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组织酶催化光学分析系统及化学发光新体系应用研究
作 者: 王周平
导 师: 章竹君
学 校: 西南师范大学
专 业: 分析化学
关键词: 组织酶催化 生物发光 光度/荧光/化学发光分析 流动注射分析 药物-蛋白相互作用 药物
分类号: O657.3
类 型: 博士论文
年 份: 2004年
下 载: 216次
引 用: 0次
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内容摘要
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近年来，基于分离纯化酶的传统酶法分析日益受到新型生物催化活性材料如微生物、动植物组织、细胞、受体等，尤其是动植物组织的挑战，催化活性稳定、寿命长、取材容易、价格低廉等特性使得这类组织酶催化生物传感器／分析系统越来越受到人们的重视，相关研究成为酶法分析和生物传感器研究领域的一大热点。生物发光菌历来在食品安全、环境监测等方面发挥着重要作用，研究发现新型生物发光菌、构建新型无试剂型生物发光传感器是这一研究领域的主要任务之一。化学发光分析因其灵敏度高、线性范围宽、分析速度快、仪器设备简单便宜以及易于实现自动化和连续分析等特点，吸引着众多分析工作者的广泛关注，已被成功地应用于生物技术、药学、分子生物学、临床医学和环境检测等领域中许多重要的无机和有机物质的分析。 本研究工作主要集中于三个方面，一方面是将组织酶催化反应与光学检测系统(光度、荧光、化学发光)在线偶合，建立了一系列基于组织酶催化的流动注射光学分析系统；另一方面是发现了新型生物发光真菌-蜜环菌，对其气相和液相生物发光行为进行研究，初步构建了一种非常简单的无试剂型气体氧和溶解氧生物传感器；第三方面是建立新的化学发光分析方法，研究在线微透析、微超滤与其结合应用于药物-蛋白相互作用 的学位论文">药物-蛋白相互作用研究和将其应用于实际样品分析的可行性。 第一章为组织酶催化光学分析系统研究。1．1部分概述了组织酶催化分析研究的相Southwest Normal University， Doetor Thesis一Z.Wang(200126)关进展. 1 .2部分研究发现富含多酚氧化酶的蘑菇组织可催化氧化儿茶酚及儿茶酚胺类物质转化为有色的醒类和红类物质，这些产物的吸收峰较之原物质吸收峰明显红移。基于此，采用非常简单的固定化方法并结合流动注射技术，建立了一种简单、灵敏、准确、廉价的无试剂型儿‘茶酚、盐酸异丙肾上腺素、盐酸多巴胺流动注射紫外一可见分光光度分析系统。该系统对上述几种物质具有良好的响应，儿茶酚、盐酸异丙肾上腺素、盐酸多巴胺物质浓度在一定范围内与吸光度分别呈良好的线性关系(儿茶酚，2x10.6一l、10.3gmr’;盐酸异丙肾上腺素，4灰20一6一sxzo.‘9 ml‘’;盐酸多巴胺，4xlo“6一sxlo‘4gml一’)。对三种物质的检测限(3a)分别为:4x 10一，9 ml一’，1 .3xlo一‘gml一‘，z.oxlo一6gml‘’。将该系统用于药物制剂中盐酸异丙肾上腺素、盐酸多巴胺含量的测定，所得结果与药典标准方法测得结果一致。同时用蘑菇组织制作的反应器具有制作简单、高酶活、使用寿命长、易更换和成本低廉等优点。 1，3部分研究发现富含多酚氧化酶的蘑菇组织可替代纯多酚氧化酶催化氧化异丙肾上腺素使其转化为异丙肾上腺素红，该物质在碱性条件下可进一步发生重排而成为具有强烈荧光特性的三轻基叫垛类物质。基于此将蘑菇组织催化氧化异丙肾上腺素反应与流动注射荧光光度分析技术结合，建立了一种非常简单、灵敏和高选择性的异丙肾上腺素蘑菇组织催化氧化一流动注射荧光分析系统。在实验选定条件下，荧光强度与异丙肾上腺素浓度在 3xlo一8一1又10一，9 ml一’范围内呈良好的线性关系，检测限为1 .0x10一8 9 mrl(3。)，相对标准偏差小于5%(n二11)。将该系统初步应用于药物制剂中异丙肾上腺素含量的测定，所得结果与药典标准方法测得结果一致。采用流动注射技术同时解决了产物荧光不稳定、常规类似测定中需加入稳定剂等问题而无须考虑反应动力学因素。用蘑菇组织制作的反应器同样具有制作简单、高酶活、使用寿命长、易更换和成本低廉等优点。没有氧化剂的引入，也符合当前绿色化学的潮流。 1.4部分构建了一种基于萝卜组织催化的流动注射化学发光谷氨酞胺分析系统。萝卜组织富含谷氨.酞胺酶，可替代纯谷氨酞胺酶定量催化谷氨酞胺生成氨的反应，将这一催化反应与NBS一二氯荧光素化学发光体系测定氨的反应在线偶合，使催化反应与化学发光检测反应在不同位置发生，既保证了二者反应都在最佳条件下进行，又使得所制作的组织反应器寿命大大延长。在实验选定的最佳条件下，化学发光强度与谷氨酞胺浓度在7x10一8一l、10一，gmr’范围内呈良好的线性关系，检测限为2.3、10一sgml一，(3。)，相对标准偏差小于5%。将该系统应用于药物制剂、水样中谷氨酞胺含量的测定，结果令人满 2竺丝竺塑鱼竺些竺竺业丝竺竺卫些全圣丛些卫丝坐丝...一…~1「_…__……~_:煞少睽意。同时试验了将该系统用于生物体液中谷氨酸胺测定的可能性，标准加入实验表明该系统可成功用于血样中谷氨酞胺的检测，并具有监测谷氨酞胺类药物服用后血药浓度变化的潜能。 第二章为生物发光真菌.蜜环菌生物发光行为研究。2.1部分概述了生物发光的原理和相关研究进展。 2，2部分研究报道了一种天然生物发光真菌一蜜环菌的气相发光行为。结果显示在酸碱度、温度和培养基质适
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全文目录
Abstract 15-23
Chapter 1 Tissue enzyme catalysis-based optical analytical systems 23-71
1.1 Introduction 23-31
1.1.1 Tissue-based biosensor 24-27
1.1.2 Other analytical procedures based on tissue enzyme catalysis 27-29
References 29-31
1.2 A mushroom tissue-based flow through spectrophotometric system for the determination of catechol and catecholamines 31-43
1.2.1 Introduction 31-33
1.2.2 Experimental 33-35
1.2.2.1 Reagents and solutions 33-34
1.2.2.2 Apparatus 34
1.2.2.3 Preparation of tissue reactor 34-35
