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

Konrad Thorsteinsson,Erik Olsén,Hudson Pace,Marta Bally,Eneas Schmidt

doi:10.1021/acs.analchem.0c02271

Konrad Thorsteinsson, Erik Olsén + Show 3 more

Open Access

https://doi.org/10.1021/acs.analchem.0c02271

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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 Förster 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 ∼109 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.

Highlights

Extracellular vesicles (EVs) produced by both eukaryotic and prokaryotic cells have been found to mediate intercellular communication in a variety of biological contexts
We have developed and validated a Förster resonance energy transfer (FRET)-based method to quantify the total surface area of membranes in a purified vesicle sample
Through the sonication-induced fusion of vesicles containing a FRET pair and nonlabeled sample vesicles. An advantage of this assay is that it can be performed without any specialized instrumentation; just a relatively cheap bath sonicator and a spectrofluorometer, generic instruments commonly found in biochemistry labs, are sufficient for its execution

Summary

Introduction

Extracellular vesicles (EVs) produced by both eukaryotic and prokaryotic cells have been found to mediate intercellular communication in a variety of biological contexts. Liposomes are artificially produced vesicles that have been widely investigated as drug delivery vehicles.[13−15] As outlined above, biological and synthetic vesicles are associated with a broad spectrum of research areas, all of which are currently hindered by the underdevelopment of methods to quantify lipid vesicle concentrations in a robust and versatile manner. The concentration of an EV sample is often reported in terms of total protein content, given in protein mass per unit volume;[16] for virus particles, the quantification of oligonucleotides can be used;[17] while for liposomes, phosphorus quantification is an extremely robust approach to determine the number of phospholipids in the sample. As a complement to biomolecular quantification approaches, methods allowing for the determination of particle concentrations, given in particles/volume, have been developed within the last decade. A constraint of this method is that the relatively weak scattering properties of vesicles in solution limit

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