水溶性CdTe纳米晶荧光探针的制备表征及应用
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摘要
半导体纳米晶标记是目前免疫分析研究中一个新兴的关键技术。在免疫分析中,常用于标记抗体的主要荧光试剂有稀土螯合物、异硫氰酸荧光素(FITC)、四乙基异硫氰酸罗丹明(TRITC)、四乙基罗丹明(RB200)。基于CdTe纳米晶具有很大的激子玻尔直径(15 mm)和独特的光电特性,例如荧光量子产率高、荧光光谱对称且半峰宽窄、吸收光谱连续、发光的连续可调性、强抗光漂白性等,有望取代有机染料成为新一代的荧光探针。用这种荧光纳米颗粒作为标记物,可显著提高分析方法的灵敏度和分辨率,为建立免疫分析方法检测环境持久性有机污染物的残留提供了可靠的纳米荧光探针。
本文主要的研究成果简述如下:
(1)采用水相合成技术制备了表面带有羧基的发射红光的CdTe纳米晶。应用单因素法探索最佳的合成条件。采用TEM、XRD、紫外-可见吸收光谱和荧光光谱等技术对所制得产品进行表征。结果表明,当Cd~(2+):HTe~-:TGA的摩尔比为1.00:0.20:2.40,反应体系的pH为11.0,96℃条件下回流4个小时,为制备发射红光的CdTe纳米晶的最佳实验条件。该红光纳米晶量子产率较高(42.54%)、半峰宽较窄(FWHM=17.3 nm、EM=696 nm),与其它制备方法相比,这种方法不但缩短了红光CdTe纳米晶的制备时间,而且提高了水溶性CdFe纳米晶的荧光量子产率。在此基础上,以硫脲为硫源,通过简单且绿色的合成方案在水溶液中成功的制备了CdTe/CdS核壳型纳米晶。CdTe纳米晶的成功制备,为其在环境监测分析中的应用提供了性能优良的荧光探针。
(2)基于铜(Ⅱ)离子在pH为7.4的磷酸盐缓冲溶液中对CdTe纳米晶的荧光具有较强的淬灭作用,实现了对铜(Ⅱ)离子的定量检测。在最佳的条件下,纳米晶的浓度(以Cd~(2+)计)为1.25×10~(-4) mol/L时,此方法对浓度在10μg/L～320μg/L的范围内,体系的相对荧光强度与铜(Ⅱ)离子的浓度呈线性关系,其线性回归方程为ΔI=0.9093+0.0032C(μg/L);检出限为1.06μg/L。本方法应用于实际样品中铜离子的测定,结果令人满意。
(3)采用巯基乙酸、L-半胱氨酸、氨基磺酸为稳定剂直接在水溶液中合成了CdTe纳米晶。在选定的实验条件下,CdTe纳米晶与牛血清白蛋白相结合,并通过光谱数据、电泳得以证实。同时系统的探讨了以巯基乙酸为稳定剂所合成的CdTe纳米晶与牛血清白蛋白之间Stern-Volrner平衡原理。CdTe-牛血清白蛋白的荧光发射峰发生蓝移,而发射谱的半峰宽不变,证明了二者的结合,是由于共价作用引起的。BSA的存在,形成了稳定的类似于核壳结构的(CdTe)_X(BSA)_(1-X)复合物,借助于其外端的羧基,它能与生物分子的胺基通过酰胺键相结合,从而达到标记生物分子的目的,为实现与抗体或抗原的共价链接奠定了理论基础。
(4)CdTe纳米晶,经过表面修饰剂的作用,表面带有了羧基,在EDC和NHS等交联剂的作用下被激活,具备了一定的生物功能。通过共价链接作用,分别链接上了实验室自制的四氯联苯(PCB_4)、多溴联苯(PBB)、菲(PH)、萘(NA)等抗体,且标记效率值F/P均在1～5之间,进一步溶液稀释比与体系的荧光强度成良好的线性关系,说明了所制的各种抗体荧光免疫试剂性能优良,从而为建立环境持久性有机污染物的荧光免疫分析方法提供了新的荧光标记物。
Luminescent quantum dots-semiconductor nanocrystals are a promising technology in the applications of immunoassay as fluorescence probes.There have been numerous reports of conjugating antibody with various different organic fluorescent dyes,including Rare-earth Chelate, FITC,TRITC,and Tetraethyl-Rhodamine.As the result of CdTe nanocrystals have greater exciton Bohr diameter of 15 um,which have unique electronic and optical properties such as high quantum yield,a narrow emission bands continuous broad absorption band,and high resistance to photo-bleaching,receive more concerns in recent years.The experiment results demonstrate that the QDs-based fluorescence probes can exhibit good sensitivity and exceptional phot(?)-stabil(?)ty, which would provide a new probe in detecting Immunoassay Environmental Persistent Organic Pollutants.
