CN109580597B - 一种基于dna纳米管的电化学发光生物传感器及其制法和应用 - Google Patents
一种基于dna纳米管的电化学发光生物传感器及其制法和应用 Download PDFInfo
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Abstract
本发明公开了一种基于DNA纳米管的电化学发光生物传感器；以及所述生物传感器的制备方法及其检测甲基化酶和黄曲霉素的分析应用。本发明的技术方案是利用DNA纳米管放大信号技术，设计了电化学发光生物传感器，通过“on‑off”检测模式，实现了对甲基化酶和黄曲霉素的灵敏分析。目标甲基化酶引发了发夹DNA的甲基化和剪切反应，将Ru(phen)3 2+标记的纳米管组装到电极上，实现了甲基化酶的电化学发光检测。进一步利用纳米管上的适体结合目标黄曲霉素，纳米管结构破坏释放出信号探针，对黄曲霉素进行ECL检测。该DNA纳米管为研制生物传感器检测多种目标开发了新的信号放大技术。
Description
技术领域：
本发明涉及一种基于DNA纳米管的电化学发光生物传感器；以及所述生物传感器的制备方法及其检测甲基化酶和黄曲霉素的分析应用。
背景技术：
异常的甲基化酶和黄曲霉素会引发癌症[Chen,S.；Lv,Y.；Shen,Y.；Ji,J.et.al.ACS appl.Mater.interfaces.2018,10,6887-6894.]，因此定量检测甲基化酶和黄曲霉素十分重要。DNA具有理想的分子识别性能和精确的碱基配对能力[Zahid,M.；Kim,B.et.al.Nanoscale Res.Lett.2013,8,119.]具有可控粒径的DNA纳米管已经成为一种很有前景的纳米材料和分子诊断工具，可以通过多种灵活的方法制备[Maier,A.M.；Bae,W.et.al.ACS Nano.2017,11,1301-1306.]。DNA自组装方法具有可控性和结构的预测性[Surwade,S.P.；Zhao,S.；Liu,H.J.Am.Chem.Soc.2011,133,11868-11871.自组装的DNA纳米管可以进行药物输送[Wilner,O.I.；Orbach,R.et.al.Nat.Commun.2011,2,540–549.]。小分子探针可以扦插到DNA纳米管的双链中，研制生物传感器[Yao,W.；Wang,L.et.al.Biosens.Bioelectron.2009,24,3269-3274.]。Ru(phen)3 2+可以扦插于双链DNA的沟槽中作为ECL探针[Zhao,Y.；He,X.W.；Yin,X.B.Chem.Commun.2011,47,6419-6421.]。MB作为电化学探针可以通过π-π堆积作用与双链DNA结合[Gill,R.；Patolsky,F.；Katz,E.；Willner,I.Angew.Chem.Int.Ed.2005,44,4630-4633.]。
电化学方法简单、快速、方便，电化学生物传感器在疾病诊断中引起了极大的兴趣[Hou,T.；Li,W.；Liu,X.J.；Li,F.Anal.Chem.2015,87,11368-11374.]。电化学发光背景低、灵敏度高、仪器简单、可控性好[Liu,Z.；Qi,W.；Xu,G.Chem.Soc.Rev.2015,44,3117-3142.]。利用Ru(phen)3 2+结合超支化滚环放大反应研制了电化学发光生物传感器检测基因突变[Zhang,Y.；Wang,L.X.et.al.Chem.Commun.2017,53,2910-2913.]
