The encapsulation of proteins and other biomolecules in sol-gel-derived glasses is an intriguing research area because it involves placing delicate biological molecules with well-defined structures in the pores of a matrix that is, by comparison, hard and disordered. The encapsulation of biomolecules in sol-
gel-derived glasses has been ongoing for more than a decade, with the first research in this area published in 1990 (7). The first studies of biodoped sol-gel materials reported alkaline phosphatase encapsulated in base-catalyzed silica, and the resulting sol-gel material was opaque with low enzymatic activity. In subsequent work, a sol-gel synthesis route that was able to produce optically transparent monoliths was developed (8). The proteins copper-zinc superoxide dismutase, cytochrome-c (cyt-c), and myoglobin were immobilized in the pores of transparent silica glasses, and the biological function and activity of these proteins were monitored spectroscopically. Since that time, a variety of biomolecules, including antibodies, enzymes, other proteins, and even bacteria, have been successfully immobilized in sol-gel matrices, many of which were optically transparent. Research results have been described and summarized in a number of published reviews (9-12).
One of the most important aspects of using the sol-gel method to encapsulate biomolecules is that the method can be tailored so as not to denature the proteins. That is, the sol-gel-encapsulated biomolecules retain their characteristic reactivities and spectroscopic properties. Some important factors that need to be considered include pH, pore size, and the presence of alcohol. A synthesis protocol that has been successfully used to fabricate optically transparent and biologically active silica gels is illustrated in Fig. 4. The protocol consists of (1) mixing alkoxide precursor(s) with water in the presence of dilute acid (catalyst); (2) sonicating the mixture to fully hydrolyze the alkoxide, usually in the absence of alcohol; (3) adding a pH-buffered solution containing the biomolecule of interest; and (4) casting the solution into the desired geometry and shape (monolith or thin film). In this protocol, alcohol concentration is minimized, and the pH of the biomolecule-doped sol is raised to a level that ensures the viability of the biomolecule.
Using the synthesis protocol described in Fig. 4, gelation occurs typically between 2 and 30 min. The gelation time depends on a variety of factors, including ionic strength of the buffer, concentration of biomolecules, addition of alcohol (or lack thereof), and temperature. To lengthen the gelation time, one can reduce the buffer ionic strength, reduce the biomolecular concentration, add alcohol, and/or lower the temperature. Another means to lengthening the gelation time is to include an additive such as polyethyelene glycol (PEG) or polyvinyl alcohol. We have successfully extended the gelation time for sols synthesized with the TMOS precursor and biological buffers by tens of minutes or even hours by altering the synthesis conditions. After gelation, poly-condensation continues, and the gel network grows in mechanical stability and strength. Retaining sol-gel materials in the wet gel state can be easily accomplished by storing the materials in sealed containers so that pore liquid is not
allowed to evaporate. If a xerogel is desired, evaporation should be performed slowly so as to prevent cracking in the matrix.
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