High‐Pressure Generator of Singlet Oxygen

Human eosinophils, stimulated with phorbol myristate acetate, were found to produce 1268 nm chemiluminescence characteristic of singlet oxygen. Singlet oxygen generation required the presence of bromide ion. A bromide ion concentration of 100 microM, comparable to the total bromine content of whole blood, was sufficient for the eosinophils to generate measurable amounts of singlet oxygen. For the conditions used (10(7) cells/ml and 10 micrograms/ml phorbol myristate acetate), the duration of the singlet oxygen generation was brief, about 5 min, and the total yield of singlet oxygen was modest, 1.0 +/- 0.1 microM. The cells remained viable after the singlet oxygen production ceased. This is the first demonstration of singlet oxygen production from living cells. The singlet oxygen generated by eosinophils likely results from a peroxidase-catalyzed mechanism, since a purified eosinophil peroxidase-hydrogen peroxide-bromide system was also shown to produce singlet oxygen. The unique properties of eosinophil peroxidase are illustrated by the fact that at p2H 7.0 and with 100 microM bromide, eosinophil peroxidase generated 20 +/- 2% of the theoretical yield of singlet oxygen, whereas under identical conditions, myeloperoxidase and lactoperoxidase produced only 1.0 +/- 0.1% and -0.1 +/- 0.1%, respectively.

Three dimensional transient analysis of bubble dynamics in centrifugal bubble singlet oxygen generator for scalable COIL

The oxidation of linoleic acid catalyzed by soybean lipoxygenase isozymes was accompanied by 1268 nm chemiluminescence characteristic of singlet oxygen. The recombination of peroxy radicals as first proposed by Russell (Russell, G.A. (1957) J. Am. Chem. Soc. 79, 3871-3877) is a plausible mechanism for the observed singlet oxygen production. Lipoxygenase-3 was the most active isozyme. Under the optimal aerobic conditions of p2H 7, 100 micrograms/ml lipoxygenase-3, 100 microM linoleic acid, 100 microM 13-hydroperoxylinoleic acid, and air-saturated buffer, the yield of singlet oxygen was 12 +/- 0.4 microM or 12% of the amount predicted by the Russell mechanism. High yields of singlet oxygen required the presence of 13-hydroperoxylinoleic acid. Systems containing lipoxygenase-2 and lipoxygenase-3 produced comparable yields of singlet oxygen without added 13-hydroperoxylinoleic acid, since the lipoxygenase-2 served as an in situ source of hydroperoxide. Lipoxygenase-1 was active only at low oxygen concentrations. Its singlet oxygen-producing capacity was greatly increased by the addition of acetone to the system. Lipoxygenase-2 did not produce detectable quantities of singlet oxygen.

Copyright: © 2013 Sarma SJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Electronically excited, highly reactive state of molecular oxygen is known as singlet oxygen. Apart from different other methods; singlet oxygen is mainly produced by irradiation of molecular oxygen in presence of photosensitizer pigments such as rose Bengal or methylene blue. Because of its potential applications in many fields including wastewater treatment, singlet oxygen remains to be one of the mostly explored research subjects of recent years. Singlet oxygen can be used as treatment mean against both pathogens and toxic organic pollutants present in wastewater [1]. For example, numbers of reports are available on oxidation of phenol and its derivatives, either in organic solvents or in aqueous solution, by the action of photosensitizer induced singlet oxygen [2]. Similarly, Schafer et al. [3] have investigated the inactivation of bacteria, such as Escherichia coli or Deinococcusradiodurans using photosensitizer technology involving singlet oxygen [3]. In addition to commonly used photosensitizer pigments, aromatic hydrocarbons (napthalenes, anthracenes, biphenyls) have also been evaluated for possible application as photosensitizer [2]. Similarly, a range of quinones can also work as sensitizers for singlet oxygen [2,4]. Even, the organic matter of an aquatic system has the potential to work as the inherent photosensitizer to support the formation of singlet oxygen as well as other reactive oxygen species [1]. Similarly, Dalrymple et al. [5] also have reported singlet oxygen production by aquatic dissolved organic matter [5]. Wastewater contains all these different types of photosensitizers; hence, degradation of different pollutants and elimination of pathogens by singlet oxygen produced during wastewater treatment process is a likely phenomenon. However, singlet oxygen has very short lifetime and they may also be involved in other intermolecular interactions, which are not relevant in pollutant degradation or pathogen inactivation [6]. Likewise, yield of singlet oxygen depends on the oxygen concentration [7]. However, the oxygen demand of wastewater treatment process is already very high. Therefore, whether the oxygen left after microbial utilization will be sufficient to generate the required amount of singlet oxygen for a significant response or not, is a question. Likewise, due to irradiation, chromophoric dissolved organic matter may be degraded to be more available for microbial utilization and it may further enhance the bacterial growth [8], including pathogens. Thus, considering additional cost involved, in the case of artificially irradiated wastewater treatment process, the purpose of pathogen inactivation and pollutant degradation using singlet oxygen may not be very effective. In this context, Mostafa & Rosario-Ortiz [1] have indicated that the concept of inherent photosensitizer (organic matter) mediated singlet oxygen generation and wastewater treatment may have more practical implication in the case of relatively cost effective sunlight-mediated nonconventional wastewater treatment processes, such as constructed wetlands [1]. Thus, in order to have a better understanding of the role of singlet oxygen in wastewater treatment, actual contribution of naturally produced singlet oxygen, in pathogen inactivation and contaminant degradation during wastewater treatment, should be precisely estimated. Further, possible enhancement of the efficiency of singlet oxygen, by the technique such as artificial irradiation and additional cost involved in such operation, should be determined. Likewise, alternative wastewater treatment processes should be designed, where maximum utilization of natural photosensitization is possible. Finally, based on the techno-economic feasibility of using artificial irradiation, the possibility of using it (in a cost effective way) to complement natural photosensitization, should also be explored.

