Nebraska Redox Biology Center Educational Portal

Singlet Oxygen

Singlet oxygen is electronically excited state of molecular oxygen. It belongs to high energy state (22 kcal/mol) and very reactive ROS involved in chemical reactions. Singlet oxygen react with many kinds of biological molecules such as DNA, proteins and lipids [ 1, 2, 3, 4, 5, 6, 7 ].

Singlet oxygen can be generated by an input of energy. Singlet oxygen is produced by light absorption by photosensitizers and, in plants, particularly by the chlorophylls and their precursors [ 8, 9, 10, 11 ]. Carotenoids are involved in removing excess light energy from chlorophyll molecules and quenching the singlet oxygen molecules directly in photosynthetic systems [ 10, 11, 12 ]. Byological systems have no any special enzymatic system for sccvenging of singlet oxigen. Beta-carotene and uric acid may work as highly effective singlet oxygen physical quenchers [ 9, 10, 11, 12 13, 14, 15 ].

Due to its highly reactive nature, singlet oxygen has very few direct methods of determination. Indirect methods using spectrophotometric, fluorescent or chemiluminescent probes have been extensively studied [ 16 ].

1) Molecular emission spectroscopy [ 16, 17, 18 ]. Direct light emission at ca. 1270 nm, being an intrinsic property of singlet oxygen, was frequently applied for its detection and characterization.

2) A chemical probe is usually used to trap the singlet oxygen and then detection and quantification can be based on absorbance measurement. 9,10-diphenylanthracene (DPA), the most frequently used spectrophotometric probe, reacts specifically with singlet oxygen to form a stable endoperoxide. The decrease in absorbance at 355 nm was proportional to singlet-oxygen production of the system under consideration [ 16, 19, 20 ].

3) Fluorescence probes are utilized to detect singlet oxygen molecules through changes in fluorescence properties (e.g., fluorescence intensity, wavelength, quantum yield or fluorescence lifetime). Fluorescence probes are sensitive, have fast response, and can afford high spatial resolution via microscopic imaging [ 16, 19, 20 ].

9-[2-(3-carboxy-9,10-dimethyl) anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX) is a fluorescent probe for singlet-oxygen detection. DMAX detection is very sensitive and very specific to singlet oxygen.

The chemical reaction scheme of DMAX with singlet oxygen [ 16 ].

Rare-earth-chelate-based luminescence probes are used for singlet oxygen detection [ 16, 21, 22 ].

The chemical structure and the reaction scheme of (a) ATTA-Eu3+, (b) PATA-Tb3+ and (c) MTTA-Eu3+ with singlet oxygen [ 16 ].

Chemiluminescence is one of the most sensitive methods in singlet-oxygen detection. Compared with fluorescence, chemiluminescence does not require excitation light, 2-methyl-6-phenyl- 3,7-dihydroimidazo [1,2-a] pyrazin-3-one (CLA), and its derivatives MCLA and FCLA. These compounds emit light spontaneously in the presence of singlet background fluorescence and scattering light interference are eliminated [ 16, 23, 24 ].

The chemical structure of CLA and its reaction scheme [ 16 ].

Tetrathiafulvalenem (TTF) derivates, the probes with a strong electron donor and an anthracene luminophore have excellent selectivity and high sensitivity for singlet-oxygen detection [ 16, 25, 26, 27 ].

The chemical structure and reaction scheme of (a) 4,4'(5')-bis [2-(9- anthryloxy)ethylthio] tetrathia-fulvalene, (b) 4,5-dimethylthio-4'-[2-(9-anthryloxy) ethylthio] tetrathiafulvalene [ 16 ].

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