Development of carbendazim and carbaryl detection method using immobilized cyclodextrin on microtiter plates

Abstract

A method was developed for the immobilization of beta-CD on polystyrene microtiter plate for the determination of benzimidazole fungicides and carbaryl insecticide based on competitive inhibition ELISA. The microtiter plate wells were coated with methyl vinyl ether-maleic anhydride copolymer (MMAC). Amino groups were introduced to the MMAC-coated plate by the addition of 1,6-hexamethylenediamine (HMDA). The beta -CD was subsequently immobilized by the use of divinyl sulfone as a cross-linking reagent. The result indicated that beta -CD was successfully immobilized on the prepared amino plate and was still able to entrap benzimidazole and carbaryl tracers, 2-succiamidobenzimidazole or 1-(5-carboxypentyl)-3-(1-naphthyl) urea conjugated to horseradish peroxidase, respectively. The immobilized beta -CD was first used for the determination of benzimidazole fungicide by competitive encapsulation with benzimidazole tracer. The optimum conditions of the assaywere determined. The benzimidazole tracer concentration at 2.5 microgram/ml was used for the competitive encapsulation into 4% (w/v) beta -CD and the loading buffer pH 2.5 was used for carbendazim encapsulation. 0.1M acetate/citric acid buffer, pH 5 containing 0.5 M NaCl was used as washing buffer. The typical standard curve of carbendazim had an IC[subscript 50] value and the detection limit of 1.56 and 0.2 microgram/ml, respectively. The competitive encapsulation approach was also applied to the determination of carbaryl. The assay condition was similar to that of carbendazim except for the pH loading buffer used for carbaryl encapsulation by beta -CD. The carbaryl standard was incubated at pH 7.4 with immobilized CD-microtiter plate and the carbaryl tracer concentration at 2.5 microgram/ml was used for competitive encapsulation. An IC[subscript 50] value obtained from the typical standard curve of carbaryl was 0.4 microgram/ml and the limit of detection was 0.15 microgram/ml. The method was employed in the detection of carbendazim and carbaryl in grape spiked samples. It was found that competitive encapsulation was affected by sample matrix, thus the standard curves in the presence of matrix were performed and they were recommended to be used in place of the typical standard curve. The overall results clearly demonstrate the flexibility of the method over ELISA for the determination of related compounds under specific conditions