CN107462579A - 一种检测汞离子的试剂盒及检测方法 - Google Patents
一种检测汞离子的试剂盒及检测方法 Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y35/00—Methods or apparatus for measurement or analysis of nanostructures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
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- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Health & Medical Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Nanotechnology (AREA)
- Plasma & Fusion (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Crystallography & Structural Chemistry (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明涉及一种检测汞离子的试剂盒及检测方法,所述试剂盒包括浓度为10~30nmol/L的纳米金颗粒溶液、浓度为5~20μmol/L的寡聚核苷酸溶液、中性盐溶液及缓冲液;所述纳米金颗粒的粒径为5~40nm;所述寡聚核苷酸溶液为T33。本发明还公开了采用上述试剂盒检测汞离子的方法。本发明所述试剂盒针对汞离子的检测,其选择性高、灵敏度高,还可即时响应,并且还可通过肉眼观测。
Description
技术领域
本发明涉及一种检测汞离子的方法及检测试剂盒,具体涉及一种基于纳米金颗粒/寡聚核苷酸为检测汞离子的方法及试剂盒。
背景技术
汞,俗称水银,是常温下唯一的液态金属,具有流动性,在常温条件下有一定的挥发性。重金属元素汞及其离子的污染向来是一个严重的环境问题。它们一旦进入环境后,由于很难被降解,可长期残留于环境中,产生持续性的污染。汞及其离子对人体的健康也存在着巨大危害,汞被人体吸收入血.经血细胞膜的扩散和携带作用到全身各器官组织中,对人的神经系统、造血系统、呼吸系统和肾脏等具有严重的破坏能力,而且这种毒性多为不可逆的(Environ.Geochem. Health 2003,25,325;Environ.Toxicol.2003,18,149;Environ.Pollut. 2004,131,323)。更为严重的是,在某些细菌和微生物的作用下,金属汞及其无机盐会被转化为汞的有机形式甲基汞、乙基汞.相比与无机汞,它们在脂肪中具有很强的亲脂性,侵入生物体后吸收率达98%,会积累在中抠神经,毒性大大增强。而且它们会在食物链的各个环节中累积(Chemosphere 1997,35(11),1875;J.Agric.Food Chem.2003,51,838;U.S.EPA,Regulatory Impact Analysis of the Clean Air Mercury Rule:EPA-452/R-05-003,2005.),给人类的健康和生存带来巨大威胁。
而汞及其化合物又是人类生活生产活动必不可少的物质。现在世界上约有80多种工业生产需要汞作原料或辅助材料以及农业上常用的各种含汞杀菌剂、军事上用起爆剂的雷汞,齿龈科填充龋齿的材料中致部分具有漂白、祛斑作用的化妆品中都有汞元素的存在,每年散失在环境中的汞估计达1.5万吨(Environ.Res.1998,77,68;Environ. Res.1998,77,73;Chemosphere 2000,40,1335)。汞的污染来源有冶金、氯碱工业、电器工业、矿物燃料的燃烧。另外,汞也是传统电池的成分之一,有人报道:l节一号电池烂在土壤里.可以使1平方米的土地失去利用价值,一个小小的纽扣电池可以污染60万升水,相当于一个人一生的饮水量。因此,我们迫切需要一种快速、简便且高效的检测方法,用来检测饮用水、环境、工业废水、废渣中残留的Hg2+。
