| 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.
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