【作者】 张诺；

【导师】 魏琴；

【作者基本信息】 济南大学 ， 分析化学， 2010， 硕士

【摘要】 DNA是活细胞中最重要的分子,它含有特定细胞的全部遗传信息,在分子生物学和生物医学领域的研究非常广泛。DNA生物传感器是当今生物传感技术的研究热点。用DNA修饰电极研究DNA与其他物质的相互作用,不仅能克服溶液电化学方法背景信号大,弱信号难以提取的缺点,而且DNA的用量大为减少,方法的灵敏度得到提高,以较少样品消耗得到多种作用参数。研究DNA与其它小分子的相互作用是生物化学和分子生物学中的重大课题,对疾病诊断、环境检测、医药研究和新药设计起着重要作用。水溶性阳离子卟啉具有优异的生物活性,可以作为潜在的抗癌、抗菌药物和核酸结构及动力学探针,其与DNA的相互作用成为研究的热门话题。对卟啉化合物与DNA结合模式的研究也有助于寻求卟啉化合物的生物活性及其与DNA的结合模式之间的关系。DNA与药物分子等相互作用的研究,有助于人们了解某些药物分子对DNA体内复制和转录的影响以及由此引起的物种性变异、各种化合物与DNA间亲和力的大小和化学核酸酶的作用机制等信息。本文合成了具有优良生物相容性和独特吸附性的羟基磷灰石(HAp),并制备了DNA-纳米羟基磷灰石修饰电极(dsDNA/HAp/GCE),用表面电化学法研究了DNA与其它小分子的相互作用。论文主要包括以下几个方面的内容:第一部分概述了DNA修饰电极制备方法的研究现状及DNA修饰电极的研究意义和应用。介绍了本论文的研究内容及特点。第二部分介绍了HAp的制备及表征。HAp [Ca10(PO4)6(OH)2]是常用的骨修复材料,其规则的立体化学结构和独特的多吸附位点使其在催化、蛋白质分离等领域倍受瞩目。本文采用共沉淀法与微乳液法方法,制备了纳米HAp,并分析了在微乳液介质中HAp的合成过程。第三部分研究了DNA与水溶性阳离子卟啉-三甲铵基苯基卟啉(TAPP)的相互作用及检测DNA损伤。利用滴涂法将具有优良生物相容性和独特吸附性的HAp修饰在玻碳电极上形成纳米薄膜。电化学实验结果证明该纳米HAp薄膜能有效地将双链DNA吸附于其表面。采用循环伏安法和交流阻抗法系统研究了固定在HAp薄膜上的DNA与TAPP之间的相互作用并将此修饰电极用于检测DNA损伤和测定卟啉类物质。实验结果表明,在50 ~ 250 mV·s-1扫描速度范围内该电极反应过程系表面吸附反应控制;在pH值为3.7 ~ 9.1的区间内,随pH值增大,TAPP的氧化还原峰都发生正移,这表明TAPP在DNA修饰电极上的氧化还原过程不仅有电子参与,而且还有质子参与;在pH = 7.0的磷酸盐缓冲溶液(PBS)中,随溶液离子强度增大,TAPP在DNA修饰电极上的表观式量电位不断正移,表明TAPP与DNA之间的相互作用主要是小沟槽的嵌入作用,二者可生成超分子化合物。根据Langmuir吸附公式,得出TAPP与DNA之间的结合常数为1.48×105 mol·L-1。第四部分研究了维生素B12与DNA的相互作用。采用循环伏安法系统研究了维生素B12与DNA在pH = 4.9的HAc-NaAc中相互作用的电化学行为,所得结论比溶液电化学法更具说服力。实验结果表明,DNA的存在能导致维生素B12还原峰电流的降低。通过测定裸玻碳电极和DNA修饰电极的一些电化学参数,证明维生素B12与DNA在该实验条件下结合生成了一种非电活性的超分子化合物,并利用一系列方程求得该超分子化合物的组成为1 : 1,结合常数β= 5.35×105 mol·L-1。第五部分研究了DNA修饰电极对氯霉素的电催化作用。利用dsDNA/HAp/GCE对氯霉素的电催化作用,建立了对氯霉素含量进行定量分析的一种电分析方法。在pH = 6.0的KH2PO4-Na2HPO4缓冲溶液和0.1 mol·L-1 KCl溶液中,氯霉素的浓度在2.80×10-7 ~ 3.60×10-6 mol·L-1范围内与峰电流呈良好的线性关系,线性回归方程和线性相关系数分别为:ip(μA) = 5.865 + 0.4784 c (μmol·L-1),r = 0.9942,检测限可达1.35×10-7 mol·L-1。利用该法对氯霉素滴眼液进行定量分析,8次平行样品分析结果的相对标准偏差小于3 %,完全满足定量分析的要求。更多还原

