以钙黄绿素为化学发光试剂的化学发光反应及其分析应用研究

【作者】 聂菲；

【导师】 吕九如；

【作者基本信息】 陕西师范大学 ， 分析化学， 2008， 博士

【摘要】 化学发光分析法以其灵敏度高、线性范围宽、分析速度快以及仪器设备相对简单便宜等诸多优点,在痕量分析领域显示出广泛的应用前景。化学发光试剂是化学发光分析的基础,应用性能稳定、发光产率高、应用范围广的化学发光试剂,对于提高化学发光分析的灵敏度、拓展化学发光分析的应用领域有着重要的意义。我们研究发现,在一定的条件,一些荧光试剂也可用作化学发光试剂,它们可以直接吸收化学反应的能量产生化学发光。通过测定发光信号可以对相关物质进行定量分析。本论文分为两部分,第一部分为综述,第二部分为研究报告。第一部分综述了近三年来化学发光分析的研究现状,主要包括化学发光分析的新技术,如化学发光分析与各种技术的联用、化学发光成像技术、纳米技术、化学发光试验装置的集成化、化学发光免疫分析等,以及各个化学发光体系的研究进展的。第二部分为具体的研究工作,系统研究了荧光试剂钙黄绿素的化学发光性质,构建了铁氰化钾-钙黄绿素、高锰酸钾-钙黄绿素、铈（Ⅳ）-钙黄绿素、N-溴代丁二酰亚胺（NBS）-钙黄绿素和N-氯代丁二酰亚胺（NCS）-钙黄绿素化学发光体系,考察了金属离子和有机物在这些体系中的化学发光行为,探索了此类化学发光反应的机理,建立了一系列物质灵敏的化学发光分析方法。研究报告由七部分组成:1.以钙黄绿素为化学发光试剂的化学发光新体系观察到铁氰化钾与钙黄绿素可以产生化学发光,一些金属离子和有机物可以增敏这一化学发光信号。基于对一些相关反应的化学发光动力学性质、化学发光光谱,以及一些物质的紫外吸收光谱和荧光光谱的研究,提出了铁氰化钾-钙黄绿素化学发光反应可能的机理以及金属离子和有机物对该体系化学发光增敏的机理。在一定的条件下,得出了一些发光活性物质的分析参数。基本构建了一个以钙黄绿素作为化学发光试剂的化学发光新体系。2.其它钙黄绿素化学发光体系的研究系统考察了多种物质（包括金属离子和一些药物）在高锰酸钾-钙黄绿素、NCS-钙黄绿素和NBS-钙黄绿素化学发光体系中的化学发光行为,发现许多物质在这些体系中都具有化学发光活性。对一些物质的分析条件进行了优化,建立了这些物质化学发光分析方法。3.铁氰化钾-钙黄绿素化学发光体系测定酮替芬的研究发现钙黄绿素可以吸收铁氰化钾氧化酮替芬反应的化学能而产生化学发光。以钙黄绿素为化学发光试剂,构建了铁氰化钾-酮替芬-钙黄绿素化学发光体系。利用此体系建立了测定酮替芬的化学发光分析新方法,方法的线性范围为2.0×10^-8～6.0×10^-6g·mL^-1,检出限为8×10^-9g·mL^-1,对浓度为5.0×10^-7g·mL^-1酮替芬溶液进行11次平行测定的RSD为2.1%。