1.2.2.4 Procedures 35
1.2.3 Results and discussion 35-41
1.2.3.1 Selection of tissue material 35-36
1.2.3.2 Absorption characteristics of the products 36-37
1.2.3.3 Optimization of reaction conditions 37-38
1.2.3.4 Analytical performance 38-39
1.2.3.5 Selectivity and lifetime of the reactor 39
1.2.3.6 Application 39-41
1.2.3.6.1 Determination of isoprenaline hydrochloride 40
1.2.3.6.2 Determination of dopamine 40-41
1.2.4 Conclusion 41-42
References 42-43
1.3 A mushroom tissue-based flow injection fluorescence system for the determination of isoprenaline 43-56
1.3.1 Introduction 43-46
1.3.2 Experimental 46-48
1.3.2.1 Reagents and Materials 46
1.3.2.2 Apparatus 46-47
1.3.2.3 Preparation of tissue reactor 47
1.3.2.4 Procedure for calibration 47-48
1.3.2.5 Procedure for pharmaceutical preparations 48
1.3.3 Results and discussion 48-53
1.3.3.1 Selection of tissue material 48
1.3.3.2 Reaction and spectral characteristics 48-49
1.3.3.3 Conditions for the system 49-51
1.3.3.3.1 Effect of temperature 49
1.3.3.3.2 Effect of pH 49-50
1.3.3.3.3 Effect of concentration of NaOH 50
1.3.3.3.4 Effect of flow rate 50-51
1.3.3.3.5 Stability of reagents 51
1.3.3.4 Performance of the proposed system 51
1.3.3.5 Stability and lifetime of the system 51-52
1.3.3.6 Selectivity of the system 52
1.3.3.7 Application 52-53
1.3.4 Conclusion 53-54
References 54-56
1.4 A palnt tissue-bascd flow injection chemiluminescnce system for glutamine 56-71
1.4.1 Introduction 56-59
1.4.2 Experimental 59-62
1.4.2.1 Reagents and materials 59-60
1.4.2.2 Apparatus 60
1.4.2.3 Preparation of tissue reactor 60-61
1.4.2.4 Procedure for calibration 61
1.4.2.5 Procedure for pharmaceutical preparations 61
1.4.2.6 Procedure for spiked human plasma 61-62
1.4.3 Results and discussion 62-67
1.4.3.1 Selection of tissue material of glutaminase 62
1.4.3.2 Conditions for enzymatic reaction in plant tissue column and flow-injection system 62-65
1.4.3.2.1 Effect of temperature on the tissue reactor and CL intensity 62
1.4.3.2.2 Selection of pH on the tissue reactor 62-63
1.4.3.2.3 Effect of flow rate of samples on the system 63-64
1.4.3.2.4 Effect of NBS concentration on the CL intensity 64
1.4.3.2.5 Effect of dichlorofluorescein concentration on the CL intensity 64
1.4.3.2.6 Effect of NaOH concentration on the CL intensity 64-65
1.4.3.3 Analytical characteristics, reproducibility and lifetime of the system 65
1.4.3.4 Selectivity of the system 65-66
1.4.3.5 Applications 66-67
1.4.4 Conclusion 67-68
References 68-71
Chapter 2 Investigation on a novel and native bioluminescent fungi-Armillariaella mellea 71-103
2.1 Introduction 71-84
2.1.1 Typical bioluminescence systems and their bioluminescent mechanisms 71-76
2.1.2 Application of bioluminescence 76-80
References 80-84
2.2 An investigation on a novel and native bioluminescent fungi-Armillariaella mellea in gas phase 84-95
2.2.1 Introduction 84-85
