CN113970580A - 一种双放大电致化学发光传感器及测定毒死蜱的方法 - Google Patents
一种双放大电致化学发光传感器及测定毒死蜱的方法 Download PDFInfo
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Abstract
本发明设计农残分析测定技术领域,特别是涉及一种基于中空Fe/Co‑MOF及Ag NPs的双放大适配体传感器以及用其测定毒死蜱的方法,包括双放大传感器制备步骤和使用该传感器测定毒死蜱的操作方法等;这种传感器利用了中空Fe/Co‑MOF对鲁米诺‑H2O2体系的催化作用,及纳米银对鲁米诺‑H2O2增强发光作用,实现联合增强ECL发光,用于毒死蜱的检测,灵敏度高,特异性好。
Description
技术领域
本发明涉及农残测定技术领域,特别是涉及一种双重信号放大电致化学发光传感器以及用于毒死蜱的方法,包括双重信号放大传感器的制备步骤和使用该传感器测定毒死蜱的操作方法等;这种双重信号放大的传感器,利用了中空Fe/Co-MOF对鲁米诺-H2O2体系的催化作用,及纳米银对鲁米诺-H2O2增强发光作用,实现联合增强ECL发光,用于毒死蜱的检测,灵敏度高,特异性好。
背景技术
随着社会生产以及健康的需求,农药残留检测受到广泛关注,传统农残检测技术成本高,时间长,催生出快速检测农残的技术至关重要。毒死蜱杂环类有机磷农药,广泛应用于小麦、大豆等作物防害,然而在防治病虫害的同时也易对生态环境造成污染,因此实现对毒死蜱农残的准确、有效检测势在必行。酶抑制法作为农残快速检测的最为成熟技术之一,其操作简单,成本较低在市场广受欢迎,但所用的酶和配套试剂有一定的特异性,对有机磷农药没有适用性且准确性、重现性有待提高;荧光光谱法是通过分析光谱间的差异来确定农药分子种类和浓度的无损检测技术,选择性和灵敏度高,但是极易受外界条件的干扰;气相色谱法是发展较早的农残检测方法,有较高的分离效率,检测灵敏度高,但样品的前处理过程较繁琐,不利于快速检测;酶联免疫吸附法具有准确性、特异性、稳定性和适用范围广、检测速度快以及费用低的特点,但是易发生交叉反应影响检测灵敏度。生物传感器相对于仪器检测方法来说,农残检测过程相对简单、不需要昂贵仪器,更适用于现场检测。目前使用的生物传感器有酶传感器、免疫传感器、适配体传感器及微生物传感器,电化学适配体传感器检测灵敏,特异性强,在农药快速检测方面具有优越性,为快速检测毒死蜱提供了新的思路和方法。适配体生物传感器是以核酸适配体作为生物识别元件,通过独特的三维结构和受体结合位点实现对多重目标物的特异性结合,从而达到精确识别目标物的目的。在电化学适配体传感器中,灵敏度是衡量传感器分析性能的标准之一。电化学传感器在构建和检测过程中,为了传感器的灵敏度,研究人员设计了多种分析策略,如位阻效应、能量转移、酶抑制反应以及各种信号放大策略,能通过多种途径放大ECL信号,但这些信号放大策略多是采用单一信号放大方式。为解决这一问题,研究者们开始采用双放大信号的方法来放大电化学信号,极大的提高了传感器测试的灵敏度和准确性。此发明利用中空Fe/Co-MOF和纳米银对鲁米诺-H2O2发光体系的联合增强作用,构建了ECL传感器用于毒死蜱的检测。
发明内容
本发明的目的就是针对上述农残传感分析中的缺点,构建一种能够用于快速、准确、高灵敏和选择性检测农药残留毒死蜱的双放大电化学适配体传感器。本发明要解决的技术问题是利用两种材料的协同作用实现在测试过程中提高ECL信号,进而提高灵敏度。两种功能材料的复合与单一信号放大方式相比,实现了ECL双重信号放大,提高发光效率,为农残毒死蜱的检测提供了一种可行的方法。
本发明的技术方案为:采用硬模板法合成中空Fe/Co-MOF,制备了中空Fe/Co-MOF-Lu-Ag的复合材料,并负载毒死蜱适配体,适配体与电极表面的毒死蜱互补链(cDNA)杂交形成双螺旋结构,构建双放大的电致化学发光传感器;过氧化氢作为鲁米诺的共反应剂,提高鲁米诺发光强度;中空Fe/Co-MOF中Co3+↔Co2+、Fe3+↔Fe2+的循环可逆反应,催化H2O2还原成更多的O2 •-,进而催化鲁米诺激发态产生,极大的提高鲁米诺发光强度;纳米银负载于中空Fe/Co-MOF后,Ag+↔Ag进一步催化更多的H2O2还原成更多的O2 •-,进一步增强发光强度,提高发光效率,实现联合增强电致化学发光。具体方案为:
1.一种双放大电化学适配体传感器测定毒死蜱的方法,合成具有大量催化位点的中空Fe/Co-MOF,负载鲁米诺发光材料及纳米银;通过中空Fe/Co-MOF中Fe3+及Co3+的循环可逆反应催化H2O2产生更多O2 •-;纳米银的氧化还原反应产生更多的O2 •-;鲁米诺与H2O2的体系,鲁米诺被氧化成鲁米诺阴离子LH-,LH-能够生成鲁米诺阴离子自由基L•-,L•-可与O2•-反应生成鲁米诺激发态AP*,AP*返回到基态AP产生发光;Lu-Ag-Fe/Co-MOF复合材料协同作用,增强发光;
2. 所述的Fe/Co-MOF复合材料,其制备方法如下:
(1)PS球的合成:30 mL超纯水置于三口烧瓶中,N2除氧后,依次加入1.5 g聚乙烯吡咯烷酮(PVP)及0.015 g过硫酸钾,完全溶解后加入3 mL苯乙烯持续70 ℃加热24 h。用水和乙醇洗涤并分离出产物,将产物分散在5 mL乙醇中得到PS球分散液,在分散液中加入0.5g PVP搅拌12 h,反应结束后将产物用乙醇、甲醇充分洗涤,后分散在10 mL甲醇溶液中,得到PVP修饰的PS球甲醇溶液;
(2)FeOOH纳米棒的合成:0.2 mL聚乙烯亚胺加入到100 mL 20 mM FeCl3•6H2O溶液中,80 ℃下加热搅拌2 h,用超纯水离心洗涤三次,得到FeOOH纳米棒。将FeOOH纳米棒分散到10 mL的PVP乙醇溶液中,室温下搅拌12 h,充分洗涤收集产物,将产物分散于15 mL的甲醇中,获得PVP修饰的FeOOH纳米棒甲醇溶液;
(3)中空Fe/Co-MOF的合成:取0.5 mL FeOOH分散液、2 mL PS球甲醇溶液和15 mL20 mM的Co(NO3)2•6H2O甲醇溶液加入于烧瓶中,超声混合,随后迅速加入25 mL 160 mM 2-甲基咪唑的甲醇溶液,搅拌5 min使其充分混合均匀,将产物在室温下静置4 h,后将产物用乙醇和甲醇各洗涤3次,随后在37 ℃下真空干燥24 h。最后将产物在N2保护下800 ℃烧结2h,得到中空Fe/Co-MOF;
3. 所述双放大电化学适配体传感器,其构建方法如下:
(1)取3 mL 5 mM AgNO3和0.5 mL 0.01M鲁米诺溶液,加入到装有9.0 mL无水乙醇和5.0 mL的超纯水的烧瓶中,然后快速加入入0.1 mL 0.1 M NaOH,搅拌4 h。当溶液颜色变成深黄色时,将混合物用乙醇充分洗涤,将产物分散到5 mL乙醇中,获得Lu-Ag乙醇溶液;
(2)取1 mL 0.125 g/L Fe/Co-MOF乙醇溶液和500 μL Lu-Ag乙醇溶液加入到烧瓶中。在震荡器上震荡30 min,随后在4 ℃下孵育12 h。获得中空Fe/Co-MOF-Lu-Ag复合材料。取80 μL 1 μM毒死蜱适配体加入到800 μL 0.125 g/L Fe/Co-MOF-Lu-Ag乙醇溶液中。在震荡器上震荡30 min,随后在4 ℃下孵育12 h,制得Apt-Ag-Lu-Fe/Co-MOF;
(3)将处理好的玻碳电极GCE在1%氯金酸溶液中-0.2 V下恒电位镀金,制得AuNPs/GCE修饰电极;
(4)在该修饰电极上滴加8 μL 1μM毒死蜱互补链cDNA,4 ℃下孵育12h,得cDNA/AuNPs/GCE,然后以MCH封闭非特异性位点;
(5)在电极表面上滴加8 μL Apt-Ag-Lu-Fe/Co-MOF,37 ℃下孵育1 h,再以pH 7.8的PBS缓冲液冲洗,得Apt-Ag-Lu-Fe/Co-MOF/MCH/cDNA/Au/GCE;
4. 所述的双放大适配体传感器用于检测毒死蜱,方法如下:
(1)将不同浓度的毒死蜱农药滴加在构建的传感器表面,37 ℃孵育1 h,以pH 7.8的PBS缓冲液冲洗;
(2)以适配体传感器为工作电极,Ag/AgCl电极为参比电极,铂电极为辅助电极;在含有0.01 M H2O2的0.1 M pH 7.8 PBS的缓冲溶液中,在0.0~0.7 V电位区间进行循环,扫描光电倍增管600 V,记录发光强度。
本发明的有益效果为:
1.本发明传感信号灵敏且传导速度快。中空多孔Fe/Co-MOF具有大比表面积、低密度、高负载能力以及质荷传递距离短等优势,能够起到提高传感器导电性,催化、放大信号的作用;
2.双放大电致化学发光传感器提高了毒死蜱检测选择性;两种功能材料的复合与单一信号放大方式相比,实现了ECL双重信号放大;
3.本发明传感器灵敏度高,选择性好,可以实现对农残毒死蜱的简单、快速、高灵敏检测;线性范围为0.1 pM~0.1 μM,检测限为0.042 pM;
4.本发明样品测定程序简单,操作简便快捷。
附图说明:
图1所示为不同浓度毒死蜱的时间-ECL光强曲线(A)和线性关系(B)
其中, 1--10-7, 2--10-8, 3--10-9, 4--10-10, 5--10-11, 6--10-12, 7--10-13,8--0 M
具体实施方式
为了更好地理解本发明,下面用具体实例来详细说明本发明的技术方案,但是本发明并不局限于此。
实施例1中空Fe/Co-MOF的制备:
(1)PS球的合成:30 mL超纯水置于三口烧瓶中,N2除氧后,依次加入1.5 g聚乙烯吡咯烷酮(PVP)及0.015 g过硫酸钾,完全溶解后加入3 mL苯乙烯持续70 ℃加热24 h。用水和乙醇洗涤并分离出产物,将产物分散在5 mL乙醇中得到PS球分散液,在分散液中加入0.5g PVP搅拌12 h,反应结束后将产物用乙醇、甲醇充分洗涤,后分散在10 mL甲醇溶液中,得到PVP修饰的PS球甲醇溶液;
(2)FeOOH纳米棒的合成:0.2 mL聚乙烯亚胺加入到100 mL 20 mM FeCl3•6H2O溶液中,80 ℃下加热搅拌2 h,用超纯水离心洗涤三次,得到FeOOH纳米棒。将FeOOH纳米棒分散到10 mL的PVP乙醇溶液中,室温下搅拌12 h,充分洗涤收集产物,将产物分散于15 mL的甲醇中,获得PVP修饰的FeOOH的纳米棒甲醇溶液;
(3)中空Fe/Co-MOF的合成:取0.5 mL FeOOH分散液、2 mL PS球甲醇溶液和15 mL20 mM的Co(NO3)2•6H2O甲醇溶液加入于烧瓶中,超声混合,随后迅速加入25 mL 160 mM 2-甲基咪唑的甲醇溶液,搅拌5 min使其充分混合均匀,将产物在室温下静置4 h,后将产物用乙醇和甲醇各洗涤3次,随后在37 ℃下真空干燥24 h。最后将产物在N2保护下800 ℃烧结2h,得到中空Fe/Co-MOF。
实施例2双放大适配体传感器制备:
(1)取3 mL 5 mM AgNO3和0.5 mL 0.01 M鲁米诺溶液,加入到装有9.0 mL无水乙醇和5.0 mL的超纯水的烧瓶中,然后快速加入入0.1 mL 0.1 M NaOH,搅拌4 h。当溶液颜色变成深黄色时,将混合物用乙醇充分洗涤,将产物分散到5 mL乙醇中,获得Lu-Ag乙醇溶液;
(2)取1 mL 0.125 g/L Fe/Co-MOF乙醇溶液和500 μL Lu-Ag乙醇溶液加入到烧瓶中。在震荡器上震荡30 min,随后在4 ℃下孵育12 h。获得中空Fe/Co-MOF-Lu-Ag复合材料。取80 μL 1 μM毒死蜱适配体加入到800 μL 0.125 g/L Fe/Co-MOF-Lu-Ag乙醇溶液中。在震荡器上震荡30 min,随后在4 ℃下孵育12 h,制得Apt-Ag-Lu-Fe/Co-MOF;
(3)将处理好的玻碳电极GCE在1%氯金酸溶液中-0.2 V下恒电位镀金,制得AuNPs/GCE修饰电极;
(4)在该修饰电极上滴加8 μL 1 μM毒死蜱互补链cDNA,4 ℃下孵育12h,得cDNA/Au NPs/GCE,然后以MCH封闭非特异性位点;
(5)在电极表面上滴加8 μL Apt-Ag-Lu-Fe/Co-MOF,37 ℃下孵育1 h,再以pH 7.8的PBS缓冲液冲洗,得Apt-Ag-Lu-Fe/Co-MOF/MCH/cDNA/Au/GCE。
实施例3 双放大适配体传感器用于检测毒死蜱的方法:
(1)将不同浓度的毒死蜱农药滴加在构建的传感器表面,37 ℃孵育1 h,以pH 7.8的PBS缓冲液冲洗;
