A NEW TRYPSIN AFFINITY MONOLITHIC CAPILLARY COLUMN PREPARED FROM GLYCERYLSILANE PRECURSOR THROUGH SOL-GEL METHOD

Abstract

A new precursor was synthesized with tetraethyl orthosilicate and glycerol using Amberlyst 15 (dry) as catalyst. The resulting precursor showed improved biocompatibility compared to tetraethyl orthosilicate. The resulting precursor was used to form monolithic silica capillary columns involving the addition of a buffered trypsin solution containing poly(ethylene glycol) 600 and 3-aminopropyltriethoxysilane. Nonselective adsorption of the silica matrix was reduced by adopting appropriate ion strength, 3-aminopropyltriethoxysilane, and tetramethylammonium chloride. The morphology of glycerylsilane derived sol-gel matrix was assessed using scanning electron microscopy. The column capacity (Bt) was determined to be 0.31nmol. The Kd of entrapped enzyme in the column was found to be 1.64 µM. The resultant affinity capillary columns showed good repeatability as well as long lifetime. The resultant affinity capillary columns can retain entrapped enzymes in an active state for a long time, minimize nonselective interactions between small molecules and the silica matrix, and also have good repeatability. Such columns provide a promising tool for development of biosensors, affinity supports, and immobilized enzyme reactors.