The Study And Application Of The Novel Catalytic And Colorimetric Molecular Beacon

As one kind of nucleic acid probes, the stem-loop molecular beacons calls big attention from scientists of the relative field for its stability and specificity in the detection of targets. Recently, combing the molecular beacon technology with other kinds of bioanalysis metods to achieve the high sensitive and rapid target determination has become a hot spot for the molecular beacon study. With the continually development of the molecular beacon technology, it will be widely used in the gene diagnosis, biochemistry, clinical medicine etc.According to the different detection signal, the molecular beacon can be classified into fluorescent molecular beacon and electrochemical molecular beacon. The traditional furoescent molecular beacon has many advantages, for example, quick operation and high sensitivity. Further more, it possesses the capability to achieve the real-time analysis and detection of DNA、RNA、protein and other kinds of small biological molecules in vivo samples. However, the weaknesses of furoescent molecular beacon, like, unstability in complex system and fluorescence bleaching, limit its further development. Compared to furoescent molecular beacon, electrochemical molecular beacon does not exsit the problem of fluorescence bleaching, in addition, it has no turbidity requirement of solution samples and possesses simple operation, low cost and high sensitivity. But, most of the electrochemical molecular beacon must be forced to be immobilized on the electrode. During the process of molecular beacon immobilizing to the electrode, because of the space effect and nonuniform immobilization, hardly can EMBs and the target DNA hybridize efficiently, resulting in slow down the development of electrochemical molecular beacon. What’s more, the immbolized electrochemical molecular beacon can not be used in the real-time detection in vivo samples.In this paper, we adopted the hemin dual labeled molecular beacon as the colorimetric active sensing assay to detect the nucleic acid and protein. To study the mutant genge has a great significance for the prevention and diagnosis of cancer. In chapter 2, we make a deep study on the mutant p53 gene based on the specific recognization and hybridization of Hs-MB with target DNA. On this basis, we take an advantage of the colorimetric Hs-MB in protein analysis. A novel protein detection assay based on aptameric exonuclease protection, nicking enzyme assisted signal amplification and DNAzyme Hs-MB is developed for highly sensitive insulin detection. All this work establishes the foundation of molecular beacon applying in vivo system.Chapter 1 PrefaceFirstly, this chapter introduces the basic related knowledge of molecular beacon, including:the structure of molecular beacons, the detection principle, classification, application, advances and prospects. Secondly, focuses on the conformation and catalysis mechanism of DNAzyme. Finally, a part of this chapter elaborates the design concept and theoretical support analysis of DNAzyme based on colorimetric molecular beacons (Hs-MB) and its purpose and significance of innovationChapter 2 Study on the p53 gene based on the colorimetric active Hs-MBA novel colorimetric molecular beacon (Hs-MB) was proposed for mutant p53 gene detection based on the sequence-specific recognition, DNA hybridization and DNAzyme. The synthesized Hs-MB by our group is particularly designed to include at its two termini two optically and electronically active hemin group. This kind of DNAzyme active Hs-MB can be applied to the specific p53 gene detection. In the absence of the target DNA, the Hs-MB keeps its hairpin-shaped structure which cannot induce a configuration change of the DNAzyme module from an inactive to an active form, generating hardly any colorimetric signal change. With the introduction of the mutant DNA, the hemin labeled Hs-MB opened the hairpin structure to form the double-stranded stem, consequently, resulting in the separate of the tightly bounding hemin particles of the stem terminus, leading to the formation of the catalytically active HRD-like DNAzyme. Therefore, after the catalyzing the H2O2-mediated oxidation of the colorless (ABTS2-) into the green radical anion ABTS+ the concentration of the target can be be distinguished by by the observation of the solution change in color. Owing to the unique character of the Hs-MB, the sensing assay is sensitive to single-base mismatched p53, reaching a low detection limit 0.08nM, which is expected to be a promising detection tool for the SNP gene determination in the complex biological system.Chapter 3 Study on the insulin based on the colorimetric active Hs-MBIn this chapter, a novel protein detection assay based on aptameric exonuclease protection, nicking enzyme assisted signal amplification and DNAzyme avtive Hs-MB is developed for highly sensitive insulin detection. We applied a special oligonucleotide probe containing a protein aptamer sequence at the 3’-terminus, which has the capacity to recognize the protein target with high affinity and specificity. Specifically, the aptamer probe is protected from exonuclease-catalyzed digestion upon binding to the insulin. The protected aptamer probe hybridizes with the dual hemin labeled molecular beacon (Hs-MB), a reporter signal oligo-DNA. Consequently, the process is triggered in the presence of a nicking enzyme, resulting in the continuous enzyme cleavage of many hybridized Hs-MBs and accumulation of hemin to form DNAzyme, providing a DNAzyme catalysis absorbance amplification detection signal for the target. This assay permits the detection of porcine insulin specifically with a detection limit as low as 2 pM without using washes or separations. Our method exhibits excellent sensitivity. The mechanism, moreover, may be generalized and used for other forms of protein analysis by changing the corresponding aptamer without changing the other conditions. So our new strategy may provide a homogeneous detection platform for many proteins and can be applied well in practice...