CN116643049B - 基于改性纳米金材料的丙溴磷农药胶体金标记物及其应用 - Google Patents
基于改性纳米金材料的丙溴磷农药胶体金标记物及其应用 Download PDFInfo
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Abstract
本发明涉及胶体金标记物制备技术领域,尤其涉及一种基于改性纳米金材料的丙溴磷农药胶体金标记物及其应用,包括以下步骤:S1:S101:柠檬酸钠还原法制纳米金溶胶;S102:将γ‑氨丙基三乙氧基硅烷加入四氢呋喃中,搅拌溶解后,再滴加HCl溶液,35‑45℃搅拌处理10‑12h;S103:将S102所得八氨基POSS前驱体溶液缓慢滴加至S101所得纳米金溶胶中,再于55‑60℃水浴搅拌反应24h,离心、洗涤、干燥;S2:将所得八氨基POSS改性纳米金颗粒加入混合缓冲液中,25℃搅拌复溶后,加入丙溴磷单克隆抗体,37℃搅拌反应1‑2h,得金标记物溶液。本发明制出的胶体金试纸对丙溴磷的检测限最低至0.01mg/kg,克服了现有胶体金试纸对丙溴磷检测限低的问题。
Description
技术领域
本发明涉及胶体金标记物制备技术领域,还涉及丙溴磷农药残留检测领域,尤其涉及一种基于改性纳米金材料的丙溴磷农药胶体金标记物及其应用。
背景技术
丙溴磷是一种有机农药,分子式为C11H15BrClO3PS,属三元不对称的非内吸性广谱杀虫剂,有触杀和胃毒作用,作用迅速,能防治危害棉花、蔬菜、果树等作物的多种昆虫,对水稻二化螟、钻心虫、稻纵卷叶螟、水稻稻飞虱同样有效。由于丙溴磷农药的大量使用,导致丙溴磷在果蔬、谷物等食品中的富集,当人在食用这种高剂量丙溴磷的食品后,会引起神经系统中枢和外周神经的损害,危害心血管系统、呼吸系统、生殖系统,严重时可危及生命。
对此,我国国家标准GB 2763-2019《食品安全国家标准食品中农药最大残留限量》中规定了不同食品中丙溴磷的残留限量。但是,现有检测丙溴磷残留的胶体金试纸的检测限度较高,对于食品中残留量低的丙溴磷无法检出,无法满足马铃薯、苹果、糙米等最大残留限量低的作物的检测需求。如申请号为 CN202010138495.X 的发明专利,公开一种检测丙溴磷的试纸条及方法,包括样品吸收垫、结合物释放垫、反应膜、吸水垫和底板,所述反应膜上具有包被有丙溴磷半抗原-载体蛋白偶联物的检测线和包被有羊抗鼠抗抗体的质控线,所述结合物释放垫上喷涂有丙溴磷单克隆抗体-胶体金标记物;所述丙溴磷单克隆抗体是以丙溴磷半抗原-载体蛋白偶联物作为免疫原制备获得;所述丙溴磷半抗原-载体蛋白偶联物由丙溴磷半抗原与载体蛋白偶联得到,所述载体蛋白为牛血清白蛋白、卵清蛋白、血蓝蛋白、甲状腺蛋白或人血清白蛋白,所述丙溴磷半抗原是以三氯硫磷为起始原料,先后与丁硫醇、乙醇、5-溴-3-氯-2-羟基苯甲酸进行缩合反应得到,该丙溴磷半抗原合成路线如下:
。
但是,该检测丙溴磷的试纸条对蔬菜水果中丙溴磷的检测限为0.1mg/kg,无法满足最大残留限量低的食品的检测需求。
发明内容
有鉴于此,本发明的目的在于提出一种基于改性纳米金材料的丙溴磷农药胶体金标记物的制备方法,以解决现有胶体金试纸(非荧光检测试纸)对丙溴磷检测限低的问题。
基于上述目的,本发明提供了一种基于改性纳米金材料的丙溴磷农药胶体金标记物的制备方法,包括以下步骤:
S1:制备八氨基POSS改性纳米金颗粒:
S101:将1wt%氯金酸溶液加热至沸腾后,再滴加3-5wt%柠檬酸钠溶液,磁力搅拌反应3-6h,得纳米金溶胶;
