Research Of Electrochemical Detection On Micro PCR Chip For Buiomedical Detection

Biological chips, which have the advantages of microminiaturization, portability, high sensitivity and low-cost, have attracted worldwide concern. Micro PCR (Polymerase Chain Reaction) chip, as a kind of biochips, which combines the functions of increasing the temperature limitation of the traditional PCR and detecting the PCR products into a single full-featured chip, makes LOC(Lab-On-a -Chip) to be possible. Moreover, the PCR technique has an irreplaceable position in biochemistry detection, therefore, there are higher requirements of microminiaturization, portability and marketization, and Micro PCR chip is the promising one which can achieve these goals.In this thesis, according to MEMS(Micro Electro Mechanical Systems) technology, micro electrode chips and static real time PCR chips are prepared, at the same time, the three modules miniature intelligent quantitative PCR system and the static real-time quantitative miniature PCR system are packaged respectively. The experiments of the performance of micro chips in biochemical environment, the cleaning of chips and the seal of 60nlmicro reaction cell are carried out systematically. For the application research of microelectrode of micro PCR chips in electrochemistry detection, especially in detecting the PCR productions, a new label-free electrochemistry detection method of the PCR products is proposed. The main content of the thesis is summarized as the following:In chapter 2, the MEMS technology is used to produce feature-standard microelectrode array chips and static real-time quantitative PCR micro-array chips on the basis of micro PCR chip design. The adaptation research of chips in biochemical environment is carried out further, which provide a new basis and suggestion for the fabrication of biochips by using MEMS technology. Meanwhile, the assembling of the two kinds of PCR systems is achieved, the temperature control module, the biochemical detection module and the data display module are included.In chapter 3, the material selection of the micro-reactor warehouse and the cover of the static real-time quantitative PCR chip are studied, the hydrophobic PDMS membrane is finally chosen as the cover. The tightness of the reaction cells whose volume is 60nl is researched through MB (Methylene blue) indicator. It proves that the sealing effect of the micro-reaction cells is good when the temperature is below 57℃, which provides a guarantee and an important basis for the reliability of the experiments that followed.In chapter 4, the electrochemistry specificity detection of the aqueous solution of formaldehyde, carbinol and ethylalcohol is studied by using the microelectrode array of chip produced in Chapter 2. It is found that the three objects have respective specific peaks at different potential and the specific peaks also appear when they are mixed together. A detection method of the PCR products of the label-free electrochemical deposition of DNA(Deoxyribonucleic acid) based on microelectrode is brought up on account of the static real-time quantitative PCR chip produced in Chapter 2. This detection method is based on the high sensitivity of microelectrode as well as the theory that the mismatch rate of PCR within 20 circles can be neglected, and the result can be quantized beyond 20 circles. Using the electronegativity of the double stranded DNA and the theory of electrostatic attraction, it is easy to deposit the double stranded DNA to the surface of the working gold electrode. And then, the detection is completed by the electrochemistry workstation. It is proved that this detection method is feasible in detecting the PCR product.In chapter 5, integration of PCR gene amplification and real-time detection is discussed by using static micro-PCR chip produced in Chapter 2. And then according to the current problems, two aspects are deeply discussed, which are seal and micro-heaters. Constructive comments are proposed for current obstacles encountered in the experiment.