Fibre optic biosensor for the determination of d-glucose based on absorption changes of immobilized glucose oxidase

Glucose oxidase was covalently attached on a nylon net and the decrease of absorption of its prosthetic group (FAD) related to its reduction by glucose was determined. The analytical range from 2 to 10 mM (0.036–0.18% w/v) of glucose was achieved; both response (10 s to 5 min) and regeneration times (2–15 min) of an enzyme layer were considerably dependent on the glucose concentration measured. Storage stability of the enzyme layer was longer than 30 months and the operational stability more than 200 analyses. The optimum pH value of the enzyme layer was 5.8, which accords well with the value of a free enzyme. Maximum activity of the enzyme layer was found at 37 °C, nevertheless, the conventional temperature of 30 °C was used for further experiments. The biosensor behaviour was tested in different buffers and 0.1 M citrate buffer was chosen as the most suitable one. The detection limit (2 mM of glucose) was influenced by dissolved oxygen: removal of oxygen by bubbling with nitrogen decreased the detection limit to 0.5 mM; on the contrary, the addition of catalase, which raises the amount of available oxygen, elevated it significantly. The biosensor prepared was tested on real samples of soft drinks; the results obtained were comparable with an amperometric and a spectrophotometric enzyme analysis.