【作者】 孙翠骥；

【导师】 徐力； 聂广军；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 有良好生物兼容性,低毒性,和稳定性的可以靶向组织或细胞纳米结构在纳米生物医学上已经引起了很大的兴趣。本文成功用“点控制”的技术在去铁蛋白内部可控合成两个协同作用的可以经激发产生荧光的金簇。并通过测定荧光光谱,证实随着金簇尺寸的增大和之间距离的减少,两金簇之间的能量共振转移(Forster resonance energy transfer, FRET)会使两个金簇总的荧光强度逐渐增强,并使荧光光谱发生红移。通过冷冻电镜(Cryo-electron microscopy)和透射电镜(transmission electron microscopy)的低分辨,高分辨模式和扫描模式分别得到成对金簇在蛋白内部的直观图像。在扫描模式下还得到金元素的X射线能量散射图谱(energy-dispersive-X-ray spectroscopy, EDX)证明确实是在去铁蛋白中组装成功双金簇。通过测定Au-Ft (apoferritin-far-red Au cluster)的X-射线近边吸收峰,证明是在去铁蛋白的两个重链上组装上的双金簇。成功的在有去铁蛋白受体的Caco-2细胞及小鼠的肾脏部位得到了特定的生物医学靶向荧光成像。这些实验结果表明,利用生物体本身存在的具有靶向功能的蛋白质和无机纳米材料组装体,有可能成为一种解决包括肿瘤在内的各种难以攻克疾病的诊断及治疗的潜在方法。相关文章已经被JACS (IF=8.58)接收。更多还原

【Abstract】 Functional nanostructures with high biocompatibility and stability, low toxicity and specificity of targeting to desired organs or cells are of great interest in nanobiology and medicine. However, the challenge is to integrate all these desired features into a single nanobiostructure, which can be applied to biomedical applications and eventually in clinical settings. In this context, we designed a strategy to assemble two gold nanoclusters at the ferroxidase active sites of ferritin heavy chain. Our studies showed that the resulting nanostructures (Au-Ft) retain not only the intrinsic fluorescence properties of noble metal, but gain enhanced intensity, show a red-shift and exhibit tunable emissions due to the coupling interaction between the paired Au clusters. Furthermore, Au-Ft possessed the well defined nanostructure of native ferritin, showed organ-specific targeting ability, high biocompatibility and low cytotoxicity. The current study demonstrates that an integrated multimodal assembly strategy is able to generate stable and effective biomolecule-noble metal complexes of controllable size and with desirable fluorescence emission characteristics. Such agents are ideal for targeted in vitro and in vivo imaging. These results thus open new opportunities for biomolecule-guided nanostructure assembly with great potential for biomedical applicationsIn this paper, for making the idea of assembling the Au clusters into apoferritin to be true, the strategy which is used is "point controlling". Apoferritin is suitable to the condition which this idea needs:firstly, having the point which the reaction needs-the histidine at the ferroxidase active sites of apoferritin can bind Au3+ secondly, having the reactor which the reaction needs-the interior cavity of apoferritin is 10 nm, and the big pore of it is about 1 nm; thirdly, the chemical and physical characteristics of apoferritin are very stable, and the apoferritin can endure strong acid and alkali. By controlling the speed of the OH" reducing Au3+, we can get the size of the Au clusters we need. Through cryo-EM, HAADF-STEM, HRTEM and so on we can prove that we have assembled paired far-red Au clusters in apoferritin..Using the characteristic of far-red fluorescence of paired far-red Au clusters, we have done the research in the biomedical imaging:(1) Paired far-red Au clusters used in the cell fluorescent imaging. We choose two kinds of cell, one is Caco-2 which has the receptor of apoferritin and the other is HepG2 which has no receptor of apoferritin. Paired far-red Au clusters within apoferritin can absorb on the surface of cell membrane of Caco-2, but can not absorb on the surface of cell membrane of HepG2.(2) Paired far-red Au clusters used in the animal targeting fluorescent imaging. We can see the fluorescent imaging of the shape of two kidneys at the back of mice after several hours by vein injecting. It clearly says that paired far-red Au clusters reached the position of kidneys.Through this paper it can confirm that far-red Au clusters within apoferritin can have a good prospect in the future.更多还原