Rapid detection of anticholinesterase insecticides by a reusable light addressable potentiometric biosensor

Abstract

A light addressable potentiometric sensor (LAPS) was used to detect organophosphate and carbamate anticholinesterases (anti-ChEs), using eel acetylcholinesterase (AChE) as the biological sensing element.Biotinylated AChE was preincubated with inhibitor or buffer alone and then captured on biotinylated nitrocellulose membrane via streptavidin cross-linking, or AChE was preimmobilized on the capture membrane and then a sample containing the anti-ChE was filtered through the capture membrane. Hydrolysis of acetylcholine (ACh) by the captured AChE resulted in a strong potentiometric signal, and the immobilized AChE retained its affinity for ACh and anti-ChEs. IC50 values for inhibition of captured AChE obtained by the LAPS agreed with those obtained by a spectrophotometric method or a fiber optic evanescent fluorosensor. Paraoxon and bendiocarb were detected at 10 nM, while higher concentrations were required for monocrotophos, dicrotophos, dichlorvos, phosdrin, diazinon, tetraethyl pyrophosphate, aldicarb, and methomyl. Important features of the LAPS for detection of anti-ChEs are speed (eight samples assayed simultaneously in minutes), precision, and reusability. INTRODUCTIONDetection of anticholinesterases (anti-ChEs) is of major concern to the agriculture chemical industry, regulating agencies (e.g., FDA, EPA), health care professionals, and the Department of Defense. In addition to the physical and chemical assay methods [e.g., gas chromatography, high-pressure liquid chromatography (Conaway, 1991;Sharma et al., 1990), and immunoassay (Kaufman and Clower, 1991)], several methods using acetylcholinesterase (AChE) have been developed. These enzymatic assays include methods dependent on a change in pH, measured electrometrically (Michel, 1949) or titermetrically (Jacobsen et al., 1957), or on a change in color [e.g., Ellman etal. (1961)]. Other methods used to assay cholinesterase (ChE) activity radiometrically (Potter, 1967;Lewis and Eldefrawi, 1974) {ire too slow and use hazardous radioactive materials, while the histochemical method (Koelle and Friedenwald, 1949) is not applicable for screening or detection purposes in body fluids or environmental samples.The advent of biosensor technology has renewed interest in developing better detection methods for anti-ChEs using AChE as the sensing element. AChE enzyme electrodes were developed by immobilizing the enzyme protein on glass electrodes and were used to detect anti-ChEs (Baum and Ward, 1971;Durand et al., 1984). More sensitive fiber optic biosensors, using ChEs as their sensing elements, have also been developed to detect anti-ChEs (Rogers et al., 1991a;Hobel and Polster, 1992;Skladal and Mascini, 1992;Marty et al., 1992).Recently, a light addressable potentiometric sensor (LAPS) (Molecular Devices Corp.,

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon

“…The finding that paraoxon is a much more potent inhibitor of AChE than DFP (Figure 3 ) confirms previous reports (1,2,19). However, paraoxon causes less aging of the phosphorylated AChE than does DFP (Table 2, Figure 3).…”

“…Alternatively, one can use any number of the eight enzyme sites on the capture membranes for replication. Although the kinetic parameters (K, and V,,,) of the immobilized purified AChE are the same as those of the membrane-bound AChE (19), it is not certain whether the former retains all of its stereochemical characteristics.…”

Sensitive Analyte Detection and Quantitation Using the Threshold Immunoassay System

“…Using some of the binding schemes described above, binding parameters (absolute and relative) such as K a and have been derived for antibody/antigen, lectin/carbohydrate, and agonist/antagonist/receptor binding pairs (11)(12)(13)(14)(15)(16)(17). We have found that very accurate values can be derived that can be corroborated by literature values or by other physical techniques.…”

Fiber-Optic Immunosensors for Detection of Pesticides

“…We have utilized a fiber optic evanescent fluorosensor (Rogers et al, 1991) and a potentiometric sensor (Fernando et al, 1993) to detect acetylcholinesterase (AChE) inhibitor insecticides. The fiber optic biosensor has the advantage of no direct electrical connections, no drift problem, suitability for continuous monitoring and easier applicability for field use.…”