罗丹明B-Ce(IV)化学发光新体系在抗坏血酸及DNA测定中的应用研究
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摘要
本工作的主要内容是将新建立的罗丹明B-Ce(Ⅳ)-硫酸的酸性化学发光体系与流动注射技术结合用于蔬菜中抗坏血酸及合成样中DNA的测定，目的是拓展该类物质的检测方法及扩大化学发光方法的应用范围。
论文包括两个部分。
第一部分 化学发光分析概述及抗坏血酸、DNA测定的研究进展。
第一章 化学发光分析概述
介绍了化学发光分析的基本原理和仪器装置，对常见的化学发光体系进行了归纳和总结，展望了化学发光分析的发展趋势和应用前景。
第二章 DNA定量技术的研究进展
主要就近几年分光光度法、荧光法、共振光散射法以及化学发光法在DNA定量分析中的应用进行了总结和评述，共引用文献62篇。
第三章 抗坏血酸测定方法的研究进展
对近年来抗坏血酸测定的常用方法如分光光度法、荧光法、电化学法、动力学方法及化学发光法等进行了总结和评述，共引用文献55篇。
第二部分 罗丹明B-Ce(Ⅳ)化学发光体系的应用研究。
工作基于罗丹明B-Ce(Ⅳ)-硫酸这一酸性化学发光体系对合成样中DNA的含量及蔬菜中抗坏血酸的含量进行了测定。主要内容如下：
第一章 罗丹明B-Ce(Ⅳ)化学发光体系测定DNA的研究
本章利用罗丹明B与Ce(Ⅳ)在硫酸介质中的化学发光反应，建立了两种测定超微量小牛胸腺DNA和鲱鱼精DNA的分析方法，并对这两种方法的发光机理分别进行了探讨。方法简便、快捷，检出限远低于其它测定方法。
§1 热变性DNA的流动注射化学发光测定研究
将DNA热变性，测定罗丹明B-Ce(Ⅳ)-热变性DNA在硫酸介质中的化学发光强度，由此建立了一种测定DNA含量的流动注射化学发光新方法。该法对小牛胸腺DNA和鲱
周敏:罗丹明B一代)(IV)化学发光新体系在抗坏血酸及DNA测定中的应用研究
鱼精DNA的检出限分别为6.5x10石产创mL和4.3x108产岁mL;线性范围分别为
2.6x10-5一0.26产创山L(r二0.9995)和5.0x10-8一5.oxlo一产岁mL(r=0.9996);对2.6x10-3
群岁mL小牛胸腺DNA和1 .0x10一6产留mL鲜鱼精DNA，相对标准偏差分别为0.93%和
0.90%(n二n)。对六种DNA合成样进行测定，回收率在100.8一108.0%之间。
互2咪哇缓冲液活化DNA的流动注射化学发光测定研究
DNA预先以咪哇一盐酸缓冲溶液印H7.0)活化，在0.8 mo呱的硫酸介质中，活化的
DNA使罗丹明B一Ce(w)体系的化学发光信号增强，增强的发光信号值与DNA浓度的对
数成线性关系，据此建立了另一种测定DNA含量的流动注射化学发光分析方法。该法
对小牛胸腺DNA和鲜鱼精DNA的检出限分别为3.5xlo一7产岁mL和&3xlo一9产留mL;线
性范围分别为2.oxio一0.2产创m环= 0.9993)和i.oxlo一0.1产岁加场二0.9997孙对2.Ox10-3
产岁mL小牛胸腺DNA和1 .0x10礴产岁mL鲜鱼精DNA，相对标准偏差分别为1.1%和
0.99%(n=n)。对六种DNA合成样进行测定，回收率在99.5一109.0%之间。
第二章罗丹明B一{e(工V)化学发光体系测定抗坏血酸的研究
罗丹明B与Ce(哟在硫酸介质中能产生很强的化学发光，抗坏血酸的加入使发光增
强，据此，本章建立了一种测定抗坏血酸的高灵敏流动注射化学发光分析方法。该法检
出限为i.ox1o一，3 mol/L;线性范围为3.sxio一‘3一i.oxio一，0 mo呱(r=0.9998)。对i.oxio一“
mo呱抗坏血酸溶液重复测定11次，相对标准偏差为0.92%。方法用于测定新鲜蔬菜中
的抗坏血酸，结果令人满意。同时本章对该发光反应的机理也进行了较为详细的讨论。
This paper presents a series of sensitive FI method utilizing a chemiluminescence(CL) system of cerium (IV) with rhodamine B in sulfuric acid for the determination of ascorbic acid in vegetables and for the quantification of ultramicro calf thymus DNA. and herring sperm DNA in synthetic samples. The present work aims to develop the detection methods for
these substances and to spread the use of the CL technique. This paper consists of two parts.
