荧光稀土纳米二氧化硅探针的制备及应用
|
摘要
近年来,随着生命科学的不断发展,生物化学分析的应用也越来越广泛。由于荧光光谱的灵敏度高、选择性好,荧光探针技术已经在蛋白质、核酸、细胞检测及免疫分析等方面发挥了重要作用,其中各种荧光稀土纳米颗粒探针则是荧光分析的有力工具。
本文采用反相微乳液法制备了掺杂型荧光(Eu、Tb)纳米二氧化硅颗粒,及共价型荧光纳米二氧化硅BHHCT-Eu-SiO2,通过X-射线衍射、红外光谱、荧光光谱等手段对制备物的结构、形态、性质进行了表征。并将BHHCT-Eu-SiO2与氯霉素抗体相连作为荧光探针应用于氯霉素的免疫层析试纸条分析。
本文所制备的掺杂型荧光(Eu、Tb)纳米二氧化硅颗粒均为非晶态或极小晶体,EDTA中的羰基与Eu3+(Tb3+)以双齿配位的形式配位,当掺杂元素为Eu时,荧光光谱分析表明,在396nm处没有出现Eu3+的本征激发峰,而在310nm处出现一较强激发峰,表明配合物的激发光谱由配体EDTA吸收能量引起,并将能量传递给Eu3+使之发出特征荧光,在310nm光激发下,发射波长位于615nm处,对应于Eu3+离子电偶极跃迁5D0→7F2,发射光谱中没有出现磁偶极跃迁5D0→7F1表明Eu3+的配合物中稀土离子处于不对称中心；当掺杂元素为Tb时,荧光光谱分析表明,在277-396nm之间有一宽而强的Tb3+离子的激发谱带,240nm处的最强激发峰是由配体EDTA吸收能量,并将能量传递给Tb3+使之发出特征荧光所引起,在240nm光激发下,发射波长位于486nm(5D4→7F6)、544nm(5D4→7F5)、582nm(5D4→7F4)、620nm(5D4→7F3)。掺杂的EDTA-Eu(Tb)与SiO2摩尔比在1：120-1：15之间时,随着EDTA-Eu(Tb)的掺杂量的增加荧光粒子在各发射波长下的最大发射荧光强度均成指数函数增长。
荧光粒子BHHCT-Eu-SiO2,激发峰分别位于231nm、274nm、318nm、340nm处,其中231、274和340nm处的激发峰由配体的n(π)→π*跃迁引起,最大激发峰318nm是由配体吸收能量,通过分子间能量传递给Eu3+,使之发出荧光所致,发射光谱中最强发射峰位于615nm(5D0→7F2)处,另外两个弱发射峰位于592nm(5D0→7F1)和649nm(5D0→7F3)处,615nm处发射峰强度远远强于592nm处发射峰,说明荧光探针中Eu3+处于非反演中心。BHHCT-Eu-SiO2-Ab作为荧光探针进行免疫层析试纸条法检测氯霉素时,对氯霉素检测限为10ng/mL,氯霉素琥珀酸盐检测限为1ng／mL,重复性良好,特异性良好,与甲砜霉素、卡娜霉素、磺胺二甲基嘧间无交叉反应。
In recent years, with the constant development of life science, biochemical analysis was uesed more and more widely. Fluorescence spectroscopy has high sensitivity and selectivity, so fluorescence probe technology has played an important role in the proteins, nucleic acids, cells and immune detection analysis. Among them, lanthanide-coated nanoparticals probe is a powerful tool.
In this paper, fluorescent doped Eu and Tb nano-silica particles were prepared by reverse microemulsion, fluorescent covalent nano-silica BHHCT-Eu-SiO2 was prepared by reverse microemulsion, too. The structure, morphology and the nature of preparing products were characterized by X-ray diffraction, infrared spectroscopy, fluorescence spectroscopy. And BHHCT-Eu-SiO2 was used in detecting chloramphenicol by immunochromatographic strip method.
In this paper, fluorescent doped Eu and Tb nano-silica particles are amorphous or very small crystals, the coordination forms of carbonyl in EDTA with Eu3+(Tb3+) is double coordination. When the doping element is Eu, fluorescence spectroscopy analysis shows that the features excitation peak at 396nm was not observed, and the excitation peak appeared at 310nm, it shows that the excitation spectrum of the complex was caused by EDTA absorbing energy and the energy was transfered to Eu3+ which make it issue features fluorescent. With the 310nm of light excitation, the emission wavelength is 615nm. The strong emission band of Eu3+ at 615nm belongs to electric dipole transition 5D0→7F2, as magnetic dipole transition 5D0→7F1 of Eu3+ was not observed, the Eu3+ in the complex was at the asymmetric center. When the doping element is Tb, fluorescence spectroscopy analysis shows that between 277 and 396nm, there is a strong and wide-band excitation, the strongest excitation occurred at 240nm, it was caused by EDTA absorbing energy and the energy was transfered to Tb3+ to make it to issue features fluorescent. With the 240nm of light excitation, the emission wavelength appeared at 486nm(5D4→7F6) 544nm(5D4→7F5)、582nm(5D4→7F4)、620nm(5D4→7F3). When the molar ratio of EDTA-Eu(Tb) and SiO2 was between 1:120 to 1:15, the fluorescence intensity in every emission wavelength will increase with the concentration of Eu(Tb), and the growth is exponential growth.
The excitation peaks of BHHCT-Eu-SiO2 appeared at 231nm,274nm,318nm, 340nm, among them the excitation peaks at 231nm,274nm,340nm are caused by n(π)→π* transition of some groups in BHHCT. The strongest excitation occurred at 318nm, it was caused by ligand absorbing energy and the energy was transfered to Eu3+ to make it to issue features fluorescent. With the 318nm of light excitation, the emission wavelength appeared at 615nm(5D0→7F2)、592nm(5D0→7F1)、649nm(5D0→7F3), and 5D0→7F2 transition is much stronger than 5D0→7F1, which indicates that the Eu3+ in the complex is at the asymmetric center. When BHHCT-Eu-SiO2-Ab as a fluorescent probe was used in detecting chloramphenicol by immunochromatographic strip method, the method detection limit of chloramphenicol is 10ng/mL, while chloramphenicol succinate salt is lng/mL. This method has good reproducibility and specificity, it has no cross reaction with thiamphenicol kanamycin sulfamethazine.
