Aqueous Sol−Gel Process for Protein Encapsulation

Porous silica materials made by low-temperature sol−gel process are promising host matrixes for encapsulation of biomolecules. To date, researchers have focused on sol−gel routes using alkoxides such as tetramethyl orthosilicate (TMOS) and tetraethyl orthosilicate (TEOS) for encapsulation of biomolecules. These routes lead to formation of alcohol as a byproduct that can have a detrimental effect on the activity of entrapped biomolecules. We have developed a novel aqueous sol−gel process to encapsulate biological molecules (such as enzymes, antibodies, and cells) that uses neutral pH and room temperature and does not generate alcohol as a byproduct. The process uses sodium silicate as precursor and is carried out in two steps: preparation of a low-pH silicate sol followed by gelation at neutral pH with a suitable buffer containing biomolecules. Two enzymes widely used in biosensing applications, horseradish peroxidase (HRP) and glucose-6-phosphate dehydrogenase (G6PDH), were used to prepare enzyme-doped silica monoliths and to investigate the effect of silica as host matrix on enzyme kinetics. The yield of the encapsulation process was close to 100% for both enzymes, and no significant leaching of enzyme molecules was observed over time. Encapsulated enzymes followed Michaelis−Menten kinetics and maintained good catalytic activity, the specific activity of encapsulated HRP and G6PDH being 73% and 36% of the specific activities of the free enzymes, respectively. The values of the Michaelis constant ( K m ) of the encapsulated enzymes were higher than those of the free enzymes, indicating the presence of partitioning and diffusional effects in the pores of the sol−gel matrix. The encapsulated enzymes also exhibited a different pH dependence of catalytic activity; the pH maxima for enzymatic activity for encapsulated enzymes were higher by 0.5−1 pH unit than those for enzymes in solution. These novel enzyme-doped silica matrixes provide promising platforms for development of biosensors, affinity supports, and immobilized enzyme reactors.