荧光探针法检测药物中氨基酸的含量及应用

发布时间：2018-05-08 11:42

本文选题：荧光探针 + 氨基酸　；参考：《河南师范大学》2017年硕士论文

【摘要】：氨基酸是一组在各种生物功能中发挥重要作用的有机分子。它们不仅是构成蛋白质的结构单元,而且还被认为是调节重要生理活动的神经递质,与很多生命活动息息相关。氨基酸的定性和定量分析在医学医药、临床诊断、食品科学等科研中具有重要的意义。目前,氨基酸的检测方法也不局限于比较老的化学法包括甲醛滴定法和凯氏定氮法、高效液相色谱法、离子交换色谱法、电化学方法、光化学分析法以及氨基酸分析仪等方法,也有许多新颖的方法被提出,例如:液相色谱与光学偶联检测、离子对液相色谱-串联质谱光谱法、亲水作用色谱-串联质谱等众多方法。但是上述方法大部分存在分析过程繁琐、分析过程和时间较长、分析耗费比较高的缺点。与之相对比,荧光探针法具有一系列的优点例如:便于操作、较高的灵敏度、具有安全性和很强的复用能力等优点。所以,研究建立一种低检测限、高灵敏度的快速荧光探针法对氨基酸的测定是很有必要的。本论文主要分为以下三个方面:1.介绍了氨基酸的简介、作用,简单叙述了近年来氨基酸分析的常用方法,概述了荧光分析法的基本原理。2.研究了几种对氨基酸定量检测的荧光探针法:(1)在pH=8.8的CL缓冲溶液和表面活性剂SDS中,Cu~(2+)能和桑色素发生反应形成一个络合物,从而使桑色素的荧光猝灭,再加入一定量的赖氨酸后,Cu~(2+)能和赖氨酸形成一个更为稳定的络合物,从而解离出来桑色素时体系的ΔF增加且增加量与赖氨酸的浓度在一定范围内具有线性关系。因此,我们建立了一种荧光探针法定量检测赖氨酸的方法。在激发狭缝宽度为为5nm,发射狭缝宽度为为5nm时,线性回归方程为:F=98.75+49.93C(mg/L),相关系数R为0.9997,线性范围为1.0~5.0mg/L,检出限为0.0057mg/L。(2)在pH=10.8的Sφrensen缓冲溶液和表面活性剂OP水溶液的存在下,Ni~(2+)能和钙黄绿素形成一个络合物从而使钙黄绿素的荧光猝灭,当再加入一定量的天冬氨酸后,天冬氨酸能使猝灭后的Ni~(2+)-钙黄绿素的荧光增加,而且增加量与天冬氨酸的浓度在一定范围内成正比。所以,我们建立了一种检测天冬氨酸的荧光探针法。在激发狭缝的宽度为3nm,发射狭缝的宽度为5nm的条件下,线性回归方程为:F=88.47+8.497C(mg/L),相关系数为R=0.9981,线性范围为1~40mg/L,检出限为0.0050mg/L。(3)在pH为9.6的Clark-Lubs缓冲溶液和表面活性剂SDS中,激发狭缝宽度为3nm,发射狭缝宽度为5nm时,不同浓度的半胱氨酸能使猝灭的Pb~(2+)-水杨基荧光酮体系的荧光不断变大且在一定范围内呈现良好的线性,线性回归方程为:F=52.03+173.2C(mg/L),相关系数为0.9986,线性范围为0.5~5.0mg/L,检出限为0.00086mg/L。3.我们通过研究体系的荧光强度和氨基酸、金属离子的含量之间的关系,探究了荧光探针与金属离子、氨基酸之间发生作用的反应机理和络合比,并进行了初步的探讨。
[Abstract]:Amino acids are a group of organic molecules that play an important role in various biological functions. They are not only the structural units of proteins, but also considered to be neurotransmitters regulating important physiological activities, which are closely related to many life activities. The qualitative and quantitative analysis of amino acids is of great significance in medical medicine, clinical diagnosis, food science and other scientific research. At present, the determination of amino acids is not limited to the older chemical methods, including formaldehyde titration and Kjeldahl nitrogen determination, high performance liquid chromatography, ion exchange chromatography, electrochemical methods, photochemical analysis and amino acid analyser, etc. Many novel methods have been proposed, such as liquid chromatography coupled with optical detection, ion-pair liquid chromatography-tandem mass spectrometry, hydrophilic interaction chromatography-tandem mass spectrometry and so on. However, most of the above methods have the disadvantages of complicated analysis process, long analysis process and time, and high analysis cost. By contrast, the fluorescence probe method has a series of advantages, such as easy to operate, high sensitivity, safety and strong reuse ability. Therefore, it is necessary to establish a rapid fluorescence probe method with low detection limit and high sensitivity for the determination of amino acids. This paper is divided into the following three aspects: 1. This paper introduces the brief introduction and function of amino acids, briefly describes the common methods of amino acid analysis in recent years, and summarizes the basic principle of fluorescence analysis. The fluorescence probe method for quantitative determination of amino acids: 1) in the CL buffer solution of pH=8.8 and surfactant SDS) can react with Morin to form a complex, which can quench the fluorescence of Morin. After adding a certain amount of lysine, the CuCuP2) can form a more stable complex with lysine, thus the 螖 F of the system increases when the mulberry pigment is dissociated, and the increase has a linear relationship with the concentration of lysine in a certain range. Therefore, we established a fluorescence probe method for quantitative determination of lysine. When the excitation slit width is 5 nm and the emission slit width is 5nm, The linear regression equation is: FG 98.7549.93Cg / L, the correlation coefficient R is 0.9997, the linear range is 1.0 ~ 5.0mg / L, the detection limit is 0.0057mg / L ~ (2) in the presence of S 蠁 rensen buffer solution of pH=10.8 and surfactant op aqueous solution, NiNi2) can form a complex with calcium xanthophyllin, which can quench the fluorescence of calcitonin. After adding aspartic acid, the fluorescence of quenched Ni~(2 can be increased, and the increase is proportional to the concentration of aspartic acid in a certain range. Therefore, we developed a fluorescence probe method for the detection of aspartic acid. Under the conditions that the width of the excited slit is 3 nm and the width of the emission slit is 5nm, the linear regression equation is: F88.47 8.497Cng / L, the correlation coefficient is 0.9981, the linear range is 1 ~ (40) mg / L, the detection limit is 0.0050 mg / L ~ (-3) in Clark-Lubs buffer solution and surfactant SDS with pH 9.6, the linear range is 0.9981, and the detection limit is 0.0050 mg / L ~ (-1). When the excitation slit width is 3 nm and the emission slit width is 5nm, different concentrations of cysteine can increase the fluorescence of quenched Pb~(2 / salicylidene fluorone system and show good linearity in a certain range. The linear regression equation is 52.03 173.2Cu mg / L, the correlation coefficient is 0.9986, the linear range is 0.5 ~ 5.0 mg / L, the detection limit is 0.00086 mg / L ~ (-3). By studying the relationship between fluorescence intensity, amino acid content and metal ion content, the reaction mechanism and complexation ratio between fluorescence probe and metal ion and amino acid were studied.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3;TQ460.72

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