摘要
对单个分子的空间分布和时间轨道进行直接观测,并对其进行识别、分类和定量描述,一直是分析工作者的梦想。上世纪七十年代由于研究生物大分子的化学动力学的需要,出现了荧光相关光谱技术。随着九十年代显微共焦技术,计算机技术和光学检测技术的发展进步,荧光相关光谱逐渐发展为一种溶液中单分子检测技术,被广泛用于DNA与蛋白质之间的相互作用研究,细胞膜上和细胞内部的粒子的扩散研究和药物的高通量筛选等方面。荧光相关光谱通过测定溶液中微区内(通常<10-15L)发光粒子由于布朗运动或化学反应而产生的荧光涨落(荧光强度的明暗变化),并对荧光强度随时间变化的函数进行相关分析,从而获得粒子浓度、化学动力学参数等有关信息。
本论文工作以减少检测体积,提高荧光收集效率,增加信/背比为理念,在总结前人工作的基础上,经过艰苦实验摸索,自主构建了一套性能优良的荧光相关光谱单分子检测系统,建立了一系列荧光纳米材料表征的新方法,用于荧光纳米材料中一些重要的基础问题研究。主要研究内容包括如下几方面:
1.荧光相关光谱系统的研制。以激光共焦构型为其主体光学结构,选用水浸物镜作为光学收集元件,针孔作为空间滤波元件,自组装了一套检测体积小于1fL,信/背比达到400的荧光相关光谱单分子检测系统。系统测试和基因的点突变分析结果表明,该系统具有高灵敏和高稳定性的特点,可以用于生物大分子的扩散动力学,生物大分子间的相互作用研究和基因点突变分析等领域。
2.采用荧光相关光谱技术建立了一种水溶液中荧光粒子的动力学半径测定的新方法。同时将荧光相关光谱技术与其它光谱技术相结合,对水相合成的碲化镉量子点的分子量和摩尔消光系数等参数进行了系统研究。并在传统的FCS测定发光量子点浓度方法的基础上,提出了一种快速测定量子点样品中发光量子点比例的新方法,并对一系列量子点样品的发光量子点比例进行了测定。实验结果发现,水相合成的量子点的量子产率与发光量子点比例成正比,而发光量子点的量子产率可以认为接近100%。同时,将荧光相关光谱用于量子点标记辣根过氧化物酶的产物的表征,发现在一个辣根过氧化物酶分子可以结合大约一个量子点。
3.将荧光相关光谱系统与微流控芯片电泳联用,建立了一种溶液中荧光粒子的荷电或表面电荷的测定方法,并对荧光素的荷电和CdTe量子点的表面电荷进行了测定。我们发现,荧光素在pH7.4和pH8.4的缓冲溶液中的带电荷分别为1.1±0.5和1.5±0.4。巯基丙酸(MPA)为稳定剂的CdTe量子点在不同pH的TBE缓冲溶液中的表面电荷分别为6.2±0.4 (pH 8.4), 6.3±0.8(pH 9.3)和7.6±0.7(pH10.0)。此外,还发现量子点的表面电荷不仅与量子点所在的缓冲溶液的pH有关,而且还跟表面配体的种类等其它因素有关。巯基丙酸(MPA)为稳定剂的CdTe量子点在高浓度的谷胱甘肽(GSH)溶液中充分交换后,表面电荷变为11.8±0.5。
4.采用荧光相关光谱和其它光谱技术,对重金属离子(Ag~+离子)对碲化镉量子点的淬灭过程和机理进行了研究。研究发现,在Ag~+离子淬灭量子点的过程中,Ag~+离子进入量子点表面未配位完全的Te原子位置,形成的AgTe结构而产生新的表面缺陷,导致量子点荧光淬灭。尤为重要的是,发现Ag~+离子在量子点表面结合后,直接将发光量子点变成不发光的量子点,从而导致量子点的荧光淬灭。因此,认为Ag~+离子淬灭量子点过程是量子点逐个淬灭过程,而不是单个量子点荧光逐步衰减的过程。同时,我们还系统地研究了透析过程对荧光量子点的荧光增强作用。实验发现,透析过程可以将部分不发光量子点转变成发光量子点,从而显著提高量子点的荧光量子产率。其原因可能是由于透析过程消除了溶液中过量的Cd-MPA复合物,并导致量子点表面结构发生重组和调整的缘故。
5.采用荧光相关光谱技术对激光照射下量子点发生团聚和光活化的过程进行了系统的研究。实验结果表明,量子点在激光照射下发生的光活化过程与量子点的团聚直接相关。量子点的光活化是由于激光照射下量子点发生团聚后,量子点表面发生重组优化的结果。而量子点的粒径对量子点在光照下能否发生团聚具有决定性作用。粒径越小的量子点在光照下其表面越容易发生光化学反应,从而导致量子点相互团聚的发生。溶液中盐类分子可以降低量子点表面能,从而抑制量子点间的团聚发生,从而抑制量子点光活化。同时还发现,光活化过程与量子点的浓度有关。只有量子点浓度超过活化临界浓度后,量子点才能在光照下发生团聚和光活化。
To directly observe distributions and time trajectories of single molecules or single particles and then to analyze, sort, and describe them, are still one of analysts’dreams. In 1970s, in order to measure molecular diffusion and reaction kinetics of biomacromolecules in the highly diluted solution, fluorescence correlation spectroscopy (FCS) technique was introduced. And with the development of confocal microscope technique, optical detection technique and computer science in 1990s, FCS gradually become an advanced single molecule detection technique in the solution. Now it has recently experienced growing popularity in the life science, such as study on the interaction of DNA and protein, diffusion of particles in the inner of cell and on the cell membrane and high throughput screening of new drug (HTS). FCS is an ultrasensitive and noninvasive detection technique that uses statistical analysis of the fluctuations of fluorescence emitted from a small, optically well-defined open volume element. The autocorrelation function can provide us some important information, such as the average number of luminescent particles in the volume and the coefficient of diffusion etc. As a new single molecule detection technique, study on FCS instrument and application is scarce.
