The ongoing development of the technique of two-beam fluorescence cross-correlation spectroscopy coupled with continuous-flow capillary electrophoresis .

摘要

This thesis describes work performed in the development and application of the technique of two-beam fluorescence cross-correlation spectroscopy coupled with continuous-flow capillary electrophoresis (2bFCCS-CFCE). In this approach, fluorescently-labeled molecules of interest are monitored as they electrophoretically migrate in continuous solution between two, spatially-separated laser foci. The work presented here illustrates a number of advantages that 2bFCCS-CFCE analysis has over more conventional capillary electrophoresis (CE) techniques. Three sets of experiments were performed that illustrate these advantages. In the first set of experiments, 2bFCCS-CFCE was used to detect and discriminate between three different analyte molecules electrophoretically migrating in opposite directions. The ability of 2bFCCS-CFCE to monitor molecules electrophoretically migrating in opposite directions without the need of sample injection steps or multiple buffers demonstrated a distinct advantage over conventional CE techniques, which require complex buffer conditions, sample injection, and can only analyze species migrating in uniform direction. The second set of experiments demonstrated 2bFCCS-CFCE on microchip platforms (2bFCCS-CFMCCE). Several design advantages of 2bFCCS-CFMCCE relative to separations-based CE microchip platforms, such as increased miniaturization and simpler fluid handling format, were demonstrated. The third set of experiments demonstrated the ability of 2bFCCS-CFCE to measure the effective charge of a single-stranded DNA (ssDNA) in the presence of magnesium ions. The measurement of effective charge requires analysis of both diffusion and electrophoretic migration simultaneously. Conventional CE experiments are not capable of making this measurement. 2bFCCS-CFCE was used to successfully measure the effective charge, revealing the interesting relationship between ssDNA charge and size.