铕、金纳米颗粒与蛋白质的相互作用及分析应用的研究
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摘要
【目的】研究嗅标记蛋白(Olfactory Marker Protein,OMP)对铕、金纳米颗粒的荧光增强效应,建立灵敏的测定蛋白质含量的分析方法,探讨铕、金纳米颗粒与蛋白质相互作用的机理。
【方法】
1.铕纳米颗粒与蛋白质的相互作用及分析应用的研究
在Eu(NO_3)_3溶液中加入还原剂单宁酸,30℃条件下剧烈搅拌30min,冷却至室温后离心、洗涤、定容即得到铕纳米颗粒。在搅拌过程中缓慢加入修饰剂硫辛酸,冷却至室温后离心、洗涤、定容,再加入嗅标记蛋白OMP,37℃条件下水浴8h,可将OMP连接到铕纳米颗粒。研究OMP对铕纳米颗粒的荧光增强效应和OMP含量与荧光强度变化的关系。同时应用共振光散射技术、吸收光谱技术、透射电子显微技术、圆二色谱技术等手段进行机理研究和讨论。
2.金纳米颗粒与蛋白质的相互作用及分析应用的研究
在煮沸搅拌条件下向氯金酸溶液中加入还原剂柠檬酸三钠,继续加热搅拌15 min后停止加热,冷却至室温后离心、洗涤、定容即得到金纳米颗粒。在搅拌过程中缓慢加入修饰剂硫辛酸,冷却至室温后离心、洗涤、定容,再加入嗅标记蛋白OMP,在37℃条件下水浴8h,可制得与OMP结合的金纳米颗粒。研究OMP对金纳米颗粒近红外荧光增强作用以及OMP含量与荧光强度增强的关系。同时应用共振光散射技术、吸收光谱技术、透射电子显微技术、圆二色谱技术等手段对荧光增强效应机理进行研究和讨论。
【结果】
1.铕纳米颗粒与蛋白质的相互作用及分析应用的研究
在271nm的激发波长下,OMP的加入能显著增强铕纳米颗粒在379nm处的荧光强度,且荧光增强程度与OMP的含量在一定范围内呈正比。在最佳实验条件下,测定OMP、HRP、P450、BSA和NSE的线性范围分别为2.0×10~(-8)-8.0×10~(-6)g/mL、2.0×10~(-8)-9.0×10~(-6)g/mL、6.0×10~(-8)-1.2×10~(-5)g/mL、6.0×10~(-8)-1.0×10~(-5)g/mL和3.0×10~(-8)-1.2×10~(-5)g/mL。它们的检出限分别为9.8×10~(-9)g/mL、1.0×10~(-8)/mL、2.9×10~(-8)g/mL、3.2×10~(-8)g/mL和1.2×10~(-8)g/mL。该方法用于模拟样品中OMP含量的测定,结果令人满意。机理研究表明,铕纳米颗粒、硫辛酸与OMP发生了相互作用,使得OMP的构象发生变化,形成了铕-硫辛酸-OMP络合物,在271 nm的激发波长下,体系可能发生了能量传递过程,即OMP中的色氨酸残基和酪氨酸残基将吸收的能量通过硫辛酸这个中介体传递给铕纳米颗粒,从而发射铕纳米颗粒的荧光峰,荧光强度明显增强。
2.金纳米颗粒与蛋白质的相互作用及分析应用的研究
实验表明,在538nm的激发波长下,OMP的加入可以显著增强金纳米颗粒在近红外区的811nm处的荧光强度,并且荧光增强程度与OMP的含量在一定范围内呈正比,可用于蛋白质含量的测定。在最佳实验条件下,检测OMP、HRP、P450、BSA和NSE的线性范围分别为2.0×10~(-8)-9.0×10~(-6)g/mL、3.0×10~(-8)-8.0×10~(-6)g/mL、8.0×10~(-8)-1.2×10~(-5)g/mL、5.0×10~(-8)-1.0×10~(-5)g/mL和5.0×10~(-8)-8.0×10~(-6)g/mL。检出限分别为6.2×10~(-9)g/mL、8.4×10~(-9)g/mL、5.5×10~(-9)g/mL、6.0×10~(-9)g/mL和9.7×10~(-9)g/mL。可见该方法有较高的灵敏度和较宽的线性范围。对模拟样品采用标准加入法测定OMP含量,并将结果和用紫外吸收法测定的结果进行比较,结果精密度和准确度均比较满意。机理研究表明,带有巯基的硫辛酸将金纳米颗粒与OMP连结起来,形成了金-硫辛酸-OMP复合物,并定向排列聚集成超晶格结构,导致表面等离子体传播特性改变,使金纳米颗粒近红外区的荧光强度显著增强。
【结论】
1.OMP可以显著增强铕纳米颗粒的荧光强度和金纳米颗粒在近红外区的荧光强度。
2.利用蛋白质对铕、金纳米颗粒的荧光增强效应可以建立测定蛋白质含量的新方法,结果令人满意。
3.在铕纳米颗粒、硫辛酸、OMP三者间的能量传递过程使铕纳米颗粒的荧光强度明显增强,而OMP对金纳米颗粒的荧光增强效应则是由表面等离子体传播特性改变引起的。
本论文的主要特点和创新点:
1.利用稀土元素良好的发光性能和金属纳米材料检测生物分子灵敏度高的特点制备了铕纳米颗粒,探索建立测定蛋白质含量的分析方法。
2.在前人工作的基础上研究了蛋白质对金纳米颗粒近红外荧光强度的增强效应,提高其测定蛋白质含量的灵敏度。
3.利用荧光技术、光散射技术、吸收光谱技术、透射电子显微技术等多种手段研究了体系作用的机理。
Objective
This research aims to study the effect of olfactory marker protein(OMP)on the fluorescence enhancement of europium and gold nanoparticles,to develop a sensitive method for the quantitative determination of proteins,and to study the interacting mechanism between europium,gold nanoparticles and proteins.
Methods
1.Interactions and applications between europium nanoparticles and proteins
