Study And Applications Of Fluorescent Signal Amplification For Biological Macromolecues Analysis

With the development of Genome Project and Protein Project, more sensitive, accurate, convenient and economic detection methods for biological macromolecules have been the focus of biology, medicine and chemistry. In recent years, signal amplification detection technologies based on electrochemical, optical, piezoelectric technologies and the use of various enzymes and nanoparticles, have realized highly sensitive detections of biological macromolecules. How to utilize the existing molecular biology techniques and tools to convert the information of biological molecules to detectable signals with high sensitivity and specificity, how to further expand the use of signal amplifying methods in molecular biology and biomedical application, and how to design novel signal conversion means and develop novel signal amplification detection technology platform to address the research process to life in the new issue, are still significant topics for researches in biomedical and analysis chemical areas.In this thesis, a series of fluorescence signal amplifying detection technology platforms based on a variety of nucleic acid enzymes and fluorescent nucleic acid probes have been developed for quantitative analysis of DNA, point mutation detection, multiple single nucleotide polymorphisms (SNPs) genotyping and protein detection. The main researches included in this dissertation are presented as following:1. The construction of a padlock probe for rolling chain amplification based on proximity-ligation and detection of point mutation. Rolling circle amplification (RCA) has a wide range of applications in the biochemical analysis and clinical diagnosis due to its high sensitivity and specificity, isothermal operation and many other advantages. But this method is still difficult to construct padlock probes, which has restricted the RCA to be used widely. In this part, we developed a novel method for construction of padlock probe based on proximity-ligation. This method avoids to syntheses cost long DNA fragment and improves the construction efficiency of padlock probes. Here, point mutant genes have been detected sensitively using this method.2. Fluorescence signal amplifying detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction with molecular beacon. In this method, the specific long-stem molecular beacon (MB) is designed as a template of polymerization reaction and fluorescence signal carrier and the target is designed as a trigger of polymerization reaction. Upon recognition and hybridization with the target ssDNA, the stem of the MB is opened. The opened MB anneals with the primer and triggers the polymerization reaction circle-after-circle, which results to amplification of fluorescence signal. This design of method is simple and its operation is easy in isothermal reaction conditions. The detecting limit is6.4×10-15mol/L. It is expected to provide a simple, fast and sensitive platform for detection and subsequent analysis of nucleic acids, and potential to be widely used in virus detection.3. Multiple fluorescence signals amplifying detection of SNPs based on nicking enzyme mediated circular strand-displacement polymerase reaction. In this part, based on nicking enzyme mediated circular strand-displacement polymerase reaction, an isothermal fluorescence signals amplifying method for multiple SNPs detection platform,is established through the use of multiplex allele-specific probes containing signal discriminating sequences, nicking enzyme sites and primer annealing site. The trick of this method is to divert the allele-specific discriminations to multiplex fluorescence signals. This method is simple and easy to operation. Furthermore, this assay could not only determine the sites of multiplex SNPs, but also identify them accurately. This approach would be a promising technology for multiple SNPs genotyping.4. Development of a simple method for protein detection based on double-stranded fluorescent aptamer probe. Aptamer has a wide range of applications for protein detection due to its easy to synthesis and label, high and specific affinity with its target. In this part, a novel method with a double-stranded fluorescent probe for protein detection has been developed. A liner range of6.0-100.0nmol/L of protein detection is achieved. The method has a detection limit of6.0nmol/L. This strategy is easy to generalize for any aptamer without prior knowledge of its secondary tertiary structure, and would be used as a simple and general tool for protein detection.5. Fluorescence signal amplifying detection of protein based on hairpin aptamer probe and strand-displacement polymerase reaction. In this part, a novel fluorescent method of protein detection has was developed using hairpin aptamer based on polymerase reaction. The hairpin aptamer was designed to be employed as the protein ligand and template of the polymerase reaction. When the aptamer was bound to the protein, it would change to a liner strand and induce the strand-displacement polymerase reaction. Then protein detection was carried out by monitoring the polymerase reaction without directly labeling with the aptamer. Then the displaced protein could bind to the aptamer again and trigger a next strand-displacement polymerase reaction, leading to fluorescence signal amplifying detection of target protein. The result showed the method with a liner range of0.5～8.0nmol/L and detection limit of0.5nmol/L. This proposed method has the potential to design other protein probe with complex structure aptamer and be used as a simple and general tool for protein detection.