| Chapte 1 PrefaceThis chapter mainly includes three parts:molecular beacon, Host-Guest recognition technology and design the purpose and significance of this paper. First of all, elaborating the related knowledge of molecular beacon, including the structure and principle of molecular beacon, classification, application, research progress and prospects. Moreover, the principle and application of Host-Guest Recognition will be introduced in detail. At the last, elaborating the design concept of the Host-Guest-based novel electrochemically active-inactive switching molecular beacon(CB[7]/Fc-MB), the significance and the innovation briefly.Chapter 2 The preparation and characterization of the novel electrochemical molecular beacon(Fc-MB)Most traditional electrochemical molecular beacons are fixed on the surface of electrode, while the detection convenient rapidly, but there are still some problems brought by the beacon on the electrode surface, such as the detection accuracy and reproducibility is poor that caused by different density of beacon on the surface of electrode. Therefore, designing a novel electrochemical molecular beacon that does not need to be fixed on the electrode surface and could directly detect the targets in homogeneous solution will be the research trend nowadays. In this chapter, we have designed a novel electrochemical activity molecular beacon(Fc-MB). In this experiment, firstly, we synthesized FcCO -NHS ester through esterification reaction, and the purification and characterization were carried out. Then, mixing uniformly the FcCO-NHS ester and MB (modified amino at both ends) to prepared Fc-MB by amide reaction, and the thorough purification and characterization were carried out. Meanwhile, HPLC and Uv-Vis absorption spectroscopy were used to monitoring the purification process. The experimental results showed that the yield and purity of synthetic FcCO-NHS ester and Fc-MB are satisfied.Chapter 3 Study on the properties of the Host-Guest-based novel electrochemically active-inactive switching molecular beacon (CB[7]/Fc-MB)With the vigorous development of the Host-Guest recognition technology, the inclusion phenomenon and mechanism between host compounds and guest molecules have become the research focus in recent years.There are many methods are used to corroborate their inclusion phenomenon, such as NMR, X-ray diffraction spectrometry, fluorescence spectrometry, Uv-Vis absorption spectrometry and so on. Among them, Uv-Vis absorption spectrometry is the most convenient. According to the previous research, the crystal study reveals significant difference in the structure of the complex (β-CD-Fc, CB[7]-Fc) between β-CD and CB[7]. Based on the detailed analysis, we combined CB [7] with Fc-MB to construct the novel electrochemically active-inactive switching molecular beacon CB[7]/Fc-MB, and explored its related properties. In this experiment, Firstly, we studied on the inclusion complex between FcCOOH and CB[7], and characterization was carried out by Uv-Vis absorption spectrometry. Then, the molar ratio method was used to calculate the inclusion constant (5 x 105), which is lower than CB [7] -Fc. Because of any modification or substituents covalently attached to Fc may influence the host guest interactions between the CB[7] and Fc guests. Moreover, electrochemical method was used to explore the influences of modification or substituents covalently attached to Fc on the electrochemical activity. The results show that compared with FcCOOH, in the same condition, the Fc-MB is more difficult to be oxidized. Finally, we studied on the thermal stability properties of the Fc-MB, and measured the Tm value is about 78 ℃, which higher than 58 ℃ (the Tm value of MB). This indicated that the thermal stability of Fc-MB was markedly improved. Therefore, Fc-MB with high stability could effectively decrease the false signal, and further improve the detection sensitivity, which laid a solid foundation for applying Fc-MB to genetic testing areas.Chapter 4 Study on the application of the Host-Guest-based novel electrochemically active-inactive switching molecular beacon (CB[7]/Fc-MB)P53 gene as one of the most important tumor suppressor genes in the human body, there are about more than 50% of human cancers closely related to mutation of P53 gene. Among the mutation forms of P53 gene, the results of studies have shown that the main mutation form is missense mutation caused by point mutations, accounts for about 80% of the total. Therefore, There is an urgent need for developing a novel method with high efficiency and accuracy for the detection of SNP. In this paper, we constructed a novel eletrochemical molecular beacon based on host-guest recognition. In the absence of target DNA, Fc-MB maintained a hairpin structure, two ferrocene group are closer each other, which shields CB[7] from recognizing the guests because of the space steric hindrance and molecular size mismatch.Thus, a higher electrochemical signal was detected. In the presence of target DNA, the hairpin structure was opened to form a rigid double helix structure, resulting two ferrocene groups at the ends of the MB apart away from each other. Meanwhile, the host-guest recognition happened between CB[7] and ferrocene group to form the inclusion complex CB[7]-Fc-DNA. Therefore, a lower electrochemical signal was detected. According to this mechanism, we achieved the goal of detection of target DNA (mutant DNA). The experimental results show that CB[7]/Fc-MB has a good specificity recognition (recognition rate is about 90%), and between the concentrations of the target DNA and the electrochemical signal changed value AI exists good linear relationship, the linear equation Y=1.73927 X-8.50589 (R2= 0.99451), the liner range 10nM-50nM, the detection limit of 7.8 nM. |
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