荧光光谱法测定生物大分子的研究及应用
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摘要
蛋白质和核酸是构成机体的两种重要的生物大分子。核酸是生物基本遗传物质,与生物生长、发育及癌变、突变等异常活动相关;蛋白质则负责各种生理功能,维持生物体新陈代谢活动,是生物性状直接表达者。它们是生物化学与生命科学中重要的两类生物大分子。因此研究机体分子结构、功能以及定性和定量分析,已成为当前生物分析化学研究的前沿热点之一。
本文以荧光、共振散射技术为主要研究手段,建立快速灵敏的检测蛋白质与核酸新方法,同时应用TEM、吸收光谱技术等手段进行机理研究和探讨。
本文共分为五部分,第一部分综述了生物大分子荧光探针以及纳米粒子作为光谱探针在生物分析中的研究进展及分析应用。
在论文的第二部分,研究了桑色素–Al(III)–蛋白质体系的荧光增强效应,建立了蛋白质测定的新方法。本研究发现,BSA的加入可以显著增加桑色素–Al(III)的荧光强度,据此建立了一种检测蛋白质的快速、灵敏、简便的荧光光谱法。在最佳实验条件下体系的荧光强度与蛋白质的浓度在一定范围内呈良好的线性关系,检出限可达10-9g mL-1,并成功应用于实际样品的检测中。机理研究表明桑色素的羟基与Al(III)配位形成桑色素–Al(III)配合物,由于Al(III)的架桥作用,产生大的聚合体,导致体系荧光强度的增强,求得能量转移率E=0.75。
在论文的第三部分,研究发现蛋白质可以显著增强槲皮素–Al(III)体系的荧光,从而建立了一个测定蛋白质的新体系。在最佳条件下,BSA与EA分别在1.0×10-8~3.0×10-7 g·ml-1和5.0×10-8~4.5×10-7 g·ml-1范围内与荧光强度呈良好的线性关系,检出限分别为2.3×10-9和1.0×10-8 g·mL-1,并且该体系稳定性好选择性高。研究了槲皮素、Al(III)与BSA的作用机理,发现槲皮素、Al(III)生成二元复合物,再与BSA结合,生成槲皮素–Al(III)–BSA聚集体。
在论文的第四部分,研究了桑色素、nanoTiO2和核酸的相互作用。研究发现核酸可以显著增强nanoTiO2–桑色素的荧光强度,且体系的荧光强度与核酸的浓度在一定范围内呈良好的线性关系。据此建立了测定核酸的新方法。ctDNA和yRNA的线性范围分别为:2.0×10-8~2.2×10-7 g·mL-1和1.0×10-8~2.5×10-7 g·mL-1;检出限分别为4.8×10-9 g·mL-1和1.2×10-9 g·mL-1,并成功应用于实际样品酵母RNA的测定。
在论文的第五部分,研究了核酸对Al(III)–nanoTiO2体系共振光散射强度的增强效应。研究发现,核酸的加入使得体系的共振光散射强度显著增强,在最佳条件下,体系的共振光散射强度增强程度和核酸的浓度在一定范围内呈良好的线性关系,检出限达到10-11 g·mL-1,并成功应用于实际样品酵母RNA的测定。机理研究表明Al(III)的吸附架桥作用,使得nanoTiO2、Al(III)与DNA三者之间形成网状聚集体,导致体系的共振光散射强度的增强。
Proteins and nucleic acids are the two kinds of biological macromolecules in our body. Nucleic acids are the basic genetic material of biological, it is related to the biological growth and development, cancer, mutation and other unusual activity. Proteins are the carriers of many physiological functions and are also the direct expresser of physiological characters. Proteins play an important role in the metabolism of human body, the most disease of human being is caused by abnormal of protein. They are two types of most important biological macromolecules in life scicence and biochemistry. Therefore study the molecular structure, qualitative and quantitative analysis of nucleic and protein has become the hot spots in bioanalytical chemistry.
This thesis focused on the development of new probes of proteins and nucleic acids, and to astablish sensitive method for the quantitative determination of them used the resonance light scattering(RLS) and fluorescence as the primary technique. The TEM, absorption techniques were also used to study the interaction mechanism.
This paper is divided into five parts, the first overview the applications of nano–particles and drug molecules as a spectral probe in the analysis of biological macromolecules(nucleic acid and protein).
