一种用于快速检测己烯雌酚的基于硝酸纤维素膜的电化学免疫传感器
|
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。
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.
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。
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.
引文
[1]李永存,李伟,吴建华.饮用水健康与饮用水处理技术问答[M].北京：中国石化出版社,2004.133-135.
[2]于智勇,蒋建国,薛庆於.外因性内分泌干扰物质对人类的危害及预防[J].生物学杂志,2010,27(2)：70-73.
[3]Kalra S P. Interacting appetite regulating pathways in the hypothalamic regulation of body weight [J]. Endocr Rev,1999,20:68-100.
[4]Strader A D. Gastrointestinal hormones and food intake [J]. Gastroenterology,2005,128: 175-191.
[5]Volkoff H, Unniappan S, Cerda-Reverter J M. Neuropeptides and the control of food intake in fish[J]. GenComp Endocrinol,2005,142:3-19.
[6]Kuo L E, Tilan J U, Li L. Neuropeptide Y acts directly in the periphery on fat tissue and mediates stress-induced obesity and metabolic syndrome [J]. Nat Med,2007,13:803-811.
[7]Bosi E M, Bazzigalupp I E, Salvi M. Fulminant autoimmune Type 1diabetes during interferon-alpha therapy: a case of Thl-mediated diseases[J]. Diabet Med,2001,18: 329-332.
[8]Crespillo A, Bermudez-Silva F J, Rivera P. Expression of cannabinoid system in muscle: effects of high fat diet and CB1 receptor blockade [J]. Biochem J, in press.
[9]Muller T D, Jarick I. Genetic variation of the ghrelin activator gene ghrelin O-acyltransferase(GOAT) is associated with anorexia nervosa [J]. J Psychiatr Res, in press.
[10]Tabb M M. New modes of action for endocrine-disrupting chemicals, Mol. Endocrinol.[J]. 2010,20:475-482.
[11]Tyler C R, Nilsen M V, Blackwell R. Monoclonal antibody enzyme-linked immunosorbent assay to quantify vitellogenin for studies on environmental estrogens in the rainbow trout (Oncorhynchus mykiss) [J]. Environ ToxicolChem 2002,21:47-54.
[12]Carnevali O M. Exposure to xenobiotic compounds:looking for new biomarkers [J]. Gen Comp Endocrinol 2003,131:203-208.
[13]Carnevali O, Speciale C, Peng C. DEHP impairs zebrafish reproduction by affecting critical factors in oogenesis [J]. PLoS One 5,2010, e10201.
[14]Henley D V. Physiological effects and mechanisms of action of endocrine disrupting chemicals that alter estrogen signaling [J]. Hormones 9,2010:191-205.
[15]Newbold R R, Jefferson W N. Impact of environmental endocrine disrupting chemicals on the development of obesity [J]. Hormones 9,2010:206-217.
[16]Sumpter J P. Xenoendocrine disrupters environmental impacts [J]. Toxicol Lett,1998, 102-103:337-342.
[17]Oka T A N, Shinkai T, Sakuma K. Bisphenol A induces apoptosis in central neural cells during early development of Xenopus laevis [J]. Biochem Biophys Res Commun,2003,312 877-882.
[18]Soverchia L, Ruggeri B, Ballarini P. Modulation of vitellogenin synthesis through estrogen receptor beta-1 in goldfish (Carassius auratus) juveniles exposed to 17-beta estradiol and nonylphenol [J]. Toxicol Appl Pharmacol,2005,15:236-243.
[19]Kaneko M, Yamamoto K, Nakamura M. Bisphenol A acts differently from and independently of thyroid hormone in suppressing thyrotropin release from the bullfrog pituitary [J]. Gen Comp Endocrinol 2008,15:574-580.
[20]Kloas W Li, Urbatzka R, Springer T. Does atrazine affect larval development and sexual differentiation of South African clawed frogs[J]. Ann NY Acad Sci,2009,1163:437-440.
[21]Newbold R R, Jefferson W N, Heindel J J. Effects of endocrine disruptors on obesity [J]. Int J Androl,2008,31:2015-2018.
[22]Newbold R R., Jefferson W N. Environmental estrogens and obesity[J]. Mol Cell Endocrinol,2009,304:84-89.
[23]Chen J, Russo J. Regulation of energy metabolism pathways by estrogens and estrogenic chemicals and potential implications in obesity associated with increased exposure to endocrine disruptors[J]. Biochimica et Biophysica Acta,2009,1793:1128-1143.
