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MICROCHIP CAPILLARY ELECTROPHORESIS: A POWERFUL TOOL FOR BINDING STUDIES AND TRACE PROTEIN ANALYSIS USING APTAMER PROBES

by GONG, MAOJUN

Abstract (Summary)
Microchip capillary electrophoresis (CE) is the miniaturized form of traditional CE, a powerful electrical separation technique performed in a capillary with micron diameters. The advantages of microchip CE over traditional CE include shorter analysis time and less consumption of chemicals and reagent. The objective of the microchip program in our group is to develop microchip CE instrumentation and methodologies for high throughput screening of pharmaceuticals. As an important step, my work was focused on the development of methodologies for binding studies and trace protein assays in single-lane glass microchips. A pair of thrombin and thrombin-binding aptamer was chosen as a model system to demonstrate the feasibility of microchip CE in binding studies. The combination of a short detection length (1.0 cm) and a high electric field (670 V/cm) succeeded in the detection of the aptamer-thrombin complex and reduction of thrombin adsorption. The limit of detection (LOD) reached 5.0 nM of thrombin. To determine the equilibrium dissociation constant of aptamer-thrombin, a simple hydrodynamic injection method in microchips was developed. This hydrodynamic injection significantly reduces sample bias although the dispensing bias still exists. Using the hydrodynamic injection method, the dissociation constant of aptamer-thrombin was determined to be 23 nM which is consistent with reported results. Moreover, an accurate and fast frontal analysis method was developed for the determination of dissociation constant with a model pair of aptamer and immunoglobulin E (IgE). The capability of microchip CE coupled with a laser-induced fluorescence (LIF) detection system in the detection of trace protein was demonstrated using a 66-mer photoaptamer to detect 165-mer vascular endothelial growth factor (VEGF165). A LOD of 1.0 nM was achieved for the detection of VEGF165 using the fluorescently-labeled photoaptamer. To enhance detection sensitivity of analyte in microchip CE, a field-amplified stacking injection (FASI) was developed in a single-lane glass microchip. This FASI method takes advantage of the pressure-induced flow as a way to supply analytes for stacking. A 100-fold detection enhancement for model analytes of fluorescein (FL), FITC and 5-carboxyfluorescein (5-FAM) using FASI was achieved relative to the commonly used pinched injection method. Sweeping is an effective and convenient way to preconcentrate analytes in micellar electrokinetic chromatography (MEKC). Several sweeping schemes in traditional and microchip CE were developed using cationic surfactants. A new sweeping carrier, aggregates of dodecyltrimethylammonium bromide (DTAB), was found to have strong sweeping power for oligonucleotides at concentrations below, instead of above, the critical micelle concentration (CMC). A 112-fold detection enhancement was achieved for 15-mer thrombin-binding aptamer in traditional CE. An unlimited-volume injection method was developed using electrokinetic stacking injection (EKSI). Equivalent injection capillary lengths of 7.3 and 8.4 m for FL and 5-FAM were achieved in 60-min EKSI using DTAB micelles. A flow control method for sweeping microchip CE was developed using tetradecyltrimethylammonium bromide (TTAB). This method successfully preconcentrated analytes of FL and 5-FAM with detection enhancements of 250- and 500-fold, respectively
Bibliographical Information:

Advisor:

School:University of Cincinnati

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:microchip capillary electrophoresis binding studies trace analysis sample stacking sweeping

ISBN:

Date of Publication:01/01/2006

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