【作者】 王东；

【导师】 王柯敏； 羊小海；

【作者基本信息】 湖南大学 ， 分析化学， 2009， 硕士

【摘要】 单分子检测能实时监测单分子行为和分子间相互作用,可揭示集团平均掩饰下的静态不均一和动态波动等个体差异性,已在分子构象变化、生物分子相互作用和超灵敏检测等方面得到广泛应用。本文基于全内反射荧光显微术（TIRFM）,在单分子和单颗粒水平上对核酸适体（aptamer）的构象变化以及量子点成像进行了研究,主要包括:1.在单分子水平研究表面固定的核酸适体的构象变化。在三磷酸腺苷（ATP）核酸适体的5’端标记生物素和荧光基团四甲基罗丹明（Tetramethylrhodamine, TMR）,3’端标记猝灭基团Dabcyl。利用生物素和亲和素相互作用,将ATP核酸适体固定在玻片表面。基于TIRFM考察了核酸适体分子在ATP或cDNA作用下的荧光强度变化。结果表明,无ATP和cDNA时,核酸适体分子荧光强度较弱,大多数处于闭环状态,少数处于无规则卷曲状态。在ATP作用下,无规卷曲状态的核酸适体分子变成闭环状态,荧光强度下降。在cDNA作用下,核酸适体分子变为双螺旋状态,荧光强度显著升高。而在cDNA和ATP同时存在下,随着ATP浓度的增加,核酸适体分子由双螺旋状态转变为闭环状态,荧光强度下降。本研究对发展基于核酸适体的生物传感器具有参考价值。2.利用单分子成像发展了基于量子点标记核酸适体的高灵敏蛋白质检测方法。目标蛋白凝血酶有两个核酸适体,结合位点亦不相同。本文将其中一条核酸适体通过生物素和亲和素的相互作用固定在玻片表面,另一条标记有量子点或荧光染料TMR。凝血酶与固定在玻片上的核酸适体以及荧光标记的核酸适体形成“三明治”结构,通过TIRFM测定荧光信号。结果表明,以量子点为荧光标记,凝血酶检测的线性范围为0.1 pM到10 pM,检测下限达到60 fM。以TMR为荧光标记,凝血酶检测的线性范围为5 pM到98 pM,检测下限为2.8 pM。可见,相比于TMR,以量子点为荧光标记的检测下限降低了47倍。这归因于量子点的高摩尔吸光系数和强光稳定性等优越的光学特性。3.基于单颗粒成像,研究了水溶性氨基化CdTe量子点与1-丁基-3-乙基咪唑四氟硼酸盐离子液体（[BEIm]·BF₄）作用后单个颗粒荧光性质变化。结果表明,量子点刚进入离子液体时,由于脂溶性离子液体的保护,量子点荧光强度和抗光漂白性能增强;随着与离子液体作用时间的延长,量子点闪烁现象变得明显,荧光强度和抗光漂白性能都有所减弱。红外光谱、荧光光谱以及量子点与NaBF₄溶液作用的结果表明,量子点闪烁现象、荧光强度和抗光漂白性能的变化可能是由于BF₄-结合到了量子点颗粒表面,并对量子点产生了刻蚀作用,使量子点的表面缺陷增加。本工作有助于了解离子液体与量子点的相互作用机理。更多还原

【Abstract】 The single-molecule behavior and the interaction between molecules can be real-time monitored by single-molecule detection (SMD) to unveil molecular properties of the static inhomogeneities and dynamic fluctuations, and the molecular properties are hidden in averaged ensemble. Single-molecule detection has been widely used in the molecular conformational change, biomolecular interactions and ultrasensitive detection and so on. In this paper, based on total internal reflection fluorescence microscopy (TIRFM), the aptamers conformation change and quantum dots imaging were studied, include:1. The configuration change of aptamers fixed on slide was studied at the single- molecule level. The adenosine triphosphate (ATP) aptamers were labled with fluorophore tetramethylrhodamine (TMR) and biotin at 5’ end, quencher Dabcyl at 3’ end. The aptamers were fixed on the slide with the interaction of biotin and avidin. Based on TIRFM, the aptamers were studied in the presence of ATP or cDNA. The results showed that without ATP and cDNA, fluorescence intensity of the aptamers was weak, and the majority of the aptamers were closed-loop, and a few of aptamers were random coil. In the presence of ATP, aptamers changed into closed loop from random coil, and the fluorescence intensity declined. In the presence of cDNA, the aptamers formed double helix, and fluorescence intensity increased. In the presence of cDNA and ATP, with the concentrations of ATP increased, the aptamers changed into closed loop from double helix, and the fluorescence intensity declined. The method provides a reference to develop the biosensors based on aptamer.2. The method for the ultrasensitive detection of protein was developed by quantum dots labeled aptamers with single-molecule imaging. Thrombin as target protein has two aptamers, and the binding sites are different. In this paper, an aptamer was fixed on slide with the interaction of biotin and avidin, and another aptamer was labeled with quantum dots or TMR. Sandwich assay was formed by thrombin and the aptamer fixed on the slide and the fluorescent-labeled aptamer, and then detected the fluorescence signal by TIRFM. The results showed that using the quantum dots as fluorescent labeling, the linear relationship for thrombin detection was obtained in the range of 0.1 to 10 pM, and the detection limit reached 60 fM. Using TMR as fluorescent labeling, the linear relationship for thrombin detection was obtained in the range of 5 to 98 pM, and the detection limit was 2.8 pM. So, compared to the TMR, quantum dots can decrease the detection limit to 47 times. It is attributed the quantum-dots superior optical properties of the large molar absorptivity and strong photostability.3. The single-particle fluorescence properties of water-soluble amino-CdTe quantum dots treated with 1-butyl-3-ethylimidazolium tetrafluoroborate ([BEIm]?BF4) ionic liquids were studied with single-molecule imaging. Quantum dots were extracted into the ionic liquids, the fluorescence intensity of quantum dots and anti-photobleaching were enhanced in the first hour, because of the protection of hydrophobic ionic liquids. Then, the blinking became apparent and anti-photobleaching capacity decreased with the time. Infrared spectroscopy, fluorescence spectroscopy and the results of quantum dots treated with NaBF4 solution showed that the changes of blinking and anti-photobleaching may be due to the BF4- binding to the surface of quantum dots and the BF4- etches quantum dots, which lead to the increase of surface defects of quantum dots. This work is contributed to investigate the interaction mechanism of quantum dots and ionic liquids.更多还原