基于纳米磁珠和双标记抗体的玉米赤霉烯酮快速、高灵敏检测方法的建立及应用
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摘要
玉米赤霉烯酮(zeralenone,ZEN)具有雌激素活性,主要污染谷物和饲料,大量聚积可导致流产和死胎,给动物和人类健康带来严重威胁。本研究通过将ZEN偶联抗原ZEN-BSA包被于纳米磁珠(magnetic nanoparticles,MNPs),制备纳米磁珠-偶联抗原复合物(MNPs-BSA-ZEN),同时使用金颗粒(Au nanoparticles,AuNPs)和辣根过氧化物酶(horseradish peroxidase,HRP)双标记的ZEN单克隆抗体,建立新型酶联免疫检测方法(MNPs-HRP-AuNPsIC-ELISA)。检测下限(IC10)达到0.03ng/mL,检测区间(IC20–IC80)为0.05–0.89ng/mL,半数抑制率(IC50)为0.22ng/mL,与ZEN类似物(α-zearalanol、zearalanone、α-zearalenol、β-zearalenol和β-zearalanol)的交叉反应性依次为19.2%、11.7%、8.3%、1.2%和4.3%,与黄曲霉毒素B1、赭曲霉毒素A、伏马毒素B1、桔青霉素和展青霉毒素几乎不存在交叉反应。在玉米、面粉和大豆样本中的加标回收率可达81.6%–113.5%,与LC-MS/MS同时对天然样本中ZEN含量的检测结果表明,两种方法相关性良好。本研究建立的MNPs-HRP-AuNPs IC-ELISA具备快速和高灵敏的双重优势,也可为其他霉菌毒素精准检测技术的开发提供参考。
Zearalenone(ZEN), a mycotoxin, is produced by Fusarium graminearum and Fusarium roseum growing primarily on corn or hay exposed to high moisture and threatens to food safety and public health.Children are more vulnerable to ZEN than adults. Sensitive, accurate and rapid analytical methods with new strategies for signal enhancement are needed for detection of ZEN. Therefore, a novel enzyme linked immunosorbent assay(MNPs-HRP-AuNPs IC-ELISA) using magnetic nanoparticles(MNPs) and double labeled monoclonal antibody complex(Anti-ZEN-HRP-AuNPs) was prototyped and used for detecting ZEN in cereal samples. The detection signal was enhanced which in turn improved the sensitivity of the assay.The lower limit of detection using MNPs-HRP-AuNPs IC-ELISA was 0.03 ng/mL while the IC50 was0.22 ng/mL. The linear working range was 0.05-0.89 ng/mL. The cross-reactivities with zeralenone analogues(α-zearalanol, zearalanone, α-zearalenol, β-zearalenol and β-zearalanol) were 19.2%, 11.7%,8.3%, 1.2% and 4.3%, respectively. No cross-reactivity(<0.01%) was observed with other co-occurring mycotoxins(AFB1, OTA, FB1, CIT and PAT). The recovery rates in spiked corn, wheat and soybean samples were 81.6%-113.5%, and the intra-day and inter-day relative standard deviations were <10%.Simultaneous analysis of commercial samples(corn, wheat, and feedstuff) showed a good correlation between MNPs-HRP-AuNPs IC-ELISA and liquid chromatography tandem mass spectrometry(LC/MS-MS).Hence, this new method can be used as a rapid, sensitive, and less time-consuming method to determine levels of zeralenone in cereal and feedstuff samples.
