金属纳米粒子增强荧光有机磷生物传感器

【作者】 黑婷婷；

【导师】 张耀东；

【作者基本信息】 陕西师范大学 ， 分析化学， 2011， 硕士

【摘要】 有机磷具有高效的杀虫能力,已经成为当今广泛使用的农药之一,有机磷农药的残留所引起的食品安全问题长期以来一直是人们所关注的焦点。因此,建立快速、可靠、灵敏、简便和实用的有机磷检测的分析方法,对于环境保护和食品安全等具有重要现实意义。生物传感器的研究和应用是实现实时、定位、在线分析的重要途径,已成为当前分析化学研究领域热点之一。基于AChE的有机磷生物传感器的研究越来越受到人们的重视,其中包括光学、电学、电化学、热学、磁学等换能器。由于荧光法检测具有灵敏度高、良好的组织穿透力、高空间分辨率和成本低等优点,因此最受人们的亲睐而且得到广泛的应用。生物缀合物是指两个或两个以上具有生物活性的分子连接起来形成一个新的复杂的生物复合物,同时具有各个组成分子的性能。本论文的研究目的是：将贵金属纳米材料(即金和银纳米粒子)和乙酰胆碱酯酶(AChE)共价结合形成生物缀合物来构建分子纳米传感器,利用金属纳米子结合于AChE对荧光探针的影响,建立高灵敏度、快速响应的有机磷检测新方法。本论文分为三章,第一章为绪论,简要综述了蛋白质荧光生物传感器和贵金属纳米材料在生物传感分析中的研究进展；介绍了乙酰胆碱酯酶的特点和应用；归纳了贵金属纳米材料的基本性质、化学修饰以及在分析化学中的应用；并论述了本论文的研究思路、目的和意义。第二章和第三章为实验部分,具体内容如下：第二章金纳米粒子增强荧光有机磷生物传感器7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO)是一类红色的荧光染料分子。利用金属纳米粒子对DDAO荧光特性的影响,本章建立了基于乙酰胆碱酯酶的有机磷检测新方法,并初步探讨了分子传感机理。首先,利用巯基与Au形成共价键的作用,将11-巯基十一烷酸(MUA)共价结合到金纳米粒子上,再通过EDC/NHS将羧基活化,然后利用乙酰胆碱酯酶分子上的自由氨基,将AChE共价固定在金纳米粒子上,形成AuNPs/AChE缀合物：利用吸收光谱、透射电镜等方法对合成的金纳米粒子、AuNPs/AChE等缀合物进行了表征,考察了温度和pH值对缀合物的影响。研究了AuNPs/AChE生物缀合物对对氧磷的生物传感器性能；还测定了DDAO与AChE的双分子结合速率常数和抑制常数,探讨了DDAO对AChE的抑制类型、对氧磷与AuNPs-AChE-DDAO生物缀合物的结合常数等；利用Autodock4.2软件,对DDAO与乙酰胆碱酯酶的分子对接进行了研究。结果表明：吸收光谱图和透射电子显微镜图片表明了AuNPs/AChE缀合物的形成。与自由酶相比,在同样的温度和酸度条件下,固定化的AChE具有更好的稳定性。当AuNPs/AChE生物缀合物存在时,DDAO的荧光强度急剧增加,对氧磷加入到此体系中后,DDAO则发生荧光猝灭,据此,建立了一种简单灵敏的测定对氧磷的方法,检出限为4×10-7M。该传感器具有很好的稳定性,响应快、灵敏度高等优点。对DDAO与AChE的相互作用研究表明,DDAO对AChE有很强的抑制作用,抑制常数的大小为Ki为1.1×10-7M,属混合型抑制。对氧磷与AuNPs-AChE-DDAO生物缀合物的结合常数为3.2×106L·mol-1,说明对氧磷与DDAO/AChE/AuNPs具有强的相互作用；分子对接的结果表明,与对氧磷结合于AChE的活性中心Ser200不同,DDAO结合于AChE的外周阴离子位点,表明了DDAO-AChE-AuNPs对对氧磷的传感性是由于其对生物缀合物中AChE分子构象的扰动,引起DDAO周围微环境的改变,造成荧光发射性能的改变。第三章银纳米粒子增强荧光有机磷生物传感器在第二章的研究中,发现了金纳米粒子对复合物中荧光探针DDAO具有增敏作用,尽管有文献报道金纳米粒子具有荧光增敏作用,但大量文献报道金纳米粒子具有荧光猝灭作用,这取决于金纳米粒子与荧光探针之间的距离,更多的文献报道银纳米粒子具有荧光增敏作用。为了进一步验证实验结果,本章将银纳米粒子标记于乙酰胆碱酯酶上进行研究。利用吸收光谱、透射电镜等方法对合成的银纳米粒子、AgNPs/AChE缀合物进行了表征,考察了温度和pH值对缀合物的影响。研究了AgNPs/AChE生物缀合物对对氧磷的生物传感器性能。我们还测定了对氧磷与AgNPs-AChE-DDAO生物缀合物的结合常数。结果表明：吸收光谱图和透射电子显微镜图片表明了AgNPs/AChE缀合物的形成。与自由酶相比,在同样的温度和酸度条件下,固定化的AChE同样具有更好的稳定性,对氧磷与AgNPs-AChE-DDAO生物缀合物的结合常数为1.7×106L·mol-1,说明对氧磷与DDAO/AChE/AgNPs也具有较强的相互作用。AgNPs/AChE生物缀合物的存在同样对DDAO具有荧光增敏作用,对氧磷加入到此体系中后,DDAO则同样发生荧光猝灭作用,说明DDAO/AChE/AgNPs生物缀合物同样可对对氧磷进行高灵敏的生物传感分析。更多还原

