Application of Fluorescence Techniques to Characterise the Preparation of Protein-Containing Sol-Gel Derived Hosts for use as Catalytic Media

Abstract

In this work we collate and review the usage that we have made of fluorescence techniques employed to follow the sol to gel transition and aging in different tetraethylorthosilicate based materials. The sol-gel method allows porous glasslike of good optical quality to be produced at relatively low (ambient) temperatures, which facilitates the incorporation of a range of molecules; from laser dyes to biomolecules. Here the use of “common” viscosity (DASPMI) and polarity (Nile red) sensitive fluorescence probes to monitor the host manufacture is made. Nile red was also used to label two catalytically active proteins (cytochrome c and subtilisin Carlsberg). These were incorporated into the different host media and the dye used to ascertain changes in protein conformation, both upon incorporation and at the end of an aging period. Complementary measurements of catalytic activity were performed. The probe emission was monitored via steady state and time-resolved fluorescence techniques and comparison made with the catalytic activity measurements to elucidate the amount of accessible and active protein. Overall it was found that the hosts became stable after an aging period approaching 20 days and that the major influence on the catalytic reaction rates was that of host mediated mass transport.