Synthesis Of Gold Nanoparticles And Gold Nanoclusters, And Studying Their Enzymatic Properties For Modification Of Glucose Oxidase

Nanometer gold particles were divided into gold nanoparticles (AuNPs) and gold nanoclusters (AuNCs) by different particle size. Because of unique optical properties, electrical properties, biological compatibility and chemical activity, etc., they had become one of hot topics in biosensors, immune response, detection of heavy metals and biological labeling applications.The preparation processes and physical and chemical properties of gold nanoparticles and gold nanoclusters were described in this paper. It studied two kinds of nanoparticles on the modification of glucose oxidase. The optimal conditions of modification were studied in detail. Meanwhile, The enzymology properties and catalytic effect were contrasted between modified glucose oxidase and free glucose oxidase in detail, details were as follows:1. Gold nanoparticles and thiol-functionalized gold nanoparticles were prepared by the improved chemistry method. It was shown that gold nanoparticles showed the characteristic surface plasmon peak at about 527 nm, the average diameter was about 6nm-12nm, and has a good dispersivity. We assigned the absorption band at 1577 cm-1 totally with the standard nanoparticles characteristic absorption peaks; meanwhile, thiol-functionalized gold nanoparticles showed the characteristic surface plasmon peak at about 535 nm, and the average diameter was about 10 nm-15 nm, it also has a good dispersivity; and IR analysis was shown that they had characteristic surface plasmon peak at 2921 cm-1,2850 cm-1 and 1699 cm-1, respectively. It demonstrated the presence of carboxylic groups on the gold surface.2. The color of the bovine serum albumin-directed synthesis of gold nanoclusters was pale yellow under visible light, and they emitted an intense red fluorescence under UV light (365 nm). They showed emission peaks and characteristic surface plasmon peak at 640 nm and 210 nm, respectively.3. The thiol-functionalized gold nanoparticles and gold nanoclusters were used to modify glucose oxidase and the best modification condition was optimized. The enzymology character and dynamic parameters of two kinds of modified enzymes were investigated respectively. The result was shown that, the optimum modification time was 2 h, the optimal EDC concentration of modified enzyme was 8mmol/L, the optimal amount of enzyme was 1 mL, the optimal reaction condition was pH 7.0, the optimal reaction temperature was 25℃, and Km value was 2.94 mg/mL. The enzyme activity of gold nanoparticles modified enzymes was the best when the EDC concentration was 12 mmol/L, modification time was 1 h, and the amout of enzyme was 1.5 mL. Meanwhile, the optimum modification time was 2 h, the optimal EDC concentration of modified enzyme was 16 mmol/L, the optimal amount of enzyme was 2 mL, the optimal reaction condition was pH 5.0, the optimal reaction temperature was 25℃, and Km value was 1.69 mg/mL. The enzyme activity of gold nanoclusters modified enzymes was the best when the EDC concentration was 12 mmol/L, the amout of enzyme was 1.5 mL, and the pH was 5.0. Contrasting enzymology characters of gold nanoparticles modified enzymes and gold nanoclusters modified enzymes, we could know that they had same optimal reaction temperature, while Km value of gold nanoclusters modified enzymes was lower than one of gold nanoparticles modified enzymes, the former had more acid resistance than the latter, but when the pH increased, the enzyme activity reduced rapidly.4. Because bovine serum albumin-directed synthesis of gold nanoclusters easily form precipitates in weak acidic environment, purified modified glucose oxidase by gold nanoclusters could be acquired with different solubility in ammonium sulfate between modified glucose oxidase by gold nanoclusters and free glucose oxidase respectively. It showed that when 40% ammonium sulfates were added into the reaction mixture, we could get the purified modified glucose oxidase by gold nanoclusters.5. The electrical graphs of modified enzymes by gold nanoclusters were analyzed by agarose gel electrophoresis under single factor. It was shown that modified enzymes by gold nanoclusters also emitted an intense red fluorescence under UV light (365 nm). There was a single electrophoresis pattern in agarose gel electrophoresis. With the increasing EDC concentration, the lager the modified enzymes were, the slower their mobility was, when the EDC concentration was 16 mmol/L, the modified enzymes have the largest molecular weight; with the different modified time, it showed different mobility, when modified time was 1h, the modified enzymes have more single electrophoresis pattern; the lager the proportion of enzyme were, the slower their mobility was. It could be concluded that modified enzymes by gold nanoclusters were acid hydrolases, the enzyme activity of modified enzymes by gold nanoclusters was better than the free enzyme's. Meanwhile, it not only has enzyme activity, but also has intense red fluorescence, which could be used as safe and non-toxic labers.