CN107505277A - 一种基于Au@Ag核壳纳米粒子的合成检测汞离子的方法 - Google Patents
一种基于Au@Ag核壳纳米粒子的合成检测汞离子的方法 Download PDFInfo
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Abstract
一种基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法,属于纳米生物技术检测领域。本发明包括:金纳米粒子修饰Hg2+的一段适配体DNA,抗坏血酸偶联Hg2+的另一段适配体DNA,在Hg2+存在的条件下形成包含抗坏血酸的T‑Hg2+‑T错配结构,Au@Ag核壳纳米粒子的形成以及紫外吸收光谱的测定。本方法操作简单方便,能够实现Hg2+高灵敏、高特异性、高实用性的检测,是一种具有重大开发前景的纳米传感检测方法。
Description
技术领域
一种基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法,属于纳米生物技术检测领域。
背景技术
汞是环境中一种生物毒性极强的重金属污染物,普遍存在于自然界中,它能够被生物富集并在生物体内累积,可以通过食物链转移到人体内,由于汞具有高毒性,因此对人体健康造成重大的危害。人体内累积的微量汞无法通过自身排泄进行代谢,将直接导致心脏、肝脏、甲状腺疾病,损伤中枢神经系统、呼吸系统,慢性汞中毒,甚至引发恶性肿瘤的形成。2001年美国环保局(EFA)发布,在饮用水中汞离子的含量不能超过2ppb(10nM),因此需要不断开发高灵敏的Hg2+检测方法。
传统的Hg2+检测方法主要是基于仪器检测,主要包括冷蒸汽原子荧光光谱(CV-AFS)、原子吸收光谱(AAS)、电感耦合等离子体原子发射光谱(ICP-AES)、电感耦合等离子体质谱(ICP-MS)。这些方法往往存在仪器昂贵、分析周期长、样品预处理复杂、检测费用高等问题,已经难以适应汞离子检测的方便、快捷、灵敏度等方面的要求。近年来随着生物传感器检测技术的不断发展,一些列简便高灵敏的生物传感器得到不断开发,并且成为今后生物检测领域的新途径和新的检测方法的发展方向。
抗Hg2+的适配体对Hg2+具有高亲和性和高特异性的识别能力,Hg2+的适配体是一对富含胸腺嘧啶碱基的单链DNA序列,在Hg2+存在的条件下,两条单链DNA分子形成T-Hg2+-T碱基错配结构,因此将Hg2+适配体结合纳米粒子载体将开发多种纳米生物传感器,例如:比色传感器、化学发光传感器、表面增强的拉曼光谱传感器、表面等离子共振传感器、磁共振传感器等。金纳米粒子具有良好的光电性质、化学性质和生物兼容性,并且易于表面修饰。不同粒径的金纳米粒子的紫外吸收峰不同,若在金纳米粒子表面包裹一层银壳结构,形成的新的Au@Ag核壳纳米粒子将同时出现金和银的紫外吸收峰。
本发明是借助于T-Hg2+-T碱基错配作用将DNA上偶联的抗坏血酸包裹在金纳米粒子的表面,通过抗坏血酸对硝酸银的还原作用,在金纳米粒子的表面形成一层银壳结构,并且不同Hg2+浓度下金纳米粒子表面的银壳厚度不同,通过测定反应后体系中紫外吸收的变化,从而实现对Hg2+含量的检测。
发明内容
要解决的技术问题:基于传统的仪器检测Hg2+的方法存在仪器昂贵、分析周期长、样品预处理复杂、检测费用高等问题,已经难以适应汞离子检测的方便、快捷、高灵敏度等方面的要求。
技术方案:本发明公开了一种基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法,包括如下具体步骤:
(1)金纳米粒子修饰Hg2+的一段适配体DNA
将新合成的金纳米粒子在12000r/min的转速条件下离心去除上清,并用pH8.0 0.01 M的Tris-HCl缓冲液将其进行重悬并浓缩四倍,测得浓缩后的金纳米粒子的浓度为10nM,以用于DNA的偶联;取1mL浓缩后的金纳米粒子,然后加入100μL 10μM的DNA1,使得DNA1的终浓度为1μM,DNA1和金纳米粒子以100:1的摩尔比在室温条件下进行过夜偶联反应,将反应后的金纳米粒子在12000r/min的转速条件下离心以除去未结合的DNA1,然后将其用pH8.00.01 M的Tris-HCl缓冲液进行重悬,从而得到修饰后的金纳米粒子;
