Preparation, Application Of Multiplex Core-shell Fluorescence Nanoparticles As Bio-labels And Novel Model Of Immuno-PCR Mediated Using Phage

This dissertation focuses on ultra-high sensitive detection technology. Time-resolved Fluoroimmunoassay (TRFIA) and immuno-PCR are two such tools that are applicable to detection of very low concentration of antigen, antibody and pathogenic microorganism. And this dissertation consists of three parts, including a brief review of ultra-high sensitive detection technology, the synthesis of BBCAP and preparation, application of multiplex core-shell fluorescence nanoparticles; and development of phage-mediated immuno-PCR.In chapter one, the basic principles and highlights of different kinds of ultra-sensitive detection technology are reviewed. The late development in TRFIA and immuno-PCR for protein detection are summarized. And finally the research proposal for this dissertation is presented.In chapter two, we first describe the synthesis of a new lanthanide chelate: 2,9-bis[N,N-bis(carboxymethyl)aminomethyl]-1,10-phenanthroline (BBCAP) could bind both Eu(III) and Tb(III) and display their respective fluorescent. BBCAP was covalently linked with 3-aminopropyl(triethoxy) silane to form silica nanoparticles in the presence of precisely controlled ratios of Eu(III) to Tb(III). The nanoparticles thus prepared could have specified ratios of luminescence intensities at two well-resolved emission under a single-wavelength excitation. After comprehensive characterization with respect to their fluorescence lifetime, photostability, thermostability, and chelate leakage, practical use of these nanoparticles was evaluated in a time-resolved immunofluorometric assay of human hepatitis B surface antigen (HBsAg). The detection antibody was labeled with the Tb(III) silica nanoparticles using a site-specific labeling strategy that allows the binding sites of the labeled antibody orient away from the nanoparticles and toward the antigen. The detection limits, calculated with the concentration corresponding to three times of the standard deviations of the background signal, was 35 pg/mL. The upper limit of the calibration curve was ca 200 ng/mL. The coefficient of variation (CV) across the whole range was below 8.9%. In comparison, under the same conditions, the detection limit of ELISA was 200 pg/mL and the upper limit of the calibration curve was ca 10 ng/mL. In chapter three, we described an ultrasensitive immunoassay for detecting HBsAg via immuno-PCR using phage-antibody complex. We chose T7 phage as a natural DNA containing nanoparticle to develop immuno-PCR. The phage was first activated with sulfo-SMCC in sodium phosphate, while antibody was thiolated using Traut's reagent in a non-amine-containing buffer. The activated phage and the thiolated antibody were mixed immediately, after reaction overnight at 4°C, the mixtures were purified by Pall filter, resulting the phage-antibody complex. Regular PCR tubes were activated using 1% glutaraldehyde, and then coated with antibody. The results showed that there's a linear range from 1000 ng/mL to 0.01 ng/mL for HBsAg. The detection limit was ca 0.1 pg/mL. HBsAg was examined in sera from Hepatitis B patients and negative control using both immuno-PCR and ELISA, and the results showed immuno-PCR correlated well with ELISA.