此法已用于药品中酮替芬含量的测定,结果与药典测定结果一致。对化学发光反应的机理也进行了初步的探讨。4.以钙黄绿素为化学发光试剂测定酮替芬的研究发现铁氰化钾可以与钙黄绿素和酮替芬的混合物产生化学发光,有趣的是痕量的Mg²⁺可以增敏这一化学发光信号,增强的化学发光信号与酮替芬的浓度相关。基于这一现象,建立了测定酮替芬的流动注射化学发光分析法,方法的线性范围为6.0×10^-9～2.0×10^-7g·mL^-1,检出限为3×10^-9g·mL^-1,对浓度为2.0×10^-8g·mL^-1酮替芬溶液进行11次平行测定的RSD为1.8%。此法已用于药品中酮替芬含量的测定。在对化学发光反应的动力学性质、化学发光光谱、紫外可见吸收光谱以及荧光光谱研究的基础上,提出了反应可能的机理。5.高锰酸钾-钙黄绿素化学发光体系测定芬氟拉明的研究发现芬氟拉明注入高锰酸钾与钙黄绿素反应后的溶液中,可以观察到后化学发光现象。基于此,建立了测定芬氟拉明的流动注射化学发光分析法。探讨了反应可能的机理。化学发光强度与芬氟拉明的浓度在1.0×10^-7～6.0×10^-6 g·mL^-1的范围内呈线性关系,方法的检出限为6×10^-8 g·mL^-1。对浓度为5.0×10^-7g·mL^-1芬氟拉明溶液进行11次平行测定的RSD为2.2%。此法已用于减肥药中芬氟拉明含量的测定。6.铈（Ⅳ）-钙黄绿素化学发光体系测定痕量铝发现铝（Ⅲ）可以极大地增敏铈（Ⅳ）-钙黄绿素产生的微弱的化学发光信号,基于此,建立了简单、灵敏和选择性的直接测定铝的化学发光分析法。方法的线性范围是2.0×10^-10～4.0×10^-8g·mL^-1,检出限为8×10^-11g·mL^-1（3σ）。对浓度为1.0×10^-9g·mL^-1的铝离子进行11次测定的相对标准偏差为2.5%。该方法已被用于水样中铝离子的测定,结果令人满意。对该化学反应的机理也进行了探讨。7.以钙黄绿素为化学发光试剂测定血浆中的硫利达嗪以钙黄绿素为化学发光试剂,构建了高锰酸钾-钙黄绿素化学发光体系,发现硫利达嗪在此体系中可以产生化学发光,甲醛可以增敏这一化学发光信号。利用此现象,建立了测定硫利达嗪的化学发光分析新方法。方法的线性范围为2.0×10^-8～6.0×10^-6g·mL^-1,检出限为8×10^-9g·mL^-1,对浓度为1.0×10^-7g·mL^-1硫利达嗪溶液进行11次平行测定的RSD为2.1%。此法已用于药品及血浆中硫利达嗪含量的测定,标准方法对照试验及加标回收试验表明,结果令人满意。对化学发光反应的机理也进行了初步的探讨。更多还原

