金纳米棒的合成、加工及作为方向探针在单分子成像上的应用
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摘要
单分子技术对基础科学研究具有非常重要的意义。这种技术不仅能够获得研究对象的共性,还能得到个体的差异,能够揭示传统方法所不能够得到的信息。金纳米棒作为单分子光学探针拥有传统荧光染料不能够比拟的优势,它不仅光学信号强,光学稳定性好,而且易加工制备。更重要的是它对光的吸收和散射是偏振性的,非常适合用作方向探针。目前,用金纳米棒作为单分子方向探针去研究生物学物理学事件的报道还不多。原因可能来自于三个方面：一是因为缺少良好的制备加工方法得到所需尺寸的金纳米棒,二是没有合适的成像技术手段来破译它的三维方向,三是没有适当的应用研究体系。
本论文围绕当前研究的难点和热点问题,借鉴前人的研究思路和成果,开展了以下工作：
(1)在第二章中,我们用H2O2作还原剂首次在碱性条件下通过种子生长法制备得到了高质量的金纳米棒。采用UV-Vis光谱和透射电子显微镜考察了溶液pH、H2O2/Au3+比例和银离子浓度对金纳米棒尺寸和形貌的影响,发现这三个因素的变化都会改变金纳米棒的长径比和相应的长轴等离子体共振峰位置。pH>～6.0是用H2O2制备金纳米棒的关键。相比于传统的用抗坏血酸作还原剂制备金纳米棒的方法,通过使用H2O2得到的金纳米棒具有良好的形貌和较好的单分散性。此外,我们的方法还有助于认识pH值影响纳米材料形貌的机制。
(2)基于合成研究的基础,在第三章中,通过再加工制备出了具有高散射信号的适合作分子方向光学探针的金纳米棒。首先,给出了金纳米棒等离子体共振能量吸收和散射理论,并模拟了金纳米棒长径比和环境介质对长轴等离子体共振的影响,证明了长径比为2.2左右的金纳米棒具有最强的光散射信号,适合用于以CCD为检测器的单分子成像研究。在加工过程中,我们先用传统方法制备出了小粒径金纳米棒,再以这些金纳米棒为种子进行二次生长,控制条件得到了所需长径比的金纳米棒。用紫外可见分光光度计研究了再生长过程中的光谱变化,通过透射电镜考察了准确的长度、直径变化。最后,在暗场显微镜下对单个金纳米棒进行光学成像,证明了它们具有较强的散射信号。通过转动光路中加入的偏振片证明了再加工后的金纳米棒散射是偏振性的,可以作为方向探针进行单分子成像研究。
(3)在第四章中,我们开发了新的光学成像技术来研究金纳米棒和细胞相互作用过程中的跨膜过程。通过理论推导给出不同照明方式下,特别是暗场斜照明方式下得到金纳米棒的三维角度的方法。对比了细胞顶部成像和细胞侧壁成像对研究金棒和细胞相互作用的影响,证明了细胞侧壁成像不仅能够准确得到三维角度,更利于得到金纳米棒和细胞膜的夹角,便于捕捉金纳米棒在整个跨膜过程中的旋转动态机制。最后,通过实时观测了金纳米棒的跨膜机制证明了双通道暗场细胞侧壁成像是获得纳米颗粒跨膜动态动力学良好的技术手段。
(4)在有了良好的材料和技术基础之上,第五章进一步研究了单个金纳米棒在细胞膜上的平动和转动行为。我们先建立了单个金纳米棒平动和转动研究的理论。重点研究了金纳米棒在膜上迁移过程中平动和转动变化,分析了可能涉及的生物学过程,初步得出了单分子平动和转动的不相关性。最后统计了41个金纳米棒的平动扩散系数和转动扩散系数,并引入相关系数为衡量参数,证实了金纳米棒在细胞膜上的平动和转动是相互独立的。
(5)通过光学技术手段得到的金纳米棒的角度是相对于晶体的偏振方向和成像平面定义的。第六章中,我们研究了极坐标系中金纳米棒的方向空间分布,通过统计分析发现,金纳米棒有三种旋转模式：部分受限,完全受限和自由转动。这三种模式和金纳米棒的细胞内吞过程是密切相关的。旋转模式、实时角度、轨迹、平动扩散系数、旋转扩散系数等参数一起对金纳米棒的细胞内吞过程作了完美的诠释。我们还发现通过受限模式的变化可以确定内吞完成的准确时刻。
(6)第七章进王步研究了蛋白包裹的单个金纳米棒在液固界面上的方向动力学。发现这些金纳米棒会有不同的方向动态,而且这些动态是与颗粒本身和界面的相互作用力大小密切相关的。为了研究解吸附过程中的旋转动态的变化,我们先在高盐浓度下将金纳米棒吸附在C18表面,然后再用低浓度的盐溶液进行洗脱。金纳米棒的旋转动态的分布是随着洗脱液盐浓度的降低而发生变化的。实时示踪单颗粒解吸附过程,进一步证明了分子从界面解吸附是先转起来再离开的。
Single molecule techniques are significant for investigating the basic science. They can obtain not only the common properties of the objects of research but also the individual events, which can reveal the unprecedented information that the traditional method can not achieve. As single molecule optical probes, gold nanorods (AuNRs) have several obvious advantages in contrast to traditional fluorescent dyes. They have strong optical signals, high photostability and can be easily synthesized and engineered. More importantly, the absorption and scattering of light by AuNRs are polarized, which make them be suitable to probe orientations. However, there are few studies to investigate the biological and physical events by using AuNRs as orientation probes. There are three crucial reasons that contribute to the result, that is, the lack of well preparing and engineering methods for obtaining AuNRs of specific sizes, the deficiency of suitable techniques to decipher their orientations and the lack of appropriate research systems.
