【作者】 赵晓寅；

【导师】 周天舒；

【作者基本信息】 华东师范大学 ， 环境科学， 2011， 硕士

【摘要】 毛细管电泳(Capillary Electrophoresis, CE)是以高压电场为驱动力,以毛细管为分离通道,依据样品中各组分之间淌度或/和分配系数的差异而实现高效、快速分离的一类新型液相分离技术。CE以其高效、快速、经济、环保、样品用量少等优点,逐渐成为无机分子、有机分子、生物大分子和对映体分离检测、生物医学、食品科学、分析化学、环境科学等领域中中最具活力、最有发展前景的分离分析科学之一。激光诱导荧光(Laser-Induced Fluorescence, LIF)检测,其突出的优点就是灵敏度高,可高达10-9～10-12mol/L。CE与LIF联用,结合了两者的优点,快速、高效、灵敏,非常适合生物样品及环境中痕量物质的分析。随着经济和人们生活水平的发展,人类赖以生存的自然环境变得越来越糟,环境问题日益突出,一些化学品的使用给环境甚至人类的健康带来了深远的危害。溴代阻燃剂(BFRs)是一种用于某些高分子聚合物材料、树脂的生产过程中以阻燃、抗燃、减少火灾可能性的物质。由于近年来BFRs,特别是四溴双酚-A(TBBPA),在人们生产生活中的广泛使用,加之其具有难降解性和高脂溶性,能够通过食物链在生物体内累积,使处于高营养级的生物受到毒害,最终导致对人体健康的危害。近年来,溴代阻燃剂的环境安全问题逐渐引起了人们的重视,但从分子角度对其生物毒性方面的研究还较少。农药残留也是一个突出的环境问题,对水、土壤、大气等环境介质造成污染,其引发的食品安全问题也令人担忧。根据上述研究现状,本论文以研究环境污染及其影响为出发点,建立了快速、准确、灵敏的检测谷胱甘肽、甲状腺激素及氨基甲酸酯类农药的毛细管电泳激光诱导荧光检测法。同时建立SD大鼠的TBBPA受损模型,初步探讨TBBPA对生物体的毒性效应。全文共分以下四个部分：1绪论(第一章)该部分首先概述了毛细管电泳的发展、原理及其应用现状；其次分别概述了溴代阻燃剂和农药残留的污染现状及其研究现状；最后详细综述了毛细管电泳在环境污染物分析中的应用。2毛细管电泳激光诱导荧光检测对还原型和氧化型谷胱甘肽分析的方法研究及其在TBBPA暴露下对大鼠体内谷胱甘肽含量影响的应用(第二章)谷胱甘肽以原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)两种形式广泛存在于生物体内。谷胱甘肽在体内起着重要的作用,比如维持血红蛋白中铁离子的状态、阻止血液中含巯基蛋白质的氧化等。GSH/GSSG则标志着体内氧化应激的程度。我们通过检测大鼠脑中和血清中GSH和GSSG的含量变化来研究TBBPA的生物毒性。本文以FITC为荧光衍生试剂,建立了用于同时分离检测还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)的毛细管电泳激光诱导荧光检测法。对衍生缓冲液和运行缓冲液的pH和浓度、进样时间、分离电压等影响测定的因素进行了优化,获得最佳测定条件为：两种化合物在pH 9.9,50 mmol/L的硼砂缓冲体系中衍生,并在pH 9.7,50mmol/L的硼砂缓冲液中进行分离检测。GSH和GSSG在3-1000 nmol/L范围内,荧光强度与浓度呈良好的线性关系,检出限分别为3.0×10-10 mol/L和1.0×10-9mol/L(S/N=3)。并将该方法用于四溴双酚-A(TBBPA)暴露后大鼠体内GSH、GSSG含量的变化,探讨其可能的生态毒理效应。3毛细管电泳激光诱导荧光法对甲状腺激素测定方法的研究及其在TBBPA暴露下大鼠体内甲状腺激素含量影响的应用(第三章)甲状腺是人体内最大的内分泌腺体,其分泌的甲状腺激素(T4)和三碘甲腺原氨酸(T3)在多种生理过程中起着极其重要的调节作用,比如个体的生长和发育、碳水化合物的新陈代谢、氧气的消耗过程、蛋白质的合成和胎儿早期的神经发育等。TBBPA与甲状腺激素具有相似的结构,作用于机体后将影响甲状腺素的正常分泌,引起甲状腺干扰效应。因此我们通过检测大鼠体内T4和T3的含量变化探讨TBBPA的甲状腺激素干扰效应,为甲状腺激素干扰物筛选方法的建立奠定基础。本文以异硫氰酸荧光素(FITC)为衍生试剂,建立了用于同时分离检测甲状腺激素(T4)和三碘甲腺原氨酸(T3)的高效、灵敏的毛细管电泳激光诱导荧光检测方法(CE-LIF).我们分别对衍生化条件如衍生缓冲液的类型、pH和浓度,以及衍生剂浓度进行了优化；同时也对影响毛细管电泳的分离条件包括运行液缓冲液、分离电压、进样时间等因素进行逐一优化以获得良好的分离效率和灵敏度。在最佳条件下,两种化合物在pH 9.2,25mmol/L的磷酸盐缓冲体系中经FITC衍生后用CE-LIF在含15%乙腈(v/v)的硼砂缓冲体系(pH 8.9,40 mmol/L)中于10 min内得到分离。在0.003-1μmol/L范围内,T4和T3的荧光强度与浓度呈良好的线性关系,检出限分别为3.0×10-9mol/L和1.0×10-9mol/L(S/N=3)。并将该方法用于四溴双酚-A (TBBPA)暴露后大鼠体内T4和T3含量的变化,探讨其可能的生态毒理效应。4毛细管电泳激光诱导荧光检测法在氨基甲酸酯类农药的理化性质研究中的应用(第四章)氨基甲酸酯类农药是农业生产中较为常用的一类农药,具有杀虫效率高、选择性强、分解快、残留期短等特点。但随着其广泛应用于农业生产,造成对环境的影响,特别是水体,直接危及到人体的健康。这类农药作用于生物体时会抑制其体内乙酰胆碱酯酶的活性,急性中毒时会出现肌肉颤动、瞳孔缩小等胆碱酯酶抑制症,危害生物体的神经系统。因此对环境中及食品中一些微量农药残留的灵敏检测及对其理化性质的研究具有非常重要的现实意义。本文通过毛细管电泳激光诱导荧光检测法对灭除威(XMC)、混杀威(2,3,5-trimethacarb)、灭虫威(mercaptodimethur)等三种氨基甲酸酯类农药的水解产物进行检测,从而测定它们的水解常数及活化能。该法基于氨基甲酸酯类农药水解产生的甲胺与荧光衍生试剂4-氯-7-硝基-2,1,3-苯并二唑(NBD-C1)进行衍生化反应,再用毛细管电泳激光诱导荧光检测法测定。文中对水解和衍生反应的时间及温度、衍生缓冲液和运行液缓冲液的pH值及浓度、分离电压、进样时间等因素进行了逐一优化以期达到检测的最佳条件。在20mmol/L pH 9.8的硼砂缓冲液中,三种农药分别发生水解反应并与NBD-C1进行衍生化反应,随后在50 mmol/LpH 8.5的硼砂运行液中经毛细管电泳激光诱导荧光法得到分离检测。三种农药的检出限分别可达3.0×10-9、1.0×10-9和3.0×10-9mol/L(S/N=3),相对标准偏差(RSD)在2.22%-3.37%(n=7)范围内。更多还原

