Single Extracellular Vesicle Protein Analysis Using Immuno‐Droplet Digital Polymerase Chain Reaction Amplification

• DOI: 10.1002/adbi.201900307
• Publication Date: 2020-03-12T10:40:27Z
• Authors: Jina Ko; Yongcheng Wang; Jonathan C. T. Ca...; Angela Marquard; Jeremy Gungabeesoon; Alain Charest; David Weitz; Mikael J. Pittet; Ralph Weissleder
• Keywords: Extracellular Vesicles; Mice; 0303 health sciences; 03 medical and health sciences; Animals; Humans; Equipment Design; Microfluidic Analytical Techniques; Single-Cell Analysis; Polymerase Chain Reaction; Cell Line; 3. Good health

AbstractThere is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno‐droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody–DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read‐out by droplet imaging. In these proof‐of‐principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses.