CN110632061B - 一种氨基三唑的可视化比色检测方法 - Google Patents
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Abstract
氨基三唑(aminotriazole,ATZ)是农业生产中最常使用的氨基甲酸酯类除草剂之一,其被广泛用于杂草清除、提高食物产量等。但是,由于氨基三唑的高水溶性及其对动物的强致癌作用,其残留物对人类健康和自然环境构成巨大威胁,因此,迫切需要针对氨基三唑发展高效检测方法。在此,我们针对氨基三唑构建了一种可视化比色检测技术:基于氨基三唑对过氧化氢酶(catalase,CAT)的抑制作用,利用H2O2作为媒介,当存在氨基三唑时,过氧化氢酶活性被抑制且不能催化分解H2O2,H2O2会氧化I‑,生成I2从而纵向刻蚀金纳米棒(AuNRs),金纳米棒溶液从深蓝色逐渐变为亮蓝色、紫色直至红色,同时,金纳米棒的紫外吸收峰蓝移,其变色程度和吸收峰蓝移波长与氨基三唑的浓度成正相关。该新方法对氨基三唑检测的良好性能及实际应用能力得到了验证,具有明显的创新性和应用价值。
Description
技术领域
本发明属于纳米技术和分析检测领域,涉及基于过氧化氢酶活性调控介导的金纳米棒刻蚀可视化比色传感器的构建及氨基三唑检测分析新方法。
背景技术
氨基三唑,即3-氨基-1,2,4-三氮唑,是一种广泛使用的非选择性杂环除草剂,被称为“杀草强”。常与其他农药混合使用,用于作物芽后除草,清除农业区灌溉渠道和路边的杂草以及控制柑橘生长期的杂草。氨基三唑在水中有很强的溶解性,常被发现于地表水及地下水系统中,这就使得氨基三唑易于在生态生物环境中积累,并通过随后的食物链中的生物放大作用,对人类健康以及整个生态系统都将构成巨大的潜在危害。美国环境保护局(EPA)已禁止将氨基三唑用于食用作物,因为它已经被证实对动物有较强的致癌作用。因此,尤其是在环境检测和食品检测方面,开发针对氨基三唑的高效检测方法十分迫切。
近几十年,可视化比色方法用于环境有害物质的检测受到越来越多的关注,因为此方法具有便捷性、可靠性及应用范围广泛的特性,大大满足了即时检测的需求。可视化比色方法是基于Beer-Lambert定律而衍生出的一种方法,通过简单的紫外—可见光光谱仪器测定相关生色团的吸光度就可以推知待测物质的准确浓度;而只需要通过肉眼对颜色的分别,也可以实现对目标物的半定量。在许多的文献报道中,金纳米材料是可视化比色传感中最常用的材料之一,因为其具有以下几个重要特征:1.超高的消光系数;2.独特的等离子体共振特性;3.敏感的颗粒间距离依赖性颜色变化;4.多样的尺寸/形状变化依赖性的颜色改变,色彩变化范围更广泛,更易被裸眼识别,为可视化比色检测提供了新的识别方法。
本发明受到农药检测的重要性及金材料尺寸依赖性可视化比色的简便性等特性的启发,构建了一种基于金纳米棒的可视化比色方法用于氨基三唑的检测。首次利用金纳米棒的尺寸改变的可视化比色方法来检测水中的氨基三唑。利用氨基三唑对过氧化氢酶的抑制作用,影响后续催化产物,从而对金纳米棒产生纵向刻蚀改变其尺寸,即可产生裸眼轻松分辨的不同颜色,达到对氨基三唑的便捷且低成本的可视化比色检测目的。
发明内容
本发明的目的在于克服传统氨基三唑检测方法耗时久、专业操作技术限制的缺点,结合金纳米棒的合成与性能优势,构建一种可视化比色传感器,将其发展为一种氨基三唑的可视化检测新方法。本方法用于氨基三唑的检测,具有简单方便、成本低廉、特异性好等优点,具有良好的社会价值和应用前景。
