Determination of hydrogen peroxide by micro-flow injection-chemiluminescence using a coupled flow cell reactor chemiluminometer

A novel flow cell reactor was developed for micro-flow injection determination of hydrogen peroxide (H2O2) using horseradish peroxide (HRP)-catalysed luminol chemiluminescence. The newly developed how cell reactor for a chemiluminometer allowed mixing of the chemiluminescent reagents in front of a photomultiplier for maximum detection of the emitted light. The rapid mixing allowed a decrease in the flow rate of the pump to 0.1-0.01 mL/min, resulting in increased sensitivity of detection of light. The how cell reactor was made by packing HRP-immobilized gels into a flow cell (Teflon tube; 6 cm x 0.98 mm i.d.) located in the cell holder of a chemiluminometer (flow-through type). The HRP-immobilized gels were made by immobilizing HRP onto the Chitopearl gel by the periodate method. H2O2 specimens (50 mu L) were injected into a stream of water delivered at a how rate of 0.1 mL/min and mixed with a luminol solution (0.56 mmol/L in Tricine buffer, pH 9.2) delivered at 0.1 mL/min in the flow cell reactor. Within-run reproducibility of the assay of H2O2 was 2.4% (4.85 mu mol/L; flow rate 0.1 mL/min, injection interval 10 min). The reproducibility of the H2O2 assay was influenced by the flow rates and the injection intervals of the H2O2 specimens. As the flow rates decreased, both the light intensity and the light duration increased. Optimal light intensity was obtained at a luminol concentration of 3-8 mmol/L, but 0.56 mmol/L was sufficient for assay of H2O2 in clinical specimens. At a luminol concentration of 0.56 mmol/L, the regression equation of the standard curve for H2O2 (0-9.7 mu mol/L) was Y = 27.5 X-2 + 394 X + 58.9 (Y = light intensity; X = concentration of H2O2) and the detection limit of H2O2 was 0.2 mu mol/L. This method was used to assay glucose (2.7-16.7 mmol/L) based on a glucose oxidase (20 U/mL, pH 7.4) reaction. The standard curve for glucose was Y = 167 X-2 - 351 X + 1484 (Y = light intensity; X = glucose). The within-run reproducibility for an aqueous glucose standard (2.7 mmol/L) and a control serum (glucose, 5 mmol/L) was 4.48% (n = 5) and 5.70% (n = 9), respectively. Copyright (C) 2000 John Wiley & Sons, Ltd.