CN111705112B - 一种基于硅量子点、荧光素标记的dna、剪切酶的汞离子荧光检测方法 - Google Patents

一种基于硅量子点、荧光素标记的dna、剪切酶的汞离子荧光检测方法 Download PDF

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CN111705112B
CN111705112B CN202010382371.6A CN202010382371A CN111705112B CN 111705112 B CN111705112 B CN 111705112B CN 202010382371 A CN202010382371 A CN 202010382371A CN 111705112 B CN111705112 B CN 111705112B
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由天艳
李文佳
刘�东
李玉叶
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Abstract

本发明属于荧光生物检测技术领域，公开了一种基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法。本发明中，我们利用硅量子点带正电并可以猝灭荧光素(Rox)的荧光信号的性质，将可以与汞离子特异性结合成双链的DNA连接在Rox上，结合exonuclease III酶剪切双链DNA，达到信号放大的目的，实现了对汞离子的高灵敏检测。本发明提出的基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法对汞离子检测的线性范围宽(2×10‑11–1×10‑ 8mol/L)，检测限低(6.67×10‑12mol/L)，并且具有良好的选择性。通过标准加入法，对镇江古运河水和农田土壤中的汞离子进行分析，获得了满意的回收率。具有良好的实际应用前景。

Description

一种基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光 检测方法
技术领域
本发明属于荧光生物检测技术领域，具体涉及一种基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法。
背景技术
汞(Hg2+)污染多年来一直被认为是一个重要的世界性问题，因为它在低浓度下仍具有很高的毒性，并且可以在人体中产生生物累积效应。因此，痕量Hg2+的检测十分必要。荧光法由于分析速度快、成本效益好、操作方便等优点，已被广泛应用于汞离子(Hg2+)的检测。为了避免其它潜在物质的干扰，科研工作者利用Hg2+与胸腺嘧啶碱基之间的配位相互作用来提高检测的选择性，具体来说，胸腺嘧啶-胸腺嘧啶(T-T)在DNA双链中的错配会吸引水溶液中的Hg2+与胸腺嘧啶(T)配对形成稳定的胸腺嘧啶-Hg2+-胸腺嘧啶(T-Hg2+-T)DNA双链。
为了提高荧光传感器对Hg2+检测的灵敏度，杂交链式反应(HCR)、滚动圆扩增(RCA)和酶辅助扩增等扩增策略被广泛应用于Hg2+检测中。其中，酶辅助扩增策略主要采用外切酶III(ExoIII)能有效地降解dsDNA的平末端和5’-突出末端，而对ssDNA或dsDNA的3’-突出末端的活性较低的性质。由于其不需要特定的识别序列而成为信号放大策略的理想候选。
发明内容
本发明旨在发展一种基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法。具体通过如下技术方案实现：利用硅量子点带正电并可以猝灭荧光素(Rox)的荧光信号的性质，将可以与汞离子特异性结合成双链的DNA连接在Rox(Rox-DNA)上，无汞离子存在时，硅量子点通过静电吸附猝灭Rox的荧光信号；当汞离子存在时，Rox-DNA通过T-Hg2+-T结构形成双链结构，Exo III剪切双链DNA，释放Rox和Hg2+达到信号放大的目的，实现了对汞离子的高灵敏检测。
一种基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法，包括如下步骤：
(1)将制备的SiQDs溶液与Rox-DNA溶液加入到一定体积的Tris-HCl溶液中，并在特定温度下孵育一定时间；
(2)向步骤(1)制备混合溶液中加入已知浓度的Hg2+溶液混合，并在特定温度下孵育一定时间；
(3)向步骤(2)制备混合溶液中加入ExoIII溶液混合，在特定温度下作用一段时间，然后在高温下灭活ExoIII，待混合液冷却后用荧光分光光度计在室温下分别检测溶液在414nm和607nm处的荧光强度I414和I607，得出荧光强度I607/I414比值与汞离子浓度对数的标准曲线；
(4)按照步骤(1)～(3)的操作，将上述已知浓度的Hg2+溶液用待测的Hg2+溶液替换，用荧光分光光度计在室温下分别检测溶液在414nm和607nm处的荧光强度I414和I607，计算此时I607/I414比值并代入步骤(3)中的标准曲线，得出待测溶液中汞离子的浓度。
步骤(1)中，合成SiQDs的具体方法为：将6.665mL 60mM的还原剂L-谷胱甘肽(L-GSH)和13.135mL去离子水加入100mL烧杯中，通入氮气10min后，加入0.2mL硅源N-[3-(三甲氧基硅基)丙基]乙二胺(DAMO)，继续通氮气10min，得到混合溶液。将装有混合溶液的烧杯放入美的家用微波炉中，在微波功率为700W的条件下，微波反应8min，得到棕黄色固体。向烧杯中加10mL去离子水溶解得到的固体物质，接着将溶液转移到离心管中放入离心机离心10min，离心过程的离心速率为10000rpm，取上清液，转移到透析袋透析处理24h。提纯后的量子点溶液经冷冻干燥、研磨得到略带黄色的硅量子点粉末，置于4℃下保存。所述透析袋的截留分子量为1000。
