Immobilization and characterization of sol-gel-encapsulated acetylcholinesterase fiber-optic biosensor

A rapid approach for preparing sol-gel based fiber-optic biosensor with encapsulated pH-sensitive 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 x 10(-5):2 and 3:5, respectively. The 1 mM pH 8.5 Tris-buffer had sufficient buffer capacity for acetylcholine preparation and the substrate concentration must be below 50 mM 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 20 mM was also obtained (R-2 = 0.98). Furthermore, a 30% inhibition can be achieved within 30 min when 0.54 muM (152 ppb) paraoxon was added into the system. This indicates that the developed biosensor is suitable for the analysis of acetylcholine and organophosphorus neurotoxins. (C) 2001 Elsevier Science B.V. All rights reserved.