Instrumentation, capillary coating, and labeling chemistry for capillary electrophoresis with laser -induced fluorescence detection.

摘要

A miniaturized Capillary Electrophoresis (CE) instrument with Laser-Induced Fluorescence (LIF) detection (CE-LIF) and its performance optimization is shown. Different injection techniques in CE are discussed and experimental results show the high sensitivity of the CE-LIF method. Limit of detection of 150 fluorescein molecules is presented. A light shield was designed for the instrument and it was shown that the major contribution to the background noise comes from the laser and not from the room light.;A method is presented for electroosmotic flow (EOF) measurement in the case that the instrument does not have a UV detector, as is the case for CE-LIF. The method validation is shown and statistical data analysis shows that this new method is as precise as the neutral marker injection method for EOF measurement. Basic principles of CE were investigated including the effect of pH, ionic strength, temperature and electric field on EOF.;Coating the capillary inner surface with a polymer to eliminate EOF is also presented. A series of polymers from the most hydrophobic to the most hydrophilic one are compared to find which polymer shows the lowest EOF and minimum adsorption of proteins and antibody (Ab) to the capillary wall. It was shown that a Si-C sub-layer is much more stable than Si-O sub-layer and acryloylaminopropanol (AAP) monomer, when polymerized on the capillary surface, has the least EOF and also the minimum adsorption of proteins.;A high-sensitive method to measure natural toxins in water (microcystins) using CE is also studied. The immunoassay technique was chosen because it was already used for this class of toxins that are liver carcinogens, but the classical immunoassay technique lacked sensitivity. The first step was to fluorescently label the analyte, called antigen in immunoassay, and this process involved studying amine labeling chemistry, developing carboxylic acid labeling, and finally synthesizing an arginine-specific labeling reagent.;Labeling chemistry for CE-LIF Immunoassay of carboxylic acids is presented. Despite being optimized by the author, amine-labeling chemistry with FQ fluorogenic dye is not discussed because it is widely used in CE and the analytes of interest lack an amine group. Microcystin lacks an amine group, but it has two carboxylic acid groups and one arginine group.;Microgram-scale organic synthesis of an arginine-specific reagent is also studied. Microcystin contains an arginine group. In this study an optimized procedure to label the arginine group is presented. Because the sample quantity is very limited, the synthesis approach was developed in a way to cut down the weight of starting material to the microgram level to reduce reagent consumption.