CN115248240A - 一种基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究 - Google Patents
一种基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究 Download PDFInfo
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Abstract
本发明公开了基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究,该方法主要设计了一种Pb2+特异性酶(Pb‑DNAzyme),并将这种酶通过Au‑S键修饰至金电极表面,当在Pb2+存在的条件下,DNAzyme被激活而将底物链切割成两部分,留在电极表面的那部分DNA片段由于Pb2+嵌入可以形成G4结构,通过结晶紫在G4表面的堆叠可以实现电化学方波伏安检测。另一方面,收集被剪切下的DNA片段,通过碱基互补配对原则可以被设计好的DNA三棱柱结构悬挂端捕获,引发杂交链(HCR)反应,随后将末端转移酶TdT、三磷酸腺嘌呤脱氧核苷酸dATP和三磷酸胸腺嘧啶脱氧核苷酸dTTP等引入电极表面,形成一个巨大的DNA网络结构,能够作为联吡啶钌(Ru(phen)3 2+)的载体进行信号放大,用于电化学发光信号测定。
Description
技术领域
本发明涉及一种双通道电化学方法及其应用,尤其是涉及基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究,属于功能生物材料和生物传感技术领域。
背景技术
铅离子(Pb2+)是剧毒的重金属之一,这种不能生物降解的剧毒重金属会在人体内蓄积,即使在低浓度下也会对人类健康造成重大影响,譬如Pb2+中毒与神经、心血管、精神、生殖和发育障碍有关,对肝、肾和其他器官造成严重损害,尤其是儿童,体内异常的Pb2+含量会使其患上健忘症、贫血、肌肉瘫痪和智力低。因此,Pb2+的准确、灵敏检测对人体健康具有重要意义,目前的分析检测方法有原子吸收光谱法(AAS)、原子荧光光谱法(AFS)、电感耦合等离子体光发射光谱法(ICP-OES)、电感耦合等离子体质谱法(ICP-MS)、比色法、荧光法以及光电化学法等。尽管这些检测方法有较高的灵敏度和精确度,但仍有一些局限性,如需要大量昂贵的仪器设备和复杂的样品预处理等缺点。而电化学方法以其成熟的理论、操作简便、高灵敏度和良好的选择性等优点而受到科学家的广泛关注。因此,开发一种新的电化学方法实现Pb2+的灵敏检测对人体健康有着十分重要的意义。
脱氧核酶(DNAzyme)是一种人工脱氧核糖核酸酶,是通过体外筛选技术获得的具有酶活性的ssDNA序列,能够和特定的金属离子相结合,一般由由底物链和酶链组成。底物链含有单个rA连接(核糖核苷腺苷)作为切割位点。这些DNA分子片段和某种特定的金属离子相结合后,该特定金属离子可作为辅助因子,催化其特定的DNA片段断裂,对金属离子表现出极强的亲和力和特异性。与蛋白质酶相比,这些金属离子特异性DNAzyme具有生产成本低、制备步骤简单、易于储存等优点,这使得金属离子特异性DNAzyme作为检测金属离子的生物传感器平台引起了科学家的广泛关注。
本发明构建了基于DNAzyme的双通道电化学方法,并将其应用在铅离子检测中,该方法主要设计了一种Pb2+特异性酶(Pb-DNAzyme),并将这种酶通过Au-S键修饰至金电极表面,当在Pb2+存在的条件下,DNAzyme被激活而将底物链切割成两部分,留在电极表面的那部分DNA片段由于Pb2+嵌入可以形成34结构,通过结晶紫在34表面的堆叠可以实现电化学方波伏安检测。另一方面,收集被剪切下的DNA片段,通过碱基互补配对原则可以被设计好的DNA三棱柱结构悬挂端捕获,引发杂交链(HCR)反应,随后将末端转移酶TdT、三磷酸腺嘌呤脱氧核苷酸dATP和三磷酸胸腺嘧啶脱氧核苷酸dTTP等引入电极表面,形成一个巨大的DNA网络结构,能够作为联吡啶钌(Ru(phen)3 2+)的载体进行信号放大,用于电化学发光信号测定。目前为止,未见基于DNAzyme的双通道电化学方法,本发明专利中联合G四联体、HCR反应、TdT延伸反应实现了Pb2+的电化学方波伏安检测和电化学发光检测,为复杂样品中Pb2+的分析检测提供了一种新思路。
