CN107167507A - 带dna分子探针的石墨烯微电极电化学检测传感器 - Google Patents

带dna分子探针的石墨烯微电极电化学检测传感器 Download PDF

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CN107167507A
CN107167507A CN201710343969.2A CN201710343969A CN107167507A CN 107167507 A CN107167507 A CN 107167507A CN 201710343969 A CN201710343969 A CN 201710343969A CN 107167507 A CN107167507 A CN 107167507A
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王德强
查克·特里里
黎学思
朱济峰
周大明
何石轩
唐鹏
谢婉谊
石彪
梁丽媛
黄绮梦
周硕
邓云生
方绍熙
余玲
鲁志松
陆文强
王炜
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Abstract

本发明涉及一种带DNA分子探针的石墨烯微电极电化学检测传感器,包括电极区域、电极和连接金线;所述电极区域由工作电极区域(9)、参比电极区域(10)和对电极区域(8)组成,工作电极区域上并列设置四个带DNA分子探针石墨烯微电极,连接石墨烯电极连接金线(6),再通过电极夹(5)与电极转向控制器(4)连接至工作电极(2),四个带DNA分子探针石墨烯微电极共用参比电极(1)和对电极(3),参比电极和对电极分别通过电极连接金线(11)与参比电极区域(10)和对电极区域(8)相接。本发明可以同时检测四种不同miRNA,达到高通量检测目的,检测快速方便,且具有高灵敏度、高选择性、高准确度、无需标记等优点。

Description

带DNA分子探针的石墨烯微电极电化学检测传感器
技术领域
本发明属于电化学生物传感系统领域,涉及一种带DNA分子探针的石墨烯微电极电化学检测传感器。
背景技术
肺癌诊断的传统方法往往依赖于胸片、胸部CT、正电子放射断层造影术(PET)、磁共振成像(MRI)、痰细胞学检查和活组织检查等方法,这些方法对癌症初期亚毫米大小的肿瘤的检测分辨率都不高,且结果具有较高的假阳性。近十余年来,随着高通量、大规模基因及蛋白质分析技术的涌现,肿瘤基因组学、蛋白质组学、代谢组学等研究方兴未艾,已筛选出了许多有前景的候选标志物(profiles)。其中,一种小型非编码RNA——miRNA,在人类体液中稳定的存在,被认为是最有前景的癌症早期诊断生物标志物。由于miRNA的片段短,同源序列多,在血液中的浓度较低以及碱基序列组成的巨大差异,使得要实现无标记检测miRNA具有极大的挑战性的。
因此,科学家提出了一些miRNA的检测方法来克服这些问题,其中主要选用Northern杂交充当miRNA早期分析的黄金标准。然而,这种方法需要大量的RNA样品和低通量分析,且不适用于大规模的使用,因此仍然受到技术上的限制。此外,定量逆转录实时聚合酶链式反应(QRT-PCR)测定法和微阵列技术已经被用于miRNA的检测。然而,这些方法要求扩增,交叉杂交,且步骤耗时长,因此容易出错;而短序列的miRNA同样使得引物的设计难度加大,从而导致其灵敏度变差。此外,所有这些方法都非常消耗人力,并需要配置完善的实验室和专业的、训练有素的操作人员。除了这些传统的miRNA检测方法,科学家已经开发了一些无需标记的分析方法用作miRNA检测,包括光子微环谐振器,纳米孔,表面增强拉曼光谱和表面等离子体共振。和传统的技术相比,这些新兴的技术提高了灵敏度和选择性,扩展了检测(LOD)极限,增加了复式检测能力,降低了成本,并实现了小型化。然而,这些方法仍然需要对目标miRNA进行化学修饰,在生理介质中不容易操作,不能进行复式检测,而且需要昂贵的设备。这些问题严重地限制了它们在资源极其有限的情况下的即时诊断能力。因此,市场上急需一个平台可以提供不需要任何放大处理就达到高灵敏度和特异性,操作流程简单,并且能够复式检测多种miRNA的高通量诊断模式。
