【作者】 杨君；

【导师】 毕彩丰； 范玉华；

【作者基本信息】 中国海洋大学 ， 海洋化学工程与技术， 2011， 博士

【摘要】 在“人类基因组项目”的推动下,人类疾病的DNA诊断及治疗的研究得以快速发展,这也大大加快了生命科学、医学检测和药物筛选等领域技术的进步。金属配合物与DNA、蛋白质、氨基酸等生物分子的作用机理研究,是治疗某些疾病的重要环节。多数金属配合物抗肿瘤药物都是通过与肿瘤细胞DNA的作用,从而破坏肿瘤细胞,表现出其抗癌活性。希夫碱配合物由于具有抑菌、抗肿瘤、抗病毒、对DNA结构的插入作用等多种生物活性,使得它们在生物医学研究等方面起着不可忽视的重要作用。近年来,希夫碱配合物与DNA相互作用的研究,越来越受到人们的重视。但是系统性对比结构相似的希夫碱配合物与DNA之间作用的报道不多。因此,有必要筛选具有特定结构的一系列希夫碱金属配合物,研究它们与DNA的作用机制,总结规律,以期为实现药物初步筛选获得一些有价值的规律。识别和检测血液、组织、体液等实际样品中特定序列的DNA片段,可以在分子水平上确定感染疾病的根源,对药物研发、食品及环境污染的控制和疾病的诊断治疗等意义重大。电化学DNA传感器检测技术不仅可以用来识别特定序列的DNA,还可以检测DNA的损伤,以及研究一些药物与DNA的作用机理,进行特定药物的设计合成,在临床诊断、体外药物筛选等方面都有应用。它提供了一种操作简便、价廉、无污染、灵敏度和选择性好的快速响应的DNA检测技术,已成为当今生物医学领域的前沿性课题。各种新颖的设计思路不断涌现,使电化学DNA传感器的性能不断得到改善,为在药物研究、医学诊断、卫生防疫、环境科学及生物工程等领域的应用中实现低浓度DNA的快速检测提供了有力的研究手段和方法。本论文是基于希夫碱配合物与DNA相互作用的研究现状及特定序列DNA检测技术的发展状况而展开的,研究了同系列希夫碱金属配合物与DNA的相互作用,同时将纳米材料技术和核酸杂交技术相结合构造了两种新颖的电化学DNA传感器。主要进行了以下几个方面的工作:（1）采用电化学方法研究了两种希夫碱配合物[CuL（CH₃COO）（H₂O）]·H₂O、[CoL（CH₃COO）（H₂O）] (L: 2-羟基^-1-萘醛)与DNA的相互作用,紫外可见光谱学方法辅助验证。实验结果表明,配合物[CuL（CH₃COO）（H₂O）]·H₂O、[CoL（CH₃COO）（H₂O）]与DNA之间均存在着一定程度的嵌插作用,两种配合物在电极上都发生不可逆氧化还原反应。而且这两种配合物与DNA的结合常数分别为3.24×10⁴ L·mol^-1、1.36×10⁴ L·mol^-1。希夫碱配合物与DNA之间的作用明显强于相应的配体;在配体相同的情况下,希夫碱铜配合物与DNA的结合常数略大于希夫碱钴配合物。同时,研究发现以[CuL（CH₃COO）（H₂O）]·H₂O作为电化学探针,能在2.0×10^-5 mol·L^-11.2×10^-4 mol·L^-1浓度范围内对溶液中的DNA进行定量检测。以[CoL（CH₃COO）（H₂O）]作为电化学探针,能在1.0×10^-5 mol·L^-19.0×10^-5 mol·L^-1浓度范围内对溶液中的DNA进行定量检测。（2）采用电化学方法研究了两种氨基酸希夫碱铜配合物[CuL1（H₂O）3] （L1: L-天冬酰胺缩邻香草醛）、和[Cu₃（L2）2（CH₃COO）2（H₂O）]·2H₂O （L2: L-谷氨酰胺缩邻香草醛）与DNA的相互作用,紫外可见光谱学方法辅助验证以上结果。实验结果表明,配合物[CuL₁（H₂O）3]与[Cu₃（L2）2（CH₃COO）2（H₂O）]·2H₂O均与DNA之间存在静电作用,两种配合物在电极上发生的电化学过程均为不可逆氧化还原反应。而且两者与DNA的结合常数分别为9.6×10³ L·mol^-1、5.46×10³ L·mol^-1。配合物与DNA之间的作用明显强于相应的配体;在与同一金属配位的情况下空间位阻大、平面性较差的氨基酸希夫碱铜配合物与DNA的结合常数略小。同时,研究发现以[CuL1（H₂O）3]作为电化学探针,能在1.0×10^-5 mol·L^{-1 9}.0×10^-5 mol·L^-1浓度范围内对溶液中的DNA进行定量检测。以[Cu₃（L₂）₂（CH₃COO）2（H₂O）]·2H₂O作为电化学探针,能在2.0×10^-5 mol·L^-19.0×10^-5 mol·L^-1浓度范围内对溶液中的DNA进行定量检测。（3）采用电化学方法研究了三种希夫碱铜配位聚合物[C₁₉H₁₁NO₅Cu]n·n CH₃CH₂OH、[C₁₆H₁₀O₆NCu]n·2nH₂O和[C₁₅H₉NO₅Cu]n·n（CH₃CH₂OH）（H₂O）与DNA的相互作用,紫外可见光谱学方法辅助验证。实验结果表明,配位聚合物[C₁₉H₁₁NO₅Cu]n·nCH₃CH₂OH、[C₁₆H₁₀O₆NCu]n·2nH₂O和[C₁₅H₉NO₅Cu]n·n（CH₃CH₂ OH）（H₂O）均与DNA之间存在着一定程度的嵌插作用,三种配位聚合物在电极上发生了不可逆氧化还原反应。配位聚合物与DNA之间的作用明显强于相应的配体;不论分子中是否存在类似萘环等具有较好平面性的结构,在生成希夫碱铜配位聚合物后,均与DNA之间存在不同程度的嵌插作用,这可能与配位聚合物的特殊结构有关。同时,研究发现以[C₁₉H₁₁NO₅Cu]n·nCH₃CH₂OH作为电化学探针,能在0.4×10^-5 mol·L^-14.0×10^-5 mol·L^-1浓度范围内对溶液中的DNA进行定量检测。以[C₁₆H₁₀O₆NCu]n·2nH₂O作为电化学探针,能在1.0×10^-5 mol·L^-11.2×10-4 mol·L^-1浓度范围内对溶液中的DNA进行定量检测。以[C₁₅H₉NO₅Cu]n·n （CH₃CH₂OH）（H₂O）作为电化学探针,能在1.0×10^-5 mol·L^-11.2×10-4 mol·L^-1浓度范围内对溶液中的DNA进行定量检测。（4）用硝酸腐蚀金银合金箔片,制成纳米多孔金片（NPG）,然后将其修饰到玻碳电极上,得到NPG修饰电极,将NPG电极作为固定DNA探针的电极并结合DNA-Au生物条形码的多次放大技术研制了一种新型电化学DNA检测传感器。其构造过程如下:将探针DNA固定在NPG电极上,然后探针DNA与目标DNA的一端互补杂交,目标DNA的另一端与修饰在30 nm Au上的连接DNA杂交。然后为了进一步放大信号,连接在NPG电极表面的30 nm Au再修饰上大量的带有信号DNA的13 nm Au。单个30 nm Au NPs修饰后可以负载多个带有信号DNA的13 nm Au NPs,而一个13 nm Au上标记有大量的信号DNA。这样检测目标DNA就可以转化为检测成倍放大后的信号DNA,通过两种不同粒径的Au NPs的联合放大作用达到提高检测灵敏度的目的。然后将层层组装后的电极放入[Ru（NH₃）6]³⁺的溶液中,单链DNA上吸附[Ru（NH₃）₆]³⁺,用计时电量法进行检测。而利用多孔金片修饰电极作为固定DNA探针的平台有两方面优势,一是相对于裸金电极,NPG电极有更大的表面积,使得它可以固定更多的DNA探针;另外,多孔金片的高导电性也进一步增强了DNA传感器的灵敏度。结合DNA-Au生物条码的多步放大和多孔金片修饰电极两方面的优势,这种传感器检测目标DNA的线性范围是8.0×10^-16 mol·L^-15.0×10^-17 mol·L^-1,检测限是2.0×10^–17 mol·L^-1。