本文采用反相微乳液法制备了掺杂型荧光(Eu、Tb)纳米二氧化硅颗粒,及共价型荧光纳米二氧化硅BHHCT-Eu-SiO2,通过X-射线衍射、红外光谱、荧光光谱等手段对制备物的结构、形态、性质进行了表征。并将BHHCT-Eu-SiO2与氯霉素抗体相连作为荧光探针应用于氯霉素的免疫层析试纸条分析。
本文所制备的掺杂型荧光(Eu、Tb)纳米二氧化硅颗粒均为非晶态或极小晶体,EDTA中的羰基与Eu3+(Tb3+)以双齿配位的形式配位,当掺杂元素为Eu时,荧光光谱分析表明,在396nm处没有出现Eu3+的本征激发峰,而在310nm处出现一较强激发峰,表明配合物的激发光谱由配体EDTA吸收能量引起,并将能量传递给Eu3+使之发出特征荧光,在310nm光激发下,发射波长位于615nm处,对应于Eu3+离子电偶极跃迁5D0→7F2,发射光谱中没有出现磁偶极跃迁5D0→7F1表明Eu3+的配合物中稀土离子处于不对称中心；当掺杂元素为Tb时,荧光光谱分析表明,在277-396nm之间有一宽而强的Tb3+离子的激发谱带,240nm处的最强激发峰是由配体EDTA吸收能量,并将能量传递给Tb3+使之发出特征荧光所引起,在240nm光激发下,发射波长位于486nm(5D4→7F6)、544nm(5D4→7F5)、582nm(5D4→7F4)、620nm(5D4→7F3)。掺杂的EDTA-Eu(Tb)与SiO2摩尔比在1：120-1：15之间时,随着EDTA-Eu(Tb)的掺杂量的增加荧光粒子在各发射波长下的最大发射荧光强度均成指数函数增长。
荧光粒子BHHCT-Eu-SiO2,激发峰分别位于231nm、274nm、318nm、340nm处,其中231、274和340nm处的激发峰由配体的n(π)→π*跃迁引起,最大激发峰318nm是由配体吸收能量,通过分子间能量传递给Eu3+,使之发出荧光所致,发射光谱中最强发射峰位于615nm(5D0→7F2)处,另外两个弱发射峰位于592nm(5D0→7F1)和649nm(5D0→7F3)处,615nm处发射峰强度远远强于592nm处发射峰,说明荧光探针中Eu3+处于非反演中心。BHHCT-Eu-SiO2-Ab作为荧光探针进行免疫层析试纸条法检测氯霉素时,对氯霉素检测限为10ng/mL,氯霉素琥珀酸盐检测限为1ng／mL,重复性良好,特异性良好,与甲砜霉素、卡娜霉素、磺胺二甲基嘧间无交叉反应。
In recent years, with the constant development of life science, biochemical analysis was uesed more and more widely. Fluorescence spectroscopy has high sensitivity and selectivity, so fluorescence probe technology has played an important role in the proteins, nucleic acids, cells and immune detection analysis. Among them, lanthanide-coated nanoparticals probe is a powerful tool.
In this paper, fluorescent doped Eu and Tb nano-silica particles were prepared by reverse microemulsion, fluorescent covalent nano-silica BHHCT-Eu-SiO2 was prepared by reverse microemulsion, too. The structure, morphology and the nature of preparing products were characterized by X-ray diffraction, infrared spectroscopy, fluorescence spectroscopy. And BHHCT-Eu-SiO2 was used in detecting chloramphenicol by immunochromatographic strip method.
In this paper, fluorescent doped Eu and Tb nano-silica particles are amorphous or very small crystals, the coordination forms of carbonyl in EDTA with Eu3+(Tb3+) is double coordination. When the doping element is Eu, fluorescence spectroscopy analysis shows that the features excitation peak at 396nm was not observed, and the excitation peak appeared at 310nm, it shows that the excitation spectrum of the complex was caused by EDTA absorbing energy and the energy was transfered to Eu3+ which make it issue features fluorescent. With the 310nm of light excitation, the emission wavelength is 615nm. The strong emission band of Eu3+ at 615nm belongs to electric dipole transition 5D0→7F2, as magnetic dipole transition 5D0→7F1 of Eu3+ was not observed, the Eu3+ in the complex was at the asymmetric center. When the doping element is Tb, fluorescence spectroscopy analysis shows that between 277 and 396nm, there is a strong and wide-band excitation, the strongest excitation occurred at 240nm, it was caused by EDTA absorbing energy and the energy was transfered to Tb3+ to make it to issue features fluorescent. With the 240nm of light excitation, the emission wavelength appeared at 486nm(5D4→7F6) 544nm(5D4→7F5)、582nm(5D4→7F4)、620nm(5D4→7F3). When the molar ratio of EDTA-Eu(Tb) and SiO2 was between 1:120 to 1:15, the fluorescence intensity in every emission wavelength will increase with the concentration of Eu(Tb), and the growth is exponential growth.
The excitation peaks of BHHCT-Eu-SiO2 appeared at 231nm,274nm,318nm, 340nm, among them the excitation peaks at 231nm,274nm,340nm are caused by n(π)→π* transition of some groups in BHHCT. The strongest excitation occurred at 318nm, it was caused by ligand absorbing energy and the energy was transfered to Eu3+ to make it to issue features fluorescent. With the 318nm of light excitation, the emission wavelength appeared at 615nm(5D0→7F2)、592nm(5D0→7F1)、649nm(5D0→7F3), and 5D0→7F2 transition is much stronger than 5D0→7F1, which indicates that the Eu3+ in the complex is at the asymmetric center. When BHHCT-Eu-SiO2-Ab as a fluorescent probe was used in detecting chloramphenicol by immunochromatographic strip method, the method detection limit of chloramphenicol is 10ng/mL, while chloramphenicol succinate salt is lng/mL. This method has good reproducibility and specificity, it has no cross reaction with thiamphenicol kanamycin sulfamethazine.
引文
[1]量子点公司.http://www.qdots.com[EB/OL].2006.