The Eu(NO_3)_3 solution was reduced by tannic acid under vigorous stirring at 30℃water bath for 30 min and cooled to ambient temperature.Europium nanoparticles were obtained after the solution was centrifuged,scrubbed and metered volume. Nanoeuropium-OMP conjugate was synthesized by the similar method.Lipoic acid was added while the solution was stirred and OMP was added to the solution at 37℃water bath for 8 hours.The fluorophotometer was used to observe the effect of OMP on the fluorescence enhancement of europium nanoparticles and relationship between fluorescence changes and the concentration of OMP.The resonance light scattering, absorption spectra,transmission electron microscope and circular dichroism spectra techniques were also used to study the interacting mechanism between europium nanoparticles and proteins.
2.Interactions and applications between gold nanoparticles and proteins
The AuCl_3 solution was reduced by trisodium citrate under vigorous stirring at boiling for 15 min and cooled to ambient temperature.Gold nanoparticles were obtained after the solution was centrifuged,scrubbed and metered volume.Nanogold-OMP conjugate was synthesized by the similar method.Lipoic acid was added while the solution was stirred and OMP was added to the solution at 37℃water bath for 8 hours.The fluorophotometer was used to observe the effect of OMP on the near-infrared fluorescence enhancement of gold nanoparticles and relationship between fluorescence changes and the concentration of OMP. The resonance light scattering,absorption spectra,transmission electron microscope and circular dichroism spectra techniques were also used to study the interacting mechanism between gold nanoparticles and proteins.
Results
1.Interactions and applications between europium nanoparticles and proteins
Under the excitation of 271 nm,the fluorescence intensity at 379 nm of europium nanoparticles can be greatly enhanced by the addition of OMP.Under the optimum conditions,there is a linear relationship between the enhancement of fluorescence intensity and the concentration of proteins in the range of 2.0×10~(-8)to 8.0×10~(-6)g/mL for OMP, 2.0×10~(-8)to 9.0×10~(-6)g/mL for HRP,6.0×10~(-8)to 1.2×10~(-5)g/mL for P450,6.0×10~(-8)to 1.0×10~(-5)g/mL for BSA and 3.0×10~(-8)to 1.2×10~(-5)g/mL for NSE,and their limits of detection are 9.8×10~(-9)g/mL,1.0×10~(-8)g/mL,2.9×10~(-8)g/mL,3.2×10~(-8)g/mL and 1.2×10~(-8)g/mL.This method can be used to determine the concentration of OMP in simulated sample and the result is satisfactory.The study of mechanism indicates that the interaction of europium nanoparticles,lipoic acid and OMP makes the construction of OMP change to form Eu-lipoic acid-OMP conjugate.Under the excitation of 271 nm,an energy transfer process may occur in this system,that is,tryptophane and tyrosine residue of OMP transmit the absorbed energy to europium nanoparticles through lipoic acid and notably enhance the fluorescence intensity.