In the second part of the paper, to study the fluorescence enhancement effect of Morin–Al (III)– protein system, and establishe a fast, sensitive and simple method for determination of protein with fluorescence spectroscopy. Under the optimum conditions, the fluorescence intensity and the concentration of protein within a certain range showed good linear relationship, the detection limit of up to 10-9g mL-1, and successfully applied to real samples. Mechanism of the system shown that the hydroxy of morin coordinate to formation of morin–Al (III)–association complex with Al (III), due to the bridging role of Al (III) in the protein binding, resulting in large aggregates and the fluorescence intensity increases, the rate of energy transfer obtained E = 0.75.
In the third part of the paper, the study found the protein can significantly enhance the system of quercetin–Al (III) fluorescence, thereby establishing a new system for determination of protein. Under the optimum conditions, BSA and EA, respectively 1.0×10-8~3.0×10-7 g·ml-1 and 5.0×10-8~4.5×10-7 g·ml-1 within the range of fluorescence intensity showed good linearity The detection limits were 2.3×10-9 and 1.0×10-8 g·mL-1, and the high selectivity and good stability of the system. And studies the mechanism of quercetin, Al (III) and BSA can found that quercetin, Al (III) generates binary complex, and then combined with the BSA, while the quercetin–Al (III) by Al (III) binary system as a bridge combined with the BSA.
In the fouth section, the Morin, nanoTiO2 and nucleic acid interactions was studied. The study found that the fluorescence of morin–nanoTiO2 significantly enhanced with nucleic acid adding, and the fluorescence intensity and the concentration of nucleic acid within a certain range showed good linearity. Based on the establishment of a new method for the determination of nucleic acids. The linear range of DNA and RNA, respectively: 2.0×10-8~2.2×10-7 g·mL-1 and 1.0×10-8~2.5×10-7 g·mL-1, detection limits were 4.8×10-9 g·mL-1 and 1.2×10-9 g·mL-1, and successfully applied to the determination of actual samples of yeast RNA.
In the fifth section, we developed nucleic acids can largery enhance the intensity of resonance light scattering of Al (III)–nanoTiO2 system, under optimum conditions, There is a better linear correlation between the enhancing extent of fluorescence intensity and the concentration of nucleic acids and the detection limit of protein is found to be 10-11 g·mL-1, and successfully applied to the determination of actual samples of yeast RNA. Mechanism of studies have shown that owing to the bridging fuction of Al (III), nanoTiO2, Al (III) and DNA form a network aggregates, resulting in the system enhancement of resonance light scattering intensity.
本文以荧光、共振散射技术为主要研究手段,建立快速灵敏的检测蛋白质与核酸新方法,同时应用TEM、吸收光谱技术等手段进行机理研究和探讨。
本文共分为五部分,第一部分综述了生物大分子荧光探针以及纳米粒子作为光谱探针在生物分析中的研究进展及分析应用。
在论文的第二部分,研究了桑色素–Al(III)–蛋白质体系的荧光增强效应,建立了蛋白质测定的新方法。本研究发现,BSA的加入可以显著增加桑色素–Al(III)的荧光强度,据此建立了一种检测蛋白质的快速、灵敏、简便的荧光光谱法。在最佳实验条件下体系的荧光强度与蛋白质的浓度在一定范围内呈良好的线性关系,检出限可达10-9g mL-1,并成功应用于实际样品的检测中。机理研究表明桑色素的羟基与Al(III)配位形成桑色素–Al(III)配合物,由于Al(III)的架桥作用,产生大的聚合体,导致体系荧光强度的增强,求得能量转移率E=0.75。
在论文的第三部分,研究发现蛋白质可以显著增强槲皮素–Al(III)体系的荧光,从而建立了一个测定蛋白质的新体系。在最佳条件下,BSA与EA分别在1.0×10-8~3.0×10-7 g·ml-1和5.0×10-8~4.5×10-7 g·ml-1范围内与荧光强度呈良好的线性关系,检出限分别为2.3×10-9和1.0×10-8 g·mL-1,并且该体系稳定性好选择性高。研究了槲皮素、Al(III)与BSA的作用机理,发现槲皮素、Al(III)生成二元复合物,再与BSA结合,生成槲皮素–Al(III)–BSA聚集体。
在论文的第四部分,研究了桑色素、nanoTiO2和核酸的相互作用。研究发现核酸可以显著增强nanoTiO2–桑色素的荧光强度,且体系的荧光强度与核酸的浓度在一定范围内呈良好的线性关系。据此建立了测定核酸的新方法。ctDNA和yRNA的线性范围分别为:2.0×10-8~2.2×10-7 g·mL-1和1.0×10-8~2.5×10-7 g·mL-1;检出限分别为4.8×10-9 g·mL-1和1.2×10-9 g·mL-1,并成功应用于实际样品酵母RNA的测定。
在论文的第五部分,研究了核酸对Al(III)–nanoTiO2体系共振光散射强度的增强效应。研究发现,核酸的加入使得体系的共振光散射强度显著增强,在最佳条件下,体系的共振光散射强度增强程度和核酸的浓度在一定范围内呈良好的线性关系,检出限达到10-11 g·mL-1,并成功应用于实际样品酵母RNA的测定。机理研究表明Al(III)的吸附架桥作用,使得nanoTiO2、Al(III)与DNA三者之间形成网状聚集体,导致体系的共振光散射强度的增强。
