CN113624729A - 一种快速检测粮食中百菌清残留的试纸条的制备方法 - Google Patents
一种快速检测粮食中百菌清残留的试纸条的制备方法 Download PDFInfo
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Abstract
一种快速检测粮食中百菌清残留的试纸条的制备方法属于检测技术领域,该方法包括以下步骤:(1)荧光探针制备;(2)试纸条检测条件筛选:偶联铕的单抗标记浓度、荧光探针用量、检测线上检测抗原浓度、检测线抗原喷涂速率(0.4~0.8μL/cm);(3)时间分辨荧光免疫层析试纸条制备;(4)样品的检测:①样品前处理;②提取时间的筛选;③样品提取液稀释比例筛选;④检测分析,定性分析:紫外灯下,通过肉眼判断定性结果;定量分析:反应结束后30s内,采用荧光读数仪读取结果,得到被测粮食中百菌清的含量。本发明建立的百菌清残留的试纸条检测方法,具有准确度、精准度和重现性良好,操作简单快速,且具有可视的检测优势。
Description
技术领域
本发明属于检测技术领域,主要涉及一种快速检测粮食中百菌清残留的试纸条的制备方法。
背景技术
我国是农药的生产大国和使用大国。据统计,我国每年可生产农药382万吨,约占世界农药总产量的十分之一;施用量也远远高于世界平均水平,每年农药总使用量高达130万吨。农药的种类很多,根据其化学结构可分为有机类农药和无机类农药,其中有机类农药又可细分为有机氯类、有机磷类、拟除虫菊酯类和氨基甲酸酯类等,是当前农业领域应用最为广泛,同时也是残留和危害最为严重的农药种类。百菌清(chlorothalonil,CTN)是一种高效、广谱的有机氯杀菌剂,对多种作物的真菌病害有良好的防治效果,因此在我国的粮食、水果、水稻等作物生产中应用非常广泛。由于百菌清在植物上的黏着性较强,不易被雨水冲刷,难以降解且药效期较长等特点,极易在粮食和水果中残留,从而对农产品的质量安全造成影响。大量的研究表明,百菌清对鱼类和水生无脊椎动物具有明显的毒性,其中许多鱼类的96h半致死量(LC50)为10~195μg/L。除此之外,人们还发现长期低剂量的百菌清暴露甚至可以抑制青春期小鼠卵巢的发育
百菌清用检测方法为高效液相色谱法(HPLC)、气相色谱法(GC)、色谱-质谱联用(GC/LC-MS)和免疫分析法。仪器方法的检测限低、专一性强,但由于其需要贵重仪器且样品前处理复杂、分析时间长,很难满足现场快速筛选、检测的要求。免疫分析方法成本低廉,选择性强,简单快速,近年来已经越来越多地应用于农药残留检测,其中时间分辨荧光免疫层析方法(Time-resolved fluorescence immunochromatographic assay,TRFICA)是以包埋了稀土元素离子螯合物的时间分辨荧光微球(Time-resolved FluorescentMicrospheres,TRFMs)为免疫探针,在层析体系中通过竞争反应体系进行检测的方法。利用其荧光衰变期长和发射光谱窄的特点,可使有效信号与非特异性的背景荧光信号区分,减少非特异性荧光的干扰,提高了灵敏度。通过荧光读数仪检测荧光强度比值,具有简单、快速和可视的检测优势。然而,时间分辨荧光微球作为免疫探针检测粮食中农药残留的免疫层析检测方法鲜有报道。本发明基于百菌清抗原抗体特异性结合反应,建立基于稀土铕荧光微球作为检测信号,可快速检测百菌清残留。
发明内容
本发明旨在针对现有背景技术中存在的不足,而提供的一种快速检测粮食中百菌清残留的试纸条的制备方法。
本发明所要解决的技术问题是通过以下技术方案来实现,具体步骤如下:
