可视化蛋白芯片法同时检测牛奶中喹诺酮类抗生素残留的含量
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摘要
目的:利用可视化蛋白芯片分析技术实现对牛奶中9个喹诺酮类抗生素残留含量的同时检测。方法:采用直接竞争法反应原理,将9个喹诺酮类药物的单克隆抗体以微阵列形式固定于微孔板底部,制备成微孔板生物芯片,并依次与纳米材料标记的喹诺酮类人工抗原、喹诺酮类药物反应,最后用银增强显色产生肉眼可见的阵列点。显色结果用可视化芯片分析仪快速扫描成像,图像采用芯片分析软件处理并提取信号,实现定量检测。结果:本法测定牛奶中9个喹诺酮类药物的加标回收率分别在70%～130%之间,且RSD均在15%以内。同时对超市购得的16个品牌牛奶样品中喹诺酮类抗生素的含量进行了检测,含量均小于1 ng·mL~(-1),符合国家规定的最大允许残留量。结论:本法能够用于牛奶样本中9个喹诺酮类药物的残留的同时快速初筛。
Objective:To develop a visual protein chip method for the simultaneous detection of quinolones antibiotic residues in milk.Methods:A 96-well microplate was used as solid support,on which monoclonal antibody of nine quinolones were immobilized,respectively.After immobilization,a mixture of artificial antigens of quinolones analytes which labled nanomaterials and either standard solutions containing the analytes or samples were added to the array reaction area.Nanosilver enhancement technique was applied to amplify the detection signals,producing black image on array spots visible with naked eyes.The signals were detected with a visual scanner,therefore the analyte residues could detect quantitatively.Results:The recovery rate of 9 quinolones in milk was in the range of 70% to 130% with RSD<15%.At the same time,the content of quinolone antibiotics in 16 brands of milk samples purchased by supermarkets was tested,and the content was less than 1 ng·ml~(-1),which met the maximum allowable residue of the state. Conclusion:These results indicate that the protein microarray method is suitable for the routine screening of nine quinolones in milk.
Objective:To develop a visual protein chip method for the simultaneous detection of quinolones antibiotic residues in milk.Methods:A 96-well microplate was used as solid support,on which monoclonal antibody of nine quinolones were immobilized,respectively.After immobilization,a mixture of artificial antigens of quinolones analytes which labled nanomaterials and either standard solutions containing the analytes or samples were added to the array reaction area.Nanosilver enhancement technique was applied to amplify the detection signals,producing black image on array spots visible with naked eyes.The signals were detected with a visual scanner,therefore the analyte residues could detect quantitatively.Results:The recovery rate of 9 quinolones in milk was in the range of 70% to 130% with RSD<15%.At the same time,the content of quinolone antibiotics in 16 brands of milk samples purchased by supermarkets was tested,and the content was less than 1 ng·ml~(-1),which met the maximum allowable residue of the state. Conclusion:These results indicate that the protein microarray method is suitable for the routine screening of nine quinolones in milk.
引文
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[21]CUI M,LIN H,WANG X,et al.5-sulfosalicylic acid dihydratebased pretreatment for the modification of enzyme-linked immunoassay of fluoroquinolones in fishery products[J].JImmunoassay Immunochem,2015,36(5):517
[22]SUI J,LIN H,CAO L,et al.Dot-immunogold filtration assay for rapid screening of three fluoroquinolones[J].Food Agric Immunol,2009,20(2):125
[23]ZHU Y,LI L,WANG Z,et al.Development of an Immunochromatography strip for the rapid detection of 12 fluoroquinolones in chicken muscle and liver[J].J Agric Food Chem,2008,56:5469
[24]HU G,SHENG W,ZHANG Y,et al.A novel and sensitive fluorescence immunoassay for the detection of fluoroquinolones in animal-derived foods using upconversion nanoparticles as labels[J].Anal Bioanal Chem,2015,407(28):8487
[25]TAO X,WANG J,XIE Y,et al.Dual-label chemiluminescence strategy for multiplexed immunoassay of 20 fluoroquinolones,15β-lactams,15 sulfonamides,and CAP in milk[J].Food Anal Method,2017,10(9):3009