传统的Hg2+检测的主要方法是光谱法,主要指原子吸收光谱方法(Anal.Chim.Acta 1988,208,151;J.Anal.At.Spectrom.2006,21, 94),近年来,科学工作者们发展了原子光谱与ICP技术的联用的,提高了检测的灵敏度,拓宽了检测的线性范围,而电感耦合等离子体质谱(ICP-MS)使检测更加灵敏,数据分析更加方便(J.Anal.Atom.Spectrosc.1998,13,659;Trends Anal.Chem.2005,24,383),以及灵敏度更高的电化学传感方法(Electroanalysis 1998,10,399;Anal. Chem.2002,74,921)。但是,这些方法不仅需要昂贵的仪器和受过专门训练的专业人员,而且还需要复杂的样品预处理过程,因此这些方法大多数只能限制在实验室中的应用。除光谱法外,最近科学工作者们还发展了一系列合成有机荧光小分子为代表的Hg2+传感体系(J. Am.Chem.Soc.2000,122,6769;J.Am.Chem.Soc.2000,122,968; Chem.Eur.J.2004,10,6247;Angew.Chem.,Int.Ed.2005,44,4405),虽然在选择性上有所突破,但存在灵敏度不高、体系水溶性差等缺陷。随着分子生物学技术的快速发展,利用生物大分子的特异性检测各种靶物质提供了有利条件。最近,各研究小组纷纷报道了以蛋白质(J. Am.Chem.Soc.2004,126,728;J.Am.Chem.Soc.2006,128,28188)、 DNA酶(DNAzymes)(Org.Biomol.Chem.2004,2,307;Angew.Chem.,Int.Ed.2007,46,7587)以及生物分子修饰的纳米粒子(Anal.Chem. 2006,78,445;Anal.Chem.2006,78,8332)为平台的Hg2+传感体系,实现了高选择性、高灵敏度检测。尤其是富含胸腺嘧啶脱氧核苷的寡聚核苷酸提供了一个特异性检测Hg2+的平台。这主要是由于胸腺嘧啶脱氧核苷酸能够选择性的与Hg2+发生相互作用,形成碱基对 (T-Hg2+-T),而且这种碱基对是热力学稳定的。自从2004年,Ono研究小组(Angew.Chem.,Int.Ed.2004,43,4300)报道通过两端标记的富含胸苷的DNA探针(一端标记荧光素基团,另一端标记荧光猝灭基团DABCYL)结合Hg2+后,体系荧光变化实现对Hg2+的特异性检测。该方法具有较高的特异性,可排除实际样品体系中大多数离子的干扰,检测限可以达到40nM,但DNA探针需要进行双标记,检测成本仍然较高,更为重要的是该方法还需要依赖于荧光仪器,没有实现可视检测。可视检测是最直接最方便的检测方法,它不需要借助任何仪器只需要通过肉眼观察信号的变化。尽管文献已经报道一些有关Hg2+的可视检测体系(Nano Lett.2001,1,165;J.Org.Chem.2005,70,2912;J.Org. Chem.2005,70,5827;J.Am.Chem.Soc.2005,127,12351;Angew. Chem.,Int.Ed.2007,46,4093;J.Am.Chem.Soc.2008,130,3244)但这些检测方法存在一些诸如水溶性差,选择性不好,灵敏度低或者复杂的表面修饰和体系设计,响应时间长等缺陷,限制了它们的应用范围。
发明内容
基于上述背景技术,本发明的第一目的在于提供一种高选择性、高灵敏度、即时响应、可视的检测汞离子的试剂盒。
所述试剂盒包括浓度为10~30nmol/L的纳米金颗粒溶液、浓度为 5~20μmol/L得寡聚核苷酸溶液、中性盐溶液及缓冲液;
所述纳米金颗粒的粒径为5~40nm;
所述寡聚核苷酸为T33。
其中,所述寡聚核苷酸T33序列为:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTT-3’本发明进一步提供的,所述纳米金颗粒溶液制备的步骤为:将质量百分比为0.005%~0.02%的HAuCl4溶液加热至沸,加入质量百分比为0.5%~0. 2%的柠檬酸三钠溶液,搅拌,沸腾6~8分钟,停止加热冷却至室温,采用0.1~0.3μm的膜过滤,滤液即为纳米金颗粒溶液。
所述纳米金颗粒溶液的制备步骤进一步优选为:将质量百分比为 0.008%~0.012%的HAuCl4溶液加热至沸,加入质量百分比为 0.8%~1.2%的柠檬酸三钠溶液,搅拌,沸腾6~8分钟,停止加热冷却至室温,采用0.2μm的膜过滤,制得16~18nmol/L的纳米金颗粒溶液;
其中所述HAuCl4溶液与柠檬酸三钠溶液的添加比例为: 25~30:1,优选为28~29:1。
所述纳米金颗粒溶液的制备过程,对仪器设备的要求较高,在制备之前,将所有的玻璃仪器与搅拌磁子经过洁净处理,避免其他物质对纳米金颗粒形成的干扰。
所述洁净处理具体为:玻璃仪器与搅拌磁子先用铬酸洗液浸泡 30分钟后,然后再用去离子水彻底清洗,烘箱烘干备用。
本发明进一步提供的所述纳米金颗粒溶液与所述寡聚核苷酸溶液的体积比为:10~15:1,优选为12~13:1;
