Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping

Abstract

BackgroundExosomes are one of the several types of cell-derived vesicles with a diameter of 30–100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define their phenotype and determine their concentration in biological fluids. To identify circulating as well as cell culture-derived vesicles, the current standard is immunoblotting or a flow cytometrical analysis for specific proteins, both of which requires large amounts of purified vesicles.MethodsBased on the technology of protein microarray, we hereby present a highly sensitive Extracellular Vesicle (EV) Array capable of detecting and phenotyping exosomes and other extracellular vesicles from unpurified starting material in a high-throughput manner. To only detect the exosomes captured on the EV Array, a cocktail of antibodies against the tetraspanins CD9, CD63 and CD81 was used. These antibodies were selected to ensure that all exosomes captured are detected, and concomitantly excluding the detection of other types of microvesicles.ResultsThe limit of detection (LOD) was determined on exosomes derived from the colon cancer cell line LS180. It clarified that supernatant from only approximately 104 cells was needed to obtain signals or that only 2.5×104 exosomes were required for each microarray spot (~1 nL). Phenotyping was performed on plasma (1–10 µL) from 7 healthy donors, which were applied to the EV Array with a panel of antibodies against 21 different cellular surface antigens and cancer antigens. For each donor, there was considerable heterogeneity in the expression levels of individual markers. The protein profiles of the exosomes (defined as positive for CD9, CD63 and CD81) revealed that only the expression level of CD9 and CD81 was approximately equal in the 7 donors. This implies questioning the use of CD63 as a standard exosomal marker since the expression level of this tetraspanin was considerably lower.

Isolation of exosomes from whole blood by integrating acoustics and microfluidics

“…Moreover, the high centrifugal force used in ultracentrifugation (100,000-200,000 × g) has been shown to cause exosome fusion, promote coagulation, and alter their structures, properties, and functions, which may impact downstream analysis (12,13,18). Other methods, including immunoaffinity capture (19,20), precipitation kits such as ExoQuick (System Biosciences) and Total Exosome Isolation (Invitrogen) (12,21), microfluidics (17,22,23), nanoscale lateral displacement arrays (24), nanostructurebased filtration (25), nanoplasmonic chip (26), magnetoelectrochemical sensor (27), and dialysis membrane filtration (28), have been implemented. However, these methods frequently suffer from drawbacks such as the need for additional reagents/labels, long processing time, low reproducibility, low exosome integrity, low exosome purity, and/or low exosome yield.…”

Carcinogenic activity of PbS quantum dots screened using exosomal biomarkers secreted from HEK293 cells

“…28 Western blotting analysis for the representative exosomal biomarkers CD63 and CD9 29,30 confirmed that the isolated vesicles were enriched in the exosomal fraction.…”

Bulk immunoassays for analysis of extracellular vesicles

“…A recent example applied to EV is the "EV array" [21,22]. The EV array utilizes slides printed with an array of antibodies to capture EV and then determines the amount of captured EV by labeling with a cocktail of biotinylated CD9, CD63 and CD81 and the use of streptavidin-Cy5 as a reporter molecule.…”

“…NTA does not detect the concentration of EV, but the concentration of all particles inside the detection size range (typically 80-500 nm [25]). Furthermore, a wide array of reference samples is used, including EV derived from different cell cultures [13,14,16,17,20,21,23,26] and/or plasma [21,27] were used. All these factors lead to considerable uncertainty in the limit of detection.…”

“…Performing a set of parallel experiments does not change this duration for any BIA if the increase in time due to pipetting actions is neglected. Most BIA require two to four hours to prepare and perform an experiment, but FLISA and TRFIA require 24 and 8 hours, respectively, due to longer sample incubation steps [21,23]. Longer incubation allows more contact between sample and capture surface, and thus longer incubation may benefit the limit of detection for ELISA, SPR, and ac-EHD.…”