新型高灵敏赭曲霉毒素A间接竞争化学发光免疫分析法
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摘要
建立了一种测定赭曲霉毒素A的新型高灵敏化学发光间接竞争酶联免疫分析方法。以酶标赭曲霉毒素A二抗上的辣根过氧化物酶催化过氧化脲氧化3-(4-羟苯基)丙酸,生成具有荧光的3-(4-羟苯基)丙酸二聚体。并利用乙腈介质中双[2,4,6-三氯苯基]草酸酯和过氧化脲在增强剂咪唑的作用下反应产生强化学发光,以发光强度确定待检物中赭曲霉毒素A含量。结果表明,在最佳条件下IC50为0. 55 ng/m L,在0. 05～6. 08 ng/m L范围内有良好的线性关系,最低检出限为0. 01 ng/m L。样品加标回收实验显示葡萄干和葡萄汁样品的平均回收率分别为84. 55%～91. 36%和73. 32%～87. 64%,批内与批间变异系数均小于10%,精密度良好。该新型化学发光方法检测赭曲霉毒素A时发光强度更大、发光时间更长,可用于食品中赭曲霉毒素A的高灵敏度痕量检测。
A novel indirect highly sensitive competitive chemiluminescent immunoassay was established to determine ochratoxin A. Oxidation of 3-(4-hydroxyphenyl) propionic acid urea peroxide was catalyzed by horseradish peroxidase that was on an enzyme-labeled ochratoxin A secondary antibody to generate a fluorescent 3-(4-hydroxyphenyl)propionic acid dimer. A strong chemiluminescence was produced by the reaction between bis(2,4,6-trichlorophenyl)oxalate and urea peroxide in acetonitrile medium under the action of imidazole. The content of ochratoxin A in samples was determined by luminescence intensity. The results showed that under the optimal conditions,the IC50 was 0. 55 ng/m L with a good linearity in the range of 0. 05-6. 08 ng/m L,and a detection limit of 0. 01 ng/m L. The average recovery rates of raisins and grape juice samples were 84. 55%-91. 36% and 73. 32%-87. 64%,respectively. The intra-assay and inter-assay variation coefficients were both less than 10% with good precision. This novel chemiluminescence method has higher luminescence intensity and longer emission time when detecting ochratoxin A. It can be used for detecting trace ochratoxin A in foods with high-sensitivity.
A novel indirect highly sensitive competitive chemiluminescent immunoassay was established to determine ochratoxin A. Oxidation of 3-(4-hydroxyphenyl) propionic acid urea peroxide was catalyzed by horseradish peroxidase that was on an enzyme-labeled ochratoxin A secondary antibody to generate a fluorescent 3-(4-hydroxyphenyl)propionic acid dimer. A strong chemiluminescence was produced by the reaction between bis(2,4,6-trichlorophenyl)oxalate and urea peroxide in acetonitrile medium under the action of imidazole. The content of ochratoxin A in samples was determined by luminescence intensity. The results showed that under the optimal conditions,the IC50 was 0. 55 ng/m L with a good linearity in the range of 0. 05-6. 08 ng/m L,and a detection limit of 0. 01 ng/m L. The average recovery rates of raisins and grape juice samples were 84. 55%-91. 36% and 73. 32%-87. 64%,respectively. The intra-assay and inter-assay variation coefficients were both less than 10% with good precision. This novel chemiluminescence method has higher luminescence intensity and longer emission time when detecting ochratoxin A. It can be used for detecting trace ochratoxin A in foods with high-sensitivity.
引文
[1] MEULENBERG E P. Immunochemical methods for ochratoxin a detection:A review[J]. Toxins,2012,4(4):244-266.
[2] PETZINGER E,ZIEGLER K. Ochratoxin A from a toxicological perspective[J]. Journal of Veterinary Pharmacology&Therapeutics,2000,23(2):91-98.