(1)荧光探针的制备:采用铕纳米材料与单抗偶联制备荧光探针,取800μL0.2moL/L的硼酸缓冲液于2mL的离心管中,加入200μL聚苯乙烯荧光微球乳胶,涡旋混匀;将离心管置于数控高功率超声仪中超声12-18min,以使乳胶材料在溶液中均匀分散;加入40μL 15mg/mL的EDC水溶液,室温下涡旋振荡10-20min;然后14000r/min,10℃条件下高速离心10min,弃上清以去除过量的EDC,沉淀物用1mL硼酸缓冲液复溶,重复超声步骤;加入一定量的抗体水溶液涡旋混匀,并转移至摇床振荡器上室温振荡12h,使抗体与微球材料充分偶联;重复离心步骤,弃上清以去除未偶联的抗体,下层沉淀用1m L含0.5%BSA的硼酸缓冲液复溶,再次重复超声步骤使其均匀分散;随后转移至摇床振荡器上室温振荡2-5h,以封闭微球材料表面的未结合位点;待反应完成后,将制得的探针于4℃条件下保存备用。。
(2)时间分辨荧光免疫层析试纸条的检测条件筛选:偶联铕的单抗标记浓度(抗CTN-单克隆抗体50-100μg/mL)、荧光探针用量(进行50、100、200、300、400和500倍的稀释使用)、检测线上检测抗原浓度(CTA-BSA 2-5mg/mL)、检测线抗原喷涂速率(0.4~0.8μL/cm)。
(3)时间分辨荧光免疫层析试纸条的制备:在NC膜上喷涂抗原CTA-BSA作为T检测线,羊抗鼠IgG作为质控线(C线),包被后置于37℃的鼓风干燥箱中烘2h,将样品垫、吸水纸、NC膜与PVC底板进行组装,切成宽3mm的试纸条后避光干燥备用。
(4)样品的检测:①样品前处理:粮食粉碎过20目筛,分装避光保存②提取时间的筛选:准确称取1.0g粮食样品,分别加入5mL提取剂70%甲醇水,充分振荡3-10min,室温离心(4000r/min,5min);取100μL上清液,用样品缓冲液稀释4倍,制得待测样品液;③样品提取液稀释比例的选择(样品提取液与反应缓释液的稀释比例设置为1∶1、1∶2、1∶3、1∶4和1∶5);④检测分析:取100μL样品缓冲液或待测样品液,加入含有荧光探针的微孔中吹打混匀,37℃孵育2-5min;将试纸条插入微孔,37℃反应5-10min,随后对荧光层析试纸条进行检测分析。定性分析:在紫外灯下通过肉眼判断定性结果,当含有一定浓度待测物时,T线不显线,C线显红色荧光,则结果为阳性;相反,当无待测物时T线和C线均显红色荧光,结果为阴性。定量分析:反应结束后30s内,将荧光层析试纸条插入荧光读数仪读取结果,得到被测粮食中百菌清的含量。
(5)准确度评价:对含有50μg/kg百菌清的空白粮食样品分别采用该时间分辨荧光免疫层析定量分析法和气相色谱串联质谱仪(GC-MS/MS)测定两种检测方法进行检测,并对检测结果的一致性进行分析。
本发明的有益效果:目前,针对检测粮食中百菌清残留的研究已有大量报道,主要有高效液相色谱-质谱法联用,高效液相色谱气相色谱法等色谱法。虽然色谱及其联用技术能检测灵敏度高、重复性好,能够实现农产品和食品当中农药残留的准确分析,但是仪器分析需要专业技术人员的培训,复杂的样品前处理步骤以及昂贵的仪器设备,无法满足现场快速检测的需要。基于稀土金属时间分辨特征的荧光免疫层析分析技术,以其高灵敏、抗干扰、检测方便、逐渐成为市场备受青睐的产品。目前针对粮食中农药残留的时间分辨荧光免疫层析检测方法鲜有报道。因此本研究建立了基于稀土铕荧光微球作为检测信号,可快速检测百菌清的时间分辨荧光免疫层析检测方法,制备一种快速检测粮食中百菌清的时间分辨荧光免疫层析试纸条,可快速准确有效地测出被测粮食中所含百菌清的含量,节省大量人力和屋里,对于农产品污染的防治起到一定的促进作用,进一步保障了农产品的安全和健康。
下面通过具体实施例,对本发明的技术方案作进一步的具体说明。
