CN112098381A - 一种铜掺杂碳点模拟酶结合荧光探针检测铬的方法 - Google Patents
一种铜掺杂碳点模拟酶结合荧光探针检测铬的方法 Download PDFInfo
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Abstract
本发明公开了一种铜掺杂碳点模拟酶结合荧光探针检测铬的方法。本发明采用乙二酸四乙酸铜钠(Na2[Cu(EDTA)])为前驱,热解法合成铜掺杂碳点(Cu‑CDs),基于Cu‑CDs的模拟酶催化活性,以邻苯二胺为底物,在Cu‑CDs模拟酶作用下,双氧水催化氧化邻苯二胺(OPD)产生具有荧光2,3‑二氨基酚嗪(DAP),铬(III)的存在,抑制该催化反应的进行,导致DAP荧光下降,铬(VI)被2'‑联氨‑双‑3‑乙基苯并噻唑啉‑6‑磺酸(ABTS)还原为铬(III),从而建立铬检测新方法,检出限为20μg/kg。将本方法应用于环境及食品中铬的检测分析,结果与标准方法相符。本方法具有灵敏度高、特异性的特点,同时具有操作简单、快速、不需要大型仪器等优点。
Description
技术领域
本发明涉及化学分析检测技术领域,具体为一种铜掺杂碳点模拟酶结合荧光探针检测铬的方法。
背景技术
作为人体内重要的微量营养元素之一,铬(Cr)是重要的血糖调节剂,也是蛋白质分解酶的成份之一,对人体的糖脂代谢、蛋白质合成有不可忽视的作用。铬主要以Cr(Ⅲ)和Cr(VI)两种形态存在,Cr(Ⅲ)可作为葡萄糖耐量因子的组成成分、体内某些代谢酶类的活化剂以及核酸的稳定剂,促进胰岛素发挥作用,维持正常糖代谢,促进胆固醇和脂肪酸的生成并促进造血机能。铬的毒性与其存在的价态有关,六价铬比三价铬毒性高100倍,并易被人体吸收且在体内蓄积,三价铬和六价铬可以相互转化。天然水不含铬;海水中铬的平均浓度为0.05μg/L;饮用水中更低。铬的污染源有含铬矿石的加工、金属表面处理、皮革鞣制、印染等排放的污水。
碳纳米材料模拟酶相比于其他纳米模拟酶,合成成本低,生物相容性好,在实际应用中存在较大的价值,因此研究出具有优良纳米结构的碳纳米模拟酶是十分的有意义和有应用前景。碳纳米材料模拟酶中的碳纳米粒子由于尺寸小,环保,又有独特的发光性能,更是成为广大科研工作者的研究对象。碳点(CDs)是一种生物相容性好、毒性低或没有毒性的纳米材料,在化学分析、生物成像和光催化等研究领域得到了广泛应用。钟青梅等研究了一种以CDs为模拟酶的葡萄糖测定新方法,展现CDs作为模拟酶的应用前景。但以金属掺杂碳点为纳米模拟酶催化氧化邻苯二胺,产生荧光2,3-二氨基酚嗪,重金属抑制该酶催化反应的研究未见报道。
本发明采用乙二酸四乙酸铜钠(Na 2[Cu(EDTA)])为前驱,热解法合成铜掺杂碳点(Cu-CDs),基于Cu-CDs的模拟酶催化活性,以邻苯二胺为底物,在Cu-CDs模拟酶作用下,双氧水催化氧化邻苯二胺(OPD)产生具有高荧光产率的荧光2,3-二氨基酚嗪(DAP),其酶活高于辣根过氧化酶50倍,铬(III)的存在,抑制该催化反应的进行,导致DAP荧光下降,铬(VI)被2'-联氨-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)还原为铬(III),从而建立铬检测新方法,检出限为20μg/kg。
发明内容
本发明的目的在于提供一种铜掺杂碳点模拟酶结合荧光探针检测铬的方法,利用铜掺杂碳点对邻苯二胺的模拟酶催化作用,铬(III)抑制该催化反应作用,建立铬(III)荧光检测新方法。
