VDisk: Microfluidic Cartridge for Multimodal High-Yield, High-Purity Isolation of Extracellular Vesicles from up to 1 mL of Plasma

Blood-derived extracellular vesicles (EVs) hold strong diagnostic potential, yet conventional methods such as ultracentrifugation (UC) and size-exclusion chromatography (SEC) struggle to efficiently separate EVs from lipoproteins and plasma proteins due to overlapping biophysical properties. Manual workflows further introduce operator-dependent variability, limiting reproducibility and hindering clinical translation. This study presents the Vesicle Disk (VDisk), an EV purification platform using centrifugal microfluidics that combines cation exchange chromatography (CEX), sequential filtration (SeqF), and multimodal chromatography (MMC) for efficient, label-free EV isolation from up to 1 mL plasma. Various VDisk configurations with different filter membranes and plasma volumes (0.1- 1.0 mL) were benchmarked against SEC for recovery, purity, reproducibility and robustness. VDisk achieved up to 84.3% EV recovery, surpassing SEC (60.0%), with excellent reproducibility (CV < 5%) and consistent performance under both fasting and postprandial sampling conditions. VDisk offers application-specific flexibility: For applications requiring high EV concentrations, processing up to 1 mL of plasma yields a two to three times higher EV concentration compared to SEC. For purity-critical applications, processing 0.1-0.5 mL of plasma achieves approximately two times higher EV/total protein ratio. These results establish VDisk as an automated, robust, scalable, and adaptable alternative to existing EV isolation methods, suitable for both research and clinical applications.