Study On Synthesis Of Environment-Sensitive Phase Separating Polymer And Rare-Earth Chelating Agent And Their Applications In Fluorescence Analysis

The fluorescence immunoassay (FIA) based on a fast homogeneous antigen-antibody immune complexation reaction and a simple heterogeneous separation process by environment-sensitive polymer as a carrier is of great interest in recent years. The development of time-resolved fluorescence immunoassay (TRFIA) using rare-earth chelate as a label has been an attractive area in last decade. The main efforts of this dissertation are thus centered on these two research directions. The dissertation consists of six chapters, which basically involves (1) preparation of environment-sensitive phase separating polymer and its application in FIA; (2) synthesis of rare-earth chelating agent and its application.In chapter 1, the basic principle, test formats and problems in FIA were introduced. The recent advances in homogeneous FIA, heterogeneous FIA, FIA with homogeneous reaction and heterogeneous separation were reviewed. The combination of FIA with amplification of fluorescence signal, high efficient separation and automatic technique was summarized. The research proposal for this dissertation was also presented.In chapter 2, A novel temperature-sensitive polymer, poly(N-isopropylacrylamide-acrylamide) [P(NIP-AA)J, was synthesized. Its lower critical solution temperature (LCST) could be regulated by changing the content of acrylamide (AA) for different purposes. The P(NIP-AA) with LCST of 37癈 was used as a separating carrier for the reactants in immunoassay. The sensitivity of this method was comparable to that using poly-N-isopropylacrylamide(PNIP) as a carrier. However, owing to the elevation of LCST of the polymer, the immunoreaction could be carried out at a temperature closer to the physiological environment of organism and the rate of immunoreaction was faster. A sandwich fluorescence immunoassay for rabbit IgG and a competitive enzyme-linked fluorescence immunoassay for human IgG were established with satisfactory results. In chapter 3, the superiority of pH-sensitive polymer to temperature-sensitive polymer as immobile phase of FIA was firstly discussed. Poly(N-isopropylacrylamide-methacrylic acid) [P(NIP-MAA)J, a linear water-soluble pH-sensitive polymer whose phase transition temperature is about 5.6 at 37℃, was synthesized and used as a novel separation carrier for the reactants in immunoassay. A fluorescence immunoassay for rabbit IgG was established. Immune reaction time was decreased to 4 min at 37℃. The pH-sensitive mechanism was simply clarified. The immobilization efficiency and non-specific adsorption of antibody on P(NIP-MAA) polymer were evaluated. If antigen or antibody was immobilized by the way of heat initiation instead of oxidation-reduction initiation, the immobilization efficiency, immunological reaction activities of biological active matter and phase transition pH range were improved. Moreover, some environment-sensitive polymer synthesized only in organic solvent or under high temperature could be used as an immunoreaction carrier by heat initiation immobilization. Two immobilization modes were compared in the determination of human IgG. As a little hydrophobic monomer butyl methacrylate (BMA) was introduced to co-polymerized with INP and MAA in organic solvent, the lower critical solution pH (LCSP) was increased to 6.0, which was more suitable to FIA. Then a competitive FIA for rabbit IgG was established using this pH-sensitive polymer.In chapter 4, Poly(N-isopropylacrylamide-N,N'-dimethyl aminopropyl methacrylamide) [P(NIP-DMAPM)], a linear novel water-soluble pH-sensitive polymer whose phase transition temperature is about 7.4 at 37℃, was synthesized. The P(NIP-DMAPM) precipitated out of water above a critical pH 7.4 at 37℃ and redissolved at pH below 7.4. P(NIP-DMAPM) was used as a novel separation carrier for the reactants in immunoassay and a fluorescence immunoassay for rabbit IgG and human IgG was built. Because of neutral LCSP, the damage to antigen-antibody immune complexation was greatly decreased in the course of separation. The nonspecific adsorption and inhibition of...