Development of a laccase based paper biosensor for the detection of phenolic compounds

Laccase (E.C.1.10.3.2, p-benzenediol: oxygen oxidoreductase) catalyzes the oxidation of various aromatic compounds, particularly phenols, which are organic pollutants, present in wastewater. With this specific function this enzyme has had a great impact on the development of biosensors for both environmentally important pollutants and clinically relevant metabolites. There are different detection procedures such as chromatographic and spectrophotometric methods to detect the presence of phenolic compounds in wastewater. However, they are time-consuming processes and require specific instruments and qualified staff. The development of a low-cost, portable and disposable biosensor by the immobilization of laccase in Whatman paper has been described. The enzyme is loaded with colouring agent MBTH that allows detection of oxidation products of phenols by forming maroon-green colours. The optimum enzyme concentration loaded onto papers forming lowest background colour and producing highest colour intensity was determined to be 2 mg/ml while the optimum MBTH concentration was 24 mM. The biosensor was responsive to L-DOPA and catechol, with the lowest detection limits of 64 mu M. The colour intensities were found to be consistent with substrate concentration and the results were reproducible.