Polyelectrolyte nanocomplex formation of heparin-photosensitizer conjugate with polymeric scavenger for photodynamic therapy.

Traditional photodynamic therapy for cancer relies on dye-photosensitized generation of singlet oxygen. However, therapeutically effective singlet oxygen generation requires well-oxygenated tissues, whereas many tumor environments tend to be hypoxic. We describe a platform for targeted enhancement of photodynamic therapy that produces singlet oxygen in oxygenated environments and hydroxyl radical, which is typically regarded as the most toxic reactive oxygen species, in hypoxic environments. The 24-subunit iron storage protein bacterioferritin (Bfr) has the unique property of binding 12 heme groups in its protein shell. We inserted the isostructural photosensitizer, zinc(II) protoporphyrin IX (ZnP), in place of the hemes and extended the surface-exposed N-terminal ends of the Bfr subunits with a peptide targeting a receptor that is hyperexpressed on the cell surface of many tumors and tumor vasculature. We then loaded the inner cavity with ∼2500 irons as a ferric oxyhydroxide polymer and finally conjugated 2 kDa polyethylene glycol to the outer surface. We showed that the inserted ZnP photosensitizes generation of both singlet oxygen and the hydroxyl radical, the latter via the reaction of photoreleased ferrous iron with hydrogen peroxide. This targeted iron-loaded ZnP-Bfr construct was endocytosed by C32 melanoma cells and localized to lysosomes. Irradiating the treated cells with light at wavelengths overlapping the ZnP Soret absorption band induced photosensitized intracellular Fe2+ release and substantial lowering of cell viability. This targeted, light-triggered production of intracellular singlet oxygen and Fenton-reactive iron could potentially be developed into a phototherapeutic adjunct for many types of cancers.

The development of activatable photodynamic therapy (PDT) has demonstrated a utility for effective photosensitizer quenchers. However, little is known quantitatively about Forster resonance energy transfer (FRET) quenching of photosensitizers, even though these quenchers are versatile and readily available. To characterize FRET deactivation of singlet oxygen generation, we attached various quenchers to the photosensitizer pyropheophorbide-alpha (Pyro) using a lysine linker. The linker did not induce major changes in the properties of the photosensitizer. Absorbance and emission wavelength maxima of the quenched constructs remained constant, suggesting that quenching by ground-state complex formation was minimal. All quenchers sharing moderate spectral overlap with the fluorescence emission of Pyro (J > or = 5.1 x 10(13) M(-1) cm(-1) nm4) quenched over 90% of the singlet oxygen, and quenchers with weaker spectral overlap displayed minimal quenching. A self-quenched double Pyro construct exhibited intermediate quenching. Consistent with a FRET deactivation mechanism, extension of the linker to a 10 residue polyproline peptide resulted in only the quenchers with spectral overlap almost 2 orders of magnitude higher (J > or = 3.7 x 10(15) M(-1) cm(-1) nm4) maintaining high quenching efficiency. Overall, there was good correlation (0.98) between fluorescence quenching and singlet oxygen quenching, implying that fluorescence intensity can be a convenient indicator for the singlet oxygen production status of activatable photosensitizers. Uniform singlet oxygen luminescence lifetimes of the compounds, along with minimal triplet state transient absorption were consistent with quenchers primarily deactivating the photosensitizer excited singlet state. In vitro, cells treated with well-quenched constructs demonstrated greatly reduced PDT induced toxicity, indicating that FRET-based quenchers can provide a level of quenching useful for future biological applications. The presented findings show that FRET-based quenchers can potently decrease singlet oxygen production and therefore be used to facilitate the rational design of activatable photosensitizers.

