VDisk: A Microfluidic Cartridge for Automated and Reproducible Label-Free Isolation of Extracellular Vesicles from Plasma

Blood-derived extracellular vesicles (EVs) hold strong diagnostic potential, yet conventional isolation methods such as ultracentrifugation and size-exclusion chromatography (SEC) involve manual handling steps and show substantial run-to-run variability, hindering clinical translation. This study presents the Vesicle Disk (VDisk), a centrifugal microfluidics-based EV purification platform that combines cation-exchange chromatography, sequential filtration, and multimodal chromatography for automated, label-free EV isolation from up to 1 mL of plasma. VDisk configurations differing in filter membrane and plasma volume (0.1-1.0 mL) are benchmarked against SEC for yield, purity, reproducibility and robustness. VDisk matches SEC in EV yield and exceeds it in EV/contaminant ratios at reduced input volumes, while achieving greater reproducibility (intra-donor CV < 5% for CD9 and CD81, versus up to 16% for SEC) and maintaining consistent yield and contaminant removal under both fasting and postprandial sampling conditions. The platform is application-tunable: processing up to 1 mL of plasma maximizes EV yield and concentration, whereas processing 0.5 mL achieves approximately 2-3-fold higher EV/total protein ratios than SEC. These findings establish the VDisk as an automated, robust and adaptable alternative to existing EV isolation methods for both research and clinical translation.