Disposable potentiometric enzyme sensor for direct determination of organophosphorus insecticides.

Abstract

A potentiometric disposable enzyme sensor for the direct and fast determination of organophosphorus (OP) insecticides was developed by using an organophosphorus hydrolase (OPH) immobilized on an ion-selective electrode. The disposable screen-printed transducer was based on double matrix membrane technology which allows easy mass production. The potentiometric device consisted of a H(+)-sensitive electrode with integrated Ag/AgCl reference electrode. The electrodes were prepared with N,N-dioctadecylmethylamine as H(+)-sensitive ionophore and pH calibration resulted in slopes of 55 mV decade-1 over a pH range from 11 to 6. OPH was isolated from recombinant Escherichia coli DH5 alpha and immobilized within poly(carbamoyl sulfonate) prepolymer on the surface of the H(+)-sensitive electrode without any further fixation membrane. OPH catalyzes the hydrolytic cleavage of OP compounds which releases protons in a concentration proportional to hydrolyzed substrate. Sensor performance was investigated with regard to enzyme load, concentration, pH and temperature of the measuring buffer using paraoxon as analyte. Best sensitivity and response time were obtained with sensors prepared with 250 U of OPH and measuring at 37 degrees C in 1.0 mM HEPES buffer, pH 9.3, containing 100 mM NaCl. The enzyme sensor exhibited a linear calibration range of 0.01-0.15 mM chlorpyrifos, 0.05-0.35 mM diazinon, 0.05-0.4 mM paraoxon and 0.007-0.05 mM parathion, respectively. For all these analytes response times to reach 95% of maximum change in potential did not exceed 5 min. Sensors stored under dry conditions at 4 degrees C still showed 60% of initial hydrolytic rate after 70 d. The sensors even when stored dry were ready for measurements after 5 min incubation in measuring buffer. A range of putative interfering substances did not influence sensor response, and suitability of measuring OPs in soil extracts was ascertained.