Fabrication And Characterization Of The Enzyme Membranes For An Amperometric Glucose Biosensors

Amperometric glucose biosensor is applied widely in many fields. A very important factor in glucose biosensor research is the immobilization of glucose oxidase (GOD). Researching and developing appropriate matrixes and relevant methods of immobilized enzyme are important in the development of novel glucose biosensors. Therefore, it is expected to find an effective immobilization strategy to retain high activity of the immobilized GOD and excellent performance of determining glucose. In this work, glucose biosensors were developed based on covalently immobilizing GOD on nitrocellulose membrane and polycarbonate membrane using the method of cross linking. The main contents of the thesis are given as follows:1. A glucose biosensor was developed based on immobilizing GOD on a nitrocellulose membrane (Φ10mm) using glutaraldehyde as a cross linking agent. FTIR was employed to study the reaction between glutaraldehyde and GOD. It showed that GOD was immobilized on the nitrocellulose membrane by covalent bonds. The activity of the GOD membranes on different immobilization conditions was examined. The optimum conditions were2.5% (v/v) glutaraldehyde solution, pH=6.5of phosphate buffer solution (PBS) and10.0g-L"1GOD solution. The resulting glucose biosensor exhibited a fast response of80-100s, high sensitivity of1.1μA-L-mmol-1and wide linear range from0.05mmol·L-1to4.0mmol·L-1. The apparent Michaelis-Menten constant was7.2mmol·L-1suggesting that the proposed GOD membrane performs good affinity with P-D-glucose. The proposed biosensor had an excellent selectivity, reproducibility, operation stability and long-term stability. The glucose concentrations in real samples were determined and the proposed method agreed quit well with the spectrophotometric method. The GOD membrane was also applied in a commercial analytic instrument and showed an excellent linearity, stability, selectivity, thermal stability as well as the fast response.2. A glucose biosensor was developed based on dropping the GOD solution on a polycarbonate membrane (Φ10mm) using the glutaraldehyde vapor as a cross linking agent. The optimum condition is dropping4μL of5g·L-1GOD solution. The GOD membrane shows a very fast response of20s, wide linear range from0.1mmol-L"1to2.5mmol·L-1, high sensitivity of1.32μA·L·mmol-1and the apparent Michaelis-Menten constant was141.95mmol·L-1. The selectivity of the GOD membrane should be improved. The GOD membrane was also applied in a commercial analytic instrument and showed an excellent linearity, stability, selectivity well as the fast response.