DNA 1: 5’-SH-AAAAAAGTGACCATTTTTGCAGTG-3’。
(2)抗坏血酸偶联Hg2+的另一段适配体DNA
称取50mg抗坏血酸于圆底烧瓶中,用2mL吡啶溶剂将其进行溶解,然后加入34mg琥珀酸酐,在室温下避光搅拌反应2天,然后将反应后的溶液用旋转蒸发仪旋干,再加入5mL pH6.5的0.01M 2-吗啉代乙磺酸缓冲液将其溶解,置于磁力搅拌反应器上,然后加入150mg碳二亚胺和100mg N-羟基琥珀酰亚胺,在磁力搅拌的状态下将抗坏血酸琥珀酰酯进行活化,反应时间为2-4h;取pH7.4 0.01M的磷酸盐缓冲液溶解的浓度为1μM的氨基修饰的DNA2 500μL,将500μL活化好的抗坏血酸琥珀酰酯加入到DNA2中,在振荡的条件下反应4-6h,得到抗坏血酸偶联的DNA2;
DNA2:5’-NH2-AAAAAACACTGCTTTTTTGGTCAC-3’。
(3)在Hg2+存在的条件下形成包含抗坏血酸的T-Hg2+-T错配结构
将1mL步骤(1)中修饰DNA1的金纳米粒子探针和1mL步骤(2)中偶联好的DNA2按照1:1的体积比混合,分装到PCR管中每管100μL,然后在每管中分别加入浓度为0.5ng mL-1、5ng mL-1、10ng mL-1、50ng mL-1、100ng mL-1、200ng mL-1的Hg2+,在室温下孵育反应4h后,使得DNA1和DNA2与Hg2+充分结合形成T-Hg2+-T错配结构;将反应体系在12000r/min的条件下离心去除未结合的DNA2,然后再向每管加入100μL超纯水将金纳米粒子进行重悬。
(4)Au@Ag核壳纳米粒子的形成以及紫外吸收光谱的测定
将步骤(3)中得到的金纳米粒子溶液的pH用NaOH水溶液调整到9.0,然后向每管中加入包含200mM聚乙烯吡咯烷酮的1M硝酸银溶液50μL,在缓慢振荡的条件下反应1-3h,借助于金纳米粒子表面的抗坏血酸作为还原剂将硝酸银进行还原,并在金纳米粒子的表面形成一层银壳,从而形成Au@Ag核壳纳米粒子;将不同Hg2+浓度下的Au@Ag核壳纳米粒子分别在520nm和400nm的波长下进行紫外测定,从而得到A520/A400的紫外吸收比值,并建立Hg2+浓度和A520/A400的对应关系。
本发明所述的基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法所用的金纳米粒子的粒径为15nm。
本发明所述的基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法步骤(2)中抗坏血酸琥珀酰酯的活化时间为3h。
本发明所述的基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法步骤(2)中抗坏血酸琥珀酰酯加入到DNA2后的反应时间为5h。
本发明所述的基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法步骤(4)中抗坏血酸还原硝酸银的时间为2h。
本发明所述的15nm的金纳米粒子通过柠檬酸三钠还原氯金酸的方法进行合成,合成步骤:将三口瓶用王水浸泡过夜,然后用超纯水清洗干净,在洁净的三口瓶中加入95mL的超纯水,再加入2.5mL质量浓度为0.4%的氯金酸,磁力搅拌并加热沸腾,7-8min后加入2.5mL质量浓度为1%的柠檬酸三钠,溶液从无色变为红色后停止加热,继续搅拌15min,即得到15nm金纳米粒子。
有益效果:本发明提供了一种基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法,借助于T-Hg2+-T碱基错配作用将DNA上偶联的抗坏血酸包裹在金纳米粒子的表面,抗坏血酸对硝酸银具有还原作用,在不同浓度Hg2+存在的条件下,金纳米粒子表面形成的银壳厚度不同,通过测定不同Hg2+浓度下A520/A400的紫外吸收比值,从而间接对Hg2+含量进行测定。
附图说明
图1 Au@Ag核壳纳米粒子的TEM图。
图2不同Hg2+浓度下Au@Ag核壳纳米粒子的紫外吸收光谱图。
图3 Hg2+检测的标准曲线。
具体实施方式
实施例1
