化学发光分析新方法、新技术的研究
摘要
自从1877年首次发现化学发光现象以来，人们对化学发光现象的认识和研究，已经历了一个极其漫长的发展过程。化学发光是在没有光、电、磁、声、热源激发的情况下，由化学反应或生物化学反应产生的一种光辐射。以此为基础的化学发光分析，由于可以进行发射光子计量，又没有外来激发光源存在时散射光背景的干扰，因而具有很高的灵敏度(检出限可达10~(-12)～10~(-21)mol)，很宽的线性范围(3～6个数量级)，同时仪器设备又很简单、廉价、易微型化，在二十世纪的最后十年发展非常迅速。化学发光分析法在无机物、有机痕量和超痕量分析领域内都得到了广泛应用。特别是近年来，化学发光分析法在多类复杂有机物质，如氨基酸、蛋白质、维生素、核酸、DNA、激素、生物碱及各类药物及毒物的检测，多种生物活性物质的分析，多种抗体和抗原的免疫分析，基因芯片、蛋白质芯片、受体芯片、酶芯片、微流控芯片研究中得到了广泛地应用，而且呈现出上升趋势。为生命科学、环境科学、材料科学的研究提供了许多新的、高灵敏度的、有效的分析手段，推动了这方面科学理论和高新技术的发展；同时，其他相关学科的研究成果也为化学发光分析法提供了许多新的技术和手段，出现了许多新的化学发光法，如纳米发光、发光成像、发光活体分析，大大促进了化学发光的发展及应用。目前，在化学发光分析研究中主要有两大方向：化学发光新体系的研究和新技术在经典化学发光体系中的应用研究。本论文的研究工作也就集中在两个方面。
本论文在全面总结和论述化学发光分析的原理、化学发光反应机理及分析应用、电化学发光分析法、化学发光免疫分析法、偶合化学发光分析法、化学发光分析法与液相色谱及毛细管电泳联用技术、化学发光传感器等方面国内外研究现状的基础上，提出并系统研究了几类新型的化学发光分析法，设计出了一系列新的高灵敏度、高选择性且稳定响应的流通式化学发光传感器，并把一
两南帅他人学博1：学位论义 化学发光分析新方江、新技术的叭穴
些新的技术引入化学发光分析中，从而把化学发光分析法应用范田扩大到药理
学研究中。其主要内容包括以下几个方面：
一、电生试剂化学发光分析法的研究
充分利用电化学和流动注射分析技术，提出了电生不稳定试剂化学发光分
析法的新思想。设计出一种新型的流通式电解池，并充分利用流动注射分析法
的特点，采用恒电流电解技术，分别在阳极氧化低价稳定的＊、Br、Co’＼Mn’＼
Ag＼Cu’”，从而在线定量地产生相应的、具有氧化性的不稳定试剂CIO。BrO、早
Co’＼Mn’＼Ag’＼Cu’－，并使这些不稳定试剂在线与试液中的待测物质发生反
应，产生化学发光。基于此原理，己成功地建立了测定异烟姘、哇宁、庆大霉．
素、毗派酸、卡托普利、金霉素、铂离于、亚硫酸根等化学发光新方法。
在这类化学发光新方法中，不稳定氧化剂是山电化学反应在线产生的，从
而消除了山其不稳定性所带来的不利影响，而且其浓度可用改变电解电流大小
来加以调控。我们还发现在设计的电解池和反应池中进行化学发光反应时，电
生试剂呈现出初生态的特点，具有很高的反应活性，能获得较强的化学发光强
度。这是用其它方法制备的相同试剂所不具有的；另外，山于电生试剂的成分
单一，试剂纯净，避兔了化学法制备试剂所引进杂质的干扰。另一方面，在这
类化学发光新方法中，我们设计出了一种新型的流通式电化学发光池，其中把
电解地与化学发光池彼此分离，设计在流路的不同位置。这样设计可以使电化
学反应和化学发光反应在不同的区域发生，能够确保它们均在各自最佳条件下
进行的，两者之间也不可能产生干扰；样品可以不进入电解池，不会对电极产
生污染；化学发光信号在发光池中检测，不存在电极对光学信号所带末的十扰。
这类化学发光新方法的提出，大大地改善了原有0 化学发光体系和 BrO
化学发光体系的分析性能，并使这些非常规氧化剂 CO’＼ Mfl’＼ Ag’＼ CLI’“首 凸
次应用于化学发光分析中。这对研究化学发光反应提供了一种新思路，并拓宽
了化学发光类型及应用范围。毛
二、新型流通式化学发光生物传感器的研究
本论文改变传统光学传感器静态晌应模式，把流动注射分析技术引入传感
器的设计中，克服以往定静态响应模式的缺点，建立动态响应模式，设计出流
通式化学发光传感器，并且在传感器分于识别系统作了研究，完成了一系列性
能优良流通式光学传感器。
一2一
的南帅他人学博卜学位论文 化学发光分析新方浊、新技术的研大
1、首次提出流通式化学发光组织传感器。组织传感器国内外都己经厂展了
研究工作。但是，目前在这类生物传感器中，换能器几乎全部是电流型的电化
学换能器。这类传感器中，一般通过物理的方法，把极其少量的生物材料固定
在电极上。山于生物材料的固定量极少，故其生物催化活性不会?
