Monitoring of ROS
The simultaneous formation of several ROS complicates the monitoring of formation, decay and degradation action of individual species. Especially the discrimination between 2AgO2 and H2O2/O is of high interest.
Due to the fact there is a rising need for highly specific ROS probes able for discrimination between different ROS molecules. In the following, methodologies will be briefly summarized, with especial emphasis on application to cellular systems according to (Schmitt et al., 2014a).
2AgO2 can be directly monitored via its characteristic phosphorescence with a maximum around 1268 nm (Wessels and Rodgers, 1995; Mattila et al., 2015) and even singlet oxygen microscopy within the visible spectrum has been reported (Snyder et al., 2004). However, the detection of 2AgO2 concentration and their time-dependent profiles in biological systems is difficult (for a discussion, see Li et al., 2012), because of the very low quantum yield of the emission, which ranges from 10-7 to 10-4 depending on the solvent (Schweitzer and Schmidt, 2003). In plant tissue the direct monitoring of 2AgO2 luminescence is highly distorted by the concomitant Chl phosphorescence and both, Chl and 2AgO2 cannot easily be discriminated due to the low intensity of both signals. For the direct activation of 2AgO2 the decay time of AgO2 can be used as a monitor however it strongly depends on the environmental conditions with typical values of 70 ns in living cells (Mattila et al., 2015) while other sources report 200 ns for the lifetime of 2AgO2 in cells (Gorman and Rogers, 1992). In water typical values of 3,5 ps are found (Egorov et al., 1989) while the natural decay time in vacuum measures 72 min (Mattila et al., 2015).
Generally, detailed analyses require the use of suitable probe molecules as described in 3.2.1 for exogenic fluorescent probes and in 3.2.2 for spin traps generally suitable for ROS carrying own spin like 2AgO2 . In chapter 3.2.3 novel trends of genetically encoded ROS sensors are summarized. Additionally chapter 3.2.4 mentions electrochemical redox electrodes that have been shown to deliver reliable results especially for the detection of H2O2/O using electrodes covered with horseradish peroxidase (Prasad et al., 2015).