Singlet Oxygen Luminescence Detection Research Articles

New insights to primary photodynamic effects – Singlet oxygen kinetics in living cells

The kinetics of chemical singlet oxygen quencher consumption inside living cells during low dose illumination was revealed via time resolved singlet oxygen luminescence detection. Deviations of the measured data from the common theoretical model for 1O 2 kinetics forced the authors to consider a one-dimensional diffusion model for description of the kinetics of singlet oxygen generated by membrane localized photosensitizers. Our observations reconcile seemingly contradictory reports presenting different values for the efficiency of singlet oxygen interaction with cells.

The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria

New antibacterial strategies are required in view of the increasing resistance of bacteria to antibiotics. One promising technique involves the photodynamic inactivation of bacteria. Upon exposure to light, a photosensitizer in bacteria can generate singlet oxygen, which oxidizes proteins or lipids, leading to bacteria death. To elucidate the oxidative processes that occur during killing of bacteria, Staphylococcus aureus was incubated with a standard photosensitizer, and the generation and decay of singlet oxygen was detected directly by its luminescence at 1,270 nm. At low bacterial concentrations, the time-resolved luminescence of singlet oxygen showed a decay time of 6 +/- 2 micros, which is an intermediate time for singlet oxygen decay in phospholipids of membranes (14 +/- 2 micros) and in the surrounding water (3.5 +/- 0.5 micros). Obviously, at low bacterial concentrations, singlet oxygen had sufficient access to water outside of S. aureus by diffusion. Thus, singlet oxygen seems to be generated in the outer cell wall areas or in adjacent cytoplasmic membranes of S. aureus. In addition, the detection of singlet oxygen luminescence can be used as a sensor of intracellular oxygen concentration. When singlet oxygen luminescence was measured at higher bacterial concentrations, the decay time increased significantly, up to approximately 40 micros, because of oxygen depletion at these concentrations. This observation is an important indicator that oxygen supply is a crucial factor in the efficacy of photodynamic inactivation of bacteria, and will be of particular significance should this approach be used against multiresistant bacteria.

Time-resolved detection of singlet oxygen luminescence in red-cell ghost suspensions: concerning a signal component that can be attributed to 1O 2 luminescence from the inside of a native membrane

For about ten years, it has been debated whether in principle it is possible to detect 1O 2 located within the cell membrane by performing experiments with cell suspensions or even in tissue. In this paper we present our investigations on photosensitized red-cell ghost suspensions (RCGSs) and our strategy for the detection of luminescence of singlet oxygen ( 1O 2) from the inside of the cell membrane. Using a very sensitive apparatus for time-resolved 1O 2 detection, a very promising sensitizer and an adequate experimental strategy, a very small amount of the detected luminescence indeed can be attributed to 1O 2 from the inside of the ghost membrane.

Time-resolved detection of singlet oxygen luminescence in red cell ghost suspensions

The photophysical properties of dyes used, or of potential use, in PDT can be changed markedly in different environments. The system ghost/sensitizer is more realistic to estimate the photophysical parameters of sensitizers as compared to systems like sensitizers/micelles or liposomes. Since singlet oxygen ( 1O 2) seems to be an important factor in PDT, the influence of temperature and buffer on the generation of 1O 2 in ghost suspensions is of interest. The influence of these parameters on ghosts containing pheophorbide (pheo) was investigated by time resolved detection of 1O 2-luminescence. An unexpected influence of exposure to light was found. During light exposure the 1O 2-luminescence intensity increases. In addition to the slow decaying luminescence signal a fast decaying signal was found that shows a different behaviour upon changes in temperature than the slow decaying component.

Detection of Near-IR Singlet Oxygen Luminescence with an FT-Raman Spectrometer

The first excited state of molecular oxygen, O2(1Δg), lying at 0.98 eV above the ground state, O2(3Σg-), is relatively long-lived as a consequence of the multiple forbiddenness of the O2(1Δg) → O2(3Σg-) transition. As a result, O2(1Δg-) shows high reactivity to organic substrates, and irreversible photo-oxidative damage may occur. A typical example is that singlet oxygen is believed to be the cytotoxic species generated in the photodynamic therapy (PDT) of cancer.

Spectroscopic studies of cutaneous photosensitizing agents--XVI. Disperse blue 35.

The photochemistry (Type I and II) of the phototoxic textile dye Disperse Blue (DB-35) and its purified components has been studied using electron spin resonance in conjunction with spin trapping technique and the direct detection of singlet oxygen (1O2) luminescence. The main components of DB-35 (which is synthesized by the successive nitration, reduction and methylation of 1,8-dihydroxy-anthraquinone) were separated by HPLC and identified by mass spectrometry and 2-D NMR as 4,5-diamino-1,8-dihydroxyanthraquinone (4,5-DDHAQ; 62% of total dye) and 2,7-diamino-1,8-dihydroxyanthraquinone (2,7-DDHAQ; 31% of total dye). Minor components included 2,5-diamino-1,8-dihydroxyanthraquinone (2,5-DDHAQ) and a monomethylated derivative of either 4,5-DDHAQ or 2,7-DDHAQ. Irradiation (624 nm) of 4,5-DDHAQ and 2,7-DDHAQ in dimethylsulfoxide resulted in the generation of superoxide which was trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Visible light irradiation of the components in ethanol generated 1O2 with the yields decreasing in the following order: 4,5-DDHAQ greater than 2,5-DDHAQ greater than 2,7-DDHAQ. These findings indicate that upon irradiation by visible light DB-35 can generate active oxygen species which may be responsible for the photocontact dermatitis caused by this dye.

Superoxide, hydrogen peroxide and singlet oxygen in hematoporphyrin derivative-cysteine, -NADH and -light systems

Hematoporphyrin derivative and light in the presence of cysteine or glutathione were found to convert oxygen to superoxide and hydrogen peroxide at pH < approx. 6.5, while at pH > 6.5 no superoxide or hydrogen peroxide production was observed. However, at pH values greater than 6.5 the rate of oxygen consumption increased. This rate paralleled the acid dissociation curve of the cysteine thiol group and is consistent with the chemical quenching of 1O 2 by cysteine. The superoxide and hydrogen peroxide formation observed below pH 6.5 appeared not to be related to the singlet oxygen production of hematoporphyrin derivative. In addition, superoxide and hydrogen peroxide production was observed with hematoporphyrin derivative and light in the presence of NADH, both above and below pH 6.5. Direct detection of singlet oxygen luminescence at 1268 nm in the hematoporphyrin derivative-light system ( 2H 2O as solvent) revealed an apparent linear increase in the singlet oxygen emission intensity as the p 2H was raised from 7.0 to 10.0. Azide efficiently quenched this observed emission. In addition, at p 2H 7.4, 1 mM cysteine resulted in a 40% reduction of the singlet oxygen luminescence, while at p 2H 9.4 the signal was quenched by over 95% (under the experimental conditions employed). In total, we interpret these results as consistent with the chemical quenching of 1O 2 by the ionized thiol group of cysteine.