[2]Xiaojun Zhao, Rovelyn Tapec-Dytioco, and Weihong Tan. Ultrasensitive DNA detection using highly fluorescent bioconjugated nanoparticles[J]. J.Amer.Chem.Sci,2003,125:11474-11475P
[3]Xiaohu Gao, Yuanyuan Cui, Richard M Levenson, et al. In vivo cancer targeting and imaging with semiconductor quantum dots[J]. Nat.Biotech, 2004,22,969-976P
[4]Evelyn B Voura, Jyoti K Jaiswal, Hedi Mattoussi, et al. Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy[J]. Nat.Med,2004,10,993-998P
[5]Xiaojun Zhao, Lisa R. Hilliard, Shelly John Mechery, et al. A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles[J]. Nat.Acad.Sci. USA,2004,101,15027-15032P
[6]Zhiqiang Ye, Mingqian Tan, Guilan Wang, et al. Preparation characterization, and time-resolved fluorometric application of silica-coated terbium(Ⅲ) fluorescent nanoparticles[J]. Anal.Chem,2004,76,513-518P
[7]Mingqian Tan, Zhiqiang Ye, Guilan Wang, et al. Preparation and time-resolved fluorometric application of luminescent europium nanoparticles[J]. Chem.Mater,2004,16,2494-2498P
[8]Zhiqiang Ye, Mingqian Tan, Guilan Wang, et al. Novel fluorescent europium chelate-doped silica nanoparticles:preparation, characterization and time-resolved fluorometric application [J]. J.Mater.Chem,2004,14, 851-856P
[9]Mingqian Tan, Guilan Wang, Xiaodan Hai, et al. Development of functionalized fluorescent europium nanoparticles for biolabeling and time-resolved fluorometric applications[J]. J.Mater.Chem,2004,14, 2896-2901P
[10]Xiaodan Hai, Mingqian Tan, G.uilian Wang, et al. Preparation and a time-resolved fluoroimmunoassay application of new europium fluorescent nanoparticles[J]. Anal.Sci,2004,20,245-246P
[11]Zhiqiang Ye, Mingqian Tan, Guilan Wang, et al. Development of functionalized terbium fluorescent nanoparticles for antibody labeling and time-resolved fluoroimmunoassay application[J]. Talanta,2005,65, 206-210P
[12]Zhiqiang Ye, Mingqian Tan, Guilan Wang, et al. Preparation, characterization and application of fluorescent terbium complex-doped zirconia nanoparticles[J]. J.Fluoresc,2005,15,499-505P
[13]Mingqian Tan, Guilan Wang, Zhiqiang Ye, et al. Synthesis and characterization of titania-based monodisperse fluorescent europium nanoparticles for biolabeling[J]. J.Lumin,2006,117,20-28P
[14]Richard R.De Haas, Nico P.Verwoerd, et al. The use of peroxidase-mediated deposition of biotin-tyramide in combination with time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ Hybridization[J]. J.Histochem.Cytochem, 1996,44(10),1091-1099P
[15]Bjartell A, Laine S, Pettersson K, et al. Time-resolved fluorescence in munocytochemical detection of prostate-specific antigen in prostatic tissue sections[J]. Histochem J,1999,31,45-52P
[16]Connally, R.Veal, D.Piper J. Time-resolved fluorescence microscopy using an improved europium chelate BHHST for the in-situ detection of Cryptosporidium and Giardia[J]. Microsc.Res.Techniq,2004,64,312-322P
[17]张立德,牟季美.纳米材料和纳米结构[M].科学出版社,2001,2：60-61 贝
[18]王世敏,许祖勋,傅晶.纳米材料制备技术[M].化学工业出版社,2002,02页
[19]唐宝莲,莫祥银.SiO2的开发应用现状[J].南京工业大学学报,2004,26(01)：98-102页
[20]Barthel H, Rosch L. Fumed silica-production, properties and applications [C]. Organosilicon Chemistry Ⅱ:From molecules to Materials, VCH, Weinheim,1996:761-778P
[21]赵宜新,王明聪.气相法白炭黑的制造方法[P].CN98114270.2,1999-0217.
[22]刘立泉,何永,张崇等.纳米二氧化硅粉体的制备[[J].电子元件与材料,2000,19(04)：2832页
[23]郑典模,苏学军.化学沉淀法制备纳米SiO2的研究[J].南昌大学学报工科版,2003,25(02)：3941页
[24]胡庆福,李国庭,王金阁等.CO2沉淀法制取高补强白炭黑[J].非金属矿,2000,23(06)：23-24页
[25]瞿其曙,何友昭,淦五二等.超细二氧化硅的制备及研究进展[J].硅酸盐通报,2000,19(05),57-63页
[26]胡兵,蒋斌波,陈纪忠.单分散性SiO2的制备与应用[J].化工进展,2005,24(06)：603-607页
[27]Zhao D Y, Fengj.L.Triblock copolymer synthesis of mesoporous silica with periodic s0 to 300 angstrom pores. Science[J].1998,279:548-552P