发明内容
本发明所要解决的技术问题是提供一种特异性好、灵敏度高、检测速度快、结果准确可靠、成本低的基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究。
本发明解决上述技术问题所采用的技术方案为:一种基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究,具体步骤如下:
(1)DNA三棱柱的制备
使用三条DNA链(L、Sa和Sb)来合成DNA纳米三棱柱。将购买的DNA干粉置于离心机中用5000rmp离心15min,然后根据DNA合成报告将其稀释到1μM。合成原则:控制摩尔比L3∶Sa∶Sb=1∶3∶3,L3的最终浓度约为200nM。
采用三步合成我们所需要的DNA三棱柱纳米结构,上半部分合成步骤如下:将L3(1μM,10μL)、Sa(1μM,30μL)和10×TAE/Mg2+(6μL)混合,加入双蒸水至总体积为50μL,混匀,退火。
下半部分合成步骤如下:将L3(1μM,10μL)、Sb(1μM,30μL)、10×TAE/Mg2+(6μL)和TCEP(40mM,2μL)混合,加入双蒸水至总体积为50μL,混匀,退火。
退火条件:95℃,5min;65℃,30min;50℃,30min;37℃,30min;22℃,30min;4℃,30min;hold,4℃。
三棱柱的组装:按照1∶1的比例混匀上半部分和下半部分溶液,进行退火组装,退火条件同以上。
其中DNA序列如下:
10×Tris-醋酸-EDTA(10×TAE/Mg2+)配比如下:
10×TAE/Mg2+缓冲溶液中:0.4M Tris,0.02M EDTA,0.2M HAc以及12.5mM Mg2+。
(2)电化学生物传感器的制备
A.将金电极(Au,直径为2mm)在麂皮上依次用粒径0.3μm、0.05μm的三氧化二铝粉末抛光0.5~5min,抛光后将电极置于超声清洗器中用超纯水超声清洗1~5min,然后用N2吹干,记为电极a;
B.方波伏安检测通道电极制备:将100μL含有0.3μM DNA2,0.2μM DNA1,50mMNaCl,10mM HEPES混合溶液于37℃下反应1h,取5μL滴于Au上,在4℃下孵育过夜,用蒸馏水缓缓冲洗电极,然后滴上5μL 1mM MCH溶液孵育30min用来封闭电极,用蒸馏水缓缓冲洗电极,记为电极b;再将5μL 0.1μM Pb2+注入电极表面反应液,于37℃下反应1.5h,随后收集电极表面溶液用于电化学发光检测通道电极制备(约4μL),用蒸馏水缓缓冲洗电极,记为电极c。取5μL浓度为1mM的结晶紫溶液(CV)滴涂于电极c上,室温下静置15min,用蒸馏水缓缓冲洗电极,记为电极d。通过方波伏安法对不同浓度的Pb2+进行电化学检测。
C.电化学发光检测通道电极制备:取5μL(1)中DNA三棱柱合成溶液滴涂于电极a’,在4℃下孵育过夜,用蒸馏水缓缓冲洗电极,记为电极b’。再将步骤B中Pb2+剪切步骤所收集到的溶液(约4μL)滴于电极b上,室温下静置1h,用蒸馏水缓缓冲洗电极,记为电极c’。制备100μL含有1μM H1,1μM H2,10mM Tris-HCl,1mM EDTA,1M NaCl的混合液作为HCR杂交溶液,混合均匀后,取5μL滴涂于电极c’上,37℃下反应2h,用蒸馏水缓缓冲洗电极,记为电极d’。将100μL含有0.5mM dATP,0.5mM dTTP,0.5U/μL TdT,TdT反应缓冲液,蒸馏水混合均匀后,取5μL滴涂于电极d’上,在37℃下反应2h,用蒸馏水缓缓冲洗电极,记为电极e’。取5μL浓度为10mM的Ru(phen)3 2+溶液,滴涂于电极e’上,室温下避光反应1h,用蒸馏水缓缓冲洗电极,记为电极f’。通过电化学发光法对不同浓度的Pb2+进行电化学发光检测。
基于以上溶液,通过改变Pb2+浓度(0.001~800nM),其他步骤同上,可实现对Pb2+的双通道检测。