各实施案例中的步骤(5)时间分辨荧光免疫层析试纸条准确度评价方法均相同,其采用的GC-MS/MS条件如下:色谱柱:DB-5ms毛细管柱(30m×0.25mm,0.25μm);载气:高纯He气;柱流量:1mL/min;PTV进样口温度:65℃;1μL分流进样,分流比为1∶20;溶剂延迟:6min;色谱柱升温程序:初始温度40℃保持4min,以25℃/min升至125℃;再以10℃/min升至300℃,保持4min。离子源(EI)温度:250℃;电离电压:70eV;多反应监测(MRM)模式扫描,保留时间、定量离子对和定性离子对见表1。
表1多反应监测扫描模式(MRM)下百菌清的保留时间、监测离子对和碰撞能
实施例1
(1)采用铕纳米材料与单抗偶联制备荧光探针的方法为:取800μL 0.2moL/L的硼酸缓冲液于2m L的离心管中,加入200μL聚苯乙烯荧光微球乳胶,涡旋混匀;将离心管置于数控高功率超声仪中超声12min,以使乳胶材料在溶液中均匀分散;加入40μL 15mg/mL的EDC水溶液,室温下涡旋振荡10min;然后14000r/min,10℃条件下高速离心10min,弃上清以去除过量的EDC,沉淀物用1mL硼酸缓冲液复溶,重复超声步骤;加入一定量的抗体水溶液涡旋混匀,并转移至摇床振荡器上室温振荡12h,使抗体与微球材料充分偶联;重复离心步骤,弃上清以去除未偶联的抗体,下层沉淀用1m L含0.5%BSA的硼酸缓冲液复溶,再次重复超声步骤使其均匀分散;随后转移至摇床振荡器上室温振荡2h,以封闭微球材料表面的未结合位点;待反应完成后,将制得的探针于4℃条件下保存备用。
(2)时间分辨荧光免疫层析试纸条的检测条件筛选:偶联铕的单抗标记浓度(抗CTN-单克隆抗体55μg/mL),百菌清荧光探针进行100倍稀释使用,检测线上CTA-BSA浓度为2.0mg/mL,检测线抗原喷涂速率为0.4μL/cm。
(3)时间分辨荧光免疫层析试纸条的制备:在NC膜上喷涂抗原CTA-BSA作为T检测线,羊抗鼠IgG作为质控线(C线),包被后置于37℃的鼓风干燥箱中烘2h,将样品垫、吸水纸、NC膜与PVC底板进行组装,切成宽3mm的试纸条后避光干燥备用。
(4)样品的检测:①样品前处理:粮食粉碎过20目筛,分装避光保存②提取时间的筛选:准确称取1.0g粮食样品,分别加入5mL提取剂70%甲醇水,充分振荡5min,室温离心(4000r/min,5min);取100μL上清液,用样品缓冲液稀释4倍,制得待测样品液;③样品提取液与反应缓释液的稀释比例设置为1∶1;④检测分析:取100μL样品缓冲液或待测样品液,加入含有荧光探针的微孔中吹打混匀,37℃孵育2min;将试纸条插入微孔,37℃反应5min,随后对荧光层析试纸条进行检测分析。定性分析:在紫外灯下通过肉眼判断定性结果,当含有一定浓度待测物时,T线不显线,C线显红色荧光,则结果为阳性;相反,当无待测物时T线和C线均显红色荧光,结果为阴性。定量分析:反应结束后30s内,将荧光层析试纸条插入荧光读数仪读取结果,得到被测粮食中百菌清的含量。
(5)准确度评价:对含有50μg/kg百菌清的空白粮食样品,分别采用该时间分辨荧光免疫层析定量分析法和气相色谱串联质谱仪(GC-MS/MS)测定两种检测方法进行检测,并对检测结果的一致性进行分析。
实施例2
(1)采用铕纳米材料与单抗偶联制备荧光探针的方法为:取800μL 0.2moL/L的硼酸缓冲液于2mL的离心管中,加入200μL聚苯乙烯荧光微球乳胶,涡旋混匀;将离心管置于数控高功率超声仪中超声10min,以使乳胶材料在溶液中均匀分散;加入40μL 15mg/mL的EDC水溶液,室温下涡旋振荡15min;然后14000r/min,10℃条件下高速离心10min,弃上清以去除过量的EDC,沉淀物用1mL硼酸缓冲液复溶,重复超声步骤;加入一定量的抗体水溶液涡旋混匀,并转移至摇床振荡器上室温振荡12h,使抗体与微球材料充分偶联;重复离心步骤,弃上清以去除未偶联的抗体,下层沉淀用1mL含0.5%BSA的硼酸缓冲液复溶,再次重复超声步骤使其均匀分散;随后转移至摇床振荡器上室温振荡2h,以封闭微球材料表面的未结合位点;待反应完成后,将制得的探针于4℃条件下保存备用。