Zearalenone(ZEN), a mycotoxin, is produced by Fusarium graminearum and Fusarium roseum growing primarily on corn or hay exposed to high moisture and threatens to food safety and public health.Children are more vulnerable to ZEN than adults. Sensitive, accurate and rapid analytical methods with new strategies for signal enhancement are needed for detection of ZEN. Therefore, a novel enzyme linked immunosorbent assay(MNPs-HRP-AuNPs IC-ELISA) using magnetic nanoparticles(MNPs) and double labeled monoclonal antibody complex(Anti-ZEN-HRP-AuNPs) was prototyped and used for detecting ZEN in cereal samples. The detection signal was enhanced which in turn improved the sensitivity of the assay.The lower limit of detection using MNPs-HRP-AuNPs IC-ELISA was 0.03 ng/mL while the IC50 was0.22 ng/mL. The linear working range was 0.05-0.89 ng/mL. The cross-reactivities with zeralenone analogues(α-zearalanol, zearalanone, α-zearalenol, β-zearalenol and β-zearalanol) were 19.2%, 11.7%,8.3%, 1.2% and 4.3%, respectively. No cross-reactivity(<0.01%) was observed with other co-occurring mycotoxins(AFB1, OTA, FB1, CIT and PAT). The recovery rates in spiked corn, wheat and soybean samples were 81.6%-113.5%, and the intra-day and inter-day relative standard deviations were <10%.Simultaneous analysis of commercial samples(corn, wheat, and feedstuff) showed a good correlation between MNPs-HRP-AuNPs IC-ELISA and liquid chromatography tandem mass spectrometry(LC/MS-MS).Hence, this new method can be used as a rapid, sensitive, and less time-consuming method to determine levels of zeralenone in cereal and feedstuff samples.
引文
Cha SH,Kim SH,Bischoff K,Kim HJ,Son SW,Kang HG,2012.Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay.Journal of Veterinary Science,13(2):119-125
Dellafiora L,Galaverna G,Dall'asta C,Cozzini P,2015.Hazard identification of cis/trans-zearalenone through the looking-glass.Food and Chemical Toxicology,86:65-71
Dong GL,Pan YH,Wang YL,Ahmed S,Liu ZL,Peng DP,Yuan ZH,2018.Preparation of a broad-spectrum anti-zearalenone and its primary analogues antibody and its application in an indirect competitive enzyme-linked immunosorbent assay.Food Chemistry,247:8-15
Efsa J,2004.Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to zearalenone as undesirable substance in animal feed.EFSA Journal,89:1-35
Hu Y,Shen GQ,Zhu HL,Jiang GX,2010.A class-specific enzyme-linked immunosorbent assay based on magnetic particles for multiresidue organophosphorus pesticides.Journal of Agricultural and Food Chemistry,58(5):2801-2806
Jiang W,Luo P,Wang X,Chen X,Zhao Y,Shi W,Wu X,Wu Y,Shen J,2012.Development of an enzyme-linked immunosorbent assay for the detection of nitrofurantoin metabolite,1-amino-hydantoin,in animal tissues.Food Control,23(1):20-25
Knutsen HK,Alexander J,Barreg?rd L,Bignami M,Brüschweiler B,Ceccatelli S,Cottrill B,Dinovi M,Edler L,Grasl-Kraupp B,2017.Scientific opinion:risks for animal health related to the presence of zearalenone and its modified forms in feed.EFSA Journal,15(7):doi:10.2903/j.efsa.2017.4851
Li Z,Li M,Li F,Zhang MZ,2018.Paper-based chemiluminescence enzyme-linked immunosorbent assay enhanced by biotin-streptavidin system for high-sensitivity C-reactive protein detection.Analytical Biochemistry,559:86-90
Mousavi Khaneghah A,Fakhri Y,Raeisi S,Armoon B,Sant'ana AS,2018.Prevalence and concentration of ochratoxin A,zearalenone,deoxynivalenol and total aflatoxin in cereal-based products:A systematic review and meta-analysis.Food and Chemical Toxicology,118:830-848
