Use of biosensors for rapid and sensitive detection of pesticides in food samples for food safety chemical risk assessment

Abstract

The utility of pesticides in the agricultural field is unquestionable, but at the same time pesticide use presents serious hazards to the environment and the human health. For that reason, detection of pesticides and their biotransformation products in food is of utmost importance. According to previous studies, esterase-based biosensors have been proposed as a viable and efficient solution for the detection of organophosphate pesticides. In this project, a double mutant of the thermostable esterase-2 (EST2) from Alicyclobacillus acidocaldarius was studied as a potential biosensor, for its ability to detect residual amounts of pesticides. Initial characterisation of the enzyme was performed, that included determination of optimal pH, thermophilicity, as well as kinetic analysis. Subsequently, the enzyme was studied by enzymatic activity assays with and without the presence of various organophosphate compounds. The effect of the organophosphates on the enzymatic activity was measured and complete inhibition of the enzyme was observed after incubation with paraoxon. These experiments were followed by an additional method involving labelling of the enzyme with a fluorescent probe. In this case, the effect of different pesticides on the EST2 enzyme was monitored by measuring the fluorescence quenching upon addition to the enzyme. Fourteen compounds were screened with this method and significant fluorescence quenching was observed in the presence of paraoxon and methyl-paraoxon when used in equimolar amounts with the enzyme in the range of nanomolar. This biosensor has been also used to test the presence of pesticides in real food samples, like fruits and juices. This research represents a starting point to develop effective fluorescence-based biosensors aiming at the screening of mutants with different pesticide selectivity profiles. The use of this enzyme-based biosensor can have applications in the field of food traceability as well as environmental monitoring, to control the presence of toxic chemicals, in particular organophosphate pesticides.

Keywords: EST2; biosensors; fluorescence; organophosphates; thermostable enzyme.

Figure 1

Crystal structure (pdb:1EVQ) of the thermophilic carboxylesterase EST2 from Alicyclobacillus acidocaldarius, with indication of the modified Cys35 residue where the fluorescence probe IAEDANS has been conjugated and the position where paraoxon interacts in the active site

Figure 2

Comparative specific activity (%) relative to control assay containing 4% of corresponding solvent. Equimolar amounts of enzyme and organophosphate were incubated for 5 min at RT. After incubation, 3 pmol of enzyme were transferred into the assay mixture and activity was measured at 405 nm

Figure 3

Real food samples used for testing model matrix by which pesticide can be extracted: (a) grapes; (b) cherries; (c) commercial juice. Paraoxon has been added and after extraction, the presence of pesticide has been detected by using the fluorescence‐labelled bioreceptor 2 m‐EST2