Part One A review on CL analysis and on the development of the determination of ascorbic acid and DNA.
Chapter One A review on CL analysis
This chapter introduces the basic principles and the instrumentation of CL analysis. Mainly it summarizes the common CL system and their analytical applications, and vistas the newly developing techniques and further trends of CL method.
Chapter Two The development of DNA quantification
In this chapter, the methods for DNA determination such as spectrophotometry, fluorometry, resonance light scattering methods and CL methods are reviewed, and 62 references are cited.
Chapter Three The development of the determination of ascorbic acid
all kinds of methods including spectrophotometry, fluorometry, electrochemical methods, kinetic methods and CL methods for the determination of ascorbic acid in recent years are reviewed, and 55 references are cited.
Part Two The application of the rhodamine B-Ce(IV) CL system
In this part, some research reports were given. The contents of ascorbic acid in vegetables and DNA (including calf thymus DNA and herring sperm DNA) in synthetic samples were determined by flow-injection(FI) methods utilizing a CL system of cerium (IV) with rhodamine B in sulfuric acid. The detailed works are as follows:
Chapter One Studies on the ultra-trace determination of DNA by FI-CL assay using Ce(IV) -rhodamine B system
In this section, two novel CL methods for the determination of ultra-trace calf thymus DNA and herring sperm DNA were presented, based on the CL reaction of rhodamine B with Ce(IV) in sulfuric acid. The proposed methods have advantages of simplicity, rapidity, reproducibility, and especially the relatively low detection limits. The possible mechanism has also been discussed respectively.
1 FI-CL assay for thermally denatured DNA
A high sensitive FI-CL method for determination of calf thymus DNA and herring sperm DNA has been developed. The method based on the CL reaction of rhodamine B-cerium (IV)-thermally denatured DNAs in sulfuric acid media. The proposed procedure allows quantitation of DNAs in the concentration range of 2.6x10-5-0.26 ug ml-1 for calf thymus DNA and 5.0xl0-8-5.0xl0-5 ug ml-1 for herring sperm DNA with correlation coefficients 0.9998 and 0.9996, respectively. The detection limits (3a) are 6.5x10-6 ug ml-1 for calf thymus DNA and 4.3x10-8 ug ml" for herring sperm DNA, accordingly. The possible CL mechanism in the system is discussed as well.
2 FI-CL assay for activated DNA by imidazole-HCl buffer solution
In this section, a new FI-CL method for determination of calf thymus DNA and herring sperm DNA in an acidic medium were developed. Imidazole-HCl buffer solution was firstly applied to activate DNA in CL methods. In 0.8 mol/L sulfuric acid media, the CL of rhodamine B-Ce (IV) system is enhanced by DNA, which activated previously by imidazole-HCl buffer solution (pH 7.0). The enhanced intensity of CL is in proportion to log DNA in the range 1.0xl0-8-0.1 ug ml"1 for herring sperm DNA and 2.0x10-6-0.2 ug ml-1 for calf thymus DNA with the detection limits of 8.3 x10-9 ug ml-1 for herring sperm DNA and 3.5x10-7ug ml-1 for calf thymus DNA, respectively. The relative standard deviation (R.S.D) for 1.0x10-4 ug ml-1 herring sperm DNA was 0.99% and for 2.0x10-3ug ml-1 calf thymus DNA was 1.1% (n=11). Using the optimized system, DNA contents in six synthetic samples has been determined with recoveries of 99.5%-109.0%.
