Is There a Simple and Easy Way to Detect Singlet Oxygen? Comparison of Methods for Detecting Singlet Oxygen and Application to Measure Scavenging Activity of Various Compounds

Reactive oxygen species (ROS) are known to exert both beneficial and harmful effects in the human body. The beneficial aspects of ROS are, for example, roles in signaling cascades and immunological defense. In contrast, overproduction of ROS has been reported to be involved in various diseases such as cancer, cardiovascular disease, and diabetes. To prevent the harmful effects of ROS, organisms have antioxidant enzymes and various antioxidative compounds. Various antioxidative compounds are present in foods, beverages, agricultural products, plants, as well as in the human body [1]. Measurement of antioxidant activity is important for developing new drugs, health foods, and nutritional supplements.


Introduction
Reactive oxygen species (ROS) are known to exert both beneficial and harmful effects in the human body. The beneficial aspects of ROS are, for example, roles in signaling cascades and immunological defense. In contrast, overproduction of ROS has been reported to be involved in various diseases such as cancer, cardiovascular disease, and diabetes. To prevent the harmful effects of ROS, organisms have antioxidant enzymes and various antioxidative compounds. Various antioxidative compounds are present in foods, beverages, agricultural products, plants, as well as in the human body [1]. Measurement of antioxidant activity is important for developing new drugs, health foods, and nutritional supplements.
Atmospheric oxygen (triplet oxygen, 3 O 2 ) is a stable form of oxygen molecule. The other form, singlet oxygen ( 1 O 2 ), is highly reactive and considered as one of the ROS, although it is not a radical molecule. 1 O 2 reacts with many kinds of biological components such as lipids, proteins, and nucleic acids [2][3][4]. 1 O 2 is short lived because it reacts with biomolecules and collisions with water molecules rapidly causing the return of 1 O 2 to the ground state. Thus, it is not easy to quantify 1 O 2 . 1 O 2 is thought to be a cause of some disorders such as photo-aging, skin damage, and erythropoietic porphyria [5]. On the other hand, cell death and tissue destruction induced by 1 O 2 are used for photodynamic therapy (PDT), a promising cancer treatment [6][7][8].
affect the quenching activity of the test compound, it is not ideal to use light or heat for 1 O 2 measurement. In this paper, we summarize and compare various methods for the measurement of 1 O 2 for the purpose of evaluating the 1 O 2 quenching activity of various test compounds. Artificial 1 O 2 generation is usually performed either by photosensitization of photosensitizers, such as rose bengal and porphyrins, or thermal decomposition of 4-methyl-1,4-etheno-2,3-benzodioxin-1(4H)-propanoic acid (EP).

UV-Vis Spectroscopy
UV-Vis absorbance measurement is simple, speedy, and the most commonly used method. Anthracene and benzofuran are known as compounds that selectively scavenge 1 O 2 . The absorbance of these aromatic molecules decreases when they are oxidized by 1 O 2 , and the amount of 1 O 2 can be estimated semi-quantitatively from the decrease in absorbance. UV-Vis spectroscopy is somewhat less sensitive than fluorescence spectroscopy. However, UV-Vis spectroscopy is less prone to artifacts, since the absorption spectrum of a compound with high absorption coefficient cannot be dominated by small amounts of contaminants. One of the most widely used 1 O 2 probes for UV-Vis spectroscopy is 1,3-diphenylisobenzofuran (DPBF, Figure 1), which is commercially available at a low cost. DPBF reacts with 1 O 2 in an essentially diffusionlimited reaction rate and thus has very high sensitivity. It traps up to 50% of 1

Fluorescence Spectroscopy
Comparing the quantitative measurements of absorbance and fluorescence, the detection limit of fluorescence is generally three orders of magnitude lower. To detect 1 O 2 , a fluorescent sensor called Singlet Oxygen Sensor (SOSG, Figure 1) [13] has been used widely, ranging from material studies to medical applications, although the probe is expensive. SOSG exhibits weak blue fluorescence with excitation peaks at 372 nm and 393 nm and emission peaks at 395 nm and 416 nm. Upon reaction with 1 O 2 , endoperoxide, the immediate product of SOSG, exhibits green fluorescence with excitation and emission peaks at 504 nm and 525 nm, respectively. Therefore, SOSG can readily detect 1 O 2 . However, it was demonstrated that SOSG can generate 1 O 2 under exposure to UV (355 nm) or visible light (532 nm) in the absence of photosensitizers [14]. It was confirmed that the irradiation of visible light induces SOSG transitions to a triplet excited state, which converts 3 O 2 to 1 O 2 [15]. Therefore, a positive error can occur with respect to the amount of 1 O 2 measured by SOSG. In  Arch Pharmacol Ther. 2020 Volume 2, Issue 2 addition, since many photosensitizers used to generate 1 O 2 have a pronounced absorption band in the visible region, if the absorption overlaps with the emission peak of the fluorophores of probes, the signal produced by the reaction with 1 O 2 will be diminished. Photosensitizers, such as rose bengal and eosin Y, exhibit an intense absorption band at 525 nm, which coincides with the emission peak of SOSG endoperoxide [16]. Incorrect use of SOSG may lead to misinterpretation of the data on the yield of 1 O 2 production by photosensitization reactions. For biological application, a new type of fluorescence probe for 1 O 2 (Si-DMA, Figure 1) was developed [17]. Si-DMA has an emission peak at 680 nm and is permeable through cell membranes. It is now commercially available and can be used for imaging of 1 O 2 produced in mitochondria and by PDT.

