CN113884477A - 一种基于牡丹状金纳米花和荧光信号放大的铅离子检测方法 - Google Patents
一种基于牡丹状金纳米花和荧光信号放大的铅离子检测方法 Download PDFInfo
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Abstract
本发明涉及了一种基于牡丹状金纳米花和荧光信号放大的铅离子检测方法，包括以下步骤：采用水热还原法制备胶体金种子溶液，利用对苯二酚合成方法，在胶体金种子溶液基础上，制备得到金纳米花。用还原剂三（2‑羧乙基）膦与cDNA反应脱二硫键，加入金纳米花与cDNA链通过金硫键结合，加入Pb2+适配体链与cDNA形成双链，离心，重悬，制备传感器。荧光基团标记的Pb2+适配体链与cDNA形成的双链靠近金纳米花，荧光猝灭，通过加入Pb2+，Pb2+与荧光标记的Pb2+适配体链形成G‑四聚体结构，使得适配体链远离金纳米花，荧光恢复，并且加入剪切酶，剪切G‑四聚体结构，释放Pb2+，释放的Pb2+进一步参与反应，实现信号放大。通过两者荧光信号的变化，得到了用于检测Pb2+的适配体传感器。同其它用于Pb2+含量检测的荧光传感器相比，所制备的适配体传感器具有灵敏度高、重复性好、准确度高的优点。
Description
技术领域
本发明涉及一种基于牡丹状金纳米花和荧光信号放大的铅离子检测方法，尤其涉及针对特异性识别Pb2+的荧光基团标记的适配体链的信号分子的制备方法。
背景技术
随着我国工业的飞速发展，大量重金属废水及废弃物未经处理直接排入江、河、湖、海、地下水等自然环境，造成水体的污染；同时，我国存在大片污灌区，重金属在污灌区的农田中长期积累，造成土壤的污染。据报道，重金属污染以镉最为严重，其次是汞、铅。动植物从环境中摄取重金属，经过食物链累积并最终进入人体，造成急性或慢性中毒反应。以铅离子（Pb2+）为例，铅中毒病症早期表现为贫血、衰弱和疲倦感，出现运动和感觉异常，严重时出现末梢神经炎等神经症状，对人体健康造成了极大危害。因此，建立简单、快速、高灵敏的Pb2+检测方法，对环境及生物样品的分析检测有重要的意义。Pb2+的常规分析方法主要包括电感耦合等离子质谱法（ICP-MS）、原子吸收光谱法（AAS）和原子发射光谱法（AES）、原子荧光光谱法（AFS）及电化学法等；这些方法可实现对铅的特异性、高灵敏检测，但大多需要昂贵精密的仪器，操作步骤复杂，需专业人员进行操作，检测成本较高，难以满足现场大批量检测的实际需要。而基于适配体的荧光传感器是利用目标分子与识别元件特异性结合过程产生的荧光信号为测量基础时，根据荧光信号强度的变化来实现对目标物的定性或定量分析，具有特异性高、操作简便、稳定性好的特点，因此，未来Pb2+的检测方法必定朝着快速灵敏化、便携化、自动化的方向发展。
发明内容
本发明涉及一种基于牡丹状金纳米花和荧光信号放大的铅离子检测方法。
一种基于牡丹状金纳米花和荧光信号放大的铅离子检测方法，包括以下步骤：
所述牡丹状金纳米花的制备：用经典的柠檬酸盐水热还原法制备了胶体金种子溶液，将1 mL氯金酸溶液加入到99 mL的超纯水中，将混合液加热至沸腾，用1~5 mL柠檬酸钠溶液在剧烈搅拌下快速加入到上述煮沸的溶液，再煮沸10 min，制得胶体金种子溶液；利用对苯二酚合成方法制备金纳米花，在搅拌条件下，向100 mL的超纯水中加入300~400 μL氯金酸溶液、200~400 μL柠檬酸钠溶液和1~2 mL胶体金种子溶液，然后将混合溶液加到1~3mL对苯二酚溶液中，孵育30 min，制得金纳米花，保存，供下一步使用；
所述荧光适配体传感器的制备：用还原剂三（2-羧乙基）膦与cDNA反应脱二硫键，加入金纳米花与cDNA链通过金硫键结合，加入Pb2+适配体链与cDNA形成双链，离心，重悬，制备得到传感器；
