EVALUATION OF THE ADSORPTIVE IMMOBILIZATION OF HORSERADISH-PEROXIDASEON PTFE TUBING IN FLOW SYSTEMS FOR HYDROGEN-PEROXIDE DETERMINATION USING FLUORESCENCE DETECTION

A simple means of immobilisation of horseradish peroxidase (HRP) on th e inner wall of PTFE tubing by physical adsorption is described. The H RP-immobilised tubing was used to form a reaction coil that was insert ed into a flow system. Various conditions influencing the reaction, im mobilisation, desorption and stability of the reaction coil were evalu ated by a flow injection method based on HRP-catalysed fluorescent rea ction of H2O2 with p-hydroxyphenylpropionic acid as fluorogenic substr ate. Re-immobilisation showed good reproducibility when 1% (w/v) sodiu m dodecyl sulphate (SDS) was used to desorb the denatured enzyme and p roteins from the wall of PTFE tubing. The immobilised reaction coil is stable for at least a month if it is kept in buffered phosphate solut ion at pH 5.8 in a refrigerator. The immobilised coil was washed with 1.0x10(-4) mol L-1 substrate solution for 40 h at a flow rate of 1.5 m t min-l without losing activity. When substrate and 200 nmol L-1 H2O2 were continuously reacted and flowed through the reaction coil, the re sponse was stable for 7 h (equivalent to about 1400 150 mu L injection s 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 fluores cence intensity and H2O2 concentration were obtained in the range 4-80 ng mL(-1) with HRP solution and with the immobilised reaction coil sy stem. The detection limit (S/N=3) was 2 nmol L-1 H2O2 in both cases. T he relative standard deviations (8 replicates) for the detection of 4 and 80 nmol L-1 H2O2 were 5% and <1%, respectively. (C) 1998 Elsevier Science B.V.