【作者】 焦嫚；

【导师】 董学芝；

【作者基本信息】 河南大学 ， 分析化学， 2011， 硕士

【摘要】 蛋白质是生命的物质基础,是构成生物体细胞组织的重要成分,是生物体发育及修补组织的原料。蛋白质在维持人体内的酸碱平衡、传递遗传信息、调节代谢、催化生物体内各种反应进行等方面都起着至关重要的作用。食品中蛋白质的分离、定性及定量分析是食品检验中最重要的工作。随着分析手段的不断进步,对食品中蛋白质含量的测定方法也正向准确和快速的方向发展。近年来,利用荧光试剂或有机染料与蛋白质作用,结合荧光光度法定量测定蛋白质含量的方法得到了很大的发展,此方法具有灵敏度高,线性范围宽,检出限低,操作简单、快速等特点,适用于常规分析。而新型荧光试剂量子点的出现,对该方法的研究起了很大的推动作用,量子点具有激发光谱宽且连续分布、发射光谱窄而对称、光谱可调谐、稳定性好、抗漂白能力强等优点,这些特殊的光学性质,使其在分子生物学、细胞生物学、基因组学、药物筛选、生物大分子相互作用等研究中有极大的应用前景,但是利用量子点测定食品中蛋白质含量的研究报道较少。本文以荧光光度法为检测手段,合成具有优异光学性质的荧光试剂,使其与蛋白质作用,探寻定量测定蛋白质更简单、更灵敏的方法,并用于样品检测。主要研究内容包括:一.综述了蛋白质测定方法,量子点合成及其在生物分子检测中的应用。二.建立了CdS-牛血清白蛋白(BSA)荧光光度法测定蛋白质含量的新方法:在pH为7.50的B-R缓冲溶液中,CdS与BSA作用,使CdS本身的荧光强度显著增强。该方法用于牛奶、蛋清中蛋白质含量的测定,并与双缩脲法作对照,结果满意。三.研究了多种表面活性剂对CdS-BSA体系荧光特性的影响:结果表明,阳离子表面活性剂如十六烷基三甲基溴化铵(CTMAB)对体系有显著的增敏作用,而阴离子表面活性剂如十二烷基硫酸钠(SDS)有猝灭作用,且有乳状沉淀产生,非离子表面活性剂如吐温-20对体系的荧光强度影响较小,在此基础上选择CTMAB与体系作用,建立了测定蛋白质含量的新方法。在pH 8.50的硼酸-硼砂缓冲溶液中,CTMAB的加入,可以使体系的荧光峰显著增强。该方法可用于肉松、豆浆中蛋白质含量的测定,与考马斯亮蓝法作对照,结果满意。四.前文中基于BSA能使CdS荧光强度显著增强,建立了测定蛋白质的方法。但是BSA直接与CdS作用,在使CdS荧光强度增强的同时,其荧光吸收峰发生蓝移,从而给测定结果造成一定的影响。本文经研究发现,先使8-羟基喹啉与CdS作用(pH 8.90的硼酸-硼砂缓冲溶液),CdS的荧光强度降低,且吸收峰蓝移至510nm,然后再加入BSA,体系的荧光吸收峰仍在510nm处不变,荧光强度显著增强,并且在一定浓度范围内,体系的荧光强度与BSA的浓度呈良好的线性关系,据此建立了CdS—8-羟基喹啉荧光光度法测定蛋白质的新方法。该法用于肉松、鸡精、奶粉等食品中蛋白质含量的测定,并与双缩脲法做对照,结果满意。五.实验以水溶法制备了具有核壳结构的CdS/ZnO微粒,研究了该微粒与牛血清白蛋白作用后体系的荧光特性,建立了CdS/ZnO荧光光度法测定蛋白质的新方法:在pH 6.10的KH2PO4-Na2HPO4缓冲溶液中,CdS/ZnO与牛血清白蛋白(BSA)反应后,荧光强度显著增强,并且荧光强度的增强与牛血清白蛋白的浓度有良好的线性关系。该法用于食品和尿液中蛋白质的测定,并与考马斯亮蓝法比较,结果满意。六.与课题相关的其它研究:(1)以乙酸锌为锌源,硫化钠为硫源,巯基乙酸为修饰剂水相合成了ZnS微粒。基于ZnS微粒能与结晶紫反应使结晶紫的吸光度降低,加入牛血清白蛋白反应后,体系的吸光度显著增强,据此建立了结晶紫-ZnS-BSA分光光度法测定蛋白质的新方法。该法用于肉松、酸奶等食品中蛋白质含量的测定,与考马斯亮蓝法做对照,结果满意;(2)研究了食品中糖精钠含量的一种新的测定方法,在pH 7.00的Britton-Robinson(B-R)缓冲溶液中,结晶紫与糖精钠作用可使结晶紫吸光度显著增强,据此建立了结晶紫-紫外光度法测定食品中糖精钠含量的新方法。该方法应用于食品中糖精钠含量的测定,与国标法-高效液相色谱法对照,结果满意。更多还原

