【作者】 张爱平；

【导师】 方禹之； 何品刚；

【作者基本信息】 华东师范大学 ， 分析化学， 2004， 硕士

【摘要】 脱氧核糖核酸(DNA)是遗传信息的承担者，DNA分子中碱基序列的变异与人类许多遗传疾病有关。因此，对特定序列的DNA分析以及对DNA链中碱基突变的检测在卫生防疫、疾病诊断、药物研究、环境科学及生物工程方面具有深远的意义。电化学DNA生物传感器是一门涉及生物化学、电化学、医学及电子学等领域的交叉学科，它提供了一种全新的DNA(基因)检测技术，具有简单、可靠、价廉、灵敏和选择性好等优点，并且与目前的DNA生物芯片技术兼容，在分子生物学、生物医学工程、环境监测、食品卫生检验等方面显示了广阔的应用前景，已成为当今生物学、医学领域的前沿性课题。 纳米材料被认为是跨世纪材料研究领域的热点，有“21世纪最有前途的材料”之美誉。当物质的结构单元进入纳米量级(1～100 nm)时，其本身具有量子尺寸效应、表面效应和宏观量子隧道效应，因而展现出许多特有的性质，在催化、光吸收、生物医药、磁介质及新材料等方面得到了广泛的应用。纳米颗粒的比表面积大、表面反应活性高、催化效率高、吸附能力强等这些优异性质，为生物医学研究提供了新的研究途径。 本论文主要是将纳米技术、核酸分子杂交技术与电化学分析技术相结合，研制具有高灵敏度高选择性的基于纳米物质电化学DNA生物传感器，成功地应用于对特定序列DNA片断快速、灵敏和准确的识别，为基因的快速分析测定提供了一种简便、快捷、廉价的检测装置。基于纳米物质发展起来这种袖珍式、电池驱动、价格低廉的DNA检测器必将给疾病早期诊断行业带来巨大的利益。 摘要 首先系统介绍了DNA生物传感器的基本结构及其分类。着重介绍了DNA电化学传感器的设计，包括DNA片段的固定方法、DNA杂交以及杂交信号的转化两方面。第二章基于硫化锡纳米团簇标记DNA电化学传感的研究 我们在此报道硫化福(Cds)纳米团簇标记的DNA生物传感器。合成表面具有自由梭基的硫化福(C ds)纳米团簇，将其标记于人工合成的5’端氨基修饰的寡聚核普酸(ODN)片段上，制备成CdS纳米团簇标记的DNA探针。在一定的条件下，使其与固定在玻碳电极表面的待测DNA序列进行杂交反应。以汞修饰的玻碳电极为工作电极，利用阳极溶出示差脉冲伏安法(ASDPV)测定Cd(II)的量，从而间接实现对互补、三碱基错配、非互补DNA片段的识别。对目标DNA序列的检测限达到0.2 pmol几数量级。第三章PbS纳米粒子标记寡聚核营酸电化学检测DNA杂交 我们报道了一种基于PbS纳米粒子标记检测DNA杂交的方法。这种方法用PbS纳米粒子标记，然后电化学测定Pb(II)。PbS纳米粒子标记到寡聚核昔酸上制成相应的DNA探针，利用特定序列杂交反应，识别固定在电极上DNA序列。检测限为0.3 pmol/L目标DNA。PbS纳米粒子很容易与DNA生物分子连接，并且ASv对Pb(II)的测定具有较高的灵敏度，因此这种方法有望开发出新的电化学DNA生物传感器。第四章多量子点用于不同目标DNA电化学同时测定 本文报道了利用多个量子点(QDs)作为标记物，对多条不同序列的DNA杂交反应电化学同时检测。一系列表面修饰梭酸基的无机溶胶(QDs)在水溶液中比较方便地合成。在EDAC作用下，自由的梭酸基与氨基修饰的寡聚核昔酸共价连接制成QDs标记的DNA探针。在一定的条件下，QDs标记的DNA探针与固定在玻碳电极表面待测DNA序列进行杂交反应，之后以汞修饰的玻碳电极为工作电极，利用阳极溶出伏安法(ASV)测定溶解的金属离子，从而对目标DNA序列间接测定。QDs释放出的金属离子具有互不干扰的、不同的电化学电摘要位，因而它们被用于对多个目标DNA同时测定。本实验中同时测定两种不同目标DNA序列的检测限为0.8 pmol/L。第五章亚甲基兰识别固定于修饰ZrO:金电极表面DNA的电化学研究 在此报道了电化学沉积二氧化错(ZrO:)薄膜于金电极表面用于ssDNA的固定，并以亚甲基兰(MB)为指示剂电化学检测DNA杂交。由于磷酸基与Zr仇的亲和作用，5气端带磷酸基的寡聚核昔酸共价固定于ZrOZ薄膜上，将固定有单链DNA(SSDNA)的电极置于互补的DNA溶液中杂交。与互补的DNA靶序列杂交后，电活性标记物MB的峰电流与杂交前(信号较强)相比较急剧下降。杂交后阴极峰电流与目标DNA浓度(2 .25 x 10一’o一2.25 x 10’8 moFL)的对数值成线性关系，检测限达到1 x 10一’0 mol几。第六章磁性纳米粒子用于DNA杂交电化学的研究 在此我们报道一种电化学DNA生物传感器。它是利用磁性纳米颗粒固定目标DNA，并以ZnS纳米粒子作为标记物电化学测定DNA杂交。在偶联活化剂EDAC作用下，夕端修饰磷酸基的目标DNA共价固定到氨基修饰的磁性纳米颗粒上。ZnS纳米粒子标记的DNA探针对固定在磁性纳米粒子上目标DNA测定。这种方法不但具有较高的灵敏度，而且消除了非特异性吸附的影响，是一种比较理想的方法。第七章DNA生物传感器的应用及展望更多还原

