Measuring hydrogen peroxide

Two techniques to measure H2O2 can be combined with the O2k: electrochemical measurement with a H2O2 electrode and fluorescence measurement using Amplex Red.

Electrochemical Measurement

For electrochemically (H2O2 electrode) measuring H2O2 (and all other amperometric methods) please consult our"NO" or "amperometric" manual. All stated there about measuring NO is technically also correct for using a H2O2 electrode together with the oxygraph. In this manual you will also find which accessories you need to use a H2O2 electrode together with the oxygraph (there are different requirements for different generations of O2ks). The electrode itself has to be bought from a third party. It is sometimes stated that the available H2O2 electrodes may not have the required sensitivity for most ROS studies. WPI states a lower detection limit of "<100 nM" for their 2mm "macro" electrode and "<10 nM" for their 0.1 mm "mini sensor". The latter value is quite similar to the stated detection limit for fluorescence based methods, however it is not clear if the "mini sensor", probable developed for direct tissue insertion, works well in macroscopic chambers. It seems a direct comparison would be helpful. Please add your experiences! Picomolar detection ranges have been reported for a methode combining Amplex red with square wave voltammetry .

Fluorescence

The other method is based on fluorescence spectroscopy, using Amplex Red (or one of its brand names). The oxygraph has been coupled to full fluorescence spectrophotometers for this purpose Anderson 2010 AJP but also quite "simple" solutions of a LED light source and a photodiode + filter have been attached to the oxygraph for the same purpose. In the second example the potentiometric channel of the O2k multisensor extension was used to directly record the signal in DatLab. In any way, this currently involves a good degree of tinkering by the user, though we will start to develop a commercial solution.