(2)时间分辨荧光免疫层析试纸条的检测条件筛选:偶联铕的单抗标记浓度(抗CTN-单克隆抗体60μg/mL),百菌清荧光探针进行500倍稀释使用,检测线上CTA-BSA浓度为2.4mg/mL,检测线抗原喷涂速率为0.5μL/cm。
(3)时间分辨荧光免疫层析试纸条的制备:在NC膜上喷涂抗原CTA-BSA作为T检测线,羊抗鼠IgG作为质控线(C线),包被后置于37℃的鼓风干燥箱中烘2h,将样品垫、吸水纸、NC膜与PVC底板进行组装,切成宽3mm的试纸条后避光干燥备用。
(4)样品的检测:①样品前处理:粮食粉碎过20目筛,分装避光保存②提取时间的筛选:准确称取1.0g粮食样品,分别加入5mL提取剂70%甲醇水,充分振荡4min,室温离心(4000r/min,5min);取100μL上清液,用样品缓冲液稀释4倍,制得待测样品液;③样品提取液与反应缓释液的稀释比例设置为1∶2;④检测分析:取100μL样品缓冲液或待测样品液,加入含有荧光探针的微孔中吹打混匀,37℃孵育2min;将试纸条插入微孔,37℃反应8min,随后对荧光层析试纸条进行检测分析。定性分析:在紫外灯下通过肉眼判断定性结果,当含有一定浓度待测物时,T线不显线,C线显红色荧光,则结果为阳性;相反,当无待测物时T线和C线均显红色荧光,结果为阴性。定量分析:反应结束后30s内,将荧光层析试纸条插入荧光读数仪读取结果,得到被测粮食中百菌清的含量。
(5)准确度评价:对含有50μg/kg百菌清的空白粮食样品,分别采用该时间分辨荧光免疫层析定量分析法和气相色谱串联质谱仪(GC-MS/MS)测定两种检测方法进行检测,并对检测结果的一致性进行分析。
实施例3
(1)采用铕纳米材料与单抗偶联制备荧光探针的方法为:取800μL 0.2moL/L的硼酸缓冲液于2m L的离心管中,加入200μL聚苯乙烯荧光微球乳胶,涡旋混匀;将离心管置于数控高功率超声仪中超声15min,以使乳胶材料在溶液中均匀分散;加入40μL 15mg/mL的EDC水溶液,室温下涡旋振荡18min;然后14000r/min,10℃条件下高速离心10min,弃上清以去除过量的EDC,沉淀物用1mL硼酸缓冲液复溶,重复超声步骤;加入一定量的抗体水溶液涡旋混匀,并转移至摇床振荡器上室温振荡12h,使抗体与微球材料充分偶联;重复离心步骤,弃上清以去除未偶联的抗体,下层沉淀用1mL含0.5%BSA的硼酸缓冲液复溶,再次重复超声步骤使其均匀分散;随后转移至摇床振荡器上室温振荡2h,以封闭微球材料表面的未结合位点;待反应完成后,将制得的探针于4℃条件下保存备用。
(2)时间分辨荧光免疫层析试纸条的检测条件筛选:偶联铕的单抗标记浓度(抗CTN-单克隆抗体80μg/mL),百菌清荧光探针进行300倍稀释使用,检测线上CTA-BSA浓度为3mg/mL,检测线抗原喷涂速率为0.8μL/cm。
(3)时间分辨荧光免疫层析试纸条的制备:在NC膜上喷涂抗原CTA-BSA作为T检测线,羊抗鼠IgG作为质控线(C线),包被后置于37℃的鼓风干燥箱中烘2h,将样品垫、吸水纸、NC膜与PVC底板进行组装,切成宽3mm的试纸条后避光干燥备用。
