CN117723526B - 一种单原子纳米酶基荧光探针快速检测草甘膦和镉方法 - Google Patents
一种单原子纳米酶基荧光探针快速检测草甘膦和镉方法 Download PDFInfo
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Abstract
本发明公开了一种单原子纳米酶基荧光探针快速检测草甘膦和镉方法,该方法以叶绿素钠盐为配体,Fe3+为中心原子,合成与天然金属酶的活性中心具有相似结构Fe‑N‑C单原子纳米酶,Fe‑N‑C具有强过氧化物酶活性,通过强电子转移产生羟基自由基(·OH),由于生成的·OH能够氧化非荧光对苯二甲酸(TA)为强荧光的2‑羟基对苯二甲酸(PTA)。当Cd2+引入到荧光体系中,Fe‑N‑C的电子转移加强,使Fe‑N‑C过氧化酶活性得到加强,体系荧光增强,而当加入草甘膦时,由于其与Fe与Cd形成配合物,Fe‑N‑C模拟过氧化酶活性受到抑制,体系荧光强度降低,由此建立高灵敏、选择性强镉及草甘膦荧光检测新方法,检出限分别都为0.049µg/kg及0.037µg/kg,能够满足国家食品安全的相关要求。
Description
技术领域
本发明涉及化学分析检测技术领域,具体为一种单原子纳米酶基荧光探针快速检测草甘膦和镉方法。
背景技术
镉广泛存在于生活环境中,被认为是危害最大的重金属之一,被列为人类致癌物;即使是低水平的暴露也会对呼吸系统、心血管系统、免疫系统、生殖系统和胚胎发育产生毒性作用。世界卫生组织规定饮用水中Cd2+的最大污染水平为3 µg/L。目前,Cd2+的测定方法主要包括原子吸收法(AAS)、电感耦合等离子体质谱(ICP-MS)、电化学法及比色探针法。草甘膦(Glyphosate, Gly)是一种常用的广谱有机磷除草剂,具有除草活性高和毒性较低的特点,被广泛应用于茶业、 粮食、果蔬、林木等的种植生产。随着抗草甘膦转基因农作物的大量种植,草甘膦的应用范围不断扩大,现已成为世界上生产和使用量最大的除草剂。草甘膦具有潜在的致癌毒性,2015 年被国际癌症研究机构(IARC)列为 2A 类 致癌物。因此,草甘膦的过量残留对人类健康和生态环境造成严重威胁。我国生活饮用水卫生标准中规定饮用水中草甘膦的最高限量浓度为0.7 mg/L。目前,草甘膦的检测方法主要包括离子色谱法、高效液相色谱-质谱法、酶联免疫吸附法、电化学法和分光光度法等。这些方法具有较高的灵敏度,但是需要精密的仪器和专业的操作人员,样品制备也较复杂,不利于草甘膦及Cd2+的快速检测。因此,迫切需要开发一种低成本且快速的检测草甘膦及Cd2+的分析方法;
酶模拟物是一类非蛋白质结构的催化剂,具有与天然酶相似的催化特性。酶模拟物具有高性价比、高稳定性、易制备、结构和组成可控等优点,已成为生物传感的重要工具。过渡金属-氮掺杂碳(M-N-C)纳米结构具有足够的活性、优异的稳定性和充分暴露的活性位点,被认为是各种领域中最有前途的催化剂。特别是具有高度分散的Fe-Nx活性位点和丰富的活性C-N基团的Fe-N-C催化剂引起了广泛的研究兴趣。受配位设计策略的启发,我们将叶绿素上的氮作为铁原子的锚定点,以防止金属原子的迁移和耦合。在一步溶剂热法中,叶绿素配合物形成原子级分散的Fe-N-C位点锚定的无定形碳材料;
本发明以叶绿素钠盐为配体,Fe3+为中心原子,合成与天然金属酶的活性中心具有相似的电子和几何结构Fe-N-C单原子纳米酶,Fe-N-C具有强的过氧化物酶活性,通过强电子转移产生羟基自由基(·OH),由于生成的·OH能够氧化非荧光对苯二甲酸为强荧光的2-羟基对苯二甲酸。当Cd2+引入到荧光体系中,由于加强了Fe-N-C的电子转移,使得Fe-N-C模拟过氧化酶活性得到加强,体系荧光增强,而当加入草甘膦时,由于其与Fe与Cd形成配合物,Fe-N-C模拟过氧化酶活性受到抑制,体系荧光强度降低,由此建立高灵敏、选择性强镉及草甘膦荧光检测新方法,检出限分别都为0.049µg/kg及0.037µg/kg,能够满足国家食品安全的相关要求;本方法具有操作简单、灵敏度高、快速等特点。
