硅基微聚合酶链式反应芯片的热设计、分析和优化
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摘要
聚合酶链式反应(PCR),即通过引物延伸核酸特定片断而进行的重复双向DNA 合成,是一种非常重要的分子生物学技术,它能够实现核酸分子的富集,以满足后续的分析与检测的需要,因此是许多临床检测与生物分析技术的先导。在微系统朝着生物领域进军的过程中,微PCR 芯片始终是研究的热点之一。本文从传热学角度对微PCR 芯片的热循环性能进行分析、设计和优化,并实验研究了硅对PCR 过程的抑制作用。
采用数值模拟和集总热容法研究了现有的微腔型PCR 芯片和微流控型PCR 芯片的热循环过程,分析了芯片几何形状、材料、换热条件等因素对芯片热循环性能的影响;讨论了两种芯片的恒温控制效果。在对两种芯片热性能分析的基础上,针对芯片的要求给出了优化设计的建议。
在对已有微PCR 芯片热循环性能的分析和比较的基础上,提出了一种新型的热循环过程的实现方法,即“加热冷却-恒温”混合热循环模式,并采用硅微加工技术加以实现,研制出硅基液滴振荡型微PCR 芯片,PCR反应液以液滴形式在三个恒温工作区之间做往返振荡运动,以此来完成对反应液内DNA 的变性、退火和延伸,实现核酸的扩增;通过对人乳头瘤病毒的PCR 扩增实验验证了本文所研制的芯片的可靠性,结果表明硅基液滴振荡型微PCR 芯片能够实现快速而准确的核酸扩增,芯片扩增所需时间仅为常规PCR 仪的1/9。
针对所研制芯片在实验过程中出现的问题和不足,给出芯片优化设计的策略;建立了硅基液滴振荡型微PCR 芯片的集总热容模型,采用该模型对芯片进行热性能优化;通过数值模拟的方法研究了优化后芯片的热循环性能;对芯片热循环和恒温控制过程的耦合仿真整定了PID 控制器参数。
最后,为了分析微PCR 芯片中硅材料对PCR 过程的抑制作用,采用实时定量PCR 的方法研究了不同氧化状态下硅纳米颗粒对核酸扩增效率的影响,并籍此探讨硅对PCR 过程的抑制作用及其机理。
Polymerase Chain Reaction (PCR) is a very important molecular biological method. A wide variety of DNA/RNA molecules can be amplified by this enzyme catalysis reaction and thereby enriched for the further analysis. In the past decades, micro PCR chip is one of the hotspots in the development of micro systems for the biological and clinical applications. In the present dissertation, thermal analysis, design and optimization of silicon-based micro PCR chips were given, and the silicon inhibition effects on nucleic acids amplification was studied experimentally.
Numerical simulation and lumped heat capacity analysis were carried out to study the thermal performances of the micro chamber PCR chip and micro continuous-flow PCR chip. Effects of the chip geometries, materials and boundary conditions on the thermal cyclings of the PCR chips were investigated. The characteristics of the constant-temperature control in the chips were also analyzed. Optimizations were suggested based on the numerical and analytical results.
Based on the studies of thermal performances of the micro chamber PCR chip and micro continuous-flow PCR chip, a novel thermal cycling model, named “heating/cooling –constant temperature”hybrid model was put forwards. And an original micro PCR chip, droplet-based micro oscillating-flow PCR chip, was fabricated by silicon microfabrication technique to realize the hybrid thermal cycling model. In the novel PCR chip, a droplet of the PCR mixture flew through three temperature zones in an oscillating model to realize the denaturation, annealing and extension processes. HPV-DNA was amplified by the present chip system, and the results demonstrated that the droplet-based micro oscillating-flow PCR chip can achieve fast and correct nucleic acids amplification, and the time needed for the chip PCR to finish the amplification is about 1/9 of that required by the conventional instrument.
A lumped heat capacity model of the droplet-based micro oscillating flow PCR chip has been built to optimize the chip geometries and the heat sink temperature in the operation. Thermal performance of the optimized chip was numerically simulated, and the results indicated that an ultra-fast heating and cooling rates were achieved with very small temperature non-uniformities. Parameters used in the constant-temperature control were also optimized by a coupled simulation of the chip thermal cycling and the control system. In order to quantificationally analyze the inhibition effects of the native silicon oxide and the untreated silicon on the PCR, silicon nano-particles with different oxidized degrees were added into the PCR mixtures. A real-time PCR was carried out to study the amplification performance of these PCR mixtures. Mechanisms of the inhibition phenomena were also analyzed based on the experimental results.
