可视化普鲁士蓝纳米酶免疫亲和柱快速检测植物源性食品中氯吡脲

Rapid detection of forchlorfenuron in plant-derived food by visual Prussian blue nanase-enzyme immunoaffinity column

目的建立快速检测植物源性食品中氯吡脲的可视化普鲁士蓝纳米酶免疫亲和凝胶柱的检测方法。方法首先通过水热法制备了类过氧化氢酶性质的普鲁士蓝纳米粒子。其次,通过3-巯基丙酸衍生反应合成了氯吡脲半抗原,通过活泼酯法原理偶联合成氯吡脲完全抗原,通过免疫小鼠成功制备了氯吡脲多克隆抗体。最后,制备了普鲁士蓝纳米酶标记抗原、琼脂凝胶闭合胶体、抗体胶,组装了普鲁士蓝纳米酶可视化免疫亲和凝胶检测柱,建立了检测方法,测试了凝胶检测柱的特异性,检测了4种实际样品,得到方法检出限。结果合成的普鲁士蓝纳米粒子呈立方体形状,分布均匀集中,粒径约为145.68 nm±12.00 nm,能够催化3,3’,5,5’-四甲基联苯胺(3,3’,5,5’-tetramethylbenzidine,TMB)和过氧化氢(hydrogen peroxide,H_(2)O_(2))混合溶液显蓝色,具有类过氧化氢酶性质。制备的氯吡脲多克隆抗体半数抑制率(50%inhibiting concentration,IC_(50))为2.35 ng/mL,抗血清效价最高为1:72000倍。凝胶检测柱的最佳组装条件为普鲁士蓝纳米酶标记抗原稀释1500倍,氯吡脲抗体胶稀释20倍、辣根过氧化物酶抗体胶稀释30倍,凝胶检测柱的检出限为5μg/L,4种实际样品的方法检出限为20μg/kg,针对化学结构相似的5种农药具有良好的特异性。结论此方法具有便捷、快速及灵敏的特点,可以作为植物源性食品中氯吡脲的快速检测方法。

Objective To establish a rapid detection method of forchlorfenuron in plant-derived food by visualizable Prussian blue nanase immunoaffinity gel column.Methods Firstly,Prussian blue nanoparticles with catalase-like properties were prepared by hydrothermal method.Secondly,the hapten antigen of forchlorfenuron were synthesized by 3-mercaptopropionic acid derivative reaction,forchlorfenuron complete antigen was synthesized by active ester method,and the polyclonal antibody of forchlorfenuron was successfully prepared by immunizing mice.Finally,Prussian blue nanase labelled antigen,agarose gel closed colloid,forchlorfenuron antibody colloid,the Prussian blue nanase visualizable immunoaffinity gel column was assembled,the detection method was established,the specificity of the gel column was tested.Four actual samples were detected,the limit of detection was acquisited.Results The Prussian blue nanoparticles had a cubic shape,evenly distributed and concentrated,the particle size was about 145.68 nm±12.00 nm.It could catalyze 3,3’,5,5’-tetramethylbenzidine(TMB)and hydrogen peroxide(H_(2)O_(2))mixed solution to develop blue color,suggested with catalase properties,the 50%inhibiting concentration(IC_(50))of the polyclonal antibody was 2.35 ng/mL,the highest titer of forchlorfenuron polyclonal antibody was 1:72000.The optimal assembly condition of the gel column was 1500-fold dilution of Prussian blue nanase labeled antigen,forchlorfenuron antibody gel diluted 20 times,horseradish peroxidase antibody gel diluted 30 times,the limit of detection of gel column was 5μg/L,the limits of detection of the 4 kinds of actual samples were 20μg/kg.Five kinds of pesticides with similar chemical structure had good specificity.Conclusion This method is convenient,rapid and sensitive,it can be used as a rapid detection method for forchlorfenuron in plant-derived food.