Flow analysis for determination of paraoxon with use of immobilized acetylcholinesterase reactor and new type of chemiluminescent reaction

A highly sensitive flow analysis method for determination of acetylcholines terase (AChE) inhibitors like organophosphorous pesticides using a new chem iluminescent reaction was developed and optimized. This method is fast, sen sitive, and cheap, because it requires only one enzyme and its substrate. T he system incorporates a reactor with immobilized AChE on controlled pore g lass (CPG) and a chemiluminometric detector. Variations in enzyme activity due to inhibition are measured from the changes of concentrations of thioch oline produced when the substrate (acetylthiocholine chloride) is pumped be fore and after the passage of the solution containing the pesticide through the immobilized AChE reactor. Thiocholine is determined by a new chemilumi nescent reaction with luminol in the presence of potassium ferricyanide. Th e percentage inhibition of enzyme activity is correlated to the pesticide c oncentration. The inhibited enzyme is reactivated by 10 mM pyridine-2-aldox ime methiodide (2-PAM). The experimental conditions were first optimized fo r activity determination of the effect of pH, flow rates, and Tris concentr ations. For the measurement of AChE inhibition, the appropriate concentrati on of the substrate is selected such that the rate of noninhibited reaction can be considered unchanged and could be used as a reference. For optimiza tion of experimental conditions for inhibition, several parameters of the s ystem are studied and discussed: flow rate, enzyme-pesticide contact time, luminol concentration, ferricyanide concentration, 2-PAM concentration, and configuration of the FIA manifold. Paraoxon, an organophosphorous pesticid e was tested. For an inhibition time of 10 min the calibration graph is lin ear from 0.1 to 1 ppm paraoxon with a relative standard deviation (n = 5) o f 4.6% at 0.5 ppm. For an inhibition time of 30 min the calibration graph i s linear from 25 to 250 ppb paraoxon. (C) 2000 John Wiley & Sons, Inc.