Single Extracellular Vesicle Analysis Using Droplet Microfluidics

Abstract

Extracellular vesicles (EVs) are lipid-bound nanometer-sized vesicles released by all cell types that contain molecular payload such as proteins and/or nucleic acids. EVs are a key facet of cell-to-cell communication and have the potential to be used in the diagnosis of numerous diseases, chief among them being cancer. However, most methods of EV analysis struggle to identify the rare, malformed proteins indicative of tumor cells as tumor EVs represent only a tiny fraction of the bulk EVs present in the bloodstream. Here, we present a method of single EV analysis, utilizing droplet microfluidics to encapsulate EVs, which are labeled with DNA barcodes linked to antibodies, in droplets with the DNA extension used to amplify the signals associated with each EV. The amplified DNA can then be sequenced to assess the protein content of individual EVs, enabling the detection of rare proteins and EV subpopulations within a bulk EV sample.

Keywords: Droplet digital PCR; Droplet microfluidics; Extracellular vesicles; High-throughput; Multiplexing; Sequencing.