为实现上述目的,本发明的技术方案是:
先将氨基三唑与过氧化氢酶混匀孵育,随后向氨基三唑—酶混合液中加入H2O2,混匀孵育。在醋酸—醋酸钠缓冲溶液中加入金纳米棒,缓慢加入KI溶液,温和混匀,使I-充分分散在体系中;再向上述溶液中加入氨基三唑—酶—H2O2混合液混匀,H2O2与I-反应生成I2,对金纳米棒产生纵向刻蚀;采用紫外—可见光光谱仪收集金纳米棒的吸收峰光谱图,根据金纳米棒的颜色变化及吸收峰的相对位置改变量,实现氨基三唑的定性与定量检测。
所述醋酸-醋酸钠缓冲溶液浓度为0.2M,pH为3.6;
所述金纳米棒为本实验室合成;
所述氨基三唑从阿拉丁试剂(上海)有限公司购买;
所述过氧化氢酶从源叶生物科技(上海)有限公司购买,与氨基三唑溶液3:1体积比混合孵育;
所述H2O2溶液的浓度为0.5-1mM,所述KI溶液的浓度为0.1-1mM,所述过氧化氢酶溶液的浓度为20-30mg/mL。
本发明所述可视化比色传感方法的反应过程优选:
(1)取氨基三唑溶液10mL加入到容积为0.5mL的EP管中,再加入30mL的过氧化氢酶溶液,混合均匀;
(2)静置5小时;
(3)向(1)中所述混合液体中加入14mL H2O2溶液;
(4)摇晃混匀后,37.5℃放置孵育60分钟。
本发明所述的一种氨基三唑的可视化比色过程优选:
(1)在以上述方法反应之后,将混合液加入到金纳米棒的醋酸缓冲液中,
其中含有6.3mL KI溶液;
(2)50℃水浴静置反应15分钟;
(3)通过紫外-可见光光谱仪或裸眼进行吸收峰谱信号或色彩信号采集,实现定量或半定量分析。
与现有技术相比,本发明的优势在于:该方法具有专一性高、操作简便、条件温和、经济实用等优点;特别是操作简便,可以克服由于传统仪器的复杂前处理导致检测时间过长,同时,也可使用裸眼实现氨基三唑的半定量检测;实现了实际水样中氨基三唑的可视化检测。因此,该发明方法具有原始创新性、良好的社会价值和应用前景。
附图说明
图1为实施例1中可视化比色检测方法用于氨基三唑检测的可行性验证;
图2为实施例2中透射电子显微镜(TEM)表征氨基三唑调控金纳米棒刻蚀;
图3为实施例3中可视化比色检测方法用于氨基三唑检测的实验条件优化;
图4为实施例4中可视化比色检测方法针对不同浓度氨基三唑的检测能力研究;
图5为实施例5中可视化比色检测方法用于氨基三唑检测的选择性研究;
图6为实施例6中可视化比色检测方法应用于实际水样的分析;
图7为实施例1中可视化比色检测方法用于氨基三唑检测的原理图。
具体实施方式
以下结合附图对本发明的实施例作详细说明:本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和过程,旨在易于理解本发明的技术方案特征,对本发明的保护范围不构成任何限制。凡采用等同变换或者是等效替换而形成的技术方案,均落在本发明权利保护范围之内。
实施例1可视化比色检测方法用于氨基三唑检测的可行性验证
配制构建可视化比色传感器所需的溶液:将合成好的金纳米棒储备溶液置于室温待用,配制500mg/mL的过氧化氢酶溶液,配制25mM的H2O2溶液,配制100mM的KI溶液,配制0.2M的醋酸—醋酸钠缓冲液(pH=3.6),放置4℃冰箱内待用。
通过单一变量法,系统地考察了各反应成分对光谱信号的影响。
