【作者】 唐尧基；

【导师】 李耀群；

【作者基本信息】 厦门大学 ， 分析化学， 2008， 博士

【摘要】 全内反射荧光法（TIRF）具有高度的界面特异性,可以有效排除大量本体溶液的干扰,获取界面信号,是一种良好的界面分析技术;生命物质在界面的性质研究在基础科学研究、生物学以及工业中都具有重要的意义,已经引起了广泛的关注。卟啉是一类具有重要生物意义的化合物,在新陈代谢中起着不可缺少的作用;蛋白质是生物体的重要组成成分,参与所有的生命活动。卟啉和蛋白质在溶液和界面的性质以及两者的相互作用一直是人们广泛关注的课题。本论文利用TIRF并结合同步荧光技术研究了卟啉、蛋白质在固/液以及液/液界面的荧光光谱、吸附特性以及两者相互作用。论文共分五章:第一章为绪论,评述了TIRF在生命物质界面分析中的研究进展,本章共分六部分。第一部分介绍了TIRF的原理、发展及其特点;第二部分对生命物质的界面研究作了简单评述,介绍界面研究的意义和常用的界面研究方法;第三部分重点介绍TIRF研究生命物质在固/液界面性质的主要研究内容和研究进展;第四部分是TIRF应用于液/液界面的研究现状;第五部分介绍TIRF与同步扫描技术的联用;最后一部分是在上述调研的基础上提出了本论文的构思。第二章研究了在十六烷基三甲基溴化铵（CTAB）存在条件下,TPPS在水溶液、亲水玻璃/水界面以及二氯二甲基硅烷修饰的疏水玻璃/水界面的荧光光谱和吸附特性,考察了影响TPPS荧光光谱和界面吸附的因素,探讨了特定条件下TPPS在界面的吸附动力学情况,对于生物相容性材料的研究有重要的参考价值。本章首先应用常规荧光、全内反射荧光并结合同步荧光技术深入探讨了TPPS在水溶液、亲水和疏水玻璃/水界面的荧光特性,并考察了TPPS在界面的吸附。TPPS有几种不同的存在型体,如双质子化、非质子化、胶束化单体、H-聚集、J-聚集以及与其它物质形成复合体等,实验结果表明,在CTAB存在条件下,无论在亲水还是在疏水玻璃/水界面,TPPS的非质子型体(TPPS^4-)均优先吸附在界面上,且在亲水界面上,只有在pH非常低的情况下有双质子型体(H₂TPPS^2-)存在,而在疏水的玻璃/水界面,在很宽的pH范围内,H₂TPPS^2-都与TPPS^4-共存在界面上。详细考察了不同类型表面活性剂及其浓度、TPPS浓度、pH值对本体溶液和界面荧光的影响,结果显示静电力在TPPS固/液界面吸附过程中有重要的作用。考察了TPPS在不同溶液条件下的吸附动力学,比较了TPPS亲水和疏水界面上不同的吸附特性,探讨了影响TPPS在固/液界面吸附的因素,为调控TPPS在界面的吸附和应用提供新的研究方法和理论依据。第三章研究了TPPS与BSA在溶液中和固/液界面上的相互作用以及BSA在玻璃/水界面的吸附特性。首先考察了TPPS对溶液中BSA内源荧光的影响,发现TPPS对BSA的内源荧光具有猝灭作用,并计算其Stern-Volmer猝灭常数以及猝灭速率常数,证明TPPS对BSA具有静态猝灭作用。研究了不同pH条件下,BSA对溶液中TPPS荧光的影响,发现两者在较低的pH范围更易于形成结合体。以TPPS作为BSA的荧光探针,提出了一种TIRF测定溶液中BSA含量的新方法,BSA浓度在1.47×10^-8～1.18×10^-7M范围与界面结合体同步荧光强度成正比,线性拟合方程为I=-3.21+14.53C_BSA(10^-8M),并应用于实际血清样的分析,与临床数据结果吻合较好。本章重点利用TIRF考察了各种因素对蛋白质在玻璃/水界面的吸附的影响,考察了不同溶液条件下BSA在固/液界面的吸附动力学,获得不同条件下BSA的吸附动力学参数,结果表明BSA的吸附动力学符合Elovich方程和双常数方程,计算了不同条件下BSA在亲水玻璃/水界面的饱和吸附量和吸附平衡常数,结果表明,BSA在玻璃/水界面的吸附过程符合Langmuir吸附模型,BSA以单分子层吸附于玻璃/水界面。初步探讨了BSA在二氯二甲基硅烷修饰的疏水玻璃/水界面的吸附特性,并与亲水玻璃/水界面的性质进行比较。第四章研究了不同pH和浓度条件下BSA与TPPS在液/液界面的结合情况,并探讨了BSA在界面的吸附情况。结果表明,在液/液界面两者主要以1:1比例相结合;比较界面与甲苯中的荧光光谱,可以推测,油/水界面的极性更接近有机相。建立了现场检测油/水双相体系分配的荧光技术,考察了BSA与TPPS在水、界面以及油相中的分配和型体特征。通过绘制全内反射同步荧光强度与溶液总浓度的关系曲线,获得BSA的临界胶束浓度为1.0×10^-4 M;提供了计算界面吸附参数的新方法,获得了BSA-TPPS在甲苯/水界面的饱和吸附量和吸附平衡常数。第五章是本论文的结语与展望。总结了本论文研究工作的创新性,并对研究工作的进一步发展进行了展望。更多还原

