CN108896639B - lncRNA MEG3双重检测电化学基因传感器，其制备方法与应用 - Google Patents
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Abstract
本发明涉及lncRNA MEG3双重检测电化学基因传感器，其制备方法与应用，基于酶辅助目标循环信号放大与DNA链杂交（HCR）信号增强策略，采用二硫化钨/树枝状金纳米复合材料修饰电极以及碱基特异性互补配对，通过双信号探针实现对双组分的超灵敏定量分析。本发明的电化学基因传感器检测双组分的线性范围为1fM~100pM，检测限低，分别为0.25fM和0.3fM；具有优异的灵敏度、重复性、特异性和稳定性，并且成本低，因而在生化研究和临床分析等领域具有较大的应用价值。
Description
技术领域
本发明属于材料制备和检测分析技术领域，具体涉及一种lncRNA MEG3双重检测电化学基因传感器，其制备方法与应用。
背景技术
lncRNA MEG3作为肺癌早期诊断和预后效果检测的一种标志物具有广阔的前景。然而，超灵敏地同时检测两个lncRNA特征序列仍具有挑战性。电化学传感器因简单高效、低成本、高灵敏度等优异的特性，常被应用核酸电化学传感器的构建。目前，对电化学生物传感器同时检测lncRNA双组分特征序列还没有相关的报道。
发明内容
本发明的目的在于提供一种lncRNA MEG3双重检测电化学基因传感器，其制备方法与应用。基于酶辅助目标循环信号放大反应，固定化lncRNA分子探针和导电纳米材料结合，使得标记了电化学活性分子的单链能够发生杂交反应，从而实现以电化学方法同时进行双组分的检测。
为实现上述目的，本发明采用如下技术方案：
一种lncRNA MEG3双重检测电化学基因传感器的制备方法，包括如下步骤：
(1)以金电极作为工作电极，取二硫化钨分散液滴于已经洁净处理过的金电极表面，采用电化学沉积法制得二硫化钨/树枝状金纳米复合材料的修饰金电极；
(2)将巯基化的捕获探针混合物溶液滴于电极表面，再用巯基己醇封闭电极表面多余的活性位点，得到捕获探针修饰金电极；
(3)制备磁性微球，将表面含羧基官能团的磁性微球与5’端氨基修饰的引物单链通过共价键结合，加入双重检测对象T1和T2温育后，添加核糖核酸酶RNase A，在RNase A作用后磁性分离，获取上清液，即得到酶辅助目标循环信号放大反应的中间探针；
(4)将步骤(2)获取的捕获探针修饰金电极浸没在步骤(3)获取的上清液中混合孵育，再与辅助探针孵育后，将修饰金电极浸没在以二茂铁和亚甲基蓝作为信号分子的信号探针溶液中进行杂交链式反应，获得所述基因传感器。
本发明所述的电化学基因传感器的制备方法，所述的步骤(1)中制备二硫化钨/树枝状金纳米复合电极的沉积电压为-0.1V，沉积电量为0.05～0.2C。优选沉积电量为0.15C。在-0.1V，0.15C条件下，二硫化钨/树枝状金纳米复合电极的亲水性最佳，有利于固定在其表面的DNA链保持活性。
所述的步骤(1)中，捕获探针的修饰时间为16小时，捕获探针溶液的最优浓度为2μM。
所述的步骤(2)中，检测对象，即lncRNA MEG3双重特征序列分别为：
T1：5’-CCUCUUAUUUAUUCUGUUCAUUCUGUUCGU-3’
T2：5’-UCCACUUGUGUUCGUUUCUGAAUAUCCUUU-3’
所述的步骤(4)中，信号探针溶液的浓度为2μM；信号链杂交最优反应时间为65分钟。
本发明的另一目的在于提供上述制备方法制备的电化学基因传感器，以及所述电化学基因传感器在lncRNA MEG3检测中的应用。
本发明的方法制备的传感器具有如下有益效果：
(1)高灵敏度。本发明利用链杂交反应、酶辅助靶标循环信号放大以及电极界面修饰纳米材料的联用，不仅放大了电化学信号的读出，而且纳米材料大大加快了电子的传递，使得传感器检测更为稳定，提高检测灵敏度。实验得出传感器的线性检测范围为1fM～100pM，检测限分别为0.25fM和0.3fM。