S102:将γ-氨丙基三乙氧基硅烷加入四氢呋喃中,搅拌溶解后,再滴加20-25wt%HCl溶液,35-45℃搅拌处理10-12h,得八氨基POSS前驱体溶液;
S103:将S102所得八氨基POSS前驱体溶液缓慢滴加至S101所得纳米金溶胶中,再于55-60℃水浴搅拌反应24h,离心、洗涤,并于40℃真空干燥6h后,得八氨基POSS改性纳米金颗粒;
S2:制备金标记物溶液:将八氨基POSS改性纳米金颗粒加入混合缓冲液中,25℃搅拌复溶后,加入丙溴磷单克隆抗体,37℃搅拌反应1-2h,得金标记物溶液。
进一步地,所述氯金酸溶液、柠檬酸钠溶液、γ-氨丙基三乙氧基硅烷、四氢呋喃、HCl溶液的用量比例为100mL:100mL:(20-45)mg:(2-5)mL:(1-3)mg。
进一步地,所述八氨基POSS改性纳米金颗粒、混合缓冲液、丙溴磷单克隆抗体的用量比例为(1-2) mg:1mL:(20-30) μg。
进一步地,所述混合缓冲液配方为:PBS缓冲液0.02M,BSA 0.5w/v%,Triton X-1000.05w/v%,羧甲基葡聚糖1w/v%,pH=7.2。
另一方面,本发明还提供所述的基于改性纳米金材料的丙溴磷农药胶体金标记物在制备用于丙溴磷农药残留检测的胶体金试纸中的应用。
进一步地,该胶体金试纸上含有金标记物包被的金标记垫。
进一步地,该胶体金试纸以PVC板为底板,且底板表面自左向右依次粘贴有样品垫、金标记垫、反应膜、吸水垫。
进一步地,所述反应膜自左向右间隔设有检测线T线和质控线C线,T线上包被有丙溴磷半抗原-OVA 偶联物,C线上包被有羊抗鼠 IgG。
进一步地,该胶体金试纸的制备方法,包括以下步骤:
A1:制备样品垫:将玻璃纤维素膜浸于含0.5w/v% BSA的0.02M、pH=7.2 PBS缓冲液中,静置浸渍1h,37℃烘干后,得样品垫;
A2:制备金标记垫:将金标记溶液喷涂于玻璃纤维素膜上,喷膜量为5-7.5μL/cm,37℃烘干后,得金标记垫;
A3:制备反应膜:
A301:用0.02M、pH=7.2 PBS缓冲液将丙溴磷半抗原-OVA 偶联物稀释至0.4mg/mL,得丙溴磷半抗原-OVA 偶联物包被液;
A302:用0.02M、pH=7.2 PBS缓冲液将羊抗鼠 IgG稀释至0.2mg/mL,得羊抗鼠 IgG包被液;
A303:以硝酸纤维素膜作为反应膜承载体,在硝酸纤维素膜上间隔划分出检测线T线和质控线C线,并用0.4mg/mL丙溴磷半抗原-OVA 偶联物包被液喷涂T线、0.2mg/mL羊抗鼠IgG包被液喷涂C线,T、C线喷膜量均为1μL/cm,37℃烘干后,得反应膜;
A4:组装胶体金试纸:将样品垫、金标记垫、反应膜、吸水垫自左向右依次粘贴在PVC底板上,且反应膜上的T线与金标记垫相邻,制得丙溴磷农药残留检测用胶体金试纸。
进一步地,所述的丙溴磷农药残留检测用胶体金试纸对果蔬谷物中的丙溴磷检测限为0.01-0.02mg/kg。
本发明的有益效果:
本发明首次利用八氨基POSS与丙溴磷单克隆抗体制备金标记物溶液,并进一步应用于丙溴磷农药残留检测的胶体金试纸,制出的胶体金试纸对果蔬谷物中丙溴磷的检测限最低至0.01mg/kg,克服了现有胶体金试纸(非荧光检测试纸)对丙溴磷检测限低的问题,满足马铃薯、苹果、糙米乃至生乳等食品中丙溴磷最大残留限量低的检测需求。
