singlet oxygen detection

Trends in Analytical Chemistry, Vol. 30, No. 1, 2011

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Recent developments in the detection of singlet oxygen with molecular spectroscopic methods Haiyan Wu, Qijun Song, Guoxia Ran, Xiaomu Lu, Baoguo Xu Singlet oxygen is a unique reactive oxygen species, as its chemical reactivity derives from its characteristic electronically-excited state. The involvement of singlet oxygen in many important atmospheric, physical, chemical, biological, and therapeutic processes has attracted intense research interest in recent years. The detection and the quantification of singlet oxygen are very important for understanding its mechanism of action in various processes. Due to its highly reactive nature, singlet oxygen has very few direct methods of determination. Only molecular phosphorescence at 1270 nm has been utilized. Indirect methods using spectrophotometric, fluorescent or chemiluminescent probes have therefore been extensively studied. This review reflects recent developments in singlet-oxygen detection with molecular spectroscopic methods. We begin with a brief introduction of the basic properties, the production and the applications of singlet oxygen. With this background information, we review the four molecular spectroscopic methods (i.e., emission, spectrophotometry, fluorescence and chemiluminescence). We pay special attention to attractive chemical probes with high selectivity and sensitivity in quantifying singlet oxygen. ª 2010 Elsevier Ltd. All rights reserved. Keywords: Chemical probe; Chemiluminescence; Emission; Fluorescence; Molecular spectroscopy; Reactive oxygen species; Singlet oxygen; Singlet-oxygen detection; Singlet-oxygen quantification; Spectrophotometry

1. Introduction Haiyan Wu, Qijun Song*, Guoxia Ran, Xiaomu Lu, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China Baoguo Xu School of Communication and Control Engineering, Jiangnan University, Wuxi 214122, China

*

Corresponding author. Fax: +86 510 85917763; E-mail: [email protected]

Singlet oxygen is a member of the general class of reactive oxygen species (ROS) that is believed to play an important role in many fields (e.g., organic synthesis, photodynamic therapies and biological processes). Molecular oxygen differs from most molecules in having P an open-shell triplet ground state 1 g (3O2). There are two low-lying excited states above P the triplet ground state, 1Dg and 1 þ g. Molecular orbital theory predicts two low-lying excited singlet states differing in only the spin and the occupancy of oxygens two degenerate anti-bonding orbitals. Due to the Pþ high energy, low stability of the 1 g state of singlet oxygen, it has a much shorter lifetime than the 1Dg state, so the term ‘‘singlet oxygen’’ often refers to the 1Dg state. The 1 generation of singlet Dg (1O2) Poxygen 3 1 from ground state g ( O2) using direct electronic excitation is spin forbidden and inherently inefficient. Photosensitized

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methods or chemical reactions are therefore more frequently utilized for singlet-oxygen production. Photosensitized generation of singlet oxygen is a simple, controllable method, requiring only oxygen, light of an appropriate wavelength, and a photosensitizer capable of absorbing and using light energy to excite oxygen to its singlet state [1–3]. Alternatively, chemical reactions can generate singlet oxygen [4,5]. A wellknown example is based on the reaction between hydrogen peroxide and hypochlorite. Other sources are also used for the generation of singlet oxygen. For example, singlet oxygen may be produced by decomposition of hydrogen peroxide, naphthalenic endoperoxides, superoxide ion, and triphenyl phosphate ozonide [5,6]. However, in many studies, the generation of singlet oxygen by using the above-mentioned chemical reactions or the photosensitized methods was regarded as complicated due to the presence of some side reactions. 133