发明内容
针对现有技术的不足,本发明提供了一种单原子纳米酶基荧光探针快速检测草甘膦和镉方法,利用本发明合成的Fe-N-C单原子纳米酶具有过氧化酶活性,产生的·OH能够氧化非荧光对苯二甲酸为强荧光的2-羟基对苯二甲酸,利用Cd2+对荧光体系活性的增强作用及草甘膦(Glyphosate, Gly)对纳米酶荧光体系的抑制作用,而建立了Cd和草甘膦的“开-关” 荧光快速检测方法。
本发明具有单原子纳米酶基荧光探针快速检测草甘膦和镉方法如下:
(1)称取0.12-0.15g 六水合三氯化铁与0.25-0.30g叶绿素钠盐混合,研磨均匀后,加入10-15mL甲醇分散,于80℃烘箱干燥老化2-3h,得到的墨绿色干燥粉末;称取 5-6g粉末于石英舟中,置于管式炉750℃ 在N2保护下煅烧2-2.5h,得到Fe-N-C单原子纳米酶,并将其分散于去离子水中,即得到Fe-N-C单原子纳米酶溶液;
(2)在Fe-N-C单原子纳米酶溶液中加入不同浓度的Cd2+标准溶液,作用5-10 min后,加入对苯二甲酸(TA)溶液、H2O2溶液,然后加入pH 4.0醋酸盐缓冲溶液定容,反应5-10min后,于405nm波长处测定荧光强度,建立荧光强度与Cd2+浓度的定量关系,绘制标准曲线,得到回归方程;
(3)在Fe-N-C单原子纳米酶溶液中加入Cd2+及不同浓度的草甘膦标准溶液,作用5-10 min后,加入对苯二甲酸(TA)溶液、H2O2溶液,然后加入pH 4.0醋酸盐缓冲溶液定容,反应5-10 min后,于405nm波长处测定荧光强度,建立荧光强度与草甘膦浓度的定量关系,绘制标准曲线,得到回归方程;
(4)消解检测样品中Cd2+及提取检测样品中草甘膦,获得样品测定液,按照步骤(2)及(3)操作测定荧光强度,代入回归方程,计算待测样品液中Cd2+及草甘膦浓度。
所述的Cd2+标准溶液的浓度为0.08~20 μg/L,草甘膦标准溶液浓度为0.16~12 μg/L;Fe-N-C单原子纳米酶溶液的浓度为0.1mg/mL,添加量为50-100μL;TA溶液浓度为50mmol/L,添加量为50-100μL,H2O2溶液浓度为50mmol/L,添加量为20-50μL。
本发明的优点和技术效果:
1、本发明以叶绿素钠盐为配体,Fe3+为中心原子,合成与天然金属酶的活性中心具有相似的电子和几何结构Fe-N-C单原子纳米酶,Fe-N-C具有强的过氧化物酶活性,通过强的电子转移产生羟基自由基(·OH),生成的·OH能够氧化非荧光对苯二甲酸为强荧光的2-羟基对苯二甲酸,由于Cd2+引入增强了Fe-N-C单原子纳米酶与底物的亲和力,使荧光体系强度增强,而加入草甘膦,降低了Fe-N-C单原子纳米酶与底物的亲和力,使荧光体系强度减弱,从而建立了了Cd和草甘膦的“开-关” 荧光快速检测新方法;
2、本发明建立的Cd2+和草甘膦荧光检测方法,具有高的检测灵敏度,检出限分别都为0.049µg/kg及0.037µg/kg,能够满足国家食品安全的相关要求,而共存的其他金属离子、其他农药及共存物不干扰测定,方法具有好的选择性。
附图说明
图1为本发明实施例1制备的Fe-N-C的TEM图;
图2为本发明实施例1中(Fe-N-C+TMB+H2O2)、(Fe-N-C+TMB+H2O2 + Cd2+)及(Fe-N-C+TMB+H2O2 + Cd2++Gly)的紫外光谱图;
图3 为实施例1中Fe-N-C氧化TMB的米氏动力学曲线;
图4 为实施例1中Fe-N-C+Cd2+氧化TMB的米氏动力学曲线;
图5为实施例1中Fe-N-C+Cd2++Gly氧化TMB的米氏动力学曲线;
