Polymer Based Continuous-Flow PCR Microfluidic Chip System And Its Application

The polymerase chain reaction (PCR) microfluidic chip is one kind of the micro chemical reaction system with micron-scalar unit reaction boundary. It has characteristics of small linear dimension, great gradient of physical quantity, high surface to volume ratio and laminar flow with low Reynolds number. It could carry out flexible production, scale-up, rapid and high-throughput screening. The PCR microfluidic chip organically combines the advantages of the PCR technology and the microfluidic chip technology. It could quickly realize the in vitro amplification of specific DNA and has become an important research field of the MEMS (Micro Electro Mechanical System) technology. Comparing to conventional PCR thermocycler, the main advantages of the PCR microfluidic chip are shorter reaction time, smaller size, lower reagent consumption, higher amplification specificity and conveniently integration with other analysis and detection instruments.To develop a practical PCR microfluidic chip system, the polymethyl methacrylate (PMMA) based continuous-flow PCR microfluidic chip system and its application technology was researched in this thesis. Excimer laser and CO2 laser direct-writing ablation techniques were both used to fabricate the microchannels on the PMMA substrate. The microchannels with rectangular, semicircular and Gaussian cross-sectional shapes were separately fabricated with appropriate laser working parameters. The PMMA substrate ablated with serpentine microchannels was bonded together with other plain PMMA cover sheet to form a closed PCR microfluidic chip by means of the hot-press bonding technique.The bottom surface of the microchannel fabricated with excimer laser direct-writing ablation technique has periodic ripples because of the pulse output character of the excimer laser. When CO₂ laser direct-writing ablation technique was used to fabricate the microchannel, some small bumps of resolidified material were attached to its surface. The excimer laser irradiation technique was utilized in this paper to polish the microchannel and improve its surface quality. The fluid could continuously and smoothly flow in the polished microchannel without generation of bubbles. Moreover, the surface hydrophilicities of PMMA microchannel and substrate were modified using excimer laser ablation and irradiation techniques. These phenomenons are useful in the application of the PCR microfluidic chip.Numerical simulations based on Computational Fluid Dynamics (CFD) method were made to analyze the characteristics flow through the microchannels with different cross-sectional shapes. Laminar flow was still valid because of the small dimension of the microchannel and the low flow rate. The velocity distributions were all non-uniform and the fastest velocity was at the center of the microchannel. This phenomenon is useful for diffusion and confusion of the PCR mixture in the microchannel. The outlet velocity distribution relative proportions in the microchannels with different cross-sectional shapes were distinct. The influence of the change in the viscosity caused by the temperature could be ignored in this paper. The pressure drop across the microchannels is proportional to the flow rate and inverse proportional to the temperature. When hydraulic diameters are same, the microchannel with Gaussian cross-section has larger area. It is more propitious to decrease friction, reduce pressure drop and enhance fluidic flowability and stability.The overall structure of the continuous-flow PCR microfluidic chip system was improved in this paper. The top-plug sampling interface was designed to reduce the dead volume of the interface and the instability of flow rate caused by the size transition between different channels. New double-sealing method was developed to increase the sealing effect and prolong the service life. The temperature distribution characteristic on the PCR microfluidic chip was meliorated with parameters optimization of temperature control system and addition of the heat insulation baffles. The heat insulation effect between different temperature zones was strengthened. An air-operated sampling device for the continuous-flow PCR microfluidic chip was developed. It could reduce reagent consumption and flexibly control the flow rate of PCR mixture in the microchannel without dead volume. Meanwhile, the sampling device could realize serially sampling and it is helpful to the multi-samples amplification in sequence.Finally, the amplification experiments of 400 bp DNA template were made to optimize the working parameters of the PMMA based continuous-flow PCR microfluidic chips system. During the amplification, the addition of polyvinylpyrrolidone (PVP) solution into the PCR mixture could make surface dynamic passivation to reduce undesired adsorption of polymerase enzyme onto inner surface of the microchannel. It could ensure realization of the amplification process. However, the concentration of the PVP solution has no obvious influence on the passivation effect in this paper. Length extensions of the pre-denaturation, the denaturation and the annealing microchannels are favorable for sufficient denaturation of DNA template and completely renaturation between the template and the primers. Proper flow rate of the PCR mixture in the microchannel and appropriate amplification cycle numbers are needed to obtain an optimal amplification result. The amplification result using the chip with the microchannel having Gaussian cross-section was worse than others, because of the minor time difference between various reaction steps of PCR mixture, caused by the non-uniform velocity distribution. Nevertheless, its amplification result also could fulfill the requirement of product gel electrophoresis detection. The surface of the microchannels ablated with excimer laser may has more carboxyl groups generated during ablation process, and its hydrophilicity was improved, so this is also propitious to reduce adsorption of the polymerase enzyme onto the surface of the microchannel and enhances the amplification effect. The minimum volume of PCR mixture is 8μL for per amplification process. Multi samples amplification could be carried out with high efficiency and steady effect utilizing this system. The amplifications of other DNA templates (180 bp DNA template of Arabidopsis thaliana and 990 bp DNA template of Pseudomonas) were successfully made to further testify the feasibility and versatility of this system.A PMMA based continuous-flow PCR microfluidic chip system was successfully developed and its amplification parameters were investigated, in this paper. The combinational use of the low-cost polymer material and flexible laser micromachining technique not only successfully realizes amplification but also remarkably reduces the fabrication cost of the PCR microfluidic chip, especially at the step of laboratory research. The use of this system could offer a little of instructions and references for further commercial use and the functional integration research of the PCR microfluidic chip.