Immobilized enzyme modulator microassay (IEMMA) for the detection of pesticide in fresh produce

A simple and novel quantitative method for the analysis of enzyme modulators in a microtiter format is described. One sample is analyzed in each well, which has its baseline level of enzyme activity predetermined. The modulator can be monitored in a fluorometric, photometric or luminescence microtiter plate reader. Analysis relies on the slope of the enzyme kinetic reaction. Thus the assay is independent of the starting time of the reaction and the exact volume of the substrate added in each well. The advantage is that multiple samples can be manipulated comfortably, while reproducibility remains high, with a standard deviation of less than 7% of the measured activity. For the detection of pesticides in fresh produce, up to 96 vegetable or fruit samples can be analyzed simultaneously. The method utilises acetycholinesterase immobilized onto microtitre wells. Vegetables such as choi sam, lettuce and tomato have been tested, showing less than 6% inhibitory effect on the immobilized AChE. The enzyme has been found to be extremely sensitive to carbofuran, detecting <0.1 mg/kg in all vegetable tested. Sensitive to methamidophos, an organophosphate insecticide extensively used in S E Asia is <3 mg/kg. This method can be used to complement existing analytical methods.