Hydroxyapatite as a novel reversible in situ adsorption matrix for enzyme thermistor-based FIA

Abstract

The application of the easily available and inexpensive chromatographic matrix hydroxyapatite for reusable and reversible immobilization of enzymes for enzyme thermistor-based flow injection analysis of glucose and urea was tested. The immobilization was achieved by simple affinity adsorption of glucose oxidase and urease by a suitable pH-induced alteration of the protein charge. A linear detection range of 0.05–8.0 mM was observed for glucose estimation depending on the sensitivity and sample loop parameters with a detection limit of 0.05 mM. A broad detection range of 0.5–50 mM was observed for urea using the flow injection calorimetric biosensor. Some real samples like commercial soft drink, syrups, honey and serum samples were analyzed. The novelty of the described work is the rapid set up of glucose analysis using hydroxyapatite as a reusable immobilization support in a flow injection thermal biosensor without any need for covalent immobilization or chemical cross-linking. The property of hydroxyapatite to adsorb and desorb proteins as a function of the buffer pH and ionic strength makes in situ enzyme reloading or exchange possible. The standard curves were obtained within few hours with a high degree of reproducibility and the enzyme remained fully active even after 3 months.