Determination of serum glucose by horseradish peroxidase‐catalysed imidazole chemiluminescence coupled to a micro‐flow‐injection system

The reactivity of flow‐injection (FI)‐horseradish peroxidase (HRP)‐catalysed imidazole chemiluminescence (CL) was studied for continuous determination of hydrogen peroxide (H 2 O 2 ) and serum glucose with immobilized glucose oxidase. Light emission by the HRP‐catalysed imidazole CL was obtained when immobilized HRP, alkaline imidazole (in Tricine solution, pH 9.3) and H 2 O 2 were reacted at room temperature. The optimal pH for the CL reaction was 9.3 and the optimal concentration of imidazole was 100 µmol/L. When no imidazole was added, the light intensity of the same H 2 O 2 specimen decreased to a level that could not be quantitatively determined. The spectrum of the light emitted by imidazole CL was in the range 400–600 nm with a peak at 500 nm. The calibration equation for determination of H 2 O 2 was y = 9860 x 2 + 3830 x + 11 700, where y = light intensity (RLU) and x = concentration of H 2 O 2 (µmol/L). The detection limit of H 2 O 2 was 5 pmol, and the reproducibility of the H 2 O 2 assay was 2.3% of the coefficient of variation (H 2 O 2 48 µmol/L, n = 13). The CL method was successfully applied to assay glucose after on‐line generation of H 2 O 2 with the immobilized glucose oxidase column, resulting in good reproducibility (CV = 3.3% and 1.0% for the standard glucose and the control serum, respectively). Copyright © 2007 John Wiley & Sons, Ltd.