Chemiluminescence (CL) is one of the luminescent phenomena and it can be defined as the light emission (ultraviolet, visible or infra-red radiation) produced by a chemical reaction. Although the first chemiluminescent phenomenon was reported in 1887, development of this research was slow before the latter of the 20th century. Application of the Chemiluminescence method in analysis became possible with appearance of the highly sensitive photomultipliers (PMT) in the 1950s, and now Chemiluminescence became one of the most sensitive detection methods. Due to the simplicity, low detection limit, large calibration ranges and rapid analysis speed, Chemiluminescence has been widely used in practical applications during the last 50 years. The most visible fact in the rapid development is reflected in the great numbers of publications devoted entirely to the discussion of Chemiluminescence.
Current research in Chemiluminescence is focussed to two general directions. One direction is the discovery of new Chemiluminescence reactions and investigation of their applicability to real samples and the other direction is the development of Chemiluminescence detection systems for newly developed separation techniques.
In this thesis, the principle of Chemiluminescence, bioluminescence and electrochemiluminescence, and research works in recent years about Chemiluminescence coupled with capillary electrophoresis and liquid chromatography, Chemiluminescence sensor and application of Chemiluminescence in biochip were reviewed. The present research work was focussed on the new Chemiluminescence systems, novel flow-through Chemiluminescence biosensors. The major content was described as follows:
1. Chemiluminescence analysis with on-line electrogenerated reagents as the oxidant
Since Abbort et al reported the CL determination of morphine and its derivation by oxidation with potassium permanganate, there have been increasing interest in the
application of inorganic oxidate-based CL reactions to analytical chemistry. Inorganic oxidants, such as H202, Br2, KMnO4, Ce(IV), [Ru(bpy)3]2; [Fe(CN)6]3+, 10;, CIO', BrO" and BrO3", were frequently used in CL reaction; however, this method can not be employed if the reagents are not stable for at least the duration of the experiment. For example, the disproportionation of CIO' and BrO" is very easy in solution, so their applications in CL analysis are limited.
The application of strongly oxidizing reagents in quantitative analysis has in general been restricted to those reagents that are not or only very slowly reduced by water, thus making possible the storage of standard solutions. Stronger oxidants, such as cobalt(III), silver(II) and manganese(III), are quickly reduced by water, so their application in quantitative analysis under normal conditions is not possible.
The success of flow injection analysis (FIA) can be attributed to the reproducibility of the operations involved in the system, such as injection, pumping and timing, allowing quantitative analysis without the necessity of completeness of the chemical reaction. As a complete chemical reaction is unnecessary, the idea arises that instability of the reagent also might be no objection as long as the reaction rate is reproducible. This would open possibilities for the application in quantitative analysis of these reagents, which are unstable under normal condition, by generating them in flow systems, starting from a stable oxidation state of the same element. In flow system, the unstable solution generated by chemical oxidation contains some superfluous oxidant, which interfere with the subsequent reaction. The electrochemical generation does not have the drawback mentioned above.
In this thesis, the investigating aim of the research work was focus on the development of new oxidants in CL analysis to extend the analytical application fields or to improve the analytical performance of some CL analysis relating to unstable oxidic reagents by combining flow-injection technique. Some reagents needed for the CL reaction are ele
本论文在全面总结和论述化学发光分析的原理、化学发光反应机理及分析应用、电化学发光分析法、化学发光免疫分析法、偶合化学发光分析法、化学发光分析法与液相色谱及毛细管电泳联用技术、化学发光传感器等方面国内外研究现状的基础上，提出并系统研究了几类新型的化学发光分析法，设计出了一系列新的高灵敏度、高选择性且稳定响应的流通式化学发光传感器，并把一
两南帅他人学博1：学位论义 化学发光分析新方江、新技术的叭穴
些新的技术引入化学发光分析中，从而把化学发光分析法应用范田扩大到药理
学研究中。其主要内容包括以下几个方面：
一、电生试剂化学发光分析法的研究
充分利用电化学和流动注射分析技术，提出了电生不稳定试剂化学发光分
析法的新思想。设计出一种新型的流通式电解池，并充分利用流动注射分析法
的特点，采用恒电流电解技术，分别在阳极氧化低价稳定的＊、Br、Co’＼Mn’＼
Ag＼Cu’”，从而在线定量地产生相应的、具有氧化性的不稳定试剂CIO。BrO、早
Co’＼Mn’＼Ag’＼Cu’－，并使这些不稳定试剂在线与试液中的待测物质发生反
应，产生化学发光。基于此原理，己成功地建立了测定异烟姘、哇宁、庆大霉．
素、毗派酸、卡托普利、金霉素、铂离于、亚硫酸根等化学发光新方法。
在这类化学发光新方法中，不稳定氧化剂是山电化学反应在线产生的，从
而消除了山其不稳定性所带来的不利影响，而且其浓度可用改变电解电流大小
来加以调控。我们还发现在设计的电解池和反应池中进行化学发光反应时，电
生试剂呈现出初生态的特点，具有很高的反应活性，能获得较强的化学发光强
度。这是用其它方法制备的相同试剂所不具有的；另外，山于电生试剂的成分