In this paper, we did several studies to attempt to address the above problems. The main investigations are as follows.
(1) In chapter2, we synthesized high-quality AuNRs by using H2O2as the reducing agent through seed-mediated methods under alkaline conditions for the first time. The impacts of solution pH value, H2O2/Au3+ratio and the concentration of silver ion on the size and morphology of AuNRs were all investigated by UV-Vis spectroscopy and transmission electron microscope (TEM). It was found the alternations of three factors would lead to the change of aspect ratio of AuNRs and the corresponding longitude localized surface plasmon resonance (LLSPR) peak. pH higher than～6.0is essential for preparing AuNRs. Compared with the AuNRs through traditional method using ascorbic acid as the reducing agent, the AuNRs by H2O2have good morphology and improved monodispersity. In addition, our approach aided to understand the mechanism that how pH variation affect the morphology of nanomaterials.
(2) Based on AuNRs synthesis, in chapter3, we further engineered AuNRs by post-processing procedures, which have high light scattering signals and are suitable for single molecular optical probes. At the beginning, the theories on light absorption and scattering of AuNRs were presented and the simulated results of aspect ratio and environmental medium on LLSPR were also demonstrated. AuNRs with aspect ratio around2.2have been proved to have strongest scattering efficiency and be suitable for single molecule imaging by using CCD. In the process, AuNRs with small diameter were synthesized first by traditional seed-mediated method and then large AuNRs were obtained through overgrowth process by using them as seeds. By controlling the synthetic conditions, the desired AuNRs were obtained. The alternations of UV-Vis spectra, the length, and the diameter were investigated by spectrophotometer and TEM. At last, single AuNRs scattering images under dark-field microscopy were obtained. By rotating a polarizer in the optical path, the question whether the scattering intensity of AuNRs through overgrowth is polarized was addressed, and the AuNRs are proved to be appropriate to probe orientations in single molecule imaging.