【Abstract】 Capillary electrophoresis (CE) is an important and powerful technique in the area of liquid-phase separations. The separation principle is based on the phenomena that ionized molecules of different mass or/and charge will travel with different velocities through a fused silica capillary under the force of a high-voltage electric field. Due to the different velocities, the analytes can be separated and then detected by the detector. Thanks to its high separation efficiency, short analysis time, low cost, environmental friendliness and small sample consumption, CE has been widely flourishing in many areas such as food analysis, pharmaceutical analysis, biochemical analysis, and environmental analysis etc. The prominent advantage of laser-induced fluorescence is its high sensitivity. CE-LIF combins their merits so that fits to the trace analysis of biological samples and environmental samples.Our living environment became worse and worse with the development of economy. Brominated flame retardants (BFRs) are used in a number of industries in the manufacture of polymers and synthetic resins to reduce the risk of fires. Recently, for BFRs especially TBBPA, great concern has been aroused because of their wide spread, stable structures and uncertain risk to human health. But research on neurotoxicity of BFRs is still less. Pesticide residue is also an important environment problem which caused contamination to water, soil, air and so on. Food security derived from pesticide residue is worrying.In this dissertation, we laid our emphasis on the research of environment pollution and its effects and established methods to determine glutathione (GSH and GSSG) and thyroid hormone (T4 and T3) after rats’exposure to TBBPA, and carbamate pesticides by capillary electrophoresis (CE) method coupled with laser-induced fluorescence (LIF) detection. The dissertation includes four chapters:Chapter 1. IntroductionIn this chapter, development, the principle and applications of capillary electrophoresis were outlined. The characteristics of BFRs and pesticide residue were introduced, and the applications of capillary electrophoresis in environmental analysis were highlighted, as well as the meanings of this paper.Chapter 2. Determination of GSH and GSSG by Capillary Electrophoresis with Laser-Induced Fluorescence Detection and its application on the Rats after Exposure to TBBPAGSH and GSSG play a great role in some biological process, and GSH/GSSG is a biomarker of oxidative stress. We study the neurotoxicity of TBBPA through determination GSH and GSSG in rats’brain and serum. A capillary electrophoresis (CE) method coupled with laser-induced fluorescence (LIF) detection was developed for the separation and detection of GSH and GSSG after derivatization by fluorescein isothiocyanate (FITC). The separation and detection conditions were optimized in terms of buffer concentrations, buffer pH, separation voltage and injection time. Under the optimum experiment conditions, GSH and GSSG were derivatized with FITC in 