(2)高特异性。多种不同序列对本检测体系均无明显的干扰。
(3)结果准确。回收率均在93～105％之间。
(4)高效率。本发明能实现对两个目标物的同时检测。
本发明的电化学基因传感器基于链杂交反应、酶辅助靶标循环信号放大以及电极界面修饰纳米材料的联用，实现对lncRNA双重特征序列片段的同时检测，具有灵敏度高、选择性好、分析快速、结果准确等优点，可以实现低浓度lncRNA的检测，具有良好的应用前景。
附图说明
图1是本发明传感器的制备方式流程图；
图2是本发明的传感器的机理验证；
图3是本发明的不同信号放大策略的传感器比较；
图4是本发明检测lncRNA双重特征序列片段的线性关系图。
具体实施方式
为使本发明的目的、技术方案和特点更加清楚，下面结合实施例对本发明作进一步的说明。
实施例1
本实施例说明本发明基因传感器的制备方法，如图1所示，包括如下步骤：
(1)采用电化学沉积方式制备二硫化钨/树枝状金纳米复合材料修饰电极；
传感器为三电极体系构建的传感器，金电极为工作电极，饱和甘汞电极为参比电极，铂电极为对电极。
金电极作为工作电极，将金电极在丙酮、乙醇、水中依次超声清洗后用氮气吹干。取20μL 1mg/mL的二硫化钨分散液滴于已经洁净处理过的金电极表面，空气中干燥，获得二硫化钨修饰的金电极，之后采用电化学沉积法，设置沉积电压-0.1V，沉积电量0.15C，制得树枝状金纳米结构。
(2)电极表面孵育捕获探针；
在电极表面滴加20μL浓度均为2μM的巯基化的捕获探针1(CP1)和捕获探针2(CP2)，探针序列如下：
CP1：SH-(CH2)6-GTATCTCCCCCTTGT
CP2：SH-(CH2)6-GCCCCTTATGTATGT
室温下培养16h，缓冲液冲洗后，在氮气流中干燥，用巯基己醇封闭电极表面多余的活性位点，得到捕获探针修饰金电极。
(3)核酸外切酶RNaseA辅助信号放大
采用溶剂热法制备Fe3O4@C磁性微球，将表面含羧基官能团的磁性微球与5’端氨基修饰的引物单链(P1和P2)通过共价键结合。加入检测对象(T1或T2)，37℃温育1h后加入核糖核酸酶RNaseA，在RNaseA作用下磁性分离，获取上清液，即得到酶辅助目标循环信号放大反应的中间探针IT1和IT2。
其中，引物序列如下：
P1：
NH2-(CH2)6-TTTTTTGGAATAACGAACAGAAUGAACAGAAU|AAAUAAGAGGACAAGGGGGAGATAC；
P2：
NH2-(CH2)6-TTTTTTCAGGACAAAGGAUAUUCAGAAACGAA|CACAAGUGGAACATACATAAGGGGC；
“|”处为RNaseA切割位点；
(4)将步骤(2)获取的修饰金电极在20μL步骤(3)获取的中间探针中孵育2h，之后在20μL浓度均为2μM的辅助探针H1和辅助探针H2混合孵育2h。
所述辅助探针H1和H2序列如下：
H1：CTCTTATTTAAGAGCGCC；
H2：CCACTTGTGGAAGAGGTG；
将含上述探针的金电极浸没在20μL浓度为2μM的信号探针溶液中进行杂交链式反应，65min，37℃下完成自组装，获得所述基因传感器。
其中信号探针选用二茂铁(Fc)和亚甲基蓝(MB)标记信号探针，探针序列如下，浓度均为2μM：
S1，Fc：Fc-(CH2)6-ACAAACAAAGAAGTGTGTTACGGCGCTCTT；
S2，Fc：Fc-(CH2)6-CACTTCTTTGTTTGTAAGAGCGCCGTAACA；
S1，MB：MB-(CH2)6-TCACAAACAAAGTTCCCGCGTCACCTCTTC；
S2，MB：MB-(CH2)6-GAACTTTGTTTGTGAGAAGAGGTGATCTAC。
实施例2
本实施例对实施例1的基因传感器进行机理验证。
设置4个实验组，分别为不加检测对象、加入单检测对象T1、加入单检测对象T2和加入双重检测对象(T1和T2)。利用DPV进行电化学检测，结果如图2所示。