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There is therefore still a need to find an environmentally-friendly singlet-oxygen generator with high quantum yield. A new method, based on the calcium peroxide diperoxohydrate (CaO2Æ2H2O2), was reported recently [7]. CaO2Æ2H2O2 can be readily prepared from calcium chloride and hydrogen peroxide. A distinct advantage of this generator, compared with other singlet-oxygen sources, is that CaO2Æ2H2O2 can be recovered by simple filtration, so it can be used in organic synthesis as an alternative to the photochemical methods. Analytical tools for obtaining the concentration, the spatial distribution and temporal information about singlet oxygen are required to understand the chemistry of singlet oxygen in various systems. Due to the short lifetime of singlet oxygen (3.5 ls in H2O and 67.0 ls in D2O) [2], it is difficult to detect the small, highly reactive singlet oxygen. There have been considerable research efforts in singlet-oxygen detection. Some of the detection techniques are well established {e.g., electron-paramagnetic resonance (EPR), microwave spectroscopy, emission spectroscopy, photoionization spectroscopy and mass spectrometry [8]}. Among these, the EPR method has been utilized more frequently to obtain information on the relative efficiency of singlet-oxygen production [9,10]. EPR not only detects singlet oxygen but also quantifies it with high sensitivity. However, EPR signals are often affected by the co-existing ions and solvent, which may lead to significant errors on many occasions. Furthermore, the expensive instrument and the relative complexity of analysis procedures have also prevented its wide application. Several excellent review articles [8,11–21] relevant to singlet-oxygen chemistry have been published. However, many of these papers discussed the formation, chemical reactions and other physical and chemical properties of singlet oxygen. The detection of singlet oxygen with fluorescence and chemiluminescence probes was briefly mentioned in review papers about ROS detection [18– 21]. There is a lack of more recent, comprehensive summaries on the methods of singlet-oxygen detection. The primary focus of this review will therefore be on the recent advances in the detection methods, particularly the molecular spectroscopic probes having high sensitivity and selectivity for singlet-oxygen detection.

2. Molecular emission spectroscopy Direct light emission at ca. 1270 nm, being an intrinsic property of singlet oxygen, was frequently applied for its detection and characterization [22–27]. This transition of singlet oxygen to the ground state is strongly forbidden, so the quantum yield is very low, 105–107, depending on the surrounding environment [28]. The simultaneous transitions of singlet-oxygen dimol (simultaneous emission from two singlet-oxygen mole134

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cules upon collision) can be observed as a red glow at 634 nm and 703 nm in the visible spectral region. The emission intensity depends on the pressure of the oxygen molecules [29]. The time dependence of singlet-oxygen luminescence was studied to gain detailed information about singlet-oxygen interaction with its environment. Time-resolved measurements can provide information about the kinetics of the production or decay of singlet oxygen. Using this tool, the singlet oxygen generated by the hematoporphyrin derivatives (HpDs) and protoporphyrin IX (PpIX) in liposomes was monitored [30,31]. The excitation-energy transfer from photosensitizers to the singlet oxygen inside lipid membranes was elucidated in detail [30]. Microscopy was also utilized to determine spatial resolution in singlet-oxygen detection, so as to gain information about the location of singlet-oxygen production. The light emission of singlet oxygen at the boundary of two immiscible solvents was studied by an optical microscope equipped with an array detector to provide spatial resolution [32,33]. The lateral resolution of 2.5 lm was close to the diffraction limit (1.5–2.0 lm), which depends on the numerical aperture of the optics used. The solvent effect on the rate constant of radiative decay of singlet oxygen was also studied and accuracy was improved by using an array detector [33]. The emission of singlet oxygen at 1270 nm was often monitored with a cryogenic germanium-diode detector. The sensitivity of this kind of detector is not high enough, so that limits wide application of emission spectroscopy. Several research groups focused on improving the light-detection systems in order to monitor singlet oxygen more effectively. Infrared (IR)-sensitive photomultipliers, with improved sensitivity, were reported [2,34,35]. The sensitivity obtained with these detectors was more than one order of magnitude higher than that of the traditional germanium-diode detector, which makes them more suitable for quantitative detection of singlet oxygen. In spite of the above progress in light-detection techniques, detection still suffers from the intrinsic low efficiency of singlet-oxygen emission. Also, the low signal-to-noise ratio is often associated with detection in the near-infrared (NIR) region, which limits the effectiveness of this technique in many applications, so some other indirect methods based on using spectrophotometric, fluorescent or chemiluminescent probes were proposed and found wide application [14].