[26]钟文英,王兴如,许丹科,等.可视化蛋白芯片法同时检测牛乳中残留的磺胺类和喹诺酮类药物[J].食品科学,2016(02):193ZHONG WY,WANG XR,XU DK,et al.Simultaneous determination of multiresidues of sulfonamides and quinolones in milk with visual protein chip[J].Food Sci,2016(2):193
[27]LI Z,LI Z,ZHAO D,et al.Smartphone-based visualized microarray detection for multiplexed harmful substances in milk[J].Biosens Bioelectron,2017,87:874
[28]王兴如,钟文英,李周敏,等.微孔板生物芯片测定蜂蜜中四环素残留方法的研究及应用[J].药物分析杂志,2015,35(7):1240WANG XR,ZHONG WY,LI ZM,et al.Visual microplate biochip for determination of tetracycline residues in honey[J].Chin JPharm Anal,2015,35(7):1240
[29]李周敏,许丹科.可视化蛋白芯片检测牛奶中庆大霉素的方法研究[J].分析科学学报,2014,20(5):687LI ZM,XU DK.Protein chips for visualized determination of gentamicin residues in milk[J].J Anal Sci,2014,20(5):687
[30]LI Z,LI Z,JIANG J,et al.Simultaneous detection of various contaminants in milk based on visualized microarray[J].Food Control,2017,73:994
[31]LI Z,LI Z,XU D.Simultaneous detection of four nitrofuran metabolites in honey by using a visualized microarray screen assay[J].Food Chem,2017,221:1813
[32]LI Z,LI Z,NIU Q,et al.Visual microarray detection for human IgEbased on silver nanoparticles[J].Sens Actuat B:Chem,2017,239:45
[33]SYAFRIZAYANTI,LUEONG SS,DI C,et al.Personalised proteome analysis by means of protein microarrays made from individual patient samples[J].Sci RepUk,2017,7:1
[34]SIEVERS S,CRETICH M,GAGNI P,et al.Performance of a polymer coated silicon microarray for simultaneous detection of food allergen-specific IgE and IgG4[J].Clin Exp Allergy,2017,47(8):1057
[35]BELUSHKIN A,YESILKOY F,ALTUG H.Nanoparticle-enhanced plasmonic biosensor for digital biomarker detection in a microarray[J].Acs Nano,2018,12(5):4453
[36]QUAN X,DING Y,FENG R,et al.Expression profile of cytokines in gastric cancer patients using proteomic antibody microarray[J].Oncol Lett,2017,14:7360
[37]OKUMA HS,KOIZUMI F,HIRAKAWA A,et al.Clinical and microarray analysis of breast cancers of all subtypes from two prospective preoperative chemotherapy studies[J].Brit J Cancer,2016,115(4):411
[38]HARALAMBIEVA IH,GIBSON MJ,KENNEDY RB,et al.Characterization of rubella-specific humoral immunity following two doses of MMR vaccine using proteome microarray technology[J].Plos One,2017,12(11):e188149
[39]LIU L,WU S,JING F,et al.Bead-based microarray immunoassay for lung cancer biomarkers using quantum dots as labels[J].Biosens Bioelectronics,2016,80:300
[40]SAUER U.Analytical protein microarrays:advancements towards clinical applications[J].Sensors-Basel,2017,17(2):256
[41]ZHOU W,YANG M,LI S,et al.Surface plasmon resonance imaging validation of small molecule drugs binding on target protein microarrays[J].Appl Surf Sci,2018,450:328
[42]LI Z,YAN M,LI Z,et al.A multiplexed screening method for agonists and antagonists of the estrogen receptor protein[J].Anal Bioanal Chem,2012,403(5):1373
[2]NAVRáTILOVáP,VYHNáLKOVáJ,VORLOVáL.Detection of fluoroquinolone residues in milk using Yersinia spp.strains:towards better sensitivity for flumequine determination[J].J Vet Res,2017,61:467
[3]PATYRA E,KOWALCZYK E,GRELIK A,et al.Screening method for the determination of tetracyclines and fluoroquinolones in animal drinking water by liquid chromatography with diode array detector[J].Pol J Vet Sci,2015,18(2):283
[4]D,ANGELO V,TESSARI F,BELLAGAMBA G,et al.Microextraction by packed sorbent and HPLC-PDA quantification of multiple anti-inflammatory drugs and fluoroquinolones in human plasma and urine[J].J Enzym Inhib Med Chem,2016,31(Sup3):110
