【作者】 丁红红；

【导师】 吴霞；

【作者基本信息】 山东大学 ， 分析化学， 2008， 硕士

【摘要】 核（苷）酸、蛋白质是生命现象的物质基础,与生物的遗传变异、肿瘤的发生、病毒的感染、射线对机体的作用有着重要关系。因此,深入研究生物分子与小分子物质（或生物分子）的相互作用机理,建立对生物分子快速、简便的分析方法,对分子水平上阐明生命的奥秘等方面具有重要的意义,是当前生物分析化学研究的前沿和热点。本论文利用荧光技术、共振光散射技术、吸收光谱技术、圆二色光谱技术、透射电镜等技术研究了生物分子与有机化合物（或生物分子）之间相互作用机理,建立了对核（苷）酸简便、快速、准确、灵敏而选择性的分析方法。论文共分五个部分。论文的第一部分综述了核（苷）酸荧光探针研究进展和发展趋势,以及小分子物质与生物大分子相互作用的研究手段等,共引用文献142篇。在论文的第二部分,以桑色素-KI为荧光探针建立了灵敏的核酸分析方法。研究表明,在中性条件下,桑色素-KI能够选择性地识别双螺旋核酸中的鱼精子DNA,从而建立了选择性检测鱼精子DNA的荧光新方法。在最佳实验条件下,hsDNA和smDNA的线性范围分别为8.0×10^-9-2.0×10^-5g mL^-1和5.0×10^-9-1.0×10^-5g mL^-1,它们的检出限分别达到了4.5ng mL^-1和3.5ng mL^-1。KI-morin与fsDNA相互作用机理研究表明:KI-桑色素与fsDNA主要以沟槽式结合,还存在分子间作用力。在低浓度下,KI对桑色素-fsDNA的荧光进一步增强主要源于更有利于发光结构的形成。论文的第三部分,用桑色素作为荧光探针,建立了灵敏而选择性测定鸟嘌呤的新方法。结果表明,在NaAc-HAc缓冲中,鸟嘌呤碱基加入到桑色素体系中,荧光强度显著增强且增强程度与鸟嘌呤碱基的浓度有良好的线性关系。该方法可用于鸟嘌呤的测定。在最佳实验条件下,鸟嘌呤的线性范围为2.0×1^-8-8.0×10^-5mol L^-1,检出限为7.0×10^-9mol L^-1,干扰实验结果表明其它碱基对鸟嘌呤的测定影响较小。机理研究认为,鸟嘌呤对桑色素的荧光增强作用源于氢键缔合物的形成,氢键作用增大了桑色素结构的共平面效应和刚性,减小了桑色素的能量耗散,导致其荧光寿命的增加和荧光强度的增强。论文的第四部分,利用荧光光谱和圆二色光谱对肾上腺素-氢氧化钠-2,3-二氨基萘体系的作用机理进行了研究。利用双倒数曲线法测定了β-CD_x对肾上腺素-氢氧化钠-2,3-二氨基萘体系和肾上腺素-氢氧化钠-丙酮-2,3-二氨基萘体系的包结常数和包结比。结果表明,在丙酮存在下,荧光产物与β-CD_x以1:1配合,包结常数平均为206L mol^-1。而无丙酮存在时,产物与β-CD_x存在多级包结。在β-CD_x低浓度区间形成的是1:1的包结物,其平衡常数为68L mol^-1;随着β-CD_x浓度的继续增加,还会与一分子的β-CD_x进行包结,最终形成2:1的包结物,其平衡常数平均为1270 L mol^-1。圆二色光谱研究表明:肾上腺素-氢氧化钠-2,3-二氨基萘体系的反应产物以轴向包结的方式进入β-CD_x的空腔体系。在这个体系中,丙酮和β-CD_x存在协同增敏效应。有机溶剂的加入使得荧光产物的吲哚环裸露在空腔外,由于空间位阻效应,荧光产物与β-CD_x形成1:1（或者2:2）包结物。在论文第五部分的研究中,利用去溶剂化法合成了BSA纳米粒子。研究发现,该蛋白质纳米粒子在430nm处产生一个新的荧光发射峰,其源于纳米粒子中蛋白质分子的聚集;并且在表面活性剂CTMAB的存在下,核酸可以明显增强该蛋白质纳米粒子的荧光强度,从而建立了检测核酸的荧光新方法。利用圆二色光谱,共振光谱以及透射电镜技术研究了蛋白质纳米粒子、CTMAB和核酸之间的相互作用。研究认为,体系以核酸为模板,通过静电引力,阳离子表面活性剂在其表面上聚集,形成预胶束,并促使带负电荷的纳米粒子在预胶束上聚集,形成新的聚集体,从而导致体系的荧光增强。本论文主要特点:1.研究发现,荧光分析中常用的猝灭剂KI在低浓度下能够增强morin-hsDNA体系的荧光,桑色素-KI可以作为荧光探针能够选择性地识别双螺旋核酸中的鱼精子DNA,由此建立了选择性测定核酸的荧光光度法,并已用于合成样品的测定。机理研究认为,在桑色素-KI主要以沟槽作用方式与fsDNA结合,在低浓度下,KI对桑色素-fsDNA的荧光进一步增强主要源于更有利于发光结构的形成。2.研究发现,在弱酸条件下鸟嘌呤能明显增强桑色素的荧光强度,而DNA中其他三种正常碱基的干扰较小,据此建立了选择性测定鸟嘌呤的荧光光度法。本方法还具有操作简便、快速、线性范围宽、灵敏度高等特点。机理研究认为,鸟嘌呤和桑色素形成匹配的分子间三氢键,这增大了桑色素结构的共平面效应和刚性,减小了桑色素能量耗散作用,从而使得桑色素的荧光寿命和荧光强度增加,并已将该体系应用于鸟嘌呤的定量测定。3.利用圆二色光谱和荧光光谱技术研究了环糊精对肾上腺素-氢氧化钠-2,3-二氨基萘体系的包结行为,确定了体系的包结比和包结常数,提出了有机溶剂丙酮在体系中荧光增敏效应的原因以及对体系包结模式的影响。4.研究发现,蛋白质纳米粒子在430nm左右能发射新的荧光峰;在表面活性剂CTMAB的存在下,核酸能明显增强体系的荧光强度,从而建立了核酸新的荧光分析方法。机理研究表明,核酸与CTMAB为蛋白质纳米粒子提供了一个疏水的微环境,并促进了体系的进一步聚集,形成大的聚集体,从而导致体系的荧光增强。更多还原

【Abstract】 Nucleic acids（nucleoside）and proteins are the material base of all life.They relate to heredity,tumor,the effect of virus,and so on.So,it is very important for researchers to expound the secrets of life,to develop new functionalized medicine to treate many difficult diseases.What we should do is to further study the interaction mechanism between biomolecules and organic molecules（or biomolecules）,to develop rapid and convince assay for biomolecules.This project is the forward position and hot point in biochemical and biophysical researches.This thesis studies the interaction mechanism between