3. Spectrophotometric probes Spectrophotometry is a more convenient option for detection of excited oxygen molecules. 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 with a reaction constant of 1.3 · 106 L/molÆs. The decrease in absorbance at 355 nm was proportional to singlet-oxygen production of the system under consideration. Other lipid-soluble traps (e.g., 2,5-dimethyfuran derivatives) were also reported [36]. Compared with DPA, these chemical traps exhibit a number of advantages in that their physical quenching effects are negligible, and medium effects on reactivity are also minimal. It is worth noting that incorporation of these compounds into the lipid bilayer depends on their structural modifications (i.e., a charged terminal trimethylammonium head group was seen to facilitate the incorporation of probes in the lipid bilayer). Unfortunately, these chemical traps are soluble in a lipid matrix only, thus limiting their use in aqueous systems. Attempts were made to overcome the poor water solubility of chemical traps. Anthracene-9,10-diyldiethyl disulfate (EAS), anthracene-9,10-bisethanesulfonic acid (AES), anthracene-9,10-divinylsulfonate(AVS), bis-9,10anthracene-(4-trimethyl-phenylammonium)dichloride (BPAA), N,N 0 -di-(2,3-dihydroxypropyl)-9,10-anthracene-dipropanamide (DHPA), and sodium 1,3-cyclohexadiene-1,4-diethanoate (CHDDE) are a few examples that have improved water solubility [37–43]. However, some water-soluble singlet-oxygen traps (e.g., EAS, AES and AVS) are anionic, and they may interact with cationic photosensitizers (e.g., methylene blue), thus prohibiting their applications in some photosensitized systems. By contrast, chemical traps (e.g., BPAA with a cationic substituent, and DHPA, being a non-ionic anthracene derivative) may be better options in such systems [42]. Although spectrophotometric probes received much attention due to their convenience in use, they have some intrinsic disadvantages (e.g., low sensitivity and poor photostability), so their usage is limited when high sensitivity was required.

4. Fluorescence probes 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 [44]. 4.1. Organic As mentioned above, DPA was used as a spectrophotometric probe for singlet-oxygen detection. However, based on measurement of absorbance, the sensitivity of

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DPA or its derivatives is not high enough for detection of singlet oxygen in many applications, so probes based on fluorescence measurement were proposed. DPAX, namely 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-one (as shown in Fig. 1a), was the first fluorescence trap for detection of singlet oxygen [45]. DPAX and its derivatives are almost non-fluorescent but their endoperoxides (DPAX-EPs) are strongly fluorescent with a quantum yield in the range 0.5–0.7. Fluorescence intensity was unchanged upon reaction with hydrogen peroxide, superoxide, or nitric oxide – which showed excellent specificity for singlet oxygen. When electron-withdrawing groups (X = Cl, F) were incorporated at the 2- and 7- positions of the xanthene chromophore, the derivatives of DPAX exhibited stable fluorescence intensity at physiological pH values. The DPAXs were successfully applied in neutral and basic aqueous systems [43,44], but they were not suitable for application in acidic conditions. Similarly, 9-[2-(3-carboxy-9,10-dimethyl) anthryl]-6hydroxy-3H-xanthen-3-one (DMAX) (as shown in Fig. 1b) was also proposed as a fluorescent probe for singlet-oxygen detection [44]. DMAX is non-fluorescent, while its endoperoxide (DMAX-EP) is highly fluorescent. Compared with DPAX, DMAX is more sensitive (being 53-fold more sensitive than DPAX) and less hydrophobic, thereby exhibiting great potential for singlet-oxygen detection in biological samples [44]. For application in organic solutions, 1,3-diphenylisobenzofuran (DPBF) may be a more appropriate choice. It shows strong fluorescence when excited at 415 nm [46] and a decrease in fluorescence intensity was observed when it reacted with singlet oxygen [47]. The efficiency of singlet-oxygen generation in photosensitized systems was evaluated by using DPBF as the probe [48–50]. However, there were also some reports indicating that DPBF was a good candidate for superoxide-anion-radical detection [51,52], so the specificity of DPBF for singlet-oxygen detection is questionable. A selective fluorescent sensor, Singlet Oxygen Sensor Green (SOSG), was commercialized recently by Invitrogen/Molecular Probes. SOSG fluorescence is practically weak blue but it becomes green upon reaction with singlet oxygen. The probe was claimed to have a good selectivity for singlet oxygen, as its response to hydroxyl radicals or superoxide was negligible [53–55], but the reagent can act as a photosensitizer under photo-irradiation, which may complicate its application in photosensitization systems. 4.2. Rare earths Rare-earth-chelate-based luminescence probes also play an important role in singlet-oxygen detection. Compared with the organic fluorescence probes, the rare-earthchelate probes exhibited some distinct advantages, including long luminescence lifetime, large Stokes shifts, http://www.elsevier.com/locate/trac

