【作者】 连子如；

【导师】 王江涛；

【作者基本信息】 中国海洋大学 ， 海洋化学， 2013， 博士

【摘要】 分子印迹技术（Molecular Imprinting Technique, MIT）是一种制备高分子聚合物的新兴技术，合成的高聚物被称为分子印迹聚合物（Molecularly ImprintedPolymer, MIP），具有选择性高、稳定性好、耐酸碱、可重复使用等优点，已经在环境监测、食品安全、分离和色谱分析等领域得到广泛应用，其中，基于传统固相萃取而发展起来的分子印迹固相萃取技术（Molecularly Imprinted Solid-phaseExtraction，MISPE），是近年来分子印迹研究的热点之一。本文选取海洋环境中典型的有机污染物作为研究对象，制备相应的分子印迹材料，通过离线模式的固相萃取，联用高效液相色谱/质谱等手段，建立相应污染物及其微藻毒素的分析方法，为海洋环境中有机污染物和麻痹性贝毒的分离检测提供一种新的思路和途径。论文的主要研究内容如下：1）选取水产品养殖中已禁用的典型渔药-三苯甲烷类物质作为印迹目标，分别制备孔雀石绿和结晶紫分子印迹聚合物，优化聚合条件，利用扫描电镜、红外光谱和紫外光谱分析等手段，对聚合物的表面形貌、模板分子与功能单体之间的相互作用力及其分子识别机理等进行研究，同时对模板分子在聚合物上的吸附容量和选择识别性能进行考察，最后以该印迹聚合物作为固相萃取填料，自制印迹固相萃取小柱，离线模式下对天然海水和鱼虾样品中的孔雀石绿和结晶紫进行分离富集和检测。结果表明，自制分子印迹固相萃取小柱能去除样品中复杂基质的干扰，快速有效地对印迹目标实现分离富集，海水样品和鱼虾样品中均有三苯甲烷类物质检出，但浓度低于国家现行标准。2）近年来，随着近岸海水养殖的迅速发展，青岛胶州湾内的水产养殖规模和养殖密度日益增大，养殖水体已有恶化的趋势。磺胺嘧啶是水产养殖中已禁用的磺胺类抗生素的一种，但因其价格低廉，存在偷偷使用的现象。本文以传统的本体聚合法制备磺胺嘧啶印迹聚合物，并制成固相萃取小柱，将其用于胶州湾海水中磺胺嘧啶的测定，加标样品的回收率为79.17%-86.54%，相对标准偏差RSD是2.43%-4.32%（n=3），大面调查的七个海水样品中，从湾西部两个站位中检出磺胺嘧啶，海湾的东部和中部均没有磺胺嘧啶检出。3）随着毒素监测在国内的广泛开展，麻痹性毒素的分离纯化，引起了人们的日益重视。膝沟藻毒素GTX2,3是我国海域产毒赤潮藻种中较为常见的一种麻痹性贝毒，毒性较强，分离提取难度较大，价格昂贵，本文采用片段印迹，选取膝沟藻毒素的结构片段咖啡因作为虚拟模板，悬浮聚合法在水相中制备了分子印迹微球，通过扫描电镜和比表面积测定仪对印迹微球的形貌进行表征，利用平衡吸附实验对膝沟藻毒素GTX2,3在印迹微球上的分子识别能力进行考察，为下一步从产毒的微藻毒株中提取膝沟藻毒素提供了研究基础。以上一步实验中合成的咖啡因分子印迹微球为填料制备固相萃取小柱，在离线模式下联用HPLC，优化淋洗和洗脱条件，建立了膝沟藻毒素GTX2,3的分离提取、检测的方法，最后，从产毒微小亚历山大藻株和塔玛亚历山大藻株的藻细胞粗提液中分离提取膝沟藻毒素GTX2,3。结果表明，GTX2,3提取率大于80%，粗提液中的其他毒素成分（如C毒、GTX1,4）经过印迹固相萃取后已经检测不到，自制印迹固相萃取小柱提取效率较高，柱效稳定，印迹微球经再生处理后，可重复利用至少7次以上。更多还原

