Immobilization of tyrosinase in chitosan film for an optical detection of phenol

An optical biosensor based on the immobilized tyrosinase enzyme in a chitosan film is described for the detection of phenol. The film was prepared by depositing a chitosan solution containing tyrosinase onto a microscope glass slide via spin coating technique. The quinone produced from the enzymatic oxidation of phenol was reacted with 3-methyl-2-benzothiazolinone hydrazone (MBTH) to produce a maroon color adduct. The color intensity of the adduct was found to increase proportionally with the increase of the substrate concentrations after 15 min exposure to the substrate. The optimum enzyme loading and chitosan concentrations were found to be 0.9 mg and 2% (w/v), respectively. The biosensor also demonstrated optimum activity at pH 6–7, optimum temperature of 45 °C and a linear response in the phenol concentration range of 2.5–70.0 μM. The response characteristics of the biosensor towards different phenolic compounds were determined. It was found that the biosensor gave a linear response in the concentration range of 2.5–50.0 μM for 4-chlorophenol, 2.5–100.0 μM for m-cresol and 12.5–400.0 μM for p-cresol. The limit of detection and the apparent Michaels–Menten constant (Km) of the biosensor were 0.9, 1.0, 1.0, 3.0 μM and 0.038, 0.037, 0.056, 0.201 mM for 4-chlorophenol, phenol, m-cresol and p-cresol, respectively. It showed good stability for at least 2 months.