【作者】 祁恒；

【导师】 左铁钏； 陈涛；

【作者基本信息】 北京工业大学 ， 光学工程， 2009， 博士

【摘要】 PCR（聚合酶链式反应）微流控芯片是一种单元反应界面为微米量级的微型化学反应系统,具有线性尺寸小、物理量梯度高、比表面积大和流动状态为低雷诺数层流等特点,可以实现柔性生产、规模放大,以及快速和高通量筛选等功能。PCR微流控芯片将PCR技术与微流控芯片技术有机地结合起来,实现了DNA的体外快速扩增,成为微机电系统（MEMS）技术发展的一个重要研究方向。与常规PCR热循环仪相比,PCR微流控芯片可以缩短反应时间,减少反应试剂消耗量,增强扩增特异性,而且便于与其他分析和检测设备进行集成。为了开发一套实用的PCR微流控芯片系统,本论文对以PMMA（聚甲基丙烯酸甲酯）为基底材料的连续流式PCR微流控芯片系统及其应用技术进行了研究。采用准分子激光和CO2激光直写刻蚀方法在PMMA材料基片表面加工微通道,通过对工艺参数进行分析与控制,分别加工出横截面形状为矩形、半圆形和高斯形的微通道,并利用热压键合方法制作出密闭的连续流式PCR微流控芯片。采用准分子激光直写刻蚀方法加工微通道时,因为准分子激光具有脉冲输出特性,所以在微通道底部存在由于光斑叠加而形成的周期性刻蚀波纹;采用CO₂激光直写刻蚀方法加工微通道时,由于在刻蚀过程中产生的熔融物质重新凝固后又附着在微通道壁上,所以会在微通道表面形成各种不规则的突起物。本论文利用准分子激光辐照的方法,对微通道表面进行抛光处理,提高了表面质量,使得流体能够在微通道中连续地流动,流动时没有气泡产生,显著提高了流动速度的稳定性。此外,准分子激光刻蚀与辐照方法还可以提高PMMA微通道与基片材料表面的亲水性,有利于其在PCR微流控芯片中的应用。为了分析微通道形状与流体在其内部流动状态之间的关系,以及温度变化的影响,本论文基于计算流体力学方法,对流体通过具有不同形状横截面的微通道时的流动状态进行了数值模拟研究。由于微通道尺度小,流速较低,所以流体在微通道内的流动呈层流状态,流速分布不均匀,在微通道中心处的流速最大,这种现象有利于PCR混合液在流动过程中的扩散与混合;微通道横截面形状的不同会导致流体速度场中不同速度的相对分布比例不同;温度变化导致的流体粘度改变,对流体流动状态的影响可以忽略不计;而流体在微通道中的压力降随着流速的增加而增大,随着温度的升高而减小;流体在微通道内的压力降会受到微通道横截面形状的影响,当水力学直径相同时,使用具有较大横截面面积的微通道（高斯形）更有利于降低流动的摩擦阻力,减小压力降,增强流体的流动性和稳定性。本论文对连续流式PCR微流控芯片系统的整体结构进行了改进与完善,设计了竖插式的进样接口,减少了接口处的死体积,减小了因通道尺寸变化造成的流速不稳定;采用新型接口密封形式提高了接口密封材料的使用寿命,延长了芯片的使用时间,稳定了实验条件;对温度控制系统进行参数优化,改善了芯片的温度分布特征,通过添加隔热挡板,增强了不同温区之间的隔热效果;搭建了一套适用于连续流式PCR微流控芯片的简易气动进样装置,该装置没有死体积,减少了样品用量,可以灵活控制PCR混合液在微通道中的流动速度,而且能够实现液滴式的多样品间断进样,有助于实现多样品的连续扩增。最后,本论文利用经过改进后的PMMA基连续流式PCR微流控芯片系统对长度为400bp的DNA模板进行扩增实验,并对扩增参数进行了优化。在扩增时,PCR混合液中应添加一定浓度的PVP（聚乙烯吡咯烷酮）溶液,对PMMA微通道表面进行动态钝化改性处理,以减少PCR混合液中聚合酶在微通道表面的吸附,从而确保扩增反应的顺利进行,但是在本论文中PVP溶液的浓度对钝化效果的影响并不显著;在进行PCR微流控芯片的微通道排布结构设计时,延长预变性通道以及增加变性通道和退火通道的长度,有利于DNA模板的充分变性和退火时引物与模板的完全复性,能够提高扩增效率;合理控制PCR混合液在微通道中的流动速度与温度循环次数,可以获得更好的扩增结果;由于高斯形横截面的微通道内流体速度场分布较差,造成PCR混合液在各阶段反应的时间存在微小差别,所以其扩增效果略差于微通道横截面为矩形和半圆形的PCR微流控芯片,但是扩增结果依然能够满足凝胶电泳的检测要求;使用准分子激光加工出的微通道,其表面的羧基基团数量在微通道刻蚀过程中可能有所增加,使得表面亲水性得到提高,因此与使用CO₂激光直写刻蚀方法加工出的微通道相比,其表面更有利于减少对PCR混合液中聚合酶的吸附,从而获得更好的扩增效果。连续流式PCR微流控芯片系统每次所需的PCR混合液最小用量约为8μL。利用多样品间断进样方法,能够实现多样品的连续扩增,扩增效率较高,结果稳定。该系统还对其它DNA模板（180bp的拟南芥DNA模板与990bp的假单胞菌种DNA模板）实现了扩增,进一步验证了该套系统的可行性与通用性。本论文搭建了一套比较完整的连续流式PCR微流控芯片系统,并对其应用技术进行了研究。将廉价的高聚物材料与快速简便的激光微加工方法应用于PCR微流控芯片的制备,同样能够实现良好的扩增效果,而且能够显著降低实验室的研发成本。该套系统的使用,对于今后PCR微流控芯片的商品化应用与集成化研究有一定的指导和借鉴意义。更多还原

【Abstract】 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.更多还原