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.
Highlights
We describe a new method for ultrasensitive detection of proteins in single extracellular vesicles (EV) that exploits antibody-basedSingle cell analysis continues to have a major impact on our immuno-droplet digital polymerase chain reaction.understanding of cell subtypes, biology, and medicine.[1,2] The The described method is sensitive and allows mulsame is likely true for analyses of single exosomes or extra- tiplexing
Barcoded antibodies were incubated with EV for labeling, followed by size exclusion chromatography (SEC) to remove unbound antibody–DNA (Ab–DNA) molecules (Figure 1A)
The concentration of labeled EV in solution was determined by nanoparticle tracking analysis (NTA), allowing us to adjust concentrations and flow rates to optimize the statistical likelihood of single EV encapsulation.[16,17,18]
Summary
Single cell analysis continues to have a major impact on our immuno-droplet digital polymerase chain reaction (iddPCR). The. EV populations can be even more heterogeneous than the labeled EV are encapsulated into 70 μm droplets in which PCR parental cells from which they are derived.[3,4] This is likely amplifies the message of the DNA barcode. Paulson School of Engineering and Applied Sciences and Department of Physics Harvard University Cambridge, MA 02138, USA. Y. Wang Department of Chemistry and Chemical Biology Harvard University Cambridge, MA 02138, USA. Weissleder Harvard Cancer Center Massachusetts General Hospital and Harvard Medical School Boston, MA 02114, USA. Charest Department of Medicine Beth Israel Deaconess Medical Center Boston, MA 02215, USA. Weissleder Department of Systems Biology Harvard Medical School 200 Longwood Ave, Boston, MA 02115, USA. We optimized the experimental conditions, validated measurements, and applied EV profiling to PD-L1 measurements in cancer-cell derived EV
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