ENFET and REFET were immersed in 10 ml of 1 mM phosphate buffer pH 8.0 at room temperature under magnetic stirring system; these experimental conditions allow to obtain the higher response of the ENFET 20 The substrate concentration BuChCl was varied by addition into the vessel of aliquots, so that a final concentration of substrate close to 20 mM was reached. For each addition, an increase of Hq concentration in enzymatic membrane is produced due to the enzymatic reaction which leads to the hydrolysis of the substrate. The steady state was obtained within 30 s and all the results have been done 1 min after each injection. For the kinetic response, the maximal slope of the response curve versus time was measured. For both the kinetic and stationary modes, three measurements for each experimental point were done. The procedure for the evaluation of the effect of the enzyme activity inhibition by pesticides on the biosensors response includes the following steps
1. The biosensor was soaked in a 1 mM phosphate buffer solution until reaching the stable baseline output signal.
2 .BuChCl was added to the measurement cell until a fixed final concentration 20 mM in our case . The corresponding steady-state output signal of the biosensor was taken as an index of the catalytic signal of the immobilized enzyme, and such a value of the biosensor response was used for further evaluation of the inhibition effect of a definite pesticide.
3. After washing, the sensor was incubated for 10 to 60 min in the solution of tested pesticide.
4. After extensive washing with the working buffer, the steady-state response of the biosensor was determined. The level of inhibition due to the action of a definite pesticide was evaluated by comparison of the steady-state levels of the biosensor before and after treatment with a pesticide solution. To reactivate the inhibited enzyme, the
biosensor was immersed for 10 to 60 min in a vigorously stirred reactivation solution consisting of a saturated solution of pyridine-2-aldoxime methiodide PAM-2 in PBS.