CN111024793A - 生物分子完全免固定的纸基电化学传感器的构建 - Google Patents

生物分子完全免固定的纸基电化学传感器的构建 Download PDF

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CN111024793A
CN111024793A CN202010021198.7A CN202010021198A CN111024793A CN 111024793 A CN111024793 A CN 111024793A CN 202010021198 A CN202010021198 A CN 202010021198A CN 111024793 A CN111024793 A CN 111024793A
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于京华
史慧慧
王衍虎
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Abstract

本发明公开了一种完全免固定高效检测miRNA的分析策略。本方法首先在微流控纸芯片原位生长金纳米，随后通过羰基和金的相互作用将葫芦[7]脲固定在纸工作电极上。由于带负电荷的磷酸二酯主链与葫芦[7]脲的负羰基之间的排斥作用，亚甲基蓝标记的探针几乎不能与葫芦[7]脲形成配合物，因此不能有效地产生电化学响应。当目标物和外切酶加入到溶液后，亚甲基蓝标记的探针被外切酶剪切释放亚甲基蓝标记的单核苷酸。由于葫芦[7]脲的超分子识别能力和单核苷酸具有超低的负电荷，亚甲基蓝标记的单核苷酸被葫芦[7]脲捕获产生可检测的电信号实现对目标物的量化检测。本方法不具有复杂的材料生长和生物标记过程，省时省力，操作简单，检测快速，为其他疾病相关生物标记物的小型化检测提供一个途径。

Description

生物分子完全免固定的纸基电化学传感器的构建
技术领域
本发明涉及纳米材料技术，生物标记技术及纸芯片技术领域，更具体的说是生物分子完全免固定的纸基电化学传感器的构建。
背景技术
作为一种小的非编码的RNA分子，miRNA在细胞增值、遗传、基因分化等生物学过程中具有重要的作用。一些miRNA还与人类癌症的发生、病毒感染和肿瘤的治疗相关，是早期疾病的诊断、发病机制的研究的重要生物标志物。到目前为止，许多分析方法-化学发光、电致化学发光、光电化学、电化学-已经实现了对miRNA的检测。其中，电化学传感由于灵敏度高、选择性好、易于微型化等优点收到广泛关注。
对于大多数的电化学传感器，电化学材料和生物识别探针通过化学或物理作用固定在电极的表面，通过DNA结构的变化或电极表面电性质的变化实现对目标物的检测。固定过程存在良好的可重复性、灵敏度高等优点，也存在许多缺点，例如耗费时间长、操作复杂、用于连接基团的试剂增加了成本、固定条件的最优化、电极表面空间位阻可能导致探针几何变化等。因此，开发无固定化的电化学传感方法实现简单、快速、低成本的生物分析是最为理想的方法。
发明内容
针对现有技术不足，本发明提供了生物分子完全免固定的纸基电化学传感器的构建。本发明通过以下措施实现：
（1）在计算机上用Adobe illustrator CS4设计纸芯片的图案，将设计好的打印图案通过蜡打印机打印在A4色谱纸上，然后将打印过的色谱纸放在烘箱中，在100 ℃加热60 s形成疏水区域和亲水的工作区域；
（2）用丝网印刷技术在步骤（1）中获得的纸芯片上印刷三电极体系（碳工作电极、碳对电极和Ag/AgCl参比电极）；
（3）利用原位还原法在碳电极的工作区域生长金纳米，其具体步骤为：首先，20 μL金纳米粒子溶液滴涂在电极表面，在室温下干燥，此过程重复三次，随后称取0.013 g盐酸羟胺溶解于1 mL去离子水中，加入1 mL质量分数1 %的氯金酸，充分摇晃后，量取20 μL滴在电极表面，室温下干燥即可，最后，将电极在1 mM葫芦[7]脲中培育1 h;
（4）取30 μL 10 μM亚甲基蓝标记的DNA 和20 μL 不同浓度的miRNA溶解在37 μLTris-HCl 缓冲液中，在37 ℃下培育2 h，然后将3 µL T7 核酸外切酶 (2 U/µL) 和 10 µL1× NEB 缓冲液加入到上述溶液中，在37 ℃下培育1 h;
（5）将步骤（4）中获得的溶液添加到磷酸盐缓冲溶液中，然后将上述溶液滴加到步骤（3）的电极的工作区域，利用三电极系统通过微分脉冲伏安法进行电化学信号检测。
本发明所述的金种子溶液合成过程是：取90 mL二次水置于三口烧瓶中并加热到90 ℃，然后加入0.5 - 0.8 mL质量分数为1 %的氯金酸溶液，持续加热至96 ℃，反应1 min后，加入2.5 mL质量分数为1 %的柠檬酸钠，在搅拌下反应15 min，得到金纳米粒子溶液。
本发明所述的1×NEB 缓冲液配制过程是取20 mM Tris-HAc缓冲液，依次加入0.2145g四水乙酸镁、0.49 g醋酸钾、0.0154 g的二硫苏糖醇，随后用醋酸调节溶液pH到7.9，定容到100 mL。
本发明的有益效果：
（1）该方法以色谱纸为基底，且不使用额外的试剂，成本低廉。
（2）该方法不具有复杂的材料生长和生物标记过程，操作简单，过程易控制。
（3）该方法分析快速，耗时少。
（4）该方法具有较高的灵敏度和可重复性，为其他疾病相关生物标记物的高效检测提供一个方式。
具体实施方式
为了进一步说明生物分子完全免固定的纸基电化学传感器的构建，本实施例按照本发明技术方案进行实施，给出具体的实施方式，但本发明不仅限于下面的实施例。
实施例1生物分子完全免固定的纸基电化学传感器的构建
（（1）在计算机上用Adobe illustrator CS4设计纸芯片的图案，将设计好的打印图案通过蜡打印机打印在A4色谱纸上，然后将打印过的色谱纸放在烘箱中，在100 ℃加热60 s形成疏水区域和亲水的工作区域；
（2）用丝网印刷技术在步骤（1）中获得的纸芯片上印刷三电极体系（碳工作电极、碳对电极和Ag/AgCl参比电极）；
（3）利用原位还原法在碳电极的工作区域生长金纳米，其具体步骤为：取90 mL二次水置于三口烧瓶中并加热到90 ℃，然后加入0.8 mL质量分数为1 %的氯金酸溶液，持续加热至96 ℃，反应1 min后，加入2.5 mL质量分数为1 %的柠檬酸钠，在搅拌下反应15 min，得到金纳米粒子溶液；取20 μL上述制备的金纳米粒子溶液滴涂在电极表面，在室温下干燥，此过程重复三次，随后称取0.013 g盐酸羟胺溶解于1 mL去离子水中，加入1 mL质量分数1 %的氯金酸，充分摇晃后，量取20 μL滴在电极表面，室温下干燥即可，最后，将电极在1 mM葫芦[7]脲中培育1 h;
（4）先配制1×NEB 缓冲液，取20 mM Tris-HAc缓冲液，依次加入0.2145g四水乙酸镁、0.49 g醋酸钾、0.0154 g的二硫苏糖醇，随后用醋酸调节溶液pH到7.9，定容到100 mL；然后取30 μL 10 μM亚甲基蓝标记的DNA 和20 μL 不同浓度的miRNA溶解在37 μL Tris-HCl 缓冲液中，在37 ℃下培育2 h，然后将3 µL T7 核酸外切酶 (2 U/µL) 和 10 µL 1× NEB 缓冲液加入到上述溶液中，在37℃下培育1 h;
（5）将步骤（4）中获得的溶液添加到磷酸盐缓冲溶液中，然后将上述溶液滴加到步骤（3）的电极的工作区域，利用三电极系统通过微分脉冲伏安法进行电化学信号检测。
序列表
<110> 济南大学
<120> 生物分子完全免固定的纸基电化学传感器的构建
<130> 2020
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
mbacccctat cacgattag 19
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<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 2
uuaaugcuaa ucgugauagg ggu 23

