高灵敏度玉米赤霉烯酮免疫层析检测卡的研制
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摘要
目的 研制高灵敏度玉米赤霉烯酮免疫层析检测卡。方法 制备一种粒径为(116.7±2.0)nm、分支多的金纳米花(AuNFs),用AuNFs标记玉米赤霉烯酮(ZEN)单克隆抗体(AuNF-ZEN-Mab),研制出用于检测粮食和饲料中的玉米赤霉烯酮金纳米花免疫层析卡(ZEN-GFICT)。结果 对比法ZEN-GFICT和消线法ZEN-GFICT对ZEN标准品的检测灵敏度分别为0.5和6 ng/mL,对实际样品检出限分别为5和60μg/kg,是传统ZEN纳米金免疫层析检测卡(ZEN-GICT)的6倍(对比法)和4.5倍(消线法)。21份样品的ZEN-GFICT判断结果(对比法和消线法)与ELISA试剂盒检测结果相比,总符合率均为90.5%。结论 ZEN-GFICT对比法和消线法可以对样品中ZEN含量做出<5、 5～60和>60μg/kg的三个区间判定。检测样品时,能够筛查ZEN污染程度低的样品。
OBJECTIVE To develop a highly sensitive immunochromatographic test card for zearalenone. METHODS Gold nanoflowers(AuNFs) with a particle size of(116.7±2.0) nm and multiple branches was prepared and labeled to the zearalenone monoclonal antibody. Based on the gold nanoflower-labeled zearalenone monoclonal antibody(AuNF-ZEN-Mab), a zearalenone gold nanoflower immunochromatographic test card(ZEN-GFICT) was developed for the detection of food and feed. RESULTS The detection sensitivities of ZEN-GFICT(contrast method) and(cancellation method) for ZEN standards were 0.5 and 6 ng/mL, respectively, and the limit of detection(LOD) for actual samples were 5 and 60 μg/kg, respectively. It was 6 times(contrast method) and 4.5 times(cancellation method) of the traditional ZEN nano gold immunochromatographic test card(ZEN-GICT). The total conformity of the ZEN-GFICT judgment result(contrast method and cancellation method) of 21 samples compared with those of the ELISA kit were 90.5% and 90.5%, respectively. CONCLUSION The ZEN content in the sample with three intervals of less than 5 μg/kg, 5-60 μg/kg and more than 60 μg/kg could be screened by the ZEN-GFICT contrast method and the cancellation method. It is possible to screen samples with a low degree of ZEN contamination.
OBJECTIVE To develop a highly sensitive immunochromatographic test card for zearalenone. METHODS Gold nanoflowers(AuNFs) with a particle size of(116.7±2.0) nm and multiple branches was prepared and labeled to the zearalenone monoclonal antibody. Based on the gold nanoflower-labeled zearalenone monoclonal antibody(AuNF-ZEN-Mab), a zearalenone gold nanoflower immunochromatographic test card(ZEN-GFICT) was developed for the detection of food and feed. RESULTS The detection sensitivities of ZEN-GFICT(contrast method) and(cancellation method) for ZEN standards were 0.5 and 6 ng/mL, respectively, and the limit of detection(LOD) for actual samples were 5 and 60 μg/kg, respectively. It was 6 times(contrast method) and 4.5 times(cancellation method) of the traditional ZEN nano gold immunochromatographic test card(ZEN-GICT). The total conformity of the ZEN-GFICT judgment result(contrast method and cancellation method) of 21 samples compared with those of the ELISA kit were 90.5% and 90.5%, respectively. CONCLUSION The ZEN content in the sample with three intervals of less than 5 μg/kg, 5-60 μg/kg and more than 60 μg/kg could be screened by the ZEN-GFICT contrast method and the cancellation method. It is possible to screen samples with a low degree of ZEN contamination.
引文
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[2] MAIORANO G,RIZZELLO L,MALVINDI M A,et al.Monodispersed and size-controlled multibranched gold nanoparticles with nanoscale tuning of surface morphology [J].Nanoscale,2011,3:2227-2232.