所述荧光适配体传感器用于铅离子的检测：在上述传感器中加入200 μL浓度范围在0~1000 nM不同浓度梯度的铅离子和剪切酶的混合溶液，充分反应，反应完成后需在65℃放置20 min，离心。荧光基团标记的Pb2+适配体链与cDNA形成的双链靠近金纳米花，荧光猝灭，通过加入Pb2+，Pb2+与荧光标记的Pb2+适配体链形成G-四聚体结构，使得适配体链远离金纳米花，荧光恢复，并且加入剪切酶，剪切G-四聚体结构，释放Pb2+，释放的Pb2+进一步参与反应，实现信号放大。两者荧光信号的变化，得到了用于检测Pb2+的适配体传感器。
所述的氯金酸的质量浓度是1%~3%，柠檬酸钠的质量浓度是1%~3%。
所述的cDNA序列为5’-SH-TTTTTTTTTACCCAGCCACCC-3’。
所述的荧光基团标记的Pb2+适配体序列为5’-AAAGGGTGGGTGGGTGGGT-3’。
所述的Pb2+适配体的3’端的荧光基团分别为Cy3，Cy5，FAM，ROX中的一种。
所述的剪切酶分别为ExoI，ExoIII，RecJf酶中的一种。
本发明涉及的传感器中，以荧光基团标记的Pb2+适配体链的荧光信号作为信号分子，同其它用于Pb2+的荧光传感器相比，所制备的基于内循环信号放大策略的适体传感器具有灵敏度高、重复性好、准确度高的优点。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是牡丹状金纳米花和荧光信号放大的铅离子检测方法。
图2是胶体金种子和牡丹状金纳米花的紫外光谱图。
图3是牡丹状金纳米花的透射电镜图。
具体实施方式
下面结合附图和具体实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1
具体步骤如下：
（1）牡丹状金纳米花的制备：用经典的柠檬酸盐水热还原法制备了胶体金种子溶液，将1 mL 1%（w/v）氯金酸溶液加入到99 mL的超纯水中，将混合液加热至沸腾，用1 mL 1%（w/v）柠檬酸钠溶液在剧烈搅拌下快速加入到上述煮沸的溶液，再煮沸10 min，制得胶体金种子溶液；利用对苯二酚合成方法制备金纳米花，在搅拌条件下，向100 mL的超纯水中加入300 μL 1%（w/v）氯金酸溶液、200 μL 1%（w/v）柠檬酸钠溶液和1 mL胶体金种子溶液，然后将混合溶液加到1 mL对苯二酚溶液中，孵育30 min，制得金纳米花（图2），保存，供下一步使用；
（2）荧光适配体传感器的制备：用还原剂三（2-羧乙基）膦与cDNA反应脱二硫键，加入金纳米花与cDNA链通过金硫键结合，加入Pb2+适配体链与cDNA形成双链，离心，重悬，制备得到传感器；
（3）荧光适配体传感器用于铅离子的检测：在上述传感器中加入200 μL浓度范围在0~1000 nM不同浓度梯度的铅离子和剪切酶的混合溶液，充分反应，反应完成后需在65℃放置20 min，离心。荧光基团标记的Pb2+适配体链与cDNA形成的双链靠近金纳米花，荧光猝灭，通过加入Pb2+，Pb2+与荧光标记的Pb2+适配体链形成G-四聚体结构，使得适配体链远离金纳米花，荧光恢复，并且加入剪切酶，剪切G-四聚体结构，释放Pb2+，释放的Pb2+进一步参与反应，实现信号放大。两者荧光信号的变化，得到了用于检测Pb2+的适配体传感器。
实施例2
具体步骤如下：
（1）牡丹状金纳米花的制备：用经典的柠檬酸盐水热还原法制备了胶体金种子溶液，将1 mL 2%（w/v）氯金酸溶液加入到99 mL的超纯水中，将混合液加热至沸腾，用3 mL 2%（w/v）柠檬酸钠溶液在剧烈搅拌下快速加入到上述煮沸的溶液，再煮沸10 min，制得胶体金种子溶液；利用对苯二酚合成方法制备金纳米花，在搅拌条件下，向100 mL的超纯水中加入350 μL 2%（w/v）氯金酸溶液、300 μL 2%（w/v）柠檬酸钠溶液和2 mL胶体金种子溶液，然后将混合溶液加到2 mL对苯二酚溶液中，孵育30 min，制得金纳米花，保存，供下一步使用；
