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酞菁化合物在分析化学中的应用研究
作 者: 陈小兰
导 师: 许金钩
学 校: 厦门大学
专 业: 分析化学
关键词: 酞菁化合物 酶催化反应 反相胶束 离子缔合作用 散射光 生物大分子.
分类号: O652.3
类 型: 博士论文
年 份: 2001年
下 载: 367次
引 用: 1次
阅 读: 论文下载
内容摘要
酞菁是一类在结构上与卟啉、叶绿素很相似的化合物。由于其合成方法简单,对光、热的稳定性好,在染料、颜料、光电材料、催化剂、光动力学疗法、荧光分析等方面得到愈来愈广泛的应用。本文在前人研究的基础上,继续开拓酞菁化合物在荧光分析中的应用。同时,也对其散射光在生物大分子测定中的应用作了一些探讨。全文共分五章。 第一章首先介绍了酞菁化合物的结构、性质和应用,着重介绍了它在荧光分析中的应用。同时也结合本文研究所涉及的领域,对辣根过氧化物酶(HRP)及其模拟酶的荧光底物的发展现状、反相胶束介质中辣根过氧化物酶的研究状况作了一些评述。 第二章研究了四氨基铝酞菁作为辣根过氧化物酶及其模拟酶的新型红区荧光底物的可行性及分析应用。首先考察了以四氨基铝酞菁为底物时辣根过氧化物酶的催化活性。实验结果表明在催化四氨基铝酞菁与过氧化氢的荧光反应中,辣根过氧化物酶表现出显著的催化活性。其次,将以四氨基铝酞菁作为供氢底物的酶催化反应体系用于痕量过氧化氢的测定。所建立的方法灵敏度高、检测限低,且因荧光激发与发射均位于红区(610/678nm),可以较好地避开背景荧光和散射光的干扰。将该体系用于人血清中葡萄糖含量和雨水中过氧化氢含量的测定,结果令人满意。最后将四氨基铝酞菁这一红区荧光底物用于HRP标记的酶联吸附免疫分析。首先考察了以四氨基铝酞菁为底物的α-甲胎蛋白(α-AFT)的竞争型酶联荧光免疫分析法。先以HRP标记α-AFT抗原,利用α-AFT与α-AFT-HRP对抗体的竞争型免疫反应,通过一步免疫反应后,再与四氨基铝酞菁-过氧化氢的荧光反应偶合,建立了α-甲胎蛋白的酶联荧光免疫分析法。该法用于测定结肠癌病人血清中α-甲胎蛋白的含量,结果令人满意。其次,研究了以四氨基铝酞菁为底物的乙肝表面抗原(HbsAg)的夹心型酶联荧光免疫分析法。首先用抗HbsAg抗体包被微孔板,加入的HbsAg与包被的抗体发生特异性结合,然后再加入HRP标记的抗体与HbsAg进行特异性反应,经过两步免疫反应后,固相上结合的HRP通过与四氨基铝酞菁-过氧化氢的荧光反应偶合来加以测定,从而测得加入的HbsAg抗原的量。并将该法用于阴性血清和乙肝病毒感染者血清中HbsAg水平的测定。 第三章研究了反相胶束介质中,辣根过氧化物酶模拟酶四磺基铁酞菁(FeTSPc)的催化行为。反相胶束是两亲分子溶解在有机相及少量水中自发形成的热力学性质稳定、光学透明的球形聚集体。以其独特的溶解能力、自组装性和相对有序的结构等特点,可为酶催化反应提供模拟生物体内酶催化的微环境。因而在酶法分析中,它不仅能使酶表现出高的活性,甚至使酶呈现“超级活性”。首先考察了AOT/水/环己烷阴离子反相胶束介质中FeTSPc催化过氧化氢氧化L-酪氨酸荧光反应的催化活性。结果表明,AOT/环己烷反相胶束介质不仅能提高FeTSPc的催化活性(其催化活性约是水溶液中的8倍),而且对其催化氧化产物二-酪氨酸的荧光亦具有一定的增敏作用。并进一步研究了水含量、pH、温度、表面活性剂浓度等对催化反应的影响。其次探讨了CTAB/正庚烷-正戊醇/水阳离子反相胶束介质中FeTSPc催化过氧化氢氧化L一酪氨酸荧光反应的催化反应特性。结果表明,FeTSPc在该介质中呈现出较高的催化活性,与相同条件下水相中的催化氧化反应相比较,其催化活性提高了近15倍。 第四章探讨了离子缔合作用在核酸测定中的应用。本章共分为两部分。第一部分将爱尔新蓝SGX与阴离于七次甲基花育染料的离子缔合作用应用于核酸的定量,建立了核酸测定的新方法,并探索了其机理。该法具有简便、快速、灵敏的特点,且发射波长在近红外区(795run),可有效避免生物样品的背景干扰。对盆栽琼棕 DNA的测定表明了本法的实用性。 第H部分将爱尔新蓝SGX和模拟酶FeTSPc催化的荧光反应相结合,用于核酸的定量测定。当爱尔新蓝SGX加人到FeTSPc一过氧化氢一对羟基苯乙酸的反应体系中时,由于爱尔新蓝SGX和FeTSPC的离子缔合作用导致FeTSPC的催化活性降低,表现为体系的荧光强度降低。当痕量核酸存在时,由于核酸和爱尔新蓝SGX之间强的相互作用,削弱了爱尔新蓝SGX和FeTSPc的离子缔合作用,导致FeTSPc的催化活性恢复,表现为体系的荧光强度增大。借助于酶的高效放大作用,用此法测定核酸可达到较高的灵敏度。 第五章探讨了酞青的共振光散射在生物大分子测定中的应用。首先研究了四磺基铝酞育共振光散射在血清总蛋白测定中的应用。四磺基铝酞育在2500~750刀 urn之间有弱的共振光散射,在酸度合适的介质中(如pH-3刀)中,蛋白能使其413.0 urn处的共振散射峰显著增强,且增强程度与蛋白浓度呈良好的线性关系。据此,建立了血清中总蛋白的共振光散射增强的分析有法。该法灵敏度高、检测限低、而且操作简单。其次探讨了四氨基铝酞警共振光散射在痕量核酸测定中的应用。我们观察到,四氨基铝酞奇在 300.0—800,0 urn之间有弱的共振光散射,痕量核酸的存在能使其400 urn处的共振散射峰显著增强,增强的程度与核酸浓度之间呈良好的线性关系。据此,建立了核酸定量测定的高灵
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全文目录
Abstract in Chinese 10-13 Abstract in English 13-17 Chapter 1 Introduction 17-44 1.1 The structure, synthesis, properties and application of Metallophthalocyanine Compounds 17-28 1.1.1 Introduction 17 1.1.2 The molecular structure of phthalocyanine compounds 17-18 1.1.3 Synthesis of metallophthalocyanine compounds 18-19 1.1.4 The general properties of phthalocyanine compounds 19-20 1.1.5 The spectral characteristics of phthalocyanine compounds 20-21 1.1.6 Applications of phthalocyanine compounds 21-28 1.1.6.1 Application of phthalocyanine compounds as a catalyst 22-25 1.1.6.1.1 Application of metallophthalocyanine compounds as a eletrocatalysts in electrochemical analysis 22 1.1.6.1.2 