Abstract
An amperometric principle-based biosensor containing immobilized enzyme laccase from Trametes versicolor was developed for detection of ortho-substituted phenolic derivatives. Different immobilization methods for Trametes versicolor laccase enzyme on cellophane membrane and the enhancement of operational stability of the immobilized enzyme electrode using various protein-based stabilizing agents were studied. Among tested methods of immobilization, co-cross-linking method with bovine serum albumin was superior to the other methods in terms of sensitivity, limit of detection, response time, and operating and thermal stability. Biosensor response reached steady state within 3 min and exhibited maximum activity at 45 °C and pH 6.8. The sensitivity of the ortho-substituted phenols for the test biosensor developed with co-cross-linking method of immobilization using bovine serum albumin as the protein-based stabilizing agent was in the order: 2-aminophenol > guaiacol(2-methoxyphenol) > catechol(2-hydroxyphenol) > cresol(2-methyl phenol) > 2-chlorophenol. Validation of the newly developed biosensor by comparison with HPLC showed good agreement in the results. A newly developed biosensor was applied for quantification of ortho-substituted phenols in simulated effluent samples.
Highlights
Phenolic compounds belong to organic pollutants, which are widely distributed in the environment
The sensitivity of the ortho-substituted phenols for the test biosensor developed with cocross-linking method of immobilization using bovine serum albumin as the protein-based stabilizing agent was in the order: 2-aminophenol [ guaiacol(2-methoxyphenol) [ catechol(2-hydroxyphenol) [ cresol(2-methyl phenol) [ 2-chlorophenol
Sensitivity of the test biosensor developed with various methods of immobilization using different protein-based stabilizing agents (PBSAs) with catechol as model substrate was found to be in the order: co-cross-linking with Bovine serum albumin (BSA) [ co-cross-linking with lysozyme [ co-cross-linking with gelatin [ hybrid [ entrapment in gelatin [ cross-linking with glutaraldehyde (Figs. 9, 10)
Summary
Phenolic compounds belong to organic pollutants, which are widely distributed in the environment. The most widely used are gas chromatography (Padilla-Sanchez et al 2011), high-performance liquid chromatography (Alcudia-Leon et al 2011) and electrochemical methods (Jing et al 2011) These methods offer proper selectivity and detection limits, but are not suitable for rapid processing of multiple samples and real-time detection. Laccases reduce oxygen directly to water in a four-electron transfer step without intermediate formation of soluble hydrogen peroxide at the expense of one-electron oxidation of a variety of substrates, e.g. phenolic compounds (Haghighi et al 2003). As far as we are aware, this is the first extensive study on enhancement of operational stability of a Trametes versicolor laccase-based biosensor for the detection of ortho-substituted phenolic compounds using various methods of enzyme immobilization with different protein-based stabilizing agents (PBSAs)
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