2.2.2 Experimental 85-89
2.2.2.1 Reagents and meterials 85-86
2.2.2.2 Cultivation of strain 86-87
2.2.2.3 Preparation of the reactor 87-88
2.2.2.4 Apparatus 88
2.2.2.5 Procedure 88-89
2.2.3 Results and discussions 89-94
2.2.3.1 Selection of culture medium 89
2.2.3.2 Conditions optimization 89-90
2.2.3.2.1 Effect of media pH 89
2.2.3.2.2 Effect of temperature 89-90
2.2.3.2.3 Effect of carrier flow rate 90
2.2.3.2.4 Effect of sample volume 90
2.2.3.3 Responses to different gases 90-91
2.2.3.4 Response time 91
2.2.3.5 Reproducibility and stability of AM biosensor 91-92
2.2.3.6 Response of the biosensor to O_2 92
2.2.3.7 Possible bioluminescent mechanisms 92-94
2.2.4 Conclusion 94
References 94-95
2.3 An investigation on a novel and native bioluminescent fungi-Armillariaella mellea in aqueous phase 95-103
2.3.1 Introduction 95
2.3.2 Experimental 95-98
2.3.2.1 Reagents and strain 95-96
2.3.2.2 Apparatus 96
2.3.2.3 Cultivation of strain 96-97
2.3.2.4 Preparation of the reactor 97-98
2.3.2.5 Preparation of standard sample 98
2.3.2.6 Procedures 98
2.3.3 Results and discussion 98-101
2.2.3.1 Selection of culture medium 98
2.3.3.2 Conditions optimization 98-100
2.3.3.2.1 Effect of media pH 98-99
2.3.3.2.2 Effect of temperature 99
2.3.3.2.3 Effect of flow rate of carrier 99-100
2.3.3.2.4 Effect of sample volume 100
2.3.3.3 Response to DO 100
2.3.3.3.1 Response time 100
2.3.3.3.2 Calibration curve 100
2.3.3.3.3 Reproducibility and stability 100
2.3.3.4 Response to other substances (interference study) 100-101
2.3.3.5 Sample analysis 101
2.3.4 Conclusion 101-102
References 102-103
Chapter 3 On-line microdialysis and microultrafiltration sampling coupled with flow-injection chemiluminescence analysis for the study of drug-protein interaction 103-141
3.1 Introduction 103-113
3.1.1 Principle of drug-protein interaction 104-106
3.1.2 Methods for studying drug-protein interaction 106-110
References 110-113
3.2 Flow-injection chemiluminescence detection for studying protein binding of terbutaline sulfate with on-line microdialysis sampling 113-127
3.2.1 Introduction 113-116
3.2.2 Experimental 116-119
3.2.2.1 Chemicals and reagents 116
3.2.2.2 Apparatus 116-117
3.2.2.3 Procedures 117-119
3.2.2.3.1 Optimization of CL system 117-118
3.2.2.3.2 Drug-protein interaction studies in vitro 118-119
3.2.3 Results and discussion 119-124
3.2.3.1 Kinetic figure of the CL reaction 119
3.2.3.2 Conditions of the CL detection system 119-121
3.2.3.2.1 Effect of H_2SO_4 concentration on the CL intensity 120
3.2.3.2.2 Effect of KMnO_4 concentration on the CL intensity 120
3.2.3.2.3 Effect of HCHO concentration on the CL intensity 120
3.2.3.2.4 Effect of flow rate on the CL intensity 120-121
3.2.3.2.5 Analytical characteristics of the CL system for terbutaline sulfate determination 121
3.2.3.3 Dialytic efficiency calibration of microdialysis probe 121-122
3.2.3.3.1 Effect of perfusate flow rate on the dialytic efficiency of microdialysis probe 121-122