【Abstract】 Protein is the material foundation of life, it is also constitute an important part of the biological somatic tissue, and is a raw material of biological development and repair tissue. Protein plays a critical role in maintaining acid-base in human body, pass genetic information, regulating metabolism, catalytic various reaction in organisms, and etc. Its separation and the qualitative and quantitative analysis is the most important work in biochemistry and other biological disciplines, such as food inspection, clinical examination, diagnose disease, biological medicine separation,purification and quality inspection. With the progress of analysis methods unceasing, the determination method of protein content in food also procress to accurately and quickly.Utilization of fluorescence reagent or organic dyes with protein reaction, and the determination of combined with fluorescence spectrophotometry quantitative determinate protein content has a great development. This method has many characteristics, such as high sensitivity, wide linear range, low detect limit, simple and fast operation and so on, so it applies the conventional application. But as a new fluorescent reagent emergence of quantum dots, it played an important role in promoting this method research, Quantum dots have many advantages, such as broad excitation spectra and the continuous distribution, narrow and symmetric emission spectrum, spectrum can be tuned, good stability and strong resistance to bleaching, and so on. This special optical properties have great prospects in molecular biology, cell biology, genomics, drug screening, the study of biological macromolecules interactions, but using quantum dots in determination of protein content has a few studies.The aim of this dissertation is to explore simpler, more sensitive determination methods of protein, through synthetic excellent optical properties of nanometer particle, with using spectrophotometry as research means, and this method had used for sample testing. The major contents are described as follows:1. Summary of the protein determination methods, quantum dot synthesis methods and its applications in biomolecular detection is given in this part. 2. Established a fluorometry for the determination of protein: The interaction between CdS and Bovine Serum Albumin(BSA) in pH 7.50 Britton-Robinson buffer solution resulted in a fluorescence intensity of CdS increasement. This method has been applied for the determination of protein in milk and egg, and compared with the biuret method the results were satisfactory.3. Investigated variety of surfactants impact on the CdS-BSA system. The results showed that cationic surfactants such as cetyltrimethylammonium bromide (CTMAB) on the system have a significant sensitizing effect, and anionic surfactants such as sodium dodecyl sulfate (SDS) quenched, and there is milky precipitate, non-ionic surfactant such as Tween -20 effect on the fluorescence intensity of less effect. So a new method for the determination of protein was established as the reaction CTMAB with system. In pH 8.50 Boric acid-borax buffer solution, The fluorescence intensity of system increasing sensitivity after joined CTMAB into the system. The method have been used for determination of protein in food such as crushed dride pork and soybean milk. Comparing with the coomassie brilliant blue method, the results were satisfactory.4. A method for the determination of protein was established based on CdS fluorescence intensity significantly increased after CdS with BSA effect in above. But BSA with CdS effect directly can make CdS fluorescence intensity of enhanced. At the same time, its fluorescence absorption peaks shift to left, and the determination results had some effect. In this paper, the study found that first make 8-hydroxyquinoline and CdS effect(pH 8.90 boric acid-borax buffer solution), CdS fluorescence intensity significantly decreased, and the fluorescence absorption peaks move to 510nm, then added to BSA, the fluorescent absorption peaks of system were still in place of 510nm, fluorescence intensity increased significantly, and the fluorescence intensity of system is a good linear relationship with BSA concentration in certain concentration range. So a new method for the determination of protein was established. The method have been used for determination of protein in food such as crushed dride pork, chichen powder, Soybean milk powder, and so on. and compared with the Biuret method, the results were satisfactory.5. CdS nanoparticles were synthesized in aqueous solution in this experiment, and established a new method of protein determination with the CdS/ZnO fluorescence spectrophotometry. The result showed the fluorescence intensity of CdS/ZnO increased after reacted with BSA in the pH 6.10 KH2PO4-Na2HPO4 buffer solution. And the fluorescence intensity of system have good linear relationship with BSA concentration. This method have been used for determination of protein in food and urine, and compared with the coomassie briliant blue method,the results were satisfactory.6. The other research related to the issue introduced: In this experiment, ZnS nanoparticles that have special spectral properties were prepared with zinc acetate as the zinc source, sodium sulfide as sulfur source, mercaptoacetic acid as modifier by hydrothermal synthesis method. Based on the decrease of absorbance of crystal violet after ZnS reacted with crystal violet and the absorbance of system increased significantly by adding different amounts of bovine serum albumin, a new method about determination of protein was established. The method have been used for determination of protein in crushed dride pork, yoghot, and others, and compared with the coomassie brilliant blue method, the results were satisfactory.Another introduced a kind of method of determination of o-Sufobenzoic Acid Imide(Na) in the food. In Britton-Robinson (B-R) buffer solution of pH 7.00, the absorption wavelength of violet markedly increased after reacting with the o-Sufobenzoic Acid Imide(Na). In view of the above, the determination of o-Sufobenzoic Acid Imide(Na) in the food by UV spectrophotometry established. It has been used to the determination of o-Sufobenzoic Acid Imide(Na) in food, comparing with Guobiao law– HPLC, the results are satisfactory.更多还原