基于量子点标记的赭曲霉毒素A快速、高灵敏荧光免疫层析检测方法的建立及应用
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摘要
赭曲霉毒素A(ochratoxin A,OTA)具有肾毒性、致畸性、致癌性和免疫毒性,广泛存在于各种粮食作物及其副产品中,是食品和饲料原料的重要污染物,可在人类及动物体内蓄积,在已知发现的真菌毒素中,重要性和危害性仅次于黄曲霉毒素。本研究通过采用量子点荧光微球(quantum dots,QDs)标记OTA单克隆抗体,并基于免疫层析原理,优化、建立了OTA高灵敏荧光免疫层析检测方法(FICGA),15min即可实现对农产品中OTA污染的快速定量检测。该方法检测下限(IC_(10))达到0.04ng/mL,检测区间(IC_(20)-IC_(80))为0.05–0.59ng/mL,半数抑制率(IC_(50))为0.18ng/mL。与OTA类似物OTB、OTC交叉反应性为7.3%和11.9%,对其他常见真菌毒素AFB_1、ZEN、FB_1和DON均无交叉反应。在玉米、面粉和大豆样本中的加标回收率可达83.2%–117.8%,与LC-MS/MS同时对天然样本中OTA含量的检测结果表明,两种方法相关性良好。本研究建立的FICGA快速、灵敏,可满足基层单位和现场的快速检测需求,具有很好的应用前景。
Ochratoxin A(OTA)produced by Aspergillus and Penicillium species,is one of the most widespread toxic members of ochratoxins and has been shown to be nephrotoxic,teratogenic,carcinogenic and immunotoxic to several animal species.Rapid detection of OTA is important to prevent contaminated food from entering into the food chain.A rapid and sensitive fluorescent immunochromatographic assay(FICGA)based on the use of quantum dots(QDs)was prototyped and applied for detecting OTA in cereal samples.The detection signal was enhanced which in turn improved the sensitivity of the assay.The limit of detection of FICGA was 0.04ng/mL while the IC_(50) was 0.18ng/mL.The linear working range was0.05-0.59ng/mL.The cross-reactivities to the OTA analogues,ochratoxin B and ochratoxin C,were 7.3%and 11.9%respectively.No cross-reactivity(<0.01%)was observed to other co-occurring mycotoxins(AFB_1,ZEN,FB_1 and DON).The recovery rates in spiked corn,wheat and soybean samples were 83.2%–117.8%,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 FICGA and liquid chromatography tandem mass spectrometry(LC/MS-MS).Hence,this developed quantum dots-based FICGA shows great potential for rapid,sensitive,simple and cost-effective quantitative detection of OTA in food safety control.
Ochratoxin A(OTA)produced by Aspergillus and Penicillium species,is one of the most widespread toxic members of ochratoxins and has been shown to be nephrotoxic,teratogenic,carcinogenic and immunotoxic to several animal species.Rapid detection of OTA is important to prevent contaminated food from entering into the food chain.A rapid and sensitive fluorescent immunochromatographic assay(FICGA)based on the use of quantum dots(QDs)was prototyped and applied for detecting OTA in cereal samples.The detection signal was enhanced which in turn improved the sensitivity of the assay.The limit of detection of FICGA was 0.04ng/mL while the IC_(50) was 0.18ng/mL.The linear working range was0.05-0.59ng/mL.The cross-reactivities to the OTA analogues,ochratoxin B and ochratoxin C,were 7.3%and 11.9%respectively.No cross-reactivity(<0.01%)was observed to other co-occurring mycotoxins(AFB_1,ZEN,FB_1 and DON).The recovery rates in spiked corn,wheat and soybean samples were 83.2%–117.8%,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 FICGA and liquid chromatography tandem mass spectrometry(LC/MS-MS).Hence,this developed quantum dots-based FICGA shows great potential for rapid,sensitive,simple and cost-effective quantitative detection of OTA in food safety control.
