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Enzyme immobilization on titania-silica-gold thin films for biosensor applications and photocatalytic enzyme removal for surface patterning

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2009
Çınar, Merve
The aim of this study was to investigate the viability of patterning by immobilization, photocatalytic removal, and re-immobilization steps of the enzyme on photocatalytically active thin films for biosensor fabrication purposes. For this aim, TiO2-SiO2-Au sol-gel colloids were synthesized and deposited on glass substrates as thin films by dip coating. Cysteamine linker was assembled on gold nanoparticles to functionalize thin films with amine groups for immobilization of model enzyme invertase. Effect of immobilization temperature, enzyme concentration of the immobilization solution and immobilization period on invertase immobilization were investigated. The immobilized invertase activity was found independent from the immobilization temperature in the range tested (4oC-room temperature). The optimum enzyme concentration and period for immobilization was determined as 10µg/ml and 12 hours respectively. The resulting invertase immobilized thin films showed high storage stability retaining more that 50% of their initial activity after 9 weeks of storage. Photocatalytic enzyme removal and re-immobilization studies were carried out by irradiating the invertase immobilized thin films with blacklight. Upon 30 minutes of irradiation, immobilized invertase was completely and irreversibly inactivated. Initial immobilized invertase activity (before the irradiation) was attained when invertase was re-immobilized on thin films that were irradiated for 5 hours. Thus it was inferred that with sufficient exposure, enzymes can be completely removed from the surfaces which makes the re-immobilization possible. The possibility of enzyme removal with photocatalytic activity and re-immobilization can pave the way to new patterning techniques to produce multi-enzyme electrode arrays.