2.?Methodology2.1. ReagentsTyrosinase (EC 1.14.18.1, 2870 U/mg solid from mushroom), monomer 2-hyroxyethyl methacrylate (HEMA) and n-butyl acrylate (nBA) were purchased from Sigma. The photoinitiator 2, 2-dimethoxy-2-phenylacetophenone (DMPP), phenol and gold nanoparticles (50-130 nm) were obtained from Aldrich. The supporting electrolyte was 0.05 M phosphate buffer, prepared from potassium dihydrogen phosphate and potassium chloride that were purchased from Systerm and Merck respectively.2.2. Apparatus and measurementsAmperometric experiments were carried out in a stirred electrochemical cell containing 5 ml of 0.05 M phosphate buffer/0.1 M KCl using an Autolab PGSTAT 12 Potentiostat.

The working electrode was a carbon paste screen-printed electrode coated with tyrosinase containing photocured methacrylic-acrylic copolymer film.

Ag/AgCl (3 M KCl) (Orion) and a glassy carbon electrode (Methrom) were used as reference electrode and counter electrode respectively. The amperometric measurements for phenol were performed at -0.10 V versus Ag/AgCl reference for electrode based on photoHEMA membrane while -0.15 V for electrodes based on photoHB91 and photoHB82 membranes.2.3. Preparation of phenol biosensorBiosensor for phenol was fabricated based on the immobilization of tyrosinase in various types of methacrylic-acrylic membranes with varied hydrophilicity. For the most hydrophilic membrane, 100% of HEMA monomer was used.

For less hydrophlilic membranes, 90% of photoHEMA and 10% of nBA monomers (w/w) (photoHB91) or 80% of HEMA and 20% of nBA monomers (w/w) (photoHB82) were prepared.

For photocuring purpose, 1.6% (w/w) of photoinitiator DMPP was added to the monomer mixtures. The enzyme tyrosinase with a concentration of 18.5 mg/mL (53.1 U/mL) was prepared in 0.05 M phosphate buffer/ 0.1 M KCl at pH 7.0. The final membrane cocktail was obtained by mixing 10��l of the monomer mixture and 10��l of tyrosinase enzyme solution. Gold nanoparticles (50-130 nm) in the following amount: 5.0 mg, 1.0 mg and 0.1 mg were added to the membrane cocktails before photocuring. The photocuring of the final cocktails was performed under ultra-violet (UV) radiation in a UV box (RS Ltd.) containing four 60 watt UV lamps.

The irradiation Anacetrapib was carried out for 5 min under a nitrogen Cilengitide atmosphere. A rigid and thin (100 ��m) polymer film coated on the screen printed electrode was obtained after exposure to the UV light.The investigation of the water absorption characteristics of various membranes was carried out by exposing membranes to 0.05 M phosphate buffer. The changes in weight of the membranes were then recorded every five min and the percentage of water content and absorption were later calculated.2.4.