发明内容
有鉴于此,本发明的目的在于提供一种带DNA分子探针的石墨烯微电极电化学检测传感器。
为达到上述目的,本发明提供如下技术方案:
1.带DNA分子探针的石墨烯微电极电化学检测传感器,其特征在于,所述传感器包括电极区域、电极和连接金线;所述电极区域由工作电极区域(9)、参比电极区域(10)和对电极区域(8)组成,工作电极区域上设有带DNA分子探针石墨烯微电极。
进一步,所述工作电极区域上并列设置四个带DNA分子探针石墨烯微电极,连接石墨烯电极连接金线(6),再通过电极夹(5)与电极转向控制器(4)连接至工作电极(2),四个带DNA分子探针石墨烯微电极共用参比电极(1)和对电极(3),参比电极和对电极分别通过电极连接金线(11)与参比电极区域(10)和对电极区域(8)相接。
进一步,所述带DNA分子探针石墨烯微电极所带DNA分子探针序列分别如SEQ IDNO:1~SEQ ID NO:4所示。
进一步,带DNA分子探针石墨烯微电极的制备方法是:将石墨烯微电极的电极导电区进行等离子体清洗预处理,并且在DNA分子探针的5’端修饰二茂铁分子(Ferrocene),采用物理吸附方式,将DNA分子探针固定在石墨烯微电极上,得带DNA分子探针石墨烯微电极。
进一步,所述石墨烯微电极的制备方法为:
(1)将硅晶片切割成边长1~1.5cm的硅片,用王水浸泡、纯净水清洗去除硅片表面污渍;
(2)在步骤(1)处理干净的硅片表面上,采用化学气相沉积方法生长一层600-1000纳米的二氧化硅,形成二氧化硅绝缘层,再通过物理气相沉积将金属薄膜沉积在二氧化硅绝缘层上;
(3)用光刻技术剥离图案化成T字形芯片基板;
(4)采用直接干法或湿法转移3~5层石墨烯到二氧化硅绝缘层表面,石墨烯与T字形金属薄膜下端部位接触并覆盖1-2mm,再在石墨烯层上沉积10纳米的二氧化硅,形成钝化层;
(5)采用图案化剥离钝化层,以露出边长为0.1cm正方形墨烯作为电极导电区。
进一步,所述金属薄膜为10nm铬或钛。
进一步,所述金属薄膜为150nm金或铂。
进一步,所述钝化层采用氮化硅制备。
2.以上所述石墨烯微电极电化学检测传感器在检测miRNA中的应用。
进一步,所述miRNA为:miRNA-21、miRNA-25、miRNA-10b或/和miRNA-155。
本发明的有益效果在于:本发明的带DNA分子探针的石墨烯微电极电化学检测传感器,可以同时检测四种不同miRNA,达到高通量检测目的,检测快速方便,且具有高灵敏度、高选择性、高准确度、无需标记等优点。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1是带DNA分子探针的石墨烯微电极电化学检测传感器示意图;其中1为参比电极;2为工作电极;3为对电极;4为电极转向控制器;5为电极夹;6为石墨烯电极连接金线;7为带DNA分子探针石墨烯微电极;8为对电极区域;9为工作电极区域;10为参比电极区域;11为电极连接金线;
图2是石墨烯微电极制备方法流程图;
图3是带DNA分子探针的石墨烯微电极检测miRNA标记物示意图;
图4是本发明miRNA-10b电化学检测电化学伏安曲线峰电流曲线图。
具体实施方式
下面将结合附图,对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法,通常按照常规条件或按照制造厂商所建议的条件。
实施例1
石墨烯微电极的制备方法:
(1)将硅晶片切割成1cm*1cm大小的硅片,采用王水浸泡、纯净水清洗去除硅片表面污渍;
(2)在步骤(1)处理干净的硅片表面上,采用化学气相沉积方法生长一层800纳米的二氧化硅,形成二氧化硅绝缘层,再通过物理气相沉积将一层10纳米铬或钛金属薄膜沉积在二氧化硅绝缘层上;
(3)用光刻技术剥离图案化成T字形0.5cm*0.5cm芯片基板;