本发明进一步提供的,所述中性盐选自NaCl、KCl、CaCl2、K2SO4、 Na2SO4、CaSO4、NaNO3、KNO3、中的一种;
所述中性盐溶液的浓度为3~6mol/L;
所述中性盐溶液的添加量为占所述纳米金颗粒溶液、所述寡聚核苷酸溶液、所述中性盐溶液及所述缓冲液体积总和的0.5%~2%,优选为10%。
本发明进一步提供的,所述缓冲液的pH值为7~8;
所述缓冲液选自Tris-盐酸溶液、三乙胺醇溶液、甘氨酸-盐酸溶液中一种;
所述缓冲液与所述寡聚核苷酸溶液的体积比为:2.5~5:1,优选为 3~4:1。
作为本发明的优选方法,提供一种检测汞离子的试剂盒,所述试剂盒包括浓度为16~18nmol/L的纳米金颗粒溶液、浓度为8~12μ mol/L得寡聚核苷酸溶液、浓度为3~6mol/L NaCl溶液及pH值为7.4的 Tris-盐酸溶液;
所述纳米金颗粒的粒径为10~20nm;
所述寡聚核苷酸溶液包含胸腺嘧啶脱氧核苷,所述胸腺嘧啶脱氧核苷的含量为每升寡聚核苷酸溶液中含有8~10μmol;
所述纳米金颗粒溶液、所述Tris-盐酸溶液、所述NaCl溶液与所述寡聚核苷酸溶液的体积比为:10~15:3~4:0.6~0.8:1;
本发明的第二目的在于,提供上述试剂盒在检测汞离子上的应用。
所述检测具体包括以下步骤:
1)将寡聚核苷酸溶液与缓冲液混合,加入纳米金颗粒溶液,混合均匀,制得混合液1;
2)将待测溶液加入步骤1)制得的混合液1中,充分混合后,加入中性盐溶液,制得混合液2,肉眼观察混合液2的颜色,判断待测溶液中是否含有汞离子,和/或采用紫外光检测混合液2,计算待测溶液中汞离子的含量。
采用紫外光检测,可以初略计算待测溶液中汞离子的含量。
由于胸腺嘧啶脱氧核苷的寡聚核苷酸能够缠绕在纳米金颗粒表面,在高盐浓度下,所述胸腺嘧啶脱氧核苷的寡聚核苷酸是稳定的状态。在混合液2中加入Hg2+,由于特异性的相互作用形成碱基对 T-Hg2+-T形式,从而导致胸腺嘧啶脱氧核苷的寡聚核苷酸结构开始折叠形成类似双螺旋的结构,失去了对纳米金颗粒的保护作用,在高盐浓度下,纳米金颗粒聚集,混合液2的颜色会从红色变为蓝色。当 Hg2+浓度高于2.6μmol/L时,红色变为蓝色的效果就非常明显,肉眼可以观察的到。
所述汞离子的浓度在高于2.6μmol/L时,通过肉眼观察颜色,判断待测溶液中含有汞离子;
所述汞离子的浓度低于170nmol/L时,采用波长为400~800nm 的紫外光检测,优选波长为500~700nm。
采用紫外光谱仪在波长与520nm时,会产生明显的峰;若待检溶液中含有汞离子,在波长为676nm时,会产生峰,汞离子含量越高峰值越明显;反之,待检溶液中未含有汞离子,波长为676nm时,则不是产生峰。
作为本发明的优选方案,一种检测汞离子的方法包括以下步骤:
1)充分清洁制备仪器,将质量百分比为0.008%~0.012%的 HAuCl4溶液加热至沸,加入质量百分比为0.8%~1.2%的柠檬酸三钠溶液,搅拌,沸腾6~8分钟,停止加热冷却至室温,采用0.2μm的膜过滤,制得16~18nmol/L的纳米金颗粒溶液;所述纳米金颗粒的粒径为10~20nm;
其中,所述HAuCl4溶液与所述柠檬酸三钠溶液的添加比为 28~29:1。
2)将8~12μmol/L寡聚核苷酸T33溶液与4~6mmol/L的Tris-HCl 的缓冲溶液混合,再加入步骤1)制得的纳米金颗粒溶液,混合均匀,制得混合液1;
3)将待测溶液加入步骤2)制得的混合液1中,充分混合后,加入3.5~4.5mol/L的NaCl溶液,制得混合液2,肉眼观察混合液2的颜色,判断待测溶液中是否含有汞离子,或采用紫外光谱仪检测混合液2,初略计算待测溶液中汞离子的含量。
本发明至少包括以下有益效果:
1)本发明不仅实现了简单、快速和可视的检测,而且该体系还具有高选择性和高灵敏度。
2)本发明是以纳米金颗粒为信号转导体,寡聚胸腺嘧啶核苷酸作为Hg2+的识别位点。当体系中存在Hg2+,寡聚胸腺嘧啶核苷酸能够从任意卷曲的形态折叠为刚性的双螺旋结构,这样的刚性结构由于不能缠绕在纳米金颗粒表面,纳米金颗粒在高盐浓度下,发生聚集,体系从红色变为蓝色。
3)本发明中寡聚胸腺嘧啶核苷酸与Hg2+的特异性相互作用,使其有着优异的灵敏度和选择性,而且本发明拥有很强的抗干扰性,碱金属碱土金属离子,过渡金属离子以及重金属离子都不会对该检测方法产生影响。
4)试剂盒中的材料简单易得,纳米金颗粒不需任何修饰,寡聚胸腺嘧啶核苷酸也不需任何标记。
5)检测方法响应迅速,只需1~2分钟,拓展了的应用范围。
附图说明
图1为基于纳米金颗粒/寡聚核苷酸的Hg2+检测体系及原理示意图;
图2为T33与T33/Hg2+体系的凝胶电泳图;
图3A所示纳米金颗粒/寡聚核苷酸的Hg2+检测体系的紫外吸收光谱:(a)没有Hg2+(b)存在Hg2+.