引文
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Brennan KM,Oh SY,Yiannikouris A,Graugnard DE,Karrow NA,2017.Differential gene expression analysis of bovine macrophages after exposure to the penicillium mycotoxins citrinin and/or ochratoxin a.Toxins(Basel),9(11):366
Bueno D,Istamboulie G,Mu?oz R,Marty JL,2015.Determination of mycotoxins in food:a review of bioanalytical to analytical methods.Applied Spectroscopy Reviews,50(9):728-774
Bui-Klimke TR,Wu F,2015.Ochratoxin a and human health risk:a review of the evidence.Critical Reviews in Food Science and Nutrition,55(13):1860-1869
European C,2002.Commission decision 2002/657/ec of 12august 2002 implementing council directive 96/23/ec concerning the performance of analytical methods and the interpretation of results.Official Journal of the European Communities L,221:8-36
Hao K,Suryoprabowo S,Song SS,Liu LQ,Kuang H,2017.Rapid detection of zearalenone and its metabolite in corn flour with the immunochromatographic test strip.Food and Agricultural Immunology,29(1):1-13
Huang XL,Aguilar ZP,Xu HY,Lai WH,Xiong YH,2016.Membrane-based lateral flow immunochromatographic strip with nanoparticles as reporters for detection:a review.Biosensor and Bioelectronics,75:166-180
Jiang LY,Liu HJ,Li M,Xing Y,Ren XQ,2016.Surface molecular imprinting on cdte quantum dots for fluorescence sensing of 4-nitrophenol.Analytical Methods,8(10):2226-2232
Kawamura O,Sato S,Kajii H,Nagayama S,Ohtani K,Chiba J,Ueno Y,1989.A sensitive enzyme-linked immunosorbent assay of ochratoxin a based on monoclonal antibodies.Toxicon,27(8):887-897
Kitidee K,Khamaikawin W,Thongkum W,Tawon Y,Cressey TR,Jevprasesphant R,Kasinrerk W,Tayapiwatana C,2016.Expedient screening for hiv-1 protease inhibitors using a simplified immunochromatographic assay.Journal of Chromatography B,1021:153-158
Kong DZ,Xie ZJ,Liu LQ,Song SS,Kuang H,Xu CL,2017.Development of ic-elisa and lateral-flow immunochromatographic assay strip for the detection of vancomycin in raw milk and animal feed.Food and Agricultural Immunology,28(3):414-426
Li X,Li PW,Zhang Q,Zhang W,Ding XX,Tang XQ,2014.Development of an immunochromatographic assay for ochratoxin a in agro-products.Chinese Journal of Oil Crop Sciences,36(5):648-652(in Chinese)
Meulenberg EP,2012.Immunochemical methods for ochratoxin a detection:a review.Toxins(Basel),4(4):244-266
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
Smith MC,Madec S,Coton E,Hymery N,2016.Natural co-occurrence of mycotoxins in foods and feeds and their in vitro combined toxicological effects.Toxins(Basel),8(4):94
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)
Hu TX,Zhang L,Wen W,Zhang XH,Wang SF,2016.Enzyme catalytic amplification of mirna-155 detection with graphene quantum dot-based electrochemical biosensor.Biosensor and Bioelectronics,77:451-456
Wang J,Cheng GH,Ruan RY,He HW,Dou ML,He XP,Xu YC,2018.Review of ochratoxin A pollution in animal-derived foods and its detection methods.Journal of Food Safety and Quality,9(16):4212-4217(in Chinese)
Wang XW,Sun GZ,Li N,Chen P,2016.Cheminform abstract:quantum dots derived from two-dimensional materials and their applications for catalysis and energy.Chemical Society Reviews,45(8):2239-2262
Wang YH,Ning G,Wu YH,Wu S,Zeng BQ,Liu GQ,He XX,Wang KM,2019.Facile combination of beta-cyclodextrin host-guest recognition with exonuclease-assistant signal amplification for sensitive electrochemical assay of ochratoxin a.Biosensor and Bioelectronics,124-125:82-88