[3] WEI Q,SONG H M,LEONOV A P,et al.Gyromagnetic imaging:dynamic optical contrast using gold nanostars with magnetic cores [J].J Am Chem Soc,2009,131(28):9728-9734.
[4] PERRAULT STEVEN D,CHAN WARREN C W.Synthesis and surface modification of highly monodispersed,spherical gold nanoparticles of 50-200 nm [J].J Am Chem Soc,2009,131(47):17042-17043.
[5] LI J,WU J,ZHANG X,et al.Controllable synthesis of stable urchin-like gold nanoparticles using hydroquinone to tune the reactivity of gold chloride [J].J Phys Chem C,2011,115(9):3630-3637.
[6] 邓振伟,于萍,陈玲.SPSS 软件在正交试验设计、结果分析中的应用[J].电脑学习,2009(5):15-17.
[7] 郝拉娣,张娴,刘琳.科技论文中正交试验结果分析方法的使用[J].编辑学报,2007,19(5):340-341.
[8] 刘瑞江,张业旺,闻崇炜,等.正交试验设计和分析方法研究[J].实验技术与管理,2010,27 (9):52-55.
[9] REVERBERI M,RICELLI A,ZJALIC S,et al.Natural functions of mycotoxins and control of their biosynthesis in fungi [J].Appl Microbiol Biot,2010,87:899-911.
[10] SAMBUU R,TAKAGI M,NAMULA Z,et al.Effects of exposure to zearalenone on porcine oocytes and sperm during maturation and fertilization in vitro [J].J Reprod Develop,2011,57(4):547-550.
[11] 梁梓森,马勇江,刘长永,等.玉米赤霉烯酮对小鼠免疫器官的毒性作用[J].中国兽医科学,2010,40(3):279-283.
[12] KAKEYA H,TAKAHASHI-ANDO N,KIMURA M,et al.Biotransformation of the mycotoain,zearalenone,to a non-estrogenic compound by a fungal strain of Clonostachys sp.[J].Biosci Biotech Bioch,2002,66(12):2723-2726.
[13] 余增丽,张立实,吴德生.玉米赤霉烯酮对MCF-7细胞肿瘤相关基因表达的影响[J].毒理学杂志,2005,19(3):175-177.
[14] 中华人民共和国国家卫生和计划委员会.食品安全国家标准食品中真菌毒素限量:GB 2761—2017 [S].北京:中国标准出版社,2017.
[15] MARIN S,RAMOS A J,CUEVAS D,et al.Fusarium verticillioides and fusarium graminearum infection and fumonisin B1 and zearalenone accumulation in resveratrol-treated corn[J].Food Sci Technol Int,2006,12(4):353-359.
[16] 赵红艳.玉米赤霉烯酮胶体金免疫层析试纸条的研制[D].南京:南京农业大学,2015.
[17] 张甄,郑丽娜,裴世春.同时检测AFB1 和ZEA 免疫层析试纸条的研制[J].黑龙江八一农垦大学学报,2010,22 (2):69-73.
[18] SUN Y N,XING G X,YANG J F,et al.Development of an immunochromatographic test strip for simultaneous qualitative and quantitative detection of ochratoxin A and zearalenone in cereal [J].J Sci Food Agr,2016,96(11):3673-3678.
[19] HOYOS-LEYVA J,BELLO-PéREZ L A,AGAMA-ACEVEDO E,et al.Potential of taro starch spherical aggregates as wall material for spray drying microencapsulation:functional,physical and thermal properties [J].Int J Biol Macromol,2018,120:237-244.
[20] 陈丽媛.2018年1-6 月饲料及原料霉菌毒素分析报告 [J].国外畜牧学 (猪与禽),2018,38(8):70-72.
[21] 王若军,苗朝华,张振雄,等.中国饲料及饲料原料受霉菌毒素污染的调查报告[J].饲料工业,2003,24(7):53-54.