The origins of microfluidics field lie in microanalytical methods such as gas chromatography, high pressure liquid chromatography and capillary electrophoresis that are revolutionizing the field of chemical analysis. Microfluidic devices have been particularly attractive for separation based chemical and biological analysis since the small length scales bring fundamental improvements in reagent volume use, analysis time, resolution, and separation efficiency. However, smaller length scales and volumes are also associated with lower detection sensitivity and therefore, this limits many applications to fluorescent analytes. This dissertation focuses on electrokinetic preconcentration and separation methods to improve the detection sensitivity on microchip platform and to extend the scope of microfluidic analysis to non-fluorescent analytes.;Isotachophoresis (ITP) is a robust sample preconcentration technique that leverages spatial mobility gradients to focus analytes into zones that are ∼10 mum wide. Such extreme focusing of analytes results in drastic improvement in the detection sensitivity and resolution of electrophoretic separation system. We present a theoretical and experimental study of dynamics of ITP preconcentration that helps to identify optimum operating parameters to achieve high sample preconcentration. The theoretical study involves development of analytical models to identify the fundamental parameters governing the focusing dynamics and development of perturbation model and dispersion model to reduce the complexity of this numerically stiff problem. We performed controlled experiments to isolate the effect of each parameter influencing the preconcentration dynamics from others. These experimental results are used for validation of theoretical models and also serve as guidelines for ITP preconcentration assay design.;We have also developed an indirect detection technique to detect non fluorescent analytes on standard microfluidic setup equipped with fluorescence detection platforms. We leverage ITP to preconcentrate and separate analytes into distinct analyte zones arranged in order of reducing electrophoretic mobility. Using a ladder of fluorescent species with different electrophoretic mobilities (termed as fluorescent mobility markers) to demarcate the boundaries of these analyte zones, we indirectly detect the non-fluorescent analytes present. We obtain ∼1 muM detection sensitivity with this assay with high repeatability and have demonstrated indirect detection of a variety of analytes such as amino acids, organic acids and environmental toxins such as phenols and cresol.;Lastly, we demonstrate simultaneous electrophoretic preconcentration and separation of analytes in a single step injection process in off-the-shelf, standard microfluidic chips using isotachophoresis (ITP). Our technique leverages an electric field gradient between the leading and trailing electrolytes to concentrate analytes into distinct non-dispersing bands. This is the first experimental study to identify that the gradient results from slow reaction kinetics of hydration and carbamation of dissolved atmospheric carbon dioxide. We use a fluorescent counterion tracer technique to study the evolution of carbonate and carbamate zones and the electric field gradient region between them. Using this assay, we have demonstrated one step focusing and separation of 25 bp DNA ladder, and the fractionation of DNA and proteins. The technique has potential to simplify and improve the detection sensitivity of many microchip electrophoresis assays.
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机译：微流体领域的起源在于微观分析方法，例如气相色谱法，高压液相色谱法和毛细管电泳法，这些方法正在彻底改变化学分析领域。微流控设备对于基于分离的化学和生物学分析特别有吸引力，因为小长度标尺带来了试剂体积使用，分析时间，分离度和分离效率的根本改善。然而，较小的长度标度和体积也与较低的检测灵敏度有关，因此，这限制了荧光分析物的许多应用。本文着重研究电动预富集和分离方法，以提高在微芯片平台上的检测灵敏度，并将微流控分析的范围扩展到非荧光分析物。等速电泳（ITP）是一种强大的样品预富集技术，它利用空间迁移率梯度来聚焦分析物进入约10毫米宽的区域。分析物的这种极端聚焦导致了电泳分离系统的检测灵敏度和分辨率的大幅提高。我们介绍了ITP预浓缩动力学的理论和实验研究，有助于确定最佳操作参数以实现高样品预浓缩。理论研究涉及分析模型的开发，以识别控制聚焦动力学的基本参数，以及开发扰动模型和色散模型以减少此数值刚性问题的复杂性。我们进行了对照实验，以分离每个参数对预浓缩动力学的影响。这些实验结果可用于理论模型的验证，也可作为ITP预浓缩测定设计的指南。我们还开发了一种间接检测技术，可在配备荧光检测平台的标准微流控装置上检测非荧光分析物。我们利用ITP进行预浓缩并将分析物分离成不同的分析物区域，以降低电泳迁移率。使用具有不同电泳迁移率的荧光物质梯子（称为荧光迁移率标记）划定这些分析物区域的边界，我们间接检测到存在的非荧光分析物。通过这种测定方法，我们获得了〜1μM的检测灵敏度，并且具有很高的重复性，并证明了间接检测多种分析物的方法，例如氨基酸，有机酸和环境毒素（例如苯酚和甲酚）。最后，我们展示了同时进行电泳预富集和分离的方法使用等速电泳（ITP）在现成的标准微流控芯片中以单步进样过程分析被分析物。我们的技术利用前后电解质之间的电场梯度将分析物浓缩为不同的非分散带。这是第一个确定梯度是由溶解的大气二氧化碳的水合和氨基甲酸酯化的缓慢反应动力学产生的实验研究。我们使用荧光抗衡离子示踪剂技术研究碳酸盐和氨基甲酸酯区的演变以及它们之间的电场梯度区域。使用该测定法，我们证明了聚焦和分离25 bp DNA阶梯的第一步，以及DNA和蛋白质的分离。该技术具有简化和提高许多微芯片电泳分析的检测灵敏度的潜力。
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