Development of sensors for direct detection of organophosphates. Part II: Sol-gel modified field effect transistor with immobilized organophosphate hydrolase

摘要

pH-sensitive field effect transistors (FET) were modified with organophosphate hydrolase (OPH) and used for direct detection of organophosphate compounds. OPH is the organophosphate degrading gene product isolated from Pseudomonas diminuta. OPH was selected as an alternative to acetylcholinesterase, which requires inhibition mode sensor operation, enzyme regeneration before reuse, long sample incubation times, and a constant source of acetylcholine substrate. OPH was covalently immobilized directly to the exposed silicon nitride gate insulator of the FET. Alternatively, silica microspheres of 20 or 200 nm were formed via a base catalyzed sol-gel process and were dip-coated onto the gate surface; enzyme was then covalently immobilized to this modified surface. All sensors were tested with paraoxon and displayed rapid response (<10 s), with a detection limit of approximately 1x10 super(-6) molar. The 200 nm sol-gel gate modification enhanced the signal of enzyme-modified devices without effecting device pH sensitivity. Sensors were stored at 4 degree C in buffer and tested multiple times. Devices coated with 200 nm silica microspheres maintained significant enzymatic activity over a period of 10 weeks while uncoated devices lost all enzyme activity during the same period. The 20 nm sol-gel modification did not enhance device response or enzyme stability. Successful reuse of sensor chips was demonstrated after stripping inactive enzyme with an RF oxygen plasma system and reimmobilizing active enzyme.