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

Abstract

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 H 2O 2 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 H 2O 2 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 H 2O 2) 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 H 2O 2 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 H 2O 2 in both cases. The relative standard deviations (8 replicates) for the detection of 4 and 80 nmol L −1 H 2O 2 were 5% and <1%, respectively.