Application Of Microchip Capillary Electrophoresis In Analysis Of Biologic Samples

Life science is the one of scientific frontier in this century. The development of it calls for the advanced analytical methods for separation and detection of biologic samples. Accordingly, life analytical chemistry has become one of the important research area in analytical chemistry.The electrophoretic analysis of biologic samples, such as DNA and single cells, is an important constituent part of life science. Electrophoresis has become a promising and potential technology applied to medicine research and early diagnosis of related diseases. Microchip Capillary electrophoresis (MCE) has emerged as an important tool for analyzing biomolecules due to its high-efficiency, high-throughput and less sample consumption.Surface modification is a key technology for improving the performance of CE separation of biologic macromolecules. The aims of this paper are : (1) development for a new permanent coating method with longevity and good resistance for CE separation of DNA /PCR products with glass microchip (2) Identification of DNA fragments and PCR products with chip-based electrophoresis. (3) simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell.In the first chapter, different approaches and techniques for surface modification of CE /MCE, analysis of DNA fragments and single cell with chip-based CE were summarized.In the second chapter, a novel approach for surface modification on glass MCE with longevity and good resistance was developed. In order to improve the coating reproducibility among the microchips, migrate times of rhodamine 123 (Rh123) and fluorescein sodium (Flu) in electropherograms were used first time as standard to evaluate the effectiveness of pretreatment. By using this standard test procedure, similar density of silanols on the inner surface of different microchips were obtained. Density of silanols was further increased by treatment with HCl: methanol (1:1) under high temperature. Robust polyacrylamide coating was covalently bonded to the silylanized surface by filling the mixture of monomer, initiators and formed linear polyacrylamide into micro-channel. This procedure provides a reproducible, resistant surfacecoating, which showed good resistance to PCR products and buffer solution. Even after 309 analysis the analytical performance did not decreased.In the third chapter, the chip-based electrophoresis was used for the separation of OX-174/HaeIII DNA restriction fragments and for identification of the DNA fragment from LC-1 ScFv on coated glass microfluidic chip with the low-viscosity sieving matrix HPMC-50. The electrophoretic conditions were optimized. Compared with gel electrophoresis, the chip-based electrophoresis provided a rapid, sensitivity method for DNA analysis with higher resolution and less sample and regents consumption.In the fourth chapter, A simple method for the detection of glutathione (GSH) in single human erythrocyte was developed using on-chip mode dynamic labeling by adding the 2,3-Naphthalene-dicarboxaldehyde (NDA) simply in microchip electrophoresis buffer. By using a combination of hydrostatic pressure and low electric field, single cell sampling speed and long-term stability were improved. After lysing, the reaction between the released GSH and NDA included in electrophoresis buffer can be completed within a migrating distance of 0.5 cm in the microchip separation channel during electrophoresis. The average separation efficiency for GSH was 2.4 x 106 plates/m, which was no significant difference from off-chip derivatization. The GSH migration times on the on-chip mode and off-chip method were 116 s and 110 s, respectively. The present method can minimize the dilution of the contents of single cell during derivatization to maintain favorable kinetics for the labeling reaction, simplify the cell treatment and save the sample and reagent. A half life of 5 days of GSH in human erythrocyte was found using this method.In the fifth chapter, a microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single cell loading, docking, lysing and capillary electrophoretic separation with laser-induced fluorescence (LIF) detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 (DHR-123) in the intact cell, while GSH was on-chiplabeled with 2,3-naphthalene-dicarboxaldehyde (NDA), which was included in the separation medium. On-chip electrical-lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir into separation during the separation phase. The structure of the sample reservoir was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells h"1. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.