【Abstract】 Organophosphate has high insecticidal abilities, which has been a far-ranging pesticide nowadays. The food safe problems resulting from damages of organophosphate are always taking people’s attentions. Consequently, there is a growing interest in fast, reliable, sensitive, simple and practical detection methods, which has important practical significance on environmental protection and food safety.Biosensors have attracted much attention in recent times because of the potential applications of these devices in the clinical diagnostics, environmental monitoring, pharmaceuticals, and food processing industries due to their fast response and ease of operation. And the use of acetylcholinesterase (AChE) as organophosphate biological recognition element in biosensor technology has gained more and more attention, in particular with respect to pesticides detection. Many kinds of biosensors using different transducer for insecticide detection involving optics, electrics, electrochemistry, calorifics, magnetics,etc, has been developed. Among those, fluorescent biosensor is widely used as a class of biological sensors because of its good physical property such as high sensitivity, good penetrability, space resolution and low cost. Herein, a kind of biological sensors has been developed using fluorescence as detection signal.Bioconjugation involves the linking of two or more molecules to form a novel complex having the combined properties of its individual components. The objective of the present work is to develop a novel nanobiosensor for insecticide detection, combining noble metal nanoparticles(e.g. AuNPs or AgNPs) and with AChE by covalent bonding to form bioconjugation. Thus, the influence of metal nanoparticles was applied to improve the sensitivity of the fluorescent probe for better detetion of organophosphate.This thesis contains three chapters. Chapter one is introduction. It firstly reviews the progress of fluorescent biosensors and the basic characteristics and their applications of nobel metal nanoparticles, then summarize the functions and applications of AChE, the properties and chemical modification method of nobel metal nanoparticles, particularly in analytical chemistry application, finally describes the method, aim and significance of this research.Chapter two and chapter three are experimental sections as follows: Chapter two Organophosphate biosensor based on fluorescence enhancement with gold nanoparticles7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO) is a red fluorescent dye with weak fluorescence. By means of metal nanoparticles affecting the fluorescence properties of DDAO, a novel method based on AChE about the detection of organophosphate has been established. The sensing mechanism has been also explored. Firstly, based on the thiol 11-mercapto-undecane acid (MUA) and Au forming a covalent bond, MUA was covalently immobilized onto the gold nanoparticles, then the carboxyl was activated through the EDC/NHS, reacted with the free amino groups of AChE via covalent binding, and form AuNPs/AChE conjugate. Finally the synthesized gold nanoparticles and AuNPs/AChE were characterized by absorption spectroscopy and transmission electron microscopy methods, and the influence of temperature and pH on enzyme activity between AChE and AuNPs/AChE was studied, then the biosensor performance of AuNPs/AChE biological conjugate on detection of paraoxon was studied. It