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 (pHFET) 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 muM with the glass electrode and 20 muM 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.