CdTe、CdHgTe纳米粒子的制备、修饰及在蛋白质检测中的应用
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摘要
半导体纳米粒子因具有独特而又优异的光学特性,近年来在其合成及应用领域均引起了广泛的关注,本论文在水相条件下,研究了具有近红外发射波长的CdHgTe纳米晶在蛋白质检测中的应用;在经典合成的基础上,为拓宽纳米粒子在生物体系中的应用,研究了不同接枝试剂对TGA-CdTe纳米粒子荧光特性的影响;利用TGA、L-cys及二者比例不同的混合稳定剂制备了不同形貌的纳米晶;结合分子印迹技术的高选择性与半导体纳米粒子荧光检测技术的高灵敏性,建立了选择性测定蛋白质的方法。本论文的主要内容如下:
(1)综述了半导体纳米粒子的相关基本知识、主要合成方法、表征手段及其在生命科学领域中的广泛应用。
(2)在水相中制备了具有近红外发射波长的CdHgTe纳米粒子,对纳米粒子的合成条件进行了系统的研究,以TEM和XRD表征了CdTe和CdHgTe的形貌特征及晶体结构,研究了含不同比例的Hg(Ⅱ)的CdHgTe的荧光光谱及紫外光谱性质。实验结果表明,CdHgTe结合了CdTe纳米粒子稳定性好、荧光强度高以及HgTe发射光谱可调性好的优点,通过调节温育时间、温育温度、成分比例等手段,可以合成具有不同近红外发射波长的纳米粒子,这种方法简便易行,也更为稳定。同时基于在蛋白质存在下CdHgTe纳米粒子的荧光强度明显降低这一现象,建立了一种以CdHgTe为近红外荧光探针测定蛋白质的方法。在最佳条件下,溶菌酶(Lyz)和血红蛋白(BHb)的工作曲线的线性范围分别为0.04-5.6×10~(-6)g·mL~(-1)和0.06-6.1×10~(-6)g·mL~(-1),检测限分别为13 ng·mL~(-1)和27 ng·mL~(-1),并对合成样品进行了测定,结果令人满意。
(3)对含有不同端基及链长的接枝试剂对TGA-CdTe纳米粒子荧光性质的影响进行了研究,以荧光偏振法探讨了不同接枝试剂与TGA-CdTe纳米粒子间的作用机理。实验结果表明,短链的接枝试剂对TGA-CdTe荧光强度的增强效果好于长链的,接枝对于TGA-CdTe的荧光发射波长没有影响。同时发现,在较宽的pH范围内,接枝后以羧基为端基的TGA-CdTe的稳定性好于以氨基为端基的TGA-CdTe。
(4)利用TGA、L-cys及二者比例不同的混合稳定剂制备了球形、棒形纳米晶,以TEM表征了不同稳定剂条件下制备的纳米晶的形貌特征,研究了不同形貌的纳米晶的荧光特性。实验结果表明,仅通过改变稳定剂的比例即可制备波长可调的不同形貌的纳米晶。随着混合稳定剂中L-cys比例的增加,CdTe纳米晶的长与直径的比值增大,发射波长明显红移。当混合稳定剂中L-cys的量为25%时,所得纳米晶荧光强度最强。
(5)结合分子印迹技术的高选择性与纳米荧光探针荧光检测技术的高灵敏性,建立了选择性测定Lyz的方法。为便于调整与处理洗脱条件、易于与检测条件结合、降低预处理的干扰,实验采用了具有环境响应和智能识别性能的N-异丙基丙烯酰胺(NIPA)作为主要功能单体,以MIP-PNIPA智能水凝胶对Lyz进行印迹。研究了印迹过程中影响印迹效果及洗脱效果的温度、盐浓度、pH值的影响。实验结果表明:MIP-PNIPA在室温、pH 8.10的Tris-HCl缓冲、NaCl浓度为20 mM条件下对Lyz有较好的吸附效果;在室温、pH 6.10的柠檬酸-柠檬酸钠缓冲条件下有较好的洗脱效果,且对荧光检测影响较小。对混合样品进行处理后,以CdTe为荧光探针,pH 8.10条件下测定了洗脱后的Lyz的浓度,回收率为90.2～102%。本章所建立的研究方法为蛋白质的测定提供了一个可供参考的新思路。
In recent years,semiconductor quantum dots(QDs) have attracted great interests in synthesis and application due to their extensive physical and chemical effects.In this thesis,a novel method for the determination of proteins with CdHgTe QDs as a near-infrared fluorescence probe was developed;Based on the synthesis,the influence of the graft reagents which have an amino or a carboxyl terminus with different chain lengthes on the fluorescence properties of water-soluble thioglycolic acid stabilized CdTe nanocrystals(TGA-CdTe) were investigated;Sphere and rod CdTe NCs were prepared in aqueous solution using thioglycolic acid(TGA), L-cysteine(L-cys) and both of them as stabilizers;Furthermore,combining MIP and the QDs could improve the selective detection of the protein.Major works of this thesis can be outlined as follows:
(1) The fundamental theory of QDs,general strategies of QDs preparation, character method and their extensive application on bioscience are reviewed.