(4)样品的检测:①样品前处理:粮食粉碎过20目筛,分装避光保存②提取时间的筛选:准确称取1.0g粮食样品,分别加入5mL提取剂70%甲醇水,充分振荡10min,室温离心(4000r/min,5min);取100μL上清液,用样品缓冲液稀释4倍,制得待测样品液;③样品提取液与反应缓释液的稀释比例设置为1∶4;④检测分析:取100μL样品缓冲液或待测样品液,加入含有荧光探针的微孔中吹打混匀,37℃孵育2min;将试纸条插入微孔,37℃反应10min,随后对荧光层析试纸条进行检测分析。定性分析:在紫外灯下通过肉眼判断定性结果,当含有一定浓度待测物时,T线不显线,C线显红色荧光,则结果为阳性;相反,当无待测物时T线和C线均显红色荧光,结果为阴性。定量分析:反应结束后30s内,将荧光层析试纸条插入荧光读数仪读取结果,得到被测粮食中百菌清的含量。
(5)准确度评价:对含有50μg/kg百菌清的空白粮食样品,分别采用该时间分辨荧光免疫层析定量分析法和气相色谱串联质谱仪(GC-MS/MS)测定两种检测方法进行检测,并对检测结果的一致性进行分析。
实施例4
(1)采用铕纳米材料与单抗偶联制备荧光探针的方法为:取800μL 0.2moL/L的硼酸缓冲液于2mL的离心管中,加入200μL聚苯乙烯荧光微球乳胶,涡旋混匀;将离心管置于数控高功率超声仪中超声18min,以使乳胶材料在溶液中均匀分散;加入40μL 15mg/mL的EDC水溶液,室温下涡旋振荡20min;然后14000r/min,10℃条件下高速离心10min,弃上清以去除过量的EDC,沉淀物用1mL硼酸缓冲液复溶,重复超声步骤;加入一定量的抗体水溶液涡旋混匀,并转移至摇床振荡器上室温振荡12h,使抗体与微球材料充分偶联;重复离心步骤,弃上清以去除未偶联的抗体,下层沉淀用1mL含0.5%BSA的硼酸缓冲液复溶,再次重复超声步骤使其均匀分散;随后转移至摇床振荡器上室温振荡2h,以封闭微球材料表面的未结合位点;待反应完成后,将制得的探针于4℃条件下保存备用。
(2)时间分辨荧光免疫层析试纸条的检测条件筛选:偶联铕的单抗标记浓度(抗CTN-单克隆抗体100μg/mL),百菌清荧光探针进行200倍稀释使用,检测线上CTA-BSA浓度为4mg/mL,检测线抗原喷涂速率为0.6μL/cm。
(3)时间分辨荧光免疫层析试纸条的制备:在NC膜上喷涂抗原CTA-BSA作为T检测线,羊抗鼠IgG作为质控线(C线),包被后置于37℃的鼓风干燥箱中烘2h,将样品垫、吸水纸、NC膜与PVC底板进行组装,切成宽3mm的试纸条后避光干燥备用。
(4)样品的检测:①样品前处理:粮食粉碎过20目筛,分装避光保存②提取时间的筛选:准确称取1.0g粮食样品,分别加入5mL提取剂70%甲醇水,充分振荡8min,室温离心(4000r/min,5min);取100μL上清液,用样品缓冲液稀释4倍,制得待测样品液;③样品提取液与反应缓释液的稀释比例设置为1∶4;④检测分析:取100μL样品缓冲液或待测样品液,加入含有荧光探针的微孔中吹打混匀,37℃孵育2min;将试纸条插入微孔,37℃反应10min,随后对荧光层析试纸条进行检测分析。定性分析:在紫外灯下通过肉眼判断定性结果,当含有一定浓度待测物时,T线不显线,C线显红色荧光,则结果为阳性;相反,当无待测物时T线和C线均显红色荧光,结果为阴性。定量分析:反应结束后30s内,将荧光层析试纸条插入荧光读数仪读取结果,得到被测粮食中百菌清的含量。
(5)准确度评价:对含有50μg/kg百菌清的空白粮食样品,分别采用该时间分辨荧光免疫层析定量分析法和气相色谱串联质谱仪(GC-MS/MS)测定两种检测方法进行检测,并对检测结果的一致性进行分析。
实施例5