Watanabe Pinhata JM,Lemes RA,Fcds S,Souza AR,Chimara E,Ferrazoli L,2018.Use of an immunochromatographic assay for rapid identification of mycobacterium tuberculosis complex clinical isolates in routine diagnosis.Journal of Medical Microbiology,67(5):683-686
Zhang X,He K,Fang Y,Cao T,Paudyal N,Zhang XF,Song HH,Li XL,Fang WH,2018.Dual flow immunochromatographic assay for rapid and simultaneous quantitative detection of ochratoxin a and zearalenone in corn,wheat,and feed samples.Journal of Zhejiang University-Science B,19(11):871-883
Zhang X,Sun MJ,Kang Y,Xie H,Wang X,Song HH,Li XL,Fang WH,2015.Identification of a high-affinity monoclonal antibody against ochratoxin a and its application in enzyme-linked immunosorbent assay.Toxicon,106:89-96
Zheng MZ,Richard JL,Johann B,2006.A review of rapid methods for the analysis of mycotoxins.Mycopathologia,161(5):261-273
Zrazhevskiy P,Sena M,Gao XH,2010.Cheminform abstract:Designing multifunctional quantum dots for bioimaging,detection,and drug delivery.Chemical Society Reviews,39(11):4326-4354
Zhou YF,Xiong SC,Jiang H,Duan H,Xiong YH,Wang A,2015.Immunochromatographic assay for quantitative detection of ochratoxin a in maize by quantum dot beads.Chinese Journal of Analytical Chemistry,43(12):1837-1843(in Chinese)
李鑫,李培武,张奇,张文,丁小霞,唐晓倩,2014.农产品中赭曲霉毒素A免疫层析试纸条快速检测技术研究.中国油料作物学报,36(5):648-652
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
王健,成桂红,阮若云,何恒纬,窦明礼,贺小萍,徐跃成,2018.动物源性食品中赭曲霉毒素A污染概况及检测方法研究进展.食品安全质量检测学报,9(16):4212-4217
周耀锋,熊斯诚,江湖,段宏,熊勇华,Andrew Wang,2015.量子点荧光微球免疫层析试纸条定量检测玉米中赭曲霉毒素A.分析化学,43(12):1837-1843
Brennan KM,Oh SY,Yiannikouris A,Graugnard DE,Karrow NA,2017.Differential gene expression analysis of bovine macrophages after exposure to the penicillium mycotoxins citrinin and/or ochratoxin a.Toxins(Basel),9(11):366
Bueno D,Istamboulie G,Mu?oz R,Marty JL,2015.Determination of mycotoxins in food:a review of bioanalytical to analytical methods.Applied Spectroscopy Reviews,50(9):728-774
Bui-Klimke TR,Wu F,2015.Ochratoxin a and human health risk:a review of the evidence.Critical Reviews in Food Science and Nutrition,55(13):1860-1869
European C,2002.Commission decision 2002/657/ec of 12august 2002 implementing council directive 96/23/ec concerning the performance of analytical methods and the interpretation of results.Official Journal of the European Communities L,221:8-36
Hao K,Suryoprabowo S,Song SS,Liu LQ,Kuang H,2017.Rapid detection of zearalenone and its metabolite in corn flour with the immunochromatographic test strip.Food and Agricultural Immunology,29(1):1-13
Huang XL,Aguilar ZP,Xu HY,Lai WH,Xiong YH,2016.Membrane-based lateral flow immunochromatographic strip with nanoparticles as reporters for detection:a review.Biosensor and Bioelectronics,75:166-180
Jiang LY,Liu HJ,Li M,Xing Y,Ren XQ,2016.Surface molecular imprinting on cdte quantum dots for fluorescence sensing of 4-nitrophenol.Analytical Methods,8(10):2226-2232
Kawamura O,Sato S,Kajii H,Nagayama S,Ohtani K,Chiba J,Ueno Y,1989.A sensitive enzyme-linked immunosorbent assay of ochratoxin a based on monoclonal antibodies.Toxicon,27(8):887-897