一种铜掺杂碳点模拟酶结合荧光探针检测铬的方法,其特征在于,包括:将纳米模拟酶铜掺杂碳点加入邻苯二胺与双氧水溶液中,加入不同浓度的Cr(III),用Tris-HCl缓冲液控制pH 7.0,室温反应5-10min,生成2,3-二氨基酚嗪,测定荧光强度,以Cr(III)浓度为横坐标,荧光强度为纵坐标,绘制标准曲线。
所述的铜掺杂碳点优选由以下方法制备得到:取0.4-1份乙二酸四乙酸铜钠放入管式炉的石英管中,以5℃/min加热速率,氮气保护,300℃锻烧2-4h,将所得产品磨碎,然后溶于100-150份去离子水中,室温下40kHz超声处理15-20min,10000r/min下离心20-25min,上清液0.22μm滤膜过滤,后用截留分子量为3000-3500Da的透析袋进行透析处理24-48h,浓缩液60℃真空干燥24-48h,得到铜掺杂碳点。
所述铜掺杂碳点模拟酶、双氧水与邻苯二胺重量比优选为1:10-50:1000-1500。
所述的2,3-二氨基酚嗪最大激发波长优选为320nm,最大发射波长优选为572nm。
所述铬测定包括样品中铬(VI)用2'-联氨-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)在酸性条件下还原为铬(III),其中的酸优选为浓盐酸,ABTS的浓度优选为0.200mM,浓盐酸与ABTS的体积比优选为5:1。
所述Cr(III)浓度优选为52-1300μg/kg。
本发明的优点在于:
1.本发明利用铜掺杂碳点的模拟酶对邻苯二胺的催化作用及铬(III)对该催化反应抑制作用,建立铬检测新方法。该方法基于一步热解法制备的铜掺杂碳点作为模拟酶,对邻苯二胺为底物的氧化反应具有高效催化作用,产生高荧光强度的2,3-二氨基酚嗪,铬(III)抑制该催化反应作用,建立铬荧光检测新方法。
2、用本发明制备的铜掺杂碳点的模拟酶对邻苯二胺的催化作用,其酶活高于常规的辣根过氧化酶50倍,铬(III)对模拟酶强抑制作用,表现出检测铬(III)的高灵敏度,检测限可以达到20ng/mL,利用2'-联氨-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)对铬(VI)的强还原作用,进行总铬的测定。
3、方法用于食品、环境中铬的检测分析,结果与标准方法相符;在相同灵敏度要求下,比相应标准方法操作简单、快速、不需要大型仪器等优点。
附图说明
图1为实施例1中Cr(III)对Cu-CDs模拟酶催化OPD为DAP抑制作用呈线性荧光猝灭,可用于间接Cr(III)荧光探针检测。
图2、图3为共存的其他阳离子及阴离子对DAP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。
图1为实施例1中Cr(III)对Cu-CDs模拟酶催化OPD为DAP抑制作用呈线性荧光猝灭,可用于间接Cr(III)荧光探针检测。图2、图3为共存的其他阳离子及阴离子对DAP的影响结果,从图中可以看出,Cr(III)对Cu-CDs模拟酶催化抑制作用有较好的选择特异性。
实施例1:烟叶中铬含量测定操作步骤如下:
1、铜掺杂碳点由以下方法制备得到:取0.4-1份乙二酸四乙酸铜钠放入管式炉的石英管中,以5℃/min加热速率,氮气保护,300℃锻烧2-4h,将所得产品磨碎,然后溶于100-150份去离子水中,室温下40kHz超声处理15-20min,10000r/min下离心20-25min,上清液0.22μm滤膜过滤,后用截留分子量为3000-3500Da的透析袋进行透析处理24-48h,浓缩液60℃真空干燥24-48h,得到铜掺杂碳点。