[24]Grun F. Endocrine disrupters as obesogens[J]. Mol Cell Endocrinol,2009,304:19-29.
[25]Gesta S, Yamamoto Y, Norris A W. Evidence for a role of developmental genes in the origin of obesity and body fat distribution[J]. PNAS,2006,103:6676-6681.
[26]Newbold R R. Cellular and molecular effects of developmental exposure to diethylstilbestrol:implications for other environmental estrogens [J]. Environ Health Perspect 1995,103:83-87.
[27]Newbold R R, Snyder R J, Phillips T M. Developmental exposure to endocrine disruptors and the obesity epidemic[J]. Reprod Toxicol,2007,23:290-296.
[28]Newbold R R, Snyder R J, Jefferson W N. Perinatal exposure to environmental estrogens and the development of obesity[J]. Mol Nutr Food Res,2007,51:912-917.
[29]Schwartz M W, Porte Jr. D, Seeley R J. Central nervous system control of food intake[J]. Nature,2000,404:661-671.
[30]Schwartz M W, Porte Jr.D, Seeley R J. Brain pathways controlling food intake and body weight[J]. ExpBiol Med,2001,226:78-81.
[31]Pfaffl M W, Dreher R. Meyer Production of clenbuterol, diethylstilbestrol and trenbolone mass standards in lyophilised bovine urine[J]. Analytica Chimica Acta,2003,483(1-2): 401-412.
[32]Wenjun Wang, Ji Li. Development of Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay to the Estrogen Diethylstilbestrol[J]. Chemistry,2006,24: 1758-1765.
[33]Stefanick M L. Est rogens and progestins:background and history, trend in use, and guidelines and regimens approved by the US Food and Drug Administ ration[J]. Am Med, 2005,19(118):64-73.
[34]王凤菊,徐爱珍.己烯雌酚的临床应用[J].中国乡村医药杂志,2003,10(7)：36-37.
[35]Waalkes M P, Ward J M. Urogenital carcinogenesis in female CD 1 mice induced by in utero arsenic exposure is exacerbated by post natal dietylstilbestrol treatment[J]. CancerRes,2006,66(3):1337-1345.
[36]Newbold R R., Efferson W N. Proliferativelesions and reproductive tract tumours in male descendants of mice exposed develop mentally to diethylstilbestrol[J]. Carciinogenesis, 2000,21(7):1355-1363.
[37]薛凌壮,徐振怀,邢清晓等.三合激素己烯雌酚治疗停产鸡试验[J].中国兽医杂志,1997,23(4)：38-39.
[38]赵国成.己烯雌酚诱导母兔发情试验[J].畜牧与兽医,1996,28(3)：117-117.
[39]李德元.己烯雌酚、维生素E综合治疗母猪乏情不孕[J].畜牧与兽医,1995,27(5)：233-233.
[40]杨胜林,蒋诚绩.己烯雌酚对山羊育肥增重效果的研究[J].当代畜牧,2001,(2)：3132.
[41]Lone K P. Natural sex steroids and their xenobiotic analogs in animal production:rowth, carcass quality, pharmacokinetics, metabolism, mode of action, residues, methods and epidemiology[J]. Criti Rev Food Sci Nutr,1997,37(2):193-209.
[42]Herbst A L, Poskanzer D C. Association of maternal stilbestrol therapy with tumor appearance in young women[J]. N Engl J Med,1971,284:878-881.
[43]Saunders E J. Physical and psychological problems associated with exposure to diethylstilbestrol (DES)[J]. Hospital Community Psychiatry,1998,39(11):73-77.
[44]殷国荣,杨亚波,单联喆等.己烯雌酚对感染弓形虫孕鼠脾脏T细胞亚群的影响[J].热带医学杂志,2002,2(4)：347-350.
[45]韩见龙,于村,葛乐瑶等.毛细管气相色谱法测定肉类食品中游离己烯雌酚的残留量[J].中国卫生检验杂志,2004,14(6)：726-728.
[46]Donoho A L, Sieck R F. Gas chromatographic determination of diethylstilbestrol and its glucuronide in cattle tissue[J]. Journal of the AOAC,1973,56(4):785-792.
[47]Coffin D E. Gas chromatographic determination of diethylstilbestrol residuesin animal tissues[J]. Journal of the AOAC,1973,56(2):352-357.
[48]Tirpenou A E, Kamarianos A P. Modified method for eletron capture gas-liquid chromatographic determination of diethylstilbestrol residues in urine of fattened bulls [J]. Journal of the AOAC,1983,66(5):1230-1233.
