Flow injection amperometric enzyme biosensor for direct determination of organophosphate nerve agents

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.