利用上述一种基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究,通道一:利用方波伏安法,设置电位范围为-0.8~-0.4V,振幅为0.025V。Pb2+不仅能够对DNAzyme进行剪切反应,还能利用Pb2+诱导G4的形成,采用结晶紫的堆叠产生电化学方波伏安响应;通道二:利用电化学发光,使用计时电流法(脉冲宽度为0.25s,脉冲周期为30s)在含有10mM TPrA的磷酸盐缓冲溶液(0.1M,pH 7.0)中对上述修饰电极进行扫描,产生并记录ECL信号。光电倍增管的电压设置为800V。利用DNA三棱柱结构捕获Pb2+剪切过程中所释放的DNA,引发杂交链HCR反应,再通过TdT延伸反应进行DNA网络结构的继续合成,利用发光体Ru的嵌入实现电化学发光方法的构建。通过通道一中电化学传感器对结晶紫(CV)的电化学响应以及通道二中的电化学发光传感器对电化学发光体Ru的ECL响应,获得一系列不同浓度的Pb2+对应的电流或是电化学发光信号大小,建立电流响应或是电化学发光信号与Pb2+浓度之间的线性关系,根据两者之间的定量关系,确定待测样品中Pb2+的含量。
发明原理:本发明是一种基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究,该方法主要利用了一种铅离子特异性DNA酶(Pb-DNAzyme),在Pb2+存在的条件下,DNAzyme被激活而将底物链切割成两部分,而且可以使特定碱基序列的DNA片段形成G4结构,通过G4与特定的电化学信号物质(结晶紫)结合,成功制备电化学传感器,利用电化学方法测量Pb2+含量;另一方面,被Pb2+剪切下的DNA片段被DNA三棱柱结构捕获,通过HCR反应扩增及TdT延长扩增,嵌入联吡啶钌(Ru(phen)3 2+),利用电化学发光方法间接地测量Pb2+含量。基于此,构建了一种简单、快速、高灵敏、高选择性、免标记的Pb2+电化学分析方法。
与现有技术相比,本发明的优点在于:本发明是基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究。显然,对于通道一来说,在浓度一定范围内,Pb2+浓度越大,产生的G4结构越多,电流响应越明显;同理,对于通道二,Pb2+浓度越大,剪切反应进行的更彻底,溶液当中释放的DNA链就越多,更能够促进后续的HCR反应,最终Ru发光体嵌入的越多,ECL响应也越明显。实验结果表明,电流的大小(ECL信号的大小)与Pb2+的浓度在一定范围内呈线性关系,从而实现对Pb2+的检测。其优点在于:
(1)双通道电化学方法的构建。基于DNAzyme的双通道电化学方法,这两种方法都会因为Pb2+浓度的变化引起信号的变化,通道一方法中Pb2+不但可以进行剪切作用,还能够利用Pb2+形成G4进行信号输出,而通道二方法中充分利用了剪切后释放的DNA,引发HCR反应和TdT延伸反应;这两通道实验结果能够进行对比,能够有效地避免假阳性信号,实验结果的可靠性增加。
(2)高灵敏度。本发明基于DNAzyme制备电化学传感器,利用G4结构结合结晶紫进行电化学输出以及利用DNA三棱柱并使Ru(phen)3 2+进行ECL信号输出,得到两条线性方程:电流响应对Pb2+浓度线性相关方程为y=0.87lgCPb2++1.89,r=0.9987,检测限为0.0036nM;ECL响应对Pb2+浓度线性相关方程为y=2120lgCPb2++6818,r=0.9994,检测限为0.00023nM;说明该传感器可对Pb2+实现高灵敏度检测。
(3)高特异性。对Pb2+检测:其他对照物质如汞离子(Hg2+)、锰离子(Mn2+)、钴离子(Co2+)、镍离子(Ni2+)、钡离子(Ba2+)、铜离子(Cu2+)、镁离子(Mg2+)对体系均无干扰。
(4)结果准确。回收率均在90%~110%之间。
(5)制备与检测方法试剂用量少、成本低。本发明只需消耗少量材料和试剂就可实现对Pb2+的高灵敏检测。
综上所述,本发明是基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究,具有灵敏度高、选择性好、操作简单、分析快速、易于操作等优点,可以实现较低浓度Pb2+的检测,具有良好的应用前景。
