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 (Km) 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.