(Ⅰ) The water-soluble CdTe nanocrystals have been synthesized by using mercapto-acetate acid as stabilizing agent in four refluxing hours.Single factor methods has been used in grouping for the best conditions of the experiment.The products were characterized by using TEM,XRD, UV-Vis and fluorescence spectra.Here,the optical molar ratio of Cd~(2+):HTe~-:TGA is found to be 1.00:0.20:2.40.Maximum fluorescence produced at pH 11.0 and 96℃,with maximum emission wavelength at 696 nm.Under optical conditions,The quantum yield of the red CdTe nanocrystals was measured to be 0.4254 and the half-peak width was.about 17 nm.Recent advances have led to the development of multifunctional nanocrystals probes that are very bright and stable under the complex condition.At the same time,CdTe / CdS core-shell nanocrystals were synthesized in aqueous solution while using the thiourea as to the source of sulfur.As the result of the CdTe nanocrystals were perfectly synthesized,so it can be used to developping a new fluorescent nanocrystals probe for Environmental monitoring and chemical analysis.
(Ⅱ)With the mercaptoethanol as stabilizing agents,the CdTe nanocrystals were prepared under wild condition.A new fluorescence method has been developed for the determination of copper(Ⅱ) based on the florescence intensity of CdTe nanocrystals in pH = 7.4 phoshate buffer solution.Under optimum condition,when the concentration was 1.25×10~(-4) mol/L, the relative fluorescence intensity of QDs is linearly proportional to the concentration of copper(Ⅱ) from 10μg/L to 320μg/L.The linear equation is△I = 0.9093 + 0.0032C(μg/L ) and the limit of detection is 1.06μg/L.The method has been used to determine trace amounts of copper(Ⅱ) in vegetation with satisfactory results.
(Ⅲ) Luminescent water-soluble CdTe nanocrystals capped with different thiol carboxylic acid were synthesized in aqueous medium and then cross linked to bovine serum albumin(BSA) with NHS.The QDs-BSA conjugates were confirmed,and their stability has been proved.Due to the electrostatic interaction between the CdTe nanocrystals and BSA,it has been investigated with ultraviolet visible absorption spectroscopy and florescence spectroscopy.In addition to the BSA can link carboxyl mercaptoacetic acid on the surface of the CdTe,there were seven adjacent disulfide and cadmium link more closely so that CdTe and BSA can form a stable CdTe / BSA Core-shell nanocrystals.It was also found that the absorption intensity of CdTe nanocrystals significantly decreased after adding BSA solution and just oppersite to the florescence intensity of CdTe nanocrystals,showing that CdTe nanocrystals were bond to BSA.So CdTe nanocrystals could be link the antibody as the fluorescence probe.
(Ⅳ) The CdTe nanocrystals has strong fluorescence emission at 696 nm after modified by mercaptoacetic acid.With the effect of EDC and NHS,the binding of the PCB_4(PBB、PH、NA) antibody with CdTe nanocrystals was prepared,and this bind had fluorescence characteristics.The results of the F / P is between one and five.Therefore,The CdTe nanocrystals showed that it could be used for determination of PCB_4(PBB,PH,NA) antibody as fluorescence immunoassay regent.
本文主要的研究成果简述如下:
(1)采用水相合成技术制备了表面带有羧基的发射红光的CdTe纳米晶。应用单因素法探索最佳的合成条件。采用TEM、XRD、紫外-可见吸收光谱和荧光光谱等技术对所制得产品进行表征。结果表明,当Cd~(2+):HTe~-:TGA的摩尔比为1.00:0.20:2.40,反应体系的pH为11.0,96℃条件下回流4个小时,为制备发射红光的CdTe纳米晶的最佳实验条件。该红光纳米晶量子产率较高(42.54%)、半峰宽较窄(FWHM=17.3 nm、EM=696 nm),与其它制备方法相比,这种方法不但缩短了红光CdTe纳米晶的制备时间,而且提高了水溶性CdFe纳米晶的荧光量子产率。在此基础上,以硫脲为硫源,通过简单且绿色的合成方案在水溶液中成功的制备了CdTe/CdS核壳型纳米晶。CdTe纳米晶的成功制备,为其在环境监测分析中的应用提供了性能优良的荧光探针。
(2)基于铜(Ⅱ)离子在pH为7.4的磷酸盐缓冲溶液中对CdTe纳米晶的荧光具有较强的淬灭作用,实现了对铜(Ⅱ)离子的定量检测。在最佳的条件下,纳米晶的浓度(以Cd~(2+)计)为1.25×10~(-4) mol/L时,此方法对浓度在10μg/L～320μg/L的范围内,体系的相对荧光强度与铜(Ⅱ)离子的浓度呈线性关系,其线性回归方程为ΔI=0.9093+0.0032C(μg/L);检出限为1.06μg/L。本方法应用于实际样品中铜离子的测定,结果令人满意。
(3)采用巯基乙酸、L-半胱氨酸、氨基磺酸为稳定剂直接在水溶液中合成了CdTe纳米晶。在选定的实验条件下,CdTe纳米晶与牛血清白蛋白相结合,并通过光谱数据、电泳得以证实。同时系统的探讨了以巯基乙酸为稳定剂所合成的CdTe纳米晶与牛血清白蛋白之间Stern-Volrner平衡原理。CdTe-牛血清白蛋白的荧光发射峰发生蓝移,而发射谱的半峰宽不变,证明了二者的结合,是由于共价作用引起的。BSA的存在,形成了稳定的类似于核壳结构的(CdTe)_X(BSA)_(1-X)复合物,借助于其外端的羧基,它能与生物分子的胺基通过酰胺键相结合,从而达到标记生物分子的目的,为实现与抗体或抗原的共价链接奠定了理论基础。
