Development of a colourimetric pH assay for the quantification of pyrethroids based on glutathione-S-transferase

Recombinant glutathione-S-transferases (GSTs) can be used as analytical tools for the development of simple insecticide quantification assays. This assay explores the ability of pyrethroids to promote inhibition of the GST-catalysing 1-chloro-2,4-dinitrobenzene (CDNB)/glutathione (GSH) conjugation reaction. The sensing scheme is based on the pH change occurring in a weak buffer system by the GST reaction, which is measured using a spectrophotometer and the dye indicator bromothymol blue (616 nm). Practical use depends on the recognition affinity of the GST for insecticides, inhibition kinetics, enzyme stability and compatibility with the detection assay. In this study we compared the recombinant GSTs AgGSTD1-6 and AdGSTD1-1 from the mosquito vectors Anopheles gambiae and Anopheles dirus, respectively, with high affinity for pyrethroids, for their suitability for detecting pyrethroids in vector disease control programmes. The results showed that AgGSTD1-6 was the most suitable enzyme with the best structural stability at higher temperatures (Tm = 57 degrees C) and pH optima in the alkaline range (pH 7.7). Using the AgGSTD1-6, we subsequently developed a pH - change colourimetric assay for detecting pyrethroids. Linear calibration curves were obtained for deltamethrin (R2 = 0.99) with useful concentration ranges of 0-50 mu g mL-1. The effect of temperature in the range 25-40 degrees C on the pyrethroid quantification assay was negligible. The assay was validated with extracts from insecticide sprayed surfaces and found to be reproducible and reliable compared with the standard reverse-phase high performance liquid chromatography (Rp-HPLC) method. The potential of the assay for monitoring insecticide residues in the frame of insecticide based malaria control interventions is discussed.