Preparation, Application of Multiplex Core-shell Fluorescence Nanoparticles as Bio-labels and Novel Model of Immuno-PCR Mediated Using Phage

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 Special shape NdFeB magnets 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 http://www.999magnet.com/ 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.