附图说明
图1为本发明通道一传感器制备过程的电化学表征图;
图2为本发明通道二传感器制备过程的电化学表征图;
图3为本发明通道一传感器对Pb2+分析检测的可行性实验图;
图4为本发明通道二传感器对Pb2+分析检测的可行性实验图;
图5为本发明通道一传感器对不同浓度Pb2+的电流响应对浓度的校准曲线图;
图6为本发明通道二传感器对不同浓度Pb2+的电流响应对浓度的校准曲线图;
图7为本发明传感器对Pb2+的选择性实验图。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
实施例1DNA三棱柱的制备
使用三条DNA链(L、Sa和Sb)来合成DNA纳米三棱柱。将购买的DNA干粉置于离心机中用5000rmp离心15min,然后根据DNA合成报告将其稀释到1μM。合成原则:控制摩尔比L3∶Sa∶Sb=1∶3∶3,L3的最终浓度约为200nM。采用三步合成我们所需要的DNA三棱柱纳米结构(prism),合成步骤如下:上半部分的合成:将L3(1μM,10μL)、Sa(1μM,30μL)和10×TAE/Mg2+(6μL)混合,加入双蒸水至总体积为50μL,混匀,退火。
下半部分的合成:将L3(1μM,10μL)、Sb(1μM,30μL)、10×TAE/Mg2+(6μL)和TCEP(40mM,2μL)混合,加入双蒸水至总体积为50μL,混匀,退火。
退火条件:95℃,5min;65℃,30min;50℃,30min;37℃,30min;22℃,30min;4℃,30min;hold,4℃。
三棱柱的组装:按照1∶1的比例混匀上半部分和下半部分溶液,进行退火组装,退火条件同以上。
实施例2电化学生物传感器的制备
A.首先,将金电极(Au,直径为2mm)在麂皮上依次用粒径0.3μm、0.05μm的三氧化二铝粉末抛光0.5~5min,抛光后将电极置于超声清洗器中用超纯水超声清洗1~5min,然后用N2吹干,记为电极a;
B.方波伏安检测通道电极制备:将100μL含有0.3μM DNA2,0.2μM DNA1,50mMNaCl,10mM HEPES混合溶液于37℃下反应1h,取5μL滴于电极a上,在4℃下孵育过夜,用蒸馏水缓缓冲洗电极,然后滴上5μL 1mM MCH溶液孵育30min用来封闭电极,用蒸馏水缓缓冲洗电极,记为电极b;再将5μL 0.1μM Pb2+注入电极表面反应液,于37℃下反应1.5h,随后收集电极表面溶液用于电化学发光检测通道电极制备(约4μL),用蒸馏水缓缓冲洗电极,记为电极c。取5μL浓度为1mM的结晶紫溶液(CV)滴涂于电极c上,室温下静置15min,用蒸馏水缓缓冲洗电极,记为电极d。通过方波伏安法对不同浓度的Pb2+进行电化学检测。
C.电化学发光检测通道电极制备:取5μL(1)中DNA三棱柱合成溶液滴涂于电极a’上,在4℃下孵育过夜,用蒸馏水缓缓冲洗电极,记为电极b’。再将步骤B中Pb2+剪切步骤所收集到的溶液(约4μL)滴于电极b’上,室温下静置1h,用蒸馏水缓缓冲洗电极,记为电极c’。制备100μL含有1μM H1,1μM H2,10mM Tris-HCl,1mM EDTA,1MNaCl的混合液作为HCR杂交溶液,混合均匀后,取5μL滴涂于电极c’上,37℃下反应2h,用蒸馏水缓缓冲洗电极,记为电极d’。将100μL含有0.5mM dATP,0.5mM dTTP,0.5U/μL TdT,TdT反应缓冲液,蒸馏水混合均匀后,取5μL滴涂于电极d’上,在37℃下反应2h,用蒸馏水缓缓冲洗电极,记为电极e’。取5μL浓度为10mM的Ru(phen)3 2+溶液,滴涂于电极e’上,室温下避光反应1h,用蒸馏水缓缓冲洗电极,记为电极f’。通过电化学发光法对不同浓度的Pb2+进行电化学发光检测。
检测以上所述电极对5mM[Fe(CN)6]3-/4-(含0.1M KCl)电解质溶液的电化学响应,如图1和图2,可看出制备的传感器相比较于其他电极,峰值电流很明显,与裸金电极相近。说明传感器对Pb2+有良好的电化学响应。
实施例3可行性实验
