金属纳米粒子在生物成像与生物医学上的应用
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摘要
随着纳米科技与纳米工艺的不断发展，贵金属纳米粒子以其优良的物理化学性质，越来越多地受到广大研究人员的关注，并被广泛应用于生物医学与生命科学等领域。金属纳米粒子作为光学探针，拥有其他传统光学探针无法比拟的优势，如光稳定性好、光学信号强、易于制备与修饰，生物兼容性良好等等，在化学分析与生物传感、生物医学与生物成像等方面具有广阔的应用前景。基于金属纳米粒子光学特性的单粒子成像与单粒子光谱技术，具有纳米尺度空间分辨率以及毫秒级时间分辨率的特点，已经成为在单细胞层面上研究生物分子的行为与功能的重要手段。具有高表面化学活性的金属纳米粒子，也被作为性能优良的纳米催化剂用于化工与环境催化、生物化学与生物医药等研究方向。然而，人们在利用金属纳米粒子进行科学研究与实际应用时，仍然面临着许多挑战，如纳米粒子其尺寸和形貌不均一引起的检测灵敏度下降，现有的分析手段难以满足实际需求、复杂体现干扰难以排除等等。
本论文围绕以上提出的问题与难点开展研究，在已有的研究工作基础之上，开展了以下主要工作：
(1)采用密度梯度离心法对纳米棒进行了分离纯化。在纳米粒子稳态沉降过程中受力分析基础之上，推导了不同尺寸与形状的纳米粒子沉降速率的计算公式。利用其在离心过程中沉降速率的差异，采用不同浓度的蔗糖溶液配制的密度梯度液对不同长径比的棒状金纳粒子成功地进行了分离，并考察了梯度液组成以及离心时间对于分离效率的影响。实验结果与计算结果均表明，尽管存在着特殊情形下无法分离的局限性，但离心分离法仍能够有效地对具有不同长径比和不同尺寸的纳米粒子进行分离纯化。(第二章)
(2)系统性地研究了金纳米棒的光学特性。考察了基于Gans理论的金纳米棒等离子共振吸收与散射光谱计算公式，探讨了产生等离子共振的条件并推导了其等离光谱偏移量与介质溶液折射率变化之间的关系式。实验结果显示，金纳米棒的光谱偏移了随着介质折射率变化的增大而发生红移，而光谱变化的灵敏度也随着金纳米棒长径比的增大而增加，与计算结果完全一致。此外，还考察了采用单粒子光谱进行检测时的各种干扰因素。(第三章)
(3)以金银壳核结构的棒状纳米粒子为探针，采用传统紫外可见吸收光谱仪和单粒子暗场光谱显微镜对硫化物进行了高灵敏度高选择性的检测，并获得了比传统方法更宽的动态检测范围和更低的检测下限。采用紫外可见吸收光谱与单粒子暗场光谱显微镜进行检测的动态范围分别为0.1μM~1mM和0.01nM~10μM，该结果说明基于单粒子光谱成像技术的检测手段比宏观光谱测量具有更高的灵敏度以及更低的检测下限。(第四章)
(4)基于金银壳核结构的棒状纳米探针与硫化物的反应动力学过程，发展了一种在单个纳米粒子水平上对活细胞内硫化氢的浓度及其变化进行实时检测的新方法。通过推导该反应动力学过程与探针光谱偏移速率之间的关系式，建立了硫化物浓度变化与探针光谱变化速率的关系。进一步，我们通过实际测量和理论计算获得了在不同浓度的硫化物作用下纳米探针的光谱变化时间曲线。以该曲线为标准曲线，通过实时测量细胞内纳米探针光谱的变化速率可对细胞内局部硫化氢浓度及其变化进行测量。采用这一方法，实时检测了在外界刺激下细胞内源性硫化氢的产生过程。(第五章)
(5)利用柱面镜的散光原理，建立了一种以金纳米颗粒为探针的三维暗场成像技术，并利用该技术对溶液中的纳米颗粒进行了长时间的三维示踪。根据散光像斑的形状我们可以对垂直方向上的运动方向进行准确判断。与已有的三维散射成像技术相比，该技术拥有更高的垂直空间分辨率(~70nm)。利用该成像技术，我们准确地测量了溶液中处于自由运动状态的金纳米粒子的三维扩散系数。(第六章)
(6)采用种子诱导生长法，制备了一种能够催化双氧水、超氧根离子等活性氧化物分解的金铂复合纳米材料。经过表面功能化修饰，该颗粒能够大量进入细胞，并具有良好的生物适应性，同时能够催化细胞内活性氧化物的分解。利用该纳米粒子的这一催化特性，成功地抑制了紫外线辐射引起的细胞内活性氧化物浓度的升高，从而实现了紫外线辐射的防护作用。(第七章)
With the continuous advancements in nanoscience and nanotechnology, noblemetal nanoparticles with unique physical and chemical properties, which attractedmore and more interests of scientific researchers, have been widely applied in variousareas such as biochemistry and life sciences. As optical probes, metal nanoparticlessuch as gold and silver nanoparticles, possess of many advantages comparing withother traditional optical probes, such as high photostability, large opticalcross-section, easy to prepare and modify, well biocompatibility. Thus, noble metalnanoparticles displayed promising prospects in the applications of chemical analysisand biological sensing, biomedical and bioimaging. Based on the optical properties ofmetal nanoparticles, single-particle imaging and single-particle spectroscopy withnanometer-scale spatial resolution and millisecond time resolution have becomeimportant means for studying the behaviors and functions of biomolecules atsingle-cell level. Due to high surface chemical activities, metal nanoparticles havealso been used as active nano-catalyst for the catalysis in chemical engineering andenvironmental protection, biochemistry and biomedicine. However, there are stillgreat challenges we have to meet during various applications of metal nanoparticles,such as the declining in detection sensitivity due to the polydispersity of particle sizeand morphology, lower selectivity and temporal-spatial resolution of current methodsfor detecting in biological environments, etc.
