【作者】 郭逸蓉；

【导师】 朱国念；

【作者基本信息】 浙江大学 ， 植物保护， 2010， 博士

【摘要】 近年来,农产品质量安全问题日益成为社会关注的焦点之一。作为食品质量安全控制和环境监测的重要手段,农药残留检测技术一直是国内外食品安全领域的一大研究热点。而分子印迹技术(MIT)为解决样品分析、产物分离过程中的选择性问题提供了一条方便、有效的途径,目前已经成为了农药残留分析与检测研究领域的热门方向之一自上世纪末以来,MIT的探究及其发展迅速。然而,目前真正实现分子印迹聚合物(MIP)产业化的种类很少,大部分MIP的研究仍处于实验阶段,其中一个主要原因是MIP的制备受到多种因素的影响,印迹体系的筛选十分复杂困难,尤其是对于报道较少的弱官能性模板分子而言。本研究首先采用计算机分子模拟软件Hyperchem(?) 7.5来预测模板分子与功能单体之间的非共价作用,通过分子力学(MM)方法和PM3半经验法优化,模拟了8种常用的拟除虫菊酯类农药和13种功能单体的分子最低能量构象,然后采用Amber MM方法分析了各菊酯农药与功能单体之间的相互作用并计算了相应的结合能,以期筛选出合适的模板分子和功能单体,用于辅助设计拟除虫菊酯类农药MIP的制备。结果发现,总体上就功能单体而言,甲基丙烯酸(MAA)和2-羟基乙基-甲基丙烯酸脂(HEMA)与各菊酯农药分子的结合能较高；就农药分子而言,氰戊菊酯与MAA和HEMA的分子作用力均较强,三氟氯氰菊酯分别与两种功能单体之间的作用均较弱,其他几种菊酯类农药分别与两种功能单体的作用力差异较大；各菊酯异构体之间与功能单体的分子作用力表现出不同程度的差异。根据上述计算机预测结果,同时考虑当前农药市场信息和残留监测要求,优先选择了与功能单体互作强度不同的三氟氯氰菊酯和氰戊菊酯为代表模板分子,采用不同的功能单体、交联剂、致孔溶剂和聚合方法,分别制备并筛选得到了能特异性结合各自模板的MIPs,确定了合适的分子识别溶剂体系,并考察了有关印迹聚合物对其他拟除虫菊酯类农药的结合选择性。试验结果发现,不同的识别溶剂体系对MIP的选择性结合有较大影响；以HEMA为功能单体、对-二乙烯苯(DVB)为交联剂、乙腈为致孔剂合成的MIP,在正己烷中对三氟氯氰菊酯的选择性识别最强,特别是沉淀聚合的MIP微球有更高的结合容量；HEMA-co-DVB三氟氯氰菊酯MIP还能对溴氰菊酯和氰戊菊酯有一定的选择性结合,这表明该类MIP能用来结合多种第二代拟除虫菊酯类农药,可望在拟除虫菊酯类农药多残留检测的样品制备方面得到应用。再则,以MAA为功能单体、DVB为交联剂,在乙腈／甲苯=3：1(v／v)的混合溶剂中沉淀聚合得到的氰戊菊酯MIP微球,能在正已烷体系中特异性识别模板分子,印迹因子可达2.16。总体而言,以氰戊菊酯为模板制备的MIP印迹效果高于三氟氯氰菊酯MIP。此外,为实现MIP在含水相中对拟除虫菊酯类农药的更高特异性结合效果,制备得到了基于环糊精包合作用的MIP,性能测试表明,该MIP适用于亲水性环境下拟除虫菊酯类农药的选择性富集和分离。试验结果能够较好地佐证计算机分子模拟的预测结果。通过本论文的研究,初步建立了拟除虫菊酯类农药的计算机辅助设计MIP研制体系。同时,对拟除虫菊酯类农药MIP的选择性识别机理进行了探讨,初步阐明了拟除虫菊酯类农药MIP识别位点的形成不仅取决于模板与功能单体的相互作用,还与印迹分子的立体结构紧密相关。本研究对开发切实有效的适合于拟除虫菊酯类农药残留检测的MIT及其产品具有较好的理论指导意义,还可为其他分析物特别是非极性化合物的MIP研制中模板分子、功能单体等参数的选择提供了有益借鉴。更多还原