[49]顾永柞,周奇志.高效液相色谱在环境分析中的应用[J].重庆环境科学,1989,11(1)：50-53.
[50]叶英.高效液相色谱法测定畜禽肉中己烯雌酚[J].热带农业工程,2002,(3)：20-21.
[51]刘军.高效液相色谱法测定化妆品中的己烯雌酚[J].适用技术,1996,12(3)：45-45.
[52]周德刚,贾涛,孙志文.高效液相色谱法检测鸡肉中的己烯雌酚[J].饲料检测,2010,2：48-50.
[53]刘虹,谢承恩.高效液相色谱法测定奶粉中己烯雌酚[J].检测技术,2006,12(6)：374-375.
[54]常碧影,张萍,袁汉成等.LC-MS测定饲料中己烯雌酚方法的研究[J].2003,1(1)：38-41.
[55]张宏,毛炯,孙成均等.气相色谱质谱法测定尿及河底泥中的环境雌激素[J].色谱,2003,21(5)：451-455.
[56]吴银良,刘素英,单吉浩等.气相色谱质谱法分析鸡肝中己烯雌酚残留量[J].中国兽药杂志,2005,39(11)：8-10.
[57]贾丽,廖栩泓.非水毛细管电泳分离和测定己烯雌酚片剂中的有效成分[J].兰州大学学报(自然科学版),2003,39(4)：41-45.
[58]林子俺,庞纪磊,黄慧等.水产品中己烷雌酚、己烯雌酚与双烯雌酚残留的毛细管电泳测定[J].分析测试学报,2010,29(1)：55-58.
[59]杨冰仪,莫金垣,赖璐.肉类中己烯雌酚的高速毛细管电泳安培法测定[J].分析测试学报,2003,22(3)：15-17.
[60]罗丽荣,周福林,章竹君.流动注射化学发光法测定己烯雌酚[J].分析试验室,25(4)：79-81.
[61]Wang J. Determination of diethylstilbestrol by enhancement of luminol-hydrogen peroxide-tetrasulfonated cobalt phthalocyanine chemiluminescence[J]. Analytica Chimica Acta,2004,508(2):171-176.
[62]林珊.运用薄层色谱法对氨基酸分析实验的改进[J].研究与探索,2010(14)：172-172.
[63]李俊锁,邱月明,王超.兽药残留分析[M].上海：上海科学技术出版社,2002,2：106-107.
[64]赵铁,菅凌燕,杨君.薄层色谱法鉴别白斑膏中己烯雌酚、克霉唑、马来酸氯苯那敏和氢化可的松[J].华西药学杂志,2004,19(5)：407-408.
[65]邢丽.薄层色谱法鉴别雌氯乳膏中氯霉素和己烯雌酚[J].华西药学杂志,2006,21(2)：212-212.
[66]赵文宽,张悟铭,王长发等.仪器分析实验[M].北京：高等教育出版社,2001,1：55-56.
[67]魏立娜.荧光分光光度计的结构与应用[J].生命科学仪器,2007,5：61-62.
[68]潘祖亭,姚礼峰,马红燕.己烯雌酚荧光性质的研究及其分析应用[J].武汉大学学报(自然科学版),1997,43(6)：701-705.
[69]林杰,黄晓蓉,郑晶等.放射免疫法快速检测猪尿样中的四环素类药物残留[J].饲料工业,2009,30(15)：45-47.
[70]贺显书.酶联免疫检测技术在猪肉安全检测中的应用[J].农业与技术,2008,28(1)：15-17.
[71]谢晓峰,张子群.应用ELISA方法对禽肉产品中己烯雌酚的残留检测[J].畜牧兽医科技信息,2005,10：60-61.
[72]赵新宇,王权,闫叶娜.建立水产品中己烯雌酚残留ELISA检测方法[J].中国畜牧兽医,2010,37(10)：43-47.
[73]Freia J. Detection of pesticides residues by enzyme immunoassay[J]. Pestic Sci,1989,26: 303-307.
[74]Breyer B, Radcliff F J. Polarographic investigation of the antigen-antibody reaction[J]. Nature,1951,167:79-79.
[75]张书圣.电化学免疫分析法研究进展[J].分析化学,1995,23(10)：1211-1217.
[76]吴守国,袁倬斌.电分析化学原理[M].合肥：中国科学技术大学出版社,2006：267-269.
[77]缪璐,刘仲明,张水华.电化学免疫传感器的研究进展[J].中国医学物理学杂志,2006,23(2)：132-134.
