【作者】 范佳；

【导师】 魏景艳； 聂广军；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 贵金属纳米材料（如金,银和铂等纳米颗粒）是纳米材料的一个重要组成部分。贵金属纳米材料具有较大的比表面积,较高的表面能和表面晶体缺陷等特点。这些特点使它们不但具有更好的催化活性和选择性,而且也带来一些意想不到的新的特性。这些性质都值得我们去深入的研究。但目前为止,在温和的反应条件下,尺寸均一、粒径可控的贵金属纳米微粒的制备仍是一个尚未得到解决的课题。为实现贵金属纳米材料在大小、形状、晶体结构上的可控合成,并探索其新的特性,我们主要进行了以下研究工作:1)选用去铁蛋白（apoFt）为材料,利用其纳米尺度的中空球型结构为模板,在它的内腔合成了大小、形状均可控的铂纳米颗粒（Pt-apoFt）。尝试采用不同还原剂和不同比例的材料合成Pt-apoFt纳米颗粒;经TEM、HRTEM、DLS、ICP-OES和紫外可见光谱等表征实验,最终确定了以NaBH4为还原剂,Pt²⁺/apoFt比为1000:1为最适合成条件,获得了尺度均一,粒径小于2 nm,分散性和稳定性良好的Pt-apoFt纳米颗粒。2)详细研究了Pt-apoFt模拟过氧化氢酶的酶学性质。Pt-apoFt对pH值和温度表现出与天然酶截然不同的依赖关系。在pH3～12或4～85℃范围内,Pt-apoFt的酶反应活性随着pH值或温度的增加逐渐增强;高温和高pH值还具有对酶活的协同增强作用。Pt-apoFt在各温度条件下都保持了较高的酶活稳定性,远远优于天然酶。此外,在生理条件下测定了Pt-apoFt对底物过氧化氢的酶促反应动力学常数Km,结果显示其与底物之间的亲和力弱于天然酶,但是强于或接近于其它纳米模拟酶。3)首次发现Pt-apoFt还具有模拟辣根过氧化物酶（HRP）的活性,可以氧化多种HRP的底物（例如TMB和DAB）;研究Pt-apoFt模拟HRP酶学活性时,发现其与天然HRP对温度和pH的关系相似,最适pH均为4,最适温度也相近。酶学稳定性实验进一步证明模拟酶Pt-apoFt对各温度和pH条件的耐受性。酶促反应动力学测定结果显示Pt-apoFt对底物TMB的亲和力要高于天然HRP。4)此外本论文还研究了另一种重要的纳米颗粒—CdTe量子点,其良好的荧光特性使其在检测领域有着广阔的应用空间,本研究将心肌肌钙（cTnI）的抗体与CdTe量子点共价偶联,合成了具有抗体活性的CdTe量子点,采用斑点免疫膜渗滤法定量检测心肌细胞损伤的标识物cTnI。此方法直观,操作简单,为量子点在检测医学中的应用奠定了基础。更多还原

【Abstract】 Manufactured nanostructures that mimic enzymes are of great interest as they potentially have improved properties relative to native enzymes, such as greater resistance to extremes of pH and temperature and lower sensitivity to proteases. Several nanostructures that possess catalytic activities have been discovered. Examples include peroxidase-like activity of ferromagnetic nanoparticles, superoxide dismutase mimetic properties of ceria nanoparticles and hydrogenation catalyzing activities of ferritin encapsulated palladium nanoparticles. Such synthetic enzymes have potential applications in various fields, including biomedicine, energy storage and bioremediation.Ferritins are a well-studied family of proteins that play an important role in iron storage. They comprise 24 subunits that assemble into a hollow nanocage with an external diameter of 12 nm in diameter and an 8 nm diameter cavity. Physiologically, iron is stored within the protein shell in a compact mineral form and one protein shell can accommodate up to 4500 atoms of iron. The channels formed at the subunit junctions are required for the transport of iron and other metal ions into and out of the protein shell. Ferritins have successfully been used as a scaffold to synthesize various protein-inorganic hybrids.Platinum （Pt） is a most widely used catalyst in chemical industries. Colloidal Pt has been showed to catalyze the decomposition of hydrogen peroxide （H₂O₂）, and Pt nanoparticles recently have been demonstrated to catalyze the scavenging of both H₂O₂ and superoxide anion （O2·-）. Thus these characteristics resemble the enzymatic activities of catalase and superoxide dismutase （SOD）. These two enzymes play important roles in maintaining redox balance in living organisms by scavenging excess reactive oxygen species （ROS）. The overproduction of ROS can lead to oxidative stress, damage to virtually all biomolecules and ultimately may induce cell death.Compared to the numbers of studies in biomimetic syntheses of gold and silver, protein-guided formation of platinum, one of the most important nobel metals, is relatively underexplored. In this study we investigated the possibility of using the apoferritin （apoFt） protein shell as a nanoreactor to control the synthesis of size-tunable Pt nanostructures. ApoFt was used as a scaffold to synthesize of 1 to 2 nm Pt nanoparticles （Pt-Ft） inside its protein shell. Pt-Ft showed catalase-like activities as reported, as well as horseradish peroxidase （HRP）-like activities. Interestingly, Pt-Ft possessed these enzymatic activities with distinctive enzymatic properties, namely different responses to pH and temperature for different enzymatic substrates. Using 3,3’,5,5’-tetramethylbenzidine （TMB） and 3,3’-diaminobenzidine （DAB） as substrates, we found that the optimal pH and temperature for the oxidation catalyzed by Pt-Ft were similar to that of native HRP （pH optimum 4; temperature optimum 37°C）. However, this was not the case for the catalase-like activity of Pt-Ft, where the optimal pH and temperature were quite different from that of native catalase, being significantly higher in each case. Compared to other engineered nanoparticles, such as iron oxide and cerium oxide, Pt-Ft had a significantly smaller nanostructural core and unique peroxidase activities for different substrates. Metal oxide nanoparticles utilize different valence states (Fe²⁺/Fe³⁺ or Ce³⁺/Ce⁴⁺) for their catalytic activity, but Pt-Ft consists of mainly, if not entirely, zero-valent Pt nanoclusters, and the changes of valence states are between zero valence and oxidized Pt. Additionally, the ferritin shells make these nanostructures biocompatible and potentially more bioactive, for example, through their possible interactions with ferritin receptors.Quantum dots have physical and optical properties that make them useful tools for high-resolution labeling immunoassay. In this work, a rapid and simple method of quantitative immunoassay for Cardiac troponin I（cTnI）was developed by using quantum dots-labeled antibodies. The monoclonal antibodies of cTnI（2F11） could be labeled with CdTe quantum dots and the coupled product（CdTe-2F11）were characterized by SDS-PAGE. The result of immunofiltration assay indicated that the CdTe-2F11maintained the antibody activity. The cTnI at the different concentrations in NC membrane could react with CdTe-2F11 and be detected by using ImageMaster to analyze the fluorescence intensity of the immunodotting. The result showed that the detection limit of cTnI was 120ng, and there was a good linear relation between concentration of cTnI and the fluorescence intensity （R²=0.9966）, in this study.更多还原