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a O 1

O

O2

COOH

HO

COOH X

X O

X

X

O

O

HO

O

X=H: DPAX-1-EP Cl: DPAX-2-EP F: DPAX-3-EP

X=H: DPAX-1 Cl: DPAX-2 F: DPAX-3

b CH3

CH3

O 1

H3C

O2

O H3C

COOH

HO

O

COOH

O

DMAX

HO

O

O

DMAX-EP

Figure 1. The chemical structure and the reaction scheme of (a) DPAXs and (b) DMAX with singlet oxygen.

and sharp emission curve, which make them suitable for time-resolved luminescence detection of singlet oxygen. Their large Stokes shift can reduce the influence of excitation light effectively. Among many known rareearth chelates, only a fraction of lanthanide complexes were successfully applied for quantitative detection of singlet oxygen generated from chemical reaction, photosensitization systems and enzyme-catalyzed oxidation systems [56–59]. Time-resolved fluorometry by using lanthanide probes also provides excellent sensitivity. After reaction with singlet oxygen, the corresponding endoperoxides show a long luminescence lifetime in the ls time scale. The short-lived background noise caused by biological samples and the optical components can be eliminated effectively by using long life-time fluorescence probes, so the sensitivity is further improved. For example, an Eu3+ complex, [4´-(9-anthryl)-2,2´:6´,200 -terpyridine-6,6’’-diyl] bis (methylenenitrilo) tetrakis (acetate)-Eu3+ (ATTAEu3+) (as shown in Fig. 2a), was studied as a time-resolved luminescence probe for singlet-oxygen detection [56]. The luminescence quantum yield of EP-ATTA-Eu3+ is 17 times greater than that of ATTA-Eu3+, because the population of the excited state of the Eu3+ ion was increased after formation of the endoperoxide. A limit of detection (LOD) as low as 2.8 nmol/L was obtained by 136


using ATTA-Eu3+ as the singlet-oxygen probe. EPATTA-Eu3+ also possesses favorable chemical stability and the conditional stability constant was determined to be at the 1020 level. No decrease in fluorescence intensity was observed, even after storage of the complex for several days at room temperature. A Tb3+ complex, N,N,N 0 ,N 0 -[2,6-bis-(3 0 -aminomethyl0 1 -pyrazolyl)-4-(900 -anthryl) pyridine]tetrakis (acetate)Tb3+ (PATA-Tb3+, as shown in Fig. 2b) was another effective fluorescent probe for singlet-oxygen detection [57]. Similarly to ATTA-Eu3+, PATA-Tb3+ can specifically react with singlet oxygen to yield a strongly fluorescent endoperoxide with a 23-fold increase in the fluorescence quantum yield. PATA-Tb3+ also exhibits a number of excellent characteristics, including high water solubility, wide pH applicable range and a long fluorescence lifetime of the endoperoxide (2.76 ms), which make it especially suitable for time-resolved fluorescence detection. An LOD of 10.8 nmol/L was achieved by using PATA-Tb3+ as the singlet-oxygen probe. A very rapid reaction rate in endoperoxide formation was observed when MTTA-Eu3+ was utilized as the probe [58], where MTTA-Eu3+ is an abbreviation of [4 0 (10-methyl-9-anthryl)-2,2 0 : 6 0 ,200 -terpyridine-6,600 -diyl] bis (methylenenitrilo) tetrakis(acetate)-Eu3+ (also see Fig. 2c). The reaction-rate constant was measured at the


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a

O O

1

N N

N

Eu3+

O2

N N

N

CO2- O2C CO2- -O2C

N

N

ATTA-Eu 3+

Eu3+

N

CO2- O2C CO2- -O2C

N

EP-ATTA-Eu 3+

O O

b

1

N N

N

N 3+

Tb

CO2- O2C CO2- O2C

PATA-Eu

O2 N N

N N N

N

3+

N

N N

3+

Tb

CO2- O2C CO2- -O2C

EP-PATA-Eu

N

3+

CH3

O CH3 O

c

1

O2

N N

N

Eu3+

N N

N

CO2- O2C CO2- -O2C

N

MTTA-Eu 3+

N

Eu3+

N

CO2- O2C CO2- -O2C

N

EP-MTTA-Eu3+

Figure 2. The chemical structure and the reaction scheme of (a) ATTA-Eu3+, (b) PATA-Tb3+ and (c) MTTA-Eu3+ with singlet oxygen.

