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

Abstract

A highly sensitive flow analysis method for determination of acetylcholinesterase (AChE) inhibitors like organophosphorous pesticides using a new chemiluminescent reaction was developed and optimized. This method is fast, sensitive, and cheap, because it requires only one enzyme and its substrate. The system incorporates a reactor with immobilized AChE on controlled pore glass (CPG) and a chemiluminometric detector. Variations in enzyme activity due to inhibition are measured from the changes of concentrations of thiocholine produced when the substrate (acetylthiocholine chloride) is pumped before and after the passage of the solution containing the pesticide through the immobilized AChE reactor. Thiocholine is determined by a new chemiluminescent reaction with luminol in the presence of potassium ferricyanide. The percentage inhibition of enzyme activity is correlated to the pesticide concentration. The inhibited enzyme is reactivated by 10 mM pyridine‐2‐aldoxime methiodide (2‐PAM). The experimental conditions were first optimized for activity determination of the effect of pH, flow rates, and Tris concentrations. For the measurement of AChE inhibition, the appropriate concentration of the substrate is selected such that the rate of noninhibited reaction can be considered unchanged and could be used as a reference. For optimization of experimental conditions for inhibition, several parameters of the system 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 pesticide was tested. For an inhibition time of 10 min the calibration graph is linear from 0.1 to 1 ppm paraoxon with a relative standard deviation (n = 5) of 4.6% at 0.5 ppm. For an inhibition time of 30 min the calibration graph is linear from 25 to 250 ppb paraoxon. © 2000 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 57: 37–42, 2000

Cholinesterase-based biosensors

“…Danet et al described a flow analysis to determine paraoxon using immobilized AChE. The authors used acetylthiocholine as a substrate and originating thiocholine was determined by a chemiluminescence reaction with luminol in the presence of ferricyanide 112 . Andreou et al developed a fiber-optic pesticide biosensor for in-field use.…”

Analysis of Pesticides by Flow Injection Coupled with Chemiluminescent Detection: A Review

“…A fast, sensitive and cheap enzymatic method for the paraoxon determination was proposed by Danet et al (Danet 2000). It was based on the use of a reactor with immobilized acetylcholinesterase (AChE) on controlled pore glass.…”

Use of Esterase Activities for the Detection of Chemical Neurotoxic Agents

“…A highly sensitive flow analysis method using a chemiluminescent reaction was developed measuring the concentrations changes of thiocholine produced when the substrate (acetylthiocholine chloride) is pumped before and after the passage of the solution containing the pesticide through an immobilized AChE reactor. Thiocholine is determined by a chemiluminescent reaction with luminol in the presence of potassium ferricyanide [102] by a chemiluminometric detector. Optical fiber biosensor holding AChE and an indicator as bromothymol blue [100,103] or bromcresol purple [104], either immobilized in a solgel film, was also described for detection of OP pesticides.…”