[78]操小栋,刘松琴.HIV-1 p24抗原电化学免疫传感器[J].分析化学,2009,37：125-125.
[79]戴小锋,白燕.丙肝电化学免疫传感器在血清检测中的应用[J].分析测试学报,2006,25(1)：49-51.
[80]赵广英,马超,励建荣.电沉积纳米金修饰4-SPCE电流型VP免疫传感器的研制[J].中国食品学报,2009,9(4)：8-13.
[81]赵广英,刑丰峰.鸡白痢电化学免疫传感器的研制[J].食品科技,2007,32(4)：115-118.
[82]胡舜钦,谢志美,雷存喜.基于褐藻酸钠改性纳米金静电排斥作用的新型电化学免疫传感器的研制[J].湖南城市学院学报(自然科学),2003,24(3)：97-99.
[83]刘志国,胡舜钦,沈国励.基于褐藻酸钠-纳米金复合物作非酶标记的新型电化学免疫传感器的研制[J].理化检验—化学分册,2004,40(8)：445-449.
[84]干宁,王峰,王鲁雁等.基于纳米磁珠修饰印刷电极的牛奶中三聚氰胺检测安培免疫传感器[J].传感技术学报,2009,22(4)：456-460.
[85]赵广英,邢丰峰.基于琼脂糖和纳米金的电流型免疫传感器快速检测副溶血性弧菌[J].传感技术学报,2007,20(8)：1697-1700.
[86]李春香,萧浪涛.基于巯基自组装单层膜的植物生长激素吲哚乙酸电化学免疫传感器的研究[J].化学学报,2003,61(5)：790-794.
[87]干宁,刘飞,倪敏君.基于铜配合物和抗体共固定修饰电极的甲胎蛋白安培免疫传感器[J].四川大学学报(自然科学版),2010,47(2)：345-350.
[88]许媛媛,边超,陈绍凤等.基于微纳米加工技术的微型电化学免疫传感器研究[J].传感技术学报,2006,19(5)：2149-2152.
[89]杨洪川,袁若,柴雅琴等.空心纳米铂银-二氧化钛纳米复合膜固定人绒毛促性腺激素免疫传感器的研究[J].2009,37：78-78.
[90]李荫,梁汝萍,邱建丁等.双层类脂膜电化学免疫传感器的研究及应用进展[J].分析测试学报,2003,22(6)：108-111.
[91]陈汉忠,蒋金书.微型电化学免疫传感器的研制及初步应用[J].分析化学研究简报,2004,32(7)：958-960.
[92]赵广英,吴淑春,王丽男等.一次性电流型酶免疫传感技术快速检测禽流感病毒抗原[J].畜牧兽医学报,2008,39(10)：1442-1448.
[93]陈汉忠,蒋金书,李义平.应用电化学免疫传感器检测大片形吸虫抗原抗体反应的研究[J].畜牧兽医学报,2004,35(3)：339-342.
[94]凌晨,漆红兰,孙静等.阵列电化学免疫传感器同时检测甲胎蛋白和癌胚抗原[J].分析化学,2009,37：128-128.
[95]沈美芳,杨利国,丁炜东.动物性食品中己烯雌酚残留量检测方法评定[J].药物检测,2004,21(5)：86-88.
[96]贾丽,邵海丽,高文华.胶束电动色谱法分离与测定己烯雌酚片剂中的有效成分[J].分析试验室,2004,23(2)：21-23.
[97]Scherr D, Vaughn E D. Diethylstilbestrol revisited:androgen deprivation,osteoporosis and prostate cancer[J]. Urol,2002,167(2):535-538.
[98]唐道鹤,谢华,吴铁等.己烯雌酚对去卵巢大鼠骨骼生长及代谢的影响[J].第一军医大学学报,2003,8：798-801.
[99]Zhuo Y, Yuan R, Chai Y, et al. A reagentless amperometric immunosensor based on gold nanoparticles/thionine/Nafion-membrane-modified gold electrode for determination of al-fetoprotein[J]. Electrochemistry Communications,2005,7(4):355-360.
[100]Hu S. A label-free electrochemical immunosensor based on gold nanoparticles for detection of paraoxon[J]. Talanta,2003,61(6):769-777.
[101]Shen L, He P. An electrochemical immunosensor based on agarose hydrogel films for rapid determination of ractopamine[J]. Electrochemistry Communications,2007,9(4):657-662.
[102]吴守国,袁倬斌.电分析化学原理[M].合肥：中国科学技术大学出版社,2006.150-151.
