Study And Application Of Enzyme-immobilized Microreactor In Enzymatic Kinetics Based On Capillary Electrophoresis Technique

In recent years, capillary electrophoresis(CE), which is a new kind of separation and analytical technique and was rapidly developed, has been widely used in many fields and has been attacted many interest in separation and analytical science. CE has shown several unique advantages, such as high separation efficiency, rapid analysis, extremely small volumes of sample consumption, various operation modes,etc.,which make it a quite suitable method to efficiently separate complex analytes as well as to sensitively detect trace compounds.Based on capillary electrophoresis, the thesis investigated enzyme raction kinetics via dual-enzyme microreactors.The new dual-enzyme microreators were fabricated by covalently immboliziing dual enzyme on the inner surface of a capillary or onto surfaces of magnetic beads. The purpose of the thesis was to establish a high efficient, rapid, sensitive method for enzyme assay based on capillary electrophoresis. We showed that the microreactors has great stability and reproducibility, high sensitivity and low limit-of-detetion, as well as high selectivety, and can be used for sensitively detection in real sample. This work was mainly focused on the following aspects:(1) By covalently co-immobilizing glutamate dehydrogenase (GDH) and glutamic pyruvic transaminase(GPT) on the inner surface of a capillary, a novely GDH-GPT dual-enzyme capillary microreactor was fabricated, and was characterized by a variety of spectroscopic methods.This capillary microreactor can be used for glutamate detection was prepared.(2) By using the GDH-GPT dual-enzyme capillary microreactor in combination with ACE, the GDH-GPT coupled reaction with glutamate ads the substrate was studied. The Km constant for glutamate with GDH-GPT coupled reaction was determined. The dual-enzyme microreactor has good linear response (R2 =0.9990) with concentration of glutamate from 0 to 100μM and had good specificity. The detection limit of the proposed method is 0.15 mM glutamate (S/N=3). Finally, this method was successfully applied to determine the glutamate content in rat plasma and serum samples.(3) A new dual-enzyme immobilized capillary microreator was developed, in which the dual enzymes were coated onto the surfaces of magnetic beads. Alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) were covalently bonded on magnetic beads surface, then coated magnetic beads were fixed at appropriate location in a capillary by strong magnetic fields.(4)The capillary microreactor with ADH and LDH coated on magnetic beads, was used to study enzymatic reactions of acetaldehyde and sodium pyruvate. The Km for acetaldehyde, sodium pyruvate, NADH was determined. And the fabricated method was successfully applied to determine the trace amount of acetaldehyde and pyruvate in six beers samples.