Analytical and Bioanalytical Chemistry

, Volume 411, Issue 20, pp 5287–5296 | Cite as

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

  • Johanna Herman
  • Sharon L. NealEmail author
Research Paper


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.


Singlet oxygen Imidazole plus RNO method p-Nitrosodimethylaniline (RNO) Photosensitization Photooxidation Relative quantum yield 



The authors thank Yinan Zhang, Micaih Murray, and Mohammed Seck for their technical assistance and Professor Dominic DiToro for discussions that prompted this investigation.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2019_1910_MOESM1_ESM.pdf (327 kb)
ESM 1 (PDF 326 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of DelawareNewarkUSA

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