Development of acetylcholinesterase biosensor for the detection of pesticides

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2010
Kavruk, Murat
Pesticides are natural or artificial molecules aimed to kill, or mitigate any harmful organism. Although their use in agriculture provides us with an increased crop yield, remains of chemicals on the products creates health concerns in society. Organophosphates and carbamates are two groups of insecticides. Although they are far more lethal against insects and small animals, they can also cause poisoning in humans through the inhibition of acetylcholinesterase enzyme (AChE) that plays an important role in human nervous system. Therefore, the detection of these compounds is crucial. The conventional methods for the detection of these compounds are expensive, time-consuming and need expertise. In this study, a fast, disposable, cheap and accurate acetylcholinesterase biosensor was developed to detect organophosphate and carbamate-based pesticide residues. By means of adsorption method, AChE, the chromophore 5,5'-Dithio-bis(2-nitrobenzoic acid) (DTNB) and artificial substrate acetylthiocholine (ATCh) were immobilized on the supporting material. In optimization studies; from 3 to 15U/mL concentrations were experimented for AChE, 1 to 5mM DTNB and 1 to 5mM ATCh concentration gradients were used. v As a result of the optimization studies 12U/mL ACHE concentration, 5mM DTNB concentration and 5mM ATCh concentration were determined for constructing a pesticide biosensor. Detection limit of malathion, an organophosphate-based insecticide was found as 2.5ppm in 5% methanol solution. The biosensor conserved its integrity between pH 4 and 8, and gave false positive results after pH 10. Stability studies showed that, biosensor retained its activity for at least 60 days at 4°C to discrimnate between positive and negative controls.