(2) CdHgTe nanoparticles(NPs) with the emission in the near-infrared region were prepared in aqueous solution,and were characterized by transmission electron microscopy,X-ray diffraction spectrometry,spectrofluorometry and ultraviolet-visible spectrophotometry.The synthesized CdHgTe NPs show much promise because their NIR wavelength can be tuned by the composition and the incubation temperature.CdHgTe NPs combined the virtue of the stable and high fluorescence intensity of CdTe NPs and the NIR wavelength tuning of HgTe NPs.On one hand,the tuning of the emission wavelength of CdHgTe NPs is easier than that of single material NPs(CdTe or HgTe) by controlling the synthetic conditions.On the other hand,CdHgTe NPs are stable than HgTe NPs. Based on the fluorescence quenching of CdHgTe NPs in the presence of proteins, a novel method for the determination of proteins with CdHgTe NPs as a near-infrared fluorescence probe was developed.Under the optimal conditions, the calibration graphs were linear in the range of 0.04×10~(-6)-5.6×10~(-6) g·mL~(-1) for lysozyme(Lyz) and 0.06×10~(-6)-6.1×10~(-6) g·mL~(-1) for bovine hemoglobin(BHb), respectively.The limits of detection were 13 ng·mL~(-1) for Lyz and 27 ng·mL~(-1) for BHb,respectively.Four synthetic samples were determined and the results were satisfied.
(3) The influences of the graft reagents which have an amino or a carboxyl terminus with different chain lengthes on the fluorescence properties of water-soluble thioglycolic acid stabilized CdTe nanocrystals(TGA-CdTe) were systematically investigated.Strong enhancement effects of the grafting on the fluorescence intensity of TGA-CdTe were observed.The experiment results demonstrated the short chain length grafting can increase the fluorescence intensity of the CdTe nanocrystals(NCs) better than that of long chain length grafting,and the grafting did not influence the emission wavelength of the CdTe NCs.The fluorescence intensity of the carboxyl grafted TGA-CdTe was more stable than that of the amino grafted TGA-CdTe at wide pH ranges(pH 5.1-10.0).
(4) Sphere and rod CdTe NCs were prepared in aqueous solution using thioglycolic acid(TGA),L-cysteine(L-cys) and mixture of them as stabilizers.By only changing the molar ratio of the two stabilizers,highly luminescent CdTe NCs can be selectively prepared.With the increasing amount of the molar fraction of L-cys in the mixed stabilizers of TGA and L-cys,the aspect ratio of the NCs was increased and the emission wavelength was shifted to long wavelength range. When the molar fraction of the L-cys in the mixed stabilizers was 25%,the fluorescence intensity of the NCs was the highest.
(5) By combining the virtue of the sensitivity of CdTe and the selectivity of the MIP,a selective and sensitive fluorescence method for the determination of Lyz was developed.Since NIPA polymer undergoes a reversible swelling-shrinking cycle in response to the external environment,the rebinding ability and elute are controllable by changing the solution environment,which was convenient for the next determination step.Different influencing factors were studied,including the pH value,concentration of NaCl and temperature.The experiment results demonstrated that the optimized adsorption conditions were as follows:room temperature,Tris-HCl buffer solution with pH 8.10,20 mM NaCl.The optimal elution condition was citric acid-triple sodium citric acid buffer with pH 6.10. The Lyz in three synthetic samples were determined with CdTe as the fluorescence probe after the synthetic samples were pretreated by MIP-PNIPA, and the recoveries were 90.2～102%.This method provided a new way to study the determination of proteins.