Based on the existing research, we setted out to provide some solutions for theproblems and challenges in previous studies and the main points of our work in thisthesis are summarized as follows:
(1) A new kind of method has been proposed for the purification ofnanoparticles by using density gradient centrifugation. On the basis of force balanceof steady sedimentation, we have deduced the expressions of sedimentation rate ofnanoparticles with different sizes and shapes. By using the rate difference in thedensity gradient centrifugation, gold nanorods with differents aspect ratios has beensuccessfully isolated and purifed. To improve the separation efficiency, the effect ofcomposition of gradient solutions and centrifugation time were studied in ourexperiments. Both experimental results and calculated results demonstrated that,although particles can not be sperated for some special cases, density gradient centrigugation is still an effective method for the separation of nanoparticles withdifferent shapes and sizes.(Chapter2)
(2) The unique optical properties of gold nanorods were systematically studied.The locallized surface plasmon resonance (LSPR) absorption and scattering of goldnanorods can be calculated according to Gans theory. In this section, the expressionfor the relationships between spectral shifts and refractive index changes of mediumsolution was derived based on the resonance conditions of gold nanorods. Theexperimental results show that, the spectral redshift of gold nanorods linearly scaledup with the increase of refractive index of the medium solution, and the sensitivity ofspectral is related with the aspect ratios of gold nanorods. These results arecompletely consistent with the calculated results. In addition, we also investigatedthe factors which can affect the accuracy of single-particle detection.(Chapter3)
(3) Detection of sulfide with high sensitivity and selectivity was achieved byusing conventional UV-visible spectroscopy and single-particle spectral microscopywith gold-silver core-shell nanorods as probes. The gold-silver core-shell nanorodswere highly selective towards sulfide than other chemical species due to ultralowsolubility of Ag2S (pKsp=50.83). Two linear ranges from0.1μM to1mM and from0.01nM to10μM were obtained by using UV-visible spectroscopy andsingle-particle spectroscopy, respectively. The limit of detection of single-particledetection is much lower than by UV-visible spectroscopy, which implies thedetection at single-particle level a much sensitive than that of bulk measurements.(Chapter4)
(4) From the study of reaction kinetics between the gold-silver core-shellnanoprobes and sulfide, we successfully measured the local concentrations ofhydrogen sulfide (H2S) and its oscillation in live cells by monitoring the spectralredshift of nanoprobs. A relationship between the reaction kinetics and shifting rateof the spectra of single nanoprobes has been established. Then the time-dependentspectral shifting curves were obtained both experimentally and theoretically when thenanoprobes were exposed to different concentrations of sulfide solutions.By usingthese curves as external calibration curves, the variations of local H2S concentrationin live cells can be determined throuth detecting the LSPR spectral of singlenanoprobes in real time.With this method, generation of endogenous H2S in live cellsby applying external stimulation were detected with the nanoprobes.(Chapter5)