[28]边蕴静.纳米材料在涂料中的应用[J].化工新型材料,2001,29(7)：31-32页
[29]王海波.超重力法制取纳米级二氧化硅[J].中国照明电器,2003(09)：8-9页
[30]贾宏,郭楷,郭奋等.用超重力法制备纳米二氧化硅[J].材料研究学报, 2001,15(01)：120-124页
[31]王平,李辽沙.粉煤灰制备白炭黑的探索性研究[J].中国资源综合利用,2004：25-27页
[32]于欣伟,张霖霖,郑成等.氟硅酸钠制备白炭黑氨解过程影响因素研究[J].化学工程,2004,32(04)：67-70页
[33]易发成,宋绵新,陈廷芳等.利用埃洛石粘土制备白炭黑的研究[J].非金属矿,1999,22(01)：17-19页
[34]胡艳海,路海源,马立军等.利用蛋白石制取白炭黑工艺研究[J].非金属矿,1999,22(02)：16-17页
[35]郭茂生,冯兴仁.利用硅灰石生产白炭黑的实用技术研究[J].贵州化工,2001,26(03)：10-11页
[36]周光,王廷吉.利用非金属矿制备白炭黑现状及发展前景[J].华东地质学院学报,2001,24(04)：298-301页
[37]侯贵华,罗驹华,陈景文.稻壳制备高纯高表面积Si02的研究[J].化学世界,2004,09(09)：458-469页
[38]张其春,叶巧明.高岭土锻烧制白炭黑[J].应用化学,1999,16(05)：91-93页
[39]周德风,郝婕,巴晓微等.稻壳的开发利用[J].长春工业大学学报(自然科学版),2004,25(01)：59-62页
[40]杨先和.稻壳内高纯度SiO2的提取及其应用前景[J].无机材料学报,1992,07(03)：379-383页
[41]马雪垅,房江育.稻壳纳米SiO2研究及开发利用[J].资源开发与市场,2005,21(05)：389-390页
[42]骆锋,阮建明,万千.微乳液法制备纳米二氧化硅粉末工艺的研究[J].硅酸盐通报,2004,23(05)：48-52页
[43]Gan L M, K Zhang, Chew C H. Preparation of silica nanoparticle from sodium orthosilicate in inverse micromulsion[J]. Colloids and SurfacesA: hysicochemical and Engineering Aspects,1996,110(02):199-206P
[44]K. Pettersson, H.Siitari, I.Hemmila, et al. Time-resolved floroimmunoassay of human choriogonadotropin[J]. Clin.Chem,1983,29:60P
[45]J. Yuan, G. Wang, H. Kimura, et al. Highly sensitive time-resolved fluoroimmunoassay of human immunoglobulin E by using a new europium fluorescent chelate as a label[J]. Anal.Biochem.1997,254(2):283-287P
[46]A.K.Saha,K.Kross,E.D.Kloszewski, et al. Time-resolved fluorescence of a new europium-chelate complex demonstration of highly sensitive detection of protein and DNA samples[J]. J.Am.Chem.Soc,1993,115:11032P
[47]Harri Takalo, Elina Hanninen, Jouko Kankare. Luminescence of Europium(Ⅲ) chelates with 4-(arylethynyl)pyridines as ligands[J]. Helv.Chim.Acta,1993,76:877P
[48]J.Yuan, K.Matsumoto, and H.Kimura. A new tetradentate β-diketonate-europium chelate that can be covalently bound to proteins for time-resolved fluoroimmunoassay [J]. Anal.Chem,1998,70,596-601P
[49]J. Yuan, G. Wang, H. Kimura, et al. Sensitive time-resolved fluoroimmunoassay of human thyroid-stimulating hormone by using a new europium fluorescent chelate as a label[J]. Anal.Sci,1998,14,421-423P
[50]M. Ikegawa, J.Yuan, K.Matsumoto, et al. Elevated plasma stromal cell-derived factor 1 protein level in the progression of HIV type 1 infection/AIDS[J]. AIDS Res.Hum.Retrov,2001,17,587-595P
[51]H. Kimura, M. Suzui, F. Nagao, et al. Highly sensitive determination of plasma cytokines by time-resolved fluoroimmunoassay:effect of bicycle exercise on plasma level of interleukin-la (IL-la), tumor necrosis factor a (TNFa), and interferon λ(IFNλ)[J]. Anal.Sci,2001,17,593-597P
[52]M.Kobayashi, H.Kimura, J.Liao, et al. Measurement of mouse urinary type IV collagen using timeresolved fluoroimmunoassay[J]. Anal.Sci,2003,19, 205-210P
[53]H.Kimura, J.Yuan, G.Wang, et al. Highly sensitive quantitation of methamphetamine by timeresolved fluoroimmunoassay using a new europium chelate as a label[J]. J. Anal.Toxicol,1999,23,11-16P
[54]G.Wang, J.Yuan, K.Matsumoto, et al. Quantitative measurement of p21 protein in human serum by time-resolved fluoroimmunoassay [J]. Anal.Sci, 2001,17,881-883P
[55]J.Yuan, G.Wang, H.Kimura, et al. Highly sensitive detection of bensulfuron-methyl by time-resolved fluoroimmunoassay using a tetradentate β-diketonate europium chelate as a label[J]. Anal.Sci.1999,15, 125-128P
[56]K.Majima, T.Fukui, J.Yuan, et al. Quantitative measurement of 17β-estradiol and estriol in river water by time-resolved fluoroimmunoassay[J]. Anal.Sci,2002,18,869-874P
[57]李庆阁,许晔.荧光稀土络合物硅纳米颗粒类标记物及其制备方法.中国专利[P].02141718.0：3.