并在单碱基错配DNA的测定上显示出良好的选择性。（5）将纳米材料技术、核酸杂交技术和电化学分析技术相结合,构造了一种高灵敏度、高选择性的新型电化学DNA传感器。其创新点主要有两方面:其一,采用羧基修饰的纳米级磁珠作为捕获和放大目标DNA的载体。磁珠本身较大的表面积使其可以捕获更多的目标DNA,同时,磁珠捕获目标DNA后,通过杂交互补作用,在其表面层层组装上两种不同粒径的修饰过的Au NPs,对目标DNA的量进行了多步放大,而检测目标DNA的量就转化为直接检测放大后的信号DNA的量,从而大大提高了检测的灵敏度。其二,信号DNA是通过二硫键连接在层层组装后的磁珠外层,加入二硫苏糖醇（Dithiothreitol,DTT）后打开二硫键,可以将信号DNA从磁珠上解离下来,与磁珠磁性分离,即得到含有信号DNA的溶液,然后用“三明治夹心法”在NPG电极上检测解离下来的信号DNA溶液,将NPG电极上固定DNA探针,与解离下来的信号DNA一端杂交,信号DNA的另一端再与DNA-Au生物条码上的探针DNA进行杂交,将层层组装后的电极放入[Ru（NH₃）₆]³⁺溶液中,在信号DNA上吸附[Ru（NH₃）₆]³⁺,用计时电量法进行检测。在最佳条件下,该DNA传感器可检测到0.12 amol·L^-1级的较低浓度的目标DNA,并在单碱基错配DNA的测定上显示出良好的选择性。更多还原

【Abstract】 Under the push of the“human genome project”, the study of DNA diagnosis and treatment are developed rapidly and various technologies in the fields of disease diagnosis or drug screening have been greatly advanced.Most anti-cancer drugs work through the interactions with DNA in tumor cells. Investigating the interaction mechanism of metal or metal complex with biomolecule, such as DNA, protein and amino acid, provides a starting point for the diagnosis of human disease. Schiff base complexes have been proved to have biological activities in many fields, such as anti-cancer, antibacterial, and interactions with DNA, which causes widespread concern in the scientific community. In recent years, several reports on the interactions of Schiff base complexes with DNA have been published. However, there are rarely reports about the comparison of the interaction mechanism between homologous series of Schiff base complexes and DNA. So to summarize differences of the interactions between homologous series of Schiff base metal complexes and DNA is valuable for drug screening.The recognition and detection of DNA in the human blood, organization or body fluid sample have become more and more important for drug research and development, diseases diagnosis and environmental pollution control. DNA electro chemical sensors hold an enormous potential for the DNA recognition, detection of DNA damage, drug screening or disease diagnosis. It provides an inexpensive, non-pollution, sensitive, easy-to-use and fast response device which therefore stays in the center of interest of many biomedical scientists. At present, many new procedures are under intense investigation in order to improve the function of DNA electrochemical sensors and to meet the new challenges connected to the simple, fast response of low levels DNA in different fields.This dissertation aims to study the interaction mechanism between homologous series of Schiff base complexes and DNA. Moreover, nanomaterials and nucleic acid hybridization technique are introduced to develop two novel DNA electrochemical sensors. The main researches are as follows:（1） The interaction mechanism of [CuL（CH₃COO）（H₂O）]·H₂O and [CoL （CH₃COO） （H₂O）] with DNA was studied by means of electrochemical methods. Uv-vis spectroscopic methods validated the results. The interaction mode of two Schiff base complexes with deoxyribonucleic acid are intercalation and binding constants are 3.24×10⁴ L·mol^-1, 1.36×10⁴ L·mol^-1, respectively. Two complexes have the irreversible electrochemical redox reaction process in electrode. The binding functions of Schiff base complexes are positively related to the concentration. The binding functions of Schiff base complexes are stronger than the ligand. The binding constant of [CuL（CH₃COO）（H₂O）]·H₂O is greater than the [CoL（CH₃COO）（H₂O）]. Meanwhile, experimental results demonstrate that using [CuL（CH₃COO）（H₂O）]·H₂O or [CoL（CH₃ COO）（H₂O）] as electrochemical probes could detect 2.0×10–5 mol·L^{-1 1}.2×10–4 mol·L^-1 or 1.0×10–5 mol·L^-19.0×10^-5 mol·L^-1 DNA in solution, respectively.（2） The interaction mechanism of [CuL₁（H₂O）3] and [Cu₃（L₂）₂（CH₃COO）₂ （H₂O）]·2H₂O with DNA was studied by means of electrochemical methods. Uv-vis spectroscopic methods validated the results. The interaction mode of two Schiff base complexes with deoxyribonucleic acid are electrostatic force and binding constants are 9.6×10³ L·mol^-1,5.46×10³ L·mol^-1, respectively. Two complexes have the irreversi ble electrochemical redox reaction process in electrode. The binding functions of Schiff base complexes are positively related to the concentration. The binding functions of Schiff base complexes are stronger than the ligand. The binding ratios are related to the planar area of the complexes. Meanwhile, experimental results demonstrate that using [CuL₁（H₂O）3] or [[Cu₃（L2）2（CH₃COO）2（H₂O）]·2H₂O as electro chemical probes could detect 1.0×10^-5 mol·L^-19.0×10^-5 mol·L^-1 or 2.0×10^-5 mol·L^{-1 9}.0×10^-5 mol·L^-1 DNA in solution, respectively.（3） The interaction mechanism of [C₁₉H₁₁NO₅Cu]n·n CH₃CH₂OH, [C₁₆H₁₀O₆NCu]n·2nH₂O and [C₁₅H₉NO₅Cu]n·n（CH₃CH₂OH）（H₂O） with DNA was studied by means of electrochemical methods. Uv-vis spectroscopic methods validated the results. The interaction mode of three Schiff base coordination polymers with deoxyribonucleic acid are intercalation. Three coordination polymers have the irreversible electro chemical redox reaction process in electrode. The binding functions of Schiff base coordination polymers are positively related to the concentration. The binding functions of Schiff base coordination polymers are stronger than the ligand. The binding constant of [CuL（CH₃COO）（H2O）]·H2O is greater than the [CoL（CH₃COO） （H2O）]. No matter whether there are some flat areas like naphthalene molecular in the structure, all the coordination polymers showed intercalation with DNA. It is speculated that this might be caused by the special structure of coordination polymer. Meanwhile, experimental results demonstrate that using [C₁₉H₁₁NO₅Cu]n·nCH₃ CH2OH, [C₁₆H₁₀O₆NCu]n·2nH2O or [C₁₅H₉NO₅Cu]n·n（CH₃CH2OH）（H2O） as electro chemical probes could detect 0.4×10^-5 mol·L^-14.0×10^-5 mol·L^-1, 1.0×10^-5 mol·L^-1 1.2×10-4 mol·L^-1 and 1.0×10^-5 mol·L^-1 1.2×10-4 mol·L^-1 DNA in solution, resp ectively.