【作者】 卓淑娟；

【导师】 朱昌青；

【作者基本信息】 安徽师范大学 ， 分析化学， 2004， 硕士

【摘要】 本论文以几种卟啉、花菁、酞菁化合物作为探针，利用共振光散射技术、荧光增强方法、离子平衡移动方法、动力学荧光方法研究了它们与核酸的相互作用，建立了多个核酸测定新方法。全文共分六章。第一章 首先对荧光分析进展作了介绍，然后对近期的荧光探针研究进行了简单综述，最后提出了论文设想。第二章 介绍了实验所用试剂、仪器及本论文所涉及的几种探针的合成。第三章 用共振光散射方法，研究在表面活性剂溴代十六烷基三甲基铵存在下，溴代十六烷基吡啶基卟啉与核酸的相互作用，建立了灵敏的共振光散射测定核酸的方法。第四章 分为两部分首先，研究了一种阳离子花菁与 DNA 的相互作用，建立了荧光增强测定核酸的方法。本法快速，灵敏、重现性好，线性范围宽（超过三个数量级）。研究表明，花菁与 DNA 的作用机理：一种可能是花菁分子以单体形式结合于 DNA 小的沟槽中，沟槽壁抑制了花菁激发态扭转和非辐射衰变；另一种可能是花菁染料在双螺旋 DNA 模板上自发组装成螺旋状 J-聚集体。其次，研究了生物大分子对花菁与聚氨基酸多肽间离子缔合平衡的影响。阴离子花菁与核酸间缺乏强烈的相互作用，但能和带正电荷的聚赖氨酸发生作用形成离子缔合物。阴离子花菁在水溶液中有强荧光，在带正电荷的质子化聚赖氨酸（pKa?9.9）存在下，花菁的荧光几乎完全猝灭，在该体系中加入核酸，花菁会回复初始荧光。根据这一事实，我们建立了测定核酸的红区荧光回升方法。本法的主要优点是荧光激发和发射波长都位于近红区（778/804 nm），大大降低了生物分子或基质背 I<WP=7>中文摘要景的可能干扰。 第五章 分为三部分 首先，利用阴离子四羧基铝酞菁和阳离子溴代十六烷基吡啶基卟啉组成离子对，实现了核酸高灵敏测定。 其次，研究了四羧基铝酞菁－聚赖氨酸－核酸离子平衡系统并用于核酸分析。本法稳定、灵敏度高，同时也具有红区测量的低干扰优势。 最后，考察了四羧基铁酞菁(FeC4Pc)-聚赖氨酸-核酸离子平衡系统对 FeC4Pc 催化活性的调控作用。FeC4Pc 能催化过氧化氢和 DL-酪氨酸氧化反应而产生一强荧光物质。适量聚赖氨酸的加入，能明显降低FeC4Pc 的催化活性而导致荧光猝灭，但核酸的加入又使荧光回升。机理研究表明，FeC4Pc 与带正电荷的聚赖氨酸通过静电作用缔合后，由于聚赖氨酸是一种多肽，在其表面单个酞菁分子能有序排列（面对面叠加）形成大的聚集，屏蔽了酞菁轴向催化位点，酞菁催化活性降低。核酸的加入，能将酞菁从聚赖氨酸上解缔成为自由的酞菁分子，催化活性恢复。在最佳条件下，回升的荧光强度与核酸浓度间存在良好的线性关系。根据这个事实，我们建立了核酸测定的动力学荧光增强方法。该法具有动力学方法所固有的高灵敏度和宽线性范围的优点。 第六章 小结了本论文工作的特色及创新点。更多还原

【Abstract】 This thesis developed several new methods for the determination of nucleic acidsemploying some porphyrin, cyanine and phthalocyanine compounds as probes, by meansof resonance light-scattering, fluorescence enhancement method, shifting theion-association equilibrium and kinetic fluorometric method. The thesis consisted of sixchapters. In chapter 1, recent progress of fluorescence analysis and probe research wasreviewed. Then, the research proposal for this thesis was presented. In chapter 2, the reagents, apparatus and synthesis of some probes used by this workwere described. In chapter 3, using resonance light-scattering (RLS) technique, the interaction oftetra-(N-hexadecylpyridiniumyl) porphyrin with nucleic acids in the presence ofsurfactant cetyltrimethylammonium bromide was investigated. And a sensitive RLSmethod for the determination of nucleic acids was developed. Chapter 4 was divided into two parts. In part one, a fluorescence enhancement method was proposed for the determinationof nucleic acids based on the enhancement effect of DNA on the fluorescence of a cationcyanine. In addition to its rapid reaction, high sensitivity and good reproducibility, themethod had a rather wide linear range (beyond three orders of magnitude). The spectrainvestigation disclosed that the fluorescence enhancement could be attributed to thefollowing possible reasons: (i) the cyanine was bound in the form of monomer into theminor groove of DNA, and the wall of the minor groove inhibited the excited-statetwisting and the nonradiative decay of the dye; (ii) the cyanine dye spontaneouslyassembled into the double-helical DNA template to form helical J-aggregates. In the second part, the effect of DNA on the ion-association equilibrium between ananion cyanine and poly-amino acids polypeptide was studied. The anion cyanine couldnot be directly employed to determine nucleic acids due to no evident interaction betweencyanine and DNA. However, it could intensely interact with opposite charged poly-lysine.The free anionic cyanine was highly fluorescent in solution. In the presence ofpositively charged polymers, protonated poly-lysine (pKa?9.9), the fluorescence ofcyanine was almost completely quenched, however, the cyanine regained its originalfluorescence if nucleic acids were added. Based on this phenomenon, a novel III<WP=9>Ａｂｓｔｒａｃｔnear-infrared (near-IR) fluorescence recovery method for the quantification of nucleicacids was presented. The main advantage of this method was that both the fluorescenceexcitation and emission were located in the near-IR region (778/804nm), so thebackground interference induced by bio-molecules or matrix could be greatly overcome. Chapter 5 was divided into three parts. In part one, high sensitive determination of nucleic was developed employing atwo-regent system which was composed of an anionic tetracarboxy aluminumphthalocyanine and a cationic tetra-(N-hexadecylpyridiniumyl) porphyrin. In part two, tetracarboxy aluminum phthalocyanine-Poly-lysine-nucleic acids ion-association equilibrium system was studied and applied in the quantification of nucleicacids. This method was characterized by good stability, high sensitivity. At the same time,the method also had the advantage of near-IR region assay of little backgroundinterference. In part three, the tune action of Iron (Ⅲ) tetracarboxy phthalocyanine (FeC4Pc)–poly–lysine–nucleic acids equilibrium system on catalytic activity of FeC4Pc wasinvestigated. The oxidation reaction between hydrogen peroxide and DL-tyrosine in thepresence of FeC4Pc gave an intensive fluorescence emission. The fluorescence wasquenched by poly-lysine at its proper concentration due to its association with FeC4Pc andconsequently the descent of the catalytic activity of FeC4Pc, but recovered by addingnucleic acids. The study of mechanism showed that the electrostatic interac更多还原