用于氯霉素、克伦特罗和雌二醇三种兽药残留检测的高通量悬浮芯片技术研究

Development of a high-throughput suspension microarray technology for detection of three kinds of veterinary drug residues： chloramphenicol, clenbuterol and 17-beta-estradiol

目的建立一种氯霉素、克伦特罗和雌二醇（17-beta-estradiol，E2）的3种兽药残留的新型高通量悬浮芯片检测技术。方法合成3种兽药的牛血清白蛋白（bovine serum albumin，BSA）结合物，并进行紫外和质谱鉴定。将3种蛋白结合物偶联于聚苯乙烯荧光微球上，在液相反应体系中3种小分子兽药抗原和微球上的结合物共同竞争液相中各自特异性的生物素化单抗，优化和筛选出微球上偶联BSA结合物和反应抗体的最适加入量。绘制出3种兽药残留检测的标准曲线；对不同浓度的干扰物和待测物分组，以此进行特异性检测和盲样测定。并用扫描电子显微镜（简称电镜）进行微球表面微观结构观察。结果3种小分子兽药可与BSA成功偶联；3种结合物的加入量和抗体的加入量分别做了优化；悬浮芯片检测的标准曲线方程和方程相应的决定系数（R^2）表现良好，R^2〉0．99；3种兽药悬浮芯片的检测区间分别为（40．00～6．25）×10^5ng／L，（50．00-7．81）×10^5ng／L和1．00×10^3～7．29×10^5ng／L；最低检出限为：40ng／L、50ng／L和1μg／L；同时，悬浮芯片的特异度测试良好，与其他药物无明显交叉反应；对盲样测定的检测浓度值与实际浓度偏差在8．09％-17．03％，可认为偏差较小。电镜对微球表面微观结构的观察也直观地确证了蛋白在微球上的成功偶联。结论高通量悬浮芯片技术操作简单，灵敏快速，成本低廉，为多种兽药残留的快速检测提供了新方法，具有广阔的应用和发展前景。

Objective To establish a novel suspension microarray technology for the detection of three kinds of veterinary drug residues： chloramphenicol, clenbuterol and 17-beta-estradiol （ CAP, CL and E2 ）. Methods The three conjugates that veterinary drug coupled with bovine serum albumin （BSA） were synthesized and identified by ultraviolet （UV）spectrophotometry and mass spectrum. The veterinary drug conjugates were immobilized on the polystyrene fluorescent microspheres/beads. There were competitive reactions between the veterinary drugs in the aqueous phase and that on the beads for combination with their specific biotinylated monoclonal antibodies. The optimum amount of the veterinary drug conjugates and the antibodies were optimized and selected. The detective standard curves were plotted. The specificity and the unknown samples were also determined by grouping according to different concentrations of the interferers and the samples. Meantime, the different microstructures of the surfaces of the beads were also observed by scanning electron microscope. Results Couplings were completed between small molecular veterinary drugs and BSA. The amounts of the three conjugates and the antibodies were optimized. The detective standard curves of the suspension array and their corresponding coefficients of determination （ R^2 ） were good （ R^2 〉 0.99） .The detection ranges of the three veterinary drugs were （40.00 -6.25 ） ×10^5 ng/L,（50. 00 -7.81 ）× 10^5 ng/L and 1.00 × 10^3 - 7.29 × 10^5 ng/L respectively. Simultaneously, the specific detection of the suspension microarray was excellent and did not indicate significant cross-reactions. Errors between the found and the real are in the range of 8. 09% - 17. 03%. It can be considerd that the relative standard deviations were relatively small. Successful couplings were also directly confirmed by the observation for microstructures of the surfaces of the beads by scanning of electron microscope and laid good foundation for the following responses. Conclusion The high-throughput suspension microarray should provide a novel method for multi-analysis of the veterinary drugs and have a wide applicative prospects with simple operation, sensitive, rapid and low cost.