Application of metallophthalocyanines as a mimetic peroxidase in fluorescence analysis 22-25 1.1.6.2 Medical applications 25-26 1.1.6.3 Analytical application 26-28 1.2 Development of fluorogenic substrates for HRP and HRP Mimics 28-31 1.2.1 Fluorophores of 2, 2 '-dihydroxydiphenyl derivatives 28-29 1.2.2 Fluorescence quenching substrates 29 1.2.3 Other substrates 29-31 1.3 Study of enzymes-catalyzed reactions in reversed mieelles 31-35 1.3.1 Characteristics of reversed micelles 31-32 1.3.2 The catalytic activity of enzyme in reversed micelles 32-34 1.3.2.1 Influence of W value on the catalytic activity of enzymes 32-33 1.3.2.2 Influence of surfactant concentration oncatalyti cactivity of enzymes 33-34 1.3.3 Progress of the study on peroxidase in reversed micelles 34-35 1.4 The objective of this dissertation 35-36 Referenees 36-44 Chapter 2 Study of tetra-substituted amino aluminum phthalocyanine as a new red-region substrate for HRP and its analytical application 44-67 2.1 Introduction 44-45 2.2 Materials and apparatus 45-46 2.2.1 Materials 45-46 2.2.2 Apparatus 46 2.3 Investigation on the potential use of TAAIPc as a fluorescent substrate for HRP 46-50 2.3.1 Experimental 46-47 2.3.1.1 Procedure for the determination of the catalytic activity of HRP with TAAIPc as a substrate 46-47 2.3.2 Results and discussion 47-50 2.3.2.1 Spectral characteristics 47-48 2.3.2.2 Measurement of constant of enzymatic reaction 48-50 2.3.3 Conclusion 50 2.4 Application of TAAIPc as a fluorogenic indicator to the determination of H_2O_2 50-59 2.4.1 Application of HRP-catalyzed reaction of H_2O_2 and TAAIPc to the determination of H_2O_2 and glucose in human serum 50-55 2.4.1.1 Experimental 50 2.4.1.1.1 Assay procedure for H_2O_2 50 2.4.1.1.2 Procedure for glucose determination 50 2.4.1.2 Results and disicussion 50-55 2.4.1.2.1 Spectral characteristics 50-51 2.4.1.2.2 Optimization of experimental parameters 51-53 2.4.1.2.2.1 Effect of pH and buffer 51-52 2.4.1.2.2.2 Reaction time and temperature 52 2.4.1.2.2.3 Effects of reagent concentrations 52-53 2.4.1.2.3 Analytical performance 53 2.4.1.2.4 Effect of foreign substances 53-54 2.4.1.2.5 Determination of glucose in serum samples 54-55 2.4.2 Study of TAAIPc as a fluorogenic substrate in a HRP mimiccatalyzed oxidation reaction for the determination of H_2O_2 55-59 2.4.2.1 Experimental 55 2.4.2.1.1 Analysis of H_2O_2 using heroin as the catalyst 55 2.4.2.1.2 Analysis of H_2O_2 using FeTSPc as the catalyst 55 2.4.2.2 Results and discussion 55-59 2.4.2.2.1 Spectral characteristics 55-56 2.4.2.2.2 Optimization of experimental parameters 56-57 2.4.2.2.2.1 Effect of pH and buffer 56 2.4.2.2.2.2 Reaction time and temperature 56-57 2.4.2.2.2.3 Optimum amounts of catalyst and TAAIPc 57 2.4.2.2.3 Features of the analytical method 57-58 2.4.2.2.4 Effects of foreign substances 58 2.4.2.2.5 Determination of H_2O_2 in rainwater samples 58-59 2.4.3 Conclusions 59 2.5 Application of TAAIPe as a fluorogenie substrate in peroxidase-mediated enzyme-linked immunosorbent assay 59-65 2.5.1 A competitive immunoassay method for