Detection Of Tumor Markers By Microchip Electrophoresis Based On Nucleic Acid Isothermal Amplification

For the past few years,the analysis instruments show the trend of miniaturization,integration and portable.Different kinds of microcomplete analysis systems(μTAS)have been proposed and applied to the analysis and detection of practical samples.Among them,microchip electrophoresis(MCE)has been applied in the fields of biology,chemistry and molecular science due to its advantages of high efficiency,large flux,simple operation,high degree of integration,low consumption of samples and reagents.However,due to the small sample size and short chip channel,the detection sensitivity is low.Nucleic acid isothermal amplification is a rapid and efficient process to achieve nucleic acid sequence accumulation under constant temperature.The experimental conditions are simple,and the combination with microchip electrophoresis has a good effect on improving the sensitivity of nucleic acid detection.The abnormal expression level of tumor markers usually represents the current state of cancer,so the detection of tumor markers is of great significance for clinical diagnosis and judgment of tumor-related diseases.This paper explores a highly sensitive detection method based on nucleic acid isothermal amplification combined with MCE for tumor markers(tumor suppressor gene p53 and mucin MUC1).This thesis is mainly composed of three parts as following:1.This paper mainly introduces the characteristics of MCE,the classification of detector,online enrichment technology,nucleic acid amplification,application and the research content and significance of this paper.2.A method based on primer exchange reaction(PER)and microchip electrophoresis(MCE)was developed to detect p53 gene with high sensitivity.The presence of p53 leads to the exposure of the Hp primer binding domain,and the combination of primer and Hp triggers PER reaction,in which a large number of ss DNA strands(products)are generated in the reaction system.Under the optimal conditions,the detection limit of p53 was 29.51 p M(S/N = 3).This method could has applications to detect p53 in human serum,showing the advantages of less sample consumption,high sensitivity,strong selectivity and simple operation.3.A highly sensitive method for the detection of MUC1 was investigated by microchip electrophoresis(MCE)combined with target cycle amplification(TRA)and chain displacement amplification(SDA).A hairpin probe(HP)can be turned on in the presence of MUC1 to initiate SDA,which is then amplified to produce large amounts of single-stranded DNA(ss DNA).Under the optimal conditions,the results showed that the detection limit of MUC1 was 0.23 pg/m L(S/N = 3).This method was also successfully applied to detect MUC1 in biological samples,showing the advantages of sensitivity,specificity,easy operation,small sample consumption,etc.,providing an alternative method for the detection of other tumor markers.