电化学DNA生物传感器设计及在医学检验中的应用进展

1、分子诊断与治疗杂志2011年1月 第3卷 第1期J Mol Diagn Ther, January 2011, Vol. 3 No. 1·46· DNA 生物传感器是当前发展最迅速的基因检测方法之一，其应用范围广泛，包括传染病快速检验、疾病基因诊断、环境监测、食品安全、法医鉴定等。用于DNA 生物传感器的检测技术包括荧光技术，石英晶体微天平1，电化学发光2，表面等离子共振光谱3和电化学方法4等。在这些方法中，电化学方法因其操作简单、特异性好、灵敏度高、检测费用低、易于微型化、可再生，并且不受样品中脂血、溶血情况干扰等优点而引起了人们的广泛关注。1电化学DNA 生物传感器的基

2、本原理电化学DNA 生物传感器是以DNA 为敏感元件或检测对象，将核酸分子特异性识别过程中产生的信号通过换能器转化为电信号，从而实现对核酸的定性或定量检测。首先将DNA 探针固定到电极表面，由于探针与溶液中目的DNA 之间的高度序列特异性，使得检测电极具有极强的分子识别能力。在适当的温度、pH 和离子强度条件下，已知序列的DNA 探针与溶液中的目的DNA 序列发生杂交，从而导致电极表面结构的变化，变化的情况可通过电化学杂交指示剂所引起电信号（如电压、电流或电导）的变化体现出来，可用循环伏安法5、溶出伏安法6、差分脉冲伏安法7、交流阻抗8等方法对电信号进行检测，进而对目的基因进行定性或定量分析，

3、如图1所示。电化学DNA 生物传感器设计及在医学检验中的应用进展李博郑磊王前贾立永摘要 电化学DNA 生物传感器将电化学方法的高灵敏度和DNA 生物传感器的高度序列特异性结合起来，是一种全新的基因检测手段。本文在介绍电化学DNA 生物传感器原理的基础上，重点阐述其设计及在医学检验中的应用进展。关键词电化学；DNA 生物传感器；医学检验；进展Progress of electrochemical DNA biosensor and application in medical laboratoryLI Bo, ZHENG Lei, WANG Qian, JIA Liyong(Laboratory

4、 Medicine Center, Nangfang Hospital, Southern Medical University, Guangdong, Guangzhou 510515, ChinaABSTRACTElectrochemical DNA biosensor was a novel technique for detection of DNA which have received wide attention for its high sensitivity and high sequence-specication. Here, we review the progress

5、 of electrochemical DNA biosensor and application in medical laboratory.KEY WORDSElectrochemical; DNA biosensor; Medical laboratory; Progress基金项目：广东省科技计划（2008A050200006；2010A030300006）作者单位：南方医科大学南方医院，广东，广州 510515通讯作者：郑磊，E-mail ：n 图1电化学DNA 生物传感器的原理图Figure 1The principle of electrochemical DNA biosens

6、or2电化学DNA 生物传感器设计进展 2.1电极的修饰电化学DNA 生物传感器常用的工作电极包括：综 述·47·分子诊断与治疗杂志2011年1月 第3卷 第1期J Mol Diagn Ther, January 2011, Vol. 3 No. 1玻碳电极、金电极、铂电极、碳糊电极、石墨电极、氧化铟锡（indium tin oxides，ITO ）电极等。纳米材料由于其独特的光学、磁学、热学、电学及化学性质成为电极修饰的主要材料。其作用主要有：增加探针在电极表面的固定量；加速电子传递；从而增强传感器的响应信号、提高检测灵敏度。常用的纳米材料主要有碳纳米管9、金属纳米粒子1

7、0、金属氧化物纳米粒子11、复合纳米粒子12等。其中复合纳米粒子的修饰性能比一般纳米粒子更好。Zhang 等12构建了基于复合钼硫碘纳米线（Mo 6S 9-xIx 纳米线）的新型电化学传感器。Mo 6S 9-xIx 纳米线是一种能显示出金属电导率（S300K=2×103 S/m）的结构松散的束状平行线。导电性能更强，且因其易于在溶剂中分散成单分子的线状，反应面积更大，从而使得复合钼硫碘纳米线更适于构建纳米级电子器件。2.2DNA 探针的固定DNA 探针的固定是电化学DNA 生物传感器制作中的首要问题，探针的固定量和活性直接影响传感器的灵敏度。近年来常用的固定方法包括：吸附法：该方法较

