Determination of anticholinesterase pesticides in real samples using a disposable biosensor

A choline amperometric biosensor based on screen-printed electrodes was assembled and used to assess the inhibitory effect of organophosphorus and carbamic pesticides on acetylcholinesterase activity both in standard solutions and real samples. Acetylcholinesterase catalyses the cleavage of acetylcholine to choline and acetate, therefore the amount of choline measured using the biosensor is directly related to the enzyme activity. The extent of enzyme inhibition can be used as an index of the amount of anticholinesterase pesticides present. The hydrophobicity of organophosphorus and carbamic pesticides led to the evaluation of organic, water miscible solvents for use in the proposed method. Borate buffer containing 1% v/v acetonitrile was selected since it exhibited the least influence on enzyme activity from the tested solvents (acetonitrile, acetone, tetrahydrofuran and ethylacetate). Other solvents (dimethylsulfoxide and methanol) were avoided as they exhibited electrochemical interferences. An inhibition calibration curve was obtained using carbofuran, a strong inhibitor of acetylcholinesterase. The lowest detectable standard solution was 2 microg/L following an incubation time of 10 min. The method was then applied to real samples (fruits and vegetables) showing its suitability as a rapid screening assay (12 min per test) for the assessment of anticholinesterase pesticides. The biosensor results were compared with a standard analytical technique (gas chromatography with nitrogen phosphorus detector, GC-NPD).