| Biosensors have attracted much attention in recent times because of the potential applications of these devices in the clinical diagnostics, environmental monitoring, Pharmaceuticals, and food processing industries due to their fast response and ease of operation. For enzyme-based biosensor, the biological recognition molecule is enzyme. The enzymatic activity and stability depend on the immobilization strategy of enzymes. So, the immobilization of enzyme is a key step for constructing an enzyme-based biosensor. The purpose of this work is to explore new immobilization strategy of enzymes. The thesis includes the following parts:PartⅠ: Chemiluminescence flow-through biosensor for glucose with eggshell membrane as enzyme immobilization platformEggshell membrane is a light pink double-layered membrane inside the eggshell, it is composed mainly of biological moleculars and high crosslinked protein fibers, and it prosesses exellent gas and water permeability. In this study, a new chemiluminescence (CL) flow-through biosensor for glucose was developed by immobilizing glucose oxidase (GOD) and horseradish peroxidase (HRP) on the eggshell membrane with glutaraldehyde as a cross-linker. The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and hydrogen peroxide (H2O2) and then H2O2 oxidizing luminol to produce CL emission in the presence of HRP. The immobilization condition (e.g., immobilization time, GOD/HRP ratio, glutaraldehyde concentration) was studied in detail. It showed good storage stability at 4℃over a 5-month period. The proposed biosensor exhibited short response time, high sensitivity, easy operation, and simple sensor assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.PartⅡ: Chemiluminescence flow-through biosensor for H2O2 based on increasing HRP activity with gold nanoparticlesA novel chemiluminescence flow-through biosensor for hydrogen peroxide is described. It is prepared by immobilizing HRP and Au nanoparticles with the sol-gel method on the flow CL cell. In the presence of Au nanoparticles, the respond of biosensor is enhanced by 50-fold. The calibration graph is linear in the range of 1×10-8～1×10-6 mol/L, and the detection limit is 4×10-9 mol/L. A complete analysis could be performed in 1 min with a relative standard deviation of less than 5% for 1×10-6 mol/L H2O2(n=9).PartⅢ: Chemiluminescence flow-through biosensor for glucose based on gold nanoparticles enhanced activities of glucose oxidase and horseradish peroxidaseIn this work, we explore the feasibility of using gold nanoparticles to increase the activity of encapsulated enzyme in sol-gel network. A novel chemiluminescence flow biosensor for glucose was proposed. Glucose oxidase, horseradish peroxidase and gold nanoparticles were immobilized with sol-gel method on the inside surface of the CL flow cell. The CL detection involved enzymatic oxidation of glucose to D-gluconic acid and H2O2 and then H2O2 oxidizing luminol to produce CL emission in the presence of HRP. It was found that gold nanoparticles could remarkably enhance the CL respond of the glucose biosensor. The immobilization condition and the CL condition were studied in detail. The CL emission intensity was linear with glucose concentration in the range of 1.0×10-5～1.0×10-3mol/L, and the detection limit was 5×10-6mol/L(3σ). The apparent Michaelis-Menten constant of GOD in gold nanoparticles/sol-gel matrix was evaluated to be 0.3 mmol l-1. The proposed biosensor exhibited short response time, easy operation, low cost and simple assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum. |
PDF Full Text Request