Organophosphorus Hydrolase-Based Assay for Organophosphate Pesticides

Abstract

We report a rapid and versatile organophosphorus hydrolase (OPH)-based method for measurement of organophosphates. This assay is based on a substrate-dependent change in pH at the local vicinity of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC), which is covalently immobilized to the enzyme. This method employs the use of poly(methyl methacrylate) beads to which the FITC-labeled enzyme is adsorbed. Analytes were then measured using a microbead fluorescence analyzer. The dynamic concentration range for the assay extends from 25 to 400 &mgr;M for paraoxon with a detection limit of 8 &mgr;M. Organophosphorus insecticides measured using this technique included ethylparathion, methylparathion, dursban, fensulfothion, crotoxyphos, diazinon, mevinphos, dichlorvos, and coumaphos. This technique was used to measure coumaphos in biodegradation samples of cattle dip wastes and showed a high correlation (r2 = 0.998) to an HPLC method.

An Organophosphate Sensor Based on Photo-crosslinked Hydrogel-entrapped E. coli

“…The detection limit of this sensor is similar to those of the spectrofluorometric method using OPH-enzyme coated beads (8 μM), 7 and the potentiometric method using E. coli with surface-expressed OPH enzymes (2 μM), 13 but higher than that of using OPH-SNAFL conjugate immobilized within hydrogel (0.16 μM).…”

“…3 Because of its specificity, the OPH enzyme has been used for sensing [5][6][7] and detoxifying OPs. 8,9 However, a major problem of this naked OPH enzyme is that it shows a significant decrease in enzyme activity and stability after immobilization of the enzyme within or on a matrix.…”

Cross-linked enzyme crystals of organophosphate hydrolase for electrochemical detection of organophosphorus compounds

“…The enzyme is specific to ester bond hydrolysis in a range of neurotoxic organophosphate pesticides, including paraoxon, parathion, methyl-parathion, coumaphos and chemical warfare agents, sarin and soman, releasing the electroactive p-nitrophenol (PNP) as the product. Several types of biosensor using OPH for detection of organophosphate compounds have been developed, based on optical (Mulchandani et al 1999b;Rogers et al 1999;Simonian et al 2005), potentiometric (Mulchandani et al 1999c), and amperometric (Mulchandani et al 2001;Chough et al 2002;Wang et al 2003;Deo et al 2005) detection. However, the commercial application of OPH-biosensor has been impeded by several factors, including high cost on enzyme production, purification and processing.…”

Modified Cholinesterase Technology in the Construction of Biosensors for Organophosphorus Nerve Agents and Pesticides Detection

“…So the organophosphorus compounds act for OPH as a substrate. These types of biosensors offer, same as the cholinesterase ones, using optical, fluorescent, potentiometric or amperometric converters (Karnati et al, 2007;Mulchandani, et al 1999aMulchandani, et al , 1999bRogers et al, 1999;Wang et al, 2003). Also a field version for in-situ monitoring has been developed Mulchandani et al, 2001).…”