本工作中利用DNA纳米管放大信号技术，设计了电化学发光生物传感器，通过“on-off”检测模式，实现了对甲基化酶和黄曲霉素的灵敏分析。目标甲基化酶引发了发夹DNA的甲基化和剪切反应，将Ru(phen)3 2+标记的纳米管组装到电极上，实现了甲基化酶的电化学发光检测。进一步利用纳米管上的适体结合目标黄曲霉素，纳米管结构破坏释放出信号探针，对黄曲霉素进行ECL检测。该DNA纳米管为研制生物传感器检测多种目标开发了新的信号放大技术。
发明内容：
本发明的目的之一提供一种基于DNA纳米管组装Ru(phen)3 2+信号探针的电化学发光生物传感器；以及所述生物传感器的制备方法及其检测甲基化酶和黄曲霉素的分析应用。
具体包括以下步骤：
步骤1.DNA纳米管-Ru(phen)3 2+探针的制备：三种DNA单链S1,S2和S3溶于TE缓冲溶液中，从95℃到室温进行退火反应，组装DNA纳米管。取50μL纳米管溶液与Ru(phen)3 2+(100μL,2mM)在37℃下温育7小时。
步骤2.ECL生物传感器的制备：将金电极用α-Al2O3粉磨光，用纯水冲洗并用氮气吹干，在0.5M H2SO4溶液中，进行电化学扫描，得到完整的循环伏安峰，冲洗干净。取20μL金胶滴加在电极表面，自然晾干。将10μL发夹DNA(1.0μM)滴在电极表面室温过夜，然后用1mMMCH反应1小时。接着将不同浓度的甲基化酶、剪切酶以及DNA纳米管-Ru(phen)3 2+信号探针顺序滴在电极表面反应2小时。再将不同浓度的黄曲霉素滴到电极表面，37℃下反应3小时。然后用PBS溶液冲洗电极。
步骤3.ECL检测：利用MPI-E电化学发光仪器，在200mM,pH 7.4含有20mM TPA的缓冲溶液中进行ECL检测，电位扫描范围是0.6V～1.6V，光电倍增管(PMT)是600V。
步骤4.电泳分析：取30μL样品1(1μM发夹DNA,8U的甲基化酶，10U的剪切酶)，20μL样品2(1μM发夹DNA,8U的甲基化酶)，20μL样品3(1μM发夹DNA,10U的剪切酶)于37℃下温育3h。接着将样品放到聚丙烯酰胺凝胶中，加入1×TAE缓冲液，先设置电压为180V，3min。然后设置电压为135V，电泳时间1.5小时。EB染色后进行电泳分析。
本发明研制了多功能电化学发光生物传感器，用于甲基化酶和黄曲霉素的分析。本发明与现有技术相比，主要优点在于，DNA纳米管能够负载大量的Ru(phen)3 2+信号分子，极大地放大了信号，提高了检测的灵敏度。利用甲基化酶的特异性剪切和黄曲霉素与适体的特异性结合检测2种目标，具有很高的选择性。该传感器具有很宽的线性范围和很低的检测限，在生物检测和实际应用中具有很高的潜力。
附图说明：
图1基于DNA纳米管信号探针检测甲基化酶和黄曲霉素的ECL生物传感器原理。
图2 DNA纳米管的透射电子显微镜(TEM)图。
图3电泳表征：(A)DNA纳米管的形成过程，(B)发夹DNA的甲基化过程。
图4(A)Ru(phen)3 2+的ECL信号-电位关系曲线，插图：Ru(phen)3 2+的ECL信号-时间关系曲线。(B)不同修饰电极的ECL信号-时间关系曲线(a)裸金电极,(b)无甲基化酶,(c)有甲基化酶,(d)有黄曲霉素。
图5(A)不同浓度目标甲基化酶对应的ECL响应。(B)ECL信号变化和甲基化酶浓度的关系，插图：测定甲基化酶的矫正曲线。
图6(A)不同浓度目标黄曲霉素对应的ECL响应。(B)ECL信号变化和甲基化酶浓度的关系，插图：测定甲基化酶的矫正曲线。
具体实施方式：
实施例1.ECL生物传感器的制备及对目标的检测
DNA纳米管-Ru(phen)3 2+探针的制备：三种DNA单链S1,S2和S3溶于TE缓冲溶液中，从95℃到室温进行退火反应，组装DNA纳米管。取50μL纳米管溶液与Ru(phen)3 2+(100μL,2mM)在37℃下温育7小时。