Near-infrared Spectroscopy
1 O 2 can be directly measured by detecting weak light emission in the near infrared region. Phosphorescence at 1270 nm emitted by 1 O 2 can be detected specifically at a low quantum yield [18]. By using an improved nearinfrared-sensitive photomultiplier tube, time-resolved 1 O 2 luminescence was measured in various solutions of aluminum tetrasulphonated phthalocyanine and Photofrin [19]. Time-resolved analysis showed a strongly reduced lifetime of 1 O 2 and an increased lifetime of triplet state of the photosensitizer in the intracellular component [19]. This method is often used for imaging in cells and animals. However, the method requires specialized instruments because of the low quantum yield.

Singlet Oxygen Absorption Capacity
Recently, a new assay method to quantify 1 O 2 absorption capacity (SOAC) was developed [20][21][22][23]. The SOAC values were measured in ethanol/chloroform/D 2 O (50:50:1, v/v/v) solution at 35°C using a UV-Vis spectrophotometer equipped with a six-channel cell positioner and a temperature control unit. The reaction of antioxidants with 1 O 2 was measured using the competition reaction method, where EP was used for 1 O 2 generation and DPBF was used as an absorbance probe. In the SOAC method, 1 O 2 quenching activity is evaluated kinetically, and the activity of various phenolic antioxidants can be compared. The method is useful for evaluating the 1 O 2 quenching activity of both lipophilic and hydrophilic antioxidants. The method allows for measurement of activity that differs in five orders of magnitude (different rate constants of 10 10 M -1 s -1 for lycopene to 10 5 M -1 s -1 for ferulic acid). However, in the SOAC assay, decrease in the absorbance is observed in 120 to 180 min, during which the test compound may become photodegraded.

Electron Spin Resonance
Electron spin resonance (ESR) is a non-destructive method that detects unpaired electrons and is specific to free radicals. Since 1 O 2 is a ROS but is not a free radical, it cannot be detected directly by ESR. A 1 O 2 detector probe, 2,2,6,6-tetramethylpiperidine (TEMP, Figure 1), was introduced in 1976 [24]. TEMP reacts with 1 O 2 to produce a stable nitroxide radical, 2,2,6,6-tetramethylpiperine-N-oxyl (TEMPO), which can be quantified by ESR spectroscopy. Other sterically hindered secondary amines, such as 2,2,6,6-tetramethyl-4-piperidinol (TEMP-OH, Figure 1) and 2,2,6,6-tetramethyl-4-piperidone (TEMPD, Figure 1), are also used as 1 O 2 detector probes [25][26][27]. Photoirradiation of rose bengal is often used as a 1 O 2 generation method. When 1 O 2 generated in the rose bengal system is detected by ESR using a hindered secondary amine probe, the signal of the nitroxide radical derived from the secondary amine probe is measured. However, rose bengal reportedly generates ROS other than 1 O 2 , such as super oxide anion radical and hydroxyl radical [26,28], which may affect the ESR signal. Indeed, when visible light was irradiated to the mixed solution of TEMP-OH and rose bengal, obvious splitting was observed in the ESR spectrum. This observation suggested the overlap of 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) [25]. It was reported that the exposure of TEMPOL to hydroxyl radical led to the appearance of a new triplet ESR signal attributable to TEMPON, along with a reduction in the intensity of TEMPOL signals [29]. The following is a possible explanation for the ESR signal splitting observed in the mixed solution of rose bengal and TEMP-OH. TEMP-OH reacts with 1 O 2 to form TEMPOL, which is oxidized to TEMPON by hydroxyl radicals generated from rose bengal. On the other hand, when EP was used for 1 O 2 generation and TEMP-OH was used as a detector probe, no splitting of the three-line ESR spectrum was obtained, although the signal intensity was very small [25]. When the mixture solution of EP and TEMP-OH was reacted at 35°C for 40 min, the ESR signal intensity was smaller than that obtained with irradiation of rose bengal with visible light for 3 min, supporting that 1 O 2 production from EP is smaller than that from rose bengal. For the detection by ESR, if EP is used as a 1 O 2 generator, it is recommended to add D 2 O in the system to increase the signal intensity. Although the amount of 1 O 2 generated from EP is small, it is possible to test the 1 O 2 scavenging activity of test compounds with a reaction time of 30~40 min in the presence of D 2 O using TEMP-OH as a detector probe.

Conclusion
Our opinions with respect to the pros and cons of the detection methods for 1 O 2 are summarized in Table 1