Development of a droplet microfluidic platform for Extracellular Vesicles purification from human plasma

Currently, medical diagnostic procedures can be invasive and cumbersome, such as the need for CT scans and surgical biopsies for conditions like tumors. These approaches not only present accessibility problems but also fail to capture the dynamics of certain diseases. In this realm, the emergence of liquid biopsy as a non-invasive technique has opened new avenues for detecting diseases and predispositions through the analysis of biomarkers present in bodily fluids, such as Extracellular Vesicles (EVs). This approach holds promise in the field of cardiovascular disease diagnostics, offering the potential for early detection and personalized treatment strategies. This thesis aims to present an advancement in this direction by introducing a novel droplet microfluidic device, serving as a platform for the direct analysis of human plasma samples. Our goal is to isolate Extracellular Vesicles using droplet microfluidics and micrometric paramagnetic beads functionalized with a target antibody to match with specific proteins present on the membrane of the vesicles. The research activity requires at first the design and fabrication of the microfluidic device using soft-lithographic techniques, followed by various characterizations. To generate droplets of human plasma, a systematic exploration of various surfactants has been performed. Then, to achieve an effective capture of EV, a crucial step involves an incubation period that ensures good mixing between the magnetic beads and the human plasma sample. Following the incubation period, the magnetic beads, enriched with captured extracellular vesicles, are efficiently extracted from the droplets using a magnetic field. The vesicles anchored on the bead surfaces undergo a lysis process to separate them from their anchor point. Finally, EVs are analyzed using the Western blot technique, a widely recognized method for protein detection and quantification, giving an estimate of the efficiency of our protocol.