【Abstract】 Molecularly imprinted polymers (MIPs) are synthetic materials with artificiallygenerated recognition sites able to specifically rebind a target molecule in preferenceto other closely related compounds. MIPs have been employed in fields where acertain degree of selectivity is required such as environmental monitoring, food safety,natural products separation and chromatographic analysis. However, nowadays theiruse in solid-phase extraction, so-called molecularly imprinted solid-phase extraction(MISPE), is by far the most advanced technical application of MIPs. This dissertationtakes typical marine organic pollutants and PSP toxins as the research objects. MIPswere prepared by molecular imprinting technology (MIT), used as solid-phaseextraction sorbents, and then combined to off-line analytical techniques for thedetection of typical marine pollutants and PSP toxins.As following was primary research to be acquired in the dissertation:1） Molecularly imprinted polymers were prepared by the bulk polymerizationusing triphenylmethane dyes (malachite green and crystal violet) as the templatemolecule. Systematic investigations of synthetic conditions were conducted. Thesurface morphology and recognition mechanism of the obtained polymers werestudied using scanning electron microscope, FT-IR and spectrophotometric analysis.MIPs showed high affinity to template molecule and were successfully applied asspecial solid-phase extraction sorbent for selective extraction of triphenylmethanedyes from natural seawater and seafood samples. An off-line MISPE method followedby HPLC with diode-array detection for the analysis of triphenylmethane dyes wasalso established. The extraction on MIPs column can successfully cleanupcomplicated matrix, allowing the extraction of the target with high selectivity.2） In this chapter, a highly selective sample cleanup procedure combined withMISPE was developed for the isolation of sulfadiazine in seawater samples fromJiaozhou Bay, China. The MIP was prepared using sulfadiazine as the templatemolecule, methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the cross-linking monomer. The MIP was used as selective sorbentfor the solid-phase extraction of sulfadiazine. An off-line MISPE method followedby HPLC with diode-array detection for the analysis of sulfadiazine was established.Good linearity on MISPE column for sulfadiazine standard solutions was obtainedwithin0-200μg L1(R2>0.99). The recoveries of spiked seawater samples were in therange79.17%-86.54%. Finally, seven samples in Jiaozhou Bay were determined andthere was no sulfadiazine found except No.2and No.5sample. SDZ was found inwestern area of Jiaozhou Bay. This may be caused by the mariculture, and mostfarming locate in the western part of Jiaozhou Bay.3） Paralytic shellfish poisoning (PSP) is a potentially fatal human healthcondition that may occur after the consumption of PSP toxins contaminated shellfish.The toxins are produced predominantly by dinoflagellates. Gonyautoxins are commonof PSP toxins in China sea. In view of the high toxicity and cost of the target, dummyimprinting is a viable strategy for the synthesis of MIPs. In this chapter, molecularlyimprinted polymer microspheres (MIPMs) for gonyautoxins2,3were preparedthrough suspension polymerization method using caffeine as the dummy templatemolecule, methacrylic acid as the functional monomer, ethylene glycol dimethacrylateas the cross-linker and polyvinyl alcohol as the dispersive reagent. Systematicinvestigations of synthetic conditions were conducted and equilibrium bindingexperiments were performed. The surface morphologies of polymers were studied byusing SEM and BET. The results show that the obtained MIPMs showed high affinityto gonyautoxins2,3and this study will provide the research foundation for the nextseparation and isolation of gonyautoxins2,3from dinoflagellate Alexandrium.A new and selective sorbent for MISPE was developed for the isolation ofgonyautoxins2,3from dinoflagellate Alexandrium minutum and A. tamarense sample,and validated by HPLC coupled to fluorescence detection. The molecularly imprintedpolymer microspheres were prepared by precipitation polymerization using caffeineas the dummy template molecule and polyvinyl alcohol as the dispersive reagent. Theimprinted polymer microspheres exhibited high affinity, selectivity for gonyautoxins2,3and used as a selective sorbent for the solid-phase extraction of gonyautoxins2,3.An off-line MISPE method was established and good linearity on MISPE column forthe gonyautoxins2,3standard solutions was obtained within0-20μg L1(R2>0.99). Furthermore, the extract samples from A. minutum and A. tamarense were analyzed.The results showed the interferences matrix in the extract were obviously cleaned byMISPE. The extraction efficiency of gonyautoxins2,3exceeded80%. The obtainMIPMs could be used up to7times after a short regeneration step and maintainedtheir adsorption capacity at an almost constant value.更多还原