【作者】 朱浩；

【导师】 陶农建；

【作者基本信息】 南京大学 ， 化学， 2020， 博士

【摘要】 电荷作为物质的一种基本物理性质,在分子识别、细胞信号、酶反应和蛋白质聚集等微纳尺度下的分子过程及电化学反应中都发挥了重要的作用。运用简单且灵敏的测量手段对表界面电荷进行定量分析有助于加深研究者对于相关化学及生物过程的认知。同时,局域电荷分布的研究可以解释界面反应的动态过程及内在机制,进一步拓展其在生物分子结合动力学研究领域的应用。目前可对表界面电荷直接进行成像分析和测定的技术相对较少,而且存在一定的不足。在这一背景下,本论文基于透射式明场以及全内反射等光学显微技术对单纳米颗粒、氧化铟锡（Indium Tin Oxide,ITO）、金片和二维材料的表面电荷及其动态变化进行了成像分析和定量研究,主要内容如下:1、单颗粒表面电荷的明场成像研究及应用对电荷的精确测量在单颗粒甚至单分子的研究中有着重要作用,并有助于我们加深对涉及电荷变化的化学和生物过程的理解。我们提出了一种基于明场光学显微镜结合傅里叶变换成像处理算法对单个纳米颗粒表面电荷的测定方法。我们通过一条聚合物链将纳米颗粒固定在ITO玻璃片的表面,该纳米颗粒在交变电场的驱动下会在ITO表面上下振荡,通过明场光学显微镜和相机可以对该动态过程进行记录。利用傅里叶变换滤波器可以将弱散射光从较强的背景中分离出来,通过图像对比度的变化得到纳米振子表面的有效电荷,其精度可达数个基元电荷量。这一高灵敏度的电荷检测方法可以对单纳米颗粒的表面电荷量进行实时测定和研究。利用该方法,我们还比较了不同粒径的纳米颗粒的表面电荷,并进一步将该方法延伸至DNA分子与纳米颗粒的结合过程研究中,为单分子成像分析提供了一种新的思路。2、单颗粒表面电荷的全内反射成像研究反射型全内反射显微镜（Reflection mode total internal reflection microscope）是在结合了全内反射荧光显微镜、全内反射暗场显微镜以及表面等离激元共振（Surface Plasmon Resonance,SPR）显微镜等多种显微技术的特点发展而来。我们构建了单个二氧化硅@金核壳结构（SiO₂@Au）纳米振子,利用反射型全内反射显微镜对其振荡过程进行成像,并结合二维傅里叶变换算法对单个SiO₂@Au纳米颗粒的表面电荷进行了测定。同时,我们还比较了金膜、ITO以及石墨烯基底的背景噪声,并通过在石墨烯基底上构建类似的纳米振子揭示了石墨烯作为透明电极在该成像模式下的潜在应用。3、氧化铟锡及金片表面电荷分布的光学成像研究电极表面的电荷分布是对其表面电学信息的直观反映,可以使我们更好地了解其表面发生的电化学反应过程。通过电化学栅电压的调制作用,我们首先对金片表面的电荷进行了SPR和明场的同步成像,分别研究了两种成像技术获得的电荷分布随调制电压振幅、频率以及栅电压的变化结果,并验证了SPR成像和明场成像这两种方法对局域电荷变化的光电信号转换机制。我们还利用明场对ITO玻璃片的表面电荷分布进行了成像,并研究了该技术对电极表面电荷变化的灵敏度。实验结果显示金片的SPR信号、金片的明场光学信号以及ITO玻璃片的明场光学信号对电荷响应的灵敏度依次下降。上述研究为进一步开展微观层面上的表界面电荷分布及动态变化的可视化分析奠定了基础。4、单层二硫化钼表面电荷分布的光学成像研究对纳米材料表界面局域电荷分布的成像分析加深了研究者们对电荷参与的动态反应过程和内在机理的理解,同时还可以用于检测生物分子的结合过程。单层二硫化钼（MoS₂）的吸光度对电荷变化具有非常高的灵敏度,我们利用这一特性对其局域电荷分布及变化进行了成像分析。首先,我们验证了在电化学栅电压控制下,单层MoS₂的光学响应,并通过改变电化学栅电压的大小来获得单层MoS₂对电荷响应的灵敏度。实验结果证明,光学衍射极限成像区域内的电荷检测限为数十个基元电荷量,而在散粒噪声下的检测限可达数个基元电荷量。我们运用电化学栅电压的调控作用和对图像进行的二维傅里叶变换得到了该材料表面高分辨率的电荷分布结果,同时可以反映其表面带电杂质的分布情况。当单层MoS₂表面结合带电分子后,会引起较大的光学变化,从而可以对表面结合分子所带电荷量进行测定。该方法可以用来表征二维材料中杂质和缺陷的分布情况,同时也为带电分子的免标记成像分析提供了一种新的手段。更多还原

