【作者】 兰敏；

【导师】 刘伟；

【作者基本信息】 郑州大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 己烯雌酚(diethylstilbestrol, DES)是一种人工合成的雌激素,自1938年起在世界范围内得到了广泛应用,包括在畜牧业和水产养殖业中作为促生物剂来提高饲料的转化率,以及在动物医学中用作雌激素替代品。但由于其致癌性和细胞毒性,从20世纪80年代起大多国家已明令禁用DES。过去20年来所报道的检测动物组织、器官、尿液中所含DES的各种技术包括气相色谱法、高效液相色谱法、气相色谱-质谱联用技术、二极管矩阵-质谱联用技术、化学发光法、薄层层析法、酶联免疫吸附法、荧光分析法、放射免疫法、放射性受体测定法及电化学免疫分析法等。DES的性质稳定且沸点高,因而用气相色谱法检测须经衍生化。高效液相色谱法检测效果较佳,但该法需要相对复杂的样品提取、浓缩和清洗过程,且所需仪器复杂。较之放射免疫法和荧光分析法,酶联免疫吸附法具有安全、快速、可靠、灵敏以及低成本等优点。电化学免疫传感器,因其兼具传统免疫分析法的高特异性以及电化学检测系统的低检测限和低成本的优势,即快速、准确、简便、易携带且成本低,近年来愈来愈受关注。许多研究者们已应用此法来检测各种具有高特异性和高灵敏性的分析物。制备免疫传感器的关键是免疫活性物在电极表面的固定：固定于电极表面膜中的生物大分子必须能展现其特异的免疫活性,保持其天然结构并具有优异的稳定性。基于这些原因,各种不同的膜,包括导电聚合物、钛溶胶-凝胶及自组装膜等都已被研究用来固定免疫分子来完成其免疫反应。其中,铸制薄膜由于其简易性、多能性及生物相容性得以广泛应用,例如纳米金和琼脂糖膜。但这些膜制作繁琐且较脆易碎。本实验采用硝酸纤维素膜,一种具有高的机械强度且易购买的蛋白印迹的理想用膜,来作为制备电化学免疫传感器的一种新型的固定基质。本论文提出了一种新型的、简单的、免标记的电化学免疫传感器,用来快速检测DES。主要是通过将己烯雌酚-牛血清白蛋白(bovine serum albumin,BSA)完全抗原吸附入修饰在金电极表面的硝酸纤维素膜上而构建成的。利用电化学技术中的循环伏安法和差分脉冲伏安法来监测此传感器在不同修饰阶段时,其氧化还原探针体系“铁氰化钾／亚铁氰化钾”K3[Fe(CN)6]/K4[Fe(CN)6]的电化学信号。随着温育液中的单抗逐步地与固定的完全抗原结合K3[Fe(CN)6]的峰电流跟着逐步减小。温育液中的待测游离半抗原与浸于其中的定量的固定完全抗原竞争结合温育液中的定量单抗,由此来检测游离的DES。我们运用此新型传感器检测加标水样中的痕量DES,证明了其良好的特异性,稳定性及灵敏度。更多还原

【Abstract】 Diethylstilbestrol (DES), a synthetic hormone, has been used worldwide since 1938 as a growth-promoting agent for increasing feed conversion efficiency in aquaculture and stockbreeding and as a treatment for estrogen-deficiency disorders in veterinary medicine. But it has been banned for fattening edible animal in most countries for its carcinogenic and toxicological properties since 1980’s. So far, several techniques have been applied during last twenty years to quantitatively monitor DES residues in tissues, organs and urine,including gas chromatography (GC), high-performance liquid chromatography (HPLC), GC-mass spectrometry (GC-MS), diode array-MS (DAD-MS), chemiluminescence (CL), thin layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA), radioimmunoassays (RIA), radioreceptor assay (RRA), electrochemical immunoassay (ECIA) and so on. DES is a stable and high boiling point compound, and thus derivation is required in GC analysis. HPLC is a suitable technique producing satisfactory results, but this method requires relatively complex sample extraction, concentration and clean-up procedures. HPLC instruments are not available in many analytical laboratories. Compared to RIA and FIA, ELISA has the advantages of safety, speed, reliability, sensitivity, and low-cost.Recently, electrochemical immunosensors have gained growing attention since they combine the high specificity of traditional immunoassay methods with the low detection limits and low expenses of electrochemical measurement system, and the fact that they are fast, accurate, simple, portable, and inexpensive. The method has been used by a number of investigators for determination of various analytes with great sensitivity and specificity. It has been reported that the crucial aspect of electrochemical immunosensor is the immobilization of immunological sensitivity compounds on the electrodes. The biological macromolecules immobilized in films on electrode surfaces should exhibit their specific immunoactivity,retain their native structure and demonstrate excellent stability. For these reasons a number of different type films, such as conducting polymer, titania sol-gel, and self-assemble film, have been used to immobilize immunogenic molecule and achieve their immune response. Among them, casting films, for example, gold nanoparticles films and agarose hydrogel films, have been widely used due to their simplicity, versatility, and biocompatibility. However, these casting films are fragile and fabricated complicatedly. In this paper, we report the employment of nitrocellulose membrane, a high mechanical strength and commercially available membrane ideal for blotting of proteins, as a novel immobilization matrix for development of electrochemical immunosensors.A novel and simple label-free electrochemical immunosensor for determination of diethylstilbestrol was constructed by electrically enhanced adsorption of diethylstilbestrol-bovine serum albumin into nitrocellulose membrane modified on a gold electrode. The electrochemical characteristics of the immunosensor were investigated by cyclic voltammetry and differential pulse voltammetry in a redox probe system of K3Fe(CN)6. The redox peak current of K3[Fe(CN)6] was reduced with the binding of the antibody to the diethylstilbestrol-bovine serum albumin immobilized on the gold electrode. The effect of amount of monoclonal antibody was explored to provide optimum analytical performance. Competition for antibody binding between the free diethylstilbestrol and the diethylstilbestrol-bovine serum albumin on the electrode was applied to detect free diethylstilbestrol. The immunosensor had very high sensitivity and specificity, and was used for the quantitative determination of trace amounts of diethylstilbestrol in spiked samples.更多还原