Enzyme-based biosensor for the direct detection of fluorine-containing organophosphates

The ability of the enzyme organophosphorus acid anhydrolase (OPAA) to selectively hydrolyze the P–F bond of fluorine containing organophosphates has been used to develop a biosensor for specific detection of these compounds. Hydrolysis rate of diisopropyl fluorophosphate (DFP), paraoxon and demeton-S, by soluble and immobilized OPAA was measured. These compounds were selected as representative substrates of OPAA hydrolysis of P–F, P–O and P–S bonds, respectively. Results indicate that hydrolysis of phosphofluoridates such as DFP is dominant while hydrolysis of phosphotriesters such as paraoxon or of phosphothiolates such as demeton-S, is negligible. Two experimental approaches were used for biosensor development. In the first, OPAA was covalently immobilized on silica gel and used in batch-mode measurements with flat glass pH electrode to detect pH changes due to P–X bond cleavage. In the second approach, the enzyme was covalently immobilized to the porous silica modified gate insulator of a pH-sensitive field effect transistor (pH-FET) and changes in pH relative to a second non-enzyme coated pH-FET were measured in stop-flow mode. Concentrations of DFP down to 25 M with the glass electrode and 20 M with the pH-FET were readily detected. No sensor response was observed with paraoxon or demeton-S indicating that such OPAA-based biosensors could be useful for direct and discriminative detection of fluorine containing organophosphorus neurotoxins (such as the G-type chemical warfare agents sarin GB and soman GD) in samples also containing multiple organophosphate pesticides. © 2001 Elsevier Science B.V. All rights reserved.