(5) Using the astigmatism phenomenon induced by a cylindrical lens, we havedeveloped a three-dimensional dark-field imaging techniques with gold nanoparticles as optical probes, and tracked the three dimentional diffusion of gold nanoparticles inthe solution. The direction of particle diffusion can be distinguished accuratelyaccording to the changes in the length of two axis of scattering spots. Comparingwith existing methods for three-dimensional imaging, a much higher vertical spatialresolution (~70nm) was obtained and the three-dimentional diffusion coefficients ofmoving gold nanoparticles have been measured in our experiments.(Chapter6)
(6) Highly active gold-platinum nanocomposites (Au-Pt NPs), which cancatalyze the decomposition of hydrogen peroxide, superoxide anion and otherreactive oxygen species, have been prepared with seed mediated growth method. TheAu-Pt NPs can be readily uptake by human skin cells after surface modification. Theresults of cell cytoxicity indicated that the Au-Pt NPs are well biocompatible, andalso the Au-Pt NPs can remarkably reduce cellular oxidation stress after treated withhydrogen peroxide. This propertiy was used for scavenging cellular reactive oxygenspecies and protecting from UV Irradiation induced cell damages in our study.(Chapter7)
本论文围绕以上提出的问题与难点开展研究，在已有的研究工作基础之上，开展了以下主要工作：
(1)采用密度梯度离心法对纳米棒进行了分离纯化。在纳米粒子稳态沉降过程中受力分析基础之上，推导了不同尺寸与形状的纳米粒子沉降速率的计算公式。利用其在离心过程中沉降速率的差异，采用不同浓度的蔗糖溶液配制的密度梯度液对不同长径比的棒状金纳粒子成功地进行了分离，并考察了梯度液组成以及离心时间对于分离效率的影响。实验结果与计算结果均表明，尽管存在着特殊情形下无法分离的局限性，但离心分离法仍能够有效地对具有不同长径比和不同尺寸的纳米粒子进行分离纯化。(第二章)
(2)系统性地研究了金纳米棒的光学特性。考察了基于Gans理论的金纳米棒等离子共振吸收与散射光谱计算公式，探讨了产生等离子共振的条件并推导了其等离光谱偏移量与介质溶液折射率变化之间的关系式。实验结果显示，金纳米棒的光谱偏移了随着介质折射率变化的增大而发生红移，而光谱变化的灵敏度也随着金纳米棒长径比的增大而增加，与计算结果完全一致。此外，还考察了采用单粒子光谱进行检测时的各种干扰因素。(第三章)
(3)以金银壳核结构的棒状纳米粒子为探针，采用传统紫外可见吸收光谱仪和单粒子暗场光谱显微镜对硫化物进行了高灵敏度高选择性的检测，并获得了比传统方法更宽的动态检测范围和更低的检测下限。采用紫外可见吸收光谱与单粒子暗场光谱显微镜进行检测的动态范围分别为0.1μM~1mM和0.01nM~10μM，该结果说明基于单粒子光谱成像技术的检测手段比宏观光谱测量具有更高的灵敏度以及更低的检测下限。(第四章)
(4)基于金银壳核结构的棒状纳米探针与硫化物的反应动力学过程，发展了一种在单个纳米粒子水平上对活细胞内硫化氢的浓度及其变化进行实时检测的新方法。通过推导该反应动力学过程与探针光谱偏移速率之间的关系式，建立了硫化物浓度变化与探针光谱变化速率的关系。进一步，我们通过实际测量和理论计算获得了在不同浓度的硫化物作用下纳米探针的光谱变化时间曲线。以该曲线为标准曲线，通过实时测量细胞内纳米探针光谱的变化速率可对细胞内局部硫化氢浓度及其变化进行测量。采用这一方法，实时检测了在外界刺激下细胞内源性硫化氢的产生过程。(第五章)
(5)利用柱面镜的散光原理，建立了一种以金纳米颗粒为探针的三维暗场成像技术，并利用该技术对溶液中的纳米颗粒进行了长时间的三维示踪。根据散光像斑的形状我们可以对垂直方向上的运动方向进行准确判断。与已有的三维散射成像技术相比，该技术拥有更高的垂直空间分辨率(~70nm)。利用该成像技术，我们准确地测量了溶液中处于自由运动状态的金纳米粒子的三维扩散系数。(第六章)
(6)采用种子诱导生长法，制备了一种能够催化双氧水、超氧根离子等活性氧化物分解的金铂复合纳米材料。经过表面功能化修饰，该颗粒能够大量进入细胞，并具有良好的生物适应性，同时能够催化细胞内活性氧化物的分解。利用该纳米粒子的这一催化特性，成功地抑制了紫外线辐射引起的细胞内活性氧化物浓度的升高，从而实现了紫外线辐射的防护作用。(第七章)
With the continuous advancements in nanoscience and nanotechnology, noblemetal nanoparticles with unique physical and chemical properties, which attractedmore and more interests of scientific researchers, have been widely applied in variousareas such as biochemistry and life sciences. As optical probes, metal nanoparticlessuch as gold and silver nanoparticles, possess of many advantages comparing withother traditional optical probes, such as high photostability, large opticalcross-section, easy to prepare and modify, well biocompatibility. Thus, noble metalnanoparticles displayed promising prospects in the applications of chemical analysisand biological sensing, biomedical and bioimaging. Based on the optical properties ofmetal nanoparticles, single-particle imaging and single-particle spectroscopy withnanometer-scale spatial resolution and millisecond time resolution have becomeimportant means for studying the behaviors and functions of biomolecules atsingle-cell level. Due to high surface chemical activities, metal nanoparticles havealso been used as active nano-catalyst for the catalysis in chemical engineering andenvironmental protection, biochemistry and biomedicine. However, there are stillgreat challenges we have to meet during various applications of metal nanoparticles,such as the declining in detection sensitivity due to the polydispersity of particle sizeand morphology, lower selectivity and temporal-spatial resolution of current methodsfor detecting in biological environments, etc.
