| Summary: | 碩士 === 東海大學 === 化學工程學系 === 96 === The promotion of the activity and the increase of the life span of enzymes used in the biofuel cells were still an important issue in the research and development of the enzymatic biofuel cellss. The promotion of activity and the increase in life time of enzymes immobilized on the mesoporous materials have been demonstrated in many application fields. In this thesis, The mesoporous carbon (MCF-C) synthesized by using mesostructured cellular foam (MCF) as templates was used to immobilize the bilirubin oxidase (BOD) for preparing the enzymatic electrodes.
Using the oil-in water micro-emulsions method to prepare the MCF with the uniform spherical cells and windows, the pore structure of MCF was affected by the Pluronic P123/TMB weight ratio, the stirring rate and time, and the aging temperature, respectively. The most important one of the factors was the Pluronic P123/TMB weight ratio, and increasing the weight ratio from 0.5 to 0.75, the cells and windows diameter were increased from 20 to 23 nm and from 9 to 11 nm, respectively. However, the pore diameters of MCF were decreased for further increase in the weight ratio greater than 0.75.
Using MCF with 23 nm cells and 11nm windows as the templates for preparing MCF-C, the pore properties of MCF-C could be adjusted by changing the ratio of the sucrose (carbon source) and the template. The pore structure of MCF was not found on the MCF-C prepared with the sucrose/MCF weight ratio of 1.5. Decreasing the weight ratio the pore structure of MCF-C was close to that of MCF (template). The MCF-C with cells of 23 nm and windows of 11 nm was obtained with the weight ratio of sucrose and MCF of 0.2.
Using the enzymatic electrode prepared by the immobilization of BOD on the home-made MCF-C as the working electrode, the characteristics of oxygen reduction reaction (ORR) were affected by the amounts of MCF-C and BOD, temperature and pH of the solution. Among of these factors the most important one was the amount of BOD. When the loading of BOD on the enzymatic electrode (working electrode) was set as 6.65 μg (0.033U), the net current of ORR was obtained to be -12.0±0.63 μA at 0 V (vs. Ag/AgCl/3M NaCl) in 1mM ABTS and pH 5.0 phosphate buffer solution (PBS) saturated with oxygen at 30oC. Increasing the BOD loading to 39.9 μg (0.198U), the current of ORR increased to -33.9±4.65 μA. However, the excess of BOD on the enzymatic electrode caused the decrease in the reproducibility and the enzymatic utility of the enzymatic electrode for ORR. The optimal enzymatic reproducibility and utility were obtained with 13.3 μg (0.066U) BOD used in the enzymatic electrode.
Using Nafion® solution to immobilize the electronic mediator (ABTS) on the enzymatic electrode, the best performance of the enzymatic electroce for ORR was obtained by mixing Nafion® solution and ABTS and then casting on the electrode located between the Au and enzyme (BOD) layers. When the amount of ABTS immobilized on the electrode was 0.125 μmole, the net steady current of ORR was obtained to be -45.1±0.96 μA at 0 V in pH 5.0 PBS saturated with oxygen at 30oC. Increasing the amount of ABTS to 1.0 μmole the current of ORR was increased to -68.3±1.46 μA, the current could but be increased with further increase in the amount of ABTS due to the limitation of BOD utility.
Based on the kinetic study, the enzyme catalytic constant of free BOD (kcat) and immobilized BOD (ks,cat) were found to be 128.3 s-1and 0.63 s-1, respectively. The decrease in the enzymatic activity found in the immobilization of BOD enzyme was demonstrated from the fact of kcat/ks,cat =204.4.
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