【作者】 曹立民；

【导师】 林洪； V.M.Mirsky；

【作者基本信息】 中国海洋大学 ， 水产品贮藏与加工， 2008， 博士

【摘要】 食品中农兽药多残留的快速筛选检测,是当前及今后一段时期内食品安全检测领域的研究难点和热点之一。本论文针对目前应用较广泛的喹诺酮类药物,研究了DNA以及抗体两种生物材料对其的亲和识别特征,建立了相关的模拟和预测体系,完成了族特异性识别材料的筛选制备;在此基础上建立了食品中喹诺酮多残留的快速筛选技术,对所制备识别材料的应用性能进行了验证和完善。主要研究结果归纳如下:（1）首次采用平衡透析方式研究确定了DNA对于喹诺酮类药物的特异性结合作用、关键参数及主要影响因素。研究表明pH以及Mg²⁺对于DNA-喹诺酮之间的结合具有显著影响,最适pH处于6—7之间,最适Mg²⁺浓度为0.5-1.0mM;单链DNA对于恩诺沙星的亲和常数以及结合比约为双链DNA的2倍,而且与恩诺沙星等6种喹诺酮呈现出非常相似的结合性能:亲和常数都处于0.94×10⁵ M^-1至2.40×10⁵ M^-1之间,而结合比在4.1—6.9之间,显示出较为明显的族特异性识别特征;以未经纯化处理的牛奶为食品样本进行了DNA-恩诺沙星结合能力的验证实验,与缓冲液中的结果没有显著差异,证明所采用的DNA对于喹诺酮具有较高的特异性,可以作为族特异性识别材料用于食品分析等。（2）以单链DNA为喹诺酮识别材料,发展了表面等离子共振生物（SPR）传感器,并以牛奶为样本对其检测性能进行了验证。研究中利用LbL多层累积手段,以PDDA为配体多聚阳离子建立了DNA-PDDA在传感器芯片表面的共固定化技术,固定化DNA数量与LbL循环反应次数之间存在显著的正相关性;适量Mg²⁺的添加以及牛奶提取液对芯片的预处理,可以有效消除非特异性结合等因素对于检测的干扰;在加标牛奶样本中进行的检测显示出较好的特异性,对于思诺沙星的检测限约为3μg mL^-1,批内变异系数＜15%,0—120μg mL^-1之间呈现较好的线性关系。（3）利用Cerius2软件等建立了分子模拟及预测模型,并在此基础上完成了具有较高族结构特征的喹诺酮半抗原的筛选。对喹诺酮分子的空间构象进行模拟和叠合的结果表明,1位取代基和7位是导致不同药物间构象差异的主要因素,1位与邻近位置间的相互作用会对分子的整体构象产生影响;首次建立了分子空间结构相似度的计算与预测模型,拟合验证表明,结构相似度与抗体交叉反应率之间存在较显著的线性相关性（R=0.7852,P＜0.0001）,证明该定量模拟模型具有较高的准确度;分子模拟结果表明,培氟沙星、恩诺沙星以及环丙沙星甲基化修饰物具有较高的族内结构相似度。（4）分别制备了培氟沙星和环丙沙星甲基化衍生物抗体,测定表明它们均具有较好的族内识别能力,与前期的分子模拟及预测结果能够较好吻合,其中培氟沙星抗体对所选用10种喹诺酮药物中的9种具有25%以上的交叉反应率,对其中6种的交叉反应率基本达到70%以上。（5）利用培氟沙星抗体为族特异性识别材料,分别建立了ELISA及DIGFA免疫检测方法,鳗鱼加标检测的结果表明,两种方法均能够对恩诺沙星等6种以上的喹诺酮残留实现有效测定,其中ELISA方法对于10μg kg^-1和20μg kg^-1加标样本的平均回收率基本达到75%以上,相对标准偏差基本处于15%以内;DIGFA检测的检测限为20μg kg^-1以下,检测时间可缩短到半小时以内,检测结果可以肉眼读取而不依赖任何仪器,显示出较突出的简便性、快速性和现场适用性。更多还原

