当前位置: X-MOL 学术J. Chromatogr. A › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
A simple method for the analysis of extracellular vesicles enriched for exosomes from human serum by capillary electrophoresis with ultraviolet diode array detection
Journal of Chromatography A ( IF 3.8 ) Pub Date : 2020-11-26 , DOI: 10.1016/j.chroma.2020.461752
Oumaima El Ouahabi , Hiba Salim , Roger Pero-Gascon , Fernando Benavente

Extracellular vesicles (EVs) are membrane enclosed vesicles (<1 µm), 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.



中文翻译:

毛细管电泳-紫外二极管阵列检测法分析人血清中富集有外来体的细胞外囊泡的简单方法

细胞外囊泡(EVs)是膜包裹的囊泡(<1 µm),例如外泌体(30-150 nm),参与细胞通讯,具有重要的生物学意义。在这项研究中,EV制剂通过聚乙二醇(PEG)沉淀富集了人血清中的外来体。通过动态光散射(DLS)和纳米颗粒跟踪分析(NTA)测量分离出的颗粒大小,可以评估PEG沉淀方法的不同变量(即PEG浓度,重悬沉淀的过滤和离心分离)。此外,开发了一种新颖的毛细管电泳-紫外二极管阵列(CE-UV-DAD)方法来获得EV制剂的特征性多波长电泳图谱。使用以10%m / v的PEG沉淀的EV制剂,pH值为7.9的0.1 M Tris和0.25 M硼酸与0.5%m / v的羟丙基纤维素(HPC)的背景电解质(BGE)可以减少EV对熔融石英分离毛细管内壁的吸附。还必须使用0.1%m / v的十二烷基硫酸钠(SDS),以提高分散性,同时使颗粒的电荷均一化,以改善HPC诱导的尺寸依赖性分离。在这些优化的条件下,获得了EV制剂的特征性电泳多波长谱和外泌体标准品,并且分离显示出优异的重现性和适当的分析时间。获得的电泳指纹图谱是用于电动汽车制剂质量控制的简单,有效和互补的工具。5%m / v的羟丙基纤维素(HPC)可以减少EV对熔融石英分离毛细管内壁的吸附。还必须使用0.1%m / v的十二烷基硫酸钠(SDS),以提高分散性,同时使颗粒的电荷均一化,以改善HPC诱导的尺寸依赖性分离。在这些优化的条件下,获得了EV制剂的特征性电泳多波长谱和外泌体标准品,并且分离显示出优异的重现性和适当的分析时间。获得的电泳指纹图谱是用于电动汽车制剂质量控制的简单,有效和互补的工具。5%m / v的羟丙基纤维素(HPC)可以减少EV对熔融石英分离毛细管内壁的吸附。还必须使用0.1%m / v的十二烷基硫酸钠(SDS),以提高分散性,同时使颗粒的电荷均一化,以改善HPC诱导的尺寸依赖性分离。在这些优化的条件下,获得了EV制剂的特征性电泳多波长谱和外泌体标准品,并且分离显示出优异的重现性和适当的分析时间。获得的电泳指纹图谱是用于电动汽车制剂质量控制的简单,有效和互补的工具。为了提高分散性,同时使颗粒的电荷均一化,以改善HPC诱导的尺寸依赖性分离,还需要1%m / v。在这些优化的条件下,获得了EV制剂的特征性电泳多波长谱和外泌体标准品,并且分离显示出优异的重现性和适当的分析时间。获得的电泳指纹图谱是用于电动汽车制剂质量控制的简单,有效和互补的工具。为了提高分散性,同时使颗粒的电荷均一化,以改善HPC诱导的尺寸依赖性分离,还需要1%m / v。在这些优化的条件下,获得了EV制剂的特征性电泳多波长谱和外泌体标准品,并且分离显示出优异的重现性和适当的分析时间。获得的电泳指纹图谱是用于电动汽车制剂质量控制的简单,有效和互补的工具。分离显示出优异的重现性和适当的分析时间。获得的电泳指纹图谱是用于电动汽车制剂质量控制的简单,有效和互补的工具。分离显示出优异的重现性和适当的分析时间。获得的电泳指纹图谱是用于电动汽车制剂质量控制的简单,有效和互补的工具。

更新日期:2020-11-27
down
wechat
bug