Size-based analysis of extracellular vesicles using sequential transfer of an evaporating droplet

We report spatial separation of extracellular vesicle (EVs) populations based on particle size by using an approach that exploits Marangoni flow and the coffee-ring effect in microdroplets. Sequential transfer of a drying droplet progressively increases the mean size of EVs in the sample by repeated subsampling of a droplet during coffee-ring formation. This method allows size-based sorting, separation, and eventual retrieval of EVs for RNA and protein analysis. To demonstrate the biomedical relevance of this method, EVs from prostate cancer patients were analyzed; results revealed that the expression of cancer-associated genes and proteins was higher in small EVs than in large EVs. This ability to sort EVs using a combination of coffee ring with Marangoni flow and sequential droplet-transfer allows analysis of subpopulations of EVs, and will facilitate further studies of EVs.