步骤(1)中，SiQDs溶液浓度为0.5mg/mL，Rox-DNA溶液的浓度为1μM，SiQDs溶液，Rox-DNA溶液及Tris-HCl的体积比为10：10：53；孵育条件为：37℃下孵育15min。
步骤(2)中，Hg2+溶液的浓度为2×10-11–1×10-8mol/L；SiQDs溶液：Hg2+溶液体积比5：2；孵育条件为：37℃下孵育40min。
步骤(3)中，ExoIII溶液的浓度为2U/μL，SiQDs溶液：ExoIII溶液体积比10：3；37℃下孵育65min；ExoIII在80℃下放置10min灭活。
步骤(3)、(4)中，所述荧光分光光度计的激发波长分别设置为350nm和550nm，激发狭缝宽度为3nm，发射狭缝宽度为3nm。
所述的Tris-HCl浓度均为10mM，pH＝7.0，10mM MgCl2。
本发明的有益效果：
(1)本发明应用酶剪切放大策略实现荧光检测方法的信号放大；
(2)本发明引入富含T碱基的DNA，与Hg2+结合形成T-Hg2+-T双链结构实现对Hg2+的特异性检测；
(3)所提出的检测方法对Hg2+表现出令人满意的分析性能，检测限低6.67×10- 12mol/L(S/N＝3)，线性范围宽2×10-11–1×10-8mol/L。
附图说明
图1是本发明荧光方法用于检测汞离子的原理示意图。
图2是SiQDs的荧光激发和发射光谱图。
图3A是SiQDs、Rox-DNA和SiQDs+Rox-DNA的Zeta电势图；B是本发明荧光方法可行性分析中不同溶液的荧光光谱图。
图4A是不同浓度Hg2+标准溶液存在时溶液的荧光光谱图；B是Hg2+浓度对数与荧光强度比值间的线性关系图。
图5是各种干扰物存在时溶液的荧光比值变化图。
具体实施方式
下面结合说明书附图和实施例对本发明内容进行详细说明：
该检测方法的检测过程如图1所示，基本原理是：
首先合成了SiQDs，其最大激发波长为350nm，最大发射波长为414nm，如图2所示。硅量子点带正电与带负电的Rox-DNA通过静电作用相吸附(如图3A所示)，并猝灭荧光素(Rox)的荧光信号，当汞离子存在时，Rox-DNA通过T-Hg2+-T结构形成双链结构，Exo III剪切双链DNA，释放Rox和Hg2+达到信号放大的目的，实现了对汞离子的高灵敏检测。
该检测方法的可行性分析如下：
为了进一步验证方案的可行性，对SiQDs溶液和Rox-DNA溶液中加入各种物质前后的荧光变化情况进行考察。如图3B所示，SiQDs在414nm处有强荧光信号，Rox-DNA在607nm处有强荧光信号；当两者混合后，SiQDs在414nm处的荧光信号强度不变，Rox-DNA在607nm处的荧光信号会被SiQDs猝灭；单独加入汞离子，Rox-DNA在607nm处的荧光信号不会恢复，当继续加入Exo III后，Exo III剪切双链DNA，释放Rox，导致Rox在607nm处的荧光信号得到恢复。证明本发明的方法可用于汞离子的检测。
实施例1
(1)将制备的SiQDs配制成0.5mg/mL溶液待用；
(2)将50μL步骤(1)SiQDs溶液和50μL Rox-DNA(1μM)的溶液加入到265μLTris-HCl溶液中，37℃下孵育15min；
(3)向步骤(2)制备的若干份混合溶液中分别加入20μL的Hg2+标准溶液，浓度分别为2×10-11、5×10-11、1×10-10、3×10-10、5×10-10、1×10-9、3×10-9、5×10-9、1×10-8mol/L，在37℃下孵育40min；
(4)向步骤(3)制备混合溶液中加入15μL ExoIII(2U/μL)溶液混合，在37℃下孵育65min。然后在80℃下放置10min使ExoIII灭活。
(5)待混合液冷却后用荧光分光光度计在室温下分别检测溶液在414nm和607nm处的荧光强度，所得的谱图如图4A所示；同时获得荧光强度I607/I414比值与汞离子浓度对数的标准曲线如图4B所示；线性方程为：I607/I414＝0.64128Log CHg2++7.50114，相关系数R2＝0.99504，检测限为6.67×10-12mol/L(S/N＝3)，线性范围2×10-11–1×10-8mol/L。
(6)按照步骤(1)～(5)的操作，将上述已知浓度的Hg2+溶液用待测的Hg2+溶液替换，用荧光分光光度计在室温下分别检测溶液在414nm和607nm处的荧光强度，计算I607/I414比值为1.49569，代入步骤(5)中的标准曲线，得出待测溶液中汞离子的浓度为4.31735×10-10mol/L。
其中，所述荧光分光光度计的激发波长分别设置为350nm和550nm，激发狭缝宽度为3nm，发射狭缝宽度为3nm。
所述的Tris-HCl浓度均为10mM，pH＝7.0，10mM MgCl2。
Hg2+检测选择性的考察：
为了研究本发明检测Hg2+的特异性，对Hg2+和其它金属离子进行检测。具体步骤如下：在1.5mL离心管中，将50μL(0.5mg/mL)SiQDs溶液和50μL Rox-DNA(1μM)溶液加入到265μL Tris-HCl溶液中，37℃下孵育15min；然后分别加入20μL Hg2+(浓度为5×10-10mol/L)或K+、Ca2+、Na+、Mg2+、Fe2+、Fe3+、Cu2+、Pb2+、Ni2+、Mn2+、Cd2+和所有金属离子的混合溶液(其它金属离子浓度均为5×10-8mol/L)，在37℃下孵育40min；最后加入15μL ExoIII(2U/μL)溶液混合，在37℃下孵育65min。然后在80℃下放置10min使ExoIII灭活。待混合液冷却后用荧光分光光度计在室温下分别检测溶液在414nm和607nm处的荧光强度。如图5所示，K+、Ca2+、Na+、Mg2+、Fe2+、Fe3+、Cu2+、Pb2+、Ni2+、Mn2+、Cd2+对荧光强度I607/I414比值基本无影响，只有加入Hg2+会使荧光强度I607/I414比值明显降低。以上结果说明该荧光传感方法可以实现Hg2+的特异性检测。
其中，所述荧光分光光度计的激发波长分别设置为350nm和550nm，激发狭缝宽度为3nm，发射狭缝宽度为3nm。

