【作者】 刘楠；

【导师】 晁福寰； 高志贤；

【作者基本信息】 中国人民解放军军事医学科学院 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 食品安全已成为全球关注的焦点,在我国存在着严峻和突出的问题。其中,环境和食品中多种农兽药残留已引起人们的广泛关注。为解决一些现有常规技术达不到对多残留快速、灵敏和准确地检测,建立一种新型多农兽药残留的高通量悬浮芯片检测技术和方法,实现快速、准确、特异和高效的定量检测,为建立悬浮芯片高通量的多农兽药残留检测技术平台奠定基础。本研究以常用的4种兽药(氯霉素、克伦特罗、雌二醇和泰乐菌素)和3种农药(阿特拉津、吡虫啉和甲萘威)共7种农兽药为靶标物,基于间接竞争法的免疫学检测原理,改进了常规悬浮芯片技术在微球上固定抗体的方法,实现了一次检测可最多同时检出7种农兽药残留靶标物的目标;该技术还摒弃了常规悬浮芯片技术步骤繁琐、实验耗时长的不足,通过优化和筛选实验条件,基本达到抗原抗体的完全反应,在实验中无需常规Luminex实验所必需的昂贵的滤膜板和抽滤泵,仅需普通96孔细胞培养板或酶标板即可,也无需进行反复多次的抽滤步骤;因此,检测简便、快捷,缩短时间,节省了实验成本,结果获得稳定可靠。研究内容包括以下几方面:1、各种待测农兽药靶分子蛋白结合物的制备和鉴定2、农兽药蛋白结合物在羧基荧光微球上的偶联、确证和优化3、农兽药残留的悬浮芯片单通道检测方法建立4、SPR生物传感技术对抗原抗体反应动力学参数的测定5、微球表面形态结构的扫描电镜表征6、多农兽药残留检测的高通量悬浮芯片技术研究7、农兽药残留检测的方法学比对研究采用活性酯法、重氮化法、混合酸酐法和碳二亚胺法等合成各种农兽药的蛋白结合物;纯化后,对合成的产物进行紫外和质谱鉴定;采用氨基偶联法将7种蛋白结合物偶联于悬浮芯片检测的固相载体——聚苯乙烯荧光微球上,并进行偶联的确证。通过紫外和质谱法对7种农兽药蛋白结合物的检测,证实了各种农兽药抗原结合物的成功合成,获得了精确的偶联比,为结合物在微球上偶联量的计算提供了依据。在此基础上,对农兽药蛋白结合物的加入量、特异性生物素化抗体加入量、激活缓冲液的选择、抗体保护剂的引入、报告荧光分子链霉亲和素-藻红蛋白的稀释度等几个重要的实验条件进行了筛选和优化;通过优化条件、摸索各种待测靶标物的浓度梯度,绘制出7种农兽药靶标物单通道检测标准曲线,建立农兽药残留的悬浮芯片单通道检测方法。同时,采用SPR生物传感技术对各种农兽药抗原和其配对抗体进行了亲和力常数的测定。分别绘制出了7种农兽药残留单通道悬浮芯片检测的Logistic回归标准曲线方程,曲线关系良好,决定系数R2>0.99,获得每种靶标物的最小可检出浓度和检测区间。结果表明,各种农兽药残留的单通道检测方法简单、灵敏、快速。通过悬浮芯片的高通量检测的特异性识别实验,证明了多种特异性抗体和抗原探针之间可准确地相互识别;而且特异性竞争明显,无明显交叉反应;在有机溶剂对荧光微球的洗脱和干扰作用的实验中,证明了在有机溶剂的容积比不超过40%的情况下,微球有一定的抗有机溶剂的洗脱和干扰能力。在单通道检测法建立的基础上,依次进行3种兽药、6种农兽药和7种农兽药同时检测的多元分析,实验结果表明,各个标准曲线方程和相应的决定系数R2表现良好;对各种靶标物的检测区间和最低检出限低于国标;在悬浮芯片多元分析的特异度方面,悬浮芯片的检测特异度测试良好,与其他结构相似和非相似的多种药物无明显交叉和干扰反应;在对含有多种农兽药残留的不同盲样的检测中,检测浓度值与实际浓度的相对偏差较小。总体说来,悬浮芯片系统可用于对多农兽药残留实际样品的初步检测,整个检测过程仅耗时1~2h,基本上满足了多农兽药残留检测的灵敏、特异、快速和高效的需求。扫描电镜对微球表面微观结构的表征,也直观地确证了各种靶标农兽药结合物在微球上的成功偶联、抗原抗体的配对反应和报告荧光分子链霉亲和素-藻红蛋白在微球上的结合等一系列事件。采用传统的常规间接竞争ELISA法和LC-MS法检测各种农兽药残留,与悬浮芯片法进行比对研究,悬浮芯片法在检测区间、高通量和灵敏度与常规ELISA法相比有一定优势;和国标LC-MS法在灵敏度上相当,在检测区间上有一定优势。悬浮芯片技术在国内外多应用于对大分子蛋白质和核酸的检测,对多农兽药残留的检测未见系统报道,属方法和理论上的集成创新。本研究通过建立高通量的多农兽残留检测的悬浮芯片技术,实现了同时对多农兽药残留的高通量多元检测分析,可初步用于对实际样品中多农兽残留的高通量测试分析,检测灵敏、特异、快速和高效,与真实值比较,误差较小,为多农兽药残留的快速检测提供了新方法,为检测平台的建立奠定了基础,具有广阔的应用和发展前景。更多还原

【Abstract】 All eyes have been focused on food safety globally especially in China are extremely and fiercely severe. Close and extensive concerns about pesticide and veterinary drug residues in environment and food are continuously arising. Some normal conventional technologies aren’t capable to detect multiple residues rapidly, sensitively and accurately. To resolve the problem, a novel high-throughput suspension array technology and method has been established for multiple pesticide and veterinary drug residues. The aim of the establishment is to implement quantitative detection of the multiple residues rapidly, accurately, specifically and efficiently and for the foundation of the technical platform.Four kinds of veterinary drugs, including chloramphenicol, clenbuterol, 17-β-estradiol, tylosin and three kinds of pesticide including atrazine, imidacloprid and carbaryl which are commonly used are regarded as the targets. The experiment was based on the theoretic indirect competitive method. The conventional suspension array method that fixation on the beads by anatibody is improved. The goal that up to 7 kinds of residues can be detected at the same time has been achieved by competition of the specific biotinylated antibodies between the multiple antigens of pesticides and veterinary drugs and conjugates which labeled on the fluorescent beads in liquid phase. In addition, the shortage of conventional suspension array technology was