（2）荧光适配体传感器的制备：用还原剂三（2-羧乙基）膦与cDNA反应脱二硫键，加入金纳米花与cDNA链通过金硫键结合，加入Pb2+适配体链与cDNA形成双链，离心，重悬，制备得到传感器；
（3）荧光适配体传感器用于铅离子的检测：在上述传感器中加入200 μL浓度范围在0~1000 nM不同浓度梯度的铅离子和剪切酶的混合溶液，充分反应，反应完成后需在65℃放置20 min，离心。荧光基团标记的Pb2+适配体链与cDNA形成的双链靠近金纳米花，荧光猝灭，通过加入Pb2+，Pb2+与荧光标记的Pb2+适配体链形成G-四聚体结构，使得适配体链远离金纳米花，荧光恢复，并且加入剪切酶，剪切G-四聚体结构，释放Pb2+，释放的Pb2+进一步参与反应，实现信号放大。两者荧光信号的变化，得到了用于检测Pb2+的适配体传感器。
所制备的荧光传感器对Pb2+的检测具有准确度高，线性范围宽（0.5×10-10~2×10-7 mol/L），检测下限低（0.1×10-10 mol/L）的特点。同时，对实际样品的检测结果表明所制备的传感器具有非常好的实际应用价值。
以上实施例只是为了说明本发明，而不是对本发明的限制。在上述说明的基础上，可以对本发明作许多改进和改变。在所附权利要求书的范围内，本发明可以有不同于上述的其它实现方式，选用其它试剂材料、调整孵育时间等方法均在本发明专利要求范围之内。
Claims (2)
1.一种基于牡丹状金纳米花和荧光信号放大的铅离子检测方法，其特征在于，包括以下步骤：
（1）牡丹状金纳米花的制备：用经典的柠檬酸盐水热还原法制备了胶体金种子溶液，将1 mL氯金酸溶液加入到99 mL的超纯水中，将混合液加热至沸腾，用1~5 mL柠檬酸钠溶液在剧烈搅拌下快速加入到上述煮沸的溶液，再煮沸10 min，制备得到胶体金种子溶液；利用对苯二酚合成方法制备金纳米花，在搅拌条件下，向100 mL的超纯水中加入300~400 μL氯金酸溶液、200~400 μL柠檬酸钠溶液和1~2 mL胶体金种子溶液，然后将混合溶液加到1~3 mL对苯二酚溶液中，孵育30 min，制得金纳米花，保存，供下一步使用；
（2）荧光适配体传感器的制备：用还原剂三（2-羧乙基）膦与cDNA反应脱二硫键，加入金纳米花与cDNA链通过金硫键结合，加入Pb2+适配体链与cDNA形成双链，离心，重悬，制备得到传感器；
（3）荧光适配体传感器用于铅离子的检测：在上述传感器中加入200 μL浓度范围在0~1000 nM不同浓度梯度的铅离子和剪切酶的混合溶液，充分反应，反应完成后需在65℃放置20 min，离心；荧光基团标记的Pb2+适配体链与cDNA形成的双链靠近金纳米花，荧光猝灭，通过加入Pb2+，Pb2+与荧光标记的Pb2+适配体链形成G-四聚体结构，使得适配体链远离金纳米花，荧光恢复，并且加入剪切酶，剪切G-四聚体结构，释放Pb2+，释放的Pb2+进一步参与反应，实现信号放大；两者荧光信号的变化，得到了用于检测Pb2+的适配体传感器。
2.根据权利要求1所述的一种基于牡丹状金纳米花和荧光信号放大的铅离子检测方法，其特征在于，步骤（2）中，所述的cDNA序列为5’-SH-TTTTTTTTTACCCAGCCACCC-3’，所述荧光基团标记的Pb2+适配体序列为5’-AAAGGGTGGGTGGGTGGGT-3’，所述的Pb2+适配体的3’端的荧光基团分别为Cy3，Cy5，FAM，ROX中的一种，所述的剪切酶分别为ExoI，ExoIII，RecJf酶中的一种。
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