Preparation, Characterization And Application Of Novel Fluorescent Chitosan Nanoparticle Probe

In this thesis, the preparation and application of novel chitosan nanoparticles labeled with organic fluorescent dye were studied. Chitosan nanoparticles could be obtained by ionical cross-linking of low molecular weight chitosan with tripolyphosphate (TPP) being crosslinking reagent. Fluorescent labeled chitosan nanoparticles could be subsequently prepared using the reaction between FITC and the amine group of the chitosan nanoparticles. The size distribution, fluorescence spectroscopy and photo-stability of the fluorescent chitosan nanoparticles were characterized. The fluorescent chitosan nanoparticles were finally used as a sensitive fluorescent labeling reagent for detecting trace level of human IgG in human serum sample by fluoroimmunoassay method.In chapter one, recent works related with high sensitive fluorescent probe, structure and character of chitosan, the methods of preparing chitosan nanoparticle and surface modification of nanoparticle were reviewed.In chapter two, chitosan with low molecular weight was prepared by oxidative degradation method, in which hydrogen peroxide was employed as the oxidant. Chitosan nanopaticles were formed by ionical cross-linking of chitosan with tripolyphosphate(TPP). The factors affecting the particle preparation were systematically studied, and the favorable operation conditions were found at: molecule weight of chitosan is about 50 thousands, the chitosan concentration is 0.5% (w/v) and the TPP is 0.25% (v/v), the volume proportion of TPP to chitosan solution is 1:3. The physical and chemical properties of the microspheres were characterized by FTIR, transmission electron microscope and so on. Results showed that the microspheres prepared here were uniform, spheric microspheres with about 40nm diameter and well dispersed in 1% acetic acid.In chapter three fluorescent chitosan nanoparticles were prepared using the reaction between the isothiocyanate group of FITC and the primary amino group of chitosan. The factors effecting the preparation were studied. Results indicated thatFITC could strongly combine with chitosan in alkaline condition. In addition, the fluorescent chitosan nanoparticles obtained were more photostable than conventional organic dyes.Chapter four was aimed at the physical properties of chitosan nanoparticles. Colloidal stability as function of pH and ionic strength of the medium was analyzed. Results showed that chitosan nanoparticles were well dispersed in solution at pH=4. The presence of KC1 at low and moderate concentrations provoked swelling > weaken chitosan-TPP ionic interaction and rapidly turned on particle disintegration.The methods of surface modification of chitosan nanoparticles were also discussed in this chapter.Chapter five presented several coupling methods between the fluorescent chitosan nanoparticles and active biological ligands. Results indicated that the best coupling reagent was glutaraldehyde. Goat-anti-human IgG was labeled by fluorescent chitosan nanoparticles,which was used to detect trace level of human IgG by fluoroimmunoassay method.