3.2.3.3.2 Effect of temperature on the dialytic effciency of microdialysis probe 122
3.2.3.4 Interaction between terbutaline sulfate and BSA 122-124
3.2.4 Conclusion 124
References 124-127
3.3 A flow-injection micro-ultrafiltration sampling Chemiluminescence system for on-line determination of drug-protein interaction 127-141
3.3.1 Introduction 127-129
3.3.2 Experimental 129-132
3.3.2.1 Chemicals and reagents 129-130
3.2.2.2 Apparatus 130-131
3.3.2.3 Procedures 131-132
3.3.2.3.1 Optimization of the CL system 131-132
3.3.2.3.2 Drug-protein interaction studies in vitro 132
3.3.3 Results and discussion 132-138
3.3.3.1 Characteristics of the CL reaction 132-134
3.3.3.2 Conditions of the CL detection system 134-135
3.3.3.2.1 Effect of fluorescein concentration on the CL intensity 134
3.3.3.2.2 Effect of NaOH concentration on the CL intensity 134
3.3.3.2.3 Effect of NBS concentration on the CL intensity 134-135
3.3.3.2.4 Chemiluminescence enhancement by surfactants' 135
3.3.3.2.5 Effect of flow rate on the CL intensity 135
3.3.3.3 Analytical characteristics of the CL system for cimetidine determination 135-136
3.3.3.4 Interference study 136-137
3.3.3.5 Interaction between cimetidine and BSA 137-138
3.3.4 Conclusion 138
References 138-141
Chapter 4 Novel chemiluminescnce systems and the applications in drugs analysis 141-217
4.1 Introduction 141-167
4.1.1 Typical chemiluminscent reaction systems 142-147
4.1.1.1 Strong chemiluminescence systems 142-146
4.1.1.2 Weak chemiluminescence systems 146-147
4.1.2 Novel chemiluminscence techniques and trends 147-155
References 155-167
4.2 Sensitive flow-injection chemiluminescence determination of terbutaline sulfate based on the enhancement of luminol-permanganate reaction 167-182
4.2.1 Introduction 167-169
4.2.2 Experimental 169-172
4.2.2.1 Reagents 169
4.2.2.2 Apparatus 169-170
4.2.2.3 Procedure for calibration 170-171
4.2.2.4 Procedure for pharmaceutical preparations 171
4.2.2.4.1 Proposed CL method 171
4.2.2.4.2 Official method 171
4.2.2.5 Procedure for biological fluids 171-172
4.2.2.5.1 Determination of terbutaline sulfate in human plasma 171-172
4.2.2.5.2 Determination of terbutaline sulfate in human urine 172
4.2.3 Results and discussion 172-179
4.2.3.1 Kinetic curve of CL reaction 172-173
4.2.3.2 Optimization of the reaction conditions 173-174
4.2.3.2.1 Effect of NaOH concentration on the CL intensity 173
4.2.3.2.2 Effect of luminol concentration on the CL intensity 173
4.2.3.2.3 Effect of KMnO_4 concentration on the CL intensity 173
4.2.3.2.4 Effect of flow rate on the CL intensity 173-174
4.2.3.2.5 Effect of sample volume on the CL intensity 174
4.2.3.3 Performance of the proposed method 174-175
4.2.3.4 Interference studies 175-176
4.2.3.5 Application 176-178
4.2.3.5.1 Analysis of pharmaceutical preparations 176
4.2.3.5.2 Analysis of spiked plasma and urine sample 176-178