In this work, based on the principles to construct one single molecule detection system: (1) a small excitation volume, to reduce the background;(2) high-efficiency collection optics;(3) the use of detectors with high quantum efficiency and low dark noise;(4) careful elimination of background fluorescence by various means, a sensitive and stable fluorescence correlation spectroscopy (FCS) setup was developed and used for study on some key problems of fluorescent nanomaterials. The paper contains the following parts:
1. One fluorescence correlation spectroscopy system was developed with laser confocal optics as main optical structure, one high numerical aperture water-immersion objective as optical collecting device and a pinhole as space filter. The measurement on the diffusion of organic dyes shows that the laser highly illuminated volume is less than 1fL and ratio of signal to background (S/B) of single dye molecule is greater than 400. The high-sensitive and stable system can be used for C677T point mutation analysis of methylenetetrahydrofolate reductase gene.
2. Combined FCS with ensemble molecular spectrometry, a new method was developed to characterize molecular weight, molar extinction coefficient and bright fraction of CdTe QDs synthesized in aqueous solution. The principle is mainly based on the measurements of hydrodynamic diameters of CdTe QDs and the number of bright QDs in a small illuminated volume element using FCS technique. Hydrodynamic diameters of a series of CdTe QDs were measured with FCS and the molecular weights were calculated assuming the measured hydrodynamic diameters as the diameters of QDs. The molar extinction coefficients of QDs at different excitonic absorption peak positions were calculated with the molecular weights. Furthermore, the fitted excitonic absorption peak dependence of extinction coefficients was used to study Ostwald ripening or defocusing of QDs in aqueous synthesis process. The bright fraction of QDs samples were characterized by measuring the concentration of the bright QDs and the total concentration of QDs, and it was observed that the bright fractions of CdTe QDs sample were proportional to quantum yields (QYs) and the ratio of the bright fraction to QY was close to 1. Our methods described herein are simple and universal, and are suitable for characterization of molecular weight, molar extinction coefficient and bright fraction of QDs synthesized in aqueous phase and organic phase. Meanwhile, the methods were used to characterize HRP protein tagged with QDs. It was observed that nearly one quantum dot was conjutated on the surface of each HRP protein.
3. A new method was described for the measurement of surface charge of fluorescent particles, including QDs, by coupling FCS with microfluidic chip electrophoresis. This method has been successfully used to determine the surface charge of fluorescein and CdTe QDs. The measured charges of fluorescein in pH 7.4 and pH 8.4 buffers are 1.1±0.5 and 1.5±0.4, respectively. The surface charge of MPA-stabilized CdTe QDs in the TB buffer of pH 8.4, 9.3 and 10.0 was 6.2±0.4, 6.3±0.8, and 7.6±0.7. Meanwhile, it was found that the surface charge of QDs was remarkably associated with the type of stabilizers on QDs surface, buffer pH and other factors. After MPA-stabilized CdTe QDs dry powder was dissolved and displaced by reduced glutathione in the 10 mM borate buffer (pH 10.0) containing 10 mM reduced glutathione, surface charge of CdTe QDs changed into 11.8±0.5. Compared to the current methods, this technique is characterized by noninvasiveness, high sensitivity, effectiveness and versatility.
4. Combination of FCS with some ensemble techniques was used to investigate the quenching process and mechanism of heavy metal ions (such as Ag+) on CdTe QDs. It is found that when silver ions (Ag+) quench QDs, the free Ag+ ions enter the trap sites, bind with bare Te atoms and form the AgTe structure in the surface, which results in the luminescent quenching of QDs. The FCS experimental results show that the quenching process is not the gradual reduction of fluorescence intensity of single QDs, but the decrease in the number of bright QDs with the addition of Ag+ ions. Furthermore, FCS was used to investigate the effect of dialysis process on CdTe QDs. It is observed that some dark QDs convert into the bright QDs in the dialysis experiments and the dialysis process can improve the brightness per QDs. And the results of FCS and fluorescence spectroscopy illustrate that the increase of the fluorescence quantum yield (QY) is mainly attributed to the removal of excess unreacted Cd-MPA complex and the possible chemical change of the QDs surface in the dialysis process. These new results can help us to further understand the complex surface structure of water-soluble QDs, improve their surface chemical features, and expand their applications in some field.
5. The aggregation and the subsequent photo-activation process of MPA-capping CdTe QDs induced by laser were systematically studied with fluorescence correlation spectroscopy (FCS). It was observed that the photo-activation process was closely related with the aggregation of QDs in solution. The brightening of QDs samples was due to laser-induced aggregation and subsequent surface reconstruction. The aggregation process of QDs was dependent on sizes of QDs. The smaller QDs were apt to be aggregated together and be photo-activated by laser. And the higher concentration of salt could reduce and suppress the aggregation significantly, and then affected photo-activation process of QDs. Meanwhile, it was found that photo-activation process was also associated with the concentration of QDs. Only when the concentrations of QDs were greater than the certain concentration (called critical concentration), the photo-activation of QDs happened under laser irradiation.
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