RESUMEN
Glyphosate [N-(phosphonomethyl)glycine] is the most frequently used herbicide to date. Due to its indiscriminate use, it has become a globally occurring pollutant of surface waters. A biosensor for glyphosate is described here that consists of a carbon nano-onion/tyrosinase conjugate immobilized in a chitosan matrix on a screen-printed electrode. The analytical principle is based on the inhibition of the enzyme tyrosinase by glyphosate. L-DOPA is used as the enzyme substrate. The presence of the carbon nano-onions has a beneficial effect on the sensitivity of the assay. Glyphosate can be amperometrically quantified in the 0.015 to 10 µM concentration range and with a 6.5 nM (1.1 µg L-1) detection limit. The biosensor is stable more than 2 months at 4 °C. It was applied to the detection of glyphosate in water and soil samples taken from irrigation of a rice field after aerial application. Results were in good agreement with data obtained by a commercial ELISA. Graphical abstract A highly sensitive amperometric biosensor for glyphosate is reported, based on the covalent immobilization of a carbon nano-onion/tyrosinase conjugate on a chitosan matrix.
Asunto(s)
Técnicas Biosensibles/métodos , Carbono/química , Glicina/análogos & derivados , Monofenol Monooxigenasa/antagonistas & inhibidores , Quitosano , Electrodos , Enzimas Inmovilizadas/antagonistas & inhibidores , Glicina/análisis , Herbicidas/análisis , Límite de Detección , Monofenol Monooxigenasa/química , Nanoestructuras , Contaminantes Químicos del Agua/análisis , GlifosatoRESUMEN
The inhibitory effect of 2,4,5-T, 2,4-D, glyphosate and paraquat on the diphenolase activity of mushroom tyrosinase for oxidation of L-DOPA has been investigated by kinetic measurements, fluorescence spectroscopy and computational docking analysis. 2,4,5-T and 2,4-D inhibit the diphenolase activity of the enzyme following a competitive mechanism, while glyphosate is a mixed inhibitor according to Lineweaver-Burk kinetic analysis. The inhibitory activity follows the order glyphosate >2,4,5-Tâ¯>â¯2,4-D with IC50 values of 65, 90 and 106⯵M, respectively. Intrinsic tyrosinase fluorescence quenching and computational docking analysis suggest that 2,4,5-T and 2,4-D interact with the active site of the enzyme through hydrophobic interactions, while glyphosate also interacts with external residues of the active site of the enzyme by hydrogen bonding and hydrophilic interactions inducing conformational changes in the protein structure.
Asunto(s)
Catecol Oxidasa/química , Monofenol Monooxigenasa/química , Plaguicidas/farmacología , Ácido 2,4,5-Triclorofenoxiacético/farmacología , Ácido 2,4-Diclorofenoxiacético/farmacología , Agaricales/enzimología , Dominio Catalítico/efectos de los fármacos , Catecol Oxidasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Cinética , Simulación del Acoplamiento Molecular , Monofenol Monooxigenasa/antagonistas & inhibidores , Oxidación-Reducción/efectos de los fármacos , Análisis Espectral , GlifosatoRESUMEN
Carbon nanomaterials have emerged as suitable supports for enzyme immobilization and stabilization due to their inherently large surface area, high electrical conductivity, chemical stability, and mechanical strength. In this paper, carbon nano-onions (CNOs) were used as supports to immobilize alkaline phosphatase, horseradish peroxidase, and glucose oxidase. CNOs were first functionalized by oxidation to generate carboxylic groups on the surface followed by the covalent linking of using a soluble carbodiimide as coupling agent. The CNO-enzyme conjugates were characterized by transmission electron microscopy and Raman spectroscopy. Thermogravimetric analysis revealed a specific enzyme load of â¼0.5 mg of protein per milligram of CNO. The immobilized enzymes showed enhanced storage stability without altering the optimum pH and temperatures. These properties make the prepared nanobiocatalyst of potential interest in biosensing and other biotechnological applications.