[103]藤岛昭,相泽益男,井上彻.电化学测定方法(陈震,姚建年泽)[M].北京：北京大学出版社,1995.200-201.
[2]于智勇,蒋建国,薛庆於.外因性内分泌干扰物质对人类的危害及预防[J].生物学杂志,2010,27(2)：70-73.
[3]Kalra S P. Interacting appetite regulating pathways in the hypothalamic regulation of body weight [J]. Endocr Rev,1999,20:68-100.
[4]Strader A D. Gastrointestinal hormones and food intake [J]. Gastroenterology,2005,128: 175-191.
[5]Volkoff H, Unniappan S, Cerda-Reverter J M. Neuropeptides and the control of food intake in fish[J]. GenComp Endocrinol,2005,142:3-19.
[6]Kuo L E, Tilan J U, Li L. Neuropeptide Y acts directly in the periphery on fat tissue and mediates stress-induced obesity and metabolic syndrome [J]. Nat Med,2007,13:803-811.
[7]Bosi E M, Bazzigalupp I E, Salvi M. Fulminant autoimmune Type 1diabetes during interferon-alpha therapy: a case of Thl-mediated diseases[J]. Diabet Med,2001,18: 329-332.
[8]Crespillo A, Bermudez-Silva F J, Rivera P. Expression of cannabinoid system in muscle: effects of high fat diet and CB1 receptor blockade [J]. Biochem J, in press.
[9]Muller T D, Jarick I. Genetic variation of the ghrelin activator gene ghrelin O-acyltransferase(GOAT) is associated with anorexia nervosa [J]. J Psychiatr Res, in press.
[10]Tabb M M. New modes of action for endocrine-disrupting chemicals, Mol. Endocrinol.[J]. 2010,20:475-482.
[11]Tyler C R, Nilsen M V, Blackwell R. Monoclonal antibody enzyme-linked immunosorbent assay to quantify vitellogenin for studies on environmental estrogens in the rainbow trout (Oncorhynchus mykiss) [J]. Environ ToxicolChem 2002,21:47-54.
[12]Carnevali O M. Exposure to xenobiotic compounds:looking for new biomarkers [J]. Gen Comp Endocrinol 2003,131:203-208.
[13]Carnevali O, Speciale C, Peng C. DEHP impairs zebrafish reproduction by affecting critical factors in oogenesis [J]. PLoS One 5,2010, e10201.
[14]Henley D V. Physiological effects and mechanisms of action of endocrine disrupting chemicals that alter estrogen signaling [J]. Hormones 9,2010:191-205.
[15]Newbold R R, Jefferson W N. Impact of environmental endocrine disrupting chemicals on the development of obesity [J]. Hormones 9,2010:206-217.
[16]Sumpter J P. Xenoendocrine disrupters environmental impacts [J]. Toxicol Lett,1998, 102-103:337-342.
[17]Oka T A N, Shinkai T, Sakuma K. Bisphenol A induces apoptosis in central neural cells during early development of Xenopus laevis [J]. Biochem Biophys Res Commun,2003,312 877-882.
[18]Soverchia L, Ruggeri B, Ballarini P. Modulation of vitellogenin synthesis through estrogen receptor beta-1 in goldfish (Carassius auratus) juveniles exposed to 17-beta estradiol and nonylphenol [J]. Toxicol Appl Pharmacol,2005,15:236-243.
[19]Kaneko M, Yamamoto K, Nakamura M. Bisphenol A acts differently from and independently of thyroid hormone in suppressing thyrotropin release from the bullfrog pituitary [J]. Gen Comp Endocrinol 2008,15:574-580.
[20]Kloas W Li, Urbatzka R, Springer T. Does atrazine affect larval development and sexual differentiation of South African clawed frogs[J]. Ann NY Acad Sci,2009,1163:437-440.
[21]Newbold R R, Jefferson W N, Heindel J J. Effects of endocrine disruptors on obesity [J]. Int J Androl,2008,31:2015-2018.
[22]Newbold R R., Jefferson W N. Environmental estrogens and obesity[J]. Mol Cell Endocrinol,2009,304:84-89.
[23]Chen J, Russo J. Regulation of energy metabolism pathways by estrogens and estrogenic chemicals and potential implications in obesity associated with increased exposure to endocrine disruptors[J]. Biochimica et Biophysica Acta,2009,1793:1128-1143.
[24]Grun F. Endocrine disrupters as obesogens[J]. Mol Cell Endocrinol,2009,304:19-29.