发明人意外地发现,采用八氨基POSS改性纳米金颗粒作为金标记物主要原料,能明显提高胶体金试纸对果蔬谷物中丙溴磷的检测性能,究其原因可能是,八氨基POSS改性纳米金颗粒能与丙溴磷单克隆抗体上的羧基发生化学键合,进而提高其与丙溴磷单克隆抗体的结合稳定性,且其特殊的笼性空间结构为纳米金颗粒提供了更多的识别位点,进而提高对丙溴磷的识别灵敏度和选择性,而其特殊的空间效应也进一步提高了金抗体标记物的稳定性,防止丙溴磷单克隆抗体的水解和氧化作用。
此外,发明人还发现,采用羧甲基葡聚糖配制混合缓冲液也能一定程度上提高胶体金试纸对果蔬谷物中丙溴磷的检测灵敏度。究其原因可能是,羧甲基葡聚糖具有良好的成膜性,进而提高金抗体标记物的稳定性,进一步保持识别灵敏度。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明用于丙溴磷农药残留检测的胶体金试纸的示意图;
图1中标记为:1、样品垫,2、金标记垫,3、反应膜,4、吸水垫。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,以下结合具体实施例,对本发明进一步详细说明。
下列实施例中所使用的丙溴磷半抗原-OVA 偶联物根据“基于单克隆抗体的胶体金免疫层析法快速检测丙溴磷[J].王兆芹等,河南农业科学,2020,49( 12) : 77-82”中的“1.3.1半抗原的合成及1.3.2包被原的制备”方法合成。
本发明提供一实施例的基于改性纳米金材料的丙溴磷农药胶体金标记物的制备方法,包括以下步骤:
S1:制备八氨基POSS改性纳米金颗粒:
S101:将1wt%氯金酸溶液加热至沸腾后,再滴加3-5wt%柠檬酸钠溶液,磁力搅拌反应3-6h,得纳米金溶胶;
S102:将γ-氨丙基三乙氧基硅烷加入四氢呋喃中,搅拌溶解后,再滴加20-25wt%HCl溶液,35-45℃搅拌处理10-12h,得八氨基POSS前驱体溶液;
S103:将S102所得八氨基POSS前驱体溶液缓慢滴加至S101所得纳米金溶胶中,再于55-60℃水浴搅拌反应24h,离心、洗涤,并于40℃真空干燥6h后,得八氨基POSS改性纳米金颗粒;
所述氯金酸溶液、柠檬酸钠溶液、γ-氨丙基三乙氧基硅烷、四氢呋喃、HCl溶液的用量比例为100mL:100mL:(20-45)mg:(2-5)mL:(1-3)mg;
S2:制备金标记物溶液:将八氨基POSS改性纳米金颗粒加入混合缓冲液中,25℃搅拌复溶后,加入丙溴磷单克隆抗体,37℃搅拌反应1-2h,得金标记物溶液;
所述八氨基POSS改性纳米金颗粒、混合缓冲液、丙溴磷单克隆抗体的用量比例为(1-2) mg:1mL:(20-30) μg;所述混合缓冲液配方为:PBS缓冲液0.02M,BSA 0.5w/v%,Triton X-100 0.05w/v%,羧甲基葡聚糖1w/v%,pH=7.2。
该实施例制出的基于改性纳米金材料的丙溴磷农药胶体金标记物在制备用于丙溴磷农药残留检测的胶体金试纸中的应用:该胶体金试纸以PVC板为底板,且底板表面自左向右依次粘贴有样品垫、金标记垫、反应膜、吸水垫;所述金标记垫上包被有金标记物;所述反应膜自左向右间隔设有检测线T线和质控线C线,T线上包被有丙溴磷半抗原-OVA 偶联物,C线上包被有羊抗鼠 IgG。具体地:
该胶体金试纸的制备方法,包括以下步骤:
A1:制备样品垫:将玻璃纤维素膜浸于含0.5w/v% BSA的0.02M、pH=7.2 PBS缓冲液中,静置浸渍1h,37℃烘干后,得样品垫;
A2:制备金标记垫:将金标记溶液喷涂于玻璃纤维素膜上,喷膜量为5-7.5μL/cm,37℃烘干后,得金标记垫;
A3:制备反应膜:
