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

Adv Biosyst. 2020 Dec;4(12):e1900307. doi: 10.1002/adbi.201900307. Epub 2020 Mar 12.

Abstract

There 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.

Keywords: droplet digital PCR; extracellular vesicles; microfluidics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Equipment Design
  • Extracellular Vesicles* / chemistry
  • Extracellular Vesicles* / metabolism
  • Humans
  • Mice
  • Microfluidic Analytical Techniques / instrumentation*
  • Polymerase Chain Reaction* / instrumentation
  • Polymerase Chain Reaction* / methods
  • Single-Cell Analysis / instrumentation