The present study aims at fabricating a laccase (LAC) based amperometric biosensor for detection of phenolic compounds. LAC was immobilized into the porous matrix of polyaniline nanofibers (PANFs) in a three-step process, consisting of enzyme adsorption, precipitation, and crosslinking (EAPC). Immobilized LAC on PANF in the form of EAPC was highly active and stable when compared to control samples of 'enzyme adsorption (EA)' and 'enzyme adsorption and crosslinking (EAC)' samples. For example, the activity of EAPC was 19.7 and 15.1 times higher than those of EA and EAC per unit weight of PANF, respectively. After 6days at room temperature, EAPC maintained 100% of its initial activity, while EA and EAC retained only 7.7% and 11% of their initial activities, respectively. When the samples were subjected to the heat treatment at 60°C over 3h, EAPC maintained 74% of its initial activity, while EA and EAC retained around 1% of their initial activities, respectively. To demonstrate the feasible application of EAPC in biosensors, the enzyme electrodes were prepared and used for detection of phenolic compounds, which are environmentally hazardous chemicals. The sensitivities of biosensors with EA, EAC, and EAPC were 20.3±5.9, 26.6±5.4 and 518±11μAmM(-1)cm(-2), respectively. At 50°C for 5h, EAPC electrode maintained 80% of its initial sensitivity, while EA and EAC electrode showed 0% and 19% of their initial sensitivities, respectively. Thus, LAC-based biosensor using EAPC protocol with PANFs showed a great promise for developing a highly sensitive and stable biosensor for detection of phenolic compounds.
Keywords: Crosslinking; Enzymatic phenol sensors; Enzyme adsorption; Laccase; Polyaniline nanofibers; Precipitation.
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