CN101936905B - 一种汞离子检测试剂及检测方法 - Google Patents
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Abstract
本发明公开了一种汞离子检测试剂及检测方法，该试剂包括纳米溶胶、拉曼活性分子标记的核酸适配体和电荷修饰剂；其检测方法是先合成纳米溶胶，再向适量新鲜纳米溶胶中加入适量适配体，并混合均匀；继续加入适量电荷修饰剂，并混合均匀；然后加入待测样品，孵育1-5分钟.最后取上述纳米溶胶，测试拉曼散射光谱信号，并进行数据处理。本发明基于Hg2+加入致使纳米粒子表面核酸适配体的结构变化，即银溶胶中纳米粒子聚集和信号分子的表面增强拉曼散射信号增强。本发明采用SERS分析技术，对Hg2+检出限在nmol/L水平，对Hg2+浓度的线性响应范围快，同时具有高选择性，适于环境水中痕量Hg2+的快速检测。
Description
技术领域
本发明涉及一种检测试剂及检测方法，具体来说是一种汞离子检测试剂及检测方法。
背景技术
常见重金属一般具有较大的毒性。人体内重金属的过量积累可影响细胞水平的新陈代谢和生物体正常运作，造成组织器官的不可逆损伤。汞作为最为普遍的重金属污染物之一，通过燃煤、冶金、火山喷发和废物丢弃等途径大量排放到环境中。和其他重金属一样，汞不仅不能被微生物降解，相反却在水生微生物作用下生成毒性更大的金属有机化合物(如甲基汞)，并在食物链的生物放大作用下，经成千百倍地富集，从而对生态系统和人类健康造成巨大危害。因此，对水中Hg2+的迁移、转化和检测研究显得尤为重要。自上世纪50年代，Hg2+对水环境的污染问题即引起了人们的关注和分析工作者的重视。目前国内Hg2+检测标准方法主要采用冷原子吸收光谱法和二硫腙比色法。前者采用氯化亚锡将水样中的Hg2+还原成单质汞，形成汞蒸汽，并以氮气或干燥空气作载体，将其吹入汞测定仪，进行冷原子吸收测定。后者则在酸性溶液中利用二硫腙与汞离子生成橙红络合物，经萃取溶于三氯甲烷，测490nm波长处吸光度。这些方法或者相对费力、费时，需要相对复杂的仪器及熟练的相关技术人员方可取得可靠的检测结果；或者属于微量分析方法，难以满足当前日益增长的环境监测要求。
水溶液中金属离子种类繁多且价态多变，常规拉曼分析方法无法直接检测，选择性检测某一种金属离子更是富有挑战性的难题。这些限制因素阻碍了常规拉曼分析方法成为检测金属离子的普适分析技术。
发明内容
本发明所要解决的技术问题是提供一种汞离子检测试剂及检测方法，它可以简便快捷、高灵敏度、可选择性检测水中Hg2+，克服了传统Hg2+检测方法及检测试剂的缺点。
为了解决上述问题，本发明采用如下技术方案：
一种汞离子检测试剂，该试剂包括纳米溶胶、拉曼活性分子标记的核酸适配体和电荷修饰剂，三者摩尔比为1～6∶100～500∶5000～20000。
纳米溶胶、拉曼活性分子标记的核酸适配体和电荷修饰剂三者的摩尔比为3∶200∶10000。
该纳米溶胶为金或银纳米溶胶；
拉曼活性分子为羧基四甲基罗丹明(6-carboxytetramethylrhodamine，TAMRA)、罗丹明B(RhodamineB)、罗丹明6G(Rhodamine 6G)等罗丹明类(Rhodamine)、德克萨斯红(Texas Red)、花青素类(Cyanine)、荧光素类(Fluorescein)等；
核酸适配体为含有胸腺嘧啶(Thymine，T)的核酸单链；
电荷修饰剂为精胺(spermine)、酸类或盐类，如氯化钠等；
使用该检测试剂检测汞离子的方法，包括如下步骤：
a.合成纳米溶胶，其粒度介于10-100纳米(nm)之间；这些溶胶是具有表面增强拉曼散射效应的纳米粒子；
b.向新鲜纳米溶胶中加入拉曼活性分子标记的核酸适配体，并混合均匀，其中按上述检测试剂中的比例确定纳米溶胶与核酸适配体量的关系；