为了证明本发明传感器可以实现对Pb2+的检测,基于实施例1和实施例2制备生物传感器。对比有无Pb2+存在时,检测所制备的传感器的电化学响应,见图3和图4,有Pb2+时,电化学传感器在PBS(100mM,pH=7.0)中电化学响应明显,而无Pb2+存在时,基本无电化学响应。对于电化学发光传感器,在Pb2+存在时,该传感器在含有10mM TPrA的PBS(100mM,pH7.0)中ECL响应明显,而无Pb2+存在时,基本无ECL响应。以上结果证明该传感器可用于Pb2+检测。
实施例4 Pb2+的检测
按上述实施例1和实施例2的传感器制备步骤,以及实施例3对Pb2+的响应,通过改变Pb2+(0.001~800nM)的浓度,分别利用通道一的电化学传感器和通道二的电化学发光传感器检测对Pb2+溶液的响应,结果如图5和图6。如图5,传感器对Pb2+的电流响应与浓度呈良好的线性关系,传感器的电流响应对Pb2+浓度线性相关方程为y=0.87lgCPb2++1.89,r=0.9987,线性范围为0.01~200nM,检测限为0.0036nM;如图6,ECL响应对Pb2+浓度线性相关方程为y=2120lgCPb2++6818,r=0.9994,线性范围为0.001~400nM,检测限为0.00023nM,说明传感器对Pb2+实现高灵敏检测。
实施例5特异性检测
为了验证该传感器的抗干扰性,按上述实施例1、2、3、4的传感器制备步骤,Pb2+的溶液中,加入其它相同浓度的干扰物,如与Pb2+相同浓度的汞离子(Hg2+)、锰离子(Mn2+)、钴离子(Co2+)、镍离子(Ni2+)、钡离子(Ba2+)、铜离子(Cu2+)、镁离子(Mg2+),检测传感器对Pb2+的特异性。结果如图4所示,说明传感器对于Pb2+的检测有很好的特异性。
当然,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变化、改型、添加或替换,也应属于本发明保护范围。
Claims (3)
1.基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究,其特征在于,机理如下:本发明构建了基于DNAzyme的双通道电化学方法,并将其应用在铅离子检测中,该方法主要设计了一种Pb2+特异性酶(Pb-DNAzyme),并将这种酶通过Au-S键修饰至金电极表面,当在Pb2+存在的条件下,DNAzyme被激活而将底物链切割成两部分,留在电极表面的那部分DNA片段由于Pb2+嵌入可以形成G4结构,通过结晶紫在G4表面的堆叠可以实现电化学方波伏安检测。另一方面,收集被剪切下的DNA片段,通过碱基互补配对原则可以被设计好的DNA三棱柱结构悬挂端捕获,引发杂交链(HCR)反应,随后将末端转移酶TdT、三磷酸腺嘌呤脱氧核苷酸dATP和三磷酸胸腺嘧啶脱氧核苷酸dTTP等引入电极表面,形成一个巨大的DNA网络结构,能够作为联吡啶钌(Ru(phen)3 2+)的载体进行信号放大,用于电化学发光信号测定。目前为止,未见基于DNAzyme的双通道电化学方法,本发明专利中联合G四联体、HCR反应、TdT延伸反应实现了Pb2+的电化学方波伏安检测和电化学发光检测,为复杂样品中Pb2+的分析检测提供了一种新思路。
2.根据权利要求1所述基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究,其特征在于:基于DNAzyme的双通道电化学方法,这两种方法都会因为Pb2+浓度的变化引起信号的变化,通道一方法中Pb2+不但可以进行剪切作用,还能够利用Pb2+形成G4进行信号输出,而通道二方法中充分利用了剪切后释放的DNA,引发HCR反应和TdT延伸反应;这两通道实验结果能够进行对比,能够有效地避免假阳性信号,实验结果的可靠性增加。
3.根据权利要求1,2所述基于DNAzyme的双通道电化学方法及其在铅离子检测中的应用研究,其特征在于:通道一利用方波伏安技术对不同浓度的Pb2+进行测定,Pb2+检测限为0.0036nM;通道二利用电化学发光法对不同浓度的Pb2+进行测定,Pb2+检测限为0.00023nM。
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