(1)采用铕纳米材料与单抗偶联制备荧光探针的方法为:取800μL 0.2moL/L的硼酸缓冲液于2mL的离心管中,加入200μL聚苯乙烯荧光微球乳胶,涡旋混匀;将离心管置于数控高功率超声仪中超声15min,以使乳胶材料在溶液中均匀分散;加入40μL 15mg/mL的EDC水溶液,室温下涡旋振荡15min;然后14000r/min,10℃条件下高速离心10min,弃上清以去除过量的EDC,沉淀物用1mL硼酸缓冲液复溶,重复超声步骤;加入一定量的抗体水溶液涡旋混匀,并转移至摇床振荡器上室温振荡12h,使抗体与微球材料充分偶联;重复离心步骤,弃上清以去除未偶联的抗体,下层沉淀用1mL含0.5%BSA的硼酸缓冲液复溶,再次重复超声步骤使其均匀分散;随后转移至摇床振荡器上室温振荡2h,以封闭微球材料表面的未结合位点;待反应完成后,将制得的探针于4℃条件下保存备用。
(2)时间分辨荧光免疫层析试纸条的检测条件筛选:偶联铕的单抗标记浓度(抗CTN-单克隆抗体90μg/mL),百菌清荧光探针进行400倍稀释使用,检测线上CTA-BSA浓度为4.5mg/mL,检测线抗原喷涂速率为0.7μL/cm。
(3)时间分辨荧光免疫层析试纸条的制备:在NC膜上喷涂抗原CTA-BSA作为T检测线,羊抗鼠IgG作为质控线(C线),包被后置于37℃的鼓风干燥箱中烘2h,将样品垫、吸水纸、NC膜与PVC底板进行组装,切成宽3mm的试纸条后避光干燥备用。
(4)样品的检测:①样品前处理:粮食粉碎过20目筛,分装避光保存②提取时间的筛选:准确称取1.0g粮食样品,分别加入5mL提取剂70%甲醇水,充分振荡6min,室温离心(4000r/min,5min);取100μL上清液,用样品缓冲液稀释4倍,制得待测样品液;③样品提取液与反应缓释液的稀释比例设置为1∶4;④检测分析:取100μL样品缓冲液或待测样品液,加入含有荧光探针的微孔中吹打混匀,37℃孵育2min;将试纸条插入微孔,37℃反应8min,随后对荧光层析试纸条进行检测分析。定性分析:在紫外灯下通过肉眼判断定性结果,当含有一定浓度待测物时,T线不显线,C线显红色荧光,则结果为阳性;相反,当无待测物时T线和C线均显红色荧光,结果为阴性。定量分析:反应结束后30s内,将荧光层析试纸条插入荧光读数仪读取结果,得到被测粮食中百菌清的含量。
(5)准确度评价:对含有50μg/kg百菌清的空白粮食样品,分别采用该时间分辨荧光免疫层析定量分析法和气相色谱串联质谱仪(GC-MS/MS)测定两种检测方法进行检测,并对检测结果的一致性进行分析。
实施例6
(1)采用铕纳米材料与单抗偶联制备荧光探针的方法为:取800μL 0.2moL/L的硼酸缓冲液于2mL的离心管中,加入200μL聚苯乙烯荧光微球乳胶,涡旋混匀;将离心管置于数控高功率超声仪中超声16min,以使乳胶材料在溶液中均匀分散;加入40μL 15mg/mL的EDC水溶液,室温下涡旋振荡20min;然后14000r/min,10℃条件下高速离心10min,弃上清以去除过量的EDC,沉淀物用1mL硼酸缓冲液复溶,重复超声步骤;加入一定量的抗体水溶液涡旋混匀,并转移至摇床振荡器上室温振荡12h,使抗体与微球材料充分偶联;重复离心步骤,弃上清以去除未偶联的抗体,下层沉淀用1mL含0.5%BSA的硼酸缓冲液复溶,再次重复超声步骤使其均匀分散;随后转移至摇床振荡器上室温振荡2h,以封闭微球材料表面的未结合位点;待反应完成后,将制得的探针于4℃条件下保存备用。
(2)时间分辨荧光免疫层析试纸条的检测条件筛选:偶联铕的单抗标记浓度(抗CTN-单克隆抗体75μg/mL),百菌清荧光探针进行50倍稀释使用,检测线上CTA-BSA浓度为3.2mg/mL,检测线抗原喷涂速率为0.7μL/cm。
(3)时间分辨荧光免疫层析试纸条的制备:在NC膜上喷涂抗原CTA-BSA作为T检测线,羊抗鼠IgG作为质控线(C线),包被后置于37℃的鼓风干燥箱中烘2h,将样品垫、吸水纸、NC膜与PVC底板进行组装,切成宽3mm的试纸条后避光干燥备用。
