Detection Of Singlet Oxygen Research Articles

Evidence of singlet oxygen and hydroxyl radical formation in aqueous goethite suspension using spin-trapping electron paramagnetic resonance (EPR)

The generation of reactive species in an aqueous goethite suspension, under room light and aeration conditions, was investigated using the electron paramagnetic resonance (EPR) technique employing spin trap agents. The trap reagents, including 5,5-dimethylpyrroline N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP), were used for the detection of OH radicals (OH ) and singlet oxygen ( 1O 2), respectively. On the addition of DMPO to the goethite suspended solution, a DMPO-OH adduct was formed, which was not decreased, even in the presence of the OH scavenger, mannitol. This result implied a false positive interpretation from the DMPO-OH EPR signal. In the presence of TEMP reagent, a TEMP-O signal was detected, which was completely inhibited in the presence of the singlet oxygen scavenger, sodium azide. With both DMPO-OH and TEMP-O radicals in the presence and absence of radical scavengers, singlet oxygen was observed to be the key species formed in the room-light sensitized goethite suspension. In the goethite/H 2O 2 system; however, both OH and singlet oxygen were generated, with significant portions of DMPO-OH resulting from both OH and singlet oxygen. In fact, the DMPO-OH resulting from OH should be carefully calculated by correcting for the amount of DMPO-OH due to singlet oxygen. This study reports, for the first time, that the goethite suspensions may also act as a natural sensitizer, such as fulvic acids, to form singlet oxygen.

Optimal discrimination threshold for the detection of singlet oxygen luminescence

Direct detection of singlet oxygen (1O2) luminescence around 1270 nm is the golden standard of 1O2 identification. In this study, the effect of the discrimination threshold on the detection of 1O2 luminescence that generated from the photoirradiation of Rose Bengal (RB) was evaluated by using a self-developed photon-counting detection system. The obtained results show that the discrimination threshold for photon counting has a significant impact on the intensity and shape of the measured 1O2 luminescence, which resulted in the variation of 1O2 lifetimes. The optimal discrimination threshold is determined to be about −0.0412 V, and the corresponding 1O2 lifetime in air-saturated distilled water is 4.26±0.06 μs.

NIR area array CCD-based singlet oxygen luminescence imaging for photodynamic therapy

In this work, a near-infrared CCD-based singlet oxygen luminescence two-dimensional imaging method is proposed to detect singlet oxygen by its 1270nm luminescence. Two-dimensional singlet oxygen images with its near-infrared luminescence during photosensitization could be obtained with a CCD integration time of 1s, without scanning. The data presented shows a linear relationship between the singlet oxygen luminescence intensity and sample concentration. This method provides a detection sensitivity of 0.00189mg/ml (Hematoporphyrin monomethyl Ether dissolved in ethanol) and a spatial resolution better than 100μm. We applied this method in vivo to demonstrate its potential in monitoring photodynamic therapy.

Two-dimensional singlet oxygen imaging with its near-infrared luminescence during photosensitization

Photodynamic therapy is a promising cancer treatment that involves activation of photosensitizer by visible light to create singlet oxygen. This highly reactive oxygen species is believed to induce cell death and tissue destruction in PDT. Our approach used a near-infrared area CCD with high quantum efficiency to detect singlet oxygen by its 1270-nm luminescence. Two-dimensional singlet oxygen images with its near-infrared luminescence during photosensitization could be obtained with a CCD integration time of 1 s, without scanning. Thus this system can produce singlet oxygen luminescence images faster and achieve more accurate measurements in comparison to raster-scanning methods. The experimental data show a linear relationship between the singlet oxygen luminescence intensity and sample concentration. This method provides a detection sensitivity of 0.0181 μg∕ml (benzoporphyrin derivative monoacid ring A dissolved in ethanol) and a spatial resolution better than 50 μm. A pilot study was conducted on a total of six female Kunming mice. The results from this study demonstrate the system's potential for in vivo measurements. Further experiments were carried out on two tumor-bearing nude mice. Singlet oxygen luminescence images were acquired from the tumor-bearing nude mouse with intravenous injection of BPD-MA, and the experimental results showed real-time singlet oxygen signal depletion as a function of the light exposure.

