Capillary and Microfluidic Gradient Elution Isotachophoresis Coupled to Capillary Zone Electrophoresis for Femtomolar Amino Acid Detection Limits
Capillary and Microfluidic Gradient Elution Isotachophoresis Coupled to Capillary Zone Electrophoresis for Femtomolar Amino Acid Detection Limits
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DOI：
10.1021/ac9006182
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发表时间：
2009-07-01
影响因子：
7.4
通讯作者：
Shackman, Jonathan G.
中科院分区：
文献类型：
--
作者：
Davis, Nejea I.;Mamunooru, Manasa;Shackman, Jonathan G.
本文将梯度洗脱等速电泳与毛细管区带电泳（GEITP-CZE）在同一微柱上联用。多阶段方法解决了GEITP中分析物分辨率困难以及CZE中浓度灵敏度差的问题。GEITP采用快速电泳聚焦在一个不连续的离子界面内的样品井通过组合电渗和流体动力学流动产生。然后，当逆流减少时，界面和富集的分析物被拉入毛细管或微通道中用于柱上检测。为了将以GEITP为重点的样品转化为基于CZE的分离，将样品溶液替换为CZE缓冲液，同时保持流体力学流动，以确保向检测器迁移。单一的溶液转换和缺乏极性反转允许可重复的分离（通常峰高的相对标准偏差< 6%，迁移时间< 0.5%）。CZE过程中的低压流体动力学流动允许灵活的分辨率调整，与迁移时间的平方根线性增加，而不改变分离柱，场强，或电解质系统。作为GEITP-CZE适用性的第一个证明，研究了一系列氨基酸，这些氨基酸将在未来的火星探测任务中作为生物生命的指标进行分析。六种氨基酸的分离，检测限低至200 fM，实现了使用毛细管格式，总分析时间为11分钟。一个玻璃为基础的微流控实现也表明，可以执行GEITP-CZE在1厘米的有效长度。
In this work, gradient elution isotachophoresis was combined with capillary zone electrophoresis (GEITP-CZE) in a single microcolumn. The multistage approach addresses the issues of analyte resolution difficulties in GEITP, as well as poor concentration sensitivity in CZE. GEITP employs rapid electrophoretic focusing at a discontinuous ionic interface within a sample well generated through combined electroosmotic and hydrodynamic flows. The interface and enriched analytes are then pulled into a capillary or microchannel as the counter-flow is reduced for on-column detection. To transform GEITP-focused samples to CZE-based separation, the sample solution is replaced with CZE buffer solution while maintaining hydrodynamic flow to ensure migration toward the detector. The single solution switch and lack of polarity inversion allows for reproducible separations (typically < 6% relative standard deviation in peak heights and < 0.5% in migration times). Low-pressure hydrodynamic flow during CZE allowed for flexible resolution adjustment, with a linear increase versus the square root of migration time, without altering the separation column, field strength, or electrolyte system. As a first demonstration of the applicability of GEITP-CZE, a series of amino acids to be assayed for in future Mars exploration missions as indicators of biological life were studied. Separation of six amino acids, with limits of detection as low as 200 fM, were achieved using a capillary format with a total analysis time of 11 min. A glass-based microfluidic implementation is also demonstrated that can perform GEITP-CZE in 1 cm effective lengths.