A301:用0.02M、pH=7.2 PBS缓冲液将丙溴磷半抗原-OVA 偶联物稀释至0.4mg/mL,得丙溴磷半抗原-OVA 偶联物包被液;
A302:用0.02M、pH=7.2 PBS缓冲液将羊抗鼠 IgG稀释至0.2mg/mL,得羊抗鼠 IgG包被液;
A303:以硝酸纤维素膜作为反应膜承载体,在硝酸纤维素膜上间隔划分出检测线T线和质控线C线,并用0.4mg/mL丙溴磷半抗原-OVA 偶联物包被液喷涂T线、0.2mg/mL羊抗鼠IgG包被液喷涂C线,T、C线喷膜量均为1μL/cm,37℃烘干后,得反应膜;
A4:组装胶体金试纸:将样品垫、金标记垫、反应膜、吸水垫自左向右依次粘贴在PVC底板上,且反应膜上的T线与金标记垫相邻,制得丙溴磷农药残留检测用胶体金试纸。
实施例1
一种基于改性纳米金材料的丙溴磷农药胶体金标记物的制备方法,包括以下步骤:
S1:制备八氨基POSS改性纳米金颗粒:
S101:将100mL的1wt%氯金酸溶液加热至沸腾后,再滴加100mL的3wt%柠檬酸钠溶液,磁力搅拌反应3h,得纳米金溶胶;
S102:将20mg γ-氨丙基三乙氧基硅烷加入2mL四氢呋喃中,搅拌溶解后,再滴加1mg 20wt%HCl溶液,35℃搅拌处理10h,得八氨基POSS前驱体溶液;
S103:将S102所得八氨基POSS前驱体溶液缓慢滴加至S101所得纳米金溶胶中,再于55℃水浴搅拌反应24h,离心、洗涤,并于40℃真空干燥6h后,得八氨基POSS改性纳米金颗粒;
S2:按以下配方配制混合缓冲液:PBS缓冲液0.02M,BSA 0.5w/v%,Triton X-1000.05w/v%,羧甲基葡聚糖1w/v%,pH=7.2;
S3:制备金标记物溶液:将1mg八氨基POSS改性纳米金颗粒加入1mL混合缓冲液中,25℃搅拌复溶后,加入20 μg丙溴磷单克隆抗体,37℃搅拌反应1h,得金标记物溶液。
实施例2
一种基于改性纳米金材料的丙溴磷农药胶体金标记物的制备方法,包括以下步骤:
S1:制备八氨基POSS改性纳米金颗粒:
S101:将100mL的1wt%氯金酸溶液加热至沸腾后,再滴加100mL的4wt%柠檬酸钠溶液,磁力搅拌反应4.5h,得纳米金溶胶;
S102:将35mg γ-氨丙基三乙氧基硅烷加入3.5mL四氢呋喃中,搅拌溶解后,再滴加2mg 25wt%HCl溶液,40℃搅拌处理12h,得八氨基POSS前驱体溶液;
S103:将S102所得八氨基POSS前驱体溶液缓慢滴加至S101所得纳米金溶胶中,再于60℃水浴搅拌反应24h,离心、洗涤,并于40℃真空干燥6h后,得八氨基POSS改性纳米金颗粒;
S2:按以下配方配制混合缓冲液:PBS缓冲液0.02M,BSA 0.5w/v%,Triton X-1000.05w/v%,羧甲基葡聚糖1w/v%,pH=7.2;
S3:制备金标记物溶液:将1.5 mg八氨基POSS改性纳米金颗粒加入1mL混合缓冲液中,25℃搅拌复溶后,加入25 μg丙溴磷单克隆抗体,37℃搅拌反应1-2h,得金标记物溶液。
实施例3
一种基于改性纳米金材料的丙溴磷农药胶体金标记物的制备方法,包括以下步骤:
S1:制备八氨基POSS改性纳米金颗粒:
S101:将100mL的1wt%氯金酸溶液加热至沸腾后,再滴加100mL的5wt%柠檬酸钠溶液,磁力搅拌反应6h,得纳米金溶胶;
S102:将45mg γ-氨丙基三乙氧基硅烷加入5mL四氢呋喃中,搅拌溶解后,再滴加3mg 25wt%HCl溶液,45℃搅拌处理12h,得八氨基POSS前驱体溶液;