Schell C,Verkoyen C,Krewet E,Müller G,Norpoth K,1993.Production and characterization of monoclonal antibodies to N7-phenylguanine.Journal of Cancer Research and Clinical Oncology,119(4):221-226
?krbi?B,?ivan?ev J,?uri?i?-Mladenovi?N,Godula M,2012.Principal mycotoxins in wheat flour from the Serbian market:levels and assessment of the exposure by wheat-based products.Food Control,25(1):389-396
Smith JE,Sapsford KE,Tan WH,Ligler FS,2011.Optimization of antibody-conjugated magnetic nanoparticles for target preconcentration and immunoassays.Analytical Biochemistry,410(1):124-132
Sun Q,Li GF,Deng QM,Liu JM,Shi GQ,2015.Development and application of a highly sensitive ELISA kit for aflatoxin B1.Environmental Chemistry,34(10):1845-1853(in Chinese)
Sun YN,Hu XF,Zhang Y,Yang JF,Wang FY,Wang Y,Deng RG,Zhang GP,2014.Development of an immunochromatographic strip test for the rapid detection of zearalenone in corn.Journal of Agricultural and Food Chemistry,62(46):11116-11121
Tanaka K,Sago Y,Zheng YZ,Nakagawa H,Kushiro M,2007.Mycotoxins in rice.International Journal of Food Microbiology,119(1-2):59-66
Ul Hassan Z,Al Thani R,F AA,Al Meer S,Migheli Q,Jaoua S,2018.Co-occurrence of mycotoxins in commercial formula milk and cereal-based baby food on the Qatar market.Food Additives&Contaminants Part B-Surveillance,11(3):191-197
Wang CQ,Qian J,Wang K,Yang XW,Liu Q,Hao N,Wang CK,Dong XY,Huang XY,2016.Colorimetric aptasensing of ochratoxin A using Au@Fe3O4 nanoparticles as signal indicator and magnetic separator.Biosensor and Bioelectronics,77:1183-1191
Wang YG,Zhao GH,Li XJ,Liu L,Cao W,Wei Q,2018.Electrochemiluminescent competitive immunosensor based on polyethyleneimine capped SiO2 nanomaterials as labels to release Ru(bpy)32+fixed in 3D Cu/Ni oxalate for the detection of aflatoxin B1.Biosensors and Bioelectronics,101:290-296
Wang YK,Yan YX,Ji WH,Wang HA,Zou Q,Sun JH,2013.Novel chemiluminescence immunoassay for the determination of zearalenone in food samples using gold nanoparticles labeled with streptavidin-horseradish peroxidase.Journal of Agricultural and Food Chemistry,61(18):4250-4256
Wang YK,Wang J,Wang YC,Chen ZF,Yan YX,Hao QW,Li SQ,Yu C,Yang CY,Sun JH,2011.Preparation of anti-zearalenone monoclonal antibodies and development of an indirect competitive ELISA for zearalenone.Microbiology China,38(12):1793-1880(in Chinese)
Yao MW,Wang LY,Fang CZ,2017.The chemiluminescence immunoassay for aflatoxin B1 based on functionalized magnetic nanoparticles with two strategies of antigen probe immobilization.Luminescence,32(4):661-665
Zhang X,Fang Y,Fu ZX,Zhou YZ,Fang WH,Song HH,2018.An efficient method for aflatoxin B1 detection based on gold nanoparticles labeled with monoclonal antibodyhorseradish peroxidase complex.Mycosystema,37(11):1516-1524(in Chinese)
Zhang X,Sun MJ,Kang Y,Xie H,Wang X,Song HH,Li XL,Fang WH,2015a.Identification of a high-affinity monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay.Toxicon,106:89-96
Zhang X,Wang X,Sun MJ,Zhang XF,Song HH,Yan YX,Sun JH,Li XL,Fang WH,2015b.A magnetic nanoparticle based enzyme-linked immunosorbent assay for sensitive quantification of zearalenone in cereal and feed samples.Toxins(Basel),7(10):4216-4231
Zhang X,Wang ZH,Fang Y,Sun RJ,Cao T,Paudyal N,Fang WH,Song HH,2018a.Antibody microarray immunoassay for simultaneous quantification of multiple mycotoxins in corn samples.Toxins(Basel),10(10):doi:10.3390/toxins/0100415
