Studio e sviluppo di un dispositivo in microfluidica di gocce per l'isolamento di vescicole extracellulari

This Ph.D. thesis aims to propose and develop a droplet microfluidic platform capable of purifying biological samples and, in particular, to isolate extracellular vesicles (EVs). These are small (less than 1 µm) biomarkers secreted by the cell membrane, that contain specific proteins and genomic material from the cells of origin, and thus are considered as cargo-ships. Being released in body fluids (e.g. blood, urine), EVs can be a good candidate for early diagnosis of tumor activity by using liquid biopsy, instead of recurring to invasive tests or tissue sampling. Unlike more conventional approaches, that rely on size or density separation, this device exploits immunoaffinity capturing of EVs on micrometric magnetic beads (diameter of 2-5 µm) within droplets, confined in microchannels. The beads are functionalized at the surface by antibodies complementary to proteins present in the vesicle membrane to promote selective isolation. Additionally, the use of droplets enhances the mixing due to spontaneous recirculation of the content, which does not occur in monophasic microfluidic approaches, affected by bead sedimentation. At first, the project is focused on the microfabrication of the microfluidic device, obtained by soft-lithographic techniques. After that, the production of droplets within microchannels is characterized and the module for the extraction of magnetic beads is optimized after devoted magnetic field simulations. Finally, the validation of the protocol is performed on samples prepurified from cell culture and resuspended in a saline solution. Preliminary tests are also carried out on more complex samples, such as cell culture medium and pre-purified plasma. The novel droplet microfluidic approach is compared with traditional methods for EV isolation: i) ultracentrifugation (UC) protocols and ii) incubation with immunomagnetic beads in conventional lab-scale storage conditions. Concerning the results, the platform isolation capability is evaluated by flow cytometry and confocal microscopy. The EV size and concentration are investigated by Nanotracking analysis, estimating a bead extraction efficiency of about 56%. Colorimetric assay (BCA) and Western Blot are used to quantify the protein content, and Polymerase Chain Reaction (PCR) protocol to verify the EV integrity, by searching for miRNAs. Overall, the platform allows processing samples of volumes up to 2 mL in 4.5 hours, increasing the overall throughput with respect to the in-batch approach and to monophasic microfluidic devices.