Integrated system for extracellular vesicles isolation and detection

Extracellular vesicles (EVs) have attracted considerable interest due to their role in cell-cell communication, disease diagnosis, and drug delivery. Despite their potential in the medical field, there is no consensus on the best method for separating micro- and nanovesicles from cell culture supernatant and complex biological fluids. Here we describe an approach for affinity isolation and in situ enrichment of target sEVs from plasma allowing separation of pure, intact, and concentrated vesicles. The released vesicle are subjected to a multiparameter interferometric analysis providing information on vesicle size, number and phenotype. Single stranded DNA tagged antibodies are immobilized on the surface of magnetic beads through complementary strands of DNA. After binding of Evs, they are released from the surface using DNase I, an enzyme that catalyzes the cleavage of the dsDNA linker between the antibody and the solid surface. In this immunocapture strategy EVs are recovered from complex biological fluids in mild conditions that preserve their integrity. Released EVs can be successfully analyzed off-line by state-of-art imaging techniques (nanoparticle tracking analysis, electron microscopy and flow cytometry) and captured on antibody microarrays for phenotyping purpose. For the capturing on the microarray, we introduce a membrane sensing peptides as new, highly efficient ligands that is not biased by surface protein composition. In particular, we use a bradykinin-derived peptidic baits to capture EVs. We analyze EVs captured on the peptide and on specific antibodies by label-free, single particle counting and sizing, and by fluorescence co-localization immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81antibodies.