可以看出，在未加入检测对象时，曲线a没有电化学信号响应。当加入单检测对象T1时，曲线b有单检测对象T1所对应的二茂铁的电化学响应。当加入单检测对象T2时，曲线c有单检测对象T2所对应的亚甲基蓝的电化学响应。同时加入检测对象T1和T2时，曲线d同时出现二茂铁和亚甲基蓝的电化学信号响应。这说明本发明的电化学基因传感器能同时检测双重lncRNA特征序列片段。
实施例3
本实施例对比了采用双重信号放大策略及不采用信号放大时传感器检测结果。
传感器的制备方式如实施例1所述，本发明采用RNaseA辅助信号放大以及IT1和IT2引起的杂交链式反应进行双重信号放大。
从图3可以看出，曲线a未采用双重信号放大策略制备的基因传感器没有明显的电化学响应，而曲线b采用双重信号放大策略制备的基因传感器同时出现优异的二茂铁和亚甲基蓝的电化学响应峰。这说明本发明的基因传感器能有效地增强电化学响应，超灵敏的检测超微量的生物分子。
实施例4
本实施例说明lncRNA双重特征序列片段检测的线性关系
分别加入不同浓度的检测对象T1/T2，检测结果如图4所示，可以看出，随检测目标物的浓度增加，差分脉冲伏安电流也逐渐增加。在1fM～100pM浓度范围内，差分脉冲伏安电流与目标物浓度呈良好的线性关系，检测限分别为0.25fM和0.3fM。
上述说明并非对本发明的限制，本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内做出的任何修改、添加或者替换，均应属于本发明保护范围。
Claims (10)
1.一种lncRNA MEG3双重检测电化学基因传感器的制备方法，其特征在于，包括如下步骤：
（1）以金电极作为工作电极，取二硫化钨分散液滴于已经洁净处理过的金电极表面，采用电化学沉积的方式制备二硫化钨/树枝状金纳米复合材料的修饰金电极；
（2）将巯基化的捕获探针混合物溶液滴于电极表面，再用巯基己醇封闭电极表面多余的活性位点，得到捕获探针修饰金电极；
（3）制备磁性微球，将表面含羧基官能团的磁性微球与5’端氨基修饰的引物单链通过共价键结合，加入双重检测对象T1和T2温育后，添加核糖核酸酶RNase A，在RNase A作用后磁性分离，获取上清液，即得到酶辅助目标循环信号放大反应的中间探针；
（4）将步骤（2）获取的捕获探针修饰金电极浸没在步骤（3）获取的上清液中混合孵育，再与辅助探针孵育后，将修饰金电极浸没在以二茂铁和亚甲基蓝作为信号分子的信号探针溶液中进行杂交链式反应，获得所述基因传感器。
2.根据权利要求1所述的制备方法，其特征在于，所述步骤（1）中，制备二硫化钨/树枝状金纳米复合电极的沉积电压为-0.1 V，沉积电量为0.05~0.2 C。
3.根据权利要求1所述的制备方法，其特征在于，所述步骤（1）中，制备二硫化钨/树枝状金纳米复合电极的沉积电压为-0.1 V，沉积电量为0.15 C。
4.根据权利要求1所述的制备方法，其特征在于，所述步骤（2）中，捕获探针的修饰时间为16小时。
5.根据权利要求1所述的制备方法，其特征在于，所述步骤（2）中，捕获探针混合物溶液的浓度为2 µM。
6.根据权利要求1所述的制备方法，其特征在于，所述步骤（3）中，检测对象，即lncRNAMEG3双重特征序列分别为：
T1：5’- CCUCUUAUUUAUUCUGUUCAUUCUGUUCGU -3’
T2：5’- UCCACUUGUGUUCGUUUCUGAAUAUCCUUU -3’。
7.根据权利要求1所述的制备方法，其特征在于，所述步骤（4）中，信号探针溶液的浓度为2 µM。
8.根据权利要求1所述的制备方法，其特征在于，所述步骤（4）中，杂交链式反应的时间为65分钟。
9.权利要求1~8任一项所述制备方法制备的电化学基因传感器。
10.权利要求9所述电化学基因传感器在lncRNA MEG3检测中的应用。
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