(3) In chapter4, we developed new techniques to study the transmembrane dynamics of single AuNRs during their endocytosis. Through theoretical calculations, we provided the approach to determine the orientation of AuNRs in three-dimensional space under different illumination schemes, especially dark-filed oblique illumination. The impacts of focus plane placed at cell top and at cell sidewall on studying the transmembrane dynamics were investigated. Cell sidewall imaging mode would improve the obtained angle accuracy, be more proper to get the angle of AuNRs against membrane and be convenient to capture the whole transmembrane process. Finally, the real-time transmembrane processes verified that cell sidewall imaging under dual-channel darkfield microscopy was versatile to study the transmembrane dynamics of single AuNRs.
(4) With excellent materials and techniques, we further investigated the translational and rotational behaviors of single AuNRs on membrane in chapter5. We presented the theories on translation and rotation of single AuNRs. We mostly focused on the variation of translation and rotation of AuNRs during their translocation on membrane, analyzed the corresponding biological events, and preliminarily revealed the decoupling of translation and rotation. At last, we given the statistical results of translational diffusion coefficients and rotational diffusion coefficients of41AuNRs on membrane and found the translation and rotation of AuNRs on membrane are independent based on the statistical parameter-correlation coefficient.
(5) The azimuthal and polar angles of AuNRs obtained through optical techniques are defined with respected to the polarization direction of crystal and the imaging plane, respectively. Therefore, in chapter6, we studied the spatial distributions of AuNRs in polar coordinate system. Three rotational modes-partially confined rotation, restricted rotation and freely Brownian rotations, were found, which were closely related to endocytosis process. The rotational modes, combined with azimuthal and polar angle, out of plane angle, trajectories, the translational and rotational diffusion coefficients perfectly disclosed the detailed endocytosis dynamics. It is found that the mode variations can determine the exact time of the end of the endocytosis.
(6) The orientation dynamics of single protein-coated AuNRs at solid-liquid interface were investigated in chapter7. There are different rotational dynamics at interface, which reflect the interaction affinity between single protein-coated AuNRs and surface. The protein-coated AuNRs were absorbed on C18surface at high concentration salt solution first and then eluted with solution containing low concentration of salt to investigate orientation dynamics during desorption process. The distributions of these dynamics states changed when decreasing the salt concentration. The real-time desorption process of single AuNRs further illustrated that the desorption of molecule from surface must be preceded by rotation.