[58]Ye Xu, Qingge Li. Multiple fluorescent labeling of silica nanoparticles with lanthanide chelate[J]. Clinical Chemistry,2007,53(8):1503-1504P
[59]谢文刚.二氧化硅生物荧光纳米颗粒的制备与应用[J].湖南农业大学学报(自然科学版),2007,33(4)：496-497页
[60]何晓晓,王柯敏,谭蔚泓.基于生物荧光纳米颗粒的新型荧光标记方法及其在细胞识别中的应用[J].科学通报,2001,46(16)：1353-1356页
[61]张宏达,胡宝成.纳米颗粒作为基因载体的应用[J].生物技术通讯,2007,18(2)：295-296页
[62]Zhu S G, Gan K, Li Z, et al. Biocompatibility of polyllysine modified silica nanoparticles[J]. cancer,2003,22(10):1114P
[63]Carsten K,Mohammad S, Elenore G. Silica nanoparticle modified with aminosilanes as carriers for plasmid DNA [J]. Int J Pharm,2000,196:257P
[64]Xiao-xiao He,Kemin Wang,Weihong Tan, et al. Bioconjugated Nanoparticles for DNA Protection from Cleavage Journal of American Chemist Society[J].2003,125(24):7168-7169P
[65]程凡亮,陈伶利,王卫等.氨基化二氧化硅颗粒固定木瓜蛋白酶研究[J].生物工程学报,2004,20(2)：287-289页
[66]石慧,何晓晓,王柯敏等.二氧化硅纳米与微米颗粒作为固定化酶载体的生物效应[J].高等学校化学学报,2007,28(9)：1690-1695页
[67]Hoar T P and Schulman J H. Distribution of attack on iron or zinc partly immersed in chloride solutions [J]. Nature.1943,152:102-110P
[68]Shinoda K and Friberg S. Microemulsion:colloidal aspects[J]. Adv colloid Interface Sci.1975,4:281-300P
[69]Schulman J H, Stoeckenius W, Prince L M. Mechanism of formation and structure of micro-emulsion by electron microscopy[J]. J.Phys.Chem,1959, 63:1677-1680P
[70]Prince L M.Microemulsion Theory and Practice[M]. New York:Academic Press,1977,6P
[71]Robbins M L.Micellization. Solubilization and microemulsion[M]. New York:Plenum press,1977,2:713-735P
[72]Michell D J, Ninham B W. Micelles,vesicles and microemulsions[J]. J. Chem. Soc. Faraday Trans,1981,2(77):601-629P
[73]崔正刚,殷福珊.微乳液技术及应用[M].北京：中国轻工业出版社,1999：85-99页
[74]Bourrel M,Schechter R S. Microemulsion and related systems:fomulation, solvency, and physical properties[M]. New York:Marcel Dekker Inc,1988, 30:1216P
[75]徐滨士.纳米表面工程[M].化学工业出版社,2004：1页
[76]王笃金,吴瑾光.反胶团或微乳液法制备超细颗粒的研究进展[J].粉体技术,1994,1(01)：30-33页
[77]施利毅,华彬,张剑平.微乳液的结构及其在制备超细颗粒中的应用[J].功能材料,1998,29(02)：136-139页
[78]陈磊,陈建敏,周惠娣.微乳化技术在无机纳米材料制备中的应用及发展[J].材料科学与工程学报,2004,22(01)：135-141页
[79]Cavjochi R E,SilbaeeR H. Landua level density of states as a function of Fermi energy in the two dimensional electrongas[J]. Solid State Communications,1992,81(10):821-825P
[80]华载文,施冠成.超微乳液涂层剂的合成及其自交联性能研究[J].印染,1999,25(07)：17-20页
[81]尹荔松,沈辉.微乳技术制备纳米微粒的研究进展[J].功能材料,2001,32(06)：580-582页
[82]刘强春,杨一军,戴建明.微乳法制备无机纳米材料的研究进展[J].淮北师院学报,2003,24(04)：26-30页
[83]张万忠,乔学亮,陈建国.微乳液法合成纳米材料的进展[J].石油化工2005,34(01)：84-88页
[84]黄贤智.荧光分析法(第二版)[M].科学出版社,1975：8-10页
[85]王正祥.稀土荧光配合物及其荧光防伪油墨的制备、荧光性能研究[A].2004,9页
[86]Bruchez M, Moronne M, Gin P, et al. Semiconductor nanocrystals as fluorescent biological labels[J]. Science,1998,281(5385):2013P
[87]Chan W C W, Nie S M. Quantum dot bioconjugates for ultrasensitive nonisotopic detection[J]. Science,1998,281(5385):2016P
[88]Taylor J R, Fang M M, Nie S M. Probing specific sequences on single DNA molecules with bioconjugated fluorescent nanoparticles[J]. Anal.Chem, 2000,72(9):1979P
[89]Mitchell G P, Mirkin C A, Letsinger R L. Programmed assembly of DNA functionalized quantum dots [J]. J.Am.Chem.Soc,1999,121(35):8122P
[90]Hayat M A. Colloidal gold:principles, methods and applications[M]. New York:Academic Press,1989.
[91]Santra S, Zhang P, Wang K M, et al. Conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers[J]. Anal.Chem,2001,73 (20):4988P
[92]Santra S, Wang K M, Tapec R, et al. Development of novel dye-doped silica nanoparticles for biomarker application[J]. J.Biomed.Opt,2001,6(2): 160P
[93]Swadeshmukul Santra, Rovelyn Tapec, Nikoleta Theodoropoulou, et al. Synthesis and characterization of silica-coated Iron oxide nanoparticles in microemulsion:the effect of nonionic surfactants [J]. Langmuir,2001, 17(10),2900-2906P
[94]李夏,李文君,杨永丽.镧(Ⅲ)-邻氯苯氧乙酸配合物的合成及性质[J].光谱学与光谱分析,1999,19(3)：450-452页
[95]许金钩,王尊本.荧光分析法(第三版)[M].科学出版社,2006：42页
[96]陆广农,唐宁.4个氮支索冠醚的铕(Ⅲ)、铽(Ⅲ)配合物的合成及表征[J].兰州大学学报(自然科学版),2008,44(2)：68页
[97]K.Binnemans, R. Van Deun, C.Gorller-Walrand, et al. Spectroscopic properties of trivalent lanthanide ions in fluorophosphates glasses[J]. Journal of Non-Crystalline Solids,1998,238:11-29P
[98]刘会州,郭晨,余江等.微乳相萃取技术及应用[M].科学出版社,2005：242页
[99]贾中兆.纳米二氧化硅颗粒的制备研究[D].南京工业大学,2006：43-44页
[100]http://sce.scnu.edu.cn/WebCai/analytical/chap_4/sect_2/know_2.html.
