Preparation Of Composite Fluorescent Silica Nano-probes And Their Applications To The Detection Of Cancer Cells And Pathogenic Bacteria

Compared with conventional single-component nanoparticles, Composite nanoparticles possess more unique chemical and ph Composite nanoparticles possess more unique chemical and physical properties due to the combination of binary or multi-component. They have gained increasing attention. Composite fluorescent silica nanoparticles have shown broad prospect in the analytical field of biology and medicine due to higher sensitivity and photostability compared with simple dyes. Aiming at the research direction of application of core-shell composite nanoparticles in molecular diagnosis, medical imaging, and biomaterial separation and so on, this thesis mainly focused on the synthesis and biomedical applications of several core-shell composite nanoparticles.In chapter one, the classification and importance of biomarker in bioanalysis are induced. Some important findings with signification of nanomaterials in analytical chemistry are reviewed. The synthesis methods characterazition studies are given. Based on these studies, the research proposal for this dissertation is also presented.In chapter two, the preparation, functionalization and utilization of Tris (2,2'-bipyridy) dichlororuthenium hexahydrate (RuBpy)-doped composite nanoparticles in the detection of the carcinoembryonic antigen (CEA) in the lung carcinoma cells are reported. Composite fluorescent silica nanoparticles were prepared using water-in-oil (W/O) microemulsion methods and primary amine groups were introduced onto the surface of the nanoparticles by condensation of tetraethyl orthosilicate (TEOS). The amine functions were transferred to carboxyl groups coupled with a linker elongation. Avidin was immobilized at the surface of the nanoparticles by covalent binding to the carboxyl linkers using EDC/NHS. The activity and binding capacity of the avidin-covered nanoparticles for ligand biotin was quantified by titration with biotin (5-fluorescein) conjugate. A rabbit anti-CEA antibody used as primary antibody to recognize CEA, biotinylated goat anti-rabbit antibody used as secondary antibody to recognize rabbit antibody, and then a composite fluorescent silica probe binding avidin was used to generate indicative fluorescent signal for microscopic examination. The lung carcinoma cells were identified easily with high efficiency using these composite fluorescent probes. By comparison with fluorescein isothiocyanate, dye-doped silica nanoparticles display dramatically increased stability of fluorescence as well as photostability, as compared to the common organic dye, when under continuous irradiation. In chapter three, the preparation, functionalization and utilization of organic fluorescent dye rhodamine B isothiocyanate (RBITC)-doped composite nanoparticles in the detection of mucinl (MUC1) in human breast carcinoma cells are reported. The preparation of RBITC-doped core-shell nanoparticles has been developed, which exploits synchronous hydrolysis of tetraethoxysilane in water-in-oil microemulsion with the combination of2-(amimoethyl)-propyltyiethoxy-silane (APTES) and RBITC and polymerization reaction with [hydroxyl (polyethyleneoxy) propyl] triethoxysilane. This method conquers the disadvantages of traditional preparation method about organic dye doped core-shell nanoparticles such as the leakage of fluorescent dyes and the low encapsulation efficiency of fluorescent dyes. Avidin was immobilized at the surface of the nanoparticles by covalent binding to the carboxyl linkers using cyanogen bromide. The MUC1in human breast carcinoma MCF-7cells were identified easily with high efficiency using these composite fluorescent probes. The results demonstrate that RBITC-doped silica nanoprobes display dramatically increased stability of fluorescence as well as photostability, as compared to the common organic dye when under continuous irradiation.In chapter four, the preparation and functionalization of fluorescein isothiocyanate (FITC)-doped composite nanoparticles are reported. A method of fluorescent nanoparticle based on indirect immunofluorescence microscopy and flow cytometry has been developed for the detection of E. coli O157:H7. Composite FITC-doped silica nanoparticles were synthesized using W/O microemulsion methods with the combination of APTES and FITC and polymerization reaction with carboxyethylsilanetriol sodium salt (CEOS). Protein A was immobilized at the surface of the nanoparticles by covalent binding to the carboxyl linkers. A rabbit anti-E. Coli O157:H7antibody was used as primary antibody to recognized E. coli O157:H7and then antibody binding protein (Protein A) labeled with FITC-doped silica nanoparticles was used to generate fluorescent signal. By using microscope imaging technique and flow cytometry assay, the detection of target E. coli O157:H7was successfully carried out in buffer and bacterial mixture, respectively.It is revealed that amplified signal intensity and higher photostability by using the composite fluorescent nanoparticles as the label are obtained than the direct using of conventional fluorescent dye. This proposed method will be promising for rapid identification of pathogenic bacteria in biology and medicine.In chapter five, a method of indirect immunohistochemistry based on composite fluorescent nano-probe has been developed for the detection of cytokeratin (CK) in tissue of liver cancer. RuBpy-doped composite nanoparticles binding molecule of H2N-PEG-COOH using Cyanogen Bromide were synthesized in W/O microemulsion and introduced of avidin onto the surface using EDC/NHS. By using indirect immunohistochemistry technique, CK in tissue of liver cancer was successfully detected based on composite fluorescent nano-probe.