采用数值模拟和集总热容法研究了现有的微腔型PCR 芯片和微流控型PCR 芯片的热循环过程,分析了芯片几何形状、材料、换热条件等因素对芯片热循环性能的影响;讨论了两种芯片的恒温控制效果。在对两种芯片热性能分析的基础上,针对芯片的要求给出了优化设计的建议。
在对已有微PCR 芯片热循环性能的分析和比较的基础上,提出了一种新型的热循环过程的实现方法,即“加热冷却-恒温”混合热循环模式,并采用硅微加工技术加以实现,研制出硅基液滴振荡型微PCR 芯片,PCR反应液以液滴形式在三个恒温工作区之间做往返振荡运动,以此来完成对反应液内DNA 的变性、退火和延伸,实现核酸的扩增;通过对人乳头瘤病毒的PCR 扩增实验验证了本文所研制的芯片的可靠性,结果表明硅基液滴振荡型微PCR 芯片能够实现快速而准确的核酸扩增,芯片扩增所需时间仅为常规PCR 仪的1/9。
针对所研制芯片在实验过程中出现的问题和不足,给出芯片优化设计的策略;建立了硅基液滴振荡型微PCR 芯片的集总热容模型,采用该模型对芯片进行热性能优化;通过数值模拟的方法研究了优化后芯片的热循环性能;对芯片热循环和恒温控制过程的耦合仿真整定了PID 控制器参数。
最后,为了分析微PCR 芯片中硅材料对PCR 过程的抑制作用,采用实时定量PCR 的方法研究了不同氧化状态下硅纳米颗粒对核酸扩增效率的影响,并籍此探讨硅对PCR 过程的抑制作用及其机理。
Polymerase Chain Reaction (PCR) is a very important molecular biological method. A wide variety of DNA/RNA molecules can be amplified by this enzyme catalysis reaction and thereby enriched for the further analysis. In the past decades, micro PCR chip is one of the hotspots in the development of micro systems for the biological and clinical applications. In the present dissertation, thermal analysis, design and optimization of silicon-based micro PCR chips were given, and the silicon inhibition effects on nucleic acids amplification was studied experimentally.
Numerical simulation and lumped heat capacity analysis were carried out to study the thermal performances of the micro chamber PCR chip and micro continuous-flow PCR chip. Effects of the chip geometries, materials and boundary conditions on the thermal cyclings of the PCR chips were investigated. The characteristics of the constant-temperature control in the chips were also analyzed. Optimizations were suggested based on the numerical and analytical results.
Based on the studies of thermal performances of the micro chamber PCR chip and micro continuous-flow PCR chip, a novel thermal cycling model, named “heating/cooling –constant temperature”hybrid model was put forwards. And an original micro PCR chip, droplet-based micro oscillating-flow PCR chip, was fabricated by silicon microfabrication technique to realize the hybrid thermal cycling model. In the novel PCR chip, a droplet of the PCR mixture flew through three temperature zones in an oscillating model to realize the denaturation, annealing and extension processes. HPV-DNA was amplified by the present chip system, and the results demonstrated that the droplet-based micro oscillating-flow PCR chip can achieve fast and correct nucleic acids amplification, and the time needed for the chip PCR to finish the amplification is about 1/9 of that required by the conventional instrument.
A lumped heat capacity model of the droplet-based micro oscillating flow PCR chip has been built to optimize the chip geometries and the heat sink temperature in the operation. Thermal performance of the optimized chip was numerically simulated, and the results indicated that an ultra-fast heating and cooling rates were achieved with very small temperature non-uniformities. Parameters used in the constant-temperature control were also optimized by a coupled simulation of the chip thermal cycling and the control system. In order to quantificationally analyze the inhibition effects of the native silicon oxide and the untreated silicon on the PCR, silicon nano-particles with different oxidized degrees were added into the PCR mixtures. A real-time PCR was carried out to study the amplification performance of these PCR mixtures. Mechanisms of the inhibition phenomena were also analyzed based on the experimental results.
引文
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