(4)采用直接干法转移4层石墨烯到二氧化硅绝缘层表面,石墨烯与T字形金属薄膜下端部位接触并覆盖2mm,再在石墨烯层上沉积10纳米的二氧化硅,形成钝化层;
(5)采用图案化剥离钝化层,以露出边长为0.1cm正方形墨烯作为电极导电区。
实施例2
(1)将硅晶片切割成1cm*1cm大小的硅片,采用王水浸泡、纯净水清洗去除硅片表面污渍;
(2)在步骤(1)处理干净的硅片表面上,采用化学气相沉积方法生长一层700纳米的二氧化硅,形成二氧化硅绝缘层,再通过物理气相沉积将一层150纳米金薄膜沉积在二氧化硅绝缘层上;
(3)用光刻技术剥离图案化成T字形0.5cm*0.5cm芯片基板;
(4)采用湿法转移3~5层石墨烯到二氧化硅绝缘层表面,石墨烯与T字形金属薄膜下端部位接触并覆盖1mm,再在石墨烯层上沉积10纳米的二氧化硅,形成钝化层;
(5)采用图案化剥离钝化层,以露出边长为0.1cm正方形石墨烯作为电极导电区。
以上技术方案步骤(2)中金薄膜也可采用铂薄膜代替实现发明目的,钝化层可以采用氮化硅制备。
图2是石墨烯微电极制备方法流程图。
实施例3
带DNA分子探针石墨烯微电极的制备方法:
将石墨烯微电极的电极导电区进行等离子体清洗,并且在DNA分子探针的5’端修饰二茂铁分子(Ferrocene),采用物理吸附方式将DNA分子探针固定在石墨烯微电极上,得到DNA分子探针石墨烯微电极。将四种DNA分子探针分别固定到石墨烯微电极上,DNA分子探针及其检测miRNA的序列如表1所示:
表1 DNA分子探针序列及其检测miRNA
实施例4
带DNA分子探针的石墨烯微电极电化学检测传感器
如图1所示,传感器为实施例2制备的四个带DNA分子探针石墨烯微电极7并列置于工作电极区域9,各带DNA分子探针石墨烯微电极7各连接石墨烯电极连接金线6,再通过电极夹5与电极转向控制器4连接至工作电极2,通过电极转向控制器控制电极夹对四个石墨烯电极连接金线的选择,可分别实现检测各DNA分子探针所检测的miRNA的电化学伏安曲线;四个带DNA分子探针石墨烯微电极共用参比电极1和对电极3,参比电极和对电极分别通过电极连接金线11与参比电极区域10和对电极区域8相接。
1——参比电极;2——工作电极;3——对电极;4——电极转向控制器;5——电极夹;6——石墨烯电极连接金线;7——带DNA分子探针石墨烯微电极;8——对电极区域;9——工作电极区域;10——参比电极区域;11——电极连接金线。
实施例5
肺癌早期标记物miRNA电化学伏安曲线分析及肺癌早期筛查和诊断
图3是带DNA分子探针的石墨烯微电极检测miRNA标记物示意图。
检测方法:以体液(血液、血清、血浆、唾液或尿液)中的肺癌标记物miRNA为研究对象,滴定在带DNA分子探针的石墨烯微电极电化学检测传感器的对电极区域8、工作电极区域9和参比电极区域10,保证各电极区域和待测体液充分接触,以备电化学信号检测;由电极转向控制器4对四个工作电极进行依次选择,分别获得四个工作电极上的待测体液的电化学伏安曲线。
肺癌miRNA标记物浓度标准曲线的绘制:在正常人体液中分别加入一定梯度浓度的早期肺癌miRNA标记物,并对其电化学伏安曲线进行测试,进而绘制出体液中miRNA浓度与电化学伏安曲线峰电流的标准曲线;按照同样的方法,绘制4种不同的早期肺癌标记物miRNA浓度与标准电化学伏安曲线峰电流曲线。
采用方波伏特安培测量法(SWV),在测试范围0.1V-0.6V,振幅0.025V,频率25Hz,灵敏度1e-5条件下,分别滴加空白缓冲液(Tris buffer:50mM Tris buffer,150mM NaCl,PH 7.4in DEPC water)和含500nM miRNA-10b的样品液(PBS buffer:10mM PB,150mMNaCl,PH 7.4in DEPC water)于实施例4所制备的电化学检测传感器上,检测结果如图4所示,在固定了DNA分子探针的石墨烯微电极电化学检测传感器上滴上空白的缓冲液时,其电化学信号峰值仅降低4.2%(小于5%),当传感器上滴定500nM miRNA-10b时(PBS buffer:10mM PB,150mM NaCl,PH 7.4in DEPC water),电化学信号峰值降低了37.3%,表明一定量的肺癌标志物miRNA与DNA分子探针杂化并离开传感器表面,使得电化学信号峰值的降低。