This paper focuses on the implementation of various diagnostics for optimizing chemical oxygen iodine laser. This paper also dwells on the measurement methodologies and instrumentation employed in these diagnostic systems. The prime diagnostics are for the measurement of vital species in form of iodine, singlet oxygen, and chlorine. Iodine concentration measurement (absorption at λ = 499 nm ), chlorine utilization (absorption at λ = 330 nm), and singlet oxygen yield (emission at λ = 1.27 μm) have been implemented based on optical absorption/emission principle. Furthermore, online Mach number determination, which is critical for supersonic gas flows, laser pulse detection (at λ = 1315 nm ), and flow rate measurement and control over wide range, have been carried out. A dedicated diagnostics and data acquisition system (DDAS) customized for parameter monitoring have been developed. The developed DDAS also serves the purpose of precise operation sequencing and parameter control. It is a 168-channel personal-computer-based system with customized interface electronics employing Visual C++ programming language with user-friendly graphical user interfaces. A detailed uncertainty analysis of various critical parameters has been also presented.

SummaryThe large driving forces produced in photosynthesis require precise control and regulation to prevent potentially destructive side reactions involving active oxygen species that damage pigments and proteins. Oxygen can be activated either by energy transfer, yielding singlet oxygen, or by reduction, yielding superoxide or hydrogen peroxide. At Photosystem I, the lifetime of the excited chlorophyll singlet state within the antenna pigment bed is short and little threat is posed by formation of highly reactive singlet oxygen. The situation is completely different in Photosystem II. Here, the lifetime of singlet chlorophyll is sufficiently long to allow significant formation of chlorophyll triplet states able to transfer energy to ground state triplet oxygen, generating singlet oxygen. Carotenoids intervene to control these processes in at least two important ways. First, pigments such as β-carotene are capable of directly quenching both triplet chlorophyll and singlet oxygen states (the so-called triplet valve mechanism). Second, the xanthophyll cycle is involved in lowering the yield of triplet chlorophyll formation by pre-emptive quenching of excited singlet state chlorophyll, a mechanism that can have a high quantum yield value for energy dissipation. The chief difference between these two mechanisms is that the xanthophyll cycle is inducible and subject to regulation, whereas the triplet valve pathway is constitutive and unregulated. Although formation of singlet oxygen must be avoided or controlled, chloroplasts have exploited the potential of oxygen chemistry to drive and regulate metabolism while minimizing the deleterious effects of uncontrolled interactions with oxygen. Hence, while the potential of the chloroplast as a source of oxidative stress is large, in reality this organelle offers minimal risk because of pre-emptive regulation and effective defense. The production of superoxide and hydrogen peroxide by the thylakoid membranes is limited by efficient control of electron transport. This regulation limits the potential for oxidative damage and prevents high rates of electron transport to oxygen. Effective antioxidant defense ensrures rapid elimination of active forms of oxygen further preventing oxidative damage.

Singlet oxygen is a high-energy molecular oxygen species. As one of the most active intermediates involved in chemical and biochemical reactions, singlet oxygen plays essential roles in plant responses to UV and strong light. Here, we report that Cle, an elicitor derived from fungal cell walls, induces the generation of singlet oxygen in cell cultures of ginseng, Panax ginseng. Cle treatment also triggers the activation of plasma membrane NADPH oxidase and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), subsequently leading to ethylene release and increased saponin synthesis, as shown by increased mRNA expression of squalene synthase (SQS) and squalene epoxidase (SQE), and accumulation of beta-amyrin synthase (beta-AS). Suppression of Cle-induced singlet oxygen generation or inhibition of ethylene production blocks saponin synthesis, whereas treatment of ginseng cells with ethylene or singlet oxygen induces the synthesis of saponin. Together, these results indicate that Cle-induced production of both singlet oxygen and ethylene is required for saponin synthesis, and that singlet oxygen may function upstream of ethylene during Cle-induced saponin synthesis.