50 mmol/L pH 9.9 of borate buffer and separated in 50 mmol/L pH 9.7 of borate buffer. Linear relationships between the peak area and concentrations of derivatized analytes were obtained (3-1000 nmol/L). The detection limits for GSH and GSSG were 1.0×10-9mol/L and 3.0×10-9mol/L(S/N=3), respectively. The method was applied to analysis of GSH and GSSG in hippocampus of rats after exposure of TBBPA.Chapter 3. Determination of T4 and T3 by Capillary Electrophoresis with Laser-Induced Fluorescence Detection and its application on the Rats after Exposure to TBBPAThe amino acid type thyroid hormones, thyroxine (T4) and triiodothyronine (T3), secreted by the pituitary gland are compounds of critical importance in regulating a number of biological processes. In this paper, a method based on capillary electrophoresis with laser-induced fluorescence has been developed for the simultaneous separation and determination of T4 and T3 after derivatization with FITC. The derivatization conditions were optimized in terms of buffer systems, ratios of derivatization, buffer concentrations and buffer pH. And the effects of several important factors about separation were also investigated, such as buffer concentrations, buffer pH, separation voltages and injection time. Under the optimum conditions, T4 and T3 were derivatized in phosphate buffer (pH 9.2,25 mmol/L), and then separated within 10 min in pH 8.9,40 mmol/L borate buffer containing 15% acetonitrile (v/v). The detection limits were as low as 3.0×10-9 mol/L and 1.0×10-9 mol/L (S/N=3) for T4 and T3, respectively. The method has been applied to analyze T4 and T3 in rats’serum and pituitary to study the toxicity of BFRs.Chapter 4. Dtermination of the rate constants and activation energy of carbamate pesticides hydrolysis by capillary electrophoresis with laser-induced fluorescence detectionCarbamates are an important pollutants found in various environmental matrices. A capillary electrophoresis (CE) with laser-induced fluorescence (LIF) method was used for determination of hydrolysates of (3,5-dimethylphenyl)N-methylcarbamate(XMC), 2,3,5-trimethacarb and mercaptodimethur. The method was based on simultaneous hydrolysis and derivatization reaction between the produced methylamine and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl). Effects of several key factors, such as time and temperature for hydrolysis and derivatization, pH values and concentrations of both running buffer and derivatization buffer, voltage applied and injection time were all investigated to obtain the optimum conditions for determination. The three analytes were respectively mixed with NBD-Cl in 20 mmol/L pH 9.8 borate buffer and the derivatives got good peaks in the running buffer of 50 mmol/L pH 8.5 borate buffer. The detection limits (LOD) obtained by the method were 3.0×10-9,1.0×10-9 and 3×10-9 mol/L, respectively. Relative standard deviations for the studied compounds varied from 2.22% to 3.37%(n=7).更多还原