Evaluation of the adsorptive immobilisation of horseradish peroxidase on PTFE tubing in flow systems for hydrogen peroxide determination using fluorescence detection

A simple means of immobilisation of horseradish peroxidase (HRP) on the inner wall of PTFE tubing by physical adsorption is described. The HRP-immobilised tubing was used to form a reaction coil that was inserted into a flow system. Various conditions influencing the reaction, immobilisation, desorption and stability of the reaction coil were evaluated by a flow injection method based on HRP-catalysed fluorescent reaction of H2O2 with p-hydroxyphenylpropionic acid as fluorogenic substrate. Re-immobilisation showed good reproducibility when 1% (w/v) sodium dodecyl sulphate (SDS) was used to desorb the denatured enzyme and proteins from the wall of PTFE tubing. The immobilised reaction coil is stable for at least a month if it is kept in buffered phosphate solution at pH 5.8 in a refrigerator. The immobilised coil was washed with 1.0×10−4 mol L−1 substrate solution for 40 h at a flow rate of 1.5 mL min−1 without losing activity. When substrate and 200 nmol L−1 H2O2 were continuously reacted and flowed through the reaction coil, the response was stable for 7 h (equivalent to about 1400 150 μL injections of 600 nmol L−1 H2O2) and retained 79% of its activity after 26 h of continuous reaction. The immobilised HRP is very sensitive to bubbles introduced into the flow system. Linear relationships between fluorescence intensity and H2O2 concentration were obtained in the range 4–80 ng mL−1 with HRP solution and with the immobilised reaction coil system. The detection limit (S/N=3) was 2 nmol L−1 H2O2 in both cases. The relative standard deviations (8 replicates) for the detection of 4 and 80 nmol L−1 H2O2 were 5% and <1%, respectively.