【Abstract】 With the increase of knowledge about the mutations of genes being responsible for numerous inherited human disorders, sequence-specific DNA detection has become a topic of substantial interest in the areas of early diagnosis, medicine, epidemic prevention, environmental protection and bioengineering. In this context, many new biological technologies emerged and found their applications in this field. Of them, electrochemical genosensors may offer a promising alternative for faster, cheaper and simpler nucleic acid assays. Consequently there is a considerable enthusiasm towards the development of electrochemical biosensors for DNA hybridization detection. Such biosensors commonly rely on the immobilization of a single stranded DNA sequence that recognizes its complementary DNA sequence by hybridization and the conversion of DNA base-pair recognization event into a useful electrical signal. Electrochemical biosensors are not only uniquely qualified for meeting the size, cost and power requirements of decentralized genetic testing but offers an elegant route for interfacing at the molecular level the DNA recognition and signal-transduction elements. Therefore, they are expected to have a broad prospect of application in clinic examination of inherited diseases and drug screening.Nano-materials, with their size in the range of 1-100 nm, are currently under intense investigation owing to their special properties. Thanks to their small size, these materials exhibit quanta-size effect, small-size effect, surface effect and tunneling effect that differ from both their bulk material and the individual atoms from which they comprised. With these unique properties, they are widely used in the fields of catalysis, optical absorption, medicine, magnetic medium, new materials synthesis and particularly attractive in biological applications.The combination of the unique properties of nanometer-sized materials with the event of Watson-Crick base-pairing interaction makes them excellent candidates for DNA sensors with high sensitivity and selectivity. This dissertation focuses on fabricating electrochemical DNA biosensors by the use of some kinds of nano-materials, developing a sensitive, sequence-specific and quantifiable gene detection method, and establishing the bases for application of electrochemical DNA biosensor to clinic diagnose.The dissertation includes seven chapters:Chapter 1 Introduce the composition and classification of DNA biosensorsThe abstract mainly focuses on the major steps involved in electrochemical biosensfng of DNA hybridization, the formation of the nucleic acid recognization layer, the actual hybridization event, and the conversion of this hybridization event into an electrical signal in detail.Chapter 2 Cadmium sulflde nanocluster-based electrochemical stripping detection ofDNA hybridizationA novel, sensitive electrochemical DNA hybridization detection assay, using cadmium sulfide (CdS) nanoclusters as the oligonucleotide labeling tag, is described. The assay relies on the hybridization of the target DNA with the CdS nanocluster--oligonucleotide DNA probe, followed by the dissolution of the CdS nanoclusters anchored on the hybrids and the indirectly determination of the dissolved cadmium ions by sensitive anodic stripping voltammetry (ASV) at a mercury-coated glassy carbon electrode (GCE). The combination of the large number of cadmium ions released from each DNA hybrid with the remarkable sensitivity of the electrochemical stripping analysis for cadmium at mercury-film GCE allows detection at levels as low as 0.2 pmol L-1 of the complementary sequence of DNA.Chapter 3 Lead sulflde nanoparticle as oligonucleotides labels for electrochemicalstripping detection of DNA hybridizationWe report a method for the detection of DNA hybridization in connection to lead sulfide (PbS) nanoparticle tags and electrochemical stripping measurement of the lead. The nanoparticle was used as a marker to label a sequence-known oligonucleotide, which was then employed as a DNA probe for identifying更多还原