图6为实施例1中Fe-N-C+TA+H2O2体系检测Cd2+线性荧光光谱图及回归方程;
图7为实施例1中Fe-N-C+TA+H2O2+Cd2+体系检测Gly线性荧光光谱图及回归方程;
图8为实施例1中为共存物质对Cd2+的影响结果;
图9为实施例1中为共存物质对Gly的影响结果。
具体实施方式
下面将结合具体的实施例对本发明的技术方案作进一步详细地描述说明,但本发明的保护范围并不仅限于此。
实施例1:白葡萄酒中Cd2+及油麦菜样品中Gly的测定
1、Fe-N-C单原子纳米酶制备:称取0.12g 六水合三氯化铁与0.25g叶绿素钠盐混合,研磨均匀后,加入10mL甲醇分散,于80℃烘箱干燥老化2h,得到的墨绿色干燥粉末;称取5g粉末于石英舟中,置于管式炉750℃ 在N2保护下煅烧2h,得到Fe-N-C单原子纳米酶,并将其分散于去离子水中,即得到Fe-N-C单原子纳米酶溶液。将制备的Fe-N-C单原子纳米酶进行透射电镜(TEM)表征,从中(图1)可以看出,Fe-N-C呈片状结构。
2、Fe-N-C单原子纳米酶过氧化物酶活性评价:研究选用四甲基联苯胺(3,3’,5,5’-Tetramethylbenzidine,TMB)作为底物验证过氧化物酶活性,取100μL浓度为50mmol/L的TMB,加入0.2 mg/mL Fe-N-C 100 μL及50mmol/L的H2O2 50μL,加入0.1mmol/L pH 4.0HAc-NaAc缓冲溶液至3mL,摇匀,静置15min,于654 nm波长处测定吸光度,同时取100μL0.2mg/mL的Fe-N-C和100μL 5μg/L Cd2+溶液,加入100μL浓度为50mmol/L的TMB 及50μL浓度为50mmol/L的H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,静置15min,于654 nm波长处测定吸光度,取100μL 0.2mg/mL的Fe-N-C和100μL 5μg/L Cd2+溶液,加入100μL 5μg/L 草甘膦(Gly),再加入100μL浓度为50mmol/L的TMB 及50μL浓度为50mmol/L的H2O2,加入0.1mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,静置15min,于654 nm波长处测定吸光度。结果如图2,Fe-N-C氧化TMB在酸性条件下表现强的过氧化物酶活性,当加入了Cd2+后,体系的吸光度得到明显增强,而加入Gly后,体系的吸光度下降。
实验还以TMB为底物,进行了米氏催化动力学参数测定(图3、图4、图5及表1),Fe-N-C对底物TMB的米氏常数K m 分别为0.158 mM,加入Cd2+后TMB的K m 为0.021mM,再加入Gly后TMB的K m 为0.174mM,反应速率常数分别为1.49×10-8 Ms-1、0.977×10-8 Ms-1和0.0944×10-8Ms-1,表明Cd2+加入增加了Fe-N-C单原子纳米酶与底物的亲和力及反应速度,而加入Gly后,降低了Fe-N-C纳米酶与底物的亲和力及反应速度。
表1米氏催化动力学参数
3、Cd2+工作曲线制作:在5mL具塞比色管中加入100μL 0.1mg/mL的Fe-N-C,浓度在0.08~20 μg/L Cd2+标准溶液,加入100μL浓度为50mmol/L的TA溶液、50μL浓度为50mmol/L的H2O2,加入0.1mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,静置15min,于300 nm激发波长、405nm发射波长处测定荧光强度,Cd2+浓度为横坐标,荧光强度为纵坐标,绘制标准曲线,得到回归方程,见图6;回归方程、相关系数、相对标准偏差、线性范围等见表2。