c.继续向纳米溶胶中加入电荷修饰剂，并混合均匀；
d.向上述体系加入待测样品，孵育1-5分钟，孵育即混合反应物并等待一定时间使反应物发生反应；
e.取上述纳米溶胶，测试拉曼散射光谱信号，并进行数据处理。这种检测是基于纳米粒子聚集度不同造成的拉曼散射强度或溶胶颜色变化。
与现有技术相比，本发明具有如下优点：
1、本发明采用拉曼散射(Surface-enhanced Raman Scattering，SERS)分析技术，，对Hg2+响应快，检出限在nmol/L水平，同时具有高选择性，整个操作过程只需10分钟，简便快捷，适于环境水中痕量Hg2+的快速检测。
2、本发明所采用的纳米溶胶合成方法(N.Leopold and B.Lendl.J.Phys.Chem.B.，2003，107：5723-5727)合成后的纳米溶胶则具有表面增强拉曼效应。
3、本发明采用新鲜的纳米溶胶，则避免了因纳米溶胶放存时间久，纳米粒子聚集，影响拉曼散射效果的增强。
4、本发明采用精胺作为电荷修饰剂，既可以使带负电的核酸适配体吸附到带负电的纳米粒子表面，又具有聚集纳米粒子的功能，起到聚集剂的作用。
本发明采用如下原理：
本发明基于Hg2+的存在引起纳米粒子聚集度和拉曼散射(Surface-enhanced Raman Scattering，SERS)信号变化这一原理，实现Hg2+检测。汞等重金属在环境水中大多以离子态形式存在，普通拉曼分析手段无法直接检测。依靠纳米粒子簇表面“热点”(hot spots)的电磁和化学增强效应的SERS检测，可获得高灵敏度的可观的检测信号，而无聚集或聚集度低的纳米溶胶无法产生有效增强。通常金、银等金属溶胶因表面负电荷(根据制备方法的不同也可以带正电荷)的排斥作用而分散地稳定存在。适量聚集剂精胺存在时，标记有拉曼活性分子(例如TAMRA)的带负电性适配体可借助精胺的电荷修饰和“桥梁”作用有效覆盖纳米粒子表面，并起到稳定溶胶的作用。这时因溶胶聚集度低，可以检测到较弱的SERS信号。Hg2+存在时，基于T-Hg2+-T配位作用，适配体与Hg2+结合并伴随构象折叠变化，故纳米粒子(例如Silver nanoparticles，AgNPs)表面部分暴露令排斥作用和位阻减小，纳米粒子在范德华力主导下聚集，“热点”数量的增多使TAMRA拉曼信号急剧增强，并随适配体与一定浓度范围下Hg2+结合数量的增加而增强，从而可以建立SERS信号和Hg2+浓度的关系。
附图说明
图1：Hg2+检测原理示意图。
图2：对空白和一定浓度Hg2+溶液的SERS响应谱图，内插图为SERS响应强度随时间的变化情况(稳定性考察)。
图3：对5nmol/L-5μmol/L浓度范围Hg2+溶液的SERS响应图谱，自下而上SERS相对强度依次增加。
图4：1651cm-1处的SERS强度与Hg2+浓度关系及响应曲线(3次测量)，其中内插图显示Hg2+在5-100nmol/L浓度范围内与SERS响应强度呈线性关系(线性相关系数为0.982)。
图5：对Hg2+的选择性实验暨其他常见金属离子的响应。
具体实施方式
实施例1：
a.室温下合成银纳米溶胶(N.Leopold and B.Lendl.J.Phys.Chem.B.，2003，107：5723-5727)：首先向含84mL去离子水的锥形瓶中加入5mL盐酸羟氨(0.03mol/L)和1mL NaOH(0.6mol/L)，并伴有强力搅拌。然后再向其中以2-3滴/秒匀速滴加AgNO3(0.01mol/L)共10mL。继续搅拌1小时后得到亮黄色AgNPs，粒度为35±8nm，作为储备液纳米粒子溶胶，备用。
b.向含200μL新鲜AgNPs中加入1μL适配体溶液(100μmol/L)，并混合均匀。适配体序列为：5’-TAMRA-TTCTT TCTTCCCTTG TTTGTT-3’。
