Abstract

Pesticides are identified as hazardous contaminants due to high toxicity and persistence in the environment. In this context, it is necessary to monitor these pollutants in the environment in situ, with the use of low cost analytical techniques and of quick response. Biosensors are presented as a complementary analytical tool to more complex techniques, such as chromatography. The present work aims to develop, in a simple and low cost way, an amperometric biosensor for the detection of simazine in aqueous samples from the inhibition of the enzyme peroxidase extracted from the pinto bean (Phaseolus vulgaris L.). For the development of the biosensor, the enzyme peroxidase was extracted in sodium phosphate buffer solution, of pH 6.0, and pre-purified by ammonium sulfate precipitation 40% (w / v) salt saturation to 50.0 ml of the protein extract. Peroxidase was characterized by electrophoresis and immobilized on silica-titanium oxide, synthesized by the sol-gel method in the proportions of 70% / 30% respectively. The biosensor showed linearity in the concentration range between 1.0 and 6.0 μgL-1 of simazine (R2 = 0.989) in the potential range 0.20 V to 0.30 V vs Ag / AgCl by the square wave voltammetry technique. The curve was obtained in the presence of 2.0 mgL-1 phenol and 3% H2O2 (v / v)), where the current density signal decreased in the presence of successive additions of the simazine inhibitor. The biosensor was applied to a standard simazine-fortified sample and detected the presence of simazine at the concentration of 35.05 μgL-1. Even showing a relative percentage error of 12.63%, when compared with the result of the analysis by high performance liquid chromatography, the biosensor is useful to monitor the presence or absence of the simazine pesticide in the solution. Key words: Simazine, Biosensor, Carioca beans (Phaseolus vulgaris L.).

Highlights

  • Simazine (2,4-Bis(ethylamino)-6-chloro-1,3,5-triazine) is a selective herbicide for weeds grouped into the triazine130 Afr

  • The peroxidase extracted from carioca beans (P. vulgaris L.) showed total activity of 2500 U/ml, with a protein content of 87.37 mg/ml, which characterizes a specific activity of 66.02 U.mg-1

  • The molecular mass of the commercial peroxidase is presented in literature as around 40 kDa (Sigma Aldrich 2017), while the peroxidase extracted from the bean (P. vulgaris L.) was identified at approximately 41.68 kDa

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Summary

Introduction

Simazine (2,4-Bis(ethylamino)-6-chloro-1,3,5-triazine) is a selective herbicide for weeds grouped into the triazine130 Afr. One of the proposition of this work is to assemble an enzymatic biosensor from the peroxidase enzyme extracted from the Carioca Bean (Phaseolus vulgaris L.) grains This legume is a rich, lowcost protein peroxidase rich source that can be extracted and applied to detect herbicides such as simazine. In order to the bio-reaction to occur, the active peroxidase site must be available and free of deformations, so that a molecule of hydrogen peroxide (natural substrate) oxidizes the ferroprotoporphyrin (Fe+3) group and produces the Fe+4/Fe+5 highly unstable intermediates (Pérez Galende et al, 2015; Aisha et al, 2016; Jang and Moon, 2011) In this step the phenol in solution can act as an electron mediator, recovering the initial oxidation state (Fe+3 from the native enzyme) and repeating the cycle. The reduction current of the quinones, product obtained from phenol oxidation, can be evaluated at the solution-electrode interface (Morales et al, 1996)

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