a-l-fetoprotein (a-AFP) 59-63 2.5.1.1 Experimental 60-61 2.5.1.1.1 Assay procedure for HRP 60 2.5.1.1.2 Immunoassay 60-61 2.5.1.2 Results and discussion 61-63 2.5.1.2.1 Selection of optimal conditions for HRP-catalyzed reaction 61 2.5.1.2.2 Calibration graph for HRP 61 2.5.1.2.3 Calibration graph for a-AFP 61-62 2.5.1.2.4 Immunoassay of AFP in human blood sera 62-63 2.5.2 A sandwich immunoassay method for the determination of hepatitis B surface antigen (HBsAg) 63-65 2.5.2.1 Experimental 63 2.5.2.1.1 Immunoassay for HBsAg 63 2.5.2.2 Results and disussion 63-65 2.5.2.2.1 Optimization of catalytic reaction conditions 63-64 2.5.2.2.2 Calibration graph for HBsAg 64 2.5.2.2.3 Immunoassay of HBsAg in blood sera 64-65 2.5.3 Conclusion 65 References 65-67 Chapter 3 Application of reversed micelles as a medium in mimetic peroxidase-catalyzed fluorescence analysis 67-84 3.1 Introduction 67 3.2 Materials and apparatus 67-68 3.2.1 Materials 67-68 3.2.2 Apparatus 68 3.3 Study of the effect of AOT reversed micelles on the FeTSPc-catalyzed fluorogenic oxidation reaction of L-tyrosine with H_2O_2 68-74 3.3.1 Experimental 68-69 3.3.1.1 Preparation of the reversed micelle 68-69 3.3.1.2 Steady-state method 69 3.3.1.3 Kinetic method 69 3.3.2 Results and discussion 69-74 3.3.2.1 Spectral Characteristics 69-70 3.3.2.2 Influence of AOT reversed m icelles on the fluorogenic reaction 70-71 3.3.2.3 Optimization of reaction conditions in A OT reversed micelles 71-74 3.4 Fluorimetric methods of H_2O_2 concentration and FeTSPc activity using an CTAB inverted micellar system 74-82 3.4.1 Experimental 74-75 3.4.1.1 Measurement of peroxidase activity of FeTSPc 74 3.4.1.2 Measurement of fiuorescence intensity with a steady-state method 74-75 3.4.1.3 The effect of reversed micelles on the fiuorescence of product 75 3.4.2 Results and discussion 75-82 3.4.2.1 Spectral characteristics 75-79 3.4.2.2 Optimum conditions for the FeTSPc-catalyzed reaction in CTAB reversed micelles 79-82 3.4.2.2.1 Effect of pH 79 3.4.2.2.2 Reaction time and temperature 79-80 3.4.2.2.3 Influence of W value on the catalytic reaction 80 3.4.2.2.4 Influence of CT,4B concentration 80-82 3.4.2.2.5 The optimum amounts of reagents 82 3.5 Analytical performance 82 3.6 Conclusion 82-83 References 83-84 Chapter 4 Determination of nucleic acids based on shifting the association equilibrium of ion-association complex 84-105 4.1 Introduction 84-86 4.2 Materials and apparatus 86-87 4.2.1 Materials 86-87 4.2.2 Apparatus 87 4.3 A novel method for the determination of nucleic acids by near infra fluorescence recovery 87-94 4.3.1 Experimental 87 4.3.1.1 General procedures 87 4.3.2 Results and discussion 87-94 4.3.2.1 Spectral characteristics offluorescence 87-88 4.3.2.2 Optimization of general procedure 88-89 4.3.2.2.1 Effect of buffer and pH 88-89 4.3.2.2.2 Influence of incubation time 89 4.3.2.2.3 Order of adding reagents 89 4.3.2.2.4 The optimum amounts of Alcian blue 8GX and HMC 89 4.3.2.2.5 Effect of salt concentration 89 4.3.2.3 Exploitation of reaction mechanism 89-92 4.3.2.3.1 Absorption spectral characteristics