【Abstract】 The fast screening of mulitiple drug residues in food stuffs is now one of the key problems in the field of food analysis.In this study deoxyribonucleic acid（DNA） and broad-selective antibodies were developed,characterized and validated as group-sepcific receptors for quinolones which are widely used in veterinary as antibiotics.The important results were summerized as following:（1） The ability of DNA to extract qinolones from model solutions and real probes of food was demonstrated and investigated quantitatively.An interaction between quinolones and different types of DNA was studied by equilibrium dialysis.The first application of this direct approach allowed us to determine binding constants and binding stoichiometries in different conditions. The binding of enrofloxacin to heating-denatured DNA（d-DNA） from herring sperm is pH and magnesium dependent;the highest fraction of bound drugs was found at pH 7 and magnesium concentration of 0.5-1 mM.Results for three types of DNA:d-DNA,double stranded DNA（ds-DNA） and single stranded DNA（ss-DNA） were compared.The unwound DNA showed almost doubled binding constants（Ka） and stoichiometries thus indicating on preferable interaction of enrofloxacin with single strand regions of DNA.The binding of other fluoroquinolones（lomefloxacin,ciprofloxacin,norfloxacin,danofloxacin and sarafloxacin） with d-DNA are very similar to that of enrofloxacin:the binding constants are in the range from 0.94×10⁵ to 2.40×10⁵ M^-1,and the stoichiometries are from 4.1 to 6.9 of quinolone molecule per 100 DNA bases.The binding properties were quantitatively the same for extraction of quinolones from model aqueous solutions or from liquid food（milk）.The results indicate on the efficiency of DNA for selective extraction of quinolones from real samples for further analysis.This selective binding also allows us to consider DNA as a natural receptor for development of analytical techniques for quinolones.（2） A DNA-based surface plasmon resonance biosensor was developed.Heating denatured DNA immobilized on the gold-coated glass surface was exploited as the broad selective receptor.The immobilization was performed by a layer-by-layer co-deposition with a cationic polymer.The sensor performance was tested with real biological probes.Direct and simple determination of enrofloxacin in milk samples was demonstrated.The sensor response obeys Langmuir binding isotherm being almost linear until about 20μg ml^-1.The detection limit in milk samples was estimated to be 3μg ml^-1.（3） A new molecular modeling mode was developed and validated for both quanlitive and quantitive cgaracterization of quinolones haptens.The quanlitive modeling of 3-D conformations showed that 1-substitute and 7-substitute of quinolones molecules have significant effect on the conformation of haptens;the conformation difference among quinolones is caused mainly by the different substitutes at 1 and 7 positions.The 8-substitute also showed some effect by its inter-reaction with 1-substitute.Then a quantitive model was for the first time developed and validated with 4 quinoloes and respective antibodies.The significant positive correlation between calculated similarity of haptens and cross reactivity of antibodies proves that the developed model can fit well with real properties,which allowed us to suggest its application for screen of hapten as well as the precision of the specificity of respective antibodies.（4） Based on molecular modeling,peifloxacin and a synthesize derivate of ciprofloxacin were chosed as haptens for their high structural similarity with other quinolones.The respective polyclonal antibodies were developed and characterized,and both of them showed expected broad selectivity to quinolones investigated.Antibodies against peifloxacin showed cross-reactivities over 25%to 9 of 10 quinolones investigated,among which the cross reactivity was approximately 70-100%to 6 quinolones.（5）With prepared anti-peifloxacin antibodies both ELISA and a dot immunogold filtration assay（DIGFA） were developed for screening of multiple quinolones residues.Using eel muscles as food samples,at least 6 quinolones drugs can be simultaneously determined.For enrofloxacin,norfloxacin,ciprofloxacin,peifloxacin,ofloxacin and danofloxacin spiked at concentrations of 10μg kg^-1 and 20μg kg^-1,the average recovery of ELISA was approximately more than 70%with a RSD less than 15%.With DIGFA the detection limit for 6 quinolones mentioned above was estimated to be lower than 20μg kg^-1;the detection can be completed in 30 min and the results can be judged by naked eyes instead of instruments, which proves its potenciality for fast field screening of quinolones in food samples.更多还原