Singlet Oxygen Detection by Chemiluminescence Probes in Living Cells

Nir Hananya, Ori Green, Ismael Gutiérrez-Fernández, Doron Shabat, Juan B. Arellano*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review


Singlet oxygen is a reactive oxygen species that causes oxidative damage to plant cells, but intriguingly it can also act as a signalling molecule to reprogram gene expression required to induce plant physiological/cellular responses. Singlet oxygen photosensitization in plants mainly occurs in chloroplasts after the molecular collision of ground-state molecular oxygen with triplet-excited-state chlorophyll. Singlet oxygen direct detection through phosphorescence emission in chloroplasts is a herculean task due to its extremely low luminescence quantum yield. Because of this, indirect alternative methods have been developed for its detection in biological systems, for example, by measuring the changes in the EPR signal or fluorescence intensity of singlet oxygen reaction–based probes. The singlet oxygen chemiluminescence (SOCL) is a chemiluminescence probe with high sensitivity and selectivity towards singlet oxygen and promising use to detect it in living cells without the inconvenience of low stability of the EPR signal of spin probes in the presence of redox compounds, spurious light scattering coming from the light source required for the excitation of fluorescence probes or the light emission of endogenous fluorescent molecules like chlorophyll in chloroplasts. The protocol presented in this chapter describes the first steps to characterizing singlet oxygen production within the biological system under study; this is accomplished through monitoring molecular oxygen consumption by SOCL using a Clark-type oxygen electrode and measuring the chemiluminescence generated by SOCL 1,2-dioxetane using a spectrofluorometer. For singlet oxygen detection within living cells, a version of SOCL with increased membrane permeability (SOCL-CPP) is described.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages17
StatePublished - 2024

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


FundersFunder number
Agricultural and Forestry Systems
Consejería de Educación, Junta de Castilla y León
European Commission
Consejo Superior de Investigaciones Científicas
European Regional Development Fund
Junta de Castilla y León_CSI260P20
Junta de Castilla y León


    • Chemiluminescence
    • Chloroplast
    • Photosynthesis
    • Schaap’s dioxetanes
    • Singlet oxygen
    • SOCL
    • Thylakoid


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