Flow Injection Amperometric Enzyme Biosensor for Direct Determination of Organophosphate Nerve Agents

Abstract

A flow injection amperometric biosensor for the determination of organophosphate nerve agents was developed. The biosensor incorporated an immobilized enzyme reactor that contains the enzyme organophosphorus hydrolase covalently immobilized on activated aminopropyl controlled pore glass beads and an electrochemical flow-through detector containing carbon paste working electrode, a silver/silver chloride reference electrode, and stainless steel counter electrode. The organophosphorus hydrolase catalyzed the hydrolysis of organophosphate with nitrophenyl substituent to generate p-nitrophenol which is then detected downstream electrochemically at the carbon paste electrode poised at 0.9 V vs the reference electrode. The amperometric response of the biosensor was linear up to 120 microM and 140 microM, with lower detection limits of 20 nM and 20 nM, for paraoxon and methyl parathion, respectively. The response was very reproducible (RSD 2%, n = 35) and stable for over 1 month when the immobilized enzyme column was stored at 4 degrees C. Each assay took ca. 2 min giving a sample throughput of 30 h(-1). The applicability of the biosensor to monitor paraoxon and methyl parathion in distilled water and simulated well water was demonstrated.

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.…”

Organophosphorus Pesticides Determination by Electrochemical Biosensors

“…The hydrolysis involves a pH change, as well as electroactive species generation, thus allowing the development of potentiometric and amperometric sensors for OP pesticides quantification (Anzai, 2006;Chough et al, 2002;Lei et al, 2007;Mulchandani et al, 2001a;Mulchandani et al, 2001b;Prieto-Simón et al, 2006;Rodriguez-Mozaz et al, 2004;Wang et al, 2003). For instance, OPH catalyzed hydrolysis of parathion, methyl parathion, paraoxon, fenitrothion, etc.…”

Enzyme vs. Bacterial Electrochemical Sensors for Organophosphorus Pesticides Quantification

“…An overview of the methods based on enzyme inhibition with emphasis on the non-ideal behavior of the enzyme inhibition-based biosensors and biosensing systems is presented by Luque de Castro (Luque de Castro & Herrera, 2003). Direct OP pesticides analysis could be achieved applying organophosphorus hydrolase (OPH) electrochemical sensors (Anzai, 2006;Chough et al, 2002;Lei et al, 2007 ;Mulchandani et al, 2001a;Mulchandani et al, 2001b;Prieto-Simón et al, 2006;RodriguezMozaz et al, 2004;Wang et al, 2003). The enzyme OPH demonstrates substrate specificity toward paraoxon, parathion, coumaphos, diazinon, dursban, methyl parathion, etc, and toward some chemical warfare agents (sarin, soman, tabun, VX, etc.)…”