2、Cr(III)标准曲线制作:将纳米模拟酶铜掺杂碳点加入邻苯二胺与双氧水溶液中,加入从0到250μM不同浓度的Cr(III),用Tris-HCl缓冲液控制pH 7.0,室温反应5-10min,生成2,3-二氨基酚嗪,测定荧光强度,以Cr(III)浓度为横坐标,荧光猝灭率(I0/I)为纵坐标,绘制标准曲线,计算回归方程,相关系数、相对标准偏差、线性范围等见表1。
3、方法特异性考察:图3是Cr(III)浓度为0.5mg/kg,ABTS、Cr2O7 2-、K+、Na+、Mg2+、Zn2 +、Mn2+、Cd2+、Cu2+、Co2+、Pd2+、Ni2+、Pb2+、Fe2+、Fe3+;Cl-、NO3 -、Br-、SO4 2-、S2O3 2-、ClO4 -、CO3 2-、HCO3 -、HPO4 2-、H2PO4 -、PO4 3-,以上干扰离子浓度为5mg/kg,分别加入铜掺杂碳点,仅有Cr(III)有明显的猝灭作用,其他物质几乎没有猝灭作用,方法具有好的选择特异性。
4、烟叶中铬含量测定
(1)烟叶样品处理:将烟叶试样自然风干,粉碎后充分混匀,准确称取样品0.5000g,置于50mL聚四氟乙烯微波消化瓶中,加入5mL浓硝酸和5mL质量分数为10%的过氧化氢溶液,置于微波消化炉中,在1kW功率下消解10min,消解完后过滤,并用体积分数为5%的硝酸定容至25mL。
(2)总铬测定:将上述消解液取5mL,加入50mM ABTS(0.6878g of ABTS铵盐溶解到25mL超纯水中)0.5mL,室温反应5-10min,生成2,3-二氨基酚嗪,用pH 7.0Tris-HCl缓冲液稀释至10mL,测定荧光强度,代入步骤2回归方程,得到总铬含量为0.30mg/kg。
表1线性方程、相关系数、相对标准偏差、线性范围
实施例2:环境水样中铬含量测定:
1、铜掺杂碳点制备:同实施例1;
2、Cr(III)标准曲线制作:同实施例1;
3、环境标准水样(GSBZ 50027-94)Cr(VI)的测定:将标准水样稀释至250mL,取5mL,加入50mM ABTS 0.5mL,室温反应5-10min,生成2,3-二氨基酚嗪,用pH 7.0Tris-HCl缓冲液稀释至10mL,测定荧光强度,代入步骤2回归方程,得到总铬含量为0.39mg/L,标准认定值为0.40±0.01mg/L,结果吻合。
实施例3:土壤质控样品中铬含量测定:
1、铜掺杂碳点制备:同实施例1;
2、Cr(III)标准曲线制作:同实施例1;
3、土壤质控样品(GBW 07401-GSS-1)中铬含量测定:准确称取0.0001g土壤质控样品,将其放入洗净的消解罐内,同时加入5mL浓HNO3对样品进行消解,然后再加入2mL HCl和1mL HF溶液,将溶液摇匀,放置于密封的微波消解系统中,按照表2设置的消解程序进行消解。
表2样品微波消解程序
步骤 | 系统功率(W) | 设定温度(℃) | 升温时间/min | 保持时间/min |
1 | 800 | 120 | 10 | 3 |
2 | 1200 | 150 | 3 | 6 |
3 | 1600 | 180 | 3 | 15 |
消解结束后,将上述样品冷却,并将消解罐中的样品转移到聚四氟乙烯罐中,用少量去离子水对样品进行洗涤,洗涤2-3次,然后将聚四氟乙烯罐置于电热板上蒸干,温度控制在110-120℃,蒸干后,冷却,然后用去离子水定容至5mL,加入50mM ABTS 0.5mL,室温反应5-10min,生成2,3-二氨基酚嗪,用pH 7.0Tris-HCl缓冲液稀释至10mL,测定荧光强度,代入步骤2回归方程,得到总铬含量为62.9mg/kg,标准认定值为62±4mg/kg,结果吻合。
实施例4:烟叶中Cr(III)与Cr(VI)的测定:
1、铜掺杂碳点制备:同实施例1;
2、Cr(III)标准曲线制作:同实施例1;
3、烟叶中铬含量测定
(1)烟叶样品处理:同实施例1;