【Abstract】 In recent years, increasing attention has been paid to the quality and safety (QS) of farm products and foodstuffs, and this issue has already become the top priority for the sake of human being’s health all over the world. As a key role for food QS control and environmental monitoring, the determination of pesticide residues becomes a hotspot in the related research field. Molecular imprinting technique (MIT) has proved to be an available and convenient alternative for solving the selectivity or specificity in sample analysis and product separation. Nowadays, it is a rising tide of study on pesticide residue analysis.MIT has been developed very quickly since the end of the last century. However, the efficient synthesis of molecularly imprinted polymer (MIP) is affected by lots of physical and chemical factors, and thus there are only a few kinds of MIPs which have been really developed for the commercial application. A majority of the reported MIPs were only on the laboratorial stage owing to the complexity of molecular imprinting system, especially for the poor functionalized templates. In the present study, a computational approach using Hyperchem(?) 7.5 molecular modelling software was firstly applied to predict the non-covalent interactions between pyrethroid pesticides and functional monomers. The minimum energy conformations of eight commonly used pyrethroid pesticides and thirteen functional monomers were simulated by molecular mechanics (MM) and then refined using PM3 semi-empirical mechanic methods. Afterward, the Amber MM method was used to analyse possible interactions between templates and functional monomers and to calculate their binding energies, so as to select the suitable templates and monomers and thus to design for pyrethroid-MIP development. Results indicated that, generally, in view of functional monomers, complexes of templates with methacrylic acid (MAA) and 2-hydroxyethyl methacrylate (HEMA) gave the relatively higher binding energies than these with other monomers. In terms of the eight pyrethroid pesticides, fenvalerate showed the relatively stronger interactions with MAA and HEMA, but cyhalothrin gave the opposite phenomenon; while other pyrethroids displayed the diverse intensity of interactions with the two monomers; for the same pyrethroid, the more or less variation of the interactions among different isomers was also found.Based on the above research and considering the current pesticide marketing information and the demand for residue monitoring, fenvalerate and cyhalothrin were selected as the typical templates which respectively showed high and low interactions with functional monomers. Using different monomers, cross-linkers, porogenic solvents and polymerization methods, various MIPs for fenvalerate and cyhalothrin were synthesized and screened. Then the appropriate solvent system for the specific rebinding of the MIPs was optimized and the selective recognition of related MIPs to other pyrethroids was also investigated. Results revealed that different rebinding solvents greatly influenced the selective absorption of MIPs to the corresponding pesticides. The cyhalothrin-MIPs prepared with HEMA (as functional monomer),p-divinylbenzene (DVB, as cross-linker) and acetonitrile (as porogen) had the better selectivity in n-hexane system than in other organic solvents, and particularly the MIP microsphere synthesized by precipitation polymerization had the much higher binding capacity. Withal, the HEMA-co-DVB cyhalothrin-MIP also had selective recognition to fenvalerate and deltamethrin, which suggested that this type of MIP could hopefully be applied to multi-residue determination of type II pyrethroids. Besides, fenvalerate-MIP prepared by precipitation polymerization in the mixture solvent of acetonitrile and toluene (3:1, v/v) using MAA (as functional monomer) and DVB (as cross-linker) could specifically recognize its template molecule in n-hexane, with imprinting factor (IF) up to 2.16. Compared with cyhalothrin-MIP, the imprinting efficiency of fenvalerate-MIP was higher. In addition, aimed at achieving higher specificity and efficient recognition of MIPs to pyrethroid pesticides in aqueous media, inclusion interaction was employed to synthesize pyrethroid MIPs withβ-cyclodextrin as functional monomer. And equilibrium rebinding test reflected that the developed MIP would be used as potential high-performance materials for the selective concentration and/or separation of trace pyrethroids from complex sample matrixes under the hydrophilic condition. Overall, it was found that, satisfactorily, the experimental results were in accord with the outcomes from computer simulation.In this study, an elementary research system of computer-assisted design for the preparation of MIPs for pyrethroid pesticides was established. Moreover, the mechanism of MIPs’selective binding to pyrethroid templates was also shallowly probed. It was illuminated that the formation of binding sites not only lied on the interaction between template and functional monomer, but also was tightly related to its stereostructure of template molecule. The present research could provide an academic guidance to the development of MIT for pyrethroid pesticide detection, and offered a useful reference to the preparation of MIPs for other analytes, especially the non-polar compounds.更多还原