(4)CdTe纳米晶,经过表面修饰剂的作用,表面带有了羧基,在EDC和NHS等交联剂的作用下被激活,具备了一定的生物功能。通过共价链接作用,分别链接上了实验室自制的四氯联苯(PCB_4)、多溴联苯(PBB)、菲(PH)、萘(NA)等抗体,且标记效率值F/P均在1～5之间,进一步溶液稀释比与体系的荧光强度成良好的线性关系,说明了所制的各种抗体荧光免疫试剂性能优良,从而为建立环境持久性有机污染物的荧光免疫分析方法提供了新的荧光标记物。
Luminescent quantum dots-semiconductor nanocrystals are a promising technology in the applications of immunoassay as fluorescence probes.There have been numerous reports of conjugating antibody with various different organic fluorescent dyes,including Rare-earth Chelate, FITC,TRITC,and Tetraethyl-Rhodamine.As the result of CdTe nanocrystals have greater exciton Bohr diameter of 15 um,which have unique electronic and optical properties such as high quantum yield,a narrow emission bands continuous broad absorption band,and high resistance to photo-bleaching,receive more concerns in recent years.The experiment results demonstrate that the QDs-based fluorescence probes can exhibit good sensitivity and exceptional phot(?)-stabil(?)ty, which would provide a new probe in detecting Immunoassay Environmental Persistent Organic Pollutants.
(Ⅰ) The water-soluble CdTe nanocrystals have been synthesized by using mercapto-acetate acid as stabilizing agent in four refluxing hours.Single factor methods has been used in grouping for the best conditions of the experiment.The products were characterized by using TEM,XRD, UV-Vis and fluorescence spectra.Here,the optical molar ratio of Cd~(2+):HTe~-:TGA is found to be 1.00:0.20:2.40.Maximum fluorescence produced at pH 11.0 and 96℃,with maximum emission wavelength at 696 nm.Under optical conditions,The quantum yield of the red CdTe nanocrystals was measured to be 0.4254 and the half-peak width was.about 17 nm.Recent advances have led to the development of multifunctional nanocrystals probes that are very bright and stable under the complex condition.At the same time,CdTe / CdS core-shell nanocrystals were synthesized in aqueous solution while using the thiourea as to the source of sulfur.As the result of the CdTe nanocrystals were perfectly synthesized,so it can be used to developping a new fluorescent nanocrystals probe for Environmental monitoring and chemical analysis.
(Ⅱ)With the mercaptoethanol as stabilizing agents,the CdTe nanocrystals were prepared under wild condition.A new fluorescence method has been developed for the determination of copper(Ⅱ) based on the florescence intensity of CdTe nanocrystals in pH = 7.4 phoshate buffer solution.Under optimum condition,when the concentration was 1.25×10~(-4) mol/L, the relative fluorescence intensity of QDs is linearly proportional to the concentration of copper(Ⅱ) from 10μg/L to 320μg/L.The linear equation is△I = 0.9093 + 0.0032C(μg/L ) and the limit of detection is 1.06μg/L.The method has been used to determine trace amounts of copper(Ⅱ) in vegetation with satisfactory results.
(Ⅲ) Luminescent water-soluble CdTe nanocrystals capped with different thiol carboxylic acid were synthesized in aqueous medium and then cross linked to bovine serum albumin(BSA) with NHS.The QDs-BSA conjugates were confirmed,and their stability has been proved.Due to the electrostatic interaction between the CdTe nanocrystals and BSA,it has been investigated with ultraviolet visible absorption spectroscopy and florescence spectroscopy.In addition to the BSA can link carboxyl mercaptoacetic acid on the surface of the CdTe,there were seven adjacent disulfide and cadmium link more closely so that CdTe and BSA can form a stable CdTe / BSA Core-shell nanocrystals.It was also found that the absorption intensity of CdTe nanocrystals significantly decreased after adding BSA solution and just oppersite to the florescence intensity of CdTe nanocrystals,showing that CdTe nanocrystals were bond to BSA.So CdTe nanocrystals could be link the antibody as the fluorescence probe.
(Ⅳ) The CdTe nanocrystals has strong fluorescence emission at 696 nm after modified by mercaptoacetic acid.With the effect of EDC and NHS,the binding of the PCB_4(PBB、PH、NA) antibody with CdTe nanocrystals was prepared,and this bind had fluorescence characteristics.The results of the F / P is between one and five.Therefore,The CdTe nanocrystals showed that it could be used for determination of PCB_4(PBB,PH,NA) antibody as fluorescence immunoassay regent.
引文
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