Immobilization Studies Of Biomolecules Based On Electrostatic Adsorption In Immunosensor

This thesis was composed of two parts.1. Studying the immobilization of biomolecules in biosensor, immobilization technologies of biomolecules are one of the most important to improve the performance of biosensor. To preserve the biological activity of bimolecular is paramount. There are many immobilization methods we can use, such as covalent bond and non-covalent bond. Electrostatic adsorption and non-specific adsorption have better character to preserve biological activity. In this paper, chitosan, activated alginate and nano-titanium dioxide were used to realize immobilization of biomolecules.1) A novel schistosoma japonicum (Sj) imunnosensor based on aggregation of chitosan and CNBr-activated alginate is developed. The complex formed by CNBr-activated alginate and antibody is aggregated to the surface of the paraffin-graphite-chitosan electrode by electrostatic adsorption (coacervation). The concentration of SjAg can be detected by determining the redox current of o-aminophenol, which oxidized by H2O2 in the presence of HRP. Moreover, the immunosensor shows some improved performances including high sensitivity, selectivity and less non-specific adsorption. The sensor exhibits a linear response to SjAg in the concentration range 0.64 to 40 μgmL-1. The equation can be expressed as: I=-17.308c+34.572 .correlation coefficient 0.985.2) A nano-titanium dioxide embedded carbon paste electrode (n-TiO2ECPE) was developed. As a model enzyme, horseradish peroxidase (HRP) was immobilized to electrode surface involving strong synergic interaction of the enzyme with nano-titanium dioxide and graphite powder. The current response to hydrogen peroxide on the electrode was measured in a phosphate buffer (pH 6.9) solution using 1,2-benzenediol to be the mediator. The response current of the HRP enzyme is linear in the range between 2.4 X l0-4and 3 X 10-7mol L-1 H2O2 with a sensitivity of 0.1101 A L mol-1 cm-2 and a correlation coefficient of 0.993 (n=13).2. Studing substrate in enzyme-link fluoroimmunoassay system. The fluorescence of substrate immediate influences the sensitivity of fluoroimmunoassay. 3,3',5,5'-Tetramethylbenzidine and 2-Chloro-10-[3-(4-methyl-l-piperazinyl)propyl] -lOH-phenothiazine (prochlorperazine) are used for the substrate of immunoassay.3) A novel enzyme-link fluoroimmunoassay system using 3,3',5,5'-Tetramethyl -benzidine as substrate to determine the complement 3(C3) was developed. HRP-labled complement 3 can catalyze the 3,3',5,5'-Tetramethylbenzidine to form non-fluorescence substence. The concentration of C3 can be determined by fluorescence decrease of TMB after competitive immunoreactions as nano-titanium dioxide to be carrier. The fluorescence intensity was measured in PBS containing2.5xl0-7M/mL TMB and 5xl0-7M/mL H2O2 at pH 6.8. The sensor exhibits a linear response to C3 in the concentration range 6.5ng/mL to 75ng/mL, correlation coefficient 0.973.4) A novel enzyme-link fluoroimmunoassay system using 2-Chloro-10 -[3-(4-methyl-l -piperazinyl)propyl] 10H-phenothiazine (prochlorperazine) as substrate to determine the Goat anti-IgG was developed by using Polystyrene(PS) as carrier. The HRP-labled Goat anti-IgG can catalyze the oxidation of prochlorperazine to cause the increasing of fluorescence. The concentration of Goat anti-IgG can be determined by fluorescence increase of PCP after competitive immunoreactions. The sensor exhibits a linear response to Goat anti-IgG in the concentration range 2ng/mL to 60ng/mL, correlation coefficient 0.981.