biomolecules and organic molecules（or biomolecules）using the techniques including fluorescence,absorption, RLS,CD and TEM.Some rapid,accurate assays with high sensitivity and selectivity are developed for nucleic acid and nucleoside.The main conclusions are listed as below:1.In the first section,we summarize recent developments of fluorescence probe for both nucleic acids and nucleoside,and comment on the experimental techniques to investigate the interaction between biomacromolecules and small molecules.142 references are cited here.2.In the second section,it is found that KI within a certain range of concentration can enhance the fluorescence intensity of morin-fsDNA system.Based on this phenomenon,a new and selective method for the determination of fsDNA is developed.Under the optimum conditions,the enhanced intensity of fluorescence is in proportion to the concentration of nucleic acids in the range of 8.0×10^-9-2.0×10^-5g mL^-1for hsDNA and 5.0×10^-9-1.0×10^-5g mL^-1for smDNA.Their detection limits are 4.5ng mL^-1and 3.5ng mL^-1,respectively.The interaction mechanism of KI-morinhsDNA system is studied.It is considered that there is the groove binding mode between KI-morin and DNA;the fluorescence enhancement effect induced by KI is attributed to the formation of the more favorable structure for the luminescence.3.In the third section,it is found that guanine can enhance the fluorescence of morin.Based on this,a new method for the determination of guanine is proposed.In NaAc-HAc buffer,the fluorescence intensity of morin system can be greatly enhanced by guanine and the enhanced intensity is in proportion to the concentration of guanine in the range 2.0×10^-8-8.0×10^-5mol L^-1,its detection limits is 7.0×10^-9mol L^-1. Interference test shows that the other three bases in nucleic acids have little effects on the determination of guanine.And the mechanism study indicates that the fluorescence enhancement of morin is considered to originate from the formation of intermolecular multi-hydrogen bonds between guanine and morin,which can increase the co-planar effect and the structural rigidity of morin and reduce its energy consumption originated from molecular movement.Therefore,the fluorescence intensity and lifetime of morin can been enhanced.4.In the fourth section,we study the interaction mechanism of Epinephrine-NaOH-Actone-DAN-β-CD_x system using the techniques including fluorescence, absorption,and CD.The constant and stoichiometric ratios in the system are calculated based on the "double reciprocal method".Results show that in the presence of acetone,the system forms 1:1 inclusion complex,the inclusion constant is 206L.mol^-1;whereas in the absence of acetone,it forms 1:1 inclusion complex in low concentration ofβ-CD_x,the inclusion constant is 68 L mol^-1,then forms 2:1 inclusion complex with the increase ofβ-CDx concentration,the inclusion constant K₂ is 1270 L mol^-1.The CD spectra indicate