(2)以适配体传感器为工作电极,Ag/AgCl电极为参比电极,铂电极为辅助电极;在含有0.01 M H2O2的0.1 M pH 7.8 PBS的缓冲溶液中,在0.0~0.7 V电位区间进行循环,扫描光电倍增管600 V,记录发光强度;
(3)检测一系列不同浓度的毒死蜱标准溶液的光强,绘制工作曲线;同时测定传感器线性范围和检测限。电致化学发光强度随着毒死蜱浓度增加而减弱,线性方程为DI=-359.57lgc+4857.5(R2=0.999),线性范围为0.1 pM~0.1 μM,检测限为0.042 pM。
实施例4 适配体传感器使用条件的优化
本发明对底液pH、H2O2浓度、中空Fe/Co-MOF与Lu-Ag复合物混合比例进行了条件优化。结果表明,当检测底液的pH小于7.8时,ECL强度随着pH的增加而增大。pH为7.8时,ECL强度达到最大。而当pH大于7.8时,ECL强度减小。当中空Fe/Co-MOF与Lu-Ag混合比例为1:1时,光强强度达到最大值。检测底液中H2O2浓度对ECL强度影响较大,随着H2O2浓度的增大,ECL强度逐渐增大。当H2O2浓度大于0.10 M时,ECL强度趋于稳定。Fe/Co-MOF与Lu-Ag复合物混合比例为1:1,溶液中H2O2浓度为0.10 M、pH为7.8为最优条件。
Claims (5)
1.一种基于中空Fe/Co-MOF复合纳米材料的双放大电化学适配体传感器,其特征在于,该传感器有着中空Fe/Co-MOF及纳米银联合放大信号的传感策略;以中空Fe/Co-MOF纳米材料用于负载鲁米诺及纳米银,通过中空Fe/Co-MOF和纳米银复合材料实现对鲁米诺-过氧化氢体系的催化和进一步增大信号作用,联合增强ECL发光,用于毒死蜱的检测。
2.权利要求1所述的中空Fe/Co-MOF复合纳米材料,其特征在于,中空Fe/Co-MOF与纳米银和鲁米诺复合形成中空Fe/Co-MOF-Lu-Ag复合材料,所述复合材料为中空多面体结构,平均尺寸为400 nm。
3.权利要求1所述传感器,其特征在于,其制备方法如下:
(1)取3 mL 5 mM AgNO3和0.5 mL 0.01M鲁米诺Lu溶液,加入到装有9.0 mL无水乙醇和5.0 mL的超纯水的烧瓶中,然后快速加入0.1 mL 0.1 M NaOH,搅拌4 h,当溶液颜色变成深黄色时,将混合物用乙醇充分洗涤,将产物分散到5 mL乙醇中,获得Lu-Ag乙醇溶液;
(2)取1 mL 0.125 g/L Fe/Co-MOF乙醇溶液和500 μL Lu-Ag乙醇溶液加入到烧瓶中,
在震荡器上震荡30 min,随后在4 ℃下孵育12 h,获得中空Fe/Co-MOF-Lu-Ag复合材料,
取80 μL 1 μM毒死蜱适配体加入到800 μL 0.125 g/L Fe/Co-MOF-Lu-Ag乙醇溶液中,在震荡器上震荡30 min,随后在4 ℃下孵育12 h,制得Apt-Ag-Lu-Fe/Co-MOF;
(3)将处理好的玻碳电极GCE在1%氯金酸溶液中-0.2 V下恒电位镀金,制得Au/GCE修饰电极;
(4)在该修饰电极上滴加8 μL 1μM毒死蜱互补链cDNA,4 ℃下孵育12h,得cDNA/Au/GCE,然后以MCH封闭非特异性位点;
(5)在电极表面上滴加8 μL Apt-Ag-Lu-Fe/Co-MOF,37 ℃下孵育1 h,再以pH 7.8的PBS缓冲液冲洗,得Apt-Ag-Lu-Fe/Co-MOF/MCH/cDNA/Au/GCE。
4.权利要求1所述的双放大适配体传感器用于检测毒死蜱。
5.根据权利要求1所述的双放大适配体传感器检测毒死蜱,其特征在于,方法如下:
(1)将不同浓度的毒死蜱农药滴加在构建的传感器表面,37 ℃孵育1 h,以pH 7.8的PBS缓冲液冲洗;
(2)以适配体传感器为工作电极,Ag/AgCl电极为参比电极,铂电极为辅助电极;在含有0.01 M H2O2的0.1 M pH 7.8 PBS的缓冲溶液中,在0.0~0.7 V电位区间进行循环,扫描光电倍增管600 V,记录发光强度。
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