[25]Gesta S, Yamamoto Y, Norris A W. Evidence for a role of developmental genes in the origin of obesity and body fat distribution[J]. PNAS,2006,103:6676-6681.
[26]Newbold R R. Cellular and molecular effects of developmental exposure to diethylstilbestrol:implications for other environmental estrogens [J]. Environ Health Perspect 1995,103:83-87.
[27]Newbold R R, Snyder R J, Phillips T M. Developmental exposure to endocrine disruptors and the obesity epidemic[J]. Reprod Toxicol,2007,23:290-296.
[28]Newbold R R, Snyder R J, Jefferson W N. Perinatal exposure to environmental estrogens and the development of obesity[J]. Mol Nutr Food Res,2007,51:912-917.
[29]Schwartz M W, Porte Jr. D, Seeley R J. Central nervous system control of food intake[J]. Nature,2000,404:661-671.
[30]Schwartz M W, Porte Jr.D, Seeley R J. Brain pathways controlling food intake and body weight[J]. ExpBiol Med,2001,226:78-81.
[31]Pfaffl M W, Dreher R. Meyer Production of clenbuterol, diethylstilbestrol and trenbolone mass standards in lyophilised bovine urine[J]. Analytica Chimica Acta,2003,483(1-2): 401-412.
[32]Wenjun Wang, Ji Li. Development of Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay to the Estrogen Diethylstilbestrol[J]. Chemistry,2006,24: 1758-1765.
[33]Stefanick M L. Est rogens and progestins:background and history, trend in use, and guidelines and regimens approved by the US Food and Drug Administ ration[J]. Am Med, 2005,19(118):64-73.
[34]王凤菊,徐爱珍.己烯雌酚的临床应用[J].中国乡村医药杂志,2003,10(7)：36-37.
[35]Waalkes M P, Ward J M. Urogenital carcinogenesis in female CD 1 mice induced by in utero arsenic exposure is exacerbated by post natal dietylstilbestrol treatment[J]. CancerRes,2006,66(3):1337-1345.
[36]Newbold R R., Efferson W N. Proliferativelesions and reproductive tract tumours in male descendants of mice exposed develop mentally to diethylstilbestrol[J]. Carciinogenesis, 2000,21(7):1355-1363.
[37]薛凌壮,徐振怀,邢清晓等.三合激素己烯雌酚治疗停产鸡试验[J].中国兽医杂志,1997,23(4)：38-39.
[38]赵国成.己烯雌酚诱导母兔发情试验[J].畜牧与兽医,1996,28(3)：117-117.
[39]李德元.己烯雌酚、维生素E综合治疗母猪乏情不孕[J].畜牧与兽医,1995,27(5)：233-233.
[40]杨胜林,蒋诚绩.己烯雌酚对山羊育肥增重效果的研究[J].当代畜牧,2001,(2)：3132.
[41]Lone K P. Natural sex steroids and their xenobiotic analogs in animal production:rowth, carcass quality, pharmacokinetics, metabolism, mode of action, residues, methods and epidemiology[J]. Criti Rev Food Sci Nutr,1997,37(2):193-209.
[42]Herbst A L, Poskanzer D C. Association of maternal stilbestrol therapy with tumor appearance in young women[J]. N Engl J Med,1971,284:878-881.
[43]Saunders E J. Physical and psychological problems associated with exposure to diethylstilbestrol (DES)[J]. Hospital Community Psychiatry,1998,39(11):73-77.
[44]殷国荣,杨亚波,单联喆等.己烯雌酚对感染弓形虫孕鼠脾脏T细胞亚群的影响[J].热带医学杂志,2002,2(4)：347-350.
[45]韩见龙,于村,葛乐瑶等.毛细管气相色谱法测定肉类食品中游离己烯雌酚的残留量[J].中国卫生检验杂志,2004,14(6)：726-728.
[46]Donoho A L, Sieck R F. Gas chromatographic determination of diethylstilbestrol and its glucuronide in cattle tissue[J]. Journal of the AOAC,1973,56(4):785-792.
[47]Coffin D E. Gas chromatographic determination of diethylstilbestrol residuesin animal tissues[J]. Journal of the AOAC,1973,56(2):352-357.
[48]Tirpenou A E, Kamarianos A P. Modified method for eletron capture gas-liquid chromatographic determination of diethylstilbestrol residues in urine of fattened bulls [J]. Journal of the AOAC,1983,66(5):1230-1233.
[49]顾永柞,周奇志.高效液相色谱在环境分析中的应用[J].重庆环境科学,1989,11(1)：50-53.