[101]胡顶飞,沈建忠.氯霉素类抗生素的残留分析[J].中国兽药杂志,2001,35(5)：55-57页
[102]Stenven R H, Andrew G B, Imelda M T, et al. Detection of ivermectin residues in bovine using an enzyme immunoassay[J]. Analyst,1998,123(2): 355P
[103]Rebecca S.Nicolich, Eduardo Werneck-Barroso, Marlice A.Sipoli Marques. Food safety evaluation:detection and confirmation of chloramphenicol in milk by high performance liquid chromatography-tandem mass spectrometry[J]. Analytica Chimica Acta,2006,565:97-98P
[104]G.Scortichini, L.Annunziata, M.N.Haouet, et al. ELISA qualitative screening of chloramphenicol in muscle, eggs,honey and milk:method validation according to the Commission Decision 2002/657/EC criteria[J]. Analytica Chimica Acta 2005,535,43-44P
[105]朱艳冰,李庆阁,王桂兰等.新型铕络合物用于HBsAg的时间分辨荧光免疫检测[J].标记免疫分析与临床,2002,9(3)：157页
[106]王雪,黄飚,金坚.氯霉素全抗原的合成及鉴定[J].生物技术通报,2008,1：178页
[2]Xiaojun Zhao, Rovelyn Tapec-Dytioco, and Weihong Tan. Ultrasensitive DNA detection using highly fluorescent bioconjugated nanoparticles[J]. J.Amer.Chem.Sci,2003,125:11474-11475P
[3]Xiaohu Gao, Yuanyuan Cui, Richard M Levenson, et al. In vivo cancer targeting and imaging with semiconductor quantum dots[J]. Nat.Biotech, 2004,22,969-976P
[4]Evelyn B Voura, Jyoti K Jaiswal, Hedi Mattoussi, et al. Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy[J]. Nat.Med,2004,10,993-998P
[5]Xiaojun Zhao, Lisa R. Hilliard, Shelly John Mechery, et al. A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles[J]. Nat.Acad.Sci. USA,2004,101,15027-15032P
[6]Zhiqiang Ye, Mingqian Tan, Guilan Wang, et al. Preparation characterization, and time-resolved fluorometric application of silica-coated terbium(Ⅲ) fluorescent nanoparticles[J]. Anal.Chem,2004,76,513-518P
[7]Mingqian Tan, Zhiqiang Ye, Guilan Wang, et al. Preparation and time-resolved fluorometric application of luminescent europium nanoparticles[J]. Chem.Mater,2004,16,2494-2498P
[8]Zhiqiang Ye, Mingqian Tan, Guilan Wang, et al. Novel fluorescent europium chelate-doped silica nanoparticles:preparation, characterization and time-resolved fluorometric application [J]. J.Mater.Chem,2004,14, 851-856P
[9]Mingqian Tan, Guilan Wang, Xiaodan Hai, et al. Development of functionalized fluorescent europium nanoparticles for biolabeling and time-resolved fluorometric applications[J]. J.Mater.Chem,2004,14, 2896-2901P
[10]Xiaodan Hai, Mingqian Tan, G.uilian Wang, et al. Preparation and a time-resolved fluoroimmunoassay application of new europium fluorescent nanoparticles[J]. Anal.Sci,2004,20,245-246P
[11]Zhiqiang Ye, Mingqian Tan, Guilan Wang, et al. Development of functionalized terbium fluorescent nanoparticles for antibody labeling and time-resolved fluoroimmunoassay application[J]. Talanta,2005,65, 206-210P
[12]Zhiqiang Ye, Mingqian Tan, Guilan Wang, et al. Preparation, characterization and application of fluorescent terbium complex-doped zirconia nanoparticles[J]. J.Fluoresc,2005,15,499-505P
[13]Mingqian Tan, Guilan Wang, Zhiqiang Ye, et al. Synthesis and characterization of titania-based monodisperse fluorescent europium nanoparticles for biolabeling[J]. J.Lumin,2006,117,20-28P
[14]Richard R.De Haas, Nico P.Verwoerd, et al. The use of peroxidase-mediated deposition of biotin-tyramide in combination with time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ Hybridization[J]. J.Histochem.Cytochem, 1996,44(10),1091-1099P
[15]Bjartell A, Laine S, Pettersson K, et al. Time-resolved fluorescence in munocytochemical detection of prostate-specific antigen in prostatic tissue sections[J]. Histochem J,1999,31,45-52P
[16]Connally, R.Veal, D.Piper J. Time-resolved fluorescence microscopy using an improved europium chelate BHHST for the in-situ detection of Cryptosporidium and Giardia[J]. Microsc.Res.Techniq,2004,64,312-322P
[17]张立德,牟季美.纳米材料和纳米结构[M].科学出版社,2001,2：60-61 贝
[18]王世敏,许祖勋,傅晶.纳米材料制备技术[M].化学工业出版社,2002,02页
[19]唐宝莲,莫祥银.SiO2的开发应用现状[J].南京工业大学学报,2004,26(01)：98-102页
[20]Barthel H, Rosch L. Fumed silica-production, properties and applications [C]. Organosilicon Chemistry Ⅱ:From molecules to Materials, VCH, Weinheim,1996:761-778P
