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基于仿生納米結(jié)構(gòu)構(gòu)建新型模擬酶研究

發(fā)布時(shí)間:2018-03-09 18:34

  本文選題:仿生 切入點(diǎn):去鐵蛋白 出處:《吉林大學(xué)》2015年博士論文 論文類型:學(xué)位論文


【摘要】:大自然的生命體歷經(jīng)了億萬(wàn)年的演化,幾乎完善了智能操控的一切過(guò)程。向自然學(xué)習(xí),是人工智能新材料和新體系發(fā)展的永恒主題。天然酶作為生物催化劑,能夠巧妙調(diào)節(jié)有機(jī)體的一切生命活動(dòng)。然而,由于天然酶的蛋白質(zhì)組成,大大限制了它的實(shí)際應(yīng)用,獲得高效穩(wěn)定的酶模擬物便成為了研究熱點(diǎn)。納米材料以其比表面積大,表面能較高和生物相容性好等特性,近年來(lái)已經(jīng)被廣泛應(yīng)用于模擬酶的構(gòu)建。本文正是在這一背景下,利用生物化學(xué),納米化學(xué),界面化學(xué)等綜合研究方法,設(shè)計(jì)合成了一系列具有天然類酶活性的納米材料和體系。推動(dòng)模擬酶系統(tǒng)具有更好的生物相容性和實(shí)際應(yīng)用價(jià)值,也進(jìn)一步為功能納米材料的應(yīng)用奠定了基礎(chǔ)。主要內(nèi)容如下: 1)以生物體內(nèi)廣泛存在的轉(zhuǎn)鐵蛋白天然外殼為材料基礎(chǔ),利用其中空球形納米結(jié)構(gòu)空腔為模板,通過(guò)點(diǎn)控制技術(shù)合成去鐵蛋白雙金簇納米粒子(Au-Ft)。經(jīng)過(guò)TEM,紫外可見(jiàn)光譜,熒光光譜表征,其結(jié)果與前期工作相符,證明成功制備了該粒子。詳細(xì)研究了Au-Ft的類過(guò)氧化物酶的酶學(xué)性質(zhì)。Au-Ft對(duì)溫度和pH均存在類似于HRP的依賴關(guān)系。Au-Ft的最適反應(yīng)條件為pH4.0,溫度45℃。對(duì)比于粒徑較大的金粒子,Au-Ft具有突出的類酶活性,并且去鐵蛋白外殼對(duì)金粒子的酶活性有促進(jìn)作用。Au-Ft具有較好的pH穩(wěn)定性,在高溫環(huán)境下仍然具有部分酶活性。進(jìn)一步的穩(wěn)態(tài)動(dòng)力學(xué)表明其具有米氏酶特性,并確定其反應(yīng)機(jī)制符合乒乓機(jī)制。Au-Ft對(duì)底物TMB的Km值低于天然酶,而對(duì)底物H2O2的Km值高于天然酶,表現(xiàn)出了不同的底物親和力。最后,我們將其與GOx偶聯(lián),構(gòu)建了簡(jiǎn)單快捷的葡萄糖檢測(cè)法。 2)以仿生智能納米通道為基礎(chǔ),通過(guò)離子徑跡和化學(xué)刻蝕相結(jié)合的方法,制備出具有二極管整流性質(zhì)的非對(duì)稱單孔材料。通過(guò)EDC/NHS修飾法,,將具有GPx活性中心的生物活性肽(SRGDU)修飾到單錐納米孔道內(nèi)表面。活性中心的Se與底物GSH和tBuOOH的反應(yīng)能夠引起孔道內(nèi)有效孔徑的變化。當(dāng)酶通道系統(tǒng)與底物GSH,tBuOOH依次作用時(shí),納米通道的整流強(qiáng)度和電流均呈現(xiàn)先減小后增大的趨勢(shì),且對(duì)tBuOOH的響應(yīng)程度隨著濃度增加而增大。在對(duì)比實(shí)驗(yàn)中,裸孔道和修飾對(duì)比活性肽的納米通道則表現(xiàn)出相反的變化。這種模擬酶體系還能夠?qū)崿F(xiàn)多次循環(huán)響應(yīng)。這種仿生智能模擬酶體系為模擬酶的發(fā)展提供了新的設(shè)計(jì)思路。 3)本論文還進(jìn)行了仿生智能孔道在生物毒素檢測(cè)方面的研究。伏馬毒素(fumonisinB1)主要存在于農(nóng)業(yè)中。它不但污染糧食和相關(guān)制品,并對(duì)家畜產(chǎn)生潛在的致癌性和毒性。我們基于仿生智能孔道材料,設(shè)計(jì)具有伏馬毒素響應(yīng)性的非對(duì)稱納米孔檢測(cè)體系。通過(guò)化學(xué)修飾法,將伏馬毒素抗體修飾到智能孔道內(nèi)表面,制備出對(duì)伏馬毒素具有特異性響應(yīng)的檢測(cè)器件。隨著對(duì)毒素響應(yīng)濃度的增大,孔道呈現(xiàn)的整流比逐漸減小。未修飾的納米孔與一系列伏馬毒素作用,未呈現(xiàn)出電流和整流比的變化。本研究基于仿生智能納米材料,探究了伏馬毒素檢測(cè)的新體系,為生物毒素的檢測(cè)提供了新的思路。 綜上所述,本論文遵循向自然學(xué)習(xí)的思路,以天然鐵蛋白為模板,制備去鐵蛋白雙金簇(Au-Ft)納米粒子,并首次發(fā)現(xiàn)Au-Ft的類過(guò)氧化物酶活性。進(jìn)一步探究了其催化反應(yīng)的條件,性質(zhì)和反應(yīng)機(jī)制。最后將其應(yīng)用于葡萄糖生理水平的檢測(cè)。然后我們?cè)诜律牧系幕A(chǔ)上,構(gòu)建了具有類GPx反應(yīng)性的模擬酶體系。通過(guò)電流和整流比的改變,來(lái)評(píng)價(jià)體系的響應(yīng)性。同時(shí),我們還將仿生納米材料用于生物毒素的檢測(cè),成功構(gòu)建了伏馬毒素檢測(cè)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.

【學(xué)位授予單位】:吉林大學(xué)
【學(xué)位級(jí)別】:博士
【學(xué)位授予年份】:2015
【分類號(hào)】:TB383.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 ;Size-dependent peroxidase-like catalytic activity of Fe_3O_4 nanoparticles[J];Chinese Chemical Letters;2008年06期

2 曹冬梅;孫安權(quán);;如何正確認(rèn)識(shí)伏馬毒素的危害[J];飼料廣角;2010年18期



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