Claims (6)

1.一种基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法，其特征在于，包括如下步骤：
(1)将制备的SiQDs溶液与Rox-DNA溶液加入到一定体积的Tris-HCl溶液中，并在37℃孵育15min；其中，DNA为富含T碱基的DNA；
(2)向步骤(1)制备混合溶液中加入已知浓度的Hg2+溶液混合，并在37℃孵育40min；
(3)向步骤(2)制备混合溶液中加入ExoIII溶液混合，在37℃孵育65min，然后在高温下灭活ExoIII，待混合液冷却后用荧光分光光度计在室温下分别检测溶液在414nm和607nm处的荧光强度I414和I607，得出荧光强度I607/I414比值与汞离子浓度对数的标准曲线；
(4)按照步骤(1)～(3)的操作，将上述已知浓度的Hg2+溶液用待测的Hg2+溶液替换，用荧光分光光度计在室温下分别检测溶液在414nm和607nm处的荧光强度I414和I607，计算此时I607/I414比值并代入步骤(3)中的标准曲线，得出待测溶液中汞离子的浓度。
2.如权利要求1所述的基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法，其特征在于，步骤(1)中，SiQDs溶液浓度为0.5mg/mL，Rox-DNA溶液的浓度为1μM，SiQDs溶液，Rox-DNA溶液及Tris-HCl的体积比为10：10：53。
3.如权利要求1所述的基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法，其特征在于，步骤(2)中，Hg2+溶液的浓度为2×10-11–1×10-8mol/L；SiQDs溶液：Hg2+溶液体积比5：2。
4.如权利要求1所述的基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法，其特征在于，步骤(3)中，ExoIII溶液的浓度为2U/μL，SiQDs溶液：ExoIII溶液体积比10：3；ExoIII在80℃下放置10min灭活。
5.如权利要求1所述的基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法，其特征在于，步骤(3)、(4)中，所述荧光分光光度计的激发波长分别设置为350nm和550nm，激发狭缝宽度为3nm，发射狭缝宽度为3nm。
6.如权利要求1所述的基于硅量子点、荧光素标记的DNA、剪切酶的汞离子荧光检测方法，其特征在于，所述的Tris-HCl浓度均为10mM，pH＝7.0，10mM MgCl2。
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