Study Of Interaction Between Protein And Chitosan By Total Internal Reflection Fluorescence Spectroscopy

As an important natural polymer, chitosan has been applied extensively in biomedicine for its biocompatibility, biodegradation, as well as non-toxicity to cellular tissues. There is good affinity between chitosan and protein. For the development of protein drugs and the research of biochemistry, it is significant to probe the interaction between chitosan and protein, and to observe changes of adsorption when both of them contact at interface. Total internal reflection fluorescence spectroscopy (TIRF) is an effective technique to investigate protein adsorption at interface. The total internal reflection fluorescence generates from interfacial fluorophore, which is selectively excited by evanescent wave. Therefore, there are several characteristics for evanescent wave inducing fluorescence, such as surface specificity, elimination of bulk interference and non-destruction. It has been successful to investigate protein adsorption in situ, real-time by this technique. This dissertation is concerned on the improvement of instrumental device of TIRF for probing solid/liquid interface, the option of suitable method for immobilizing chitosan, and the analysis of interaction between model proteins and ultrathin chitosan film which was immobilized on silica substrate. In addition, the adsorption behavior of rhodamine 6G (R6G) at silica/water interface was also concerned. The dissertation is composed of following parts:In the first chapter, the application of total internal reflection fluorescence technique in the research of biological systems and the advance on the study of interaction between chitosan and protein were reviewed. The principle and application of TIRF were introduced briefly. The research on biological systems, especially on the study of protein adsorption at solid/water interface, was described in detail. Then the property and application of chitosan were introduced, as well as the hot topics on the study of chitosan-protein interaction. The plan of dissertation was put forward at the last part of this chapter.In the second chapter, a new TIRF cell was developed based on the present experimental conditions. Microfluid cell made up of silica slices, of which detection volume was minimized to 200μL, was more easily for modification and cleaning. The immobility of chitosan films was monitored by total internal reflection synchronous fluorescence (TIRSF). Two methods (spin coating and chemical bonding) were employed to immobilize chitosan onto the silica substrate. We finally applied chemical bonding method to fabricate chitosan film, which utilized self-assembly membrane to introduce active group for surface grafting. The protocols of grafting were optimized as well. As a result, the uniform ultrathin chitosan film was obtained.In the third chapter, the interactions between model FITC labeled proteins (BSA, Fibrinogen, Lysozyme) and ultrathin chitosan film were investigated by TIRF, respectively. The competitive adsorption between BSA and fibrinogen was studied as well.Firstly, it was successful to label model proteins with FITC, whose labeling ratios were between 0.6 to 3.8. Red shifts were observed both in absorption and fluorescence spectra. There was 6-10 times increase in the value of fluorescent anisotropy of labeled proteins.Secondly, with increase of concentration of FITC-BSA in bulk solution, two growth regions of interfacial intensity of FITC-BSA were present on chitosan film. It is suggested that FITC-BSA will adsorb on chitosan film with the orientations changing from horizontal to vertical, and as multilayer structure at high concentration (more than 400μg/mL).The adsorption of FITC-BSA depended with pH variations. With pH increasing, there was an extreme value of interfacial intensity approximately at pH 6.5. It is illustrated that the electrostatic attraction was the main driving force to adsorb BSA. Increasing of ionic strength screened the repulsive forces between protein-chitosan, protein-protein at pH 7.4. As a result, the amount of adsorbates was increased. However, higher concentration of supporting electrolyte induced the decrease in BSA adsorption for chitosan chains tended to curling to be conglobation, which resulted in fewer sites to contact with protein.Thirdly, we investigated the concentration effects and adsorption kinetics of other two model proteins. For fibrinogen, interfacial intensity was increased with the initial concentration. At low concentration, Double Constant Equation was fitted greatly to the adsorption kinetics curves. At high concentration, Pseudo-second order model appeared as the best fitter to adsorption kinetics curves. For lysozyme, interfacial intensity changing with concentration of FITC-Lyz was in the same shape as Langmuir adsorption isotherm. The affinity of lysozyme to chitosan was much better than that of BSA for the structure of chitosan was similar to substrate of enzyme. Pseudo-second order model could describe adsorption processes of lysozyme on chitosan film exactly. With increase of initial concentration of lysozyme, there was one-order magnitude increase in adsorption rate constant (k).And then, the study of competitive adsorption of BSA and fibrinogen on chitosan film at physiological condition was carried out. It was revealed that the relative competitive adsorption capacity of fibrinogen was much larger than that of BSA. The result confirmed that chitosan, as other common haemostatic, would adsorb blood protein and activate platelet and coagulation factor for clotting.In the fourth chapter, total internal reflection synchronous fluorescence (TIRSF) was developed as a direct and successful tool to investigate a xanthene dye (R6G) at the silica/water interface. From analysis of fluorescence spectra both in bulk and interface, 5 nm red shift of fluorescence spectra at hydrophilic silica/water interface were observed. The bathochromic shift was mainly due to the limitation of rotational movements of the dye. The rigidity of the silica surface restricted the freedom of the molecules. The lower polarity of microenvironment at the silica/water interface than that in aqueous environment should also make a contribution to the phenomenon. Meanwhile, the effects of bulk concentration, pH, and ionic strength on the adsorption of R6G were studied as well.