某些植物激素和药物的检测方法及与生物大分子相互作用的研究
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摘要
植物激素的超微量及原位检测方法的建立对于研究植物激素及其分子作用机理具有重要的意义。进行药物的质量标准及药物动力学研究是药物分析的重要内容之一。本论文应用高灵敏度的化学发光、电致化学发光及荧光等手段建立了某些植物激素及药物的分析方法，并对这类物质与蛋白质等生物大分子之间的相互作用进行了研究。
论文总共九章。第一章绪论介绍了化学发光与电致化学发光常用的体系及一些新技术在药物分析上的应用，对研究蛋白质与小分子之间相互作用的一些常用方法和细胞分裂素检测技术的进展作了概述，同时还对本课题的研究目的、内容及意义做了概括。
论文的第二至第六章主要应用了化学发光与电致化学发光等手段建立了某些细胞分裂素及药物的检测方法。第二章对几种细胞分裂素对钌联吡啶电致化学发光的影响进行了研究，建立了一种检测细胞分裂素的新方法，同时对相关机理进行了探讨。第三章在激动素对硫化镉修饰玻碳电极-过硫酸钾体系电致化学发光有抑制作用的基础上建立了检测激动素的新方法，该方法的检测限低至4.2×10~(-10)mol/L，这一方法对于激动素的原位检测具有一定的意义。第四章中对石墨烯这种新型纳米材料在电致化学发光中的应用进行了研究，以石墨烯/Nafion/葡萄糖氧化酶修饰玻碳电极为工作电极，利用葡萄糖在酶催化下产生的过氧化氢可增强鲁米诺的电致化学发光的性质，建立了检测葡萄糖的方法，并应用于血清中葡萄糖的测定。第五章建立了检测泛昔洛韦的毛细管电泳–钌联吡啶电致化学发光新方法，并应用于泛昔洛韦胶囊的测定，并研究了相关机理。第六章建立了以钌联吡啶-硫酸铈铵流动注射化学发光体系检测美洛昔康的新方法并应用于美洛昔康片的含量测定，方法的检出限达3.7×10~(-10)g/mL。
论文的第七至第九章主要应用了荧光分析方法测定某些药物的含量及进行相互作用分析。第七章基于美洛昔康与浓硫酸反应后的水解产物具有荧光的特性，建立了测定美洛昔康的同步荧光分析方法并应用于美洛昔康片的含量测定，方法的检出限为1.9×10~(-9)mol/L。第八章利用他米巴罗汀对牛血清白蛋白的荧光具有猝灭作用，对两者相互作用时的猝灭类型、结合力、结合位点数与结合常数及构象变化等进行了分析，并建立了测定模拟试样中他米巴罗汀的荧光猝灭法。论文的第九章应用荧光光谱法与紫外光谱法对四种细胞分裂素与牛血清白蛋白、酪蛋白、大豆蛋白之间的相互作用进行分析，为细胞分裂素的分子作用机理研究及药代动力学研究提供一定的理论基础。
Establishing supertrace and in situ determination method for plant hormones havegreat significance in the study of plant hormones and its molecular mechanism.Thereaseach of quality standards for drugs and drug kinetics is an important part of druganalysis. In this thesis, high-sensitivity determination methods such aschemiluminescence, electrochemiluminescence(ECL) and fluorescence method wereestablished for some plant hormones and drugs analysis, and the interactions betweenthese substances and biological macromolecules had been studied also.
The thesis is divided into nine chapters. The first chapter introduces thecommonly used chemiluminescence and ECL system and some new technologyapplications in drug analysis. The commonly methods used for studying the interactionsbetween protein and small molecule were introduced and the progress in cytokinindetection were reviewed. Furthermore, the purpose，contents and significance ofthe thesis were summed up.
