Optimization of Acetylcholinesterase based on Fiber-Optic Biosensor for Real-Time Monitoring of Organophosphorus Pesticides

• Main Authors: Hsiao-chung Tsai, 蔡曉忠
• Other Authors: Ruey-an Doong
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/02492711709550683843

碩士 === 國立清華大學 === 原子科學系 === 87 === Organophosphorus pesticides (OPPs) are widely used as insecticides for agricultural or household purposes since 1970s. Although they can be quickly decomposed in the environment, their highly acute toxicity to acetylcholinesterase (AChE) received highly attention. Therefore, the development of high efficient analytical techniques for real-time monitoring the residues of OPPs in the environments is needed. The purpose of this study is to apply the sol-gel immobilization technology for developing a fiber-optic biosensor using AChE as biological recognition molecule for the detection of OPPs. Eight kinds of fluorescence dyes were used as the candidates for pH sensitive indicators. Also, the optimization in sol-gel formation and substrate preparation will be included. Results obtained in this study show that FITC-dextrans is a suitable pH sensitive fluorescence indicator for AChE based biosensor due to its low leaching ability in continuos system. The optimized ratio of TMOS:HCl:H2O was 1:3.6’10-5:2. The mixing ratio of enzyme-dye solution and sol solution was in the range of 3 to 5. By using 5mM pH8.9 Tris-HCl as immobilization buffer, a higher initial fluorescent intensity was observed. The buffer capacity for substrate preparation was 1mM pH8.5 Tris-HCl and the substrate concentration must below 50mM to get a stable pH value. Moreover, this homemade fiber-optic biosensor was highly stable in analyzing the acetylcholine with the relative standard deviation (RSD) of 3.5% (n=8). A good linearity of acetylcholine in the range from 0.5 to 20mM was also obtained (R2=0.98). A 30% inhibition can be achieved when 152ppb paraoxon was added into the system. This suggests the developed biosensor system is suitable for real-time analysis of organophosphorus pesticides.