Photochemical and phototoxic properties of ethyl 1,4-dihydro-8-nitro-4-oxoquinoline-3-carboxylate, a new quinoline derivative

The present study demonstrates photoinduced generation of superoxide radical anion and singlet oxygen upon UVA irradiation of ethyl 1,4-dihydro-8-nitro-4-oxoquinoline-3-carboxylate ( DNQC), and its cytotoxic/phototoxic effects on murine leukemia L1210 cells. The formation of reactive oxygen species (ROS) was investigated by EPR spectroscopy using in situ spin trapping technique and 4-hydroxy-2,2,6,6-piperidine (TMP) for singlet oxygen ( 1O 2) detection. The EPR spectra monitored upon photoexcitation of aerated solutions of DNQC in dimethylsulfoxide evidenced the efficient activation of molecular oxygen via Types I and II mechanisms. The cytotoxic/phototoxic effects of DNQC, analysis of cell cycle, induction of apoptosis/necrosis, DNA damage and molecular mechanism of apoptotic death of L1210 cells in dark and in the presence of UVA irradiation were compared. DNQC induced a different cytotoxic/phototoxic effect, which was concentration- and time-dependent. The four highest tested concentrations of non-photoactivated and photoactivated DNQC induced immediate cytotoxic/phototoxic effect after 24 h cultivation of L1210 cells. This effect decreased with the time of treatment. The irradiation increased the sensitivity of leukemia cell line on DNQC, but the cell sensitivity decreased with time of processing. Quinolone derivative DNQC significantly induced direct DNA strand breaks in L1210 cells, which were increased with the irradiation of cells. The DNA damage generated by DNQC alone/with combination of UVA irradiation induced cell arrest in G 0/G 1 and G 2/M phases, decrease in the number of L1210 cells in S phase and apoptotic cell death of certain part of cell population after 24 h of influence. DNQC alone/with combination of UVA irradiation induced apoptosis in L1210 cells through ROS-dependent mitochondrial pathway.

A helpful technology – the luminescence detection of singlet oxygen to investigate photodynamic inactivation of bacteria (PDIB)

Photodynamic inactivation of bacteria (PDIB) is considered a new approach for the struggle against multiresistant bacteria. To achieve a sufficient level of bacteria killing, the photosensitizer must attach to and/or penetrate the bacteria and generate a sufficiently high amount of singlet oxygen. To optimize PDIB, the direct detection and quantification of singlet oxygen in bacteria is a helpful tool. Singlet-oxygen luminescence is a very weak signal, in particular in living bacteria. We first performed experiments in aqueous photosensitizer solution to optimize the luminescence system. We eliminated non-singlet-oxygen photons, which is important for the quantification of singlet oxygen and its rise and decay rates. This procedure is even more important when the laser excitation beam is scattered by bacteria (diameter 1 microm). In suspensions with both Gram-positive and Gram-negative bacteria we then clearly detected singlet oxygen by its luminescence and determined the respective rise and decay times. The decay times should provide an indication of localization of singlet oxygen and hence of the photosensitizer even in small bacteria.

Detection of singlet oxygen in blood serum samples of clinically healthy lambs and lambs suffering from alveld disease