A gas-sampling diagnostic has been developed to determine the molecular composition in real time of the gaseous flow emanating from a 1(Delta) g oxygen generator. This excited state oxygen is the energy source for a chemical oxygen-iodine laser (COIL). The major constituents in the flow, helium, oxygen, chlorine and water are drawn from the generator and transported to a quadrupole mass spectrometer for analysis. Information derived from this instrument such as chlorine utilization, water vapor content and flow impurities will advance our understanding of the oxygen generator performance. Precise measurements of water content are particularly important because of its energy quenching effect on I*, the lasing species in a COIL. This diagnostic may also be used to measure the mixing distribution of molecular iodine in the supersonic excited oxygen flow downstream of the nozzles. This paper discusses the criteria for the design of the gas-sampling diagnostic.

The generator of singlet oxygen (SOG) remains still a challenge for a chemical oxygen-iodine laser (COIL). Hitherto, only chemical generators based on the gas-liquid reaction system (chlorine-basic hydrogen peroxide) can supply singlet oxygen, O₂(¹Δ), in enough high yields and at pressures to maintain operation of the high power supersonic COIL facilities. Employing conventional generators of jet-type or rotating disc-type makes often problems resulting mainly from liquid droplets entrained by an O₂ (¹Δ) stream into the laser cavity, and a limited scalability of these generators. Advanced generator concepts investigated currently are based on two different approaches: (i)O₂(¹Δ) generation by the electrical discharge in various configurations, eliminating thus a liquid chemistry, and (ii) O₂(¹Δ) generation by the conventional chemistry in novel configurations offering the SOG efficiency increase and eliminating drawbacks of existing devices. One of the advanced concepts of chemical generator - a spray SOG with centrifugal separation of gasliquid phases - has been proposed and investigated in our laboratory. In this paper we present a description of the generator principle, some essential results of theoretical estimations, and interim experimental results obtained with the spray SOG.

Functional singlet oxygen generators based on phthalocyanines

The singlet oxygen generator (SOG) generates singlet oxygen for a chemical oxygen iodine laser (COIL), using the gas-liquid reaction between basic hydrogen peroxide (BHP) and Cl2 gas. The Jet-SOG has been widely used, wherein jet BHP from small orifices reacts with Cl2 gas, and the BHP utilization is less than 1% in a single pass through the reaction zone. To improve BHP utilization, the reaction surface with Cl2 gas should be increased, and the droplet diameter of BHP should be decreased. In this study, two types of mist generators were tested for the SOG, with which 65-µm- and 15-µm-diameter droplets were generated. In the 65 µm mist generator, BHP utilization was 22.5% at the Cl2 flow rate of 8.3 mmol/s, and in the 15 µm mist generator, BHP utilization was 41.5% at the Cl2 flow rate of 9.0 mmol/s, that is, BHP utilization of the new SOG, Mist-SOG, markedly exceeded that of the conventional Jet-SOG.

We demonstrate the potential application of silver-pectin nanoparticles on photodynamic therapy, on a solution-base platform. Photodynamic therapy is a medical technique which uses a combination of photosensitizing drugs and light to induce selective damage on the target tissue, by electronically excited and highly reactive singlet state of oxygen. Metal enhanced singlet oxygen generation in riboflavin water solution with silver-pectin nanoparticles was observed and quantified. Here 13 nm silver nanospheres enclosed by a pectin layer were synthesized and it interaction with riboflavin molecule was analyzed. Pectin, a complex carbohydrate found in plants primary cell walls, was used to increase the biocompatibility of the silver nanoparticles and to improve metal enhanced singlet oxygen generation (28.5 %) and metal-enhanced fluorescence (30.7 %) processes at room temperature. The singlet oxygen sensor fluorescent green reagent was used to quantify the enhancement of the riboflavin singlet oxygen production induced by the silver colloid. We report a 1.7-fold increase of riboflavin emission and a 1.8-fold enhancement of singlet oxygen production.