that theβ-CDx include the guest with axial type in epinephrine-NaOH-DAN-β-CDx system.A large cooperative effect ofβ-CDx and low acetone concentration together produces a much larger enhancement.The addition of acetone makes the indole ring group of fluorescent condensate expose outside of the cavity ofβ-CDx,resulting in the formation of 1:1（or 2:2）inclusion complex because of stereo-hindrance effect.5.In the fifth section,it is found that BSA nanoparticles synthesized by a desolvation technique can emit the fluorescence peak at 430nm,which is considered to be the fluorescence peak of BSA aggregate;its intensity is greatly enhanced by nucleic acid in the presence of CTMAB.Based on this,a new method for the determination of nucleic acid is proposed.The interaction mechanism of this system is studied.It is considered that the nucleic acid acts as the template in the system,the positive charged CTMAB will assemble on the nucleic acid to form.positive charged pre-micelle.Hence to promote the assembly of negative charged BSA anoparticles on the pre-micelle and form large aggregate of nucleic-CTMAB- BSA nanoparticles.So the fluorescence intensity is greatly enhanced.The chief characteristics of this thesis are as follows:1.It is found that KI commonly used as fluorescent quenching agent can enhance the fluorescence intensity of morin-fish sperm DNA（fsDNA）system,so KI-morin can selectively recognize fsDNA in double-strand nucleic acids.Based on this,a new sensitive and selective method of determination of fsDNA is developed.The fluorescence enhancement effect induced by KI is considered to originate from the formation of the more favorable structure for the luminescence.2.We find that guanine can enhance the fluorescence of morin.Based on this,a new method for the determination of guanine is proposed.The method is simple, rapid,sensitive and selective.The mechanism study indicates that the fluorescence enhancement of morin is considered to originate from the formation of intermolecular multi-hydrogen bonds between guanine and morin.3.We study the interaction mechanism of Epinephrine-NaOH-Actone-DAN -β-CD_x using the techniques including fluorescence,absorption,and CD.The inclusion constant and stoichiometric ratios in the system are calculated based on the "double reciprocal method".In addition,the action of acetone in both the fluorescence enhancement and the inclusion of this system are proposed.4.It is found that the BSA nanoparticles can emit new fluorescence peak at 430nm,which is considered to be the fluorescence peak of BSA aggregate,its intensity is greatly enhanced by nucleic acids in the presence of CTMAB.Based on this,a new method for the determination of nucleic acid is proposed.The fluorescence enhancement of the system is considered to originate from both the formation of large BSA aggregate and the hydrophobic microenvironment provided by nucleic acid and CTMAB for BSA nanoparticles.更多还原