多环芳烃暴露标志物1羟基芘检测新方法及应用研究.pdf

南华大学硕士学位论文 多环芳烃暴露标志物1-羟基芘检测新方法及应用研究 IV多环芳烃暴露标志物 1-羟基芘 检测新方法及应用研究 研究生：欧阳运富 导 师：王永生 教授 摘 要 多环芳烃(Polycyclic Aromatic Hydrocarbons，PAHs)是一类普遍存在的持久性有机污染物，在职业高PAHs暴露环境下，容易诱发肺癌、皮肤癌等癌症，已被各国列为优先控制的污染物对PAHs的暴露评价可为流行病学研究和污染物风险评价等提供依据 由于暴露途径的复杂化， 采用尿样中1-羟基芘作为标志物来综合评价人体对PAHs的内暴露情况已经成为研究的热点因而研究1-羟基芘检测的新方法及1-羟基芘与染料或蛋白质的作用机制具有重要的现实意义 本文第二章应用共振光散射技术建立了 1-羟基芘检测的新方法研究表明：在 pH8.0 的 Tris-HCl 缓冲溶液中，乙基紫与 1-羟基芘反应形成离子缔合物，导致体系的共振光散射增强，最大共振光散射峰位于 396nm加入阴离子表面活性剂十二烷基苯磺酸钠可起增稳增敏作用在最佳实验条件下，共振光散射的增强(∆IRLS)在 4.0~982.0µg/L 范围内与 1-羟基芘浓度成线性关系，r=0.9994，方法的检出限为 1.2µg/ L，RSD=4.5%~5.3%，加标回收率为 93.3%~102.3%。

方法简便、灵敏，已应用于实际样品的测定，结果与常规的高效液相色谱法一致 本文第三章研究了 1-羟基芘对吖啶橙- 罗丹明 6G 混合体系的荧光猝灭效应和作用机制，建立了共振能量转移荧光猝灭法检测 1-羟基芘的新方法实验表明，在 λex/λem= 470/556nm，十二烷基苯磺酸钠存在下，吖啶橙- 罗丹明 6G 能够发生有效的能量转移，使罗丹明 6G 的荧光大大增强；1-羟基芘的加入使罗丹明 6G 的荧光猝灭其猝灭机制为静态荧光猝灭过程在 21.3—982.0µg/L 范围内，1-羟基芘浓度与罗丹明 6G 的荧光猝灭强度成线性关系，方法的检出限为 6.4µg/L，相对标准偏差为 0.98%~2.03%，加标回收率为 96.0%~104.4%该方法简便、快速，应南华大学硕士学位论文 多环芳烃暴露标志物1-羟基芘检测新方法及应用研究 V用于人尿中痕量 1-羟基芘的测定，结果满意 本文第四章应用荧光光谱法研究了 1-羟基芘与牛血清白蛋白分子之间的作用机制及能量转移关系，求得它们之间的结合常数 K=3.15×104L/mol，结合位点数为 0.97(t=20)℃ 根据 FÖrster 非辐射能量转移理论求得能量转移效率 E=0.43，供体－受体之间的距离 r=3.08nm；确定了 1-羟基芘与血清白蛋白分子之间的作用力主要为氢键和范德华力。

在此基础上建立了共振能量转移荧光法测定 1-羟基芘的新方法，该方法准确、灵敏，用于人尿中痕量 1-羟基芘的测定，结果与高效液相色谱法一致 关键词关键词：生物标志物，多环芳烃，1-羟基芘，共振光散射，荧光共振能量转移，乙基紫，吖啶橙，牛血清白蛋白 南华大学硕士学位论文 多环芳烃暴露标志物1-羟基芘检测新方法及应用研究 VINovel Methods for determination of 1-Hydroxypyrene as a Biomarker of Exposure to Polycyclic Aromatic Hydrocarbons and Their Application Abstract Polycyclic aromatic hydrocarbons (PAHs) are the ubiquitous persistent organic pollutants which may induce lung and skin cancers in occupational settings and have been listed in preferred controlling pollutants by many countries. The evaluation of exposure to PAHs can provide evidence for epidemiological study and risk assessment of the pollutants. However, due to the complexity of exposure, urinary 1-hydroxypyrene has been widely used as biomarkers of internal dose to evaluate the exposure to PAHs for people. So, there is important practical significance to develop novel methods for determination of 1-hydroxypyrene and study the mechanism of interaction between 1-hydroxypyrene and dyes or proteins. In the chapter 2, a new detection method for 1-hydroxypyrene has been developed using a resonance light scattering (RLS) technique. It is found that the system’s RLS intensity was significantly enhanced due to the formation of ion-associate complex between ethyl violet and 1-hydroxypyrene in Tris-HCl buffer solution of pH8.0 and the maximum RLS peak was located at 396 nm. The addition of anion surfactant—sodium dodecylbenzene sulfonate (SDBS) may increase the stability and sensitivity of the systems. Under the optimum conditions, it was found that the enhanced RLS intensity was linear over the concentration of 1-hydroxypyrene in the range of 4.0~982.0µg/L with a correlation coefficient of 0.9994. The detection limit was 1.2µg/L, RSD=4.5%~ 5.3% and the recoveries of 1-hydroxypyrene were 93.3%~102.3%. The proposed method was simple and sensitive, succeed in the analysis of human urine samples. The results of 1-hydroxypyrene were in agreement with those obtained by the method of high-performance liquid chromatography. 南华大学硕士学位论文 多环芳烃暴露标志物1-羟基芘检测新方法及应用研究 VIIIn the chapter 3, the fluorescence quenching effect of 1-hydroxypyrene to acridine orange-rhodamine 6G was studied and a novel detection method for 1-hydroxypyrene has been developed with the technology of fluorescence resonance energy transfer. It was found that the effective energy transfer could occur between acridine orange and rhodamine 6G in the presence of sodium dodecylbenzene sulfonate solution at λex/λem= 470/556nm and the fluorescence intensity of rhodamine 6G was increased markedly. 1-Hydroxypyrene can diminish the fluorescence intensity of rhodamine 6G and it was proved that static quenching exits between 1-hydroxypyrene and rhodamine 6G. The quenching value of the fluorescence intensity of rhodamine 6G was linear to the concentration of 1-hydroxypyrene in the range of 21.3~982.0µg/L and the detection limit was 6.4µg/L. The relative standards deviation (RSD) were 0.98%~2.03% and the recoveries of 1-hydroxypyrene was 96.0%~104.4%. The developed method was simple and rapid and applied to the determination of 1-hydroxypyrene in human urine with satisfactory results. In the chapter 4, the mechanism of interaction and correlation of energy transfer between bovine serum albumin (BSA) and 1-hydroxypyrene has been studed by spectrofluorometric method. The results showed that the combination constant K= 3.15×105L/mol, the number of binding sites n=0.97(t=20)℃ . The transfer efficiency of energy (E) and binding distance (r) between 1-hydroxypyrene and BSA were obtained (E=0.43, r=3.08nm) based on Försterۥs nonradiative energy transfer theory. It was found that hydrogen bond and vander Waals force played a major role in the binding reaction. And a novel method w。