2.Interactions and applications between gold nanoparticles and proteins
Experiment indicates that the near-infrared fluorescence intensity at 811 nm of gold nanoparticles can be significantly enhanced by the presence of OMP under the excitation of 538 nm.Based on this,a sensitive method for the quantitative determination of proteins was established.Under the optimum conditions,there is a linear relationship between the enhancement of fluorescence intensity and the concentration of proteins in the range of 2.0×10~(-8)to 9.0×10~(-6)g/mL for OMP,3.0×10~(-8)to 8.0×10~(-6)g/mL for HRP,8.0×10~(-8)to 1.2×10~(-5)g/mL for P450,5.0×10~(-8)to 1.0×10~(-5)g/mL for BSA and 5.0×10~(-8)to 8.0×10~(-6)g/mL for NSE,and their limits of detection are 6.2×10~(-9)g/mL,8.4×10~(-9)g/mL,5.5×10~(-9)g/mL, 6.0×10~(-9)g/mL and 9.7×10~(-9)g/mL.This method can be used to determine the concentration of OMP in simulated sample and the result is satisfactory.The mechanism research shows that lipoic acid connects gold nanoparticles and OMP by hydrosulfuryl to form Au-lipoic acid-OMP compound.This conjugate has crystal lattice construction and causes the change in transmission property of surface plasma,thus notably enhances the near-infrared fluorescence intensity of gold nanoparticles.
Conclusions
1.OMP can greatly enhance the fluorescence intensity of europium nanoparticles as well as the near-infrared fluorescence intensity of gold nanoparticles.
2.The effect of OMP on the fluorescence enhancement of europium nanoparticles and gold nanoparticles can be used to determine the concentration of proteins and the result is satisfactory.
3.The energy transfer among europium nanoparticles,lipoic acid and OMP may enhance the fluorescence intensity of europium nanoparticles,while the fluorescence enhancement of gold nanoparticles by OMP is caused by the change in transmission property of surface plasma.
The main features and creative points of this thesis are as follows:
1.Make use of good luminescent property of lanthanide and high sensitivity of nano-metal material in biomolecule detection to prepare europium nanoparticles and establish a novel method for protein determination.
2.Study the effect of OMP on the near-infrared fluorescence enhancement of gold nanoparticles based on past research to raise sensitivity of protein determination.
3.Use many research techniques,such as fluorescence,resonance light scattering, absorption spectra,transmission electron microscope to investigate the interacting mechanism of these systems.
【方法】
1.铕纳米颗粒与蛋白质的相互作用及分析应用的研究
在Eu(NO_3)_3溶液中加入还原剂单宁酸,30℃条件下剧烈搅拌30min,冷却至室温后离心、洗涤、定容即得到铕纳米颗粒。在搅拌过程中缓慢加入修饰剂硫辛酸,冷却至室温后离心、洗涤、定容,再加入嗅标记蛋白OMP,37℃条件下水浴8h,可将OMP连接到铕纳米颗粒。研究OMP对铕纳米颗粒的荧光增强效应和OMP含量与荧光强度变化的关系。同时应用共振光散射技术、吸收光谱技术、透射电子显微技术、圆二色谱技术等手段进行机理研究和讨论。
2.金纳米颗粒与蛋白质的相互作用及分析应用的研究