4、Gly工作曲线制作:在5mL具塞比色管中加入100μL 0.1mg/mL的Fe-N-C,5μg/LCd2+溶液100μL,加入100μL浓度为50mmol/L的TA溶液、50μL浓度为50mmol/L的H2O2,加入浓度在0.16~12 μg/L Gly标准溶液,加入0.1mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,静置15min,于300 nm激发波长、405nm发射波长处测定荧光强度,Gly浓度的对数为横坐标,荧光强度的负对数为纵坐标,绘制标准曲线,得到回归方程,见图7;回归方程、相关系数、相对标准偏差、线性范围等见表2。
表2线性方程、相关系数、相对标准偏差、线性范围
5、方法特异性考察:将Cd2+和其他共存离子混合,检测共存离子在上述检测体系中对Cd2+的影响,Cd2+浓度为25μg/L,以上干扰物质浓度为200μg/L,图8是共存离子(Na+、K+、Ca2+、Mg2+、Cu2+、Zn2+、Pb2+、Fe3+、Cr3+、Ag+、CO3 2-、NO3 -、SO4 2-、Cl-等)对Cd2+的影响结果,从图中可以看出,Fe-N-C检测体系有较好的选择特异性,仅有Cd2+有明显的增强氧化反应,其它物质几乎没有,方法测定Cd2+具有好的选择特异性;同时考察了测定Gly的选择性,将Gly和其他可能共存物质混合,检测金属离子对检测体系中的影响,Gly浓度为50 μg/L,其余干扰物质浓度为200 μg/L,图9是常见的有机磷农药(草铵膦、毒死蜱、马拉硫磷、杀螟硫磷、除草剂扑草净胺)等对Gly的影响结果,从图中可以看到,仅有Gly对纳米酶催化活性有明显的抑制作用,其它物质几乎没有作用,方法具有好的选择特异性。
6、白葡萄酒中Cd2+的测定
(1)样品处理:准确称取 5.0g 白葡萄酒样品烧杯中,在100℃电热板上蒸发至0.5mL 左右,加入1mL硝酸,放置100℃电热板上消解至冒白烟,消化液呈无色透明或略带黄色时,取出冷却,用1% 的硝酸溶液转移定容至10mL容量瓶,混匀备用。同时做试剂空白;
(2)白葡萄酒中Cd2+的测定:在5mL具塞比色管中加入100μL 0.1mg/mL的Fe-N-C,上述待测液1mL,加入100μL浓度为50mmol/L的TA、50μL浓度为50mmol/L的H2O2 及50μL浓度为5mmol/L的对苯二甲酸(TA),加入0.1mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,静置15min,于300nm激发波长、405nm发射波长处测定荧光强度,代入回归方程,Cd2+未检出;
(3)回收率与精密度实验:在白酒样品中分别添加3个不同浓度的Cd2+标准溶液;每个浓度平行测定3次,计算加标回收率,并计算出相对标准偏差RSD,结果见表3;测得Cd2+的加标回收率在96.2%~103.0%,RSD在3.12%~4.68%,本方法有好的的准确性和精密度。
表3 样品加标回收率及RSD(n = 3)
7、油麦菜样品中Gly的测定
(1)样品处理:取2 g样品加入10 mL 纯净水,涡旋3 min,之后,将这些样品进行超声处理10 min,并在5000 rpm下离心8 min,收集上清液,加入0.2 g PSA填料,涡旋3 min,除去有机酸、色素、脂质化合物和金属离子。此后,5000 rpm离心8 min,并在收集的上清液中加入0.2 g IRA - 400 - OH树脂去除无机阴离子。最后,收集上清液,使用0.22 µm PES微孔滤膜过滤后,为样品测定液;
