Enzymes are used in a wide range of industries for various different chemical processes. Optimizing the performance of enzymes remains an area of high interest in many research labs. An enzyme is often immobilized on or within a support structure, which allows for the biocatalyst to be relatively easy to recover post-reaction. Immobilization can also increase the structural stability of the enzyme, which is beneficial from a cost standpoint because pure enzymes can be expensive. By choosing the appropriate support and immobilization chemistry, it is possible to maximize the efficiency of a biocatalyst. The purpose of this work was to design, fabricate and utilize inverse opal structures as a support for the immobilization of enzymes. We have developed a flow-through silica inverse opal structure that was used for the immobilization of biocatalysts. The inverse opal structures were created using polystyrene nanospheres as a template, sol-gel chemistry to deposit silica in the interstitial spaces between the nanospheres, and solvent dissolution to remove the template. Scanning electron microscopy and dynamic light scattering were used to characterize the nanospheres and structures. The silica inverse opal structure has a relatively high surface area, and a surface that is amenable to a wide range of surface modification reactions. Two enzymes were chosen to evaluate our catalyst support structure; glucose oxidase and alkaline phosphatase. Absorbance and fluorescence measurements were used for the enzyme assays. Our results show an enhancement in reactivity that is associated with enzyme immobilization and nano-confinement, and also underscore limitations inherent to this approach. Three different reaction formats were examined: solution phase, immobilized enzymes on planar supports, and enzymes immobilized on the flow-through inverse opal structure. Glucose oxidase exhibited an increase in reactivity when comparing planar vs. solution phase and inverse opal vs. planar structural formats. This finding indicated an enhancement due to the immobilization process and due to the nanoconfinement of the enzyme within the inverse opal structure. In contrast, alkaline phosphatase exhibited a reduced activity when comparing solution phase vs. enzyme immobilized on planar and inverse opal structures. This finding illustrated the importance of identifying immobilization chemistry that maintains the enzyme in an active form and binds the enzyme in a way that leaves the reactive site accessible. An enhancement was observed for the inverse opal structure vs. the planar support, indicating that there remains the positive effect associated with nano-confinement of the enzyme. This project proved to be enlightening by showing the enhancements in activity for glucose oxidase, and also by showing that there are limitations that need to be addressed in the alkaline phosphatase results. There will be continued work to further characterize and optimize the flow-through inverse opal structures. In addition, it may be useful to examine the use of other materials for the inverse opal support itself. The results of this work are promising for the utilization of inverse opal structures to immobilize and optimize the performance of enzymes.
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