c.向上述溶液中加入5μL精胺(100μmol/L)，并混合均匀(D.Graham，W.E.Smith，A.M.T.Linacre，C.H.Munro，N.D.Watson and P.C.White.Anal.Chem.，1997，69：4703-4707)。
d.分别向90μL溶胶中加入一定浓度的Hg2+溶液10μL，孵育5min。
e.采用玻璃毛细管吸取少许溶胶，收集拉曼散射信号。检测过程收集拉曼散射的仪器参数分别为：激光波长-632.8nm，激光功率-12微瓦，积分时间-10秒(此条件下的检测原理参见图1)；对空白和一定浓度Hg2+溶液的SERS响应谱图不同，而且SERS响应强度随时间的稳定性很好(参见图2)。
f.定量实验：重复实验步骤d-e，得到不同浓度Hg2+的SERS响应信号(参见图3)，以1651cm-1处拉曼峰为标准峰，进行数据处理，可得到Hg2+浓度在一定范围内与SERS强度呈线性关系(参见图4)。
g.选择性实验：实施过程与实验步骤的a-e步骤相同，将待测溶液由Hg2+分别变为等浓度的其它金属离子。这些金属离子分别为：K+，Mg2+，Ca+，Zn2+，Fe3+，Cu2+，Co2+，Ni2+，Ba2+，Cd2+，Pb2+。本发明用于Hg2+检测具有高选择性(参见图5)。
该实施例对Hg2+检出限低于5nmol/L，对Hg2+浓度的线性响应范围为5-100nmol/L，同时具高选择性，整个检测过程只需5-10分钟。
实施例2：
实施过程中采用实施例1银溶胶合成法得到新鲜银溶胶后，将其离心，加入少量水得到粒子浓度为初始浓度2.5倍的AgNPs。向含200μL新鲜AgNPs中加入2.5μL适配体溶液(100μmol/L)，并混合均匀。适配体序列与实施例1中相同。向上述溶液中加入10μL精胺(100μmol/L)，并混合均匀。分别向上述溶胶中加入高浓度Hg2+溶液，1∶1(体积比)混合，孵育5min。采用玻璃毛细管吸取少许溶胶，收集拉曼散射信号。实验条件与实施例1相同。以1651cm-1处拉曼峰强度为标准峰，进行数据处理。该实施例对Hg2+检出限低于5nmol/L，对Hg2+浓度的线性响应范围为5-150nmol/L，同时具高选择性，整个检测过程只需10-15分钟。
实施例3：
a.同实施例1；b.向含200μL新鲜AgNPs中加入1μL适配体溶液(100μmol/L)，并混合均匀。适配体序列为：5’-TAMRA-TTTTTTTTTTTTTTTTTTTTT-3’；c-g同实施例1。该实施例对Hg2+检出限低于2nmol/L，对Hg2+浓度的线性响应范围为5-80nmol/L，同时具高选择性，整个检测过程只需5-10分钟。
Claims (2)
1.一种汞离子检测试剂，其特征在于：该试剂包括纳米溶胶、拉曼活性分子标记的核酸适配体和电荷修饰剂；
所述试剂的配制为：向200μL纳米溶胶中加入1μL拉曼活性分子标记的核酸适配体溶液，并混合均匀，再加入5μL电荷修饰剂，并混合均匀；
纳米溶胶为银纳米溶胶；拉曼活性分子为羧基四甲基罗丹明、罗丹明B、或罗丹明6G；核酸适配体为含有胸腺嘧啶的核酸单链；电荷修饰剂为精胺；
所述纳米溶胶浓度为1000μmol/L，拉曼活性分子标记的核酸适配体溶液浓度为100μmol/L；电荷修饰剂浓度为100μmol/L。
2.使用权利要求1所述的检测试剂检测汞离子的方法，包括如下步骤：
a.合成纳米溶胶；
b.向新鲜纳米溶胶中加入拉曼活性分子标记的核酸适配体，并混合均匀；
c.继续向纳米溶胶中加入电荷修饰剂，并混合均匀；
d.向上述体系加入待测样品，孵育5分钟；
e.取步骤d得到的纳米溶胶，测试拉曼散射光谱信号，并进行数据处理。
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