Proteins and nucleic acids are the two kinds of biological macromolecules in our body. Nucleic acids are the basic genetic material of biological, it is related to the biological growth and development, cancer, mutation and other unusual activity. Proteins are the carriers of many physiological functions and are also the direct expresser of physiological characters. Proteins play an important role in the metabolism of human body, the most disease of human being is caused by abnormal of protein. They are two types of most important biological macromolecules in life scicence and biochemistry. Therefore study the molecular structure, qualitative and quantitative analysis of nucleic and protein has become the hot spots in bioanalytical chemistry.
This thesis focused on the development of new probes of proteins and nucleic acids, and to astablish sensitive method for the quantitative determination of them used the resonance light scattering(RLS) and fluorescence as the primary technique. The TEM, absorption techniques were also used to study the interaction mechanism.
This paper is divided into five parts, the first overview the applications of nano–particles and drug molecules as a spectral probe in the analysis of biological macromolecules(nucleic acid and protein).
In the second part of the paper, to study the fluorescence enhancement effect of Morin–Al (III)– protein system, and establishe a fast, sensitive and simple method for determination of protein with fluorescence spectroscopy. Under the optimum conditions, the fluorescence intensity and the concentration of protein within a certain range showed good linear relationship, the detection limit of up to 10-9g mL-1, and successfully applied to real samples. Mechanism of the system shown that the hydroxy of morin coordinate to formation of morin–Al (III)–association complex with Al (III), due to the bridging role of Al (III) in the protein binding, resulting in large aggregates and the fluorescence intensity increases, the rate of energy transfer obtained E = 0.75.
In the third part of the paper, the study found the protein can significantly enhance the system of quercetin–Al (III) fluorescence, thereby establishing a new system for determination of protein. Under the optimum conditions, BSA and EA, respectively 1.0×10-8~3.0×10-7 g·ml-1 and 5.0×10-8~4.5×10-7 g·ml-1 within the range of fluorescence intensity showed good linearity The detection limits were 2.3×10-9 and 1.0×10-8 g·mL-1, and the high selectivity and good stability of the system. And studies the mechanism of quercetin, Al (III) and BSA can found that quercetin, Al (III) generates binary complex, and then combined with the BSA, while the quercetin–Al (III) by Al (III) binary system as a bridge combined with the BSA.
In the fouth section, the Morin, nanoTiO2 and nucleic acid interactions was studied. The study found that the fluorescence of morin–nanoTiO2 significantly enhanced with nucleic acid adding, and the fluorescence intensity and the concentration of nucleic acid within a certain range showed good linearity. Based on the establishment of a new method for the determination of nucleic acids. The linear range of DNA and RNA, respectively: 2.0×10-8~2.2×10-7 g·mL-1 and 1.0×10-8~2.5×10-7 g·mL-1, detection limits were 4.8×10-9 g·mL-1 and 1.2×10-9 g·mL-1, and successfully applied to the determination of actual samples of yeast RNA.
In the fifth section, we developed nucleic acids can largery enhance the intensity of resonance light scattering of Al (III)–nanoTiO2 system, under optimum conditions, There is a better linear correlation between the enhancing extent of fluorescence intensity and the concentration of nucleic acids and the detection limit of protein is found to be 10-11 g·mL-1, and successfully applied to the determination of actual samples of yeast RNA. Mechanism of studies have shown that owing to the bridging fuction of Al (III), nanoTiO2, Al (III) and DNA form a network aggregates, resulting in the system enhancement of resonance light scattering intensity.
引文
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