Zhang X,Wang Z,Xie H,Sun R,Cao T,Paudyal N,Fang W,Song H,2018b.Development of a magnetic nanoparticles-based screen-printed electrodes(mnps-spes)biosensor for the quantification of ochratoxin a in cereal and feed samples.Toxins(Basel),10(8):doi:10.3390/toxins/0080317
Zhou J,Du LP,Zou L,Zou YC,Hu N,Wang P,2014.An ultrasensitive electrochemical immunosensor for carcinoembryonic antigen detection based on staphylococcal protein a-au nanoparticle modified gold electrode.Sensors and Actuators B:Chemical,197(7):220-227
孙清,李谷丰,邓乾民,刘杰民,时国庆,2015.高灵敏黄曲霉毒素B1酶联免疫试剂盒的研制及应用.环境化学,34(10):1845-1853
王元凯,王君,王雨晨,陈志飞,严亚贤,郝倩雯,李树清,于翠,杨翠云,孙建和,2011.玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的建立.微生物学通报,38(12):1793-1800
章先,方云,付子贤,周一钊,方维焕,宋厚辉,2018.基于纳米金和辣根过氧化物酶双标记抗体的黄曲霉毒素B1高灵敏检测方法的建立及应用.菌物学报,37(11):1516-1524
Dellafiora L,Galaverna G,Dall'asta C,Cozzini P,2015.Hazard identification of cis/trans-zearalenone through the looking-glass.Food and Chemical Toxicology,86:65-71
Dong GL,Pan YH,Wang YL,Ahmed S,Liu ZL,Peng DP,Yuan ZH,2018.Preparation of a broad-spectrum anti-zearalenone and its primary analogues antibody and its application in an indirect competitive enzyme-linked immunosorbent assay.Food Chemistry,247:8-15
Efsa J,2004.Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to zearalenone as undesirable substance in animal feed.EFSA Journal,89:1-35
Hu Y,Shen GQ,Zhu HL,Jiang GX,2010.A class-specific enzyme-linked immunosorbent assay based on magnetic particles for multiresidue organophosphorus pesticides.Journal of Agricultural and Food Chemistry,58(5):2801-2806
Jiang W,Luo P,Wang X,Chen X,Zhao Y,Shi W,Wu X,Wu Y,Shen J,2012.Development of an enzyme-linked immunosorbent assay for the detection of nitrofurantoin metabolite,1-amino-hydantoin,in animal tissues.Food Control,23(1):20-25
Knutsen HK,Alexander J,Barreg?rd L,Bignami M,Brüschweiler B,Ceccatelli S,Cottrill B,Dinovi M,Edler L,Grasl-Kraupp B,2017.Scientific opinion:risks for animal health related to the presence of zearalenone and its modified forms in feed.EFSA Journal,15(7):doi:10.2903/j.efsa.2017.4851
Li Z,Li M,Li F,Zhang MZ,2018.Paper-based chemiluminescence enzyme-linked immunosorbent assay enhanced by biotin-streptavidin system for high-sensitivity C-reactive protein detection.Analytical Biochemistry,559:86-90
Mousavi Khaneghah A,Fakhri Y,Raeisi S,Armoon B,Sant'ana AS,2018.Prevalence and concentration of ochratoxin A,zearalenone,deoxynivalenol and total aflatoxin in cereal-based products:A systematic review and meta-analysis.Food and Chemical Toxicology,118:830-848
Schell C,Verkoyen C,Krewet E,Müller G,Norpoth K,1993.Production and characterization of monoclonal antibodies to N7-phenylguanine.Journal of Cancer Research and Clinical Oncology,119(4):221-226
?krbi?B,?ivan?ev J,?uri?i?-Mladenovi?N,Godula M,2012.Principal mycotoxins in wheat flour from the Serbian market:levels and assessment of the exposure by wheat-based products.Food Control,25(1):389-396
Smith JE,Sapsford KE,Tan WH,Ligler FS,2011.Optimization of antibody-conjugated magnetic nanoparticles for target preconcentration and immunoassays.Analytical Biochemistry,410(1):124-132
Sun Q,Li GF,Deng QM,Liu JM,Shi GQ,2015.Development and application of a highly sensitive ELISA kit for aflatoxin B1.Environmental Chemistry,34(10):1845-1853(in Chinese)
