On-line&Real Time Capillary Chip Electrophoresis Separation And Laser Induced Fluorescence Detection Of Chip PCR Products

Dierect on-line and/or real time detection of polymerse chain reaction (PCR) products is highly important in the practice of qualitative and quantitative analysis of DNA, and this is also a major and challenging subject in microfluidic chip analytical techniques. In this thesis, interfacing of chip PCR and capillary chip electropHoresis was realized taking advantage of fracture injection, static chip PCR technique and a homemade laser induced fluorescence detection system. On-line PCR products sampling, separation and detection was demonstrated with the system, and its capability in real time analysis of the PCR products was also shown.In chapter one, the principle of PCR and recent development was summarized, with empHasis on real time fluorescence quantitative PCR. Integrated chip electropHoresis related substrate materials, sample introduction methods, as well as detection methods are also reviewed.In chapter two, a homemade laser induced fluorescence (LIF) detection system was introduced, which was build based on a inverted microscope and diode laser sources (487/532nm), the performance was assessed, and the minimum detectable concentration of1×10-12mol/L for rhodamine B was achieved.In chapter three, a capillary chip electrophoresis system was established using fracture sampling and the homemade LIF. The performance such as sample injection reproducibility, resolution etc were evaluated with a fluorescence probe. Peak height reproducibility was0.95%(n=9), and separation efficiency was2.66×105plate/m. This chip system is easy to fabricate and operate, highly efficient and could be easily automated and so on.In chapter four, the capillary chip electrophoresis system was successfully used for DNA separation, including DNA marker, λDNA and a sex related DYZ-1type gene products directly amplified from plasma sample, with high separation efficiency (2.91×105plate/m), high reproducibility. The chip electrophoresis system was on-line joined to a static chip PCR system, and on-line sampling and analysis of the PCR products was implemented, and preliminary results also showed its capability in real time sampling and analysis of the products during PCR process.The system introduced in this work could become the basis of novel quantitative PCR method with its potential capabilities in on-line and real time sampling, fast separation and sensitive detection.