Chemiluminescence and electrochemiluminescence detection method had beenapplied in chapter2-7. In Chapter2, based on the facts that some of cytokinins canenhance the ECL intensities of Ru(bpy)32+, a sensitive ECL method for cytokinins hasbeen proposed, the assay conditions of cytokinins electrochemiluminescence wereoptimized, and the possible mechanisms had also been discussed. In Chapter3, the ECLassay method for Kinetin was established base on the facts that Kinetin can inhibit theECL of CdS modified glassy carbon electrode-potassium persulfate system, the thedetection limit was as low as4.2×10~(-10)mol/L, the methods has some significance forin situ detection. In Chapter4, Glucose oxidase(GOD) was encapsulated in theGraphene/Nafion film modified glassy carbon electrode(GCE) and used as an ECLsensor for glucose.The GOD retains its bioactivity after being immobilized into thecomposite film. The sensor gives a good responds to glucose with a linear response inthe range of2.0×10-6~1.0×10-4mol/L and with a detection limit of1.0×10-6mol/L. InChapter5, a capillary electrophoresis-ECL method had been developed for famciclovir assay, and which had been applied to determine the compound of todeterminatefamciclovir in capsule, the possible mechanism was studied also. In Chapter6, a newflow injection chemiluminescence method for meloxicam determination had beenproposed, the detection limit is3.7×10~(-10)g/mL, and the method was apply to thedetermination of meloxicam tablet.
Fluorescence analysis method had been applied in Chapter7～9.In Chapter7,based on the fact that the hydrolysis reaction product of meloxicam with sulfuric acidhas strong fluorescence properties, a synchronous fluorescence analysis method for thedetermination of meloxicam in tablets was established, the detection limit was1.9×10~(-9)mol/L. In Chapter8, the fluorescence quenching effect of tamibarotin to bovineserum albumin had been applied to analysis the quenching types, interaction force,binding sites number, binding constant and the conformational changes whentamibarotin interact with bovine serum albumin.A fluorescence quenching method forthe analysis of tamibarotin in simulated samples was also established. In Chapter9,fluorescence spectroscopy and UV spectra was used to analysis the interaction betweenfour cytokinins and bovine serum albumin, casein, soybean protein, the study providesthe theretical basis for molecular mechanism of cytokinin and pharmacokinetics.
论文总共九章。第一章绪论介绍了化学发光与电致化学发光常用的体系及一些新技术在药物分析上的应用，对研究蛋白质与小分子之间相互作用的一些常用方法和细胞分裂素检测技术的进展作了概述，同时还对本课题的研究目的、内容及意义做了概括。
论文的第二至第六章主要应用了化学发光与电致化学发光等手段建立了某些细胞分裂素及药物的检测方法。第二章对几种细胞分裂素对钌联吡啶电致化学发光的影响进行了研究，建立了一种检测细胞分裂素的新方法，同时对相关机理进行了探讨。第三章在激动素对硫化镉修饰玻碳电极-过硫酸钾体系电致化学发光有抑制作用的基础上建立了检测激动素的新方法，该方法的检测限低至4.2×10~(-10)mol/L，这一方法对于激动素的原位检测具有一定的意义。第四章中对石墨烯这种新型纳米材料在电致化学发光中的应用进行了研究，以石墨烯/Nafion/葡萄糖氧化酶修饰玻碳电极为工作电极，利用葡萄糖在酶催化下产生的过氧化氢可增强鲁米诺的电致化学发光的性质，建立了检测葡萄糖的方法，并应用于血清中葡萄糖的测定。第五章建立了检测泛昔洛韦的毛细管电泳–钌联吡啶电致化学发光新方法，并应用于泛昔洛韦胶囊的测定，并研究了相关机理。第六章建立了以钌联吡啶-硫酸铈铵流动注射化学发光体系检测美洛昔康的新方法并应用于美洛昔康片的含量测定，方法的检出限达3.7×10~(-10)g/mL。
论文的第七至第九章主要应用了荧光分析方法测定某些药物的含量及进行相互作用分析。第七章基于美洛昔康与浓硫酸反应后的水解产物具有荧光的特性，建立了测定美洛昔康的同步荧光分析方法并应用于美洛昔康片的含量测定，方法的检出限为1.9×10~(-9)mol/L。第八章利用他米巴罗汀对牛血清白蛋白的荧光具有猝灭作用，对两者相互作用时的猝灭类型、结合力、结合位点数与结合常数及构象变化等进行了分析，并建立了测定模拟试样中他米巴罗汀的荧光猝灭法。论文的第九章应用荧光光谱法与紫外光谱法对四种细胞分裂素与牛血清白蛋白、酪蛋白、大豆蛋白之间的相互作用进行分析，为细胞分裂素的分子作用机理研究及药代动力学研究提供一定的理论基础。