abandoned by optimization and screening the experimental conditions. Conventional millipore filter plate and pump were non-necessary during the whole procedures by antigen-antibody complete reactions. Furthermore, ordinary 96-hole cell culture plates or ELISA plates can satisfy the requirement and without repeated pumping. Therefore, it’s simple, rapid, saving lots of time, low-cost and reliable in the experiment. The followings have been concerned:1. Preparation and identification of the conjugates of pesticide and veterinary drugs2. Coupling, validation and optimization of the conjugate on fluorescent bead3. Determination of kinetic parameters of antigen-antibody reactions by SPR biosensor technology4. Characterization of the surface microstructure of the bead by scanning electron microscope5. Simultaneous detection for multiple pesticide and veterinary drug residues by high-throughput suspension array technology6. Methodological comparison of the detection for pesticide and veterinary drug residues Various chemical treatments such as active ester method, diazotization method, mixed anhydride and carbodiimide method, etc were carried out for the synthesis of the protein conjugates of the seven targets. The synthetic products were identified by ultraviolet spectrum and mass spectrum after purification of all the antibodies. The protein conjugates of the targets were identified by ultraviolet spectrum and mass spectrum. And the exact coupling ratios are obtained which provided criterion of conjugates amounts on the beads. And then, the seven protein conjugates were coupled and validated on the solid carrier-the polystyrene fluorescent beads by amino-coupling procedure.Afterwards, the important conditions such as adding amounts of the conjugates and the specific biotinylated antibodies, the activation buffers, antibody preservative and the dilution of streptavidin-phycoerythrin, etc are selected and optimized. By means of optimizing the condition and exploring a variety of the tested target concentration gradient, the standard curves of the single channel suspension array have been polted and the suspension array technique of single channel for the detection of pesticide and veterinary drug residues has been established. Logistic regression standard curves were able to be plotted respectively for the seven targets. All the coefficients of determination-R2 were high than 0.99, the minimum detection concentration and the detection arranges could be acquired respectively. The single channel detection method for pesticide and veterinary drug residue is simple, sensitive and rapid. At the same time, SPR biosensor technology was employed for the determination of antigen-antibody affinity constants of a variety of targets and their complementary antibodies.Specific recognition was carried on and it proved excellent recognition between various paired antigen-antibody on the beads. Meanwhile, specific competition occurred and no obvious cross-reactions with others in the high-throughput detection of suspension array. In the experiment of quenching and interfering by organic solvents, it indicated that when the volume ratio is not more than 40%, there is a degree of anti-quenching and anti-interference with the fluorescent beads.In the case of good response for the single channel of the suspension array, high-throughput detection and multi-analysis of suspension array for three kinds of veterinary drug