【Abstract】 As a basic physical property of matter,charge plays a vital role at micro-nano scales in electrochemical reactions and molecular processes including molecular recognition,cell signaling,enzyme reaction and protein aggregation.A simple and sensitive measurement of charge on a surface will contribute to the understanding of the related chemical and biological processes.In addition,mapping local surface charge distribution can reveal the dynamic process and internal mechanism of surface interaction,and further expand its applications in biomolecular binding dynamics.At present,there are relatively few techniques that can directly perform charge imaging analysis and measurement on the surface interface,and most of the existing approaches process some deficiencies.In this dissertation,imaging approaches and dynamic changes determination of the single nanoparticles,indium tin oxide（ITO）,gold electrode as well as two-dimentional materials are developed based on both optical transmission and total internal reflection microscopy.The main contents are as follows:1.Surface charge measurement of individual nanoparticles and its applications based on bright-field imagingA precise charge measurement plays an important role for individual nanoparticles and even single molecules,and it contributes to our comprehension of the various chemical and biological processes involving charge change.Here we propose a method to measure the surface charge of an individual nanoparticle using a conventional bright field optical microscope combined with a fast Fourier transform（FFT）imaging processing algorithm.The nanoparticle is tethered to an ITO coated glass slide surface with a polymer chain and driven into oscillation with an alternating electric field,the process of which was captured by a camera.The weak scattered light is separated from the intense bright field background with a FFT filter.The surface effective charge of the nanoparticle can be obtained from the changes of the image contrast with precision of a few elementary electron charges.The high-sensitivity charge detection method can be used to monitor the surface charge of an individual nanoparticle in real time.Using this method,we compared the surface charge of nanoparticles with different sizes,and further apply it to detect the binding of DNA to individual nanoparticles,thus demonstrating a new tool for the imaging at single-molecule level.2.Surface charge measurement of individual nanoparticles based on total internal reflection imagingThe reflection mode total internal reflection microscope（Reflection-TIRM）is developed from total internal reflection fluorescence microscope,total internal reflection dark field microscope and surface plasma resonance（SPR）microscope.Herein we constructed a single silica@gold core-shell structure（SiO₂@Au）nano-oscillator,imaged it with a Reflection-TIRM,and determined the surface charge of the individual SiO₂@Au nanoparticle by combining with two-dimentional FFT algorithm.In addition,we compared the background noise of gold film,ITO and graphene substrate with the present experimental setup,and revealed the potential application of graphene as a transparent electrode in this imaging mode by constructing a similar nano-oscillator on the graphene substrate.3.Optical imaging of surface charge distribution on indium tin oxide and gold electrode The surface charge distributions of the electrodes reflect their electrical property directly,which allows us a better understanding of the electrochemical reaction process on their surface.By applying an electrochemical gate modulation,we first performed simultaneous SPR and optical transmission imaging of the surface charge on a gold film coated glass slide.The photoelectric signal conversion mechanism of both SPR and optical transmission imaging to surface charge was demonstrated by analyzing the optical changes at different modulated potential amplitudes,different modulated frequencies,and different gate voltages,respectively.In addition,we mapped the surface charge of an ITO coated glass slide with optical transmission technique,and obtained its optical sensitivity to the surface charge.From the results,SPR response of the gold film coated glass slide,optical transmission response of the gold film coated glass slide,and optical transmission response of the ITO coated glass slide show the sequential decrease optical sensitivity to the surface charge.This laid the foundation for further visual research of the charge distribution and dynamic at the micro-level.4.Optical imaging of surface charge distribution with monolayer molybdenum disulfideThe observation of local surface charge distribution of the nanomaterials is helpful for our comprehension of the charge related dynamic reaction process and internal mechanism,and it is useful for the detection of the molecules binding to the surface.The optical absorption of monolayer molybdenum disulfide（MoS₂）is highly sensitive to charge change,based on which we demonstrate optical imaging of local surface charge distribution and charge change with this ultrathin material.We first validate the optical response of the monolayer MoS₂ and perform charge sensitivity calibration with an electrochemical gate voltage.With the current experimental setup,the charge detection limit for an imaging area defined by optical diffraction is a few tens of elementary electron charges,while the detection limit due to shot noise is only a few elementary electron charges.We have shown that a high-contrast charge images can be obtained with a modulated voltage by using two-dimensional FFT algorithm,which reflects the local charged impurity distribution in the material.We further show that binding of charged molecules to the monolayer MoS₂ leads to a large change in the image contrast,allowing us to determine charge of the adsorbed molecules.This capability opens possibilities for impurities and defects characterization in two-dimensional materials and for label-free optical detection and imaging analysis of charged molecules.更多还原