1010 L/molÆs level and the probe was successfully applied to monitor the time-dependence of singlet oxygen generated in living cells. For quantitative detection of singlet oxygen, an LOD of 3.8 nmol/L was obtained, comparable to that obtained by ATTA-Eu3+. In conclusion, lanthanide probes have many distinct advantages (e.g., low background interference, widely

applicable pH range and excellent water solubility). We expect these probes to be useful for visualizing temporal and spatial distribution of singlet oxygen in aqueous systems. A main drawback of these lanthanide probes is that they need ultraviolet (UV) light excitation, which may cause damage to cells, thus limiting their application in some biological systems. http://www.elsevier.com/locate/trac

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of CLA and its derivatives is that they lack selectivity for singlet oxygen. The detection of singlet oxygen using CLA, MCLA and FCLA were extensively discussed in previous reviews [16,17,20]. We therefore evaluate only other more recently developed chemiluminescence probes, including tetrathiafulvalenem (TTF)-substituted anthracene probes and probes based on stable dioxetane precursors.

4.3. Other transition-metal complexes In order to overcome the problem associated with the UV light excitation, an ReI complex, Re(CO)3Cl(aeip){aeip= 2-(anthracen-9-yl)-1-ethyl-imidazo[4,5-f] [1,10], phenanthroline (as shown in Fig. 3), was synthesized [60]. The ReI complex is not luminescent in its native state, but it becomes highly fluorescent in the presence of singlet oxygen. The enhancement in luminescence is due to the formation of endoperoxide via the reaction of its anthracene skeleton with singlet oxygen in both neutral and alkaline media. Compared with the other fluorescent probes, the ReI complex shows a high molar absorption at 410 nm and the fluorescence can be initiated with this wavelength of light. The LODs obtained with this compound in neutral and alkaline media are 4.9 nmol/L and 10.5 nmol/L, respectively, which are comparable with that obtained with Eu3+ and Tb3+-complexes [60]. The advantage of visible light excitation may allow the ReI complex to be used in biological systems. However, fluorescence probes with long lifetime and visible light excitation are still very rare, and the search for this type of probe will certainly be the main direction for future research.

5.1. TTF-substituted anthracene probes The probes with a strong electron donor, tetrathiafulvalenem (TTF), and an anthracene luminophore have excellent selectivity and high sensitivity for singlet-oxygen detection. The superb characteristics of these probes may be accounted for by the ability of the anthracene skeleton to react specifically with singlet oxygen to form the highly luminescent endoperoxide. The TTF unit is a strong electron donor that enhances its reaction with singlet oxygen, which is strongly electrophilic. The incorporation of the TTF with the anthracene skeleton can therefore further promote the reactivity of the molecule to singlet oxygen. 4,4 0 (5 0 )-bis[2-(9-anthryloxy)ethylthio] tetrathiafulvalene (as shown in Fig. 5a) was one reported example of such probe [64,65]. The probe exhibits a highly sensitive response to singlet oxygen only, demonstrating a much better selectivity than CLA. 4,5-dimethylthio-4 0 -[2-(9-anthryloxy) ethylthio] tetrathiafulvalene (as shown in Fig. 5b) was another, similar chemiluminescence probe [66]. A good linear relationship between chemiluminescence intensity and the amount of singlet oxygen was established in the H2O2/ClO system and an LOD of 76 nmol/L was obtained for singlet oxygen. The probe was also applied to detect singlet oxygen generated in Fe2+-catalyzed decomposition of tert-butyl hydroperoxide. However, the application of these two probes was restricted to a mixed solvent of tetrahydrofuran and H2O. By constrast, tetrathiafulvalene-anthracene dyad 1 (as shown in Fig. 5c) can be dissolved easily in ethanol and methanol, and that may permit detection of singlet oxygen under a relative mild medium, although the new probe still lacks water solubility [67].