[3] MARIN S,RAMOS A J,CANOSANCHO G,et al. Mycotoxins:occurrence,toxicology,and exposure assessment[J]. Food&Chemical Toxicology An International Journal Published for the British Industrial Biological Research Association,2013,60(10):218-237.
[4] SORRENTI V,GIACOMO C D,ACQUAVIVA R,et al.Toxicity of ochratoxin A and its modulation by antioxidants:A review[J]. Toxins,2013,5(10):1 742-1 766.
[5] GIROMINI C,REBUCCI R,FUSI E,et al. Cytotoxicity,apoptosis,DNA damage and methylation in mammary and kidney epithelial cell lines exposed to ochratoxin A[J].Cell Biology&Toxicology,2016,32(3):249-258.
[6] SCHWERDT G,HOLZINGER H,KONIGS M,et al.Effect of ochratoxin A on cell survival and collagen homeostasis in human mesangial cells in primary culture[J].Food&Chemical Toxicology,2009,47(1):209-213.
[7] IARC. Monographs on the evaluation of carcinogenic risks to humans[J]. Iarc Monogr Eval Carcinog Risks Hum,1992,55:1-316.
[8] The commission of the european communities. Commission Regulation(EU)No 594/2012[S]. 2012.
[9]中华人民共和国国家卫生和计划生育委员会,国家食品药品监督管理总局. GB 2761—2017,食品中真菌毒素限量[S].北京:中国标准出版社,2017.
[10]王龑,王刘庆,刘阳.食品中主要真菌毒素生物合成途径研究进展[J].食品安全质量检测学报,2016,7(6):2 158-2 167.
[11] RUAN Changqing,DIAO Xue,LI Na,et al. Determination of ochratoxin A and citrinin in fruits using ultrasoundassisted solvent extraction followed by dispersive liquidliquid microextraction with HPLC with fluorescence detection[J]. Analytical Methods,2016,8(7):1 586-1 594.
[12] LUCI G,INTORRE L,FERRUZZI G,et al. Determination of ochratoxin A in tissues of wild boar(Sus scrofa,L.)by enzymatic digestion(ED)coupled to high-performance liquid chromatography with a fluorescence detector(HPLC-FLD)[J]. Mycotoxin Research,2017,3:1-8.
[13] LI Peng,PEI Fei,LIU Qin,et al. Magnetic solid-phase extraction for the determination of ochratoxin A in wine and beer by HPLC-FLD[J]. Current Analytical Chemistry,2018,14(2):129-134.
[14] CANPONE L,PICCINELLI A L,CELANO R,et al.Rapid and automated on-line solid phase extraction HPLCMS/MS with peak focusing for the determination of ochratoxin A in wine samples[J]. Food Chemistry,2018,244:128-135.
[15] WANG Chengke,TAN Rong,CHEN Dan. Fluorescence method for quickly detecting ochratoxin A in flour and beer using nitrogen doped carbon dots and silver nanoparticles[J]. Talanta,2018,182:363-370.
[16]舒文祥,徐炜,李艳,等.胶体金免疫层析法快速检测赭曲霉毒素A研究[J].粮食与油脂,2011(10):20-22.
[17] SOARES R R G,RAMADAS D,CHU V,et al. An ultrarapid and regenerable microfluidic immunoassay coupled with integrated photosensors for point-of-use detection of ochratoxin A[J]. Sensors&Actuators B Chemical,2016,235:554-562.
[18]熊勇华,陈雪岚,许杨.检测赭曲霉毒素A(OTA)的酶联免疫吸附法(ELISA)体系的建立[J].食品科学,2006,27(5):30-35.
[19] KIM S,LIM H B. Chemiluminescence immunoassay using magnetic nanoparticles with targeted inhibition for the determination of ochratoxin A[J]. Talanta,2015,140:183-188.
[20]薛盼. TCPO-H_2O_2-3-(4-羟苯基)丙酸二聚体化学发光体系免疫分析的研究[D].西安:陕西师范大学,2011:9-15.