(2)总铬及Cr(III)测定:将上述消解液取5mL,分别加入50mM ABTS(0.6878g ofABTS铵盐溶解到25mL超纯水中)0.5mL及不加入ABTS,室温反应5-10min,生成2,3-二氨基酚嗪,用pH 7.0Tris-HCl缓冲液稀释至10mL,测定荧光强度,代入步骤2回归方程,得到总铬及Cr(III)含量为31.4μg/kg及218.2μg/kg,Cr(VI)186.8μg/kg。
Claims (7)
1.一种铜掺杂碳点模拟酶结合荧光探针检测铬的方法,其特征在于,包括:将纳米模拟酶铜掺杂碳点加入邻苯二胺与双氧水溶液中,加入不同浓度的Cr(III),用Tris-HCl缓冲液控制pH 7.0,室温反应5-10min,生成2,3-二氨基酚嗪,测定荧光强度,以Cr(III)浓度为横坐标,荧光强度为纵坐标,绘制标准曲线。
2.根据权利要求1所述的铜掺杂碳点模拟酶结合荧光探针检测铬的方法,其特征在于:所述的铜掺杂碳点由以下方法制备得到:取0.4-1份乙二酸四乙酸铜钠放入管式炉的石英管中,以5℃/min加热速率,氮气保护,300℃锻烧2-4h,将所得产品磨碎,然后溶于100-150份去离子水中,室温下40kHz超声处理15-20min,10000r/min下离心20-25min,上清液0.22μm滤膜过滤,后用截留分子量为3000-3500Da的透析袋进行透析处理24-48h,浓缩液60℃真空干燥24-48h,得到铜掺杂碳点。
3.根据权利要求1所述的铜掺杂碳点模拟酶结合荧光探针检测铬的方法,其特征在于:所述铜掺杂碳点模拟酶、双氧水与邻苯二胺重量比为1:10-50:1000-1500。
4.根据权利要求1所述的铜掺杂碳点模拟酶结合荧光探针检测铬的方法,其特征在于:所述的2,3-二氨基酚嗪最大激发波长为320nm,最大发射波长为572nm。
5.根据权利要求1所述的铜掺杂碳点模拟酶结合荧光探针检测铬的方法,其特征在于:所述铬测定包括样品中铬(VI)用2'-联氨-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)在酸性条件下还原为铬(III),其中的酸为浓盐酸,ABTS的浓度为0.200mM,浓盐酸与ABTS的体积比为5:1。
6.根据权利要求1所述的铜掺杂碳点模拟酶结合荧光探针检测铬的方法,其特征在于:所述Cr(III)浓度为52-1300μg/kg。
7.根据权利要求1所述的铜掺杂碳点模拟酶结合荧光探针检测铬的方法,其特征在于:所述的铜掺杂碳点由以下方法制备得到:取0.4-1份乙二酸四乙酸铜钠放入管式炉的石英管中,以5℃/min加热速率,氮气保护,300℃锻烧2-4h,将所得产品磨碎,然后溶于100-150份去离子水中,室温下40kHz超声处理15-20min,10000r/min下离心20-25min,上清液0.22μm滤膜过滤,后用截留分子量为3000-3500Da的透析袋进行透析处理24-48h,浓缩液60℃真空干燥24-48h,得到铜掺杂碳点;
所述铜掺杂碳点模拟酶、双氧水与邻苯二胺重量比为1:10-50:1000-1500;
所述的2,3-二氨基酚嗪最大激发波长为320nm,最大发射波长为572nm;
所述铬测定包括样品中铬(VI)用2'-联氨-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)在酸性条件下还原为铬(III),其中的酸为浓盐酸,ABTS的浓度为0.200mM,浓盐酸与ABTS的体积比为5:1;
所述Cr(III)浓度为52-1300μg/kg。
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