【Abstract】 DNA is the most important molecule in living cell, it contains all the genetic information of a specific cell. DNA biosensor is the hot topic in bio-sensor technology today. Compared with solution electrochemical methods, using DNA modified electrode to study interactions between DNA and other species can not only overcome shortcomings of the big background signal of the solution electrochemical methods and the weak signal difficult to extract, but also have a significant reduction in the amount of DNA. The method can improve the sensitivity and make people obtain a variety of parameters with less sample consumption. Studying the interactions between DNA and other elements is a major research topic in biochemistry and molecular biology, which plays an important role in disease diagnosis, environmental testing, and pharmaceutical research and in the design of new drugs. Water-soluble cationic porphyrins have excellent bioactivity, and they can be used as potential anticancer or antibacterial agents and probes for the structure and dynamics of DNA. Studies on the interaction of DNA with porphyrin can help to seek the relationship between the biological activity of porphyrin and the binding model. Investigations on the interaction of DNA with drug molecules help people to understand the effect of some drug molecule to the copying and transcription of DNA, and variation caused by this, which can also provide important information on the affinity between DNA and other compounds, and the mechanism of some nuclease.In this article, nano-hydroxyapatite (HAp) was prepared by combining co-precipitation with microemulsion method, which exhibited strong adsorption for DNA based on the advantages of its excellent biocompatibility and particular adsorbability. DNA and HAp were easily modified onto glassy carbon electrode (GCE) by simple and convenient“tip-coating”method. We studied the interaction of DNA with other small molecules by surface electrochemical method. The main contents of the paper were provided as followed.In the first part, we described the research significance and status of the preparation methods and application of DNA modified electrod briefly. The research contents and characteristics of this paper were also included. The second part is about the preparation and characterization of HAp. HAp (Ca10(PO4)6(OH)2), is widely used as implants or as coatings on prostheses in the field of medicine. The stereoscopic chemistry structure and unique locus of adsorption make HAp focused in the field of protein separation and catalytic. We prepared HAp by combining co-precipitation with microemulsion method, and analyzed the synthesis process of HAp in microemulsion medium.The interaction of DNA with meso-tetra-(4-trimethylaminophenyl) porphyrin by DNA-hydroxyapatite modified electrode (dsDNA/HAp/GCE) and detection of DNA damage were studied in the third part. Cyclic voltammetry was used to investigate the interaction of meso-tetra-(4-trimethylaminophenyl) porphyrin (TAPP) with DNA immobilized on HAp film as well as the detection of DNA damage and determination of porphyrin. The redox currents of TAPP on DNA modified GCE increased with scan rate from 50 to 250 mV·s-1, showing a linear relationship as expected for a surface-controlled process. Both reduction and oxidation peak potentials of TAPP underwent a positive shift with increasing pH from 3.7 to 9.1, which indicated that the redox process of TAPP had both electron and proton involved. In phosphate buffer solution (PBS) with pH 7.0, the featured potential for TAPP on dsDNA/HAp/GCE shifted positively with the increase of ionic strength of solution, suggesting an intercalation mode existed between TAPP and DNA, with a formation of supramolecular compounds. According to Langmuir adsorption formula, the binding constant of TAPP to DNA was calculated to be 1.48×105 mol·L-1.And in the fourth part, the interaction between DNA and Vitamin B12 was investigated. Cyclic voltammetry was used to investigate the interaction of DNA immobilized on the HAp film with vitamin B12 (VB12). The existence of DNA led to the decrease of reduction current of VB12. Both the electron transfer coefficient (α) and the standard rate constant (ks) were different obtained on GCE and dsDNA/HAp/GCE, which indicated the formation of an electrochemical inactive super molecular complex DNA-nVB12. The equilibrium constant of this complex was calculated to be 5.35×105 mol·L-1 and the binding number between DNA and VB12 of the complex was determined to be 1.At last, we setted up an electroanalysis method of determining Chloramphenicol (CAP) based on the eletrocalysis property of DNA modified electrod. In KH2PO4-Na2HPO4 buffer solution with pH 6.0 cotaining 0.1 mol·L-1 KCl, the peak current was proportional to the concentration of CAP in the range of 2.80×10-7 to 3.60×10-6 mol·L-1, with a detection limit of 1.35×10-7 mol·L-1. The linear regress equation was ip (μA) = 5.865 + 0.4784 c (μmol·L-1), and the linear correlation coffcient was r = 0.9942. The method has been successfully applied to the quantitative analysis of CAP in real Chloramphenicol eye drops samples, which totally meet the demand of microanalysis. The relative standard deviation of eight samples analysis results was 1ess than 3%.更多还原