PROJECT SUMMARY / ABSTRACT Extracellular vesicles (EVs) are small lipid bilayer particles secreted by most cell types, which encapsulates biomolecules including lipids, proteins, and nucleic acids from cells of origin. Profiling of EV-derived biomarkers provides a promising approach for diagnosis of various diseases. However, one of the primary challenges for the research and application of EVs is a lack of a reliable method for efficient isolation of EVs and their subpopulations from complex biofluids. Researchers in this field suffer from tedious workflow, long processing time, high cost, and poor EV quality. To overcome the challenges, WellSIM proposes in this Phase II project, which built upon the promising results generated from our Phase I project, to complete development of a Label- free Extracellular-vesicle Automated Purification System (LEAP System) for isolation of total EVs or EV subpopulations from a variety of biofluids in a non-invasive and cost-effective manner. Once developed, the integrated LEAP System will offer excellent EV quality, throughput, and flexibility for EV isolation that can outperform ultracentrifugation and other isolation techniques.