Claims (3)

1.生物分子完全免固定的纸基电化学传感器的构建，其特征在于该方法包括以下步骤：
（1）在计算机上用Adobe illustrator CS4设计纸芯片的图案，将设计好的打印图案通过蜡打印机打印在A4色谱纸上，然后将打印过的色谱纸放在烘箱中，在100 ℃加热60 s形成疏水区域和亲水的工作区域；
（2）用丝网印刷技术在步骤（1）中获得的纸芯片上印刷三电极体系（碳工作电极、碳对电极和Ag/AgCl参比电极）；
（3）利用原位还原法在碳电极的工作区域生长金纳米，其具体步骤为：首先，20 μL金纳米粒子溶液滴涂在电极表面，在室温下干燥，此过程重复三次，随后称取0.013 g盐酸羟胺溶解于1 mL去离子水中，加入1 mL质量分数1 %的氯金酸，充分摇晃后，量取20 μL滴在电极表面，室温下干燥即可，最后，将电极在1 mM葫芦[7]脲中培育1 h;
（4）取30 μL 10 μM亚甲基蓝标记的DNA 和20 μL 不同浓度的miRNA溶解在37 μLTris-HCl 缓冲液中，在37℃下培育2 h，然后将3 µL T7 核酸外切酶 (2 U/µL) 和 10 µL1× NEB 缓冲液加入到上述溶液中，在37 ℃下培育1 h;
（5）将步骤（4）中获得的溶液添加到磷酸盐缓冲溶液中，然后将上述溶液滴加到步骤（3）的电极的工作区域，利用三电极系统通过微分脉冲伏安法进行电化学信号检测。
2.根据权利要求1所述的生物分子完全免固定的纸基电化学传感器的构建，其特征是金种子溶液合成过程是：取90 mL二次水置于三口烧瓶中并加热到90 ℃，然后加入0.5 -0.8 mL质量分数为1 %的氯金酸溶液，持续加热至96 ℃，反应1 min后，加入2.5 mL质量分数为1 %的柠檬酸钠，在搅拌下反应15 min，得到金纳米粒子溶液。
3.根据权利要求1所述的生物分子完全免固定的纸基电化学传感器的构建，其特征是1×NEB 缓冲液配制过程是取20 mM Tris-HAc缓冲液，依次加入0.2145g四水乙酸镁、0.49 g醋酸钾、0.0154 g的二硫苏糖醇，随后用醋酸调节溶液pH到7.9，定容到100 mL。
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