在煮沸搅拌条件下向氯金酸溶液中加入还原剂柠檬酸三钠,继续加热搅拌15 min后停止加热,冷却至室温后离心、洗涤、定容即得到金纳米颗粒。在搅拌过程中缓慢加入修饰剂硫辛酸,冷却至室温后离心、洗涤、定容,再加入嗅标记蛋白OMP,在37℃条件下水浴8h,可制得与OMP结合的金纳米颗粒。研究OMP对金纳米颗粒近红外荧光增强作用以及OMP含量与荧光强度增强的关系。同时应用共振光散射技术、吸收光谱技术、透射电子显微技术、圆二色谱技术等手段对荧光增强效应机理进行研究和讨论。
【结果】
1.铕纳米颗粒与蛋白质的相互作用及分析应用的研究
在271nm的激发波长下,OMP的加入能显著增强铕纳米颗粒在379nm处的荧光强度,且荧光增强程度与OMP的含量在一定范围内呈正比。在最佳实验条件下,测定OMP、HRP、P450、BSA和NSE的线性范围分别为2.0×10~(-8)-8.0×10~(-6)g/mL、2.0×10~(-8)-9.0×10~(-6)g/mL、6.0×10~(-8)-1.2×10~(-5)g/mL、6.0×10~(-8)-1.0×10~(-5)g/mL和3.0×10~(-8)-1.2×10~(-5)g/mL。它们的检出限分别为9.8×10~(-9)g/mL、1.0×10~(-8)/mL、2.9×10~(-8)g/mL、3.2×10~(-8)g/mL和1.2×10~(-8)g/mL。该方法用于模拟样品中OMP含量的测定,结果令人满意。机理研究表明,铕纳米颗粒、硫辛酸与OMP发生了相互作用,使得OMP的构象发生变化,形成了铕-硫辛酸-OMP络合物,在271 nm的激发波长下,体系可能发生了能量传递过程,即OMP中的色氨酸残基和酪氨酸残基将吸收的能量通过硫辛酸这个中介体传递给铕纳米颗粒,从而发射铕纳米颗粒的荧光峰,荧光强度明显增强。
2.金纳米颗粒与蛋白质的相互作用及分析应用的研究
实验表明,在538nm的激发波长下,OMP的加入可以显著增强金纳米颗粒在近红外区的811nm处的荧光强度,并且荧光增强程度与OMP的含量在一定范围内呈正比,可用于蛋白质含量的测定。在最佳实验条件下,检测OMP、HRP、P450、BSA和NSE的线性范围分别为2.0×10~(-8)-9.0×10~(-6)g/mL、3.0×10~(-8)-8.0×10~(-6)g/mL、8.0×10~(-8)-1.2×10~(-5)g/mL、5.0×10~(-8)-1.0×10~(-5)g/mL和5.0×10~(-8)-8.0×10~(-6)g/mL。检出限分别为6.2×10~(-9)g/mL、8.4×10~(-9)g/mL、5.5×10~(-9)g/mL、6.0×10~(-9)g/mL和9.7×10~(-9)g/mL。可见该方法有较高的灵敏度和较宽的线性范围。对模拟样品采用标准加入法测定OMP含量,并将结果和用紫外吸收法测定的结果进行比较,结果精密度和准确度均比较满意。机理研究表明,带有巯基的硫辛酸将金纳米颗粒与OMP连结起来,形成了金-硫辛酸-OMP复合物,并定向排列聚集成超晶格结构,导致表面等离子体传播特性改变,使金纳米颗粒近红外区的荧光强度显著增强。
【结论】
1.OMP可以显著增强铕纳米颗粒的荧光强度和金纳米颗粒在近红外区的荧光强度。
2.利用蛋白质对铕、金纳米颗粒的荧光增强效应可以建立测定蛋白质含量的新方法,结果令人满意。
3.在铕纳米颗粒、硫辛酸、OMP三者间的能量传递过程使铕纳米颗粒的荧光强度明显增强,而OMP对金纳米颗粒的荧光增强效应则是由表面等离子体传播特性改变引起的。
本论文的主要特点和创新点:
1.利用稀土元素良好的发光性能和金属纳米材料检测生物分子灵敏度高的特点制备了铕纳米颗粒,探索建立测定蛋白质含量的分析方法。
2.在前人工作的基础上研究了蛋白质对金纳米颗粒近红外荧光强度的增强效应,提高其测定蛋白质含量的灵敏度。
3.利用荧光技术、光散射技术、吸收光谱技术、透射电子显微技术等多种手段研究了体系作用的机理。
Objective
This research aims to study the effect of olfactory marker protein(OMP)on the fluorescence enhancement of europium and gold nanoparticles,to develop a sensitive method for the quantitative determination of proteins,and to study the interacting mechanism between europium,gold nanoparticles and proteins.
Methods
1.Interactions and applications between europium nanoparticles and proteins
The Eu(NO_3)_3 solution was reduced by tannic acid under vigorous stirring at 30℃water bath for 30 min and cooled to ambient temperature.Europium nanoparticles were obtained after the solution was centrifuged,scrubbed and metered volume. Nanoeuropium-OMP conjugate was synthesized by the similar method.Lipoic acid was added while the solution was stirred and OMP was added to the solution at 37℃water bath for 8 hours.The fluorophotometer was used to observe the effect of OMP on the fluorescence enhancement of europium nanoparticles and relationship between fluorescence changes and the concentration of OMP.The resonance light scattering, absorption spectra,transmission electron microscope and circular dichroism spectra techniques were also used to study the interacting mechanism between europium nanoparticles and proteins.