[21] MARAGOS C M,BUSMAN M. Rapid and advanced tools for mycotoxin analysis:a review[J]. Food Addit Contam Part A Chem Anal Control Expo Risk Assess,2010,27(5):688-700.
[22]支正良,于山江,华万森,等.过氧草酸酯类化学发光体系的研究进展[J].化学世界,1997(12):619-624.
[23] DU Jianxiu,WANG Yadi,ZHANG Weimin. Gold nanoparticles-based chemiluminescence resonance energy transfer for ultrasensitive detection of melamine[J]. Spectrochimica Acta Part A Molecular&Biomolecular Spectroscopy,2015,149:698-702.
[24] TAO Liang,ZHANG Chunmei,ZHANG Jinpeng,et al.Sensitive chemiluminescence immunoassay for staphylococcal enterotoxin C1 based on the use of dye-encapsulated mesoporous silica nanoparticles[J]. Microchimica Acta,2016,183(7):2 163-2 168.
[25] WANG Zhihua,LIU Feng,LU Chao. Evolution of biogenic amine concentrations in foods through their induced chemiluminescence inactivation of layered double hydroxide nanosheet colloids.[J]. Biosensors&Bioelectronics,2014,60(6):237-243.
[26]王权,闫叶娜,黄克和.抗大田软海绵酸单克隆抗体的制备与间接竞争ELISA的建立[J].畜牧与兽医,2010,42(8):1-5.
[27]中华人民共和国国家卫生和计划生育委员会,国家食品药品监督管理总局.食品中赭曲霉毒素A的测定:GB 5009. 96—2016[S].北京:中国标准出版社,2016:1-6.
[28] EVERSE J. Peroxidases in chemistry and biology. Volume II.[J]. Progress in Lipid Research,1990,17(3):279-318.
[29]陈列,常文保,慈云祥.过氧化物酶催化反应的底物结构的研究[J].高等学校化学学报,1995,16(5):683-687.
[2] PETZINGER E,ZIEGLER K. Ochratoxin A from a toxicological perspective[J]. Journal of Veterinary Pharmacology&Therapeutics,2000,23(2):91-98.
[3] MARIN S,RAMOS A J,CANOSANCHO G,et al. Mycotoxins:occurrence,toxicology,and exposure assessment[J]. Food&Chemical Toxicology An International Journal Published for the British Industrial Biological Research Association,2013,60(10):218-237.
[4] SORRENTI V,GIACOMO C D,ACQUAVIVA R,et al.Toxicity of ochratoxin A and its modulation by antioxidants:A review[J]. Toxins,2013,5(10):1 742-1 766.
[5] GIROMINI C,REBUCCI R,FUSI E,et al. Cytotoxicity,apoptosis,DNA damage and methylation in mammary and kidney epithelial cell lines exposed to ochratoxin A[J].Cell Biology&Toxicology,2016,32(3):249-258.
[6] SCHWERDT G,HOLZINGER H,KONIGS M,et al.Effect of ochratoxin A on cell survival and collagen homeostasis in human mesangial cells in primary culture[J].Food&Chemical Toxicology,2009,47(1):209-213.
[7] IARC. Monographs on the evaluation of carcinogenic risks to humans[J]. Iarc Monogr Eval Carcinog Risks Hum,1992,55:1-316.
[8] The commission of the european communities. Commission Regulation(EU)No 594/2012[S]. 2012.
[9]中华人民共和国国家卫生和计划生育委员会,国家食品药品监督管理总局. GB 2761—2017,食品中真菌毒素限量[S].北京:中国标准出版社,2017.
[10]王龑,王刘庆,刘阳.食品中主要真菌毒素生物合成途径研究进展[J].食品安全质量检测学报,2016,7(6):2 158-2 167.
[11] RUAN Changqing,DIAO Xue,LI Na,et al. Determination of ochratoxin A and citrinin in fruits using ultrasoundassisted solvent extraction followed by dispersive liquidliquid microextraction with HPLC with fluorescence detection[J]. Analytical Methods,2016,8(7):1 586-1 594.
