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

Abstract

due to selective shedding of proteins and nucleic acid cargo, the much smaller size and payload capacity of EV, and stochastic cellular effects. In order to use EV more efficiently as biomarkers of disease, we need a better understanding of their composition and heterogeneity. A number of single EV analytical methods have been proposed. These include analysis by microscopic imaging of immobilized vesicles (SEA), [5,6] modified flow cytometry, [5,7-10] and digital detection using ELISA [11] or nucleic acid-based amplification. [28] In spite of this progress, it remains challenging to detect rare proteins in single EV, given the inherent signal/background limitations of direct fluorescence imaging and relatively modest enzyme mediated signal amplification in ELISA. Here we describe a new method for ultrasensitive detection of proteins in single EV that exploits antibody-based immuno-droplet digital polymerase chain reaction (iddPCR). The described method is not only sensitive but also allows multiplexing (currently up to three proteins). We used uniquely designed DNA barcoded antibodies for protein recognition. The labeled EV are encapsulated into 70 µm droplets in which PCR amplifies the message of the DNA barcode. Using different 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.

Modeling EV Kinetics for Use in Early Cancer Detection

“…One way to solve the problem is to develop single (“digital”) EV analysis techniques, which are now emerging. [ 8–11 ] Armed with these technological advances, key biological questions regarding the utility of EVs as biomarkers can now be answered.…”

“…[ 5 ] For a typical tumor, all of these approaches are predicted to be able to detect lesions smaller than 1 cm 3 (Figure 5C). For single‐EV resolution or near‐single‐EV resolution assays such as single EV analysis (SEA), [ 8 ] digital EV screening technology (DEST), [ 26 ] or digital droplet polymerase chain reaction, [ 11 ] it may even be possible to detect tumors as small as 1 × 10 −5 cm 3 (10 µg or about 10 000 tumor cells). The challenge, however, is to find EV biomarkers (or combinations, i.e., “signatures”) that can distinguish tEVs unequivocally from other host cell EVs based on molecular markers.…”

“…[ 26 ] Finally, a new method for ultrasensitive protein‐based single EV analysis is immunodigital droplet polymerase chain reaction (iddPCR) described in a companion article in this issue. [ 11 ] Using these approaches it should be possible to detect human cancers as small as 0.01 mm 3 .…”

Clinical relevance of extracellular vesicles in hematological neoplasms: from liquid biopsy to cell biopsy

“…Moreover, mass spectrometry, as above mentioned, and SOMA-scan, an affinitybased proteomic analysis technique, are used for highly sensitive and specific protein detection in human blood and other biomatrices possibly finding novel protein biomarker candidates [14]. In addition, immune-droplet digital PCR amplification method allows multiplex protein analysis in single EV [104].…”

Biomimetic Nanostructure Platform for Cancer Diagnosis Based on Tumor Biomarkers

“…Monteiro et al have detected the human epidermal receptor protein-2 (HER2), using SPR (Monteiro et al, 2016). Immunostaining (including a conjugated fluorescence antibody, aptamer, a molecular beacon) platforms (e.g., microarrays, microfluidics, and chips) can also reveal clinical information (Lau et al, 2010;Davies et al, 2012;Yoo et al, 2012;Ko et al, 2020;Rima et al, 2020). Though these platforms exhibit robustness, most of them suffer from unsatisfactory accuracy and low sensitivity and specificity due to the heterogeneity of substances, a dearth of biomarkers, and contamination (Willms et al, 2018).…”