Chapter Two Flow-injection chemiluminescence determination of ascorbic acid by use of the Ce(IV) -rhodamine B system
In this work, a highly se
论文包括两个部分。
第一部分 化学发光分析概述及抗坏血酸、DNA测定的研究进展。
第一章 化学发光分析概述
介绍了化学发光分析的基本原理和仪器装置，对常见的化学发光体系进行了归纳和总结，展望了化学发光分析的发展趋势和应用前景。
第二章 DNA定量技术的研究进展
主要就近几年分光光度法、荧光法、共振光散射法以及化学发光法在DNA定量分析中的应用进行了总结和评述，共引用文献62篇。
第三章 抗坏血酸测定方法的研究进展
对近年来抗坏血酸测定的常用方法如分光光度法、荧光法、电化学法、动力学方法及化学发光法等进行了总结和评述，共引用文献55篇。
第二部分 罗丹明B-Ce(Ⅳ)化学发光体系的应用研究。
工作基于罗丹明B-Ce(Ⅳ)-硫酸这一酸性化学发光体系对合成样中DNA的含量及蔬菜中抗坏血酸的含量进行了测定。主要内容如下：
第一章 罗丹明B-Ce(Ⅳ)化学发光体系测定DNA的研究
本章利用罗丹明B与Ce(Ⅳ)在硫酸介质中的化学发光反应，建立了两种测定超微量小牛胸腺DNA和鲱鱼精DNA的分析方法，并对这两种方法的发光机理分别进行了探讨。方法简便、快捷，检出限远低于其它测定方法。
§1 热变性DNA的流动注射化学发光测定研究
将DNA热变性，测定罗丹明B-Ce(Ⅳ)-热变性DNA在硫酸介质中的化学发光强度，由此建立了一种测定DNA含量的流动注射化学发光新方法。该法对小牛胸腺DNA和鲱
周敏:罗丹明B一代)(IV)化学发光新体系在抗坏血酸及DNA测定中的应用研究
鱼精DNA的检出限分别为6.5x10石产创mL和4.3x108产岁mL;线性范围分别为
2.6x10-5一0.26产创山L(r二0.9995)和5.0x10-8一5.oxlo一产岁mL(r=0.9996);对2.6x10-3
群岁mL小牛胸腺DNA和1 .0x10一6产留mL鲜鱼精DNA，相对标准偏差分别为0.93%和
0.90%(n二n)。对六种DNA合成样进行测定，回收率在100.8一108.0%之间。
互2咪哇缓冲液活化DNA的流动注射化学发光测定研究
DNA预先以咪哇一盐酸缓冲溶液印H7.0)活化，在0.8 mo呱的硫酸介质中，活化的
DNA使罗丹明B一Ce(w)体系的化学发光信号增强，增强的发光信号值与DNA浓度的对
数成线性关系，据此建立了另一种测定DNA含量的流动注射化学发光分析方法。该法
对小牛胸腺DNA和鲜鱼精DNA的检出限分别为3.5xlo一7产岁mL和&3xlo一9产留mL;线
性范围分别为2.oxio一0.2产创m环= 0.9993)和i.oxlo一0.1产岁加场二0.9997孙对2.Ox10-3
产岁mL小牛胸腺DNA和1 .0x10礴产岁mL鲜鱼精DNA，相对标准偏差分别为1.1%和
0.99%(n=n)。对六种DNA合成样进行测定，回收率在99.5一109.0%之间。
第二章罗丹明B一{e(工V)化学发光体系测定抗坏血酸的研究
罗丹明B与Ce(哟在硫酸介质中能产生很强的化学发光，抗坏血酸的加入使发光增
强，据此，本章建立了一种测定抗坏血酸的高灵敏流动注射化学发光分析方法。该法检
出限为i.ox1o一，3 mol/L;线性范围为3.sxio一‘3一i.oxio一，0 mo呱(r=0.9998)。对i.oxio一“
mo呱抗坏血酸溶液重复测定11次，相对标准偏差为0.92%。方法用于测定新鲜蔬菜中
的抗坏血酸，结果令人满意。同时本章对该发光反应的机理也进行了较为详细的讨论。
This paper presents a series of sensitive FI method utilizing a chemiluminescence(CL) system of cerium (IV) with rhodamine B in sulfuric acid for the determination of ascorbic acid in vegetables and for the quantification of ultramicro calf thymus DNA. and herring sperm DNA in synthetic samples. The present work aims to develop the detection methods for
these substances and to spread the use of the CL technique. This paper consists of two parts.