8、简单，主要是利用DNA 片段中带负电的磷酸骨架与带正电的固体基质表面的静电相互作用而将DNA 固定在支持物上的，可直接或利用恒电位13将探针吸附到电极表面。生物素-亲和素法：生物素与亲和素的结合具有专一、迅速和稳定的特点。Chu 等14用链霉亲和素-生物素与无垢薄膜组装结合固定DNA 探针。通过严格控制薄膜的厚度和化学性质，嵌入其中的链霉亲和素便能够与生物素化的寡核苷酸探针特异结合，并防止其他物质的非特异吸附。共价结合法：这种固定化方法首先要在电极表面或探针上修饰一些活性官能团，如氨基、羧基等，通过这些官能团之间的共价作用固定探针。Li 等15将NH 2ssDNA序列成功地通过环氧化物/氨基耦

9、合反应共价固定在用溶胶-凝胶技术和自组装技术直接耦合的金电极表面，构建出的电化学DNA 生物传感器选择性好，且能再生。自组装法：分子之间在平衡条件下，通过非共价键作用力自发聚集形成热力学稳定、结构确定、性能特殊的聚集体的过程称为自组装。通过分子的自组作用, 在固体表面自然形成高度有序的单分子层膜。Hejazi 等16在金电极表面自组装修饰包含14碱基肽核酸（peptide nucleic acid，PNA ）探针和6-巯基-1-己醇的单分子层膜，构建针对HCV 病毒3a 基因型（pHCV3a ）核心/E1区目的基因的电化学DNA 生物传感器。2.3DNA 杂交的指示电化学DNA 生物传感器通过

10、电化学杂交指示剂将杂交信号转化为可测定的电信号指示DNA 杂交，进而实现特定序列DNA 的定性或定量分析。根据来源的不同，电化学杂交指示剂可分为内源型和外源型两类。内源型指示剂主要是指DNA 分子中可被氧化还原而产生电化学信号的碱基，主要是指鸟嘌呤和腺嘌呤。外源型杂交指示剂又可根据与固定DNA 之间的关系分为标记型和非标记型。其中，标记型指示剂主要是指通过吸附、化学修饰或生物亲和等方法在基因片段上固定一些具有电活性或催化活性的无机或生物粒子，如纳米粒子17、二茂铁18、聚吡咯19、生物酶20等；杂交反应结束后会在电极表面上形成带有电活性官能团或生物酶的杂交分子，利用对其电信号的测定达到测定DN

11、A 的目的。Fang 等18通过离子锆（Zr ）将目的DNA 的磷酸根基团和二茂铁羧酸氧化还原探针的羧酸基团连接起来，实现对目的DNA 的检测。虽然这种方法灵敏性较高，但检测过程涉及合成、标记、分离纯化等，步骤较为繁琐且在操作过程中容易造成样品损失。非标记型电化学指示剂是一类以非共价形式与单链DNA 或双链DNA 分子选择性结合的电化学活性化合物。基于其与单链或双链DNA 分子结合能力的差异，通过电压、电流、电阻等可测指标对DNA 杂交过程发生与否及发生程度进行指示。最常用非标记型电化学杂交指示剂是：亚甲蓝（methyleneblue ，MB ）16、二茂铁及其衍生物21，22和各种金属配合物

12、（如钌23、铜24等）。除此之外，还有一些药物小分子（如阿霉素25）及荧光染料等。3电化学DNA 传感器在医学检验中的应用进展3.1感染性疾病的诊断分子诊断与治疗杂志2011年1月 第3卷 第1期J Mol Diagn Ther, January 2011, Vol. 3 No. 1·48·DNA 进行检测。Ansari 等26用20-碱基的淋病奈瑟菌的特异性硫醇化寡核苷酸探针（th-ssDNA ）包被在通过溶胶-凝胶方法浸涂在铟锡氧化物玻璃衬底上的氧化锌（ZnO ）膜上，构建检测淋病的DNA 生物传感器。该传感器在60 min内即完成检测，检测的目的DNA 线性范围是0.

