ABSTUCTDisposable conductometric microbiosensors based on immobilization of acetyl and butyrylcholinesterases (BuChEs) on interdigitated electrodes were fabricated. Enzymes were immobilized by crosslinking with bovine serum albumin and the differential output between the pair of electrodes covered with the enzyme membrane and the pair covered with albumin only was recorded. On the basis of these biosensors, a biochemical assay for pesticides in liquids was developed. Respective detection limits were 5 X lo-" M for diisopropyl fluorophosphate (DFP), lo-' M for trichlorfon, and M for paraoxon-methyl and paraoxon-ethyl for the assavs based on the enzymes tested. The presence of pesticides in the sample in concentration exceeding the detection limits can be detected after only 10 minutes of incubation, whereas, in order to extend the range of the determined pesticide concentration and to improve the accuracy of the assay, it is better to use incubation times longer than 30 minutes. Use of pyridin-2-aldoximide methiodide as reactivating agent allowed one to achieve the complete recovery of the sensor response when it was incubated in lowconcentrated pesticide solutions, e.g., less than M for paraoxon-methyl and during a short time, e.g., 10 to 20 minutes, for M paraoxon-methyl solution.
A glucose-sensitive field effect transistor was fabricated by immobilizing glucose oxidase on the gate of a pH-sensitive field effect transistor. Calibration curves of the biosensor were measured in phosphate and TRIS buffers in the presence of potassium ferricyanide. The use of the latter as an oxidizing substrate in the biocatalytic oxidation of glucose leads to an increase of the acidification rate of the solution inside the enzymatic layer, because three protons are now generated per one molecule of glucose instead of only one when the natural oxidizing cosubstrate, oxygen, is used. Depending on the concentration of ferricyanide we observe a 10-100 times increase of the biosensor response in concentrated buffer solutions and a substantial extension of its dynamic range. At sufficiently high concentrations of ferricyanide, the calibration curves in both buffers have a sigmoidal shape in linear coordinates with local pH changes on the surface of the field effect transistor reaching about two pH units in the saturation range. The resulting saturation of the curves at higher glucose concentrations is due to the inhibition of the activity of glucose oxidase at acidic pH by Cl- ions present in the solution. The proposed approach may be extended to allow the detection of a wide range of analytes using enzyme field effect transistors based on the enzymes for which reoxidation of the cofactor (coenzyme) leads to a liberation of H+ ions.
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