Based on the existing research, we setted out to provide some solutions for theproblems and challenges in previous studies and the main points of our work in thisthesis are summarized as follows:
(1) A new kind of method has been proposed for the purification ofnanoparticles by using density gradient centrifugation. On the basis of force balanceof steady sedimentation, we have deduced the expressions of sedimentation rate ofnanoparticles with different sizes and shapes. By using the rate difference in thedensity gradient centrifugation, gold nanorods with differents aspect ratios has beensuccessfully isolated and purifed. To improve the separation efficiency, the effect ofcomposition of gradient solutions and centrifugation time were studied in ourexperiments. Both experimental results and calculated results demonstrated that,although particles can not be sperated for some special cases, density gradient centrigugation is still an effective method for the separation of nanoparticles withdifferent shapes and sizes.(Chapter2)
(2) The unique optical properties of gold nanorods were systematically studied.The locallized surface plasmon resonance (LSPR) absorption and scattering of goldnanorods can be calculated according to Gans theory. In this section, the expressionfor the relationships between spectral shifts and refractive index changes of mediumsolution was derived based on the resonance conditions of gold nanorods. Theexperimental results show that, the spectral redshift of gold nanorods linearly scaledup with the increase of refractive index of the medium solution, and the sensitivity ofspectral is related with the aspect ratios of gold nanorods. These results arecompletely consistent with the calculated results. In addition, we also investigatedthe factors which can affect the accuracy of single-particle detection.(Chapter3)
(3) Detection of sulfide with high sensitivity and selectivity was achieved byusing conventional UV-visible spectroscopy and single-particle spectral microscopywith gold-silver core-shell nanorods as probes. The gold-silver core-shell nanorodswere highly selective towards sulfide than other chemical species due to ultralowsolubility of Ag2S (pKsp=50.83). Two linear ranges from0.1μM to1mM and from0.01nM to10μM were obtained by using UV-visible spectroscopy andsingle-particle spectroscopy, respectively. The limit of detection of single-particledetection is much lower than by UV-visible spectroscopy, which implies thedetection at single-particle level a much sensitive than that of bulk measurements.(Chapter4)
(4) From the study of reaction kinetics between the gold-silver core-shellnanoprobes and sulfide, we successfully measured the local concentrations ofhydrogen sulfide (H2S) and its oscillation in live cells by monitoring the spectralredshift of nanoprobs. A relationship between the reaction kinetics and shifting rateof the spectra of single nanoprobes has been established. Then the time-dependentspectral shifting curves were obtained both experimentally and theoretically when thenanoprobes were exposed to different concentrations of sulfide solutions.By usingthese curves as external calibration curves, the variations of local H2S concentrationin live cells can be determined throuth detecting the LSPR spectral of singlenanoprobes in real time.With this method, generation of endogenous H2S in live cellsby applying external stimulation were detected with the nanoprobes.(Chapter5)
(5) Using the astigmatism phenomenon induced by a cylindrical lens, we havedeveloped a three-dimensional dark-field imaging techniques with gold nanoparticles as optical probes, and tracked the three dimentional diffusion of gold nanoparticles inthe solution. The direction of particle diffusion can be distinguished accuratelyaccording to the changes in the length of two axis of scattering spots. Comparingwith existing methods for three-dimensional imaging, a much higher vertical spatialresolution (~70nm) was obtained and the three-dimentional diffusion coefficients ofmoving gold nanoparticles have been measured in our experiments.(Chapter6)
(6) Highly active gold-platinum nanocomposites (Au-Pt NPs), which cancatalyze the decomposition of hydrogen peroxide, superoxide anion and otherreactive oxygen species, have been prepared with seed mediated growth method. TheAu-Pt NPs can be readily uptake by human skin cells after surface modification. Theresults of cell cytoxicity indicated that the Au-Pt NPs are well biocompatible, andalso the Au-Pt NPs can remarkably reduce cellular oxidation stress after treated withhydrogen peroxide. This propertiy was used for scavenging cellular reactive oxygenspecies and protecting from UV Irradiation induced cell damages in our study.(Chapter7)
引文
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