was also calculated the bimolecular rate constants and inhibition constants of paraoxon combination with AuNPs-AChE-DDAO, and binding constants of paraoxon and AuNPs-AChE-DDAO biological conjugates. The inhibition type between AChE and DDAO was also tested. Using autodock4.2 software, the interactions between DDAO and AChE was studied by molecular docking. The absorption spectra and transmission electron microscopy images showed that the AgNPs/AChE conjugate had formed. Compared with the free enzyme, the immobilized AChE had better stability in the same temperature and acidity. In the presence of AuNPs/AChE bio-conjugates, the fluorescence intensity of DDAO had greatly enhanced,then the quenching phenomenon of DDAO fluorescence occured when the paraoxon was gradually added in this system. Consequently, a simple and sensitive method for determination of paraoxon was established according to the change of fluorescence intensity.The detection limit was 4×10-7 M. The sensor had good stability, fast response, high sensitivity. The interaction studies between AChE and DDAO showed that DDAO strongly inhibited the activity of AChE, the inhibition constant Ki was 1.1×10-7 M and resulted in a mixed inhibition type. Binding constant was 3.2×106 L-mol-1, which showed that there was a strong interaction between paraoxon and DDAO/AChE/AuNPs. The results of molecular docking showed that it was different from paraoxon binding to Ser200 in the active center of AChE. DDAO interacts with peripheral anion sites of AChE. It reasoned that the high sensing performance between paraoxon and DDAO-AChE-AuNPs was due to disturbance of the conformation of AChE conjugates. It ultimately lead to the changes of the micro-environment of DDAO and resulted in the changes of fluorescence emission performance.Chapter three Organophosphate biosensor based on fluorescence enhancement with silver nanoparticles In the second chapter, it was found that the gold nanoparticles could improve fluorescence of fluorescent probe DDAO. Many literatures reported that gold nanoparticles had fluorescence quenching features, few literatures reported that gold nanoparticles had a fluorescent enhancement effect.On the contrary, more studied showed that silver nanoparticles resulted in the fluorescent enhancement effect. To validate the experimental results, AChE the labeled silver nanoparticles was synthesized and characterized by absorption spectroscopy and transmission electron microscopy. The influence of temperature and pH value on enzyme activity of free AChE and AuNPs/AChE bioconjugate was studied. Then, the biosensing performance of AgNPs/AChE bioconjugate on detection of paraoxon was tested. The binding constants of AgNPs-AChE-DDAO bioconjugates and paraoxon was also calculated. The absorption spectra and images of transmission electron micros-copy showed that the AgNPs/AChE conjugate had been fabricated. At the same temperature and acidity, the immobilized AChE was more stabe than free AChE. The calculatesd binding constant was 1.7×106 L·mol-1, which also showed that there was a strong interaction between paraoxon and DDAO/AChE/AgNPs. In the presence of AgNPs/AChE bio-conjugates, the fluorescence intensity of DDAO had greatly enhanced, then the quenching phenomenon of DDAO fluorescence occured when the paraoxon was gradually added. It indicated that DDAO/AChE/AgNPs bioconjugate was also sensitive for biosensing paraoxon.更多还原