[50]叶英.高效液相色谱法测定畜禽肉中己烯雌酚[J].热带农业工程,2002,(3)：20-21.
[51]刘军.高效液相色谱法测定化妆品中的己烯雌酚[J].适用技术,1996,12(3)：45-45.
[52]周德刚,贾涛,孙志文.高效液相色谱法检测鸡肉中的己烯雌酚[J].饲料检测,2010,2：48-50.
[53]刘虹,谢承恩.高效液相色谱法测定奶粉中己烯雌酚[J].检测技术,2006,12(6)：374-375.
[54]常碧影,张萍,袁汉成等.LC-MS测定饲料中己烯雌酚方法的研究[J].2003,1(1)：38-41.
[55]张宏,毛炯,孙成均等.气相色谱质谱法测定尿及河底泥中的环境雌激素[J].色谱,2003,21(5)：451-455.
[56]吴银良,刘素英,单吉浩等.气相色谱质谱法分析鸡肝中己烯雌酚残留量[J].中国兽药杂志,2005,39(11)：8-10.
[57]贾丽,廖栩泓.非水毛细管电泳分离和测定己烯雌酚片剂中的有效成分[J].兰州大学学报(自然科学版),2003,39(4)：41-45.
[58]林子俺,庞纪磊,黄慧等.水产品中己烷雌酚、己烯雌酚与双烯雌酚残留的毛细管电泳测定[J].分析测试学报,2010,29(1)：55-58.
[59]杨冰仪,莫金垣,赖璐.肉类中己烯雌酚的高速毛细管电泳安培法测定[J].分析测试学报,2003,22(3)：15-17.
[60]罗丽荣,周福林,章竹君.流动注射化学发光法测定己烯雌酚[J].分析试验室,25(4)：79-81.
[61]Wang J. Determination of diethylstilbestrol by enhancement of luminol-hydrogen peroxide-tetrasulfonated cobalt phthalocyanine chemiluminescence[J]. Analytica Chimica Acta,2004,508(2):171-176.
[62]林珊.运用薄层色谱法对氨基酸分析实验的改进[J].研究与探索,2010(14)：172-172.
[63]李俊锁,邱月明,王超.兽药残留分析[M].上海：上海科学技术出版社,2002,2：106-107.
[64]赵铁,菅凌燕,杨君.薄层色谱法鉴别白斑膏中己烯雌酚、克霉唑、马来酸氯苯那敏和氢化可的松[J].华西药学杂志,2004,19(5)：407-408.
[65]邢丽.薄层色谱法鉴别雌氯乳膏中氯霉素和己烯雌酚[J].华西药学杂志,2006,21(2)：212-212.
[66]赵文宽,张悟铭,王长发等.仪器分析实验[M].北京：高等教育出版社,2001,1：55-56.
[67]魏立娜.荧光分光光度计的结构与应用[J].生命科学仪器,2007,5：61-62.
[68]潘祖亭,姚礼峰,马红燕.己烯雌酚荧光性质的研究及其分析应用[J].武汉大学学报(自然科学版),1997,43(6)：701-705.
[69]林杰,黄晓蓉,郑晶等.放射免疫法快速检测猪尿样中的四环素类药物残留[J].饲料工业,2009,30(15)：45-47.
[70]贺显书.酶联免疫检测技术在猪肉安全检测中的应用[J].农业与技术,2008,28(1)：15-17.
[71]谢晓峰,张子群.应用ELISA方法对禽肉产品中己烯雌酚的残留检测[J].畜牧兽医科技信息,2005,10：60-61.
[72]赵新宇,王权,闫叶娜.建立水产品中己烯雌酚残留ELISA检测方法[J].中国畜牧兽医,2010,37(10)：43-47.
[73]Freia J. Detection of pesticides residues by enzyme immunoassay[J]. Pestic Sci,1989,26: 303-307.
[74]Breyer B, Radcliff F J. Polarographic investigation of the antigen-antibody reaction[J]. Nature,1951,167:79-79.
[75]张书圣.电化学免疫分析法研究进展[J].分析化学,1995,23(10)：1211-1217.
[76]吴守国,袁倬斌.电分析化学原理[M].合肥：中国科学技术大学出版社,2006：267-269.
[77]缪璐,刘仲明,张水华.电化学免疫传感器的研究进展[J].中国医学物理学杂志,2006,23(2)：132-134.
[78]操小栋,刘松琴.HIV-1 p24抗原电化学免疫传感器[J].分析化学,2009,37：125-125.