[21]赵宜新,王明聪.气相法白炭黑的制造方法[P].CN98114270.2,1999-0217.
[22]刘立泉,何永,张崇等.纳米二氧化硅粉体的制备[[J].电子元件与材料,2000,19(04)：2832页
[23]郑典模,苏学军.化学沉淀法制备纳米SiO2的研究[J].南昌大学学报工科版,2003,25(02)：3941页
[24]胡庆福,李国庭,王金阁等.CO2沉淀法制取高补强白炭黑[J].非金属矿,2000,23(06)：23-24页
[25]瞿其曙,何友昭,淦五二等.超细二氧化硅的制备及研究进展[J].硅酸盐通报,2000,19(05),57-63页
[26]胡兵,蒋斌波,陈纪忠.单分散性SiO2的制备与应用[J].化工进展,2005,24(06)：603-607页
[27]Zhao D Y, Fengj.L.Triblock copolymer synthesis of mesoporous silica with periodic s0 to 300 angstrom pores. Science[J].1998,279:548-552P
[28]边蕴静.纳米材料在涂料中的应用[J].化工新型材料,2001,29(7)：31-32页
[29]王海波.超重力法制取纳米级二氧化硅[J].中国照明电器,2003(09)：8-9页
[30]贾宏,郭楷,郭奋等.用超重力法制备纳米二氧化硅[J].材料研究学报, 2001,15(01)：120-124页
[31]王平,李辽沙.粉煤灰制备白炭黑的探索性研究[J].中国资源综合利用,2004：25-27页
[32]于欣伟,张霖霖,郑成等.氟硅酸钠制备白炭黑氨解过程影响因素研究[J].化学工程,2004,32(04)：67-70页
[33]易发成,宋绵新,陈廷芳等.利用埃洛石粘土制备白炭黑的研究[J].非金属矿,1999,22(01)：17-19页
[34]胡艳海,路海源,马立军等.利用蛋白石制取白炭黑工艺研究[J].非金属矿,1999,22(02)：16-17页
[35]郭茂生,冯兴仁.利用硅灰石生产白炭黑的实用技术研究[J].贵州化工,2001,26(03)：10-11页
[36]周光,王廷吉.利用非金属矿制备白炭黑现状及发展前景[J].华东地质学院学报,2001,24(04)：298-301页
[37]侯贵华,罗驹华,陈景文.稻壳制备高纯高表面积Si02的研究[J].化学世界,2004,09(09)：458-469页
[38]张其春,叶巧明.高岭土锻烧制白炭黑[J].应用化学,1999,16(05)：91-93页
[39]周德风,郝婕,巴晓微等.稻壳的开发利用[J].长春工业大学学报(自然科学版),2004,25(01)：59-62页
[40]杨先和.稻壳内高纯度SiO2的提取及其应用前景[J].无机材料学报,1992,07(03)：379-383页
[41]马雪垅,房江育.稻壳纳米SiO2研究及开发利用[J].资源开发与市场,2005,21(05)：389-390页
[42]骆锋,阮建明,万千.微乳液法制备纳米二氧化硅粉末工艺的研究[J].硅酸盐通报,2004,23(05)：48-52页
[43]Gan L M, K Zhang, Chew C H. Preparation of silica nanoparticle from sodium orthosilicate in inverse micromulsion[J]. Colloids and SurfacesA: hysicochemical and Engineering Aspects,1996,110(02):199-206P
[44]K. Pettersson, H.Siitari, I.Hemmila, et al. Time-resolved floroimmunoassay of human choriogonadotropin[J]. Clin.Chem,1983,29:60P
[45]J. Yuan, G. Wang, H. Kimura, et al. Highly sensitive time-resolved fluoroimmunoassay of human immunoglobulin E by using a new europium fluorescent chelate as a label[J]. Anal.Biochem.1997,254(2):283-287P
[46]A.K.Saha,K.Kross,E.D.Kloszewski, et al. Time-resolved fluorescence of a new europium-chelate complex demonstration of highly sensitive detection of protein and DNA samples[J]. J.Am.Chem.Soc,1993,115:11032P
[47]Harri Takalo, Elina Hanninen, Jouko Kankare. Luminescence of Europium(Ⅲ) chelates with 4-(arylethynyl)pyridines as ligands[J]. Helv.Chim.Acta,1993,76:877P
[48]J.Yuan, K.Matsumoto, and H.Kimura. A new tetradentate β-diketonate-europium chelate that can be covalently bound to proteins for time-resolved fluoroimmunoassay [J]. Anal.Chem,1998,70,596-601P
[49]J. Yuan, G. Wang, H. Kimura, et al. Sensitive time-resolved fluoroimmunoassay of human thyroid-stimulating hormone by using a new europium fluorescent chelate as a label[J]. Anal.Sci,1998,14,421-423P
[50]M. Ikegawa, J.Yuan, K.Matsumoto, et al. Elevated plasma stromal cell-derived factor 1 protein level in the progression of HIV type 1 infection/AIDS[J]. AIDS Res.Hum.Retrov,2001,17,587-595P
[51]H. Kimura, M. Suzui, F. Nagao, et al. Highly sensitive determination of plasma cytokines by time-resolved fluoroimmunoassay:effect of bicycle exercise on plasma level of interleukin-la (IL-la), tumor necrosis factor a (TNFa), and interferon λ(IFNλ)[J]. Anal.Sci,2001,17,593-597P
[52]M.Kobayashi, H.Kimura, J.Liao, et al. Measurement of mouse urinary type IV collagen using timeresolved fluoroimmunoassay[J]. Anal.Sci,2003,19, 205-210P
[53]H.Kimura, J.Yuan, G.Wang, et al. Highly sensitive quantitation of methamphetamine by timeresolved fluoroimmunoassay using a new europium chelate as a label[J]. J. Anal.Toxicol,1999,23,11-16P
[54]G.Wang, J.Yuan, K.Matsumoto, et al. Quantitative measurement of p21 protein in human serum by time-resolved fluoroimmunoassay [J]. Anal.Sci, 2001,17,881-883P
[55]J.Yuan, G.Wang, H.Kimura, et al. Highly sensitive detection of bensulfuron-methyl by time-resolved fluoroimmunoassay using a tetradentate β-diketonate europium chelate as a label[J]. Anal.Sci.1999,15, 125-128P
[56]K.Majima, T.Fukui, J.Yuan, et al. Quantitative measurement of 17β-estradiol and estriol in river water by time-resolved fluoroimmunoassay[J]. Anal.Sci,2002,18,869-874P
[57]李庆阁,许晔.荧光稀土络合物硅纳米颗粒类标记物及其制备方法.中国专利[P].02141718.0：3.
