Desenvolvimento de um biossensor amperométrico baseado na enzima glutationa s-transferase para detecção de agrotóxicos piretroides em amostras ambientais

Abstract

ABSTRACT The use of pesticides has generated several disorders and environmental problems. Considering the need to monitor these chemicals in nature, this work aims to develop an amperometric biosensor for the detection of pyrethroid insecticides in environmental samples. In this sense, in silico analyzes were carried out on the potential for contamination of surface and groundwater and molecular docking of the enzyme glutathione S-transferase (GST) with nine pyrethroid compounds. In vitro analyzes were also performed, such as the determination of enzymatic activity through spectrophotometric analysis and electrochemical GST inhibition assays against pyrethroid insecticides. On that occasion, screen-printed biosensors based on GST were prepared with working electrode modified with the mediators Meldola blue with Reinecke's salt - MBRS and cobalt phthalocyanine - CoPC. For electrochemical analysis and characterization of these devices, cyclic voltammetry techniques with a potential range of -0.8 V to 0.8 V and a sweep rate of 50 mV s-1 and chronoamperometry with a fixed potential of 200 mV s-1 were used. The results obtained indicated medium potential for transport of insecticides in association with sediment and low potential for transport of these substances dissolved in water, for most of the insecticides under study. All compounds tested during docking showed negative binding energy with moldlock scores ranging from -75 to -91, indicating affinity with the catalytic site of the enzyme. In the spectrophotometric analysis, values from 0.12 µmol.L-1 to 20 µmol.L-1 were found for the inhibition constant - Ki, for the compounds fenpropathrin and deltamethrin, respectively. For the detection limits-LDs, values from 0.03 mg.L-1 to 0.36 mg.L-1 were found for deltamethrin and cypermethrin, respectively. The electrochemical analyzes were performed using a biosensor modified with MBRS, which presented better electrochemical performance in relation to the CoPC mediator. With this device, LD values from 3.72 µg.L-1 to 83.54 µg.L-1 were recorded, respectively for lambda-cyhalothrin (linear range from 1 to 50 µg.L-1, R2 = 0.99) and cypermethrin (linear range from 8 to 200 µg.L-1, R2 = 0.98). With the results obtained, it is considered that the elaborated biosensor proved to be effective for application in the analysis of samples of agricultural products and water analysis. It also presented relevant characteristics, such as adequate sensitivity, low cost for its development, simplified construction and handling process, good reproducibility and stability over time of analysis. It should be noted that the association of in silico and in vitro methodologies applied here constituted an important strategy for carrying out this research, considering that the prototype developed can help in the monitoring and environmental control of pyrethroid agrochemicals. It is also noteworthy that the present study generated publications of initial data in scientific journals and other articles were submitted to national and international journals and are awaiting an opinion. An innovation patent was generated as a product of this Thesis and deposited at the National Institute of Industrial Property – INPI, even before the date of defense of this study.