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Preparation and characterization of titania-silica-gold thin films over ito substrates for laccase immobilization

Eker, Zeynep
The aim of this study was to immobilize the redox enzyme laccase over TiO2-SiO2-Au thin film coated ITO glass substrates in order to prepare electrochemically active surfaces for biosensor applications. Colloidal TiO2-SiO2-Au solution was synthesized by sol-gel route and thin film was deposited onto the substrates by dipcoating method. The cysteamine was utilized as a linker for immobilization of enzyme covalently through gold active sites. Preliminary studies were conducted by using invertase as model enzyme and Pyrex glasses as substrates. The effect of immobilization parameters such as immobilization temperature, concentration of enzyme deposition solution, immobilization time for laccase were examined. Leakage studies were conducted and storage stability of immobilized laccase was determined. Highest laccase activity was achieved when immobilization was performed with 50 µg/ml solution at 4°C for 2 hours. Laccase activity decreased after 4 hours of impregnation in enzyme solution. Laccase leakage was observed in the first usage of substrates and 55% activity decrease was determined in the subsequent use which might be attributed to the presence of uncovalently adsorbed enzyme on the fresh samples. In air and in buffer storage stabilities were also tested. It was found that the activity of samples almost vanished after 6 days regardless of storage conditions. Both enzymes had more activity on ITO substrate.