| Immunoblotting, also known as Western blot, is a common technology used in separation and identification of specific proteins in complex samples. It was characterized by high resolution, good practicability, strong specificity and high sensitivity. Therefore, it was usually applied in the qualitative and semi-quantitative analysis of protein. However, there are still some disadvantages in this technology,such as tedious steps, time-consuming process and short lasting imaging caused by low stability of fluorescence in organic fluorescent chromophore. In addition,different chromophores require different excitation light sources, which lead to the complication of imaging and the lack of detection of multiple proteins simultaneous.In this paper, quantum dots(connected with a primary or secondary antibody) were used as an excellent fluorescence imaging agent in immunoblot analysis. This technology possesses a series of advantages, including strong light stability, high sensitivity, capacity of qualitative and quantitative detection of protein, fluorescent detection without chemiluminescent reagent addition, simple imaging system, easy operation and so on. These characterizations would give it more support on a new means of qualitative and quantitative protein analysis method. The thesis is mainly divided into four parts:Chapter 1: ReviewIn this chapter, the preparation methods of three types quantum dots were briefly reviewed and a comparison between them was carried out. Furthermore, the common technique and systematic composition of quantum dots coupling biomolecules were introduced. Additionally, the application of quantum dots used in Western blot was reviewed, which exhibited an excellent property in the quantitative analysis of multi-component proteins.Chapter 2: Preparation and Characterization of water-soluble Cd Te quantum dotsIn this chapter, tellurium and cadmium chloride were used as raw materials to prepare water-soluble Cd Te quantum dots with water legal system. The influence of different reaction conditions(such as ratio of cadmium and tellurium, MPA content,p H value, refluxing time and reaction temperature) on the growth rate and fluorescence intensity of Cd Te quantum dots were studied systematically, and the prepared Cd Te quantum dots were characterized by various techniques, including fluorescence spectroscopy, UV spectrum, agarose gel electrophoresis, laser particle size analyzer, high performance liquid chromatography(HPLC) and so on, and its fluorescence quantum yield was also evaluated. The preparation method of Cd Te quantum dots established in this study was as follows: p H value of 9 and oil bath temperature of 130°C. Moreover, the strongest fluorescence intensity and highest fluorescence quantum yield of Cd Te quantum dots with homogeneous size(about 10nm) were obtained in the condition of refluxing for 3 h.Chapter 3: Preparation and Characterization of CdTe quantum dots-protein conjugatesIn this chapter, quantum dots-biological protein conjugates were prepared by covalent coupling. Carboxyl on the surface of mercaptopropionic acid modified water-soluble Cd Te quantum dots were conjugated with the amine of biological protein using EDC and NHS coupling agents. All the materials reacted at 37°C for 2-4h in the alkalescence environment. Then the Cd Te quantum dots-protein conjugates with high fluorescence intensity and good stability were obtained, followed by the characterization using fluorescence spectroscopy, HPLC, agarose gel electrophoresis and laser particle size analyzer. The results showed that the quantum dots prepared under optimum conditions possessed high fluorescence intensity and good stability.Chapter 4: Application of CdTe quantum dots-protein conjugates in Western blotIn this chapter, three methods(traditional Western blot detection method,quantum dots labeled first antibody detection method, and quantum dots labeled secondary antibody detection method) were used to detect β-HCG protein, and their methodology validation. The results showed that there was a larger error in both linear relationship and precision of traditional method, which would lead to inaccurate data in the quantitative detection of proteins. Conversely, the results of quantum dots labeled first antibody detection method showed better linear relationship and precision than traditional method. In addition, quantum dots labeled secondary antibody detection method exhibited the same amplified fluorescent signal ability and sensitivity with traditional method, but better linear relationship and stability, as well as higher precision and recovery. All these properties implied that this method possesses great potential in the practice application. |
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