Reusable optical sensing microplate for hydrogen peroxide using a fluorescent photoinduced electron transfer probe (HP Green)

The first optical sensing microplate is described for repetitive high-throughput screening of hydrogen peroxide (H2O2) in solutions of physiological pH. The fluorescent probe HP Green which shows a photoinduced electron transfer (PET) was incorporated in a polyurethane polymer matrix (type D4) on the bottom of microplate wells. Enzymatic oxidation of H2O2 Green in the presence of horseradish peroxidase (HRP) enables the determination of H2O2 concentrations up to 300 μM with a LOD as low as 8.0 μM. The calibration plots show high correlation coefficients (R2 = 0.996). The new sensing microplate (V-bottom) enables parallel and rapid (96 samples in 20 min) detection of H2O2 with a standard fluorescence microplate reader. The response time is in the range of 10–20 min, and the response is reversible. Finally, the H2O2 sensing microplate has successfully been applied to the determination of H2O2 in colored cell nutrition medium as real biological sample with a strong matrix effect.