Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.

Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid‐packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a “universal” marker, alternative or complementary to traditional, characteristic, surface‐associated proteins. Here, we introduce the use of membrane‐sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label‐free, single‐particle counting and sizing, and by fluorescence co‐localisation immune staining with fluorescent anti‐CD9/anti‐CD63/anti‐CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti‐tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis.