Efficiency comparison of the imidazole plus RNO method for singlet oxygen detection in biorelevant solvents

Singlet oxygen (¹O₂) is the focus of study in many fields, including phototoxicity, antioxidant activity, pollutant weathering, photodynamic therapy, and water disinfection. The imidazole plus RNO (Imd/RNO) method, originated by Kraljic and El Mohsni, is commonly used to monitor singlet oxygen production. In this method, ¹O₂ is quenched by an acceptor, imidazole (Imd), during the formation of a trans-annular peroxide intermediate that bleaches the sensor, p-nitrosodimethylaniline (RNO). Though the method has been widely used, including to monitor ¹O₂ production in complex environments, such as surfactants and cells, studies reporting the efficiency of the assay in complex solvents have not been reported. In this research, the Imd/RNO method in complex, biorelevant solvents, i.e., sodium dodecyl sulfate, octanol, and phosphate buffer-saturated octanol, was compared with reference solvents, i.e., phosphate buffer, ethanol, and methanol, for monitoring ¹O₂ produced by Rose Bengal photosensitization using time-resolved, broadband UV-Vis absorbance measurements. Rates of sensor bleaching and sensitizer photodegradation were simultaneously monitored in each solvent to investigate correlations between the disappearance rates of sensor and sensitizer. The quantum yields of ¹O₂ production (ϕ_∆) in each solvent were calculated using a relative actinometric method. The dependence of sensor bleaching and sensitizer degradation on acceptor concentration and solvent polarity, and the results of assay controls suggest mechanistic differences underlying the reactions comprising the Imd/RNO method. These results demonstrate the need for caution and controls when using the method in complex samples including those containing cells, tissues, or nanoscale particles.