Instrument and Method to Determine the Electrophoretic Mobility of Nanoparticles and Proteins by Combining Electrical and Flow FieldFlow Fractionation
Instrument and Method to Determine the Electrophoretic Mobility of Nanoparticles and Proteins by Combining Electrical and Flow FieldFlow Fractionation
复制标题
DOI：
10.1021/ac504712n
复制
发表时间：
2015-04-21
影响因子：
7.4
通讯作者：
Roesch, Ulrich
中科院分区：
文献类型：
--
作者：
Johann, Christoph;Elsenberg, Stephan;Roesch, Ulrich
提出了一种新的FFF方法，它将非对称流FFF（AF 4）和电FFF（ElFFF）结合在一个通道中，形成电非对称流FFF（EAF 4），克服了纯ElFFF的限制。它允许测量作为尺寸的函数的电泳迁移率（μ）。该方法提供绝对值，不需要校准。两种颗粒标准品的μ结果与通过相分析光散射（帕尔斯）测定的值非常一致。EAF 4不需要低离子强度载体。这克服了ElFFF的主要限制之一，使其能够在生理条件下测量蛋白质。EAF 4能够确定单个群体的mu，这些群体被分解为单独的峰。这是证明了三个聚苯乙烯乳胶颗粒的混合物具有不同的大小，以及为单体和二聚体的BSA和抗体。实验装置由具有附加电极的AF 4通道组成;一个电极放置在底壁处的玻璃料下方，另一个电极覆盖上通道板的内部。这种设计通过保持颗粒远离电极来最小化来自电解反应的污染。此外，所施加的电压范围低（1.55 V），这将气态电解产物的量降低到低于干扰层流轮廓或检测器信号的阈值。除了测量μ之外，与纯流动FFF相比，该方法可用于改善样品组分之间的分离。对于两种蛋白质（BSA和单克隆抗体），通过施加电场来实现单体和二聚体的增强分辨率。
A new FFF method is presented which combines asymmetrical flow-FFF (AF4) and electrical FFF (ElFFF) in one channel to electrical asymmetrical flow-FFF (EAF4) to overcome the restrictions of pure ElFFF. It allows for measuring electrophoretic mobility (mu) as a function of size. The method provides an absolute value and does not require calibration. Results of mu for two particle standards are in good agreement with values determined by phase analysis light scattering (PALS). There is no requirement for low ionic strength carriers with EAF4. This overcomes one of the main limitations of ElFFF, making it feasible to measure proteins under physiological conditions. EAF4 has the capability to determine mu for individual populations which are resolved into separate peaks. This is demonstrated for a mixture of three polystyrene latex particles with different sizes as well as for the monomer and dimer of BSA and an antibody. The experimental setup consists of an AF4 channel with added electrodes; one is placed beneath the frit at the bottom wall and the other covers the inside of the upper channel plate. This design minimizes contamination from the electrolysis reactions by keeping the particles distant from the electrodes. In addition the applied voltage range is low (1.55 V), which reduces the quantity of gaseous electrolysis products below a threshold that interferes with the laminar flow profile or detector signals. Besides measuring mu, the method can be useful to improve the separation between sample components compared to pure flow-FFF. For two proteins (BSA and a monoclonal antibody), enhanced resolution of the monomer and dimer is achieved by applying an electric field.