【作者】 陈小明；

【导师】 罗和安；

【作者基本信息】 湘潭大学 ， 化学工程， 2005， 博士

【摘要】 共振光散射技术是Pastemack等于1993年建立的新的分析技术，并对生物大分子的识别、组装和聚集进行了研究，从而引起了人们的极大兴趣。核酸是最基本的生命物质之一，在生命活动中扮演着重要角色。目前，关于核酸与有机分子之间相互作用的研究是核酸研究的一个重要领域。它对于研究核酸的生物化学反应，发展核酸医药制品，了解抗癌药物的治疗机理，简便快速地进行体外筛选抗癌药物，指导设计和合成新药以及对疾病的诊断，防治和合理用药均有重要的意义。本文根据实验室的实验条件，选择共振光散射光谱法并结合吸光光度法和荧光法等方法，研究了在阳离子表面活性剂存在下，核酸与有机阴离子小分子或抗癌药物之间的三元体系的相互作用前后的共振光散射光谱特征，吸光光谱特征和荧光光谱特征等，根据共振光散射光谱信号的改变值与核酸的浓度呈正比的特点，建立了几种测定纳克级核酸的新方法。这些方法具有简单、快速，灵敏高和重现性好等特点，结果令人满意。本论文由八章组成，主要内容如下：第一章概述了核酸的研究进展，共振光散射技术的原理和定量分析基础，及其在核酸研究和分析中的应用。第二章研究了核酸与阴离子染料甲基蓝及阳离子表面活性剂十六烷基三甲基溴化铵（CTMAB）三元体系的共振光散射光谱特性及其分析应用，结合吸收光谱以及相对粘度等方法初步探讨了它们的作用机理，建立了一种纳克级核酸的检测方法。在pH=9.0的Tris（三羟甲基氨基甲烷）-HCl介质中，阴离子染料甲基蓝对核酸与阳离子表面活性剂十六烷基三甲基溴化铵（CTMAB）的共振光散射光谱有协同增强作用，产生最大散射波长为334nm的共振光散射信号。在最佳实验条件下，体系的ΔIRLS值与yDNA、ctDNA、fsDNA和yRNA在一定浓度范围内均呈良好的线性关系。检测限最低可达2.7ng／mL。对于大多数生物体系中金属离子和氨基酸的抗干扰能力都很好。这一研究使阴离子染料在核酸测定中有了新的应用，同时为选择核酸测定的新方法提供了更广阔的范围。第三章研究了茜素绿（AG）-十六烷基三甲基溴化铵（CTMAB）-核酸三元体系的RLS光谱特征、影响因素和最佳反应条件。机理研究表明体系的△I_RLS的增加是由于茜素绿通过CTMAB的桥梁作用（静电作用和疏水作用）与DNA形成大聚集体的结果。在最佳条件下，体系的△I_RLS值与yDNA和fsDNA分别在浓度为50-1200μg／L和50-1000μg／L范围内呈良好的线性关系，其线性回归方程分别为△I=24.156+0.698C（C：μg／L）和△I=29.374+0.752C（C：μg／L），相关系数分别为0.9996和0.9991，检测下限分别为yDNA3.1ng／mL，fsDNA8.2ng／mL。第四章研究了二甲酚橙（XO）-十六烷基三甲基溴化铵（CTMAB）-核酸三元体系的RLS光谱特征、影响因素和最佳反应条件。机理研究表明体系的△I_RLS的增加是由于二甲酚橙通过CTMAB的桥梁作用（静电作用和疏水作用）与DNA形成大聚集体的结果。在最佳条件下，体系的△I_RLS值与yDNA、fsDNA、ctDNA和RNA分别在一定的浓度范围内呈良好的线性关系，检测下限最低可达5.31ng／mLL。第五章研究了研究了百里酚蓝（TB）-十六烷基三甲基溴化铵（CTMAB）-核酸三元体系的RLS光谱特征、影响因素和最佳反应条件；探讨了体系的反应机理。在最佳条件下，体系的△I_RLS与yDNA、ctDNA和fsDNA在一定的浓度范围内呈线性关系。检测限最低可达1.30ng／mL。第六章研究了溴甲酚绿（BCG）-十六烷基三甲基溴化铵（CTMAB）-核酸三元体系的RLS光谱特征、影响因素和最佳反应条件。机理研究表明体系的△I_RLS的增加是由于溴甲酚绿通过CTMAB的桥梁作用（静电作用和疏水作用）与DNA形成大聚集体的结果。该体系的△I_RLS值与yDNA在0.05-0.90 ng／mL、fsDNA在0.05-0.80 ng／mL和ctDNA0.05-0.80 ng／mL在分别在一定的浓度范围内呈良好的线性关系，yDNA的检测下限为12.7ng／mL第七章，我们研究发现：在pH=9.30的HMTA-HCl缓冲溶液中，抗肿瘤药物甲氨蝶呤MTX与核酸、CTMAB反应，产生增强的共振散射光散射信号，结合荧光法和粘度法探讨了该三元体系的反应机理，研究了MTX-CTMAB与yDNA，fsDNA，ctDNA和RNA共振光散射的最佳反应条件，试图寻找了MTX能够阻断DNA复制的最小剂量，和DNA能够承受的最大剂量。MTX和DNA的反应研究有望进一步研究病变部位的DNA与抗癌物质的反应。第八章，总结。更多还原

【Abstract】 The Resonance Light Scattering (RLS) technique was set up by Pastemack and co-workers using common fluorimetric instrument in 1993. They studied distinguishing, assembly, and aggregation on bimolecular by RLS technique. People follow the RLS with great interest.Nucleic acid is one of the fundamental materials of life. It is the carrier of inheritance information and the base of gene expression. With the effort of a great deal of scientists, people have made much breakthrough in the studies on nucleic acid during the last century. Today, the study field of nucleic acid has been extended. In the chemical studies on nucleic acid, the field of small molecular is always very brighten, because it can be used as the medical treatment of cancer, the coloring agents and the probe to determinate the structure of nucleic acid. Armed with the advanced equipment and sensitive determination methods, analytical chemistry plays an important role in the studies on the interaction between DNA and small molecular for the quantitative analysis of DNA, and for the investigation of the mechanism.Based on a lot of documents and conditions of our laboratory, this thesis establishes several new determination