(1)综述了半导体纳米粒子的相关基本知识、主要合成方法、表征手段及其在生命科学领域中的广泛应用。
(2)在水相中制备了具有近红外发射波长的CdHgTe纳米粒子,对纳米粒子的合成条件进行了系统的研究,以TEM和XRD表征了CdTe和CdHgTe的形貌特征及晶体结构,研究了含不同比例的Hg(Ⅱ)的CdHgTe的荧光光谱及紫外光谱性质。实验结果表明,CdHgTe结合了CdTe纳米粒子稳定性好、荧光强度高以及HgTe发射光谱可调性好的优点,通过调节温育时间、温育温度、成分比例等手段,可以合成具有不同近红外发射波长的纳米粒子,这种方法简便易行,也更为稳定。同时基于在蛋白质存在下CdHgTe纳米粒子的荧光强度明显降低这一现象,建立了一种以CdHgTe为近红外荧光探针测定蛋白质的方法。在最佳条件下,溶菌酶(Lyz)和血红蛋白(BHb)的工作曲线的线性范围分别为0.04-5.6×10~(-6)g·mL~(-1)和0.06-6.1×10~(-6)g·mL~(-1),检测限分别为13 ng·mL~(-1)和27 ng·mL~(-1),并对合成样品进行了测定,结果令人满意。
(3)对含有不同端基及链长的接枝试剂对TGA-CdTe纳米粒子荧光性质的影响进行了研究,以荧光偏振法探讨了不同接枝试剂与TGA-CdTe纳米粒子间的作用机理。实验结果表明,短链的接枝试剂对TGA-CdTe荧光强度的增强效果好于长链的,接枝对于TGA-CdTe的荧光发射波长没有影响。同时发现,在较宽的pH范围内,接枝后以羧基为端基的TGA-CdTe的稳定性好于以氨基为端基的TGA-CdTe。
(4)利用TGA、L-cys及二者比例不同的混合稳定剂制备了球形、棒形纳米晶,以TEM表征了不同稳定剂条件下制备的纳米晶的形貌特征,研究了不同形貌的纳米晶的荧光特性。实验结果表明,仅通过改变稳定剂的比例即可制备波长可调的不同形貌的纳米晶。随着混合稳定剂中L-cys比例的增加,CdTe纳米晶的长与直径的比值增大,发射波长明显红移。当混合稳定剂中L-cys的量为25%时,所得纳米晶荧光强度最强。
(5)结合分子印迹技术的高选择性与纳米荧光探针荧光检测技术的高灵敏性,建立了选择性测定Lyz的方法。为便于调整与处理洗脱条件、易于与检测条件结合、降低预处理的干扰,实验采用了具有环境响应和智能识别性能的N-异丙基丙烯酰胺(NIPA)作为主要功能单体,以MIP-PNIPA智能水凝胶对Lyz进行印迹。研究了印迹过程中影响印迹效果及洗脱效果的温度、盐浓度、pH值的影响。实验结果表明:MIP-PNIPA在室温、pH 8.10的Tris-HCl缓冲、NaCl浓度为20 mM条件下对Lyz有较好的吸附效果;在室温、pH 6.10的柠檬酸-柠檬酸钠缓冲条件下有较好的洗脱效果,且对荧光检测影响较小。对混合样品进行处理后,以CdTe为荧光探针,pH 8.10条件下测定了洗脱后的Lyz的浓度,回收率为90.2～102%。本章所建立的研究方法为蛋白质的测定提供了一个可供参考的新思路。
In recent years,semiconductor quantum dots(QDs) have attracted great interests in synthesis and application due to their extensive physical and chemical effects.In this thesis,a novel method for the determination of proteins with CdHgTe QDs as a near-infrared fluorescence probe was developed;Based on the synthesis,the influence of the graft reagents which have an amino or a carboxyl terminus with different chain lengthes on the fluorescence properties of water-soluble thioglycolic acid stabilized CdTe nanocrystals(TGA-CdTe) were investigated;Sphere and rod CdTe NCs were prepared in aqueous solution using thioglycolic acid(TGA), L-cysteine(L-cys) and both of them as stabilizers;Furthermore,combining MIP and the QDs could improve the selective detection of the protein.Major works of this thesis can be outlined as follows:
(1) The fundamental theory of QDs,general strategies of QDs preparation, character method and their extensive application on bioscience are reviewed.
