【作者】 庄宏琳；

【导师】 黄承志；

【作者基本信息】 西南大学 ， 药物分析， 2013， 硕士

【摘要】 荧光共振能量转移(fluorescence resonance energy transfer, FRET)是一种非辐射能量跃迁,它十两个荧光发色基团在小于10m时,当供体分子吸收一定频率的光子后被激发到史高的电子能态,在该电子回到基态前,通过偶极子相互作用,实现了能量向邻近的受体分子转移(即发生共振能量转移)的过程。基于共振能量转移的方法已应用于生物分子、有机物、无机离子的分析检测中,在传统的荧光共振能量转移体系中,能量供体和受体通常为染料分子。随着科学技术的发展,Swathi等科学家通过理论计算发现,当用石墨烯作为能量受体时,能量转移的速率(k)与供受体距离(R)的四次方成反比(k∝R-4),且荧光共振能量转移可发生在供体与石墨烯之间的距离达30nm处,即所谓的长程共振能量转移(Long-rang resonance energy transfer, LrRET),这种长程共振能量转移理论扩展了荧光共振能量转移的应用范围,但在分析方面的应用还很有限。本文用氧化石墨烯作为能量受体,与带有核酸适配子的能量供体之间通过长程共振能量转移实现了细胞型朊蛋白的灵敏检测。具体包括以下两方面内容：(1)利用氧化石墨烯为能量受体,荧光染料单标记的核酸适配子灯标为能量供体,通过长程共振能量转移原理检测细胞型朊蛋白。当体系中没有细胞型朊蛋白时,核酸适配子灯标与氧化石墨烯通过π-π相互作用,使染料的荧光被猝灭；当加入细胞型朊蛋白后,朊蛋白与核酸适配子高特异性结合,使适配子灯标远离氧化石墨烯表面,从而染料的荧光得以恢复,据此检测细胞型朊蛋白,检测限达0.309μg/mL。该方法相对于其它检测朊蛋白的方法,具有灵敏、简单、特异性高的优势。(2)利用双适配子策略,通过氧化石墨烯与量子点之间的长程共振能量转移检测细胞型朊蛋白。将偶联有朊蛋白的一段适配子的氧化石墨烯作为能量受体,而将偶联有另一段适配子的量子点作为能量供体,当朊蛋白存在时,其与两段适配子同时结合,形成氧化石墨烯-朊蛋白-量子点的三明治结构,使量子点靠近氧化石墨烯表面,量子点的荧光被猝灭,实现对朊蛋白的检测。并且采用高分辨透射电镜表征了这种三明治结构的形成,初步估算了量子点到氧化石墨烯之间的距离约为19-27nm。该方法简单、快速,成功实现了细胞型朊蛋白的高灵敏检测。更多还原

【Abstract】 Fluorescence resonance energy transfer (FRET) is a nonradiative process whereby an excited state donor (D, usually a fluorophore) transfers energy to a proximal ground state acceptor (A) through long-range dipole-dipole interactions. Recently, FRET has been widely applied for the detetion of biomolecules, organisms, inorganic ions. In traditional FRET, however, both the energy donor and acceptor are dye molecules. The energy transfer in traditional FRET between the molecular dye donor and acceptor can effectively occur in the distance within1-10nm, and the energy transfer rate (k) has (R)-6dependence on the relative distance (R) between the donor and the acceptor in the Forster equation. As Swathi and coworkers theoretically predicted, if a two dimensional (2-D) graphene sheet acts as the acceptor, the rate of energy transfer from dye to the surface of graphene has a R-4dependence (k∝R-4). For the fluorescence energy transfer from donor to graphene, FRET is found to be appreciable up to a distance of30nm. This is the theory of long-rang resonance energy transfer (LrRET). In this contribution, graphene oxide (GO) is introduced to the detetion of prion protein through LrRET between GO and fluorescence donor labeled aptamer.(1) By introducing GO as energy acceptor, a LrRET strategy for the detection of cell prion protein (PrPC) is explored. In the absence of PrP, TAMRA labeled molecule aptamer beacon (MAB) can be adsorbed on the surface of GO, and the fluorescence of TAMRA is quenched by GO following LrRET process. In the presence of PrC, however, the MAB forms a ligand-binding structure, leaving the surface of GO owing to the π-π interaction between the rings of the nucleotide bases and the honeycomb surface of GO. As a result, fluorescence emission from TAMRA gets restored. It was found that a detection limit of0.309mg/mL could be achieved. The strategy is sensitive, simple and specific.(2) A dual-aptamer strategy by LrRET between GO and quantum dots (QDs) for PrPC detection was constructed. Two aptamers (Aptl and Apt2), which can recognize their two corresponding distinct epitopes of PrPC, were coupled to GO and QDs, to make GO-Aptl and QDs-Apt2ready first, which then could be coassociated together through the specific recognitions of two aptamers with their two corresponding distinct epitopes of PrPC, forming a sandwich structure of GO-PrPc-QDs and quenching the fluorescence of QDs. To ensure the formation of the sandwich structure, High resolution transmission electron microscope (HRTEM) imaging was performed, and large compact3-dimensional (3-D) GO-PrPc-QDs complexes were observed clearly. Energy donor-acceptor separation distance ranges from19to27nm.更多还原