FRET-Based Assay for the Quantification of Extracellular Vesicles and Other Vesicles of Complex Composition

Abstract

Research in the field of extracellular vesicles is rapidly expanding and finding footholds in many areas of medical science. However, the availability of methodologies to quantify the concentration of membrane material present in a sample remains limited. Herein, we present a novel approach for the quantification of vesicle material, specifically the quantification of the total lipid membrane surface area, found in a sample using Forster resonance energy transfer (FRET). In this assay, sonication is used to drive the fusion between vesicles in the sample to be quantified and liposomes containing a pair of FRET fluorophores. The change in emission spectrum upon vesicle fusion is directly related to the total membrane surface area of the sample added, and a calibration curve allows for the quantification of a variety of vesicle species, including enveloped viruses, bacterial outer membrane vesicles, and mammalian extracellular vesicles. Without extensive optimization of experimental parameters, we were able to quantify down to ∼10 9 vesicles/mL, using as little as 60 μL of the sample. The assay precision was comparable to that of a commercial nanoparticle tracking analysis system. While its limit of detection was slightly higher, the FRET assay is superior for the detection of small vesicles, as its performance is vesicle-size-independent. Taken together, the FRET assay is a simple, robust, and versatile method for the quantification of a variety of purified vesicle samples.

Recruitment of apolipoprotein E facilitates Herpes simplex virus 1 attachment, entry, and release

“…All centrifugations were done at 4°C. Concentration of NMV material was measured using a FRET based assay described previously (Thorsteinsson et al, 2020).…”

Research progress in membrane fusion-based hybrid exosomes for drug delivery systems

“…The specific method is to drive the fusion of the sample with a liposome containing a pair of FRET fluorophores by sonication, and then use a calibration curve to quantify the concentration of various types of vesicles including enveloped viruses, mammalian extracellular vesicles and so on. This detection method is simple and easy to implement with high sensitivity ( Thorsteinsson et al, 2020 ).…”

“… Characterization of membrane fusion-based hybrid exosomes. (A) FRET assay to characterize membrane fusion ( Thorsteinsson et al, 2020 ) (B) Immunomagnetic bead method to characterize membrane fusion ( Lv et al, 2020 ) (C) TEM & confocal fluorescence microscopy analysis to visualize membrane fusion ( Singh et al, 2021 ). …”

Progress in Nanomaterials-Based Optical and Electrochemical Methods for the Assays of Exosomes

“…Exosome can be labeled with dyes or dye-modified biorecognition elements through various targeting strategies and then determined by fluorescence imaging or spectroscopy. 50,[65][66][67][68][69][70][71][72] However, the low fluorescence intensities of dyes always limits the sensitivity for exosome detection. 73 Thus, several signal amplification strategies have been proposed to improve the detection sensitivity.…”