[5]WANG Q,WANG Y,ZHANG Z,et al.Waxberry-like magnetic porous carbon composites prepared from a nickel-organic framework for solid-phase extraction of fluoroquinolones[J].Microchim Acta,2017,184(10):4107
[6]WANG GN,YANG K,LIU HZ,et al.Molecularly imprinted polymer-based solid phase extraction combined high performance liquid chromatography for determination of fluoroquinolones in milk[J].Anal Methods-Uk,2016,8(27):5511
[7]WANG N,WANG Y,OMER AM,et al.Fabrication of novel surface-imprinted magnetic graphene oxide-grafted cellulose nanocrystals for selective extraction and fast adsorption of fluoroquinolones from water[J].Anal Bioanal Chem,2017,409(28):6643
[8]SEYHAN BOZKURT S,ERDOGAN D,ANTEP M,et al.Use of ionic liquid based chitosan as sorbent for preconcentration of fluoroquinolones in milk,egg,fish,bovine,and chicken meat samples by solid phase extraction prior to HPLC determination[J].J Liquid Chromatogr Related,2016,39(1):21
[9]KARAMI-OSBOO R,SHOJAEE MH,MIRI R,et al.Simultaneous determination of six fluoroquinolones in milk by validated QuEChERS-DLLME HPLC-FLD[J].Anal Methods,2014,6(15):5632
[10]SUN X,WANG J,LI Y,et al.Novel dummy molecularly imprinted polymers for matrix solid-phase dispersion extraction of eight fluoroquinolones from fish samples[J].J Chromatogr A,2014,1359:1
[11]NEVES M,SILVA G,BRITO N,et al.Aqueous ultrasoundassisted extraction for the determination of fluoroquinolones in mangrove sediment by high-performance liquid chromatography and fluorescence detector[J].J Brazil Chem Soc,2018,29(1):24
[12]VAKH C,ALABOUD M,LEBEDINETS S,et al.An automated magnetic dispersive micro-solid phase extraction in a fluidized reactor for the determination of fluoroquinolones in baby food samples[J].Anal Chim Acta,2018,1001:59
[13]CHEN L,HUANG X.Sensitive Monitoring of Fluoroquinolones in Milk and Honey Using Multiple Monolithic Fiber Solid-Phase Microextraction Coupled to Liquid Chromatography Tandem Mass Spectrometry[J].J Agric Food Chem,2016,64(45):8684
[14]BARRETO F,RIBEIRO CBD,HOFF RB,et al.Development and validation of a high-throughput method for determination of nine fluoroquinolones residues in muscle of different animal species by liquid chromatography coupled to tandem mass spectrometry with low temperature clean up[J].J Chromatogr A,2017,1521:131
[15]JANUSCH F,SCHERZ G,MOHRING SAI,et al.Determination of fluoroquinolones in chicken feces-A new liquid-liquid extraction method combined with LC-MS/MS[J].Environ Toxicol Pharm,2014,38:792
[16]LUA X,ZHOU Y,ZHANG J,et al.Determination of fluoroquinolones in cattle manure-based biogas residue by ultrasonic-enhanced microwave-assisted extraction followed by online solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry[J].Chromatogr B,2018(1086):166
[17]LIU N,ZHAO Z,TAN Y,et al.Simultaneous Raising of Rabbit Monoclonal Antibodies to Fluoroquinolones with Diverse Recognition Functionalities via Single Mixture Immunization[J].Anal Chem,2015,88(2):1246
[18]LIU YZ,ZHAO GX,WANG P,et al.Production of the broad specific monoclonal antibody against sarafloxacin for rapid immunoscreening of 12 fluoroquinolones in meat[J].J Environ Sci Health B,2013,48(2):139
[19]LI Y,JI B,CHEN W,et al.Production of new class-specific polyclonal antibody for determination of fluoroquinolones antibiotics by indirect competitive ELISA[J].Food Agr Immunol,2008,19(4):251
[20]ZHANG B,DU D,MENG M,et al.A magnetic particle-based competitive enzyme immunoassay for rapid determination of ciprofloxacin:a potential method for the general detection of fluoroquinolones[J].Anal Lett,2014,47(7):1134
[21]CUI M,LIN H,WANG X,et al.5-sulfosalicylic acid dihydratebased pretreatment for the modification of enzyme-linked immunoassay of fluoroquinolones in fishery products[J].JImmunoassay Immunochem,2015,36(5):517
