Gradient elution isotachophoresis for enrichment and separation of biomolecules
Gradient elution isotachophoresis for enrichment and separation of biomolecules
复制标题
DOI：
10.1021/ac070857f
复制
发表时间：
2007-09-01
影响因子：
7.4
通讯作者：
Ross, David
中科院分区：
文献类型：
--
作者：
Shackman, Jonathan G.;Ross, David
本文介绍了一种新的毛细管等速电泳（ITP）方法-梯度洗脱ITP（GEITP）。GEITP将最近描述的梯度洗脱移动边界电泳（GEMBE）的电泳分离技术与ITP富集步骤合并。GEMBE利用连续进样与压力控制逆流相结合;随着逆流减少，分析物依次洗脱到分离柱上并作为边界界面进行检测。通过将前导电解质并入逆流中并将终止电解质并入样品基质中，可以在毛细管入口附近形成离子界面。不连续缓冲系统在毛细管外形成高度富集的分析物区，然后随着逆流减少，分析物区被洗脱到分离毛细管上。荧光分析物的分离是通过添加到样品中的离散电解质间隔物或通过使用两性电解质混合物形成连续的间隔物来实现的。由于ITP过程发生在柱外，因此可以实现极短长度的分离，如30 μ m的分离所示。GEITP富集过程中的各种参数的影响进行了研究，包括初始逆流速率，电场，领先的电解质浓度，和逆流加速度，这是一个可调的参数，允许高度灵活的分离。检测限和灵敏度的典型增强大于10000倍，并在不到2分钟内实现，产生低皮摩尔的检测限，使用弧光灯照明和低成本的CCD检测。一个优化的系统提供了大于100000倍的改进，在检测羧基荧光素在8分钟。具体的例子，富集和分离证明包括以下内容：小染料分子，DNA，氨基酸的混合物，和蛋白质的混合物。
A novel format for performing capillary isotachophoresis (ITP) is described-gradient elution ITP (GEITP). GEITP merges the recently described electrophoretic separation technique of gradient elution moving boundary electrophoresis (GEMBE) with an ITP enrichment step. GEMBE utilizes a combination of continuous sample injection with a pressure-controlled counterflow; as the counterflow is reduced, analytes are sequentially eluted onto the separation column and detected as boundary interfaces. By incorporating leading electrolytes into the counterflow and terminating electrolytes into the sample matrix, an ionic interface can be formed near the capillary inlet. The discontinuous buffer system forms highly enriched analyte zones outside of the capillary, which are then eluted onto the separation capillary as the counterflow is reduced. Separation of fluorescent analytes was achieved either through discrete electrolyte spacers added to the sample or by using ampholyte mixtures to form a continuum of spacers. As the ITP process occurs off-column, extremely short length separations can be achieved, as demonstrated by a separation in 30 mu m. The effects of various parameters on the GEITP enrichment process are investigated, including initial counterflow rates, electric field, leading electrolyte concentration, and counterflow acceleration, which is an adjustable parameter allowing for highly flexible separations. Typical enhancements in limits of detection and sensitivity were greater than 10000-fold and were achieved in less than 2 min, yielding low-picomolar detection limits using arc lamp illumination and low-cost CCD detection. An optimized system afforded greater than 100000-fold improvement in detection of carboxyfluorescein in 8 min. Specific examples of enrichment and separation demonstrated include the following: small dye molecules, DNA, amino acid mixtures, and protein mixtures.