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实时荧光PCR芯片系统设计及其生物学应用

Study on a Real-Time Fluorescent PCR Chip System and Its Biological Applications

【作者】 罗磊

【导师】 于军;

【作者基本信息】 华中科技大学 , 微电子学与固体电子学, 2009, 博士

【摘要】 生物芯片技术是20世纪末发展起来的一项新技术。生物芯片是在微小面积上,利用微加工技术,并结合有关的化学合成技术制造而成的一种具有一定分子生物学检验功能的微型器件。分析和解释生物芯片上得到的信息,将在DNA结构与功能之间架起一道桥梁,进而推进生命科学的迅速发展。首次将实时荧光PCR技术与微流控芯片技术相结合,设计了一款实时荧光PCR芯片。在此芯片中设计了一个坝状结构,芯片键合封装后在坝下面会形成一个1um的罅缝,此罅缝能从血液中分离出白细胞,细胞分离后再往此芯片中注入PCR反应液,实现在同一腔体中进行PCR反应。由于它在很小的腔体内进行PCR反应,能大大节省样品和试剂的使用量。在此芯片上利用微加工技术,集成制作了微加热器和温度传感器。由于芯片本身是由硅片和玻璃片经阳极键合而成,有非常好的导热效果,而且没有使用具有较大热容的外置加热器件,从而提高了PCR反应的升降温速度,缩短了PCR反应总时间。加热器和温度传感器的集成化,使得我们设计的芯片温控、荧光检测平台得以微型化。同时自主设计了针对此芯片的专用检测平台,该平台由注射泵系统、实时荧光信号采集系统和PCR温度控制系统等组成,能实现样品进样,PCR温度控制,实时荧光检测、检测结果分析及处理等功能。最后,将此实时荧光PCR芯片及其检测平台应用于强直性脊柱炎相关基因HLA-B27的检测,实验证明该芯片系统能快速准确的检测出血液中的HLA-B27基因,从而验证了此芯片系统的实用价值。

【Abstract】 Biochip technology is a new technology, which developed at the end of the 20th century. Biochip is in the small area, using of micro-processing technology, combined with the chemical synthesis technology to create micro-devices, which have the function of molecular biology test. Analysis and interpretation the information of biological chips will build a bridge between the structure and function of DNA, and push forward the rapid development of life sciences.In this paper, we introduce a novel real-time fluorescent polymerase chain reaction (PCR) chip, which integrated the pretreatment of biological sample, the PCR reaction and the real-time fluorescent detection. We have built a dam in the chamber of the chip, and formed a crack underneath it to separate the white blood cell from the whole blood. Due to its small cavity in the body for PCR reaction, the sample and the use of reagents can be reduced substantially.We have produced a micro-heater and temperature sensor, using micro-chip processing technology. As the biochip is combined with silicon and glass by anodic bonding, there is a very good thermal conductivity effect. And no use of larger heat capacity of the external heater parts, so as to enhance the heating and cooling rate of the PCR reaction, reducing The total time of the PCR reaction. Heater and temperature sensor integrated, allow us to miniaturize the platform of the chip temperature controling, and fluorescence detecting.We have designed the corresponding examination system in view of this chip, which is composed of syringe pump module, temperature control module and fluorescence sampling module.We also used the constructed chip for the examination of HLA-B27 related to ankylosing spondylitis and the results show that the chip is available for the accurate and rapid analysis of HLA-B27 in the whole blood.

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