图3B为纳米金颗粒/寡聚核苷酸的Hg2+检测体系的彩色照片:(a) 没有Hg2+(b)存在Hg2+;
图4为纳米金颗粒/寡聚核苷酸的Hg2+检测体系的的透射电镜图:图4A没有Hg2+ 图4B存在Hg2+;
图5所示检测体系的灵敏度实验
图5A为纳米金颗粒/寡聚核苷酸检测体系在不同浓度Hg2+下的紫外吸收归一化图;
图5B为纳米金颗粒/寡聚核苷酸检测体系在不同浓度Hg2+下在 676nm与520nm处的紫外吸收比例图;
图6所示检测体系的选择性实验
图6A为纳米金颗粒/寡聚核苷酸检测体系在不同金属离子下在 676nm与520nm处的紫外吸收比例图;
图6B为纳米金颗粒/寡聚核苷酸检测体系在不同金属离子下的彩色照片;
图7所示检测体系的抗干扰实验
图7A为纳米金颗粒/寡聚核苷酸检测体系在金属混合离子中对 Hg2+响应的紫外吸收光谱图
图7B为纳米金颗粒/寡聚核苷酸检测体系在金属混合离子中对 Hg2+响应的紫外吸收在676nm与520nm处的比例图;
图7C为纳米金颗粒/寡聚核苷酸检测体系在金属混合离子中对 Hg2+响应的彩色照片
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
本发明采用了以下仪器与试剂:
紫外光谱仪:JASCO V-550
透射电镜(TEM):JEOL JEM-1010透射电镜仪
四氯金酸(HAuCl4·3H2O)购自国药集团上海化学试剂有限公司,柠檬酸三钠和其它的金属盐均购自北京北化化学试剂公司,纯度均为分析纯。寡聚核苷酸T33购自上海英俊生物工程和技术服务有限公司。DNA的浓度根据DNA在160μl石英比色皿中在260nm处的吸收计算。实验中所用水均为经过Millipore过滤系统处理的去离子水。
透射电镜样品的制备:样品的水溶液通过微量移液器滴在标准铜网上,避光条件下自然晾干。
以下实施例所采用的纳米金颗粒采用柠檬酸三钠还原HAuCl4的方法制备。
在制备之前,将所有的玻璃仪器与搅拌磁子都需经过洁净处理,具体的处理方法是:玻璃仪器与搅拌磁子先用铬酸洗液浸泡30分钟后,然后再用去离子水彻底清洗,烘箱烘干备用。
在250ml的烧杯中加入100mL 0.01%HAuCl4溶液,煮沸,迅速加入3.5mL 1.0%柠檬酸三钠溶液,并充分搅拌。继续加热混合液沸腾7分钟,在此过程中可见溶液的颜色逐渐变为红色。停止加热并让溶液自然冷却至室温,溶液再用0.2μm的膜过滤。经过膜过滤的纳米金颗粒用紫外吸收光谱表征,最大吸收波长在520nm,纳米金颗粒的摩尔消光系数(~108M-1cm-1,520nm),用Beer法则计算出纳米金颗粒的浓度大约为17nM。纳米金颗粒用透射电镜表征,纳米金颗粒的粒径大约为15nm。
实施例1
把141μL Tris-HCl缓冲溶液(5mM,pH=7.4)和42μL寡聚核苷酸T33(10μM,5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTT-3’) 室温下加入500μL 17nM金纳米颗粒,充分混合,得到检测汞离子的试剂盒。
混合操作1分钟后,加入7μL不同浓度的Hg2+,具体汞离子浓度为:0×10-6mol/L、1×10-6mol/L、2×10-6mol/L、3×10-6mol/L、4×10-6mol/L、 5×10-6mol/L、6×10-6mol/L、7×10-6mol/L、8×10-6mol/L、9×10-6mol/L、10×10-6mol/L。平衡1分钟后,加入10μL 4M NaCl溶液,检测体系最终的体积为700μL,最后利用紫外光谱仪和肉眼观测混合溶液的变化。空白的控制实验和其它金属离子的响应实验均采用相同的方式,使检测体系的最终的体积均为700μL。
