Measuring hydrogen peroxide
Two techniques to measure H2O2 concentration and H2O2 production can be combined with the O2k: The fluorescence measurement using Amplex Red, and the electrochemical measurement with a H2O2 electrode.
Fluorescence
The fluorimetric measurement of H2O2 in the O2k is based on the O2k-Fluorescence Module, using Amplex Red (or one of its brand names).[1] The O2k-Fluorescence Module consists of a LED light source and a photodiode + filter attached to the O2k-glass window.[2] The potentiometric channel of the O2k-MultiSensor System is used to directly record the signal in DatLab (Hickey 2011 JCPB; MitoCom. In addition, the Oxygraph-2k has been coupled to full fluorescence spectrophotometers for this purpose, with a light guide inserted through the black PEEK stopper Anderson 2010 AJP.
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 O2k-MultiSensor System (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 2 mm "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. A direct comparison would be helpful. Please add your experiences!
Picomolar detection ranges have been reported for a method combining Amplex Red (R) with square wave voltammetry.[3]
References
- ↑ Sumbalova Z, Harrison DK, Gradl P, Fasching M, Gnaiger E (2011) Mitochondrial membrane potential, coupling control, H2O2 production, and the upper limit of mitochondrial performance. Abstract Kagoshima.
- ↑ Fasching M, Harrison DK, Tretter L, Gnaiger E (2011) Combination of high-resolution respirometry and fluorimetry for continuous monitoring of hydrogen peroxide production by mitochondria with resolution in the nanomolar range. Abstract Berlin
- ↑ Lyon J L, Stevenson K J (2006) Picomolar peroxide detection using a chemically activated redox mediator and square wave voltammetry. Anal. Chem. 78: 8518-8525