Kitidee K,Khamaikawin W,Thongkum W,Tawon Y,Cressey TR,Jevprasesphant R,Kasinrerk W,Tayapiwatana C,2016.Expedient screening for hiv-1 protease inhibitors using a simplified immunochromatographic assay.Journal of Chromatography B,1021:153-158
Kong DZ,Xie ZJ,Liu LQ,Song SS,Kuang H,Xu CL,2017.Development of ic-elisa and lateral-flow immunochromatographic assay strip for the detection of vancomycin in raw milk and animal feed.Food and Agricultural Immunology,28(3):414-426
Li X,Li PW,Zhang Q,Zhang W,Ding XX,Tang XQ,2014.Development of an immunochromatographic assay for ochratoxin a in agro-products.Chinese Journal of Oil Crop Sciences,36(5):648-652(in Chinese)
Meulenberg EP,2012.Immunochemical methods for ochratoxin a detection:a review.Toxins(Basel),4(4):244-266
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
Smith MC,Madec S,Coton E,Hymery N,2016.Natural co-occurrence of mycotoxins in foods and feeds and their in vitro combined toxicological effects.Toxins(Basel),8(4):94
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)
Hu TX,Zhang L,Wen W,Zhang XH,Wang SF,2016.Enzyme catalytic amplification of mirna-155 detection with graphene quantum dot-based electrochemical biosensor.Biosensor and Bioelectronics,77:451-456
Wang J,Cheng GH,Ruan RY,He HW,Dou ML,He XP,Xu YC,2018.Review of ochratoxin A pollution in animal-derived foods and its detection methods.Journal of Food Safety and Quality,9(16):4212-4217(in Chinese)
Wang XW,Sun GZ,Li N,Chen P,2016.Cheminform abstract:quantum dots derived from two-dimensional materials and their applications for catalysis and energy.Chemical Society Reviews,45(8):2239-2262
Wang YH,Ning G,Wu YH,Wu S,Zeng BQ,Liu GQ,He XX,Wang KM,2019.Facile combination of beta-cyclodextrin host-guest recognition with exonuclease-assistant signal amplification for sensitive electrochemical assay of ochratoxin a.Biosensor and Bioelectronics,124-125:82-88
Watanabe Pinhata JM,Lemes RA,Fcds S,Souza AR,Chimara E,Ferrazoli L,2018.Use of an immunochromatographic assay for rapid identification of mycobacterium tuberculosis complex clinical isolates in routine diagnosis.Journal of Medical Microbiology,67(5):683-686
Zhang X,He K,Fang Y,Cao T,Paudyal N,Zhang XF,Song HH,Li XL,Fang WH,2018.Dual flow immunochromatographic assay for rapid and simultaneous quantitative detection of ochratoxin a and zearalenone in corn,wheat,and feed samples.Journal of Zhejiang University-Science B,19(11):871-883
Zhang X,Sun MJ,Kang Y,Xie H,Wang X,Song HH,Li XL,Fang WH,2015.Identification of a high-affinity monoclonal antibody against ochratoxin a and its application in enzyme-linked immunosorbent assay.Toxicon,106:89-96
Zheng MZ,Richard JL,Johann B,2006.A review of rapid methods for the analysis of mycotoxins.Mycopathologia,161(5):261-273
Zrazhevskiy P,Sena M,Gao XH,2010.Cheminform abstract:Designing multifunctional quantum dots for bioimaging,detection,and drug delivery.Chemical Society Reviews,39(11):4326-4354
Zhou YF,Xiong SC,Jiang H,Duan H,Xiong YH,Wang A,2015.Immunochromatographic assay for quantitative detection of ochratoxin a in maize by quantum dot beads.Chinese Journal of Analytical Chemistry,43(12):1837-1843(in Chinese)
李鑫,李培武,张奇,张文,丁小霞,唐晓倩,2014.农产品中赭曲霉毒素A免疫层析试纸条快速检测技术研究.中国油料作物学报,36(5):648-652
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
王健,成桂红,阮若云,何恒纬,窦明礼,贺小萍,徐跃成,2018.动物源性食品中赭曲霉毒素A污染概况及检测方法研究进展.食品安全质量检测学报,9(16):4212-4217
周耀锋,熊斯诚,江湖,段宏,熊勇华,Andrew Wang,2015.量子点荧光微球免疫层析试纸条定量检测玉米中赭曲霉毒素A.分析化学,43(12):1837-1843