考察AuNPs/T33体系中有无Hg2+存在紫外吸收光谱的变化,如图3A所示,曲线a是AuNPs/T33体系中不存在Hg2+时的紫外吸收,可以看到在520nm处的吸收峰,此时,体系保持红色,如图3B中a;说明可以任意卷曲的T33对纳米金颗粒的保护作用。当体系中存在Hg2+时,在676nm处出现一新的吸收峰,为图3A中曲线b,相应的体系的颜色变为蓝色,如图3B中b;说明在Hg2+存在下,T33的结构发生改变致使纳米金颗粒发生聚集,体系颜色改变。
通过透射电镜研究了AuNPs/T33体系中有无Hg2+存在时,纳米金颗粒的聚集状态。当AuNPs/T33检测体系中未有Hg2+时,纳米金颗粒比较散落,如图4A所示;当在AuNPs/T33检测体系加入5μM Hg2+,纳米金颗粒明显聚集,呈现立体的团簇状态,如图4B所示。实验结果直接证明了AuNPs/T33体系可视检测Hg2+的可行性和工作机理。
对于Hg2+检测体系来说,灵敏度是检测体系的基本要素。基于此,设计了在体系中加入不同浓度Hg2+,观测体系紫外吸收的变化情况,从而考察体系灵敏度。如图5A所示,当体系中Hg2+浓度不断增加时,在676nm处的吸收不断增强。利用紫外吸收,计算得到该体系的检测限为170nM(信噪比为3时),这与其它利用寡聚胸腺嘧啶核苷酸的Hg2+检测体系接近。图5B表示的是体系在676nm与520nm处的紫外吸收值之比随Hg2+浓度变化关系曲线,我们发现随着Hg2+浓度的不断增加(0~10μM),工作曲线呈现一个粗略的Σ型。值得一提的是当Hg2+浓度为2.6μM时,体系由红色变为蓝色,这说明该体系的可视检测限为2.6μM,这与其它利用纳米金颗粒作为信号转导体的 Hg2+检测体系相当。
实施例2
采用实施例1同样的实验条件,检测体系对其它金属离子Ba2+, Ca2+,Co2+,Cu2+,Fe2 +,Fe3+,Gd2+,K+,Li+,Mg2+,Mn2+,Ni2+,Pb2+和Zn2+的响应性。如图6A所示,当金属离子浓度均为10μM时,体系在 676nm和520nm紫外吸收值之比,可发现,体系对Hg2+的响应程度大大超过其它的金属离子。说明AuNPs/T33体系对Hg2+有着很好的选择性。图6B是体系对各种金属离子响应的彩色照片,只有存在Hg2+的体系,颜色变为蓝色,其它的均未变色,这直观的说明了AuNPs/ T33体系对Hg2+有着优异的选择性。因为聚胸腺嘧啶核苷酸能与Hg2+形成热力学稳定的T-Hg2+-T的碱基对,而其它诸如Ba2+,Ca2+,Co2+, Cu2+,Fe2+,Fe3+,Gd2+,K+,Li+,Mg2+,Mn2+,Ni2 +,Pb2+和Zn2+等离子不能形成类似的稳定结构。
实施例3
考察AuNPs/T33体系的抗干扰性,在检测体系中先加入干扰离子,然后再加入Hg2+,观测此时体系的变化情况。
采用实施例1同样的实验条件,选择三组干扰离子:第一组为重金属离子组(mix1:Ba2+,Cu2+,Mn2+和Pb2+,浓度均为5μM);第二组为碱金属和碱土金属离子组(mix 2:Ca2+,K+,Mg2+,Na+,浓度均为5μM);第三组为过渡金属离子组(mix 3:Fe2+,Co2+,Ni2+,Zn2+,浓度均为5μM)。如图7A所示,当体系中加入干扰离子(mix 1:Ba2+,Cu2+, Mn2+和Pb2+,浓度均为5μM)时,体系的紫外吸收几乎没有变化(与空白实验对比),再加入5μMHg2+时,在676nm处出现了很明显的吸收峰。说明重金属干扰离子对AuNPs/T33检测体系无影响。碱金属和碱土金属离子与过渡金属离子对体系也无干扰。图7B和图7C分别展现了在不同干扰离子存在下,体系对Hg2+的响应紫外吸收变化和可视彩色照片。实验结果再次证明体系对Hg2+有着优异的选择性。