[12] LUCI G,INTORRE L,FERRUZZI G,et al. Determination of ochratoxin A in tissues of wild boar(Sus scrofa,L.)by enzymatic digestion(ED)coupled to high-performance liquid chromatography with a fluorescence detector(HPLC-FLD)[J]. Mycotoxin Research,2017,3:1-8.
[13] LI Peng,PEI Fei,LIU Qin,et al. Magnetic solid-phase extraction for the determination of ochratoxin A in wine and beer by HPLC-FLD[J]. Current Analytical Chemistry,2018,14(2):129-134.
[14] CANPONE L,PICCINELLI A L,CELANO R,et al.Rapid and automated on-line solid phase extraction HPLCMS/MS with peak focusing for the determination of ochratoxin A in wine samples[J]. Food Chemistry,2018,244:128-135.
[15] WANG Chengke,TAN Rong,CHEN Dan. Fluorescence method for quickly detecting ochratoxin A in flour and beer using nitrogen doped carbon dots and silver nanoparticles[J]. Talanta,2018,182:363-370.
[16]舒文祥,徐炜,李艳,等.胶体金免疫层析法快速检测赭曲霉毒素A研究[J].粮食与油脂,2011(10):20-22.
[17] SOARES R R G,RAMADAS D,CHU V,et al. An ultrarapid and regenerable microfluidic immunoassay coupled with integrated photosensors for point-of-use detection of ochratoxin A[J]. Sensors&Actuators B Chemical,2016,235:554-562.
[18]熊勇华,陈雪岚,许杨.检测赭曲霉毒素A(OTA)的酶联免疫吸附法(ELISA)体系的建立[J].食品科学,2006,27(5):30-35.
[19] KIM S,LIM H B. Chemiluminescence immunoassay using magnetic nanoparticles with targeted inhibition for the determination of ochratoxin A[J]. Talanta,2015,140:183-188.
[20]薛盼. TCPO-H_2O_2-3-(4-羟苯基)丙酸二聚体化学发光体系免疫分析的研究[D].西安:陕西师范大学,2011:9-15.
[21] MARAGOS C M,BUSMAN M. Rapid and advanced tools for mycotoxin analysis:a review[J]. Food Addit Contam Part A Chem Anal Control Expo Risk Assess,2010,27(5):688-700.
[22]支正良,于山江,华万森,等.过氧草酸酯类化学发光体系的研究进展[J].化学世界,1997(12):619-624.
[23] DU Jianxiu,WANG Yadi,ZHANG Weimin. Gold nanoparticles-based chemiluminescence resonance energy transfer for ultrasensitive detection of melamine[J]. Spectrochimica Acta Part A Molecular&Biomolecular Spectroscopy,2015,149:698-702.
[24] TAO Liang,ZHANG Chunmei,ZHANG Jinpeng,et al.Sensitive chemiluminescence immunoassay for staphylococcal enterotoxin C1 based on the use of dye-encapsulated mesoporous silica nanoparticles[J]. Microchimica Acta,2016,183(7):2 163-2 168.
[25] WANG Zhihua,LIU Feng,LU Chao. Evolution of biogenic amine concentrations in foods through their induced chemiluminescence inactivation of layered double hydroxide nanosheet colloids.[J]. Biosensors&Bioelectronics,2014,60(6):237-243.
[26]王权,闫叶娜,黄克和.抗大田软海绵酸单克隆抗体的制备与间接竞争ELISA的建立[J].畜牧与兽医,2010,42(8):1-5.
[27]中华人民共和国国家卫生和计划生育委员会,国家食品药品监督管理总局.食品中赭曲霉毒素A的测定:GB 5009. 96—2016[S].北京:中国标准出版社,2016:1-6.
[28] EVERSE J. Peroxidases in chemistry and biology. Volume II.[J]. Progress in Lipid Research,1990,17(3):279-318.
[29]陈列,常文保,慈云祥.过氧化物酶催化反应的底物结构的研究[J].高等学校化学学报,1995,16(5):683-687.