Pinched-flow fractionation-based extracellular vesicle isolation by ExoFAST and its analytical benchmarking with...

The growing interest in liquid biopsy biomarkers for non-invasive diagnostic approaches has spot-lighted extracellular vesicles (EVs), particularly exosomes, as promising candidates. However, robust and reproducible isolation of EVs with well-defined analytical performance remains a challenge for translating EV-based biomarkers into clinical practice. This study evaluates the performance of an automated microfluidic workflow for EV isolation in comparison with commercially available methods. We present ExoFAST, a microfluidic device that implements pinched-flow fractionation based size-selective separation of small EVs from apoptotic bodies using a tunable microfluidic architecture. The analytical performance of ExoFAST was evaluated using standardized metrics including particle concentration, total particle yield, size distribution, and exosomal marker expression in vesicles isolated from cell culture media. Performance was benchmarked against size-exclusion chromatography (Exo-spin™), polymer-based precipitation (ExoQuick®), nanoporous membrane filtration (EXODUS), and ultracentrifugation. The ExoFAST device achieved total particle recovery comparable to ultracentrifugation, with the particle concentration within a similar range, while providing reproducible, operator-independent processing. Additionally, we demonstrated ExoFAST device's feasibility in isolating exosomes from diverse sample types, including pig plasma, pig serum, and mouse plasma. By emphasizing automated operation, controlled fractionation, and transparent interpretation of analytical metrics, this study positions ExoFAST as a validated, size-based EV enrichment workflow suitable for integration into broader EV characterization pipelines.