4.2.3.6 Discussion of possible mechanism 178-179
4.2.4 Conclusion 179
References 179-182
4.3 Flow-injection inhibition chemiluminescence determination of indapamide based on luminol-ferricyanide reaction 182-193
4.3.1 Introduction 182-184
4.3.2 Experimental 184-186
4.3.2.1 Reagents 184
4.3.2.2 Apparatus 184-185
4.3.2.3 Procedure for calibration 185
4.3.2.4 Procedure for pharmaceutical preparations 185-186
4.3.3 Results and discussion 186-191
4.3.3.1 Condition optimization of the CL system 186-188
4.3.3.1.1 Selection of oxidant and the effect of its concentration on the CL intensity 186-187
4.3.3.1.2 Effect of luminol concentration on the CL intensity 187
4.3.3.1.3 Effect of NaOH concentration on the CL intensity 187-188
4.3.3.1.4 Effect of flow rate on the CL intensity 188
4.3.3.1.5 Effect of Na_2CO_3 on the CL emission 188
4.3.3.2 Performance of the proposed method 188
4.3.3.3 Interference study 188-189
4.3.3.4 Applications 189-190
4.3.3.5 Possible mechanism of the CL system 190-191
4.3.4 Conclusion 191
References 191-193
4.4 Sensitive Flow-injection chemiluminescence determination of metformin based on N-bromosuccinimide-fluorescein system 193-205
4.4.1 Introduction 193-195
4.4.2 Experimental 195-197
4.4.2.1 Reagents 195-196
4.4.2.2 Apparatus 196
4.4.2.3 Procedure for calibration 196-197
4.4.2.4 Procedure for pharmaceutical preparations 197
4.4.3 Results and discussion 197-202
4.4.3.1 Effect of fluorescein concentration 197
4.4.3.2 Effect of sodium hydroxide concentration 197-198
4.4.3.3 Effect of NBS concentration 198
4.4.3.4 Chemiluminescence enhancement by surfactants 198-199
4.4.3.5 Effect of flow rate 199
4.4.3.6 Performance of the proposed method for metformin measurements 199-200
4.4.3.7 Interference study 200
4.4.3.8 Application 200-201
4.4.3.9 Possible mechanism of the chemiluminescence reaction 201-202
4.4.4 Conclusion 202-203
References 203-205
4.5 N-bromosuccinimide-fluorescein based sensitive flow-injection chemiluminescence determination of phenformin 205-217
4.5.1 Introduction 205-207
4.5.2 Experimental 207-209
4.5.2.1 Reagents 207
4.5.2.2 Apparatus 207-208
4.5.2.3 Procedure 208-209
4.5.3 Results and discussion 209-215
4.5.3.1 Characteristics of the CL reaction 209-210
4.5.3.2 Optimization of conditions 210-213
4.5.3.2.1 Effect of fluorescein concentration 210
4.5.3.2.2 Effect of sodium hydroxide concentration 210-211
4.5.3.2.3 Effect of NBS concentration 211-212
4.5.3.2.4 Chemiluminescence enhancement by surfactants 212
4.5.3.2.5 Effect of flow rate 212-213
4.5.3.3 Performance of the proposed method for phenformin measurements 213
4.5.3.4 Interference study 213-214
4.5.3.5 Application 214-215
4.5.4 Conclusion 215
References 215-217
Publications and presentations 217-219
Acknowledgements 219
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中图分类: > 数理科学和化学 > 化学 > 分析化学 > 仪器分析法（物理及物理化学分析法） > 光化学分析法（光谱分析法）
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