residues, six kinds of pesticide and veterinary drug residues and seven pesticide and veterinary drug residues are respectively carried out. Standard curves of the multi-analysis were plotted and the coefficients of determination-R2 were investigated and performed well. All the detection range and the lowest detection limits of the targets were determined respectively. And in the aspect of specificity for multiplex analysis, it showed no significant cross-reaction and interference with other structurally similar and non-similar drugs. In the tests of different bland samples with multiple pesticide and veterinary drug residues, the relative standard deviations of the found and the real values are relatively small. Overall, suspension array can be employed for primary detection of real samples of pesticides and veterinary drug residues. It spends only 1 to 2 hours in the whole process. And the requirements for sensitive, specific, rapid and efficient detection for residues are basically satisfied.Scanning electron microscopy was employed for characterization on the micro-structure surface of the beads. It directly confirmed a series of events occurred, such as successful coupling of the conjugates on the beads, antigen-antibody reaction and the fluorescent reporter molecules-streptavidin-phycoerythrin in a combination on beads by biotinylated antibodies.In comparison with the conventional indirect ELISA, suspension array method takes relatively notable advantages in lowest detection limits, high-thought and spends less time. In comparison with LC-MS method, the lowest detection limits of the targets determined by suspension array are lower than national standard except Atrazine and Tylosin. When compared with LC-MS in practice, suspended array have a distinct advantage in the aspects of the lowest detection limit and detection range.The lowest detection limits of suspension array were lower than which of national standards by 2 order of magnitudes, and the detection ranges of suspension array exceeds 1~3 order of magnitudes which of the LC-MS method for imidacloprid and carbaryl detection. It can be concluded that suspension array technology takes the advantage in the detection of multiple pesticide and veterinary drug residues.Traditional indirect competitive ELISA method and LC-MS method have been employed for the detection of multiple pesticide and veterinary drug residues in comparison with suspension array. Compared with traditional indirect competitive ELISA method, suspension array has the advantage over the fomer method by broad detection ranges, high-throughput and sensitivities. Suspension array corresponds with LC-MS method in sensentivity and exceeds it in detection range. It hasn’t been systemically reported at home and abroad yet, and to be considered as integrated innovation methodologically and theoretically.By virtue of establishment of multiple pesticide and veterinary drug residues by high-throughput suspension array technology, simultaneous high-throughput multi-analysis has been achieved and can be primarily employed on sensitive, specific, rapid and efficient determination of real samples and the elative standard deviations of detection values are relatively small by comparison with real values. It provides a novel method and foundation of the technical platform for multiple pesticide and veterinary drug residues with broad and promising application and development prospects.更多还原