【作者】 张凌燕；

【导师】 郑鹄志；

【作者基本信息】 西南大学 ， 分析化学， 2011， 硕士

【摘要】 量子点(又称半导体纳米晶,quantum dots,QDs)是近年来发展起来的新型荧光纳米材料。由于其直径小(与核酸、蛋白质等相近)、光学性能卓越(宽吸收、窄发射、光稳定性好等)、生物相容性良好等优点,在生命科学研究中备受青睐,被广泛应用于细胞和活体成像、荧光共振能量转移的研究、生物大分子检测等领域。朊蛋白(Proin Protein)是一种能在动物和人类中引起多种神经变性型疾病的传染性蛋白,其致病机理是正常的朊蛋白发生错误折叠,形成富含β折叠的抗蛋白酶水解的致病型朊蛋白。不稳定的致病型朊蛋白继续发生强烈的聚集,进而白组装成低聚物、纤维前体和淀粉样纤维结构。本文主要的研究工作如下：(1)合成了荧光性能优良的巯基乙酸修饰的CdTe QDs,以重组人朊蛋白(rPrP)为模式蛋白,研究了朊蛋白诱导的CdTe QDs的聚集和CdTe QDs对朊蛋白纤维化的影响。(2)基于朊蛋白诱导CdTe QDs发生聚集的现象,以CdTe QDs为荧光探针,对朊蛋白进行了可视化定性、半定量的检测,并利用CdTe QDs与朊蛋白相互作用后上清荧光强度的降低和上清荧光最大发射波长的蓝移分别对朊蛋白进行了定量检测。共存物质的干扰试验表明该检测方法选择性好,灵敏度高。同时也检测了表达朊蛋白的大肠杆菌裂解液中朊蛋白的含量。透射电子显微镜(TEM)实验表明,在二者聚集产生的沉淀中存在蛋白质纤维和淀粉样斑块,证实CdTe QDs的聚集与朊蛋白纤维化过程有关。(3)基于朊蛋白优先导致大粒径CdTe QDs聚集并引起上清最大发射峰蓝移的现象,以朊蛋白做“生物分离试剂”,对三种不同粒径的CdTe QDs进行了分离,包括三种CdTe QDs的两两分离和三种混合QDs的彼此分离。同时计算了分离回收率,并探讨了分离机制。更多还原

【Abstract】 Quantum dots, also known as semiconductor nanocrystals, are a novel type of fluorescent nanomaterials developed in recent years. They attracted a growing notice in biological research due to small diameter (similar with nucleic acids and proteins), perfect optical property (broad absorption, narrow emission, strong light stability, etc.), biocompatibility, etc., and they have been widely applied to cell and in vivo imaging, the study of fluorescence resonance energy transfer, and detection of biological macromolecules. Prion protein is a kind of infectious protein which could cause many neurodegenerative diseases in animal and human. The pathogenic mechanism is the misfolding of normal PrPc which form theβ-sheep-rich and protease-resistant PrPSc. The unstable PrPSc possesses a high tendency to aggregate and form oligomers, the precursor of amyloid fiber and amyloid plaques. The main work on this thesis can be summarized as follows:(1) CdTe QDs with excellent fluorescent properity were synthesized and modified by mercaptoacetic acid, and high purity of prion protein was purified from Escherichia coli. Subsequently, we discussed the aggregation of CdTe QDs induced by prion protein and the effects of CdTe QDs on the progress of fibrosis of prion protein.(2) Based on the aggregation of CdTe QDs induced by prion protein, CdTe QDs were used as a fluorescent probe for colorimetric qualitative and semi-quantitative detection of prion protein. Meanwhile, both the decrease in fluorescence intensity and the blue-shift of maximal emission peak of the supernatant could be used for quantitative detection of prion protein. Interference test showed that this detection had high selectivity and sensitivity. To demonstrate the potential application of this detection system, we also detected prion protein in the diluted solution of Escherichia coli lysate. Finally, transmission electron microscopy results showed that fibrils existed in the precipitates and were partly transformed to amyloid plaques.(3) According to the phenomenon that prion protein precipitated with larger CdTe QDs preferentially and caused the blue-shift of maximal emission peak of the supernatant, we used prion protein as a "biological separating reagents" to separate three different sizes of CdTe QDs, including the separation of two kinds of CdTe QDs, and three kinds of CdTe QDs from each other. In addition, the recoveries of these separation methods were calculated and the mechanism was discussed.更多还原