Sun YN,Hu XF,Zhang Y,Yang JF,Wang FY,Wang Y,Deng RG,Zhang GP,2014.Development of an immunochromatographic strip test for the rapid detection of zearalenone in corn.Journal of Agricultural and Food Chemistry,62(46):11116-11121
Tanaka K,Sago Y,Zheng YZ,Nakagawa H,Kushiro M,2007.Mycotoxins in rice.International Journal of Food Microbiology,119(1-2):59-66
Ul Hassan Z,Al Thani R,F AA,Al Meer S,Migheli Q,Jaoua S,2018.Co-occurrence of mycotoxins in commercial formula milk and cereal-based baby food on the Qatar market.Food Additives&Contaminants Part B-Surveillance,11(3):191-197
Wang CQ,Qian J,Wang K,Yang XW,Liu Q,Hao N,Wang CK,Dong XY,Huang XY,2016.Colorimetric aptasensing of ochratoxin A using Au@Fe3O4 nanoparticles as signal indicator and magnetic separator.Biosensor and Bioelectronics,77:1183-1191
Wang YG,Zhao GH,Li XJ,Liu L,Cao W,Wei Q,2018.Electrochemiluminescent competitive immunosensor based on polyethyleneimine capped SiO2 nanomaterials as labels to release Ru(bpy)32+fixed in 3D Cu/Ni oxalate for the detection of aflatoxin B1.Biosensors and Bioelectronics,101:290-296
Wang YK,Yan YX,Ji WH,Wang HA,Zou Q,Sun JH,2013.Novel chemiluminescence immunoassay for the determination of zearalenone in food samples using gold nanoparticles labeled with streptavidin-horseradish peroxidase.Journal of Agricultural and Food Chemistry,61(18):4250-4256
Wang YK,Wang J,Wang YC,Chen ZF,Yan YX,Hao QW,Li SQ,Yu C,Yang CY,Sun JH,2011.Preparation of anti-zearalenone monoclonal antibodies and development of an indirect competitive ELISA for zearalenone.Microbiology China,38(12):1793-1880(in Chinese)
Yao MW,Wang LY,Fang CZ,2017.The chemiluminescence immunoassay for aflatoxin B1 based on functionalized magnetic nanoparticles with two strategies of antigen probe immobilization.Luminescence,32(4):661-665
Zhang X,Fang Y,Fu ZX,Zhou YZ,Fang WH,Song HH,2018.An efficient method for aflatoxin B1 detection based on gold nanoparticles labeled with monoclonal antibodyhorseradish peroxidase complex.Mycosystema,37(11):1516-1524(in Chinese)
Zhang X,Sun MJ,Kang Y,Xie H,Wang X,Song HH,Li XL,Fang WH,2015a.Identification of a high-affinity monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay.Toxicon,106:89-96
Zhang X,Wang X,Sun MJ,Zhang XF,Song HH,Yan YX,Sun JH,Li XL,Fang WH,2015b.A magnetic nanoparticle based enzyme-linked immunosorbent assay for sensitive quantification of zearalenone in cereal and feed samples.Toxins(Basel),7(10):4216-4231
Zhang X,Wang ZH,Fang Y,Sun RJ,Cao T,Paudyal N,Fang WH,Song HH,2018a.Antibody microarray immunoassay for simultaneous quantification of multiple mycotoxins in corn samples.Toxins(Basel),10(10):doi:10.3390/toxins/0100415
Zhang X,Wang Z,Xie H,Sun R,Cao T,Paudyal N,Fang W,Song H,2018b.Development of a magnetic nanoparticles-based screen-printed electrodes(mnps-spes)biosensor for the quantification of ochratoxin a in cereal and feed samples.Toxins(Basel),10(8):doi:10.3390/toxins/0080317
Zhou J,Du LP,Zou L,Zou YC,Hu N,Wang P,2014.An ultrasensitive electrochemical immunosensor for carcinoembryonic antigen detection based on staphylococcal protein a-au nanoparticle modified gold electrode.Sensors and Actuators B:Chemical,197(7):220-227
孙清,李谷丰,邓乾民,刘杰民,时国庆,2015.高灵敏黄曲霉毒素B1酶联免疫试剂盒的研制及应用.环境化学,34(10):1845-1853
王元凯,王君,王雨晨,陈志飞,严亚贤,郝倩雯,李树清,于翠,杨翠云,孙建和,2011.玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的建立.微生物学通报,38(12):1793-1800
章先,方云,付子贤,周一钊,方维焕,宋厚辉,2018.基于纳米金和辣根过氧化物酶双标记抗体的黄曲霉毒素B1高灵敏检测方法的建立及应用.菌物学报,37(11):1516-1524