Electrochemical detection of extracellular hydrogen peroxide in Arabidopsis thaliana: A real-time marker of oxidative stress

An electrochemical approach to directly measure the dynamic process of H₂O₂ release from cultures of Arabidopsis thaliana cells is reported. This approach is based on H₂O₂ oxidation on a Pt electrode in conjunction with continuous measurement of sample pH. For [H₂O₂] <1mm, calibration plots were linear and the amperometric response of the electrode was maximum at pH6. At higher concentrations ([H₂O₂] >1mm), the amperometric response can be described by Michaelian-type kinetics and a mathematical expression relating current intensity and pH was obtained to quantitatively determine H₂O₂ concentration. At pH5.5, the detection limit of the sensor was 3.1μm (S/N=3), with a response sensitivity of 0.16Am^-1cm^-2 and reproducibility was within 6.1% in the range 1-5×10^-3m (n=5). Cell suspensions under normal physiological conditions had a pH between 5.5-5.7 and H₂O₂ concentrations in the range 7.0-20.5μm (n=5). The addition of exogenous H₂O₂, as well as other potential stress stimuli, was made to the cells and the change in H₂O₂ concentration was monitored. This real-time quantitative H₂O₂ analysis is a potential marker for the evaluation of oxidative stress in plant cell cultures.