(2) CdHgTe nanoparticles(NPs) with the emission in the near-infrared region were prepared in aqueous solution,and were characterized by transmission electron microscopy,X-ray diffraction spectrometry,spectrofluorometry and ultraviolet-visible spectrophotometry.The synthesized CdHgTe NPs show much promise because their NIR wavelength can be tuned by the composition and the incubation temperature.CdHgTe NPs combined the virtue of the stable and high fluorescence intensity of CdTe NPs and the NIR wavelength tuning of HgTe NPs.On one hand,the tuning of the emission wavelength of CdHgTe NPs is easier than that of single material NPs(CdTe or HgTe) by controlling the synthetic conditions.On the other hand,CdHgTe NPs are stable than HgTe NPs. Based on the fluorescence quenching of CdHgTe NPs in the presence of proteins, a novel method for the determination of proteins with CdHgTe NPs as a near-infrared fluorescence probe was developed.Under the optimal conditions, the calibration graphs were linear in the range of 0.04×10~(-6)-5.6×10~(-6) g·mL~(-1) for lysozyme(Lyz) and 0.06×10~(-6)-6.1×10~(-6) g·mL~(-1) for bovine hemoglobin(BHb), respectively.The limits of detection were 13 ng·mL~(-1) for Lyz and 27 ng·mL~(-1) for BHb,respectively.Four synthetic samples were determined and the results were satisfied.
(3) The influences of the graft reagents which have an amino or a carboxyl terminus with different chain lengthes on the fluorescence properties of water-soluble thioglycolic acid stabilized CdTe nanocrystals(TGA-CdTe) were systematically investigated.Strong enhancement effects of the grafting on the fluorescence intensity of TGA-CdTe were observed.The experiment results demonstrated the short chain length grafting can increase the fluorescence intensity of the CdTe nanocrystals(NCs) better than that of long chain length grafting,and the grafting did not influence the emission wavelength of the CdTe NCs.The fluorescence intensity of the carboxyl grafted TGA-CdTe was more stable than that of the amino grafted TGA-CdTe at wide pH ranges(pH 5.1-10.0).
(4) Sphere and rod CdTe NCs were prepared in aqueous solution using thioglycolic acid(TGA),L-cysteine(L-cys) and mixture of them as stabilizers.By only changing the molar ratio of the two stabilizers,highly luminescent CdTe NCs can be selectively prepared.With the increasing amount of the molar fraction of L-cys in the mixed stabilizers of TGA and L-cys,the aspect ratio of the NCs was increased and the emission wavelength was shifted to long wavelength range. When the molar fraction of the L-cys in the mixed stabilizers was 25%,the fluorescence intensity of the NCs was the highest.
(5) By combining the virtue of the sensitivity of CdTe and the selectivity of the MIP,a selective and sensitive fluorescence method for the determination of Lyz was developed.Since NIPA polymer undergoes a reversible swelling-shrinking cycle in response to the external environment,the rebinding ability and elute are controllable by changing the solution environment,which was convenient for the next determination step.Different influencing factors were studied,including the pH value,concentration of NaCl and temperature.The experiment results demonstrated that the optimized adsorption conditions were as follows:room temperature,Tris-HCl buffer solution with pH 8.10,20 mM NaCl.The optimal elution condition was citric acid-triple sodium citric acid buffer with pH 6.10. The Lyz in three synthetic samples were determined with CdTe as the fluorescence probe after the synthetic samples were pretreated by MIP-PNIPA, and the recoveries were 90.2～102%.This method provided a new way to study the determination of proteins.
引文
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