urine remains technically challenging due to the presence of abundant soluble proteins and non-vesicular nanoparticles. Here, we report a label-free microfluidic pillar-array platform for size-selective enrichment of urinary sEVs based on diffusion-dominated transport under low-Reynolds-number laminar flow conditions. In contrast to conventional ultracentrifugation or affinity-based approaches, the present platform offers a passive and membrane free enrichment strategy. Recent advances in microfluidic EV isolation technologies have emphasized. The present platform integrates rational microchannel design, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and mass spectrometry based proteomic profiling to validate performance. Sequential processing resulted in progressive enrichment of particles <150 nm. This suggests reduced contribution from non-vesicular proteins and preservation of vesicle-associated proteomic signatures. Collectively, these findings demonstrate a reproducible and proteomics compatible microfluidic strategy for urinary sEVs enrichment and support its potential application in non-invasive biomarker discovery.