【Abstract】 Total internal reflection fluorescence （TIRF） spectroscopy is a highly selective and sensitive way to study the interfacial region. The interfacial behavior of biomaterial is important in fundamental research, basic industry and biological sciences. This dissertation uses TIRF and its combination with synchronous fluorescence technique to study the behavior of protein and porphyrin at solid/liquid interface and liquid/liquid interface. The paper consists of five chapters.In chapter one, the research progress and application of TIRF in biomaterial study are reviewed. This chapter consists of introduction of TIRF, study of biomaterial interfacial behavior, behavior of biomaterial at solid/liquid interface and liquid/liquid interface by TIRF, the combination of TIRF and synchronous scanning technique, and the research objective for this dissertation.In chapter two, TIRF was used to investigate the adsorption behavior of meso-tetrakis（p-sulfonatophenyl）porphyrin （TPPS） at the glass/water interface in the presence of a cationic surfactant （cetyltrimethylammonium bromide, CTAB） far below the critical micelle concentration. We proposed the adsorption model of TPPS, which was different from the adsorption of TPPS in the presence of micelles of CTAB at glass/water interface. TPPS and CTAB did not form stabile complex in diluent system at the interface. The interfacial species of TPPS were analyzed by comparing the spectra of TPPS at the glass/water interface and in the aqueous phase. The influences of the TPPS concentration, the CTAB concentration, and the pH values on the interfacial fluorescence spectra and intensities were studied. It demonstrated that electrostatic interaction and hydrophobicity had important effect on the adsorption of TPPS in the presence of CTAB. The different effects of TPPS concentration on the adsorption behaviour of TPPS at different pH were observed for the first time. It was found that the adsorption isotherms of TPPS at glass/water interface could fit Freundlich equation at pH 7.1. At hydrophobic glass/water interface, diprotonated TPPS and unprotonated TPPS both exist at the interface in the wide pH range. It indicated the behavior of TPPS at different interface is different. This study provided theoretical base for interfacial adsorption of TPPS.In chapter three, the interaction of TPPS and BSA in solution and at the glass/water interface was studied by fluorescence spectroscopy. It was found that TPPS quenched the BSA intrinsic fluorescence. The Stern-Volmer quenching constant and quenching rate constant were calculated and it indicated TPPS had static quenching for BSA fluorescence. TPPS was used as a BSA fluorescence probe and a TIRF method for determining the concentration of BSA in solution was proposed. The method had been applied to real blood serum with satisfying results. Several factors on the effect of BSA adsorption onto solid/liquid interface were researched by TIRF. The adsorption equilibrium constant and the maximum amount of adsorption were calculated by Langmuir adsorption isothermal model. The adsorption kinetics of BSA in different solution conditions was studied and the adsorption kinetics parameters were gained by several adsorption kinetics models. The adsorption of BSA at hydrophobic glass/water interface modified with dichlorindimethylsilane was further studied. This chapter provides a new way for the studying of adsorption kinetics at solid/liquid interface.In chapter four, the combination of BSA and TPPS with different pH concentration, and its adsorption behaviour on liquid/liquid was studied. Under the experimental condition, the components on the interface were dominated by their combination with proportion of 1:1. Compared with the spectra of TPPS resolved in toluene, it was concluded that the polarity of oil/water interface is closer to organic phase than water, and BSA provided unpolar microenvironment for TPPS under certain condition. According to the curve relation between the interfacial fluorescence intensity and the total concentration, the critical micelle concentration （cmc） of BSA was obtained with a value of 1.0×10^-4 M. The adsorption equilibrium constant and the maximum amount of adsorption were also gained by proposed method.In chapter five, the innovation of the paper was concluded and the prospect of this research was given.更多还原