（4） An novel electrochemical DNA detection method was developed based on the Nanoporous Gold Electrode （NPG） and singal multiple amplification with two different diameters of DNA-Au bio-barcodes. The target DNA signal amplification assay used here was fabricated by immobilizing probe DNA on the NPG electrode and then being hybridized with one end of target DNA. The other end of target DNA was further hybridized with the linker DNA loaded on the carboxyl group modified Au NPs of 30 nm. To further amplify the target DNA signals, the immobilized 30 nm Au NPs was also modified with 13 nm DNA-Au bio-barcodes which were labelled many reporter DNA. In this means, one target signal could be transformed into multiple signals of the markers since a single 30 nm Au NP could be loaded with dozens of 13 nm Au NPs, and an 13 nm Au NPs could be loaded with thousands of signal DNA. Electrochemical signals of [Ru（NH₃）₆]³⁺ bound to the signal DNA via electrostatic interactions were measured by chronocoulometry （CC）. Using NPG electrode as the platform for fixing DNA probes has two advantages, one is because NPG electrode has larger surface area than the gold electrodes which makes it fixed more DNA probes. In addition, NPG electrode has high conductivity which further enhances the sensor sensitivity. Combining the two advantages of DNA-Au bio-barcodes and the NPG electrode, this assay could detect as low as amol·L^-1 target DNA and exhibited excellent selectivity against one-base mismatched DNA. This DNA biosensor could detect DNA target quantitatively in the range of 8.0×10^-16 mol·L^-1 to 5.0×10^-17 mol·L^-1, with the detection limit of 2.0×10^–17 mol·L^-1 and exhi bited excellent selectivity even for single-mismatched DNA detection.（5） An ultrasensitive electrochemical DNA detection method was developed based on multiple amplification of Nanoporous Gold Electrode, DNA-Au bio-barcodes and dithiothreitol-induced oligonucleotide releasing from bio-barcodes. Its innovations has two aspects: first, using carboxyl modified nanoscale magnetic beads as the carrier of probe DNA in order to capture more target DNA. The probe DNA which was immobilized in the magnetic beads captured the specific target DNA and then assembled layer-by-layer with the two different diameters of modified Au NPs. And the detection of target DNA is transformed into directly measure the amplified amount of reporter DNA, which greatly improve the detection sensitivity. Second, this assay relied on the ability to liberate the adsorbed thiolated oligonucleotides from the gold nanoparticle surface with dithiothreitol （DTT）. DTT was added to break up disulfide bonds and remove the reporter DNA from the surface of the layer-by-layer assembly magnetic beads. After magnetic separation, DTT-released reporter DNA were subsequently detected using the“sandwich-type”NPG-DNA assay by chronocou lometry （CC） analysis. The“sandwich-type”NPG-DNA assay was fabricated by immobilizing probe DNA on the NPG electrode and being hybridized with DTT-released reporter DNA which further hybridized with the barcode DNA loaded on the Au NPs. Electrochemical signals of [Ru（NH₃）₆]³⁺ bound to the reporter DNA via electrostatic interactions were measured by chronocoulometry （CC）. Under the optimum conditions, this assay could detect as low as 0.12 amol·L^-1 target DNA and exhibited excellent selectivity against one-base mismatched DNA.更多还原