(4)样品的检测:①样品前处理:粮食粉碎过20目筛,分装避光保存②提取时间的筛选:准确称取1.0g粮食样品,分别加入5mL提取剂70%甲醇水,充分振荡10min,室温离心(4000r/min,5min);取100μL上清液,用样品缓冲液稀释4倍,制得待测样品液;③样品提取液与反应缓释液的稀释比例设置为1∶5;④检测分析:取100μL样品缓冲液或待测样品液,加入含有荧光探针的微孔中吹打混匀,37℃孵育2min;将试纸条插入微孔,37℃反应10min,随后对荧光层析试纸条进行检测分析。定性分析:在紫外灯下通过肉眼判断定性结果,当含有一定浓度待测物时,T线不显线,C线显红色荧光,则结果为阳性;相反,当无待测物时T线和C线均显红色荧光,结果为阴性。定量分析:反应结束后30s内,将荧光层析试纸条插入荧光读数仪读取结果,得到被测粮食中百菌清的含量。
(5)准确度评价:对含有50μg/kg百菌清的空白粮食样品,分别采用该时间分辨荧光免疫层析定量分析法和气相色谱串联质谱仪(GC-MS/MS)测定两种检测方法进行检测,并对检测结果的一致性进行分析。
实验结果
用制备的时间分辨荧光免疫层析试纸条和GC-MS/MS测定两种检测方法对含有50μg/kg百菌清的小麦米和大米空白样品进行检测,通过回收率和相对标准偏差(RSD)评估其准确度及精确度,平行测定6次,以下是部分实验结果:
表2 GC-MS/MS组及各实施例的测定结果
根据表1可以得出,六组实施例的小麦米样品的百菌清回收率在96%以上,大米的回收率在94%以上,二者的相对标准偏差(RSD)均在5%以下,表明该时间分辨荧光免疫层析定量分析法准确度、精准度和重现性良好,可满足实际检测需求。同时,与GC-MS/MS所测得的结果相比,六组实施例中小麦米和大米样品的百菌清回收率和相对标准偏差(RSD)均与之接近。此外,该时间分辨荧光免疫层析试纸条的检测时间最多只需20min,操作简单快速,且具有可视的检测优势。
Claims (9)
1.一种快速检测粮食中百菌清残留的试纸条的制备方法,其特征在于,该方法包括以下步骤:
(1)荧光探针的制备:采用铕纳米材料与单抗偶联制备荧光探针,取800μL 0.2moL/L的硼酸缓冲液于2mL的离心管中,加入200μL聚苯乙烯荧光微球乳胶,涡旋混匀;将离心管置于数控高功率超声仪中超声12-18min,以使乳胶材料在溶液中均匀分散;加入40μL 15mg/mL的EDC水溶液,室温下涡旋振荡10-20min;然后14000r/min,10℃条件下高速离心10min,弃上清以去除过量的EDC,沉淀物用1mL硼酸缓冲液复溶,重复超声步骤;加入一定量的抗体水溶液涡旋混匀,并转移至摇床振荡器上室温振荡12h,使抗体与微球材料充分偶联;重复离心步骤,弃上清以去除未偶联的抗体,下层沉淀用1mL含0.5%BSA的硼酸缓冲液复溶,再次重复超声步骤使其均匀分散;随后转移至摇床振荡器上室温振荡2-5h,以封闭微球材料表面的未结合位点;待反应完成后,将制得的探针于4℃条件下保存备用。
(2)时间分辨荧光免疫层析试纸条的检测条件筛选:偶联铕的单抗标记浓度(抗CTN-单克隆抗体50-100μg/mL)、荧光探针用量(进行50、100、200、300、400和500倍的稀释使用)、检测线上检测抗原浓度(CTA-BSA 2-5mg/mL)、检测线抗原喷涂速率(0.4~0.8μL/cm)。
(3)时间分辨荧光免疫层析试纸条的制备:在NC膜上喷涂抗原CTA-BSA作为T检测线,羊抗鼠IgG作为质控线(C线),包被后置于37℃的鼓风干燥箱中烘2h,将样品垫、吸水纸、NC膜与PVC底板进行组装,切成宽3mm的试纸条后避光干燥备用。