[22]SUI J,LIN H,CAO L,et al.Dot-immunogold filtration assay for rapid screening of three fluoroquinolones[J].Food Agric Immunol,2009,20(2):125
[23]ZHU Y,LI L,WANG Z,et al.Development of an Immunochromatography strip for the rapid detection of 12 fluoroquinolones in chicken muscle and liver[J].J Agric Food Chem,2008,56:5469
[24]HU G,SHENG W,ZHANG Y,et al.A novel and sensitive fluorescence immunoassay for the detection of fluoroquinolones in animal-derived foods using upconversion nanoparticles as labels[J].Anal Bioanal Chem,2015,407(28):8487
[25]TAO X,WANG J,XIE Y,et al.Dual-label chemiluminescence strategy for multiplexed immunoassay of 20 fluoroquinolones,15β-lactams,15 sulfonamides,and CAP in milk[J].Food Anal Method,2017,10(9):3009
[26]钟文英,王兴如,许丹科,等.可视化蛋白芯片法同时检测牛乳中残留的磺胺类和喹诺酮类药物[J].食品科学,2016(02):193ZHONG WY,WANG XR,XU DK,et al.Simultaneous determination of multiresidues of sulfonamides and quinolones in milk with visual protein chip[J].Food Sci,2016(2):193
[27]LI Z,LI Z,ZHAO D,et al.Smartphone-based visualized microarray detection for multiplexed harmful substances in milk[J].Biosens Bioelectron,2017,87:874
[28]王兴如,钟文英,李周敏,等.微孔板生物芯片测定蜂蜜中四环素残留方法的研究及应用[J].药物分析杂志,2015,35(7):1240WANG XR,ZHONG WY,LI ZM,et al.Visual microplate biochip for determination of tetracycline residues in honey[J].Chin JPharm Anal,2015,35(7):1240
[29]李周敏,许丹科.可视化蛋白芯片检测牛奶中庆大霉素的方法研究[J].分析科学学报,2014,20(5):687LI ZM,XU DK.Protein chips for visualized determination of gentamicin residues in milk[J].J Anal Sci,2014,20(5):687
[30]LI Z,LI Z,JIANG J,et al.Simultaneous detection of various contaminants in milk based on visualized microarray[J].Food Control,2017,73:994
[31]LI Z,LI Z,XU D.Simultaneous detection of four nitrofuran metabolites in honey by using a visualized microarray screen assay[J].Food Chem,2017,221:1813
[32]LI Z,LI Z,NIU Q,et al.Visual microarray detection for human IgEbased on silver nanoparticles[J].Sens Actuat B:Chem,2017,239:45
[33]SYAFRIZAYANTI,LUEONG SS,DI C,et al.Personalised proteome analysis by means of protein microarrays made from individual patient samples[J].Sci RepUk,2017,7:1
[34]SIEVERS S,CRETICH M,GAGNI P,et al.Performance of a polymer coated silicon microarray for simultaneous detection of food allergen-specific IgE and IgG4[J].Clin Exp Allergy,2017,47(8):1057
[35]BELUSHKIN A,YESILKOY F,ALTUG H.Nanoparticle-enhanced plasmonic biosensor for digital biomarker detection in a microarray[J].Acs Nano,2018,12(5):4453
[36]QUAN X,DING Y,FENG R,et al.Expression profile of cytokines in gastric cancer patients using proteomic antibody microarray[J].Oncol Lett,2017,14:7360
[37]OKUMA HS,KOIZUMI F,HIRAKAWA A,et al.Clinical and microarray analysis of breast cancers of all subtypes from two prospective preoperative chemotherapy studies[J].Brit J Cancer,2016,115(4):411
[38]HARALAMBIEVA IH,GIBSON MJ,KENNEDY RB,et al.Characterization of rubella-specific humoral immunity following two doses of MMR vaccine using proteome microarray technology[J].Plos One,2017,12(11):e188149
[39]LIU L,WU S,JING F,et al.Bead-based microarray immunoassay for lung cancer biomarkers using quantum dots as labels[J].Biosens Bioelectronics,2016,80:300
[40]SAUER U.Analytical protein microarrays:advancements towards clinical applications[J].Sensors-Basel,2017,17(2):256
[41]ZHOU W,YANG M,LI S,et al.Surface plasmon resonance imaging validation of small molecule drugs binding on target protein microarrays[J].Appl Surf Sci,2018,450:328
[42]LI Z,YAN M,LI Z,et al.A multiplexed screening method for agonists and antagonists of the estrogen receptor protein[J].Anal Bioanal Chem,2012,403(5):1373