| Recent years, capillary electrophoresis(CE) has been developed as a powerful platform for online enzyme analysis, duo to its unique advantages, such as high efficiency, rapid analysis, less sample consumption and the ability to utilize several detection methods. CE has become a powerful tool for the quantitative study of enzyme-catalyzed reactions and has been widely applied in nearly all aspects of enzyme assays, including measurement of enzyme activity, screening of inhibitors, and proteome analysis. Three commonly used technologies for online enzyme assay based on CE technology are capillary electrophoresis-immobilized enzyme reactor(CE-IMER), electrophoretically mediated microanalysis(EMMA) and sequential online monitoring of enzymatic reactions. CE-IMER is an enzymatic reaction device, in which the enzymatic reaction and analyte separation are carried out in the same capillary. Sequential online monitoring of enzymatic reactions is vitally important to fully understand the metabolic enzyme functions and fundamental dynamic cellular metabolic reactions. The study of this work focused on the investigation and application of the above two kinds of CE technologies for online enzyme assays. The contents were listed below:(1) We reported a novel fabrication approach of CE-IMERs by packing 20-μm with 1-μm pore size enzyme-immobilized silica particles in the capillary with the 110-μm spherical single particles with wide throughpores(2-μm) as the frits. The fabrication procedure was simple and rapid, and the length and enzyme loading amount of the CE-IMER can be easily adjusted. The Michaelis constant(Km) and the maximum velocity(Vmax) were determined to be 1.23 ± 0.04 m M and 2.99 ± 0.10 m M/s, respectively. The results are in good agreement with those obtained using off-line enzyme assay, indicating the preparation process of immobilized enzyme reactor did not affect the enzyme structure and activity. The packed CE-IMERs exhibited excellent run-to-run reproducibility for enzyme assay, with RSDs(n=100) of 4.0% and 4.1% for the peak area of substrate and product, respectively, and 3.7% and 3.8% for the migration time, respectively. Moreover, it can still remain 90% of the initial activity after ten days with total 50 runs.(2) The packed CE-IMER was applied for trypsin inhibition study and online digestion of protein samples. Under the optimal conditions, the Ki value and IC50 value of the inhibition were determined to be 3.2 ± 0.2 m M and 5.0 ± 0.3 m M, which were in good agreement with those obtained using traditional off-line enzyme assay method. Furthermore, online trypsin digestion of myoglobin and BSA proteins were also investigated using the packed CE-IMERs. ESI-MS analysis showed that 10 peptides identified and 77% coverage of the sequence for digestion of myoglobin and 22 peptides identified with 35% coverage of the sequence for digestion BSA, which was very comparable with those obtained using free trypsin. It is indicated that the method has good application prospect in protein analysis.(3) Studies of theoretical and experimental on sequential two-diffusional sample injection for CE. We fabricated a simple and easy-to-operate sequential CE analysis system, which was constructed by coaxially aligning two capillaries through a sample vial with a distance of 5μm between the capillary ends. Direct online sample injection and sequential CE analysis were easily achieved by free diffusion(without derivatization or other chemical reaction) at the interface of the capillaries and periodically switching the high-voltage power supply off and on. We developed a two-dimensional(2-D) sequential diffusion injection(SDI) model and derived the formula of the absolute injection amount(Ainj), initial sample concentration(C0), the injection time(t), and the distance of the capillary gap(d) based on the Fick’s second law. We also carried out systemic experiments to demonstrate the model using standard NADH. The experimental results indicate that the system is a two-dimensional(2-D) diffusion system both has longitudinal and transverse directions, and enhance the injection efficiency in comparison to classical concentration diffusion on the plane interface. The results show good repeatability of sequential injection with RSD(n = 20) of 2.08% and 1.83% for peak area and peak width, respectively.(4) The SDI method has been applied for analysis of enzymatic reaction and complex samples. Using the SDI system, we detected the product NADH every 40 s and achieved online monitoring of glucose-6-phosphate dehydrogenase-catalyzed reaction. The Michaelis constant(Km) determined was in good agreement with that obtained using traditional off-line enzymatic assay method, indicating the accuracy of the present method for online enzyme assay. In the study of kinetics of enzyme reaction inhibition, we successfully obtained alanine aminotransferase inhibition kinetics parameters, which show its application prospect in the fields of inhibition study and screening inhibitors. In addition, the system was successfully applied in sequential CE analysis of amino acids mixture, which exhibits good repeatability for sequential CE analysis, with RSDs(n = 9) less than 3.45% and 3.96% for resolution and theoretical plates, respectively. The above results indicate the 2-D SDI system based on CE is a powerful tool for sequential analysis, which not only for online monitoring various bioprocesses but also for sequential analysis complex samples. |
PDF Full Text Request