聚合物量子点单粒子荧光性质及调控研究

发布时间：2018-07-31 14:36

【摘要】：共轭聚合物量子点(Pdots)作为一种新型的生物荧光探针,在生物荧光成像及长期示踪等领域的应用备受关注。其光学物理性质的表征通常局限在测试吸收、发射光谱等,这些表征方法获得的是聚合物量子点悬浮溶液整体的平均效应。当应用于单粒子荧光成像以及各类新兴的超分辨成像实验时,粒子的分布及应用过程并不是整体平均分布。因此,研究聚合物量子点单粒子的荧光性质具有重要的意义。本文选用具有优异的光学特性及较好的生物相容性的荧光共轭聚合物PFBT材料,通过优化传统“纳米再沉淀法”的制备条件,制备出一系列具有一定尺寸梯度的聚合物量子点。根据平均尺寸分布,将相应尺寸的聚合物量子点分别命名为PFBT12、PFBT21、PFBT37和PFBT58。使用全内反射荧光显微镜(TIRF-M)系统,探索了聚合物量子点单粒子荧光亮度随尺寸变化的依赖关系。根据PFBT量子点的光谱测试可知,本文所用的四种尺寸分布的PFBT量子点荧光量子效率约为30%。将四种尺寸分布的PFBT量子点相邻两种尺寸作为一组进行平行对比,共分为三组实验。每组量子点在相同的实验条件下采集并统计单粒子荧光亮度,以PFBT12的单粒子荧光亮度作为基数,对另外三种不同尺寸分布的PFBT量子点的单粒子荧光亮度进行成比例放大计算。实验结果表明,聚合物量子点单粒子荧光亮度随粒子尺寸的增加呈二次方增加的关系,并给出了一条单粒子亮度随尺寸变化的校准曲线。在给定的成像条件下,通过校准曲线与单个粒子的亮度进行比较提取出粒子尺寸。分析研究了PFBT量子点单粒子尺寸依赖的光漂白过程。PFBT量子点光漂白过程大致可归为三类。进一步,光漂白速率常数以及发射的总光子数结果表明,对于小尺寸(30nm)粒子,其光漂白速率常数随尺寸的变化并不明显;而大尺寸(30nm)粒子的光漂白过程出现了快速衰减的部分,然后表现出相对缓慢的漂白过程。PFBT发射的总光子数在粒子尺寸小于30nm时,随着尺寸的增加呈现二次方关系的增长。然而,超过30nm的粒子发射总光子数并没有呈现出预期的二次方增长关系。在生物荧光检测以及单粒子荧光成像和追踪应用中,给出了最优的聚合物量子点的尺寸选择范围(约30nm)。在进行聚合物量子点单粒子荧光稳定性追踪实验时,发现聚合物量子点的荧光会出现快速衰减的部分。为此,本文制备了一种基于酶促反应的除氧系统,在葡萄糖氧化酶的催化下,葡萄糖能够快速高效的除去光氧化产生的氧化物等荧光淬灭剂。实验结果表明,该除氧系统能够有效提高聚合物量子点单粒子的荧光稳定性。使聚合物量子点在单粒子荧光成像及追踪应用中能够具有更加优异的表现。
[Abstract]:Conjugated polymer quantum dot (Pdots), as a novel biological fluorescence probe, has attracted much attention in many fields, such as biological fluorescence imaging and long term tracer. The characterization of their optical and physical properties is usually confined to the measurement of absorption and emission spectra. These characterization methods obtain the average effect of the polymer quantum dot suspension solution as a whole. When it is applied to single particle fluorescence imaging and various new superresolution imaging experiments, the particle distribution and application process are not uniform. Therefore, it is of great significance to study the fluorescence properties of polymer quantum dots. In this paper, a series of polymer quantum dots with a certain size gradient were prepared by optimizing the preparation conditions of the traditional "nanometer reprecipitation method" using fluorescent conjugated polymer PFBT materials with excellent optical properties and good biocompatibility. According to the average size distribution, the corresponding size polymer quantum dots were named PFBT12, PFBT21, PF37 and PFBT58, respectively. The dependence of single particle fluorescence intensity on the size of polymer quantum dots was investigated by means of total internal reflection fluorescence microscope (TIRF-M). According to the spectral measurements of PFBT quantum dots, the fluorescence quantum efficiency of the four PFBT quantum dots used in this paper is about 30%. The two adjacent dimensions of four PFBT quantum dots with different size distributions were compared in parallel and divided into three groups. Under the same experimental conditions, each group of quantum dots collected and counted the single particle fluorescence luminance. Based on the single particle fluorescence luminance of PFBT12, the single particle fluorescence luminance of the other three PFBT quantum dots with different size distribution was scaled up and calculated. The experimental results show that the luminescence of single particle in polymer quantum dots increases to a quadratic degree with the increase of particle size, and a calibration curve of the change of single particle brightness with size is given. Under given imaging conditions, the particle size is extracted by comparing the calibration curve with the brightness of a single particle. The single particle size dependent photobleaching process of PFBT quantum dots. The photobleaching process of PFBT quantum dots can be classified into three categories. Furthermore, the photobleaching rate constant and the total photon number emitted by the photobleaching rate constant show that the change of the photobleaching rate constant with the size is not obvious for the small size (30nm) particles, while the photobleaching rate constant of the large size (30nm) particles decreases rapidly. Then it shows a relatively slow bleaching process. When the particle size is smaller than 30nm, the total number of photons emitted by PFBT increases with the increase of particle size. However, the total photon number of particles above 30nm does not show the expected quadratic growth relationship. The optimal size selection range of polymer quantum dots (about 30nm) is given for bioluminescence detection and single particle fluorescence imaging and tracking applications. In the experiment of single particle fluorescence stability tracing of polymer quantum dots, it is found that the fluorescence of polymer quantum dots attenuates rapidly. Therefore, a deoxidizing system based on enzymatic reaction was prepared in this paper. Under the catalysis of glucose oxidase, glucose can rapidly and efficiently remove the fluorescence quenching agent such as the oxide produced by photooxidation. The experimental results show that the system can effectively improve the fluorescence stability of polymer quantum dots. Polymer quantum dots can perform better in single particle fluorescence imaging and tracking applications.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R318.51

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