methods of DNA by means of novel resonance light-scattering technique, studies the RLS signal of nucleic acid reacts with anionic small molecular or antineoplastic in the presence of cationic surfactant cetyltrimethylammoniumbromide (CTMAB), and the interaction mechanisms of the triatomic system are investigated by various spectra. These methods are simple and rapid with excellent reappearance, selectivity, sensitivity and wide linear coverage. The thesis has some reference values to the studies on interaction mechanism between drug and small molecular, the design of the structure of drugs, the determination of the dose of drugs, and the extension of the application of resonance light-scattering technique.This paper has eight chapters.In the first chapter, we summarized the development of nucleic acids, the theory of RLS and the foundation of quantitative analysis, the application of RLS in the nucleic acids investigation and the analysis.In the second chapter, we studied the spectral characteristic of the triatomic system of anionic dye methyl blue-cation surfactant cetyltrimethylammonium bromide (CTMAB)-nucleic acid, and the elementary reaction mechanism with the ways of absorption spectra and relative viscosity. Another new determination method of DNA at nanogram level was established. At pH=9.00, nucleic acids and CTMAB could cooperatively enhance the RLS intensity of methyl blue and produce RLS with the maximum scattering peak located at 334 nm. Under the optimum conditions, the enhanced RLS intensity was proportional to the concentration of yDNA、ctDNA、fsDNA and RNA in the appropriate range. The lowest detection limit was 2.70ng/ml. This method was simple, rapid, high sensitivity and good selectivity. The resistant to most of melt and amino acid in biology system was well. And it had been applied to determination of DNA in synthetic samples with satisfactory results.In the third chapter, we studied in the hexamethylene tetramine(HMTA) buffer(pH=6.75), anionic dye alizarin green (AG) react with deoxyribonucleic acid (DNA) and cation surfactant cetyltrimethylammonium bromide (CTMAB) to form a kind of large aggregate, Which result in significant enhancement of RLS intensity. The spectral characteristics of RLS, the effective factors and optimum conditions of the reaction have been investigated. Mechanistic studies showed that the enhanced RLS comes from the aggregation of AG on DNA through the bridged and synergistic effect of CTMAB. Under the optimum conditions, the enhanced RLS intensity is proportional to the concentration of DNA in the range 50 to 1200μg/L for yeast DNA (yDNA) and 50 to 1000μg/L for fish DNA (fsDNA). The linear equation are△I= 24.156+0.698C(C:μg/L)and△I=29.347+0.752 C(C:μg/L)respectively, The correlation coefficient are 0.9996 and 0.9991respectively, the detection limits are3.1ngm/L and 8.2ngmL respectively. This method is simple, rapid and has been applied satisfactorily to determination of DNA in synthetic samples.In the fourth chapter, we studied in the hexamethylene tetramine(HMTA) buffer(pH=7.30), anionic dye xylenol orange (XO) react with deoxyribonucleic acid(DNA) and cation surfactant cetyltrimethylammonium bromide (CTMAB) to form a kind of large particles of complex, Which result in significant enhancement of RLS intensity. The spectral characteristics of RLS, the effective factors and optimum conditions of the reaction have been investigated. Under the optimum conditions, the enhanced RLS intensity is proportional to the concentration of nucleic acids in the appropriate range. This method is simple, rapid and has been applied satisfactorily to determination of DNA in synthetic samples.In the fifth chapter, we studied in the hexamethylene tetramine(HMTA) buffer(pH=7.60), anionic dye thymol blue (TB) react with nucleic acid and cation surfactant cetyltrimethylammonium bromide (CTMAB) to form a kind of large particles of complex Which result in significant enhancement of RLS intensity. The spectral characteristics of RLS, the effective factors and optimum conditions of the reaction have been investigated. Mechanistic studies showed that the enhanced RLS comes from the aggregation of TB on DNA through the bridged and synergistic effect of CTMAB. Under the optimum conditions, the enhanced RLS intensity is proportional to the concentration of nucleic acids in the appropriate range. The detection limit is 1.30ng/mL, This method is simple, rapid and has been applied satisfactorily to determination of DNA in synthetic samples.In the sixth chapter, we studied in the hexamethylene tetramine(HMTA) buffer(pH=11.00), anionic dye bromocresol green (BCG) react with nucleic acids and cation suffactant cetyltrimethylammonium bromide (CTMAB) to form a kind of large aggregate Which results in significant enhancement of RLS intensity. The spectral characteristics of RLS, the effective factors and optimum conditions of the reaction have been investigated. Mechanistic studies showed that the enhanced RLS comes from the aggregation of BCG on DNA through the bridged and synergistic effect of CTMAB. Under the optimum conditions, the enhanced RLS intensity is proportional to the concentration of DNA and RNA in the corresponding ranges respectively. This method is simple, rapid and has been applied satisfactorily to determination of DNA in synthetic samples.In the seventh chapter, Methotrexate (MTX) as an antifolate, which is widely used as chemotherapeutic drugs. A high-dose MTX therapy has a direct toxicity influence on the non-germinal cells, especially the liver cells. We investigated the interaction of methotrexate (MTX) and nucleic acids in aqueous solution by a resonance light scattering (RLS) technique with a common spectrofluorometer in the present of the cationic surfactant, the optimum dosage of MTX which enhanced the RLS intensities to the maximum is 4.54μg, while the inject dose for adults is 10-30mg and is half for children for routine use. In hexamethylenetetramine (HMTA) buffer (pH9.30), MTX and nucleic acids react with cetyltrimethylammonium bromide (CTMAB) to result in strong enhanced RLS signals. With the enhanced RLS signals, the enhanced RLS intensity is proportional to the concentration of nucleic acids in an appropriate range. The synthetic samples of nucleic acids were analyzed and obtained recovery values between 96 and 102%. The eighth chapter is the conclusion.更多还原