虽然,上文中已经用一般性说明、具体实施方式及试验,对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
SEQUENCE LISTING
<110> 北京欧凯纳斯科技有限公司
<120> 一种检测汞离子的试剂盒及检测方法
<130> 2017
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 33
<212> DNA
<213> 人工序列
<400> 1
tttttttttt tttttttttt tttttttttt ttt 33
Claims (10)
1.一种检测汞离子的试剂盒,其特征在于,所述试剂盒包括浓度为10~30nmol/L的纳米金颗粒溶液、浓度为5~20μmol/L的寡聚核苷酸溶液、中性盐溶液及缓冲液;
所述纳米金颗粒的粒径为5~40nm;
所述寡聚核苷酸为T33。
2.根据权利要求1所述的试剂盒,其特征在于,所述纳米金颗粒溶液通过以下步骤制备而得:将质量百分比为0.005%~0.02%的HAuCl4溶液加热至沸,加入质量百分比为0.5%~0.2%的柠檬酸三钠溶液,搅拌,沸腾6~8分钟,停止加热冷却至室温,采用0.1~0.3μm的膜过滤,滤液即为纳米金颗粒溶液。
3.根据权利要求2所述的试剂盒,其特征在于,所述HAuCl4溶液与柠檬酸三钠溶液的体积比例为:25~30:1,优选为28~29:1。
4.根据权利要求1-3任一所述的试剂盒,其特征在于,所述纳米金颗粒溶液与所述寡聚核苷酸溶液的体积比为:10~15:1,优选为12~13:1。
5.根据权利要求1-4任一所述的试剂盒,其特征在于,所述中性盐选自NaCl、KCl、CaCl2、K2SO4、Na2SO4、CaSO4、NaNO3、KNO3、中的一种;
所述中性盐溶液的浓度为3~6mol/L;
所述中性盐溶液的添加量为占所述纳米金颗粒溶液、所述寡聚核苷酸溶液、所述中性盐溶液及所述缓冲液体积总和的0.5%~2%,优选为10%。
6.根据权利要求1-5任一所述的试剂盒,其特征在于,所述缓冲液的pH值为7~8;
所述缓冲液选自Tris-盐酸溶液、三乙胺醇溶液、甘氨酸-盐酸溶液中一种;
所述缓冲液与所述寡聚核苷酸溶液的体积比为:2.5~5:1,优选为3~4:1。
7.根据权利要求1所述的试剂盒,其特征在于,所述试剂盒包括浓度为16~18nmol/L的纳米金颗粒溶液、浓度为8~12μmol/L得寡聚核苷酸T33溶液、浓度为3~6mol/L NaCl溶液及pH值为7.4的Tris-盐酸溶液;
所述纳米金颗粒的粒径为10~20nm;
所述纳米金颗粒溶液、所述Tris-盐酸溶液、所述NaCl溶液与所述寡聚核苷酸溶液的体积比为:10~15:3~4:0.6~0.8:1。
8.权利要求1-7任一所述试剂盒在检测汞离子上的应用。
9.根据权利要求8所述的应用,其特征在于,所述检测具体包括以下步骤:
1)将寡聚核苷酸溶液与缓冲液混合,加入纳米金颗粒溶液,混合均匀,制得混合液1;
2)将待测溶液加入步骤1)制得的混合液1中,充分混合后,加入中性盐溶液,制得混合液2,肉眼观察混合液2的颜色,判断待测溶液中是否含有汞离子,和/或采用紫外光检测混合液2,计算待测溶液中汞离子的含量。
10.根据权利要求9所述的应用,其特征在于,所述汞离子的浓度在高于2.6μmol/L时,通过肉眼观察颜色,判断待测溶液中含有汞离子;
所述汞离子的浓度低于170nmol/L时,采用波长为400~800nm的紫外光检测,优选波长为500~700nm。
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