S103:将S102所得八氨基POSS前驱体溶液缓慢滴加至S101所得纳米金溶胶中,再于60℃水浴搅拌反应24h,离心、洗涤,并于40℃真空干燥6h后,得八氨基POSS改性纳米金颗粒;
S2:按以下配方配制混合缓冲液:PBS缓冲液0.02M,BSA 0.5w/v%,Triton X-1000.05w/v%,羧甲基葡聚糖1w/v%,pH=7.2;
S3:制备金标记物溶液:将2 mg八氨基POSS改性纳米金颗粒加入1mL混合缓冲液中,25℃搅拌复溶后,加入30 μg丙溴磷单克隆抗体,37℃搅拌反应2h,得金标记物溶液。
实施例4
利用实施例1制出的基于改性纳米金材料的丙溴磷农药胶体金标记物制备
丙溴磷农药残留检测用胶体金试纸:该胶体金试纸以PVC板为底板,且底板表面自左向右依次粘贴有样品垫、金标记垫、反应膜、吸水垫;所述金标记垫上包被有金标记物;所述反应膜自左向右间隔设有检测线T线和质控线C线,T线上包被有丙溴磷半抗原-OVA 偶联物,C线上包被有羊抗鼠 IgG。具体地:
该胶体金试纸的制备方法,包括以下步骤:
A1:制备样品垫:将玻璃纤维素膜浸于含0.5w/v% BSA的0.02M、pH=7.2 PBS缓冲液中,静置浸渍1h,37℃烘干后,得样品垫;
A2:制备金标记垫:将金标记溶液喷涂于玻璃纤维素膜上,喷膜量为5μL/cm,37℃烘干后,得金标记垫;
A3:制备反应膜:
A301:用0.02M、pH=7.2 PBS缓冲液将丙溴磷半抗原-OVA 偶联物稀释至0.4mg/mL,得丙溴磷半抗原-OVA 偶联物包被液;
A302:用0.02M、pH=7.2 PBS缓冲液将羊抗鼠 IgG稀释至0.2mg/mL,得羊抗鼠 IgG包被液;
A303:以硝酸纤维素膜作为反应膜承载体,在硝酸纤维素膜上间隔划分出检测线T线和质控线C线,并用0.4mg/mL丙溴磷半抗原-OVA 偶联物包被液喷涂T线、0.2mg/mL羊抗鼠IgG包被液喷涂C线,T、C线喷膜量均为1μL/cm,37℃烘干后,得反应膜;
A4:组装胶体金试纸:将样品垫、金标记垫、反应膜、吸水垫自左向右依次粘贴在PVC底板上,且反应膜上的T线与金标记垫相邻,制得丙溴磷农药残留检测用胶体金试纸。
实施例5
利用实施例1制出的基于改性纳米金材料的丙溴磷农药胶体金标记物制备丙溴磷农药残留检测用胶体金试纸:制备方法同实施例4,区别在于:A2步骤中,金标记溶液的喷膜量为6.5μL/cm。
实施例6
利用实施例1制出的基于改性纳米金材料的丙溴磷农药胶体金标记物制备丙溴磷农药残留检测用胶体金试纸:制备方法同实施例4,区别在于:A2步骤中,金标记溶液的喷膜量为7.5μL/cm。
对比例1与实施例1相同,区别在于:以纳米金颗粒代替八氨基POSS改性纳米金颗粒;且纳米金颗粒制备方法为:将100mL的1wt%氯金酸溶液加热至沸腾后,再滴加100mL的3wt%柠檬酸钠溶液,磁力搅拌反应3h,得纳米金溶胶,再于55℃水浴搅拌反应24h,离心、洗涤,并于40℃真空干燥6h后,得纳米金颗粒。
对比例2与实施例1相同,区别在于:以八甲基POSS改性纳米金颗粒代替八氨基POSS改性纳米金颗粒;且八甲基POSS改性纳米金颗粒制备方法为:
S101:将100mL的1wt%氯金酸溶液加热至沸腾后,再滴加100mL的3wt%柠檬酸钠溶液,磁力搅拌反应3h,得纳米金溶胶;
S102:将20mg 甲基三乙氧基硅烷加入2mL四氢呋喃中,搅拌溶解后,调节pH至9.0,35℃搅拌处理10h,得八甲基POSS前驱体溶液;
S103:将S102所得八甲基POSS前驱体溶液缓慢滴加至S101所得纳米金溶胶中,再于55℃水浴搅拌反应24h,离心、洗涤,并于40℃真空干燥6h后,得八甲基POSS改性纳米金颗粒。