13、000524 fmol0.524 nmol，检测最低限为0.000704 fmol。Mach 等27将针对几种常见尿路感染细菌的16S r RNA的DNA 探针包被在电化学传感器微阵列表面，构建了检测尿路细菌感染的多通道电化学生物传感系统，并率先进行了床旁检测（point-of-care testing，POCT ）的前瞻性临床试验：对纳入试验的116例患者的109份合格尿标本进行检测，并与金标准尿培养作对比。该方法特异度和阳性预测值为100%，敏感度为89%，阴性预测值为76%。除此之外，结核杆菌28、小肠耶尔森菌9等也可用电化学DNA 生物传感器进行检测。随着医学研究的深入，人们发现许多疾

14、病，如癌症、遗传病甚至一些临床常见疾病的发生都与遗传基因有关。针对特定序列的电化学检测对于疾病的快速诊断、病程监测和预后都有重大意义。Marin 等34将膀胱纤维化相关DNA 序列夹心于两个DNA 探针之间。其中一个探针通过生物素-链霉亲和素连接于磁珠（magnetic, bead, MB），另一个通过硫醇连接于硫化镉量子点（CdS QD）作为标签。用方波电压（square-wave voltammetry，SWV ）进行电化学检测，得出很好的波形和灵敏的分析信号。Liao 等35构建了一种超敏感的检测甲状腺乳头状癌相关BRAF 突变基因的电化学方法。将30个核苷酸的生物素化DNA 探针固定在

15、一个链霉亲和素修饰的96孔微量板上，封闭之后，加入生物素化的目的DNA ，杂交30 min。然后加入链霉亲和素标记的金纳米颗粒，用方波溶出电压（square wave stripping voltammetry ，SWSV ）技术对杂交过程进行监测。这种方法能很好地识别出现在223个核苷酸DNA 上的野生型和突变型BRAF 。此外，近年来电化学DNA 传感器在点突变36和单核苷酸多态性（single nucleotide polymorphism，SNPs ）37，38检测中的应用也越来越广泛。Luo 等37报道一种不需固定探针检测序列特异性DNA 和SNP 的多通道电化学方法。利用标记有电活

16、性指示剂的电中性的PNA 探针和带负电荷的ITO 电极来实现免固定目的DNA 的检测。多种不同序列的PNA 探针标记上不同的电活性指示剂从而实现对多种DNA 或SNP 的检测。且对多种目的DNA 和SNP 同时检测时，相互之间没有干扰。这种简单而有效的免标记技术能够应用于许多领域，特别是POCT 。3.3药物应用分析许多药物通过与DNA 的相互作用而发挥疗效，因此电化学DNA 生物传感器可以作为药物应用分析的重要研究工具。有研究表明39药物与DNA 的相互作用能通过鸟嘌呤电化学信号的变化反映出来，进而能够说明药物的作用机制、DNA-药物复合物的性质、结合常数、结合部位及药物作用过程中产生自由基

17、的作用。用电化学DNA 生物传感器和差分脉冲电压技术可进行HIV-1反转录酶抑制药依法韦仑（Efavirenz ，EFV ）的药物剂型分析。EFV 使固定在石墨电极表面的鱼精双链DNA 发生不可逆的氧化作用，通过氧化电信号监测血浆中EFV 的浓度。此方法敏感、快速、简单且成本较低。·49·分子诊断与治疗杂志2011年1月 第3卷 第1期J Mol Diagn Ther, January 2011, Vol. 3 No. 14展望电化学DNA 生物传感器因其制作简单、重现性好、灵敏度高、成本低、易于实现微型化等诸多优点得到研究者的广泛青睐。未来电化学DNA 生物传感器的研究热

18、点将集中在：传感器设计的的优化：寻求电子性能更好的纳米材料进行电极的修饰，更稳定的探针固定方法及更优良的杂交指示剂；多目标同时检测：可以用一个器件同时测量多种目的基因片段，同时避免相互干扰；微型化：实现可携带式床旁检测（POCT ）。在此基础上，进一步拓展其临床应用范围，进行规范方法学评价，加强质量控制措施，相信电化学DNA 生物传感器能够成为未来临床POCT 的重要检测手段之一。参考文献1 Chomean S, Potipitak T, Promptmas C, et al. Quartz crystalmicrobalance-based biosensor for the detecti

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44、rug using electrochemical DNA biosensor and adsorptive stripping voltammetric determination on disposable pencil graphite electrodeJ. Biosens Bioelectron, 2009, 24(8: 2358-2364.电化学DNA生物传感器设计及在医学检验中的应用进展 作者： 作者单位： 刊名： 英文刊名： 年，卷(期： 李博， 郑磊， 王前， 贾立永， LI Bo， ZHENG Lei， WANG Qian， JIA Liyong 南方医科大学南方医院,广东

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