Alveld is a disease in lambs of domestic sheep (Ovis aries L.), characterized by a combination of photosensitivity and liver damage. Generation of singlet oxygen play a major role in phototoxicity reactions. The compound phylloerythrin (phytoporphyrin) is so far assumed to be the main photodynamic agent in hepatogenous photosensitivity diseases in sheep. Phylloerythrin is a potent photosensitizer and an efficient source of singlet oxygen. The compound accumulates in the peripheral circualtion upon liver damage. Liver dysfunction is also likely to cause an increase in the blood level of bilirubin. Formation of singlet oxygen by bilirubin is reported. In the present work the photosensitizing potential of serum has been measured and related to the bilirubin- and phylloerythrin levels in lambs suffering from alveld and in clinically healthy controls. The singlet oxygen level of the serum was taken as a measure of the photosensitizing potential. The observed singlet oxygen values in serum from alveld lambs were significantly higher than the corresponding values observed in clinically healthy control lambs. This indicates that the serum of the alveld lambs contains an elevated concentration of photosensitizer. The singlet oxygen level was not correlated to the concentration of bilirubin or phylloerythrin. The results indicate that the photosensitizing mechanism is quite complex and may involve other sensitizer(s) than phylloerythrin.

Detection of singlet oxygen in photoexcited porous silicon nanocrystals by photoluminescence measurements

Luminescence of gas-phase singlet oxygen optically sensitized by microporous silicon at room temperature is detected for the first time. At the same time, a photoinduced increase in the photoluminescence intensity of defects at the sample surface in oxygen atmosphere is observed. It is shown that mechanical grinding of porous silicon layers yields a decrease in the amount of photogenerated singlet oxygen.

Direct measurement of singlet oxygen produced by four chlorin-ringed chlorophyll species in acetone solution

We developed an optical system to detect singlet oxygen produced by chlorin-ringed chlorophyll (Chl) species, i.e., Chl a, Chl b, Chl d, and di-vinyl-Chl a, with a high sensitivity and examined the relationship between molecular structures and reaction rates. Chl a in acetone was the lowest producer of singlet oxygen and the most effective quencher; in contrast, Chl b exhibited the opposite properties. These results showed that replacement of side chain(s) from a methyl group to a formyl group on the R7 position on a chlorin ring induced a higher production and lower quenching of singlet oxygen in Chl molecules.

Advances in Photodynamic Therapy Dosimetry*

Photodynamic therapy (PDT) is an emerging therapeutic modality based on the interaction of light, photosensitizer and molecular oxygen. As PDT techniques continue to develop and find more potential clinical indications, accurate dosimetry becomes a critical factor for achieving satisfied individual treatments. The current status of strategies and corresponding technologies for PDT dosimetry are reviewed. Typical PDT dosimetry estimations include the direct measurement of three critical ingredients of PDT (i.e. fluence rate, photosensitizer concentration, and tissue oxygen pressure), indirect determination of the photosensitizer fluorescence photobleaching and photoproduct formation, monitoring biological responses, and detecting singlet oxygen production with its near-infrared luminescence around 1 270 nm. The advantages and limitations of each PDT dosimetric model will be presented and the main challenges in clinical PDT dosimetry will be discussed.

Photoinduced Nitric Oxide and Singlet Oxygen Release from ZnPC Liposome Vehicle Associated with the Nitrosyl Ruthenium Complex: Synergistic Effects in Photodynamic Therapy Application

Under continuous photolysis at 675 nm, liposomal zinc phthalocyanine associated with nitrosyl ruthenium complex [Ru(NH.NHq)(tpy)NO](3+) showed the detection and quantification of nitric oxide (NO) and singlet oxygen ((1)O(2)) release. Photophysical and photochemical results demonstrated that the interaction between the nitrosyl ruthenium complex and the photosensitizer can enable an electron transfer process from the photosensitizer to the nitrosyl ruthenium complex which leads to NO release. Synergistic action of both photosensitizers and the nitrosyl ruthenium complex results in the production of reactive oxygen species and reactive nitrogen species, which is a potent oxidizing agent to many biological tissues, in particular neoplastic cells.

Photosensitized production of singlet oxygen: spatially-resolved optical studies in single cells

Singlet molecular oxygen, O(2)(a(1)Delta(g)), can be created in photosensitized experiments with sub-cellular spatial resolution in a single cell. This cytotoxic species can subsequently be detected by its 1270 nm phosphorescence (a(1)Delta(g)--> X(3)Sigma). Cellular responses to the creation of singlet oxygen can be monitored using viability assays. Time- and spatially-resolved optical measurements of both singlet oxygen and its precursor, the excited state sensitizer, reflect the complex and dynamic morphology of the cell. These experiments help elucidate photoinduced, oxygen-dependent events that compromise cell function and ultimately lead to cell death. In this perspective, recent work on the photosensitized production and detection of singlet oxygen in single cells is summarized, highlighting the advantages and current limitations of this unique experimental approach to study an old problem.

