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Quantification of Small Extracellular Vesicles by Size Exclusion Chromatography with Fluorescence Detection
Rui Xu1, Austin Fitts1, Xiangtang Li1
1Department of Chemistry and Biochemistry, Jackson State University , Jackson, Mississippi 39217, United States.
Analytical Chemistry
|October 1, 2016
Summary
A new size exclusion chromatography with fluorescence detection (SEC-FD) method offers a cost-effective way to quantify small extracellular vesicles (sEVs). This technique enables precise sEV analysis for disease diagnostics and treatment monitoring.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Nanotechnology
Background:
- Small extracellular vesicles (sEVs) in body fluids are promising biomarkers for noninvasive disease diagnosis and treatment evaluation.
- Accurate quantification of sEVs is crucial but hindered by the lack of cost-effective analytical methods.
Purpose of the Study:
- To develop and validate a facile and cost-effective method for quantifying small extracellular vesicles (sEVs) using size exclusion chromatography with fluorescence detection (SEC-FD).
Main Methods:
- Developed a SEC-FD assay involving CM-Dil fluorescent labeling of sEVs in body fluid samples.
- Separated labeled sEVs using a Sepharose CL-4B column and detected them via fluorescence monitoring (Ex553 nm/Em570 nm).
- Validated the method using liposomes and albumin-FITC, assessing separation efficiency, repeatability, and linearity.
Main Results:
- Achieved high separation efficiency with liposomes eluting before albumin-FITC.
- Demonstrated excellent repeatability for liposome retention time (1.4% RSD) and sEV quantification (3.2% intraday RSD).
- Established linear calibration curves (r² = 0.996) with a detection limit of 2.9 × 10⁷ particles/mL.
Conclusions:
- The SEC-FD method provides a reliable, cost-effective, and repeatable approach for sEV quantification.
- This assay is suitable for analyzing sEV secretion dynamics, as demonstrated in TK6 cell cultures.