(1)取醋酸—醋酸钠缓冲液(0.2M,pH=3.6)250mL加入到容积为0.5mL的EP管中;
(2)加入250mL的金纳米棒储备溶液;
(3)加入6.3mL的KI溶液(100mM),温和混匀,50℃水浴放置15min;
(4)加入14mL的H2O2溶液(25mM),温和混匀,50℃水浴放置15min;
(5)加入6.3mL的KI溶液(100mM)及14mL的H2O2溶液(25mM),温和混匀,50℃水浴放置15min。
(6)加入30mL的过氧化氢酶溶液(500mg/mL),温和混匀,50℃水浴放置15min;
(7)加入30mL的过氧化氢酶溶液(500mg/mL)、6.3mL的KI溶液(100mM)及14mL的H2O2溶液(25mM),温和混匀,50℃水浴放置15min;
(8)最后加入30mL的过氧化氢酶溶液(500mg/mL)、10mL氨基三唑溶液(200mM)、6.3mL的KI溶液(100mM)及14mL的H2O2溶液(25mM),温和混匀,50℃水浴放置15min。
测试并分别记录上述各步骤在400nm-800nm范围内的的紫外-可见光光谱。
结果分析:从图1的紫外谱图中可以看出,同时加入H2O2和KI时,具有明显的峰谱改变及颜色变化,说明二者发生氧化还原反应生成I2对金纳米棒产生了刻蚀,实现了吸收峰的蓝移及颜色的转变;当在该比色体系中引入过氧化氢酶时,吸收峰不会发生改变,颜色仍保持蓝色,说明过氧化氢酶分解H2O2使得其与KI的反应不会发生,从而不会刻蚀金纳米棒;当继续加入氨基三唑时,吸收峰又发生明显蓝移同时伴随颜色向红色的转变,说明氨基三唑对过氧化氢酶产生抑制,过氧化氢酶无法分解H2O2,从而发生氧化还原反应生成I2,对金纳米棒有刻蚀效果。
实施例2透射电子显微镜(TEM)表征ATZ调控金纳米棒刻蚀
使用不同浓度的氨基三唑溶液,直观的分析考察不同浓度氨基三唑对金纳米棒的影响。
(1)取30mL的过氧化氢酶溶液(500mg/mL)和10mL氨基三唑溶液加入到容积为0.5mL的EP管中,混匀,室温孵育5h;
(2)向(1)中混合溶液中加入14mL的H2O2溶液(25mM),温和混匀,37.5℃水浴孵育60min;
(3)取醋酸—醋酸钠缓冲液(0.2M,pH=3.6)250mL加入到容积为0.5mL的EP管中;
(4)加入250mL的金纳米棒储备溶液及6.3mL的KI溶液(100mM),温和混匀;
(5)将(2)中混合液加入(4)温和混匀,50℃水浴放置15min,测试并记录400nm-800nm范围内的紫外-可见光光谱图。
结果分析:从图2中的透射电子显微镜照片更加直观地显示氨基三唑浓度对金纳米棒的刻蚀影响。随着氨基三唑的浓度增加,过氧化氢酶活性减弱,H2O2与I-发生反应,从而刻蚀金纳米棒使其长度逐渐变短,说明达到氨基三唑对过氧化氢酶的抑制效果。
实施例3可视化比色检测方法用于氨基三唑检测的实验条件优化
通过单一变量法,分别考察确定了各反应因素对紫外光谱信号的影响。
(1)H2O2浓度优化:取醋酸—醋酸钠缓冲液(0.2M,pH=3.6)250mL加入到容积为0.5mL的EP管中;随后加入250mL的金纳米棒储备溶液及6.3mL的KI溶液(100mM),温和混匀;最后加入不同体积的H2O2溶液(25mM),温和混匀,50℃放置15分钟;直接测量并记录各组溶液在400nm-800nm范围内的吸收峰位置值,得到不同浓度的H2O2对可视化比色传感器的形成及紫外光谱的影响,重复三次试验,取平均值。