[58]Ye Xu, Qingge Li. Multiple fluorescent labeling of silica nanoparticles with lanthanide chelate[J]. Clinical Chemistry,2007,53(8):1503-1504P
[59]谢文刚.二氧化硅生物荧光纳米颗粒的制备与应用[J].湖南农业大学学报(自然科学版),2007,33(4)：496-497页
[60]何晓晓,王柯敏,谭蔚泓.基于生物荧光纳米颗粒的新型荧光标记方法及其在细胞识别中的应用[J].科学通报,2001,46(16)：1353-1356页
[61]张宏达,胡宝成.纳米颗粒作为基因载体的应用[J].生物技术通讯,2007,18(2)：295-296页
[62]Zhu S G, Gan K, Li Z, et al. Biocompatibility of polyllysine modified silica nanoparticles[J]. cancer,2003,22(10):1114P
[63]Carsten K,Mohammad S, Elenore G. Silica nanoparticle modified with aminosilanes as carriers for plasmid DNA [J]. Int J Pharm,2000,196:257P
[64]Xiao-xiao He,Kemin Wang,Weihong Tan, et al. Bioconjugated Nanoparticles for DNA Protection from Cleavage Journal of American Chemist Society[J].2003,125(24):7168-7169P
[65]程凡亮,陈伶利,王卫等.氨基化二氧化硅颗粒固定木瓜蛋白酶研究[J].生物工程学报,2004,20(2)：287-289页
[66]石慧,何晓晓,王柯敏等.二氧化硅纳米与微米颗粒作为固定化酶载体的生物效应[J].高等学校化学学报,2007,28(9)：1690-1695页
[67]Hoar T P and Schulman J H. Distribution of attack on iron or zinc partly immersed in chloride solutions [J]. Nature.1943,152:102-110P
[68]Shinoda K and Friberg S. Microemulsion:colloidal aspects[J]. Adv colloid Interface Sci.1975,4:281-300P
[69]Schulman J H, Stoeckenius W, Prince L M. Mechanism of formation and structure of micro-emulsion by electron microscopy[J]. J.Phys.Chem,1959, 63:1677-1680P
[70]Prince L M.Microemulsion Theory and Practice[M]. New York:Academic Press,1977,6P
[71]Robbins M L.Micellization. Solubilization and microemulsion[M]. New York:Plenum press,1977,2:713-735P
[72]Michell D J, Ninham B W. Micelles,vesicles and microemulsions[J]. J. Chem. Soc. Faraday Trans,1981,2(77):601-629P
[73]崔正刚,殷福珊.微乳液技术及应用[M].北京：中国轻工业出版社,1999：85-99页
[74]Bourrel M,Schechter R S. Microemulsion and related systems:fomulation, solvency, and physical properties[M]. New York:Marcel Dekker Inc,1988, 30:1216P
[75]徐滨士.纳米表面工程[M].化学工业出版社,2004：1页
[76]王笃金,吴瑾光.反胶团或微乳液法制备超细颗粒的研究进展[J].粉体技术,1994,1(01)：30-33页
[77]施利毅,华彬,张剑平.微乳液的结构及其在制备超细颗粒中的应用[J].功能材料,1998,29(02)：136-139页
[78]陈磊,陈建敏,周惠娣.微乳化技术在无机纳米材料制备中的应用及发展[J].材料科学与工程学报,2004,22(01)：135-141页
[79]Cavjochi R E,SilbaeeR H. Landua level density of states as a function of Fermi energy in the two dimensional electrongas[J]. Solid State Communications,1992,81(10):821-825P
[80]华载文,施冠成.超微乳液涂层剂的合成及其自交联性能研究[J].印染,1999,25(07)：17-20页
[81]尹荔松,沈辉.微乳技术制备纳米微粒的研究进展[J].功能材料,2001,32(06)：580-582页
[82]刘强春,杨一军,戴建明.微乳法制备无机纳米材料的研究进展[J].淮北师院学报,2003,24(04)：26-30页
[83]张万忠,乔学亮,陈建国.微乳液法合成纳米材料的进展[J].石油化工2005,34(01)：84-88页
[84]黄贤智.荧光分析法(第二版)[M].科学出版社,1975：8-10页
[85]王正祥.稀土荧光配合物及其荧光防伪油墨的制备、荧光性能研究[A].2004,9页
[86]Bruchez M, Moronne M, Gin P, et al. Semiconductor nanocrystals as fluorescent biological labels[J]. Science,1998,281(5385):2013P
[87]Chan W C W, Nie S M. Quantum dot bioconjugates for ultrasensitive nonisotopic detection[J]. Science,1998,281(5385):2016P
[88]Taylor J R, Fang M M, Nie S M. Probing specific sequences on single DNA molecules with bioconjugated fluorescent nanoparticles[J]. Anal.Chem, 2000,72(9):1979P
[89]Mitchell G P, Mirkin C A, Letsinger R L. Programmed assembly of DNA functionalized quantum dots [J]. J.Am.Chem.Soc,1999,121(35):8122P
[90]Hayat M A. Colloidal gold:principles, methods and applications[M]. New York:Academic Press,1989.
[91]Santra S, Zhang P, Wang K M, et al. Conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers[J]. Anal.Chem,2001,73 (20):4988P
[92]Santra S, Wang K M, Tapec R, et al. Development of novel dye-doped silica nanoparticles for biomarker application[J]. J.Biomed.Opt,2001,6(2): 160P
[93]Swadeshmukul Santra, Rovelyn Tapec, Nikoleta Theodoropoulou, et al. Synthesis and characterization of silica-coated Iron oxide nanoparticles in microemulsion:the effect of nonionic surfactants [J]. Langmuir,2001, 17(10),2900-2906P
[94]李夏,李文君,杨永丽.镧(Ⅲ)-邻氯苯氧乙酸配合物的合成及性质[J].光谱学与光谱分析,1999,19(3)：450-452页
[95]许金钩,王尊本.荧光分析法(第三版)[M].科学出版社,2006：42页
[96]陆广农,唐宁.4个氮支索冠醚的铕(Ⅲ)、铽(Ⅲ)配合物的合成及表征[J].兰州大学学报(自然科学版),2008,44(2)：68页
[97]K.Binnemans, R. Van Deun, C.Gorller-Walrand, et al. Spectroscopic properties of trivalent lanthanide ions in fluorophosphates glasses[J]. Journal of Non-Crystalline Solids,1998,238:11-29P
[98]刘会州,郭晨,余江等.微乳相萃取技术及应用[M].科学出版社,2005：242页
[99]贾中兆.纳米二氧化硅颗粒的制备研究[D].南京工业大学,2006：43-44页
[100]http://sce.scnu.edu.cn/WebCai/analytical/chap_4/sect_2/know_2.html.
[101]胡顶飞,沈建忠.氯霉素类抗生素的残留分析[J].中国兽药杂志,2001,35(5)：55-57页
[102]Stenven R H, Andrew G B, Imelda M T, et al. Detection of ivermectin residues in bovine using an enzyme immunoassay[J]. Analyst,1998,123(2): 355P
[103]Rebecca S.Nicolich, Eduardo Werneck-Barroso, Marlice A.Sipoli Marques. Food safety evaluation:detection and confirmation of chloramphenicol in milk by high performance liquid chromatography-tandem mass spectrometry[J]. Analytica Chimica Acta,2006,565:97-98P
[104]G.Scortichini, L.Annunziata, M.N.Haouet, et al. ELISA qualitative screening of chloramphenicol in muscle, eggs,honey and milk:method validation according to the Commission Decision 2002/657/EC criteria[J]. Analytica Chimica Acta 2005,535,43-44P
[105]朱艳冰,李庆阁,王桂兰等.新型铕络合物用于HBsAg的时间分辨荧光免疫检测[J].标记免疫分析与临床,2002,9(3)：157页
[106]王雪,黄飚,金坚.氯霉素全抗原的合成及鉴定[J].生物技术通报,2008,1：178页