基于仿生纳米结构构建新型模拟酶研究

发布时间：2018-03-09 18:34

本文选题：仿生　切入点：去铁蛋白　出处：《吉林大学》2015年博士论文　论文类型：学位论文

【摘要】：大自然的生命体历经了亿万年的演化，几乎完善了智能操控的一切过程。向自然学习，是人工智能新材料和新体系发展的永恒主题。天然酶作为生物催化剂,能够巧妙调节有机体的一切生命活动。然而，由于天然酶的蛋白质组成，大大限制了它的实际应用，获得高效稳定的酶模拟物便成为了研究热点。纳米材料以其比表面积大，表面能较高和生物相容性好等特性，近年来已经被广泛应用于模拟酶的构建。本文正是在这一背景下，利用生物化学，纳米化学，界面化学等综合研究方法，设计合成了一系列具有天然类酶活性的纳米材料和体系。推动模拟酶系统具有更好的生物相容性和实际应用价值，也进一步为功能纳米材料的应用奠定了基础。主要内容如下： 1)以生物体内广泛存在的转铁蛋白天然外壳为材料基础，利用其中空球形纳米结构空腔为模板，通过点控制技术合成去铁蛋白双金簇纳米粒子（Au-Ft）。经过TEM,紫外可见光谱，荧光光谱表征，其结果与前期工作相符，证明成功制备了该粒子。详细研究了Au-Ft的类过氧化物酶的酶学性质。Au-Ft对温度和pH均存在类似于HRP的依赖关系。Au-Ft的最适反应条件为pH4.0，温度45℃。对比于粒径较大的金粒子，Au-Ft具有突出的类酶活性，并且去铁蛋白外壳对金粒子的酶活性有促进作用。Au-Ft具有较好的pH稳定性，在高温环境下仍然具有部分酶活性。进一步的稳态动力学表明其具有米氏酶特性，并确定其反应机制符合乒乓机制。Au-Ft对底物TMB的Km值低于天然酶，而对底物H2O2的Km值高于天然酶，表现出了不同的底物亲和力。最后，我们将其与GOx偶联，构建了简单快捷的葡萄糖检测法。 2)以仿生智能纳米通道为基础，通过离子径迹和化学刻蚀相结合的方法，制备出具有二极管整流性质的非对称单孔材料。通过EDC/NHS修饰法，，将具有GPx活性中心的生物活性肽（SRGDU）修饰到单锥纳米孔道内表面。活性中心的Se与底物GSH和tBuOOH的反应能够引起孔道内有效孔径的变化。当酶通道系统与底物GSH，tBuOOH依次作用时，纳米通道的整流强度和电流均呈现先减小后增大的趋势，且对tBuOOH的响应程度随着浓度增加而增大。在对比实验中，裸孔道和修饰对比活性肽的纳米通道则表现出相反的变化。这种模拟酶体系还能够实现多次循环响应。这种仿生智能模拟酶体系为模拟酶的发展提供了新的设计思路。 3)本论文还进行了仿生智能孔道在生物毒素检测方面的研究。伏马毒素（fumonisinB1）主要存在于农业中。它不但污染粮食和相关制品，并对家畜产生潜在的致癌性和毒性。我们基于仿生智能孔道材料，设计具有伏马毒素响应性的非对称纳米孔检测体系。通过化学修饰法，将伏马毒素抗体修饰到智能孔道内表面，制备出对伏马毒素具有特异性响应的检测器件。随着对毒素响应浓度的增大，孔道呈现的整流比逐渐减小。未修饰的纳米孔与一系列伏马毒素作用，未呈现出电流和整流比的变化。本研究基于仿生智能纳米材料，探究了伏马毒素检测的新体系，为生物毒素的检测提供了新的思路。综上所述，本论文遵循向自然学习的思路，以天然铁蛋白为模板，制备去铁蛋白双金簇（Au-Ft）纳米粒子，并首次发现Au-Ft的类过氧化物酶活性。进一步探究了其催化反应的条件，性质和反应机制。最后将其应用于葡萄糖生理水平的检测。然后我们在仿生材料的基础上，构建了具有类GPx反应性的模拟酶体系。通过电流和整流比的改变，来评价体系的响应性。同时，我们还将仿生纳米材料用于生物毒素的检测，成功构建了伏马毒素检测的新方法。
[Abstract]:The life of nature after millions of years of evolution, almost perfect all the process of intelligent control. Learn from nature, is the eternal theme of the development of new materials and new artificial intelligence system. The natural enzyme as a biological catalyst, can skillfully adjust organism life activities. However, due to the natural enzyme protein greatly limits its practical application, to obtain high stability of enzyme mimics has become a research hotspot. Nano materials with large specific surface area, surface energy and good compatibility and biological characteristics such as high building, in recent years has been widely used in analog enzyme. This article is in this context, the use of biological chemistry nano chemistry, surface chemistry and other research methods, a series of natural enzymes of nanometer material and system were designed and synthesized. To promote the simulation of enzyme system compatibility and practical application has better biological The value also lays the foundation for the application of functional nanomaterials. The main contents are as follows:
1) natural transferrin shell to organism widely exists as the material basis, the hollow spherical nanostructure cavity as a template by point control technology to synthesis of ferritin double gold cluster nanoparticles (Au-Ft). After TEM, UV Vis, fluorescence spectroscopy, the results are consistent with previous work, to prove that the particles the enzymatic properties of.Au-Ft were prepared successfully. A detailed study of the class of peroxidase Au-Ft on temperature and pH were similar to the HRP.Au-Ft dependence of the optimum reaction conditions of pH4.0, temperature of 45 degrees Celsius. Compared to the larger size of gold nanoparticles, Au-Ft enzyme activity is prominent, and