Sun Xuefei, Farnsworth Paul B, Tolley H Dennis, Warnick Karl F, Woolley Adam T, Lee Milton L
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA.
J Chromatogr A. 2009 Jan 2;1216(1):159-64. doi: 10.1016/j.chroma.2008.11.031. Epub 2008 Nov 19.
Electric field gradient focusing (EFGF) is a technique used to simultaneously separate and concentrate biomacromolecules, such as proteins, based on the opposing forces of an electric field gradient and a hydrodynamic flow. Recently, we reported EFGF devices fabricated completely from copolymers functionalized with poly(ethylene glycol), which display excellent resistance to protein adsorption. However, the previous devices did not provide the predicted linear electric field gradient and stable current. To improve performance, Tris-HCl buffer that was previously doped in the hydrogel was replaced with a phosphate buffer containing a salt (i.e., potassium chloride, KCl) with high mobility ions. The new devices exhibited stable current, good reproducibility, and a linear electric field distribution in agreement with the shaped gradient region design due to improved ion transport in the hydrogel. The field gradient was calculated based on theory to be approximately 5.76 V/cm(2) for R-phycoerythrin when the applied voltage was 500 V. The effect of EFGF separation channel dimensions was also investigated; a narrower focused band was achieved in a smaller diameter channel. The relationship between the bandwidth and channel diameter is consistent with theory. Three model proteins were resolved in an EFGF channel of this design. The improved device demonstrated 14,000-fold concentration of a protein sample (from 2 ng/mL to 27 microg/mL).
电场梯度聚焦（EFGF）是一种基于电场梯度和流体动力流的相反作用力，用于同时分离和浓缩生物大分子（如蛋白质）的技术。最近，我们报道了完全由聚乙二醇功能化的共聚物制成的EFGF装置，该装置对蛋白质吸附表现出优异的抗性。然而，先前的装置没有提供预测的线性电场梯度和稳定电流。为了提高性能，将先前掺杂在水凝胶中的Tris-HCl缓冲液替换为含有具有高迁移率离子的盐（即氯化钾，KCl）的磷酸盐缓冲液。由于水凝胶中离子传输的改善，新装置表现出稳定的电流、良好的重现性以及与成形梯度区域设计一致的线性电场分布。当施加电压为500 V时，根据理论计算，R-藻红蛋白的场梯度约为5.76 V/cm²。还研究了EFGF分离通道尺寸的影响；在较小直径的通道中实现了更窄的聚焦带。带宽与通道直径之间的关系与理论一致。在这种设计的EFGF通道中分离出了三种模型蛋白质。改进后的装置将蛋白质样品浓缩了14000倍（从2 ng/mL到27 μg/mL）。