一种基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法,包括如下具体步骤:
(1)金纳米粒子修饰Hg2+的一段适配体DNA
将新合成的粒径为15nm金纳米粒子在12000r/min的转速条件下离心去除上清,并用pH8.0 0.01 M的Tris-HCl缓冲液将其进行重悬并浓缩四倍,测得浓缩后的金纳米粒子的浓度为10nM,以用于DNA的偶联;取1mL浓缩后的金纳米粒子,然后加入100μL 10μM的DNA1,使得DNA1的终浓度为1μM,DNA1和金纳米粒子以100:1的摩尔比在室温条件下进行过夜偶联反应,将反应后的金纳米粒子在12000r/min的转速条件下离心以除去未结合的DNA1,然后将其用pH8.0 0.01 M的Tris-HCl缓冲液进行重悬,从而得到修饰后的金纳米粒子;
DNA 1: 5’-SH-AAAAAAGTGACCATTTTTGCAGTG-3’。
(2)抗坏血酸偶联Hg2+的另一段适配体DNA
称取50mg抗坏血酸于圆底烧瓶中,用2mL吡啶溶剂将其进行溶解,然后加入34mg琥珀酸酐,在室温下避光搅拌反应2天,然后将反应后的溶液用旋转蒸发仪旋干,再加入5mL pH6.5的0.01M 2-吗啉代乙磺酸缓冲液将其溶解,置于磁力搅拌反应器上,然后加入150mg碳二亚胺和100mg N-羟基琥珀酰亚胺,在磁力搅拌的状态下将抗坏血酸琥珀酰酯进行活化,反应时间为3h;取pH7.4 0.01M的磷酸盐缓冲液溶解的浓度为1μM的氨基修饰的DNA2 500μL,将500μL活化好的抗坏血酸琥珀酰酯加入到DNA2中,在振荡的条件下反应5h,得到抗坏血酸偶联的DNA2。
DNA2:5’-NH2-AAAAAACACTGCTTTTTTGGTCAC-3’。
(3)在Hg2+存在的条件下形成包含抗坏血酸的T-Hg2+-T错配结构
将1mL步骤(1)中修饰DNA1的金纳米粒子探针和1mL步骤(2)中偶联好的DNA2按照1:1的体积比混合,分装到PCR管中每管100μL,然后在每管中分别加入浓度为0.5ng mL-1、5ng mL-1、10ng mL-1、50ng mL-1、100ng mL-1、200ng mL-1的Hg2+,在室温下孵育反应4h后,使得DNA1和DNA2与Hg2+充分结合形成T-Hg2+-T错配结构;将反应体系在12000r/min的条件下离心去除未结合的DNA2,然后再向每管加入100μL超纯水将金纳米粒子进行重悬。
(4)Au@Ag核壳纳米粒子的形成以及检测灵敏度研究
将步骤(3)中得到的金纳米粒子溶液的pH用NaOH水溶液调整到9.0,然后向每管中加入包含200mM聚乙烯吡咯烷酮的1M硝酸银溶液50μL,在缓慢振荡的条件下反应2h,借助于金纳米粒子表面的抗坏血酸作为还原剂将硝酸银进行还原,并在金纳米粒子的表面形成一层银壳,从而形成Au@Ag核壳纳米粒子;分别测定不同Hg2+浓度下的Au@Ag核壳纳米粒子在520nm和400nm的波长下的紫外吸收值,以Hg2+浓度为横坐标,A520/A400为纵坐标建立两者之间的标准曲线,并通过计算得出Hg2+的最低检测限为0.3ng mL-1。
(5)特异性研究
将其它五种重金属离子(Zn2+、Mg2+、Cu2+、Pb2+、Mn2+)用此方法进行检测,测定0.5 ngmL-1的检测浓度下,观察金纳米粒子表面银壳的形成结构,并测定反应体系中紫外吸收的变化,结果显示,金纳米粒子表面没有形成银壳结构,从而说明其它重金属离子没有与T-T错配碱基相互作用,不能用于其他重金属离子的检测,因此此方法对Hg2+检测具有较高的特异性。
(6)添加回收实验
在含有0.5 ng mL-1 Hg2+的饮用水中,测定了添加不同浓度Hg2+后的添加回收结果,在1.5、2.8、5.4、6.2和10.5ng mL-1的添加浓度下,Hg2+的回收率在94.3%-97.6%范围内,回收结果表明此方法可以用于实际水样品中的Hg2+含量分析。
Claims (5)
1.一种基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法,其特征在于包括如下具体步骤:
(1)金纳米粒子修饰Hg2+的一段适配体DNA