(2)过氧化氢酶浓度优化:取不同浓度过氧化氢酶溶液(30mL)与14mL的H2O2溶液(25mM)混匀,37.5℃孵育1h;取醋酸—醋酸钠缓冲液(0.2M,pH=3.6)250mL加入到容积为0.5mL的EP管中;随后加入250mL的金纳米棒储备溶液及6.3mL的KI溶液(100mM),温和混匀;最后将上述不同浓度酶—H2O2混合液加入,温和混匀,50℃放置15分钟;直接测量并记录各组溶液在400nm-800nm范围内的吸收峰位置值,得到不同浓度的过氧化氢酶对可视化比色传感器的形成及紫外光谱的影响,重复三次试验,取平均值。
(3)过氧化氢酶催化分解H2O2时长优化:分别取30mL的过氧化氢酶溶液(500mg/mL)与14mL的H2O2溶液(25mM)混匀,在37.5℃水浴中孵育不同时间;取醋酸—醋酸钠缓冲液(0.2M,pH=3.6)250mL加入到容积为0.5mL的EP管中;随后加入250mL的金纳米棒储备溶液及6.3mL的KI溶液(100mM),温和混匀;最后将不同孵育时间的混合液加入,温和混匀,50℃放置15分钟;直接测量并记录各组溶液在400nm-800nm范围内的吸收峰位置值,得到不同酶促时间对可视化比色传感器的形成及紫外光谱的影响,重复三次试验,取平均值。
(4)氨基三唑抑制过氧化氢酶活性时长优化:分别取30mL的过氧化氢酶溶液(500mg/mL)与10mL的氨基三唑溶液(200mM)混匀,在室温放置不同时间;向上述溶液中加入14mL的H2O2溶液(25mM)混匀,在37.5℃水浴中孵育1h;取醋酸—醋酸钠缓冲液(0.2M,pH=3.6)250mL加入到容积为0.5mL的EP管中;随后加入250mL的AuNRs储备溶液及6.3mL的KI溶液(100mM),温和混匀;最后将上述氨基三唑—酶—H2O2混合液加入,温和混匀,50℃放置15分钟;直接测量并记录各组溶液在400nm-800nm范围内的吸收峰位置值,得到不同酶抑制时间对可视化比色传感器的形成及紫外光谱的影响,重复三次试验,取平均值。
结果分析:从图3可以看出,在多组不同浓度的H2O2中,0.5-1mM为刻蚀反应的平台值,刻蚀效果明显。体系过氧化氢酶浓度在20-30mg/ml时,达到刻蚀的理想效果。过氧化氢酶对H2O2的分解在60min时达到最大,用于后续研究。氨基三唑的抑制时间在300min时达到最佳的刻蚀效果。上述实验条件研究用于后续实验。
实施例4可视化比色检测方法针对不同浓度氨基三唑的检测能力研究
分别取30mL的过氧化氢酶溶液(500mg/mL)与10mL不同浓度的氨基三唑溶液混匀,在室温放置300min;向上述溶液中加入14mL的H2O2溶液(25mM)混匀,在37.5℃水浴中孵育1h;取醋酸—醋酸钠缓冲液(0.2M,pH=3.6)250mL加入到容积为0.5mL的EP管中;接着加入250mL的金纳米棒储备溶液及6.3mL的KI溶液(100mM),温和混匀;将上述氨基三唑-酶-H2O2混合液加入,温和混匀,50℃放置15分钟;直接测量并记录各组溶液在400nm-800nm范围内的吸收峰位置值,得到不同浓度氨基三唑对刻蚀的影响情况。
结果分析:从图4A的紫外—可见光光谱图中可以看出,随着氨基三唑浓度的增大,金纳米棒的吸收峰位置不断蓝移,这表明金纳米棒的吸收峰位置蓝移量跟氨基三唑浓度呈正相关。从图4B可以得出,该可视化比色传感器对氨基三唑检测在5-70mM浓度范围具有较好的线性关系。
