Abstract
Many alternative strategies to immobilize and stabilize enzymes have been investigated in recent years for applications in biosensors. The entrapment of enzymes within silica‐based nanospheres formed through silicification reactions provides high loading capacities for enzyme immobilization, resulting in high volumetric activity and enhanced mechanical stability. Here we report a strategy for chemically associating silica nanospheres containing entrapped enzyme to a silicon support. β‐galactosidase from E. coli was used as a model enzyme due to its versatility as a biosensor for lactose. The immobilization strategy resulted in a three‐dimensional network of silica attached directly at the silicon surface, providing a significant increase in surface area and a corresponding 3.5‐fold increase in enzyme loading compared to enzyme attached directly at the surface. The maximum activity recovered for a silicon square sample of 0.5 × 0.5 cm was 0.045 IU using the direct attachment of the enzyme through glutaraldehyde and 0.16 IU when using silica nanospheres. The immobilized β‐galactosidase prepared by silica deposition was stable and retained more than 80% of its initial activity after 10 days at 24°C. The ability to generate three‐dimensional structures with enhanced loading capacity for biosensing molecules offers the potential to substantially amplify biosensor sensitivity. Biotechnol. Bioeng. 2008;99: 261–267. © 2007 Wiley Periodicals, Inc.