兽药多残留悬浮芯片技术检测研究

发布时间：2018-06-05 18:00

本文选题：微球 + 兽药　；参考：《中国计量学院》2015年硕士论文

【摘要】：悬浮芯片技术是最近几十年兴起的多元分析方法,广泛应用于蛋白质和核酸检测。微球是悬浮芯片技术的重要载体。本文尝试先利用分散聚合方法合成单分散聚苯乙烯微球,再用沉降分离法分离获得目标微球,最后用后修饰法在微球表面修饰羧基,成功制得粒径约为5435nm,带羧基的单分散聚苯乙烯微球。这为以后研制悬浮芯片系统配套使用的荧光微球奠定扎实的基础。实验证明二抗上标记的PE荧光信号能被悬浮芯片系统所识别,并成功地将兽药抗原偶联到微球上。之后优化了反应条件,偶联时七种兽药抗原的最佳加入量为:甲硝唑4μg,呋喃唑酮8μg,莱克多巴胺4μg,沙丁胺醇2μg,克伦特罗4μg,磺胺二甲嘧啶4μg,磺胺喹恶啉8μg;反应时,兽药抗体的最佳加入量为:莱克多巴胺5ng,沙丁胺醇10ng,克伦特罗20ng,磺胺喹恶啉10ng,磺胺二甲嘧啶20ng,甲硝唑200ng,呋喃唑酮100ng;羊抗兔PE标记二抗、羊抗鼠PE标记二抗的最佳稀释倍数分别为300、150;而最适的一抗孵育、二抗孵育时间分别为60min、45min。基于优化后的实验参数,根据间接竞争法,兽药抗原偶联微球作为探针与靶分子共同竞争兽药抗体,再以PE标记二抗作为报告信号,建立了莱克多巴胺、克伦特罗、沙丁胺醇、磺胺二甲嘧啶、磺胺喹恶啉、呋喃唑酮代谢物、甲硝唑七种兽药的悬浮芯片技术单一检测方法。该单一兽药检测方法灵敏度高,线性范围宽,特异性强,回收率均在73.2%~108.3%之间。其中,莱克多巴胺、克伦特罗、沙丁胺醇、磺胺二甲嘧啶、磺胺喹恶啉、呋喃唑酮代谢物、甲硝唑的检出限分别达到0.81、0.094、0.83、0.112、0.066、0.28、0.95ng/m L。基于单一兽药残留悬浮芯片技术检测方法和特异性识别实验,建立了该七种兽药同时测定的多残留悬浮芯片技术分析方法。该多残留分析方法对莱克多巴胺、克伦特罗、沙丁胺醇、磺胺二甲嘧啶、磺胺喹恶啉、呋喃唑酮代谢物、甲硝唑的检测范围分别为1.00~200、0.20~100、1.00~100、0.20~100、0.10~100、1.00~50、1.00~100ng/m L,检出限分别为0.93、0.16、0.85、0.16、0.094、0.80、0.89ng/m L。七种兽药的结构类似物对检测无明显干扰反应。除了AOZ,其余兽药的回收率均在70%~120%之间。这证明本多残留分析方法具有灵敏度高、特异性强、线性范围宽、回收率良好等优点。与酶联免疫吸附分析法相比较,悬浮芯片技术不仅具有多元分析的特性,而且在检测范围、最低检测限等许多方面也优于ELISA。综上,悬浮芯片技术是一种优秀的兽药多残留分析的快速检测方法,为食品中污染物多残留快速分析提供全新的方法。
[Abstract]:Suspension chip technology is a multivariate analysis method developed in recent decades, which is widely used in protein and nucleic acid detection. Microspheres are important carriers of suspension chip technology. In this paper, we try to synthesize monodisperse polystyrene microspheres by dispersion polymerization, then separate them by sedimentation separation method to obtain target microspheres. Finally, we modify carboxyl groups on the surface of microspheres by post-modification method. Monodisperse polystyrene microspheres with carboxyl groups were successfully prepared with a diameter of about 5435 nm. This will lay a solid foundation for the future development of fluorescent microspheres used in the suspension chip system. The results show that the PE fluorescence signal labeled by the second antibody can be recognized by the suspension chip system and the veterinary drug antigen is successfully coupled to the microspheres. After that, the optimum reaction conditions were optimized. The optimal dosages of seven veterinary antigens were as follows: metronidazole 4 渭 g, furazolidone 8 渭 g, ractopamine 4 渭 g, salbutamol 2 渭 g, clenbuterol 4 渭 g, sulfamdimethazine 4 渭 g, sulfamethoxaline 8 渭 g. The optimum dosage of veterinary drug antibody was ractopamine 5 ng, salbutamol 10 ng, clenbuterol 20 ng, sulfamethoxaline 10 ng, sulfadiazine 20 ng, metronidazole 200 ng, furazolidone 100 ng, sheep anti PE second antibody, The optimum dilution times of sheep anti-mouse PE labeled second antibody were 300150, and the optimal incubation time of the first antibody was 60 min and 45 min respectively. Based on the optimized experimental parameters, according to indirect competition law, veterinary antigen-coupled microspheres were used as probes to compete with target molecules for veterinary drug antibodies, and PE labeled second antibody was used as report signal to establish ractopamine, clenbuterol and salbutamol. Single determination of sulfadiazine, sulfamethoxaline, furazolidone metabolites and metronidazole by suspension chip technique. The method has the advantages of high sensitivity, wide linear range and strong specificity, and the recovery rate is between 73.2% and 108.3%. The detection limits of ractopamine, clenbuterol, salbutamol, sulfadimethazine, sulfamethoxaline, furazolidone metabolite and metronidazole were 0.81ng / m, 0.81ng/ mL, 0.83ng/ mL, 0.830.09ng/ m, respectively, and the detection limits of metronidazole were 0.81ng/ m. Based on the detection method of single veterinary drug residue suspension chip and the specific identification experiment, a multi-residue suspension chip analysis method for the simultaneous determination of seven veterinary drugs was established. The detection limits of ractopamine, clenbuterol, salbutamol, sulfadimidine, sulfamethoxaline, furazolidone metabolite and metronidazole were 1.000.200.20ng / ml, 0.101.001.001.00501.00100ng / mL, respectively, and the detection limit was 0.93U 0.160.160.850.160.094U 0.800.89ngmL / L, respectively. The structural analogues of seven veterinary drugs had no obvious interference reaction to the detection. With the exception of AOZ, the recoveries of all veterinary drugs ranged from 70% to 120%. This method has the advantages of high sensitivity, high specificity, wide linear range and good recovery. Compared with enzyme-linked immunosorbent assay (Elisa), suspension chip technology not only has the characteristics of multivariate analysis, but also is superior to ELISAs in many aspects, such as detection range, minimum detection limit and so on. In summary, suspension chip technology is an excellent rapid detection method for multiresidue analysis of veterinary drugs, which provides a new method for rapid analysis of contaminants in food.
【学位授予单位】：中国计量学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.84

【相似文献】