对比例3与实施例1相同,区别在于:以双端氨基PEG改性纳米金颗粒代替八氨基POSS改性纳米金颗粒;且双端氨基PEG改性纳米金颗粒制备方法为:将100mL的1wt%氯金酸溶液加热至沸腾后,再滴加100mL的3wt%柠檬酸钠溶液,磁力搅拌反应3h,得纳米金溶胶,加入20mg双端氨基PEG,再于55℃水浴搅拌反应24h,离心、洗涤,并于40℃真空干燥6h后,得双端氨基PEG改性纳米金颗粒。
对比例4与实施例1相同,区别在于:混合缓冲液中不含有羧甲基葡聚糖。
对比例5与实施例1相同,区别在于:配制混合缓冲液时,利用葡聚糖代替羧甲基葡聚糖。
对比例6与实施例1相同,区别在于:以纳米金颗粒代替八氨基POSS改性纳米金颗粒;且混合缓冲液中不含有羧甲基葡聚糖。
对比例7-12
分别利用对比例1-6制出的基于改性纳米金材料的丙溴磷农药胶体金标记物制备丙溴磷农药残留检测用胶体金试纸:制备方法同实施例4;制出的胶体金试纸对应记作对比例7-12,下表列出对比例7-12的胶体金试纸所用的金标记物溶液原料:
对比例7 | 对比例8 | 对比例9 | 对比例10 | 对比例11 | 对比例12 | |
金标记物溶液 | 对比例1 | 对比例2 | 对比例3 | 对比例4 | 对比例5 | 对比例6 |
试验一:果蔬谷物中丙溴磷的检测
1.1样品前处理
1.1.1糙米的前处理:将糙米研磨粉碎至100目后,称取1.00±0.05g糙米粉,加入至10mL聚苯乙烯离心管中,加入6mL(0.02M、pH=7.2 PBS)缓冲液,盖上盖子,手动振荡30s,静置1min,取上层清液作为糙米样本液;
1.1.2马铃薯的前处理:将马铃薯洗净后,切成0.5cm*0.5cm*0.5cm,在用粉碎机粉碎后,称取1.00±0.05g马铃薯渣,加入至10mL聚苯乙烯离心管中,加入6mL(0.02M、pH=7.2PBS)缓冲液,盖上盖子,手动振荡30s,静置1min,取上层清液作为马铃薯样本液;
1.13苹果的前处理:同1.1.2。
1.2检测结果判定
阴性(-):T线显色比C线显色深或与C线显色一致,表示样本中丙溴磷浓度低于检测限,则检测结果为阴性(-);
阳性(+):T线显色比C线显色浅或T线不显色,表示样本中丙溴磷浓度等于或高于检测限,则检测结果为阳性(+);
无效:未出现C线,表明检测操作过程不规范或胶体金试纸已变质失效,检测结果无效。
1.3检测限试验
用胶体金试纸检测:将胶体金试纸中样品垫端浸入空白样本液中,浸渍30s,再取出后,反应5min,判定结果。
向空白糙米样本液、空白马铃薯样本液、空白苹果样本液中,分别添加丙溴磷至终浓度为5μg/kg、0.01mg/kg、0.02mg/kg、0.03mg/kg、0.04mg/kg、0.05mg/kg、0.06mg/kg、0.07mg/kg、0.08mg/kg、0.09mg/kg、0.1mg/kg,取试纸条进行检测,每个样本重复测定三次。
根据胶体金试纸显示结果确定实施例4-6及及对比例7-12制备出的胶体金试纸的检测限,结果如下表所示:
糙米中丙溴磷的检测限/mg/kg | 马铃薯中丙溴磷的检测限/mg/kg | 苹果中丙溴磷的检测限/mg/kg | |
实施例4 | 0.02 | 0.02 | 0.02 |
实施例5 | 0.01 | 0.02 | 0.01 |
实施例6 | 0.01 | 0.01 | 0.01 |
对比例7 | 0.09 | 0.09 | 0.09 |
