Abstract
An amperometric biosensor was designed for the determination of sulcotrione, a β-triketone herbicide, based on inhibition of hydroxyphenylpyruvate dioxygenase (HPPD), an enzyme allowing the oxidation of hydroxyphenylpyruvate (HPP) in homogentisic acid (HGA). HPPD was produced by cloning the hppd gene from Arabidopsis thaliana in E. coli, followed by overexpression and purification by nickel-histidine affinity. The electrochemical detection of HPPD activity was based on the electrochemical oxidation of HGA at +0.1V vs. Ag/AgCl, using a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate-modified screen-printed electrode. Assays were performed at 25°C in 0.1M phosphate buffer pH 8 containing 0.1M KCl. The purified HPPD was shown to display a maximum velocity of 0.51µMHGAmin−1, and an apparent KM of 22.6µM for HPP. HPPD inhibition assays in presence of sulcotrione confirmed a competitive inhibition of HPPD, the calculated inhibition constant KI was 1.11.10−8M. The dynamic range for sulcotrione extended from 5.10−10M to 5.10−6M and the limit of detection (LOD), estimated as the concentration inducing 20% of inhibition, was 1.4.10−10M.
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