将新合成的金纳米粒子在12000r/min的转速条件下离心去除上清,并用pH8.0 0.01 M的Tris-HCl缓冲液将其进行重悬并浓缩四倍,测得浓缩后的金纳米粒子的浓度为10nM,以用于DNA的偶联;取1mL浓缩后的金纳米粒子,然后加入100μL 10μM的DNA1,使得DNA1的终浓度为1μM,DNA1和金纳米粒子以100:1的摩尔比在室温条件下进行过夜偶联反应,将反应后的金纳米粒子在12000r/min的转速条件下离心以除去未结合的DNA1,然后将其用pH8.00.01 M的Tris-HCl缓冲液进行重悬,从而得到修饰后的金纳米粒子;
DNA 1: 5’-SH-AAAAAAGTGACCATTTTTGCAGTG-3’;
(2)抗坏血酸偶联Hg2+的另一段适配体DNA
称取50mg抗坏血酸于圆底烧瓶中,用2mL吡啶溶剂将其进行溶解,然后加入34mg琥珀酸酐,在室温下避光搅拌反应2天,然后将反应后的溶液用旋转蒸发仪旋干,再加入5mL pH6.5的0.01M 2-吗啉代乙磺酸缓冲液将其溶解,置于磁力搅拌反应器上,然后加入150mg碳二亚胺和100mg N-羟基琥珀酰亚胺,在磁力搅拌的状态下将抗坏血酸琥珀酰酯进行活化,反应时间为2-4h;取pH7.4 0.01M的磷酸盐缓冲液溶解的浓度为1μM的氨基修饰的DNA2 500μL,将500μL活化好的抗坏血酸琥珀酰酯加入到DNA2中,在振荡的条件下反应4-6h,得到抗坏血酸偶联的DNA2;
DNA2:5’-NH2-AAAAAACACTGCTTTTTTGGTCAC-3’;
(3)在Hg2+存在的条件下形成包含抗坏血酸的T-Hg2+-T错配结构
将1mL步骤(1)中修饰DNA1的金纳米粒子探针和1mL步骤(2)中偶联好的DNA2按照1:1的体积比混合,分装到PCR管中每管100μL,然后在每管中分别加入浓度为0.5ng mL-1、5ng mL-1、10ng mL-1、50ng mL-1、100ng mL-1、200ng mL-1的Hg2+,在室温下孵育反应4h后,使得DNA1和DNA2与Hg2+充分结合形成T-Hg2+-T错配结构;将反应体系在12000r/min的条件下离心去除未结合的DNA2,然后再向每管加入100μL超纯水将金纳米粒子进行重悬;
(4)Au@Ag核壳纳米粒子的形成以及紫外吸收光谱的测定
将步骤(3)中得到的金纳米粒子溶液的pH用NaOH水溶液调整到9.0,然后向每管中加入包含200mM聚乙烯吡咯烷酮的1M硝酸银溶液50μL,在缓慢振荡的条件下反应1-3h,借助于金纳米粒子表面的抗坏血酸作为还原剂将硝酸银进行还原,并在金纳米粒子的表面形成一层银壳,从而形成Au@Ag核壳纳米粒子;将不同Hg2+浓度下的Au@Ag核壳纳米粒子分别在520nm和400nm的波长下进行紫外测定,从而得到A520/A400的紫外吸收比值,并建立Hg2+浓度和A520/A400的对应关系。
2.根据权利要求1所述的一种基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法,其特征在于所述的金纳米粒子的粒径为15nm。
3.根据权利要求1所述的一种基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法,其特征在于所述的步骤(2)中抗坏血酸琥珀酰酯的活化时间为3h。
4.根据权利要求1所述的一种基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法,其特征在于所述的步骤(2)中抗坏血酸琥珀酰酯加入到DNA2后的反应时间为5h。
5.根据权利要求1所述的一种基于Au@Ag核壳纳米粒子的合成检测Hg2+的方法,其特征在于所述的步骤(4)中抗坏血酸还原硝酸银的时间为2h。
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CN108519358A (zh) * | 2018-03-14 | 2018-09-11 | 华东师范大学 | 一种基于新型金纳米颗粒检测Hg2+的方法 |
CN115902196A (zh) * | 2023-03-03 | 2023-04-04 | 山东康华生物医疗科技股份有限公司 | 一种chi3l1检测试剂盒及其制备方法 |
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