Diaminorhodamine and Light-Activatable NO Donors: Photorelease Quantification and Potential Pitfalls

Light-activatable nitric oxide (NO) donors have become of interest in the recent years. They produce NO when illuminated by light, which enables the control of its local concentration and is promising for biomedical applications. Several successful prototypes of photodonors have been published, but further research is needed to improve their properties such as water-solubility, activation wavelength, biocompatibility etc. One of major challenges on this way is to evaluate the efficiency of NO generation. Several methods may be used to track NO, including spin traps, specific electrodes and fluorescence-based probes. We have studied the applicability of well-known fluorescent reporter, diaminorhodamine (DAR-2), for the evaluation of NO production by photodonors. Our results indicate that DAR-2 can be used for the quantification of NO photorelease if this process is not accompanied by the singlet oxygen formation. Otherwise the oxidation of probe results in huge fluorescence increase, which interferes with signal due to reaction with NO. This issue should be taken into account when studying hybrids releasing both NO and ¹O₂, which are promising for photodynamic therapy.