对比例8 | 0.08 | 0.08 | 0.08 |
对比例9 | 0.06 | 0.06 | 0.06 |
对比例10 | 0.04 | 0.04 | 0.04 |
对比例11 | 0.02 | 0.03 | 0.03 |
对比例12 | 0.1 | 0.1 | 0.1 |
由上表可知,相比于对比例7-9,实施例4-6采用八氨基POSS改性纳米金颗粒作为金标记物主要原料,能明显提高胶体金试纸对果蔬谷物中丙溴磷的检测性能,检测限低至0.01-0.02mg/kg,且比较实施例4与对比例10-11发现,采用羧甲基葡聚糖配制混合缓冲液也能一定程度上提高胶体金试纸对果蔬谷物中丙溴磷的检测灵敏度。究其原因可能是,八氨基POSS改性纳米金颗粒能与丙溴磷单克隆抗体上的羧基发生化学键合,进而提高其与丙溴磷单克隆抗体的结合稳定性,且其特殊的笼性空间结构为纳米金颗粒提供了更多的识别位点,进而提高对丙溴磷的识别灵敏度和选择性,且其特殊的空间效应也进一步提高了金抗体标记物的稳定性,防止丙溴磷单克隆抗体的水解和氧化作用;而羧甲基葡聚糖能在一定程度上提高胶体金试纸对果蔬谷物中丙溴磷的检测灵敏度,考虑其羧甲基葡聚糖对金抗体标记物稳定性的影响,通过其良好的成膜性提高金抗体标记物的稳定性,进而保持识别灵敏度。
试验二:假阳性率、假阴性率试验
取空白糙米样本液、空白马铃薯样本液、空白苹果样本液及添加丙溴磷至终浓度为0.02mg/kg的糙米样本液、马铃薯样本液、苹果样本液各20份,用3个批次生产的实施例4-6制备出的胶体金试纸分别进行检测,计算其阴阳性率。
结果表明:用3个批次生产的实施例4-6制备出的胶体金试纸检测阳性样本时,观察到胶体金试纸上的T线显色比C线显色浅或T线不显色,结果全为阳性,阳性符合率为100%,假阴性率为0;检测阴性样本时,观察到胶体金试上的T线显色比C线显色深或与C线显色一致,结果全为阴性,可知阴性符合率为100%,假阳性率为0。
试验三:特异性试验
取对硫磷添加至PBS缓冲液至其终浓度为2mg/kg,得2mg/kg对硫磷样本液;2mg/kg噻虫胺、吡虫啉、三唑磷、克百威样本液的制备方法同2mg/kg对硫磷样本液;
用3个批次生产的实施例4-6制备出的胶体金试纸分别对上述样本液进行检测,每个样本重复测定三次。
结果表明:用3个批次生产的实施例4-6制备出的胶体金试纸检测2mg/kg对硫磷、噻虫胺、吡虫啉、三唑磷、克百威时,观察到胶体金试纸上的T线显色比C线显色深或与C线显色一致,结果全为阴性,说明本发明制出的胶体金试纸对于对硫磷、噻虫胺、吡虫啉、三唑磷、克百威等农药无交叉反应。
所属领域的普通技术人员应当理解:以上任何实施例的讨论仅为示例性的,并非旨在暗示本发明的范围(包括权利要求)被限于这些例子;在本发明的思路下,以上实施例或者不同实施例中的技术特征之间也可以进行组合,步骤可以以任意顺序实现,并存在如上所述的本发明的不同方面的许多其它变化,为了简明它们没有在细节中提供。
本发明旨在涵盖落入所附权利要求的宽泛范围之内的所有这样的替换、修改和变型。因此,凡在本发明的精神和原则之内,所做的任何省略、修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种基于改性纳米金材料的丙溴磷农药胶体金标记物的制备方法,其特征在于,包括以下步骤:
S1:制备八氨基POSS改性纳米金颗粒:
S101:将1wt%氯金酸溶液加热至沸腾后,再滴加3-5wt%柠檬酸钠溶液,磁力搅拌反应3-6h,得纳米金溶胶;
