RESUMEN
Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alternative to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl beta-D-galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the standard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.
Asunto(s)
Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Galactósidos/química , Pruebas de Mutagenicidad/métodos , Mutágenos/toxicidad , 4-Nitroquinolina-1-Óxido/análisis , 4-Nitroquinolina-1-Óxido/toxicidad , Electrodos , Monitoreo del Ambiente , Genes Reporteros , Quinolinas/análisis , Quinolinas/toxicidad , Respuesta SOS en Genética/efectos de los fármacos , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/enzimología , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The purpose of this study was to investigate the long-term stabilization of the liver S9-fraction that is widely used in genotoxicity assays in order to mimic bio-activating processes of xenobiotics in vitro. A successful long-term stabilization of the S9-fraction meets the growing demand for the construction of a lab independent device for the detection of genotoxic compounds in field studies with an integrated module for the metabolic activation of pre-genotoxic compounds. The carbohydrates sucrose, trehalose and raffinose were tested in different concentrations or mixtures in order to increase the product stability of the S9-fraction during and after freeze-drying. The activity of the freeze-dried S9-samples was evaluated by means of their potential to activate pre-genotoxic compounds. The successful long-term stabilization of enzymes of the rodent liver S9-fraction for 6 weeks at room temperature by freeze-drying in the presence of 250 mM trehalose is presented.