Research On Optical Biosensor Based On Nucleic Acid Signal Amplification Technique

Optical biosensor has been widely used in nucleic acid,protein and biological small molecules because of the advantages of less electromagnetic interference,fast response,large amount of information available and simultaneous detection of various objects.The amplification technique of nucleic acid signal can greatly improve the sensitivity of detection and have gained the attention of many researchers.Combining with the advantages of fluorophore-labeled nucleic acid probes and nucleic acid amplification technology,this Master's thesis developed some new methods for visualization of in situ mRNA point mutation and microRNA expression with high sensitivity and specificity.The main contents are described as following:Ultrasensitive and highly specific in situ imaging analysis of gene expression is of great significance for molecular medical research and early clinical diagnosis.In Chapter 2,we developed a new fluorescence in situ hybridization strategy by coupling a ligase-mediated specific discrimination of point mutation with a novel branched hybridization chain reaction,which is used to visualize single nucleotide variations in mRNA.In vitro results reveal that this strategy provides high specificity for discrimination of single nucleotide mutation in mRNA,and it is more sensitive than conventional HCR method due to higher amplification efficiency.Imaging results show that the highly bright spot-like signals produced by ligation-bHCR can be used for intracellular visualization and localization of miRNA at the single-molecule level,and spot signals of different colors are highly specific in genotyping point mutation of individual mRNA.Therefore,this strategy has great potential in application for early diagnosis of various diseases associated with the abnomal expression or single nucleotide mutations of mRNA.MicroRNAs(miRNAs)is a non-coded small RNA widely existed in animals and plants and viruses,which plays an important role in regulating gene expression.The studies found that abnormal expression of miRNAs is closely related to the occurrence of various diseases including cancer,and miRNAs is promising to be new biomarkers for clinical diagnosis,advanced drug development and target therapy.In Chapter 3,we use a fluorescence in situ imaging technique based on a branched hybridization chain reaction(bHCR-FISH)to detect miRNA in the tumor cells.In order to reduce the fluidity and loss of miRNA in cells fixed by poly formaldehyde(PFA),miRNAs were covalently cross-linked to the intracellular protein by EDC after PFA fixing the cells.Then,under stringent hybridization condition,the target miRNA act as the initiation strand to trigger the branched hybridization chain reaction for highly sensitive and specific in situ detection of the expression level of miRNA.We use this method to realize the single molecule visualization in situ detection of miRNA-21 in Hela and HEK293 cells with different expression levels,and the control experiments reveal that the selectivity of the method is decent.Compared with traditional FISH technology,this strategy greatly improved the sensitivity of in situ imaging analysis.The bHCR-FISH strategy based on self-triggered target can be used to visualize miRNA at single molecule resolution in single cell,which has great potential application value in the research of basic biology and clinical diagnosis.