[79]戴小锋,白燕.丙肝电化学免疫传感器在血清检测中的应用[J].分析测试学报,2006,25(1)：49-51.
[80]赵广英,马超,励建荣.电沉积纳米金修饰4-SPCE电流型VP免疫传感器的研制[J].中国食品学报,2009,9(4)：8-13.
[81]赵广英,刑丰峰.鸡白痢电化学免疫传感器的研制[J].食品科技,2007,32(4)：115-118.
[82]胡舜钦,谢志美,雷存喜.基于褐藻酸钠改性纳米金静电排斥作用的新型电化学免疫传感器的研制[J].湖南城市学院学报(自然科学),2003,24(3)：97-99.
[83]刘志国,胡舜钦,沈国励.基于褐藻酸钠-纳米金复合物作非酶标记的新型电化学免疫传感器的研制[J].理化检验—化学分册,2004,40(8)：445-449.
[84]干宁,王峰,王鲁雁等.基于纳米磁珠修饰印刷电极的牛奶中三聚氰胺检测安培免疫传感器[J].传感技术学报,2009,22(4)：456-460.
[85]赵广英,邢丰峰.基于琼脂糖和纳米金的电流型免疫传感器快速检测副溶血性弧菌[J].传感技术学报,2007,20(8)：1697-1700.
[86]李春香,萧浪涛.基于巯基自组装单层膜的植物生长激素吲哚乙酸电化学免疫传感器的研究[J].化学学报,2003,61(5)：790-794.
[87]干宁,刘飞,倪敏君.基于铜配合物和抗体共固定修饰电极的甲胎蛋白安培免疫传感器[J].四川大学学报(自然科学版),2010,47(2)：345-350.
[88]许媛媛,边超,陈绍凤等.基于微纳米加工技术的微型电化学免疫传感器研究[J].传感技术学报,2006,19(5)：2149-2152.
[89]杨洪川,袁若,柴雅琴等.空心纳米铂银-二氧化钛纳米复合膜固定人绒毛促性腺激素免疫传感器的研究[J].2009,37：78-78.
[90]李荫,梁汝萍,邱建丁等.双层类脂膜电化学免疫传感器的研究及应用进展[J].分析测试学报,2003,22(6)：108-111.
[91]陈汉忠,蒋金书.微型电化学免疫传感器的研制及初步应用[J].分析化学研究简报,2004,32(7)：958-960.
[92]赵广英,吴淑春,王丽男等.一次性电流型酶免疫传感技术快速检测禽流感病毒抗原[J].畜牧兽医学报,2008,39(10)：1442-1448.
[93]陈汉忠,蒋金书,李义平.应用电化学免疫传感器检测大片形吸虫抗原抗体反应的研究[J].畜牧兽医学报,2004,35(3)：339-342.
[94]凌晨,漆红兰,孙静等.阵列电化学免疫传感器同时检测甲胎蛋白和癌胚抗原[J].分析化学,2009,37：128-128.
[95]沈美芳,杨利国,丁炜东.动物性食品中己烯雌酚残留量检测方法评定[J].药物检测,2004,21(5)：86-88.
[96]贾丽,邵海丽,高文华.胶束电动色谱法分离与测定己烯雌酚片剂中的有效成分[J].分析试验室,2004,23(2)：21-23.
[97]Scherr D, Vaughn E D. Diethylstilbestrol revisited:androgen deprivation,osteoporosis and prostate cancer[J]. Urol,2002,167(2):535-538.
[98]唐道鹤,谢华,吴铁等.己烯雌酚对去卵巢大鼠骨骼生长及代谢的影响[J].第一军医大学学报,2003,8：798-801.
[99]Zhuo Y, Yuan R, Chai Y, et al. A reagentless amperometric immunosensor based on gold nanoparticles/thionine/Nafion-membrane-modified gold electrode for determination of al-fetoprotein[J]. Electrochemistry Communications,2005,7(4):355-360.
[100]Hu S. A label-free electrochemical immunosensor based on gold nanoparticles for detection of paraoxon[J]. Talanta,2003,61(6):769-777.
[101]Shen L, He P. An electrochemical immunosensor based on agarose hydrogel films for rapid determination of ractopamine[J]. Electrochemistry Communications,2007,9(4):657-662.
[102]吴守国,袁倬斌.电分析化学原理[M].合肥：中国科学技术大学出版社,2006.150-151.
[103]藤岛昭,相泽益男,井上彻.电化学测定方法(陈震,姚建年泽)[M].北京：北京大学出版社,1995.200-201.