Study On Methodologies And Applications Of Microchip Electrophoresis Coupled With Laser-Induced Fluorescence Detection

Micro total analysis system is a field that has been growing ever since the early 1990s. Microfluidic devices originated from the integrated circuits (IC) industry, and early microfluidic devices are mostly silicon based analyzers consisting of channels for species separation in a carrier fluid. However, with recent advances in microfabrication and device design innovative components and platforms are coming to fore with applications in biology, medicine, pharmaceutics, and food and environmental monitoring. "Micro total analysis systems" (microTAS) or "lab-on-a-chip" (LOC) are becoming a reality where entire chemical analyses in miniaturized volumes are performed with high sensitivities and in shorter time spans.Owing to its inherent selectivity and excellent sensitivity, laser-induced fluorescence (LIF) has been becoming the most popular detection scheme in conjunction with microchip electrophoresis. Various LIF detection systems based on different optical arrangements were developed and have been successfully and broadly applied in the microfluidic chip-based analysis systems. The optical arrangements of hitherto three major types of LIF detection system employed in chip devices, the confocal LIF system, nonconfocal LIF detection system and orthogonal optical arrangement system.In this dissertation, a LIF detection system for microfluidic chips with a simple orthogonal optical arrangemen was developed. The technologies about microchip electrophoresis were systemic studied, including chip microfabrication, separation DNA marker to investigate the performance of the sieve, detection of the mutations in p53 gene and determination of amino acids in milk. The work was involved in the typical manipulation, such as the preconcentration, reaction, separation and detection. And the application of the LIF detection systems was realized.The topics of this dissertation are presented below:Chapter 1 reviews the research and development on microchip-based analytical systems. Then the new projects were carried out.Chapter 2 states an integrated system involving microfluidic chip, high voltage power supply, laser-induced fluorescence (LIF) detection and control system was developed to perform microchip electrophoresis separation. A simple and convenient approach for accurately locating the chip in the integrated system was developed by using a substrate plate as the chip flat added a base spring to support the position of the chip. An orthogonal optical arrangement was employed in the LIF detection system to simplify the structure of the system with a limit of detection of 10-11 mol/L for Cy5 dye.Chapter 3 states copolymers of poly(vinylpyrrolidone) (PVP) and hydroxyethylcellulose (HEC) were synthesized, with PVP to HEC molar ratios of 3:1, 2:1 and 1:1. The copolymers were tested as separation media in DNA fragment separation analysis by microchip electrophoresis (MCE). Separation efficiency over 3.8x105 for 118 bp has been reached by using the bare channels without the additional polymer coating step. Under optimized separation conditions for longer read length DNA sequencing, the separation ability of the copolymers decreased with decreasing (PVP-co-HEC) molar ratio from 3:1,2:1 and 1:1. In comparison with (PVP-co-HEC) 1:1, the copolymer with (PVP-co-HEC) 3:1 ratio showed high separation efficiency. By using a 2.5% w/v copolymer with (PVP-co-HEC) 3:1 ratio, FX174-Haeâ…¢digest DNA marker was successfully separated within 3 min.Chapter 4 states a simple and robust static adsorptive coating process in glass microchips for DNA separation was developed by using mixed polymer consisted of 2% hydroxyethylcellulose and 4% poly (vinylpyrrolidone) as surface modifier of microfluidic channels. This surface modifier was also used as the sieving matrix for the DNA separation. In this study, FX174-HaeIII digest sample was used to investigate the performance of the polymer mixture in microchip electrophoresis with laser-induced fluorescence detection. It was also found that adding 30 nm gold nanoparticles to the mixed polymer was very useful to achieve better resolution and reproducibility. Under the optimum conditions, the DNA fragments could be successfully separated within 4 min and the relative standard deviation values of the migration times were less than 2.51%(n=5) during the one week.Chapter 5 states a form of single-strand DNA-conformation polymorphism analysis (SSCP) employing nondenaturing slab gel electrophoresis is applicable to the genetic diagnosis of mutations at exons 7,8 and 9 of the p53 gene. Recently, microchip electrophoresis (ME) systems have been used in SSCP analysis instead of conventional slab gel electrophoresis in terms of speed, sensitivity and automation. The aim of the present study was to investigate the application of SSCP and ME analysis as a rapid and effective method for detection of mutations for exons 7,8 and 9 of the p53 gene. It was found that using the electric field strength 260 V/cm and the sieving matrix of 0.5% w/v poly(ethylene oxide) was very useful to achieve better resolution and fast detection of mutations at exons 7,8 and 9 of p53 gene. Under the optimized conditions, mutations at exons 7-9 of p53 gene were analyzed within 60 s and the relative standard deviation values of the migration times were less than 5.81% (n=5). The detection limit can be as low as 1 ng L-1.Chapter 6 states a method for determination of amino acids in milk by microchip electrophoresis (ME) coupled with the detection of laser-induced fluorescence (LIF) has been developed. The amino acids in milk which derivatized with fluorescein isothiocyanate were finely separated and determinated by ME-LIF with borate buffer. The separation parameters were investigated to give an optimal experimental condition:borate buffer (100 mmol/L, pH 9.7); applied separation fleld 475 V/cm. All amino acids were separated within 150 s. All amino acid-Cy5 derivatives was linear in the appropriate concentration (R20.9904-0.9984). The relative standard deviations (n=5) of the method were 2.1%-4.5% for migration time and 2.3%-5.4% for peak area. This method allows to determining the amino acids in milk with high sensitivity.