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Flow analysis for determination of paraoxon with use of immobilized acetylcholinesterase reactor and new type of chemiluminescent reaction
Author(s) -
Danet Andrei F.,
Badea Mihaela,
Marty JeanLouis,
AboulEnein Hassan Y.
Publication year - 2000
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/(sici)1097-0282(2000)57:1<37::aid-bip6>3.0.co;2-x
Subject(s) - chemistry , paraoxon , ferricyanide , chemiluminescence , potassium ferricyanide , luminol , substrate (aquarium) , chromatography , acetylthiocholine , acetylcholinesterase , immobilized enzyme , flow injection analysis , enzyme , biochemistry , detection limit , inorganic chemistry , aché , oceanography , geology
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 m M 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