On-chip isotachophoresis assays for high sensitivity electrophoretic preconcentration, separation, and indirect detection

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.