【Abstract】 It is well known that chemiluminescence（CL） method is an important detection method,with the advantages of low detection limit,wide linear dynamic range and inexpensive instruments.The chemiluminescent reagent is the basis of CL analysis.A chemiluminescent reagent with steady application performance,high CL ratio and wide application range is very important to improve the analytical sensitivity and widen the application range of the CL analysis.To our preliminary investments,some fluorescent reagents could also be used as CL reagent. They could absorb the energy of certain reaction producing CL and related substances can be determined.This thesis includes a review and a research section.The review summarized the status of the CL study,includes the application of CL,such as the combination of CL with other techniques,CL imaging technique,nanometer technique,the integration of the experiment apparatus and the CL immunoassay,and the development of each CL systems.In the research section,the CL characteristics of calcein,a fluorescent reagent,was studied. The CL system of potassium ferricyanide-calcein,potassium permanganate-calcein, cerium（Ⅳ）-calcein,N-bromosuccinimide（NBS）-calcein and N-chlorosuccinimide（NCS）-calcein was developed.The CL behaviors of some metal ions and organic substances were studied and the mechanisms of these CL reactions were discussed.The CL methods of some substances were established.The research section includes seven parts.1.Novel chemiluminescence system with calcein as chemiluminescent reagentChemiluminescence was observed when calcein reacted with potassium ferricyanide.Several metal ions and organic compounds could enhance this CL signal.The possible mechanisms for the CL reaction and the enhance effect were discussed via the studies of the CL kinetic characteristics, the CL spectra of some related reactions,the UV absorption spectra and the fluorescence spectra of some substances.The merits of figures for some substances with CL activity were obtained.A new CL system with calcein as CL reagent was established preliminarily.2.Study on other calcein chemiluminescence systemsThe CL behaviors of some substances includes metal ions and some pharmaceutical preparations were investgated in potassium permanganate-calcein CL system,NCS-calcein CL system and NBS-calcein CL system.A lot of substances were found had CL activities.The CL methods of some substamces were established under the optimized conditions.3.Determination ofketotifen with potassium ferricyanide-calcein chemiluminescence systemIt was found that calcein could absorb the chemical energy of the oxidizing reaction between potassium ferricyanide and ketotifen accompanied with CL emission.For the first time,CL system with calcein as CL reagent was established.Employing this CL system,a CL method to determine ketotifen was founded.The linear range of the method was 2.0×10^-8～6.0×10^-6 g·mL^-1 with a detection limit of 8×10^-9 g·mL^-1.The relative standard deviation was 2.1%for 5.0×10^-7 g·mL^-1 ketotifen（n=11）.Furthermore,this method was applied to the determination of ketotifen in the tablets successfully.The mechanism of the CL reaction was discussed simply.4.Determination of ketotifen by using calcein as chemiluminescence reagentChemiluminescence was observed when potassium ferricyanide reacted with the mixture of calcein and ketotifen.Interestedly,the CL intensity would be enhanced by trace amounts of Mg²⁺ and the CL intensity was strongly dependent on ketotifen concentration.Based on this phenomenon, a flow injection CL method was established for the determination of ketotifen.The possible CL mechanism is proposed based on the kinetic characteristic of the CL reaction,CL spectrum, ultraviolet（UV） spectra and fluorescent spectra.The CL intensity was correlated linearly with concentration of ketotifen over the range of 6.0×10^-9 to 2.0×10^-7 g·mL^-1 and the detection limit was 3×10^-9 g·mL^-1.The relative standard deviation was 1.8%for 2.0×10^-8 g·mL^-1 ketotifen（n=11）. This method was applied to the determination of ketotifen in the tablets successfully.5.Determination of fenfluramine based on potassium permanganate-calcein chemiluminescence systemPost-chemiluminescence was observed when fenfluramine was injected into the mixture after the CL reaction of calcein-potassium permanganate.Based on this phenomenon,a flow injection CL method was established for the determination of fenfluramme.The possible CL mechanism was proposed.The CL intensity was correlated linearly with the concentration of fenflummine over the range of 1.0×10^-7 to 6.0×10^-6 g·mL^-1 and the detection limit was 6×10^-8 g·mL^-1.The relative standard deviation was 2.2%for 5.0×10^-7 g·mL^-1 fenfluramine（n=11）.This method was applied to the determination of fenfluramine in weight-reducing tonic successfully.6.Determination of trace aluminum（Ⅲ） using a novel cerium（Ⅳ）-calcein chemiluminescence detectionA simple,sensitive and selective CL method was developed for the direct determination of aluminum（Al）.This method is based on that the weak CL of cerium（Ⅳ）-calcein can be greatly enhanced by Al（Ⅲ）.The calibration curve was linear over the range 2.0×10^-10～4.0×10^-8 g·mL^-1 with a detection limit of 8×10^-11 g·mL^-1（3σ）.The RSD was 2.5%by 11 replicated determinations of 1.0×10^-9 g·mL^-1 Al（Ⅲ）.The proposed method has been used to determine the concentration of Al （Ⅲ） in real water samples with satisfactory results.The mechanism of the CL reaction was also discussed.7.Determination ofthioridazine with potassium permanganate-calcein chemiluminescence systemPotassium permanganate-calcein CL system was established with calcein as CL reagent. Thioridazine was found to be able to produce CL in this system and formaldehyde could enhance this CL signal.Based on this,a CL method to determine thioridazine was established.The linear range of the method was 2.0×10^-8～6.0×10^-6 g·mL^-1 with a detection limit of 8×10^-9 g·mL^-1.The relative standard deviation was 2.1%for 1.0×10^-7 g·mL^-1 thioridazine（n=11）.Furthermore,this method was applied to the determination of thioridazine in the tablets and plasma.The results were satisfactory after the control experiment with the standard method and the recovery test.The mechanism of the CL reaction was discussed simply.更多还原