Establishing supertrace and in situ determination method for plant hormones havegreat significance in the study of plant hormones and its molecular mechanism.Thereaseach of quality standards for drugs and drug kinetics is an important part of druganalysis. In this thesis, high-sensitivity determination methods such aschemiluminescence, electrochemiluminescence(ECL) and fluorescence method wereestablished for some plant hormones and drugs analysis, and the interactions betweenthese substances and biological macromolecules had been studied also.
The thesis is divided into nine chapters. The first chapter introduces thecommonly used chemiluminescence and ECL system and some new technologyapplications in drug analysis. The commonly methods used for studying the interactionsbetween protein and small molecule were introduced and the progress in cytokinindetection were reviewed. Furthermore, the purpose，contents and significance ofthe thesis were summed up.
Chemiluminescence and electrochemiluminescence detection method had beenapplied in chapter2-7. In Chapter2, based on the facts that some of cytokinins canenhance the ECL intensities of Ru(bpy)32+, a sensitive ECL method for cytokinins hasbeen proposed, the assay conditions of cytokinins electrochemiluminescence wereoptimized, and the possible mechanisms had also been discussed. In Chapter3, the ECLassay method for Kinetin was established base on the facts that Kinetin can inhibit theECL of CdS modified glassy carbon electrode-potassium persulfate system, the thedetection limit was as low as4.2×10~(-10)mol/L, the methods has some significance forin situ detection. In Chapter4, Glucose oxidase(GOD) was encapsulated in theGraphene/Nafion film modified glassy carbon electrode(GCE) and used as an ECLsensor for glucose.The GOD retains its bioactivity after being immobilized into thecomposite film. The sensor gives a good responds to glucose with a linear response inthe range of2.0×10-6~1.0×10-4mol/L and with a detection limit of1.0×10-6mol/L. InChapter5, a capillary electrophoresis-ECL method had been developed for famciclovir assay, and which had been applied to determine the compound of todeterminatefamciclovir in capsule, the possible mechanism was studied also. In Chapter6, a newflow injection chemiluminescence method for meloxicam determination had beenproposed, the detection limit is3.7×10~(-10)g/mL, and the method was apply to thedetermination of meloxicam tablet.
Fluorescence analysis method had been applied in Chapter7～9.In Chapter7,based on the fact that the hydrolysis reaction product of meloxicam with sulfuric acidhas strong fluorescence properties, a synchronous fluorescence analysis method for thedetermination of meloxicam in tablets was established, the detection limit was1.9×10~(-9)mol/L. In Chapter8, the fluorescence quenching effect of tamibarotin to bovineserum albumin had been applied to analysis the quenching types, interaction force,binding sites number, binding constant and the conformational changes whentamibarotin interact with bovine serum albumin.A fluorescence quenching method forthe analysis of tamibarotin in simulated samples was also established. In Chapter9,fluorescence spectroscopy and UV spectra was used to analysis the interaction betweenfour cytokinins and bovine serum albumin, casein, soybean protein, the study providesthe theretical basis for molecular mechanism of cytokinin and pharmacokinetics.
引文
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