(2)样品测定:在5mL具塞比色管中加入100μL 0.1mg/mL的Fe-N-C,5μg/L Cd2+溶液100μL,加入100μL浓度为50mmol/L的TA、50μL浓度为50mmol/L的H2O2 及50μL浓度为5mmol/L的对苯二甲酸(TA),加入样品测定液1mL,加入0.1mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,静置15min,于300nm激发波长、405nm发射波长处测定荧光强度,代入回归方程,Gly未检出;
(3)回收率与精密度实验:在样品中分别添加3个不同浓度的Gly标准溶液;每个浓度平行测定3次,计算加标回收率,并计算出相对标准偏差RSD,结果见表4;测得Gly的加标回收率在95.0%~104.2%,RSD在3.48%~4.76%,本方法有好的的准确性和精密度。
表4 样品加标回收率及RSD(n = 3)
实施例2:红茶样品中Cd2+及咖啡样品中Gly的测定
1、Fe-N-C单原子纳米酶制备:称取0.15g 六水合三氯化铁与0.30g叶绿素钠盐混合,研磨均匀后,加入15mL甲醇分散,于80℃烘箱干燥老化3h,得到的墨绿色干燥粉末;称取6g粉末于石英舟中,置于管式炉750℃ 在N2保护下煅烧2.5h,得到Fe-N-C单原子纳米酶,并将其分散于去离子水中,即得到Fe-N-C单原子纳米酶溶液。
2、Cd2+工作曲线制作:同实施例1。
3、Gly工作曲线制作:同实施例1。
4、红茶样品中Cd2+含量的测定
(1)样品前处理方法:称取约0.5 g,加入5.0 mL浓HNO3和1.0 mL 30 % H2O2,在180℃下微波消解20 min,加水稀释至50.0 mL备用;
(2)样品中Cd2+测定:同实施例1,小麦样品中Cd2+为5.8μg/kg。
5、咖啡样品中Gly的测定
(1)样品前处理:同实施例1;
(2)样品测定:同实施例1,咖啡样品中Gly的含量为3.5 μg/kg。
本发明建立的Cd2+及Gly测定法具有处理步骤少,所用时间短,处理成本低,操作简便,不需要大型仪器设备,在实际检测中具有较强优势。
Claims (2)
1.一种单原子纳米酶基荧光探针快速检测草甘膦和镉方法,其特征在于,包括以下步骤:
(1)称取0.12-0.15g 六水合三氯化铁与0.25-0.30g叶绿素钠盐混合,研磨均匀后,加入10-15mL甲醇分散,于80℃烘箱干燥老化2-3h,得到的墨绿色干燥粉末;称取 5-6g粉末于石英舟中,置于管式炉750℃ 在N2保护下煅烧2-2.5h,得到Fe-N-C单原子纳米酶,并将其分散于去离子水中,即得到Fe-N-C单原子纳米酶溶液;
(2)在Fe-N-C单原子纳米酶溶液中加入不同浓度的Cd2+标准溶液,作用5-10 min后,加入对苯二甲酸溶液、H2O2溶液,然后加入pH 4.0醋酸盐缓冲溶液定容,反应5-10 min后,于405nm波长处测定荧光强度,建立荧光强度与Cd2+浓度的定量关系,绘制标准曲线,得到回归方程;
(3)在Fe-N-C单原子纳米酶溶液中加入Cd2+及不同浓度的草甘膦标准溶液,作用5-10min后,加入对苯二甲酸溶液、H2O2溶液,然后加入pH 4.0醋酸盐缓冲溶液定容,反应5-10min后,于405nm波长处测定荧光强度,建立荧光强度与草甘膦浓度的定量关系,绘制标准曲线,得到回归方程;
(4)消解检测样品中Cd2+及提取检测样品中草甘膦,获得样品测定液,按照步骤(2)及(3)操作测定荧光强度,代入回归方程,计算待测样品液中Cd2+及草甘膦浓度。
2.根据权利要求1所述的方法,其特征在于:Cd2+标准溶液的浓度为0.08~20 μg/L,草甘膦标准溶液浓度为0.16~12 μg/L;Fe-N-C单原子纳米酶溶液的浓度为0.1mg/mL,添加量为50-100μL;对苯二甲酸溶液浓度为50mmol/L,添加量为50-100μL;H2O2溶液浓度为50mmol/L,添加量为20-50μL。
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