(4)样品的检测:①样品前处理:粮食粉碎过20目筛,分装避光保存;②提取时间的筛选:准确称取1.0g粮食样品,分别加入5mL提取剂70%甲醇水,充分振荡3-10min,室温离心(4000r/min,5min);取100μL上清液,用样品缓冲液稀释4倍,制得待测样品液;③样品提取液稀释比例的选择(样品提取液与反应缓释液的稀释比例设置为1∶1、1∶2、1∶3、1∶4和1∶5);④检测分析:取100μL样品缓冲液或待测样品液,加入含有荧光探针的微孔中吹打混匀,37℃孵育2-5min;将试纸条插入微孔,37℃反应5-10min,随后对荧光层析试纸条进行检测分析。定性分析:在紫外灯下通过肉眼判断定性结果,当含有一定浓度待测物时,T线不显线,C线显红色荧光,则结果为阳性;相反,当无待测物时T线和C线均显红色荧光,结果为阴性。定量分析:反应结束后30s内,将荧光层析试纸条插入荧光读数仪读取结果,得到被测粮食中百菌清的含量。
(5)准确度评价:对含有50μg/kg百菌清的空白粮食样品分别采用该时间分辨荧光免疫层析定量分析法和气相色谱串联质谱仪(GC-MS/MS)测定两种检测方法进行检测,并对检测结果的一致性进行分析。
2.根据权利要求1所述的一种快速检测粮食中百菌清残留的试纸条的制备方法,其特征在于:步骤(1)采用铕纳米材料与单抗偶联制备荧光探针的方法为:取800μL 0.2moL/L的硼酸缓冲液于2mL的离心管中,加入200μL聚苯乙烯荧光微球乳胶,涡旋混匀;将离心管置于数控高功率超声仪中超声12-18min,以使乳胶材料在溶液中均匀分散;加入40μL 15mg/mL的EDC水溶液,室温下涡旋振荡10-20min;然后14000r/min,10℃条件下高速离心10min,弃上清以去除过量的EDC,沉淀物用1mL硼酸缓冲液复溶,重复超声步骤;加入一定量的抗体水溶液涡旋混匀,并转移至摇床振荡器上室温振荡12h,使抗体与微球材料充分偶联;重复离心步骤,弃上清以去除未偶联的抗体,下层沉淀用1mL含0.5%BSA的硼酸缓冲液复溶,再次重复超声步骤使其均匀分散;随后转移至摇床振荡器上室温振荡2-5h,以封闭微球材料表面的未结合位点;待反应完成后,将制得的探针于4℃条件下保存备用。
3.根据权利要求1所述的一种快速检测粮食中百菌清残留的试纸条的制备方法,其特征在于:步骤(1)荧光探针的制备方法中荧光微球乳胶溶液的超声时间为12-18min,荧光微球的震荡活化时间为10-20min,加入抗体后的震荡反应时间为2-5h。
4.根据权利要求1所述的一种快速检测粮食中百菌清残留的试纸条的制备方法,其特征在于:步骤(2)时间分辨荧光免疫层析试纸条的检测条件中抗CTN-单克隆抗体标记浓度50-100μg/mL。
5.根据权利要求1所述的一种快速检测粮食中百菌清残留的试纸条的制备方法,其特征在于:步骤(2)时间分辨荧光免疫层析试纸条的检测条件中百菌清探针分别进行50、100、200、300、400和500倍的稀释使用。
6.根据权利要求1所述的一种快速检测粮食中百菌清残留的试纸条的制备方法,其特征在于:步骤(2)时间分辨荧光免疫层析试纸条的检测条件中检测线抗原喷涂速率为0.4~0.8μL/cm。
7.根据权利要求1所述的一种快速检测粮食中百菌清残留的试纸条的制备方法,其特征在于:步骤(4)样品提取液稀释比例的选择:样品提取液与反应缓释液的稀释比例设置为1∶1、1∶2、1∶3、1∶4和1∶5。
8.根据权利要求1所述的一种快速检测粮食中百菌清残留的试纸条的制备方法,其特征在于:步骤(4)提取时间的选择:称取样品,加入提取剂后充分振荡2-5min,按步骤制得待测样品液。
9.根据权利要求1所述的一种快速检测粮食中百菌清残留的试纸条的制备方法,其特征在于:步骤(4)检测分析时,样品缓冲液或待测样品液与荧光探针混匀后,在37℃条件下孵育2-5min;将试纸条插入微孔后,需在37℃条件下反应5-10min。
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