本论文围绕当前研究的难点和热点问题,借鉴前人的研究思路和成果,开展了以下工作：
(1)在第二章中,我们用H2O2作还原剂首次在碱性条件下通过种子生长法制备得到了高质量的金纳米棒。采用UV-Vis光谱和透射电子显微镜考察了溶液pH、H2O2/Au3+比例和银离子浓度对金纳米棒尺寸和形貌的影响,发现这三个因素的变化都会改变金纳米棒的长径比和相应的长轴等离子体共振峰位置。pH>～6.0是用H2O2制备金纳米棒的关键。相比于传统的用抗坏血酸作还原剂制备金纳米棒的方法,通过使用H2O2得到的金纳米棒具有良好的形貌和较好的单分散性。此外,我们的方法还有助于认识pH值影响纳米材料形貌的机制。
(2)基于合成研究的基础,在第三章中,通过再加工制备出了具有高散射信号的适合作分子方向光学探针的金纳米棒。首先,给出了金纳米棒等离子体共振能量吸收和散射理论,并模拟了金纳米棒长径比和环境介质对长轴等离子体共振的影响,证明了长径比为2.2左右的金纳米棒具有最强的光散射信号,适合用于以CCD为检测器的单分子成像研究。在加工过程中,我们先用传统方法制备出了小粒径金纳米棒,再以这些金纳米棒为种子进行二次生长,控制条件得到了所需长径比的金纳米棒。用紫外可见分光光度计研究了再生长过程中的光谱变化,通过透射电镜考察了准确的长度、直径变化。最后,在暗场显微镜下对单个金纳米棒进行光学成像,证明了它们具有较强的散射信号。通过转动光路中加入的偏振片证明了再加工后的金纳米棒散射是偏振性的,可以作为方向探针进行单分子成像研究。
(3)在第四章中,我们开发了新的光学成像技术来研究金纳米棒和细胞相互作用过程中的跨膜过程。通过理论推导给出不同照明方式下,特别是暗场斜照明方式下得到金纳米棒的三维角度的方法。对比了细胞顶部成像和细胞侧壁成像对研究金棒和细胞相互作用的影响,证明了细胞侧壁成像不仅能够准确得到三维角度,更利于得到金纳米棒和细胞膜的夹角,便于捕捉金纳米棒在整个跨膜过程中的旋转动态机制。最后,通过实时观测了金纳米棒的跨膜机制证明了双通道暗场细胞侧壁成像是获得纳米颗粒跨膜动态动力学良好的技术手段。
(4)在有了良好的材料和技术基础之上,第五章进一步研究了单个金纳米棒在细胞膜上的平动和转动行为。我们先建立了单个金纳米棒平动和转动研究的理论。重点研究了金纳米棒在膜上迁移过程中平动和转动变化,分析了可能涉及的生物学过程,初步得出了单分子平动和转动的不相关性。最后统计了41个金纳米棒的平动扩散系数和转动扩散系数,并引入相关系数为衡量参数,证实了金纳米棒在细胞膜上的平动和转动是相互独立的。
(5)通过光学技术手段得到的金纳米棒的角度是相对于晶体的偏振方向和成像平面定义的。第六章中,我们研究了极坐标系中金纳米棒的方向空间分布,通过统计分析发现,金纳米棒有三种旋转模式：部分受限,完全受限和自由转动。这三种模式和金纳米棒的细胞内吞过程是密切相关的。旋转模式、实时角度、轨迹、平动扩散系数、旋转扩散系数等参数一起对金纳米棒的细胞内吞过程作了完美的诠释。我们还发现通过受限模式的变化可以确定内吞完成的准确时刻。
(6)第七章进王步研究了蛋白包裹的单个金纳米棒在液固界面上的方向动力学。发现这些金纳米棒会有不同的方向动态,而且这些动态是与颗粒本身和界面的相互作用力大小密切相关的。为了研究解吸附过程中的旋转动态的变化,我们先在高盐浓度下将金纳米棒吸附在C18表面,然后再用低浓度的盐溶液进行洗脱。金纳米棒的旋转动态的分布是随着洗脱液盐浓度的降低而发生变化的。实时示踪单颗粒解吸附过程,进一步证明了分子从界面解吸附是先转起来再离开的。
Single molecule techniques are significant for investigating the basic science. They can obtain not only the common properties of the objects of research but also the individual events, which can reveal the unprecedented information that the traditional method can not achieve. As single molecule optical probes, gold nanorods (AuNRs) have several obvious advantages in contrast to traditional fluorescent dyes. They have strong optical signals, high photostability and can be easily synthesized and engineered. More importantly, the absorption and scattering of light by AuNRs are polarized, which make them be suitable to probe orientations. However, there are few studies to investigate the biological and physical events by using AuNRs as orientation probes. There are three crucial reasons that contribute to the result, that is, the lack of well preparing and engineering methods for obtaining AuNRs of specific sizes, the deficiency of suitable techniques to decipher their orientations and the lack of appropriate research systems.
In this paper, we did several studies to attempt to address the above problems. The main investigations are as follows.