Fluorescence, Cyto-, And Photo-toxicity, And Structural Studies Of Substituted Piperidones: Potential Sensitizers For Two-photon Photodynamic Therapy

Two-photon photodynamic therapy has the advantages of being highly localized in its effects and allows for deeper tissue penetration, when compared to one-photon photodynamic therapy. N-alkylated 3,5-bis(arylidene)-4-piperidones, with a donor-pi-acceptor-pi-donor structure, have the potential to be useful two-photon sensitizers. We have measured two-photon cross sections (using femtosecond excitation), fluorescence quantum yields, fluorescence lifetimes, and x-ray crystal structures for a number of these compounds. Most two-photon cross sections are comparable to or larger than that of Rhodamine B. However, the fluorescence quantum yields are low (all less than 10%) and the fluorescence lifetimes are less than 1 ns, suggesting that there may be a significant energy transfer to the triplet state. This would encourage singlet oxygen formation and increase cellular toxicity. Results of dark cyto-toxicity studies with a number of human cancer cell lines are presented. White light photo-toxicity results are also presented, and suggest that increasing the number of double bonds, from one to two, in the piperidone “wings” increases the photo-toxicity with little corresponding change in the dark cyto-toxicity. Two-photon photo-toxicity studies are also underway (exposure in the range of 740 - 860 nm) as well as singlet oxygen detection studies(detection at about 1270 nm).

Visible‐Light‐Excited Singlet‐Oxygen Luminescence Probe Based on Re(CO)3Cl(aeip)

AbstractA new rhenium(I) complex, Re(CO)3Cl(aeip) [where aeip =2‐(anthracen‐9‐yl)‐1‐ethyl‐imidazo[4,5‐f][1,10]phenanthroline], was designed and synthesized as a luminescence probe under visible light excitation at 410 nm for detection of singlet oxygen (1O2) in aqueous media. The new complex can specifically react with 1O2 in neutral and alkaline media, which results in remarkable luminescence enhancements, with 8‐ and 18.7‐fold increases in the luminescence quantum yields, respectively. The visible light excitation may allow the complex to be useful for biosystems. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Time-resolved methods in biophysics. 7. Photon counting vs. analog time-resolved singlet oxygen phosphorescence detection

Two recent advances in optoelectronics, namely novel near-IR sensitive photomultipliers and inexpensive yet powerful diode-pumped solid-state lasers working at kHz repetition rate, enable the time-resolved detection of singlet oxygen (O2(a1Deltag)) phosphorescence in photon counting mode, thereby boosting the time-resolution, sensitivity, and dynamic range of this well-established detection technique. Principles underlying this novel approach and selected examples of applications are provided in this perspective, which illustrate the advantages over the conventional analog detection mode.

No single way to understand singlet oxygen signalling in plants

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated simultaneously in various intracellular compartments and these can cause oxidative damage or act as signals. The conditional flu mutant of Arabidopsis, which generates singlet oxygen in plastids during a dark-to-light transition, has allowed the biological activity of singlet oxygen to be determined, and the criteria to distinguish between cytotoxicity and signalling of this particular ROS to be defined. The genetic basis of singlet-oxygen-mediated signalling has been revealed by the mutation of two nuclear genes encoding the plastid proteins EXECUTER (EX)1 and EX2, which are sufficient to abrogate singlet-oxygen-dependent stress responses. Conversely, responses due to higher cytotoxic levels of singlet oxygen are not suppressed in the ex1/ex2 background. Whether singlet oxygen levels lower than those that trigger genetically controlled cell death activate acclimation is now under investigation.

