单颗粒暗场光散射成像：灵敏度、准确度提升及多靶物分析应用研究

发布时间：2018-03-09 01:34

本文选题：暗场光散射成像　切入点：单纳米颗粒　出处：《西南大学》2017年博士论文　论文类型：学位论文

【摘要】：独特的斜射照明方式使暗场光散射成像具有无背景信号、信噪比高及分辨率高等独特优势。贵金属纳米颗粒作为等离子体探针具有光学信号强、光稳定性高、光学性质可调及易标记等优势。将单个贵金属纳米颗粒作为光学探针,与暗场光散射成像技术结合后获得单颗粒暗场光散射成像技术。单颗粒暗场光散射成像技术具有传统成像技术无法比拟的优势,如高灵敏度、高分辨率及精准的局域信息,因此,在单纳米颗粒水平监控动态反应过程和空间分辨方面具有广阔的应用前景。然而,单颗粒暗场光散射成像技术仍存在一定局限性,例如对单纳米颗粒局域表面等离子体共振性质研究不完善、暗场成像结果准确度不高及无法实现定量分析多靶物等。本论文围绕上述研究难点和问题,在单颗粒暗场光散射成像技术的成像性能改进及多靶物分析检测应用方面开展了如下具体工作:1.径向比对单个金纳米棒光散射分析灵敏度的影响首先,我们制备出具有不同形貌的金纳米颗粒(AuNPs),利用暗场光学显微镜与扫描电子显微镜对同一批次纳米颗粒进行共定位,研究形貌与其散射光性质的关系。结果显示,在暗场显微镜下散射绿光的为球形或者立方纳米颗粒,散射黄光的为椭球形颗粒,散射红光的为棒状纳米颗粒,并且径向比越大的纳米颗粒其散射光的红色程度越大。接下来,我们将纳米颗粒置于具有不同折光指数的介质中观察其散射光的变化,发现径向比越大的金纳米棒(Au NRs)对折光指数的变化响应越灵敏。该研究为进一步筛选具有高灵敏度的探针颗粒提供了理论基础。2.内标纳米颗粒提高暗场光学成像的准确度手工操作会在暗场光学成像结果中引入一些不可避免的误差。为了校准这一部分误差,我们引入内标纳米颗粒(IR),利用IR的散射光信号来校准探针纳米颗粒的散射光信号。我们选用未经修饰的AuNPs作为IR,选择银纳米颗粒(AgNPs)或者修饰后的AuNPs作为探针纳米颗粒。首先,我们考察了这种内标法的使用是否可行。结果表明,在不发生化学反应或者经受外界物理刺激的情况下,IR与探针纳米颗粒散射光强度的比值保持不变,证明这种方法是可行的。接下来,我们做了三组实验考察引入IR对暗场下探针光学信号准确度的影响:(1)室温下监控AgNPs在空气中的氧化过程;(2)利用AuNPs催化葡萄糖产生的过氧化氢(H2O2)降低AgNPs散射光强度来测定葡萄糖的浓度;(3)研究AuNPs与染料四甲基罗丹明(TAMRA)或者Cy3发生等离子体共振能量转移(PRET)后散射光的变化。结果表明,未经修饰的AuNPs作为IR可以大大提升探针纳米颗粒暗场光散射成像结果的准确度。3.金纳米颗粒与染料发生等离子体共振能量转移效率与供受体之间距离的关系我们将内标法进一步引入到对PRET的理论研究中。粒径为50 nm的AuNPs的散射峰位于550 nm左右,而染料TAMRA和Cy3在550 nm左右具有强吸收。当纳米颗粒与染料之间距离足够近时,两者之间能够发生PRET。其中,AuNPs作为供体,染料作为受体。我们将纳米颗粒与染料利用双链DNA连接在一起,通过调节双链DNA的长度来调控供受体之间的距离,并考察不同距离下AuNPs散射光变化情况,这样就能得到供受体之间的距离对PRET效率的影响。时域有限差分方法(FDTD)模拟结果和实验结果均表明,供受体之间的距离确实会影响PRET效率,两者之间距离越近,则PRET效率越高。4.双色光学探针对两种疾病标志物的同时检测我们在载玻片表面修饰甲胎蛋白(AFP)抗体和癌胚抗原(CEA)抗体。加入AFP和CEA后,修饰有AFP抗体的AuNPs和修饰有CEA抗体的AgNPs通过形成三明治免疫夹心结构而结合到玻片表面。金纳米颗和银纳米颗在暗场下分别散射绿光和蓝光,通过计数暗场光散射成像图片中绿色光斑和蓝色光斑的数量就能实现同时检测AFP和CEA,检测范围为0.5-100 ng/m L。我们进一步考察了AFP和CEA在同时检测中的相互干扰,发现当两者浓度在0.5-10 ng/m L时不存在相互干扰。并且我们构建的这种方法能够用于多靶物检测,只要使用散射多种颜色的纳米光学探针,便能实现对多种靶物的同时灵敏检测。总之,本论文对单颗粒暗场光散射成像分析从灵敏度、准确度、靶物检测三方面做了改进,并研究了在暗场光散射成像分析中占有重要地位的PRET的理论部分,这对于实现更简便、更灵敏的分析检测方法具有重要意义。
[Abstract]:The unique way of making oblique illumination dark field light scattering imaging with background signal, high signal-to-noise ratio and high resolution. The unique advantages of noble metal nanoparticles as plasma probe with optical signal intensity, high light stability, tunable optical properties and easy marking and other advantages. The single noble metal nanoparticles as optical probes, and combined with the dark light single particle scattering imaging obtained after dark field light scattering imaging technique. The single particle dark field light scattering imaging technique can not be compared with the traditional imaging techniques, such as high sensitivity, high resolution and accurate information, the local area therefore has a broad application prospect in the resolution of single nanoparticle level monitoring dynamic reaction process and space. However, single particle, dark field light scattering imaging technology still has some limitations, such as the single nanoparticles localized surface plasmon resonance properties research. Good, dark field imaging results is not accurate and can not achieve the quantitative analysis of multi targets. This paper focuses on the research of difficulties and problems, analysis of application carried out the following specific work in the imaging performance of improved single particle dark field light scattering imaging technique and multi targets: 1. radial alignment of single gold nanorods light scattering analysis the sensitivity of the first effect, we prepared gold nanoparticles with different morphologies (AuNPs), were Co located on the same batch of nano particles using dark field optical microscopy and scanning electron microscopy, to study the relationship between morphology and light scattering properties. The results showed that, in the light scattering under dark field microscope are spherical or cubic nanoparticles. The yellow light scattering of ellipsoidal particles, the scattering light is the rod like nano particles, nano particles and red degree greater than the radial scattering light more. Next, I We will observe the change of light scattering of nano particles with different refractive index in the medium, it is found that the radial ratio of gold nanorods (Au NRs) on the change of refractive index response more sensitive. This study provides improved optical imaging accuracy error field manual operation will introduce some inevitable in the dark field optical imaging results the theoretical basis of internal standard.2. nanoparticles for further screening of probe particles with high sensitivity. In this part the calibration error, we introduce the internal standard (IR), nano particle scattering light signal to calibrate the probe using nano particle scattering light signal IR. We use the unmodified AuNPs as IR, the choice of silver nano particles (AgNPs) or modified AuNPs nanoparticles as probe. Firstly, we investigated the use of internal standard method is feasible. The results show that the chemical reaction does not occur in or after From external physical stimulus and the ratio of IR and probe nano particle scattering light intensity remains unchanged, this method is proved to be feasible. Next, we do three experiments the effects of introducing IR on the optical signal under dark field probe accuracy: (1) the oxidation process under room temperature monitoring AgNPs in air; (2) produced by glucose catalyzed AuNPs hydrogen peroxide (H2O2) decreased the concentration of AgNPs scattering light intensity to the determination of glucose; (3) AuNPs and dye methyl four Luo Danming (TAMRA) or Cy3 energy transfer plasmon resonance (PRET) changes in light scattering. The results showed that the unmodified AuNPs as IR can greatly enhance the nanoparticle probe dark field light scattering imaging accuracy.3. gold nanoparticles and dye energy plasmon resonance transfer efficiency and the distance of the relationship between donor and recipient will further our standard method Introduction to the theory of PRET. The particle size is 50 nm AuNPs scattering peak located at about 550 nm, while TAMRA and Cy3 dyes have strong absorption at around 550 nm. When the distance between the nanoparticles and the dye is close enough, which can occur between PRET., AuNPs as the donor dye as a receptor. We will dye nanoparticles and connected together using a double stranded DNA, double stranded DNA by adjusting the length to control the distance between donor and acceptor, and the effects of different distance under AuNPs scattering light changes, so that we can get the distance between donor and acceptor influence on efficiency of PRET. The finite difference time domain method (FDTD) simulation results and experimental results show that the distance between donor and acceptor will indeed affect the efficiency of the PRET, between the distance closer, the higher the efficiency of PRET.4. double color optical probe of two markers of disease at the same time, we detected the surface of a glass slide Modification of alpha fetoprotein (AFP) antibody and carcinoembryonic antigen (CEA) antibody. Adding AFP and CEA, AuNPs modified and modified AFP antibody CEA antibody AgNPs by forming a sandwich structure and binding to the sandwich glass surface. Gold nanoparticles and silver nanoparticles in dark field scattering are green and blue. Simultaneous detection of AFP and CEA by the number of green and blue light spot can count dark field light scattering imaging in the picture, the detection range is 0.5-100 ng/m L., we further investigated the AFP and CEA at the same time interference detection, found that when the two concentrations do not interfere with each other in the 0.5-10 ng/m L. This method and we construct can be used for multiple target detection, as long as the use of a variety of colors of the nano scattering optical probe, can achieve a variety of targets and sensitive detection. In short, this paper analysis of the single particle dark field light scattering imaging From the sensitivity, accuracy, target detection three improvements have been made part of theory and research plays an important role in the analysis of dark field light scattering imaging in PRET, to realize the more simple, is of great importance to the analysis of more sensitive detection methods.

【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3;O644.1

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