Monitoring the Distribution of Covalently Tethered Sugar Moieties in Sol−Gel-Based Silica Monoliths with Fluorescence Anisotropy: Implications for Entrapped Enzyme Activity

Biocompatible silica derived from diglycerylsilane (DGS) and the sugar modified silane N-(3-triethoxysilylpropyl)gluconamide (GLS) has been shown to have great utility for the entrapment of a number of delicate proteins. To further understand the nature of the DGS/GLS composite material, it is important to characterize the local microenvironment and properties of the silica surface within the DGS/GLS monoliths. In this work, we have monitored both the steady-state and time-resolved anisotropy of the fluorescent probe rhodamine 6G (R6G) to determine the distribution of GLS within DGS/GLS-derived materials where varying levels of GLS were added either to the sol or to previously formed DGS-based gels. The data suggest that the addition of GLS to an evolving sol results in preferential coating of silica nanoparticles, which remain present in the gelled material and slowly associate with the continuous polymer network. The preferential presence of GLS on the surface of free particles results in inefficient coating of the monolithic material with the sugarsilane and causes most of the added R6G to associate with the silica skeleton. On the other hand, the addition of GLS to a preformed monolith results in preferential modification of the monolithic silica surface and a much increased level of free dye. A significant amount of R6G-bound nanoparticles remain within these materials over an extended time, suggesting that the nanoparticles do not associate with the GLS-modified silica monolith. The activity of the enzyme horseradish peroxidase was evaluated in DGS derived materials that had GLS added to either the sol or the monolith, and it was determined that optimal activity was obtained in cases where GLS was added to the sol. These results highlight the importance of controlling the time of addition of organosilane additives to evolving sols and suggest that this factor might provide a means to control the properties of the resulting nanocomposite material.