Detection of Hydrogen Peroxide in River Water via a Microplate Luminescence Assay with Time-Resolved (“Gated”) Detection

Abstract.

Hydrogen peroxide (HP) in river water was determined using the highly luminescent Eu³⁺-tetracycline hydrogen peroxide (EuTc-HP) complex. A 15-fold increase in luminescence intensity of Eu³⁺-tetracycline (EuTc) is observed at 616 nm after formation of EuTc-HP. In order to eliminate interferences by other fluorescent substances that exist in river water, a time-resolved scheme is employed: EuTc-HP has a main decay time of 61 µs, while background fluorescence decays within a few ns. Thus, by performing measurements after a delay time of typically 30 µs, the signal becomes highly specific for EuTc-HP. Under optimized conditions (pH 7 in MOPS buffer), the calibration curve for the determination of HP was linear over the range of 2 to 160 µmol L⁻¹. The limit of detection is 1.1 µmol L⁻¹ HP. An average recovery of 99.5% was obtained for measurements of river water samples spiked with HP. The relative standard deviation of less than 2% indicates the high accuracy and precision of the method.