ECL生物传感器的制备及目标检测：将金电极用α-Al2O3粉磨光，用纯水冲洗并用氮气吹干，在0.5M H2SO4溶液中，进行电化学扫描，得到完整的循环伏安峰，冲洗干净。取20μL金胶滴加在电极表面，自然晾干。将10μL发夹DNA(1.0μM)滴在电极表面室温过夜，然后用1mM MCH反应1小时。接着将不同浓度的甲基化酶、剪切酶以及DNA纳米管-Ru(phen)3 2+信号探针顺序滴在电极表面反应2小时。再将不同浓度的黄曲霉素滴到电极表面，37℃下反应3小时。然后用PBS溶液冲洗电极。在200mM,pH 7.4含有20mM TPA的缓冲溶液中进行ECL信号检测。
实施例2.ECL生物传感器的制备及对目标的检测
将“三种DNA单链S1,S2和S3溶于TE缓冲溶液中，从95℃到室温进行退火反应，组装DNA纳米管。取50μL纳米管溶液与Ru(phen)3 2+(100μL,2mM)在37℃下温育7小时。”改为“三种DNA单链S1,S2和S3溶于TE缓冲溶液中，从95℃到室温进行退火反应，组装DNA纳米管。取50μL纳米管溶液与Ru(phen)3 2+(100μL,2mM)在37℃下温育5小时。”制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感器。对甲基化酶和黄曲霉素检测的结果同实施例1。
实施例3.ECL生物传感器的制备及对目标的检测
将“接着将不同浓度的甲基化酶、剪切酶以及DNA纳米管-Ru(phen)3 2+信号探针顺序滴在电极表面反应2小时。”改为“接着将不同浓度的甲基化酶、剪切酶以及DNA纳米管-Ru(phen)3 2+信号探针顺序滴在电极表面反应3小时。”制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感器。对甲基化酶和黄曲霉素检测的结果同实施例1。
实施例4.ECL生物传感器的制备及对目标的检测
将“再将不同浓度的黄曲霉素滴到电极表面，37℃下反应3小时。”改为“再将不同浓度的黄曲霉素滴到电极表面，37℃下反应2小时。”。制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感器。对甲基化酶和黄曲霉素检测的结果同实施例1。
Claims (1)
1.一种基于DNA纳米管的电化学发光生物传感器在甲基化酶和黄曲霉素检测中的应用，其特征是：利用DNA纳米管放大信号技术，设计了电化学发光生物传感器，通过“on-off”检测模式，实现了对甲基化酶和黄曲霉素的灵敏分析；目标甲基化酶引发了发夹DNA的甲基化和剪切反应，将Ru(phen)3 2+标记的纳米管组装到电极上，实现了甲基化酶的电化学发光检测；进一步利用纳米管上的适体结合目标黄曲霉素，纳米管结构破坏释放出信号探针，对黄曲霉素进行ECL检测；
具体如下：
步骤1.DNA纳米管-Ru(phen)3 2+探针的制备：三种DNA单链S1,S2和S3溶于TE缓冲溶液中，从95℃到室温进行退火反应，组装DNA纳米管；取50μL纳米管溶液与100μL、2mM的Ru(phen)3 2+在37℃下温育7小时；
步骤2.ECL生物传感器的制备及目标检测：将金电极用α-Al2O3粉磨光，用纯水冲洗并用氮气吹干，在0.5M H2SO4溶液中，进行电化学扫描，得到完整的循环伏安峰，冲洗干净；取20μL金胶滴加在电极表面，自然晾干；将10μL、1.0μM的发夹DNA滴在电极表面室温过夜，然后用1mM MCH反应1小时；接着将不同浓度的甲基化酶、剪切酶以及DNA纳米管-Ru(phen)3 2+信号探针顺序滴在电极表面反应2小时；再将不同浓度的黄曲霉素滴到电极表面，37℃下反应3小时；然后用PBS溶液冲洗电极；在200mM,pH 7.4含有20mM TPA的缓冲溶液中进行ECL信号检测。
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