单一，试剂纯净，避兔了化学法制备试剂所引进杂质的干扰。另一方面，在这
类化学发光新方法中，我们设计出了一种新型的流通式电化学发光池，其中把
电解地与化学发光池彼此分离，设计在流路的不同位置。这样设计可以使电化
学反应和化学发光反应在不同的区域发生，能够确保它们均在各自最佳条件下
进行的，两者之间也不可能产生干扰；样品可以不进入电解池，不会对电极产
生污染；化学发光信号在发光池中检测，不存在电极对光学信号所带末的十扰。
这类化学发光新方法的提出，大大地改善了原有0 化学发光体系和 BrO
化学发光体系的分析性能，并使这些非常规氧化剂 CO’＼ Mfl’＼ Ag’＼ CLI’“首 凸
次应用于化学发光分析中。这对研究化学发光反应提供了一种新思路，并拓宽
了化学发光类型及应用范围。毛
二、新型流通式化学发光生物传感器的研究
本论文改变传统光学传感器静态晌应模式，把流动注射分析技术引入传感
器的设计中，克服以往定静态响应模式的缺点，建立动态响应模式，设计出流
通式化学发光传感器，并且在传感器分于识别系统作了研究，完成了一系列性
能优良流通式光学传感器。
一2一
的南帅他人学博卜学位论文 化学发光分析新方浊、新技术的研大
1、首次提出流通式化学发光组织传感器。组织传感器国内外都己经厂展了
研究工作。但是，目前在这类生物传感器中，换能器几乎全部是电流型的电化
学换能器。这类传感器中，一般通过物理的方法，把极其少量的生物材料固定
在电极上。山于生物材料的固定量极少，故其生物催化活性不会?
Chemiluminescence (CL) is one of the luminescent phenomena and it can be defined as the light emission (ultraviolet, visible or infra-red radiation) produced by a chemical reaction. Although the first chemiluminescent phenomenon was reported in 1887, development of this research was slow before the latter of the 20th century. Application of the Chemiluminescence method in analysis became possible with appearance of the highly sensitive photomultipliers (PMT) in the 1950s, and now Chemiluminescence became one of the most sensitive detection methods. Due to the simplicity, low detection limit, large calibration ranges and rapid analysis speed, Chemiluminescence has been widely used in practical applications during the last 50 years. The most visible fact in the rapid development is reflected in the great numbers of publications devoted entirely to the discussion of Chemiluminescence.
Current research in Chemiluminescence is focussed to two general directions. One direction is the discovery of new Chemiluminescence reactions and investigation of their applicability to real samples and the other direction is the development of Chemiluminescence detection systems for newly developed separation techniques.
In this thesis, the principle of Chemiluminescence, bioluminescence and electrochemiluminescence, and research works in recent years about Chemiluminescence coupled with capillary electrophoresis and liquid chromatography, Chemiluminescence sensor and application of Chemiluminescence in biochip were reviewed. The present research work was focussed on the new Chemiluminescence systems, novel flow-through Chemiluminescence biosensors. The major content was described as follows:
1. Chemiluminescence analysis with on-line electrogenerated reagents as the oxidant
Since Abbort et al reported the CL determination of morphine and its derivation by oxidation with potassium permanganate, there have been increasing interest in the
application of inorganic oxidate-based CL reactions to analytical chemistry. Inorganic oxidants, such as H202, Br2, KMnO4, Ce(IV), [Ru(bpy)3]2; [Fe(CN)6]3+, 10;, CIO', BrO" and BrO3", were frequently used in CL reaction; however, this method can not be employed if the reagents are not stable for at least the duration of the experiment. For example, the disproportionation of CIO' and BrO" is very easy in solution, so their applications in CL analysis are limited.
The application of strongly oxidizing reagents in quantitative analysis has in general been restricted to those reagents that are not or only very slowly reduced by water, thus making possible the storage of standard solutions. Stronger oxidants, such as cobalt(III), silver(II) and manganese(III), are quickly reduced by water, so their application in quantitative analysis under normal conditions is not possible.
The success of flow injection analysis (FIA) can be attributed to the reproducibility of the operations involved in the system, such as injection, pumping and timing, allowing quantitative analysis without the necessity of completeness of the chemical reaction. As a complete chemical reaction is unnecessary, the idea arises that instability of the reagent also might be no objection as long as the reaction rate is reproducible. This would open possibilities for the application in quantitative analysis of these reagents, which are unstable under normal condition, by generating them in flow systems, starting from a stable oxidation state of the same element. In flow system, the unstable solution generated by chemical oxidation contains some superfluous oxidant, which interfere with the subsequent reaction. The electrochemical generation does not have the drawback mentioned above.
In this thesis, the investigating aim of the research work was focus on the development of new oxidants in CL analysis to extend the analytical application fields or to improve the analytical performance of some CL analysis relating to unstable oxidic reagents by combining flow-injection technique. Some reagents needed for the CL reaction are ele
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