期刊
JOURNAL OF CHROMATOGRAPHY A
卷 1635, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.chroma.2020.461752
关键词
Capillary electrophoresis; dynamic light scattering; exosomes; extracellular vesicles; nanoparticle tracking analysis; polyethylene glycol precipitation
资金
- Spanish Ministry of Economy and Competitiveness [RTI2018-097411-B-I00]
- Cathedra UB Rector Francisco Buscarons Ubeda (Forensic Chemistry and Chemical Engineering)
- UB Solidarity Foundation
- Global Platform for Syrian Students
Extracellular vesicles (EVs) were enriched for exosomes from human serum using polyethylene glycol (PEG) precipitation in this study. A novel capillary electrophoresis-ultraviolet diode array (CE-UV-DAD) method was developed to obtain characteristic electrophoretic profiles of the EV preparations. Under optimized conditions, characteristic electrophoretic multiwavelength profiles of the EV preparation and exosome standard were obtained, showing excellent reproducibility and appropriate analysis times.
Extracellular vesicles (EVs) are membrane enclosed vesicles (<1 mu m), such as exosomes (30-150 nm), involved in cell communication, which have important biological implications. In this study, EV preparations were enriched for exosomes from human serum by polyethylene glycol (PEG) precipitation. Different variables of the PEG precipitation method (i.e. concentration of PEG, filtration and centrifugation of the resuspended pellets) were evaluated by measuring the size of the isolated particles by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). In addition, a novel capillary electrophoresis-ultraviolet diode array (CE-UV-DAD) method was developed to obtain characteristic multiwavelength electrophoretic profiles of the EV preparations. Using EV preparations precipitated with 10% m/v of PEG, a background electrolyte (BGE) of 0.1 M Tris and 0.25 M boric acid at pH 7.9 with 0.5% m/v of hydroxypropyl cellulose (HPC) allowed reducing the adsorption of the EVs to the inner wall of the fused silica separation capillary. Sodium dodecyl sulfate (SDS) at 0.1% m/v was also necessary to enhance dispersibility, while homogenizing the charge of the particles to improve the size-dependent separation induced by HPC. Under these optimized conditions, a characteristic electrophoretic multiwavelength profile of the EV preparation and a standard of exosomes was obtained, and separation showed excellent reproducibility and appropriate analysis times. The obtained electrophoretic fingerprints are a simple, effective and complementary tool for the quality control of EV preparations. (C) 2020 Elsevier B.V. All rights reserved.
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