(1) In chapter2, we synthesized high-quality AuNRs by using H2O2as the reducing agent through seed-mediated methods under alkaline conditions for the first time. The impacts of solution pH value, H2O2/Au3+ratio and the concentration of silver ion on the size and morphology of AuNRs were all investigated by UV-Vis spectroscopy and transmission electron microscope (TEM). It was found the alternations of three factors would lead to the change of aspect ratio of AuNRs and the corresponding longitude localized surface plasmon resonance (LLSPR) peak. pH higher than～6.0is essential for preparing AuNRs. Compared with the AuNRs through traditional method using ascorbic acid as the reducing agent, the AuNRs by H2O2have good morphology and improved monodispersity. In addition, our approach aided to understand the mechanism that how pH variation affect the morphology of nanomaterials.
(2) Based on AuNRs synthesis, in chapter3, we further engineered AuNRs by post-processing procedures, which have high light scattering signals and are suitable for single molecular optical probes. At the beginning, the theories on light absorption and scattering of AuNRs were presented and the simulated results of aspect ratio and environmental medium on LLSPR were also demonstrated. AuNRs with aspect ratio around2.2have been proved to have strongest scattering efficiency and be suitable for single molecule imaging by using CCD. In the process, AuNRs with small diameter were synthesized first by traditional seed-mediated method and then large AuNRs were obtained through overgrowth process by using them as seeds. By controlling the synthetic conditions, the desired AuNRs were obtained. The alternations of UV-Vis spectra, the length, and the diameter were investigated by spectrophotometer and TEM. At last, single AuNRs scattering images under dark-field microscopy were obtained. By rotating a polarizer in the optical path, the question whether the scattering intensity of AuNRs through overgrowth is polarized was addressed, and the AuNRs are proved to be appropriate to probe orientations in single molecule imaging.
(3) In chapter4, we developed new techniques to study the transmembrane dynamics of single AuNRs during their endocytosis. Through theoretical calculations, we provided the approach to determine the orientation of AuNRs in three-dimensional space under different illumination schemes, especially dark-filed oblique illumination. The impacts of focus plane placed at cell top and at cell sidewall on studying the transmembrane dynamics were investigated. Cell sidewall imaging mode would improve the obtained angle accuracy, be more proper to get the angle of AuNRs against membrane and be convenient to capture the whole transmembrane process. Finally, the real-time transmembrane processes verified that cell sidewall imaging under dual-channel darkfield microscopy was versatile to study the transmembrane dynamics of single AuNRs.
(4) With excellent materials and techniques, we further investigated the translational and rotational behaviors of single AuNRs on membrane in chapter5. We presented the theories on translation and rotation of single AuNRs. We mostly focused on the variation of translation and rotation of AuNRs during their translocation on membrane, analyzed the corresponding biological events, and preliminarily revealed the decoupling of translation and rotation. At last, we given the statistical results of translational diffusion coefficients and rotational diffusion coefficients of41AuNRs on membrane and found the translation and rotation of AuNRs on membrane are independent based on the statistical parameter-correlation coefficient.
(5) The azimuthal and polar angles of AuNRs obtained through optical techniques are defined with respected to the polarization direction of crystal and the imaging plane, respectively. Therefore, in chapter6, we studied the spatial distributions of AuNRs in polar coordinate system. Three rotational modes-partially confined rotation, restricted rotation and freely Brownian rotations, were found, which were closely related to endocytosis process. The rotational modes, combined with azimuthal and polar angle, out of plane angle, trajectories, the translational and rotational diffusion coefficients perfectly disclosed the detailed endocytosis dynamics. It is found that the mode variations can determine the exact time of the end of the endocytosis.
(6) The orientation dynamics of single protein-coated AuNRs at solid-liquid interface were investigated in chapter7. There are different rotational dynamics at interface, which reflect the interaction affinity between single protein-coated AuNRs and surface. The protein-coated AuNRs were absorbed on C18surface at high concentration salt solution first and then eluted with solution containing low concentration of salt to investigate orientation dynamics during desorption process. The distributions of these dynamics states changed when decreasing the salt concentration. The real-time desorption process of single AuNRs further illustrated that the desorption of molecule from surface must be preceded by rotation.
引文
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