S102:将γ-氨丙基三乙氧基硅烷加入四氢呋喃中,搅拌溶解后,再滴加20-25wt%HCl溶液,35-45℃搅拌处理10-12h,得八氨基POSS前驱体溶液;
S103:将S102所得八氨基POSS前驱体溶液缓慢滴加至S101所得纳米金溶胶中,再于55-60℃水浴搅拌反应24h,离心,然后洗涤,并于40℃真空干燥6h后,得八氨基POSS改性纳米金颗粒;
S2:制备金标记物溶液:将八氨基POSS改性纳米金颗粒加入混合缓冲液中,25℃搅拌复溶后,加入丙溴磷单克隆抗体,37℃搅拌反应1-2h,得金标记物溶液。
2.根据权利要求1所述的一种基于改性纳米金材料的丙溴磷农药胶体金标记物的制备方法,其特征在于,所述氯金酸溶液、柠檬酸钠溶液、γ-氨丙基三乙
氧基硅烷、四氢呋喃、HCl溶液的用量比例为100mL:100mL:(20-45)mg:(2-5)mL:
(1-3)mg。
3.根据权利要求1所述的一种基于改性纳米金材料的丙溴磷农药胶体金标记物的制备方法,其特征在于,所述八氨基POSS改性纳米金颗粒、混合缓冲液、丙溴磷单克隆抗体的用量比例为(1-2) mg:1mL:(20-30) μg。
4.根据权利要求1或2所述的一种基于改性纳米金材料的丙溴磷农药胶体金标记物的制备方法,其特征在于,所述混合缓冲液配方为:PBS缓冲液0.02M,BSA 0.5w/v%,Triton X-100 0.05w/v%,羧甲基葡聚糖1w/v%,pH=7.2。
5.一种根据权利要求1-3任一项所述的基于改性纳米金材料的丙溴磷农药胶体金标记物在制备用于丙溴磷农药残留检测的胶体金试纸中的应用。
6.根据权利要求5所述的应用,其特征在于:该胶体金试纸上含有金标记物包被的金标记垫。
7.根据权利要求6所述的应用,其特征在于:该胶体金试纸以PVC板为底板,且底板表面自左向右依次粘贴有样品垫、金标记垫、反应膜、吸水垫。
8.根据权利要求7所述的应用,其特征在于:所述反应膜自左向右间隔设有检测线T线和质控线C线,T线上包被有丙溴磷半抗原-OVA 偶联物,C线上包被有羊抗鼠 IgG。
9.根据权利要求6-8任一项所述的应用,其特征在于,该胶体金试纸的制备方法,包括以下步骤:
A1:制备样品垫:将玻璃纤维素膜浸于含0.5w/v% BSA的0.02M、pH=7.2 PBS缓冲液中,静置浸渍1h,37℃烘干后,得样品垫;
A2:制备金标记垫:将金标记溶液喷涂于玻璃纤维素膜上,喷膜量为5-7.5μL/cm,37℃烘干后,得金标记垫;
A3:制备反应膜:
A301:用0.02M、pH=7.2 PBS缓冲液将丙溴磷半抗原-OVA 偶联物稀释至0.4mg/mL,得丙溴磷半抗原-OVA 偶联物包被液;
A302:用0.02M、pH=7.2 PBS缓冲液将羊抗鼠 IgG稀释至0.2mg/mL,得羊抗鼠 IgG包被液;
A303:以硝酸纤维素膜作为反应膜承载体,在硝酸纤维素膜上间隔划分出检测线T线和质控线C线,并用0.4mg/mL丙溴磷半抗原-OVA偶联物包被液喷涂T线,0.2mg/mL羊抗鼠 IgG包被液喷涂C线,T线和C线喷膜量均为1μL/cm,37℃烘干后,得反应膜;
A4:组装胶体金试纸:将样品垫、金标记垫、反应膜、吸水垫自左向右依次粘贴在PVC底板上,且反应膜上的T线与金标记垫相邻,制得丙溴磷农药残留检测用胶体金试纸。
10.根据权利要求9所述的应用,其特征在于,所述的丙溴磷农药残留检测用胶体金试纸对果蔬谷物中的丙溴磷检测限为0.01-0.02mg/kg。
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