早期肺癌的筛查和诊断:以体液(血液、血清、血浆、唾液或尿液)中的肺癌标记物miRNA为研究对象,滴定在带DNA分子探针的石墨烯微电极电化学检测传感器的对电极区域8、工作电极区域9和参比电极区域10,保证各电极区域和待测体液充分接触,以备电化学信号检测;由电极转向控制器4对四个工作电极进行依次选择,分别获得四个工作电极上的待测体液的电化学伏安曲线;再与对应的标记物miRNA建立的标准电化学伏安曲线峰电流曲线进行比对分析,获得四种肺癌早期标记物miRNA的浓度,并以此浓度为依据,结合临床信息,对早期肺癌进行筛查和诊断。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
<110> 重庆石墨烯研究院有限公司,中国科学院重庆绿色智能技术研究院
<120> 带DNA分子探针的石墨烯微电极电化学检测传感器
<130>
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Claims (10)

1.带DNA分子探针的石墨烯微电极电化学检测传感器,其特征在于,所述传感器包括电极区域、电极和连接金线;所述电极区域由工作电极区域(9)、参比电极区域(10)和对电极区域(8)组成,工作电极区域上设有带DNA分子探针石墨烯微电极。
2.如权利要求1所述的石墨烯微电极电化学检测传感器,其特征在于,所述工作电极区域上并列设置四个带DNA分子探针石墨烯微电极,连接石墨烯电极连接金线(6),再通过电极夹(5)与电极转向控制器(4)连接至工作电极(2),四个带DNA分子探针石墨烯微电极共用参比电极(1)和对电极(3),参比电极和对电极分别通过电极连接金线(11)与参比电极区域(10)和对电极区域(8)相接。
3.如权利要求2所述的石墨烯微电极电化学检测传感器,其特征在于,所述带DNA分子探针石墨烯微电极所带DNA分子探针序列分别如SEQ ID NO:1~SEQ ID NO:4所示。
4.如权利要求1所述的石墨烯微电极电化学检测传感器,其特征在于,带DNA分子探针石墨烯微电极的制备方法是:将石墨烯微电极的电极导电区进行等离子体清洗预处理,并且在DNA分子探针的5’端修饰二茂铁分子,采用物理吸附方式,将DNA分子探针固定在石墨烯微电极上,得带DNA分子探针石墨烯微电极。
5.如权利要求4所述的石墨烯微电极电化学检测传感器,其特征在于,所述石墨烯微电极的制备方法为:
(1)将硅晶片切割成边长1~1.5cm的硅片,用王水浸泡、纯净水清洗去除硅片表面污渍;
(2)在步骤(1)处理干净的硅片表面上,采用化学气相沉积方法生长一层600-1000纳米的二氧化硅,形成二氧化硅绝缘层,再通过物理气相沉积将金属薄膜沉积在二氧化硅绝缘层上;
(3)用光刻技术剥离图案化成T字形芯片基板;
(4)采用直接干法或湿法转移3~5层石墨烯到二氧化硅绝缘层表面,石墨烯与T字形金属薄膜下端部位接触并覆盖1-2mm,再在石墨烯层上沉积10纳米的二氧化硅,形成钝化层;
(5)采用图案化剥离钝化层,以露出边长为0.1cm正方形墨烯作为电极导电区。
6.如权利要求5所述的石墨烯微电极电化学检测传感器,其特征在于,所述金属薄膜为10nm铬或钛。
7.如权利要求5所述的石墨烯微电极电化学检测传感器,其特征在于,所述金属薄膜为150nm金或铂。
8.如权利要求5-7任一项所述的石墨烯微电极电化学检测传感器,其特征在于,所述钝化层采用氮化硅制备。
9.权利要求1所述的石墨烯微电极电化学检测传感器在检测miRNA中的应用。
10.如权利要求9所述的应用,其特征在于,所述miRNA为:miRNA-21、miRNA-25、miRNA-10b或/和miRNA-155。
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