实施例5可视化比色检测方法用于氨基三唑检测的选择性研究
分别取30mL的过氧化氢酶溶液(500mg/mL)与10mL不同的三唑类农药溶液(200mM)混匀,在室温放置300min;向上述溶液中加入14mL的H2O2溶液(25mM)混匀,在37.5℃水浴中孵育1h;取醋酸—醋酸钠缓冲液(0.2M,pH=3.6)250mL加入到容积为0.5mL的EP管中;接着加入250mL的金纳米棒储备溶液及6.3mL的KI溶液(100mM),温和混匀;将上述氨基三唑—酶—H2O2混合液加入,温和混匀,50℃放置15分钟;直接测量并记录各组溶液在400nm-800nm范围内的吸收峰位置值,得到不同三唑类农药对过氧化氢酶的抑制影响。
结果分析:从图5可以看出,从左到右分别代表三唑酮、己唑醇、腈菌唑、氟硅唑、恶醚唑、丙环唑、戊唑醇,只有氨基三唑的加入才会引起可视化比色传感器的紫外光谱信号的显著改变,其它对照物质不存在干扰。说明该可视化比色传感器对氨基三唑检测具有很好的选择性。
实施例6可视化比色检测方法应用于实际水样的分析
实际水样取自河水(湘江水),用不同浓度的氨基三唑进行人为污染,制备成被污染的试剂水样。按照实施例4所涉及步骤制备可视化比色传感器,加入不同浓度氨基三唑污染的水样,检测金纳米棒吸收峰波长值,重复三次,取平均值,计算回收率。
结果分析:从表1的回收率实验结果可以得出,针对各水样检测的回收率分布在90%-110%,并且具有较小的偏差,说明该可视化比色传感器可用于试剂水样中氨基三唑的快速检测分析。
Claims (1)
1.一种氨基三唑的可视化检测方法,其特征是,以过氧化氢酶作为调停物质,利用氨基三唑对过氧化氢酶的抑制作用,将过氧化氢作为媒介,当存在氨基三唑时,由于过氧化氢酶活性的抑制,其对H2O2不能催化分解,再利用过氧化氢与KI中的I-发生氧化还原反应产生I2,随后加入金纳米棒,I2对金纳米棒快速刻蚀,使得金纳米棒的吸收峰蓝移及溶液颜色逐渐由蓝色变向红色,实现氨基三唑的可视化分析检测;
所述氨基三唑的可视化检测方法,包括如下步骤:先将氨基三唑与过氧化氢酶按体积比1:3混匀孵育,室温放置5小时,随后向氨基三唑与过氧化氢酶混合液中加入H2O2,37.5℃混匀孵育60分钟得到混合溶液一;在pH为3.6浓度为0.2M的醋酸-醋酸钠缓冲溶液中加入金纳米棒溶液,再缓慢加入KI溶液,温和混匀,使I-充分分散在体系中,得到混合溶液二;将混合溶液一加入混合溶液二中混匀,50℃水浴放置15分钟,H2O2与I-反应生成I2,金纳米棒被纵向刻蚀;采用紫外—可见光光谱仪收集400nm-800nm范围内的金纳米棒吸收峰谱,根据吸收峰的相对位置改变量对氨基三唑实现定量检测,根据金纳米棒溶液的颜色变化实现对氨基三唑的可视化检测,随着氨基三唑浓度的增大,金纳米棒的吸收峰位置不断蓝移,金纳米棒的吸收峰位置蓝移量跟氨基三唑浓度呈正相关,可视化检测中氨基三唑在5-70mM浓度范围具有线性关系;
所述H2O2浓度为0.5-1 mM;I-浓度为0.1-1 mM;过氧化氢酶浓度为20-30μg/mL,过氧化氢酶对H2O2的分解在60min时达到最大,氨基三唑的抑制时间在300 min时达到最佳的刻蚀效果。
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