5. Chemiluminescence probes Chemiluminescence is believed to be one of the most sensitive methods in singlet-oxygen detection. Compared with fluorescence, chemiluminescence does not require excitation light, so background fluorescence and scattering light interference are eliminated. Consequently, the signal-to-noise ratio can be improved and the possible damage to living cells, caused by UV irradiation in fluorescence measurement, can be avoided. A number of chemiluminescence probes were developed in recent years for singlet-oxygen detection. The set of widely-used chemiluminescence probes for singlet oxygen includes 2-methyl-6-phenyl- 3,7-dihydroimidazo [1,2-a] pyrazin-3-one (CLA), and its derivatives MCLA and FCLA (Fig. 4a–c) [61–63]. These compounds emit light spontaneously in the presence of singlet oxygen. However, the probes react not only with singlet oxygen but also with superoxide anion, so the major drawback

N

N

1

Re(CO)3Cl N

N

ReI complex

O2

O O

N

N

N

N

Re(CO)3Cl

ReI complex-EP

Figure 3. The ReI complex and its reaction with singlet oxygen.

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a O N

O

CH3 N

O 1

O2

N

NH

Trends

O CH3

*O N

-CO2

CH3

O

NH

N

+

N H

N

H

N

CH3 NH

+ hν (380 nm)

N

CLA O N

c

b O N

CH3 N

S N H

HNCNH

CH3

O

N COONa

N H H3CO

HO MCLA

O

O

FCLA

Figure 4. (a) The chemical structure of CLA and its reaction scheme with singlet oxygen; (b) the chemical structure of FCLA and (c) the chemical structure of MCLA.

5.2. Stable dioxetane precursors The chemistry of 1,2-dioxetanes offers promise for selective and sensitive detection of singlet oxygen [14,68–72]. Dioxetanes, generated by the reaction of

singlet oxygen with a sporoadamantyl-substituted vinyl ether through the [2+2] mechanism, were exploited for trap-and-trigger chemiluminescence probes [70,71]. The singlet-oxygen concentration can be accurately

a

b

c

Figure 5. The chemical structure and reaction scheme of (a) 4,4 0 (5 0 )-bis [2-(9- anthryloxy)ethylthio] tetrathia-fulvalene, (b) 4,5-dimethylthio-4 0 [2-(9-anthryloxy) ethylthio] tetrathiafulvalene with singlet oxygen, and (c) the chemical structure of tetrathiafulvalene-anthracene dyad 1.


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measured at the 5 · 1013 mol/L level with these probes. The principle is based on the reaction of singlet oxygen with the precursor to form the dioxetane (trap of singlet oxygen), which is then quantified by its chemiluminescence signal after adding a chemical trigger. Stable dioxetane precursors, as trap-and-trigger chemiluminescent probes, were successfully applied to measure the microheterogeneous distribution of singlet oxygen in irradiated humic-acid solutions [72]. The concentrations of singlet oxygen in the hydrophobic interior of chromophoric dissolved organic matter (CDOM) were found to be 2–3 orders of magnitude higher than that of the bulk aqueous phase, leading to a higher photochemical degradation rate of hydrophobic pollutants. Although several examples of chemiluminescence probes have been reported for singlet-oxygen detection in recent years, exploring new chemiluminescence probes that possess new characteristics (e.g., high sensitivity, low toxicity or non-toxicity, and environmentalfriendly nature) would provide the impetus for further studies in this area.

6. Conclusions and future prospects Singlet oxygen, as a highly reactive form of molecular oxygen, plays an important role in many environmental and biomedical processes. Sensitive and selective detection and quantification of singlet oxygen provide vital information for understanding its involvement and action mechanism in these processes. A mature analytical tool, EPR has a major disadvantage of requiring an expensive instrument and complicated operating procedures. As a specific method, direct emission of singlet oxygen at about 1270 nm is useful for singlet-oxygen detection, but the low efficiency of the emission results in weak signals. Low sensitivity is also a limitation for the spectrophotometric method. Fluorescence and chemiluminescence probes, exhibiting very high sensitivity and desirable selectivity, therefore have great potential for singlet-oxygen determination. Their time and spatial resolution can provide detailed information on the site and the kinetics of singlet-oxygen production or decay. We can foresee that synthesis of new probes with improved analytical characteristics will continue into the future.

Acknowledgements This work was supported by the National Science Foundation of China (Project Nos. 20977042 and 30971689) and the Research Fund for the Doctoral Program of Jiangnan University (No. 104205020 5091590).

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