FET-Based Biosensors for The Direct Detection of Organophosphate Neurotoxins

Recent world-wide terrorist events associated with the threat of hazardous chemical agent proliferation, and outbreaks of chemical contamination in the food supply has demonstrated an urgent need for sensors that can directly detect the presence of dangerous chemical toxins. Such sensors must enable real-time detection and accurate identification of different classes of pesticides (e.g., carbamates and organophosphates) but must especially discriminate between widely used organophosphate (OP) pesticides and Gand V-type organophosphate chemical warfare nerve agents. Present field analytic sensors are bulky with limited specificity, require specially-trained personnel, and, in some cases, depend upon lengthy analysis time and specialized facilities. Most bioanalytical based systems are biomimetic. These sensors utilize sensitive enzyme recognition elements that are the in-vivo target of the neurotoxic agents which the sensor is attempting to detect. The strategy is well founded; if you want to detect cholinesterase toxins use cholinesterase receptors. However, this approach has multiple limitations. Cholinesterase receptors are sensitive to a wide range of non-related compounds and require lengthy incubation time. Cholinesterase sensors are inherently inhibition mode and therefore require baseline testing followed by sample exposure, retest and comparison to baseline. Finally, due to the irreversible nature of enzyme-ligand interactions, inhibition-mode sensors cannot be reused without regeneration of enzyme activity, which in many cases is inefficient and time-consuming. In 1996, we pioneered a new “kinetic” approach for the direct detection of OP neurotoxins based on agent hydrolysis by the enzyme organophosphate hydrolase (OPH; EC 3.1.8.2; phosphotriesterase) and further identified a novel multienzyme strategy for discrimination between different classes of neurotoxins. The major advantage of this sensor strategy is it allows direct and continuous measurement of OP agents using a reversible biorecognition element. We also investigated incorporation of enzymes with variations in substrate specificity (e.g., native OPH, site-directed mutants of OPH, and OPAA (EC 3.1.8.1), based upon preferential hydrolysis of P O, P F and P S bonds to enable discrimination among chemically diverse OP compounds. Organophosphate hydrolase enzymes were integrated with several different transduction platforms including conventional pH electrodes, fluoride ion-sensitive electrodes, and pH-responsive fluorescent dyes. Detection limit for most systems was in the low ppm concentration range. This article reviews our integration of organophosphate hydrolase enzymes with pH sensitive field effect transistors (FETs) for OP detection.