Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer.
Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer.
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DOI：
10.1021/acs.analchem.1c00253
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发表时间：
2021-04-13
影响因子：
7.4
通讯作者：
Chiu, Daniel T.
中科院分区：
文献类型：
--
作者：
Andronico, Luca A.;Jiang, Yifei;Jung, Seung-Ryoung;Fujimoto, Bryant S.;Vojtech, Lucia;Chiu, Daniel T.
细胞外小泡(EVS)是体内大多数细胞释放的膜性颗粒，参与许多细胞间的信号传递过程。鉴于电动汽车的纳米尺寸和异质性，单分子分辨率的高灵敏度方法是研究其生物物理性质的基础。在这里，我们使用具有单分子灵敏度的基于荧光的流动分析仪来演示细胞外小泡(EVS)的大小。使用一种选择性地分配到囊泡膜上的染料，我们证明了囊泡的荧光强度与其直径成正比。我们讨论了样品制备中的限制，这些限制是用荧光膜染料测定纳米级囊泡的固有限制，并提出了几个改善数据一致性的指导方针。在优化染色条件后，我们能够测量出~35-300 nm范围内的囊泡大小，覆盖了EV大小的光谱。最后，我们开发了一种方法，通过在高采样率(10 KHz)下操作并测量粒子穿过激光光束所需的时间，根据每个囊泡在微流控通道内的移动速度来校正来自每个囊泡的信号强度。使用这一校正，我们在电动汽车尺寸测量中获得了3倍的精度，精度为±15-25%。
Extracellular vesicles (EVs) are membranous particles released by most cells in our body, and which are involved in many cell-to-cell signaling processes. Given the nanometer sizes and heterogeneity of EVs, highly sensitive methods with single-molecule resolution are fundamental to investigating their biophysical properties. Here, we demonstrate the sizing of extracellular vesicles (EVs) using a fluorescence-based flow analyzer with single-molecule sensitivity. Using a dye that selectively partitions into the vesicle’s membrane, we show that the fluorescence intensity of a vesicle is proportional to its diameter. We discuss constraints in sample preparation which are inherent to sizing nanoscale vesicles with a fluorescent membrane dye, and propose several guidelines to improve data consistency. After optimizing staining conditions, we were able to measure the size of vesicles in the range ~35–300 nm, covering the spectrum of EV sizes. Lastly, we developed a method to correct the signal intensity from each vesicle based on its travelling speed inside the microfluidic channel, by operating at a high sampling rate (10 kHz) and measuring the time required for the particle to cross the laser beam. Using this correction, we obtained a 3-fold greater accuracy in EV sizing, with a precision of ±15–25%.
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影响因子：
3.3
作者：
Gurtovenko, Andrey A.;Anwar, Jamshed
通讯作者：
Anwar, Jamshed
影响因子：
3.9
作者：
Sezgin, Erdinc;Sadowski, Tomasz;Simons, Kai
通讯作者：
Simons, Kai
影响因子：
0.7
作者：
Shulyak, I. V.;Grushova, E. I.;Semenchenko, A. M.
通讯作者：
Semenchenko, A. M.
DOI：
10.1007/978-1-4939-2550-6_15
发表时间：
2015-01-01
期刊：
PROTEOMIC PROFILING: METHODS AND PROTOCOLS
影响因子：
--
作者：
Greening, David W.;Xu, Rong;Simpson, Richard J.
通讯作者：
Simpson, Richard J.
DOI：
10.1007/978-1-62703-776-1_19
发表时间：
2014
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Guo, Fei;Jiang, Wen
通讯作者：
Jiang, Wen