Immobilization and characterization of sol-gelencapsulated acetylcholinesterase fiber-optic biosensor

A rapid approach for preparing sol-gel based fiber-optic biosensor with encapsulated pHsensitive fluorophore-acetylcholinesterase conjugates is developed for the determination of acetylcholine. The sensor consists of a pH-sensitive fluorescent indicator encapsulated with enzyme in a sol-gel network on a glass cap that can be fixed on an optical fiber and then integrated with a flow-through reactor for continuous monitoring. The sol-gel preparation procedure has been modified for maintaining a stable pH value and enzyme activity. Among the nine fluorescent indicators tested, FITC-dextran was the most suitable candidate for biosensor. The optimized ratio of TMOS:HCl:H2O and enzyme-dye:sol solution for the rapid preparation of sol-gel film were 1:3.6×10-5:2 and 3:5, respectively. The 1mM pH 8.5 Trisbuffer had sufficient buffer capacity for acetylcholine preparation and the substrate concentration must be below 50mM to obtain a stable pH value. The response of the developed biosensor to acetylcholine was highly reproducible (R.S.D.=3.5%, n=8). A good linearity of acetylcholine in the range from 0.5 to 20mM was also obtained (R2=0.98). Furthermore, a 30% inhibition can be achieved within 30min when 0.54µM (152ppb) paraoxon was added into the system. This indicates that the developed biosensor is suitable for the analysis of acetylcholine and organophosphorus neurotoxins.