the enzyme activity of ferritin the shell of gold particles have good pH stability promoting effect of.Au-Ft, in a high temperature environment still has some enzyme activity. The steady-state kinetics further showed that it has Michaelis enzymatic properties, and determined its reaction mechanism The system accords with the ping-pong mechanism. The Km value of substrate TMB is lower than that of natural enzyme, while the Km value of substrate H2O2 is higher than that of natural enzyme. It shows different substrate affinity. Finally, we establish a simple and fast glucose detection method by coupling.Au-Ft with GOx.
2) with intelligent bionic nano channel based method combined by chemical etching and ion track phase, preparation of asymmetric single hole material with diode rectifier properties. By modified EDC/NHS method, bioactive peptides will have GPx Activity Center (SRGDU) to modify the single cone nano pore surface active center. The Se and the substrate GSH and tBuOOH reaction can cause changes in pore aperture. When the enzyme substrate GSH and tBuOOH channel system, in effect, rectification intensity and current nano channel were decreased first and then increased, and the degree of response to tBuOOH with the concentration increasing. In the experiment, bare nano channel pore and modified comparative peptides showed opposite changes. This simulation system can also realize the enzyme cyclic response. The development of bionic intelligent simulation system for the simulation of enzyme enzyme provides a new Design ideas.
3) this paper also carried out on pore bionic intelligent biological toxin detection. Fumonisins (fumonisinB1) mainly exist in agriculture. It is not only the pollution of food and related products, and produce carcinogenic and toxic potential of livestock. Our intelligent porous materials based on the bionic design, with fumonisin responses of the non symmetric nano hole the detection system. Through the method of chemical modification, the modification of fumonisin antibody to the smart pore surface, prepared with a detection device specific response of fumonisins. With the increasing concentration of the toxin response, the channel rectification ratio decreases gradually. The unmodified nano hole and a series of fumonisins, did not show the variation of the current and the rectification ratio. This study is based on the intelligent bionic nano materials, exploring a new system of fumonisin detection, and provides a new idea for the detection of biological toxins.
In summary, this paper follows the natural learning method, using natural ferritin as template, the preparation of iron protein double gold clusters (Au-Ft) nanoparticles, and found that the peroxidase activity of Au-Ft for the first time. To further explore the catalytic reaction conditions, properties and reaction mechanism. Finally applied to detect glucose at physiological level. Then we based on biomimetic materials, construct the simulation system with GPx enzyme reaction. The current and the rectification ratio changes in response to the evaluation system. At the same time, we will also biomimetic nano materials for the detection of biological toxins, construct a new method of fumonisin detection.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TB383.1

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