Part One A review on CL analysis and on the development of the determination of ascorbic acid and DNA.
Chapter One A review on CL analysis
This chapter introduces the basic principles and the instrumentation of CL analysis. Mainly it summarizes the common CL system and their analytical applications, and vistas the newly developing techniques and further trends of CL method.
Chapter Two The development of DNA quantification
In this chapter, the methods for DNA determination such as spectrophotometry, fluorometry, resonance light scattering methods and CL methods are reviewed, and 62 references are cited.
Chapter Three The development of the determination of ascorbic acid
all kinds of methods including spectrophotometry, fluorometry, electrochemical methods, kinetic methods and CL methods for the determination of ascorbic acid in recent years are reviewed, and 55 references are cited.
Part Two The application of the rhodamine B-Ce(IV) CL system
In this part, some research reports were given. The contents of ascorbic acid in vegetables and DNA (including calf thymus DNA and herring sperm DNA) in synthetic samples were determined by flow-injection(FI) methods utilizing a CL system of cerium (IV) with rhodamine B in sulfuric acid. The detailed works are as follows:
Chapter One Studies on the ultra-trace determination of DNA by FI-CL assay using Ce(IV) -rhodamine B system
In this section, two novel CL methods for the determination of ultra-trace calf thymus DNA and herring sperm DNA were presented, based on the CL reaction of rhodamine B with Ce(IV) in sulfuric acid. The proposed methods have advantages of simplicity, rapidity, reproducibility, and especially the relatively low detection limits. The possible mechanism has also been discussed respectively.
1 FI-CL assay for thermally denatured DNA
A high sensitive FI-CL method for determination of calf thymus DNA and herring sperm DNA has been developed. The method based on the CL reaction of rhodamine B-cerium (IV)-thermally denatured DNAs in sulfuric acid media. The proposed procedure allows quantitation of DNAs in the concentration range of 2.6x10-5-0.26 ug ml-1 for calf thymus DNA and 5.0xl0-8-5.0xl0-5 ug ml-1 for herring sperm DNA with correlation coefficients 0.9998 and 0.9996, respectively. The detection limits (3a) are 6.5x10-6 ug ml-1 for calf thymus DNA and 4.3x10-8 ug ml" for herring sperm DNA, accordingly. The possible CL mechanism in the system is discussed as well.
2 FI-CL assay for activated DNA by imidazole-HCl buffer solution
In this section, a new FI-CL method for determination of calf thymus DNA and herring sperm DNA in an acidic medium were developed. Imidazole-HCl buffer solution was firstly applied to activate DNA in CL methods. In 0.8 mol/L sulfuric acid media, the CL of rhodamine B-Ce (IV) system is enhanced by DNA, which activated previously by imidazole-HCl buffer solution (pH 7.0). The enhanced intensity of CL is in proportion to log DNA in the range 1.0xl0-8-0.1 ug ml"1 for herring sperm DNA and 2.0x10-6-0.2 ug ml-1 for calf thymus DNA with the detection limits of 8.3 x10-9 ug ml-1 for herring sperm DNA and 3.5x10-7ug ml-1 for calf thymus DNA, respectively. The relative standard deviation (R.S.D) for 1.0x10-4 ug ml-1 herring sperm DNA was 0.99% and for 2.0x10-3ug ml-1 calf thymus DNA was 1.1% (n=11). Using the optimized system, DNA contents in six synthetic samples has been determined with recoveries of 99.5%-109.0%.
Chapter Two Flow-injection chemiluminescence determination of ascorbic acid by use of the Ce(IV) -rhodamine B system
In this work, a highly se
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