Department of Mechanical Engineering , University of Utah , 1495 E 100 S , Room 1550, Salt Lake City , Utah 84112 , United States.
Department of Pharmacology and Toxicology & James Graham Brown Cancer Center , University of Louisville , Kosair Charities Clinical & Translational Research Building, Room 411, 505 South Hancock Street , Louisville , Kentucky 40202 , United States.
Anal Chem. 2018 Nov 6;90(21):12783-12790. doi: 10.1021/acs.analchem.8b03146. Epub 2018 Oct 22.
The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, 1× phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at ∼1.3 V AC. A375 melanoma exosome recovery was approximately 70-80% after each buffer substitution, and recovery was independent of whether the sample was substituted into 1× PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using 1× PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from ∼-1.6 to -0.1 μm cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from ∼-5.5 to -0.1 μm cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.
首次显示了缓冲液替代和稀释效应对外泌体大小和电泳迁移率的影响。在各种替代流体中循环电场流动分级（Cy-El-FFF）被应用于外泌体和其他颗粒。测试的载流液为去离子（DI）水、1×磷酸盐缓冲盐水（PBS）、0.308 M海藻糖和 2%异丙醇（IPA），它们影响 Cy-El-FFF 介导的 A375 黑色素瘤外泌体的分离。所有的馏分图都显示出保留时间随着电压的增加呈新月形趋势，最大保留时间约为 1.3 V AC。每次缓冲液替代后，A375 黑色素瘤外泌体的回收率约为 70-80%，并且回收率与样品是否替代为 1×PBS 或 DI 水无关。在外泌体在去离子水中的稀释产生了对电泳迁移率的 U 形依赖性。使用 1×PBS 缓冲液稀释的效果显示出外泌体的电泳迁移率从约-1.6 到-0.1 μm cm/s V 非常缓慢的变化，随着外泌体浓度的降低。这与使用 DI 水不同，在相同的稀释范围内观察到从约-5.5 到-0.1 μm cm/s V 的大变化。在两种替代的低离子强度缓冲液中分离的 A375 黑色素瘤外泌体的馏分图与合成颗粒的馏分图进行了比较。总的来说，Cy-El-FFF 根据外泌体的大小和电荷分离外泌体的能力是一种很有前途的、无需标记的方法，可以用于初步对外泌体亚型进行分类和纯化，以用于生物库存储，以及进一步对外泌体亚型进行研究。
