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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

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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  20. 前向散射式能见度检测技术研究,P412.17
  21. 多羟基化合物硅胶基质材料在毛细管电泳和亲水色谱中的应用,O658.9

中图分类: > 数理科学和化学 > 化学 > 分析化学 > 分析作业方法与技术 > 试剂、反应
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