of HMC in the presence of Alcian blue 8GX 89-90 4.3.2.3.2 Absorption spectral characteristics of the Alcian blue 8GX-HMC system in the presence of nucleic acids 90-91 4.3.2.3.3 Absorption spectral characteristics of Alcian blue 8GX in the presence of nucleic acids 91-92 4.3.2.4 Tolerance of foreign substances 92-93 4.3.2.5 Analytical performance 93-94 4.3.2.6 Applying to sample determination 94 4.4 Application of FeTSPc-catalyzed fluorescence reaction to the determination of nucleic acids, based on shifting the association equilibrium between FeTSPc and Alcian blue 8GX 94-103 4.4.1 Experimental 94-95 4.4.1.1 General procedures 94-95 4.4.2 Results and discussion 95-103 4.4.2.1 Spectral characteristics of fluorescence 95 4.4.2.2 Quenching effect of Alcian blue 8GX on the fluorescence of the FeTSPc-p-HPA-H_2O_2 system 95-99 4.4.2.3 Fluorescence enhancing effect of nucleic acids on the Alcian blue 8GX-FeTSPc-p-HPA-H_2O_2 system 99 4.4.2.4 Optimization of experimental parameters 99-101 4.4.2.4.1 Effect of pH and buffer 99-100 4.4.2.4.2 Influence of final incubation time 100 4.4.2.4.3 The optimum amounts of reagents 100-101 4.4.2.5 Calibration graphs and limits of detection 101 4.4.2.6 Influence of foreign substances 101-102 4.4.2.7 Determination of DNA in real sample 102-103 4.5 Conclusion 103 References 103-105 Chapter 5 Application of the resonance light scattering of phthalocyanines to the determination of biomacromolecules 105-121 5.1 Introduction 105-106 5.2 Materials and apparatus 106-107 5.2.1 Materials 106-107 5.2.2 Apparatus 107 5.3 Determination of proteins at nanogram levels by a resonance light-scattering technique with tetra-substituted sulphonated aluminum phthaloeyanine 107-114 5.3.1 Experimental 107 5.3.1.1 Procedures 107 5.3.2 Results and discussion 107-113 5.3.2.1 Molecluar structure of AlS_4Pc 107-108 5.3.2.2 Spectral characteristics 108-110 5.3.2.3 Reaction and mixing sequence 110 5.3.2.4 Optimization of experimental conditions 110-112 5.3.2.4.1 Effect of pH and buffer 110-111 5.3.2.4.2 Optimum amounts of AlS_4Pc 111-112 5.3.2.5 Interference 112 5.3.2.6 Calibration graphs and the determination of clinical samples 112-113 5.3.2.7 Determinations of total protein in serum samples 113 5.3.3 Conclusions 113-114 5.4 A new resonance light-scattering method for the determination of nucleic acids at nanogram levels with tetra-substituted amino aluminum phthalocyanine 114-119 5.4.1 Experimental 114 5.4.1.1 Standard procedure 114 5.4.2 Results and Discussion 114-119 5.4.2.1 Spectral characteristics 114-116 5.4.2.2 Reaction time and adding sequence of reagents 116 5.4.2.3 Optimization of the general procedure 116-117 5.4.2.3.1 Effect of pH and buffers 116 5.4.2.3.2 Eject of TAAlPc concentration 116-117 5.4.2.4 Tolerance of foreign substances 117-118 5.4.2.5 Calibration curves 118-119 5.4.2.6 Determination of practical sample 119 5.4.3 Conclusion 119 References 119-121 Acknowledgement in English 121-122 Appendix: Publications & Presentations during Author's Ph.D. Study 122-125
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