2.Interactions and applications between gold nanoparticles and proteins
The AuCl_3 solution was reduced by trisodium citrate under vigorous stirring at boiling for 15 min and cooled to ambient temperature.Gold nanoparticles were obtained after the solution was centrifuged,scrubbed and metered volume.Nanogold-OMP conjugate was synthesized by the similar method.Lipoic acid was added while the solution was stirred and OMP was added to the solution at 37℃water bath for 8 hours.The fluorophotometer was used to observe the effect of OMP on the near-infrared fluorescence enhancement of gold nanoparticles and relationship between fluorescence changes and the concentration of OMP. The resonance light scattering,absorption spectra,transmission electron microscope and circular dichroism spectra techniques were also used to study the interacting mechanism between gold nanoparticles and proteins.
Results
1.Interactions and applications between europium nanoparticles and proteins
Under the excitation of 271 nm,the fluorescence intensity at 379 nm of europium nanoparticles can be greatly enhanced by the addition of OMP.Under the optimum conditions,there is a linear relationship between the enhancement of fluorescence intensity and the concentration of proteins in the range of 2.0×10~(-8)to 8.0×10~(-6)g/mL for OMP, 2.0×10~(-8)to 9.0×10~(-6)g/mL for HRP,6.0×10~(-8)to 1.2×10~(-5)g/mL for P450,6.0×10~(-8)to 1.0×10~(-5)g/mL for BSA and 3.0×10~(-8)to 1.2×10~(-5)g/mL for NSE,and their limits of detection are 9.8×10~(-9)g/mL,1.0×10~(-8)g/mL,2.9×10~(-8)g/mL,3.2×10~(-8)g/mL and 1.2×10~(-8)g/mL.This method can be used to determine the concentration of OMP in simulated sample and the result is satisfactory.The study of mechanism indicates that the interaction of europium nanoparticles,lipoic acid and OMP makes the construction of OMP change to form Eu-lipoic acid-OMP conjugate.Under the excitation of 271 nm,an energy transfer process may occur in this system,that is,tryptophane and tyrosine residue of OMP transmit the absorbed energy to europium nanoparticles through lipoic acid and notably enhance the fluorescence intensity.
2.Interactions and applications between gold nanoparticles and proteins
Experiment indicates that the near-infrared fluorescence intensity at 811 nm of gold nanoparticles can be significantly enhanced by the presence of OMP under the excitation of 538 nm.Based on this,a sensitive method for the quantitative determination of proteins was established.Under the optimum conditions,there is a linear relationship between the enhancement of fluorescence intensity and the concentration of proteins in the range of 2.0×10~(-8)to 9.0×10~(-6)g/mL for OMP,3.0×10~(-8)to 8.0×10~(-6)g/mL for HRP,8.0×10~(-8)to 1.2×10~(-5)g/mL for P450,5.0×10~(-8)to 1.0×10~(-5)g/mL for BSA and 5.0×10~(-8)to 8.0×10~(-6)g/mL for NSE,and their limits of detection are 6.2×10~(-9)g/mL,8.4×10~(-9)g/mL,5.5×10~(-9)g/mL, 6.0×10~(-9)g/mL and 9.7×10~(-9)g/mL.This method can be used to determine the concentration of OMP in simulated sample and the result is satisfactory.The mechanism research shows that lipoic acid connects gold nanoparticles and OMP by hydrosulfuryl to form Au-lipoic acid-OMP compound.This conjugate has crystal lattice construction and causes the change in transmission property of surface plasma,thus notably enhances the near-infrared fluorescence intensity of gold nanoparticles.
Conclusions
1.OMP can greatly enhance the fluorescence intensity of europium nanoparticles as well as the near-infrared fluorescence intensity of gold nanoparticles.
2.The effect of OMP on the fluorescence enhancement of europium nanoparticles and gold nanoparticles can be used to determine the concentration of proteins and the result is satisfactory.
3.The energy transfer among europium nanoparticles,lipoic acid and OMP may enhance the fluorescence intensity of europium nanoparticles,while the fluorescence enhancement of gold nanoparticles by OMP is caused by the change in transmission property of surface plasma.
The main features and creative points of this thesis are as follows:
1.Make use of good luminescent property of lanthanide and high sensitivity of nano-metal material in biomolecule detection to prepare europium nanoparticles and establish a novel method for protein determination.
2.Study the effect of OMP on the near-infrared fluorescence enhancement of gold nanoparticles based on past research to raise sensitivity of protein determination.
3.Use many research techniques,such as fluorescence,resonance light scattering, absorption spectra,transmission electron microscope to investigate the interacting mechanism of these systems.
引文
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