Photoprotection by Porcine Eumelanin Against Singlet Oxygen Production†

Melanin, a major pigment found in retinal pigment epithelium (RPE) cells, is considered to function in dual roles, one protective and one destructive. By quenching free radical species and reactive oxygen species (ROS) melanin counteracts harmful redox stress. However, melanin is also thought to be capable of creating ROS. In this destructive role, melanin increases redox strain in the cell. This study uses readily available eumelanin extracted from porcine RPE cells as a more authentic model than synthetic melanin to determine specific mechanisms of melanin activity with regard to singlet oxygen in the presence and absence of rose bengal, a singlet-oxygen photosensitizer. Optical detection of singlet-oxygen was determined by monitoring the bleaching of p-nitrosodimethylaniline in the presence of histidine. Production of singlet oxygen in aqueous oxygen-saturated solutions of rose bengal without eumelanin was readily accomplished. In contrast, detection of singlet oxygen in oxygen-saturated solutions of eumelanin without rose bengal failed, consistent with results of others. However, a significant decrease in singlet oxygen production by rose bengal was observed in the presence of eumelanin. After correction for light absorption and chemical bleaching of eumelanin, the results show that eumelanin also provides a photoprotective mode arising from chemistry, that is, not just the physical process of light absorption followed by energy dissipation as heat.

Direct Time-Resolved Detection of Singlet Oxygen in Zeolite-Based Photocatalysts

Singlet oxygen has been characterized spectroscopically as a product of the exposure of suspensions of zeolites containing oxidation catalysts. Spectroscopic and lifetime studies show that a part of the singlet oxygen formed reacts within the zeolite porous structure, while a significant fraction escapes and becomes available for reaction in the bulk media. The liquid phase plays a key role in determining intra- and extracavity dynamics.

Optical detection of singlet oxygen produced by fatty acids and phospholipids under ultraviolet A irradiation

Ultraviolet A (UVA) radiation has been known to generate reactive oxygen species, such as singlet oxygen, in skin, leading to the oxidation of lipids and proteins. This oxidation influences cellular metabolism and can trigger cellular signaling cascades, since cellular membranes and the stratum corneum contain a substantial amount of fatty acids and lipids. Using highly sensitive IR-photomultiplier technology, we investigated the generation of singlet oxygen by fatty acids and lipids. In combination with their oxidized products, the fatty acids or lipids produced singlet oxygen under UVA radiation at 355 nm that is directly shown by luminescence detection. Linolenic or arachidonic acid showed the strongest luminescence signals, followed by linoleic acid and docohexaenoic acid. The amount of singlet oxygen induced by lipids such as phosphatidylcholine was significantly higher compared to the corresponding fatty acids within phospholipids. This result indicates a synergistic process of oxygen radicals and singlet oxygen during irradiation. UVA radiation initiates singlet oxygen generation, which subsequently oxidizes other fatty acids that in turn produce additional singlet oxygen. This leads to an enhancement of UVA-induced damage of fatty acids and lipids, which must enhance the oxidative damages in cells.

Singlet Oxygen Photosensitization by EGFP and its Chromophore HBDI

The photosensitization of reactive oxygen species and, in particular, singlet oxygen by proteins from the green fluorescent protein (GFP) family influences important processes such as photobleaching and genetically targeted chromophore-assisted light inactivation. In this article, we report an investigation of singlet oxygen photoproduction by GFPs using time-resolved detection of the NIR phosphorescence of singlet oxygen at 1275 nm. We have detected singlet oxygen generated by enhanced (E)GFP, and measured a lifetime of 4 μs in deuterated solution. By comparison with the model compound of the EGFP fluorophore 4-hydroxybenzylidene-1,2-dimethylimidazoline (HBDI), our results confirm that the β-can of EGFP provides shielding of the fluorophore and reduces the production of this reactive oxygen species. In addition, our results yield new information about the triplet state of these proteins. The quantum yield for singlet oxygen photosensitization by the model chromophore HBDI is 0.004.