Isoelectric Focusing at the Preparative Scale and Isotachophoresis at the Analytical Scale to Separate and Concentrate Molecules of Interest
Bottenus, Danny R.
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Electrophoresis is a technique to separate and/or concentrate charged analytes under an applied electric field. This thesis is divided into seven chapters. The first chapter outlines the dissertation with an introduction of two different electrophoresis techniques, isoelectric focusing (IEF) at the preparative scale andisotachophoresis (ITP) at the analytical scale. The next two chapters examine the advancements in electric field strength of IEF techniques at the preparative scale and the incorporation of a single-point optical fiber detection system to monitor protein concentrations during IEF runs in a preparative device. The next three chapters discuss both anionic and cationic ITP to concentrate negatively and positively charged low abundant molecules at the analytical scale by experimentation and simulation. The first of these three chapters examines a 3-D numerical simulation of ITP in a microfluidic channel with experimental results to validate the simulation. The next two chapters use cationic ITP to isolate a relavant biomarker, cardiac troponin I (cTnI), and concentrate that same biomarker in both buffer and depleted human serum. The final chapter concludes the research examined in the dissertation and comments on the ongoing and future work.In the first two sections, IEF, which is a powerful technique to separate proteins and other amphoteric solutes in a pH gradient according to their isoelectric points, is used to process several milligrams of proteins at voltages as high as 15 kV. Also, a novel fiber optic detection system has been described that can monitor protein concentrations in real-time.In the next three chapters, ITP is executed to concentrate low abundant molecules at the analytical scale in a 3-D microchip platform. ITP has the ability to concentrate charged analytes by several orders of magnitudes. Included in these sections is a 3-D numerical simulation of ITP in a cascade microfluidic chipthat includes a 50-100x reduction in cross-sectional area. In addition, ITP can be used to concentrate and isolate both positively and negatively charged molecules of interest in a cascade microchip. Sensitivity of a relevant cardiac biomarker, cTnI, in both buffer and depleted human serum was observed in our microchip platform.