Evaluation of cold atmospheric pressure plasma irradiation of water as a method of singlet oxygen generation

Abstract

We used cold atmospheric pressure plasma jet to examine in detail ¹O₂ generation in water. ESR with 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide, a secondary amine probe, was used for the detection of ¹O₂. Nitroxide radical formation was detected after cold atmospheric pressure plasma jet irradiation of a 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide solution. An ¹O₂ scavenger/quencher inhibited the ESR signal intensity induced by cold atmospheric pressure plasma jet irradiation, but this inhibition was not 100%. As 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide reacts with oxidizing species other than ¹O₂, it was assumed that the signal intensity inhibited by NaN₃ corresponds to only the nitroxide radical generated by ¹O₂. The concentration of ¹O₂ produced by cold atmospheric pressure plasma jet irradiation for 60 s was estimated at 8 μM. When this ¹O₂ generation was compared to methods of ¹O₂ generation like rose bengal photoirradiation and 4-methyl-1,4-etheno-2,3-benzodioxin-1(4H)-propanoic acid (endoperoxide) thermal decomposition, ¹O₂ generation was found to be, in decreasing order, rose bengal photoirradiation ≥ cold atmospheric pressure plasma jet > endoperoxide thermal decomposition. Cold atmospheric pressure plasma jet is presumed to not specifically generate ¹O₂, but can be used to mimic states of oxidative stress involving multiple ROS.

Introduction

Plasma, the fourth state of matter, is a partially ionized gas consisting of electrons, photons, atoms, radicals, and various excited and non-excited molecules. Advances in plasma technology have enabled plasma generation at near atmospheric pressure and room temperature, referred to as cold atmospheric pressure plasma (CAP). CAP has potential in biomedical applications such as chronic wound treatment,^(1–4) infection control,^(5–8) and cancer therapy.^(9–12) CAP induces, in the liquid phase and inside cells, reactive oxygen species (ROS) formation, such as hydroxyl radicals (^•OH), superoxide anion radicals (^•O₂⁻), atomic oxygen and reactive nitrogen species (RNS), such as nitric oxide (NO), nitrogen dioxide (NO₂) and peroxynitrite(ONOO⁻).^(13–16) Previously we reported the generation of reactive species such as ^•OH, ^•H, and H₂O₂ in water by cold atmospheric pressure plasma jet (CAPPJ) irradiation.⁽¹⁷⁾

Atmospheric molecular oxygen (triplet oxygen) is a stable form of molecular oxygen. Singlet oxygen (¹O₂) is, however, highly reactive and considered a ROS. In living organisms, ¹O₂ is considered responsible for certain disorders, including photo-aging, skin damage, and erythropoietic porphyria.^(18–20) On the other hand, the cell death and tissue destruction induced by ¹O₂ are used in photodynamic therapy,^(21,22) which shows promise as a cancer therapeutic. Thus, ¹O₂ itself and/or ¹O₂ scavengers/quenchers may be used as treatments. From this perspective, it is important to establish easy and reliable methods for ¹O₂ measurement and generation. Several measurement methods exist, including electron spin resonance (ESR) spectroscopy with sterically hindered secondary amine probes, near-infrared luminescence, spectrophotometric analysis with diphenylbenzofuran, and fluorescence measurement with specific fluorescent probes.^(23–29) Along with measuring ¹O₂, facile ¹O₂ generation is also important. Widely used methods for ¹O₂ generation include photoirradiation of photosensitizers such as rose bengal ^(30,31) and porphyrins, and by endoperoxide thermal decomposition.

CAPPJ irradiation of water generates various ROS ^(17,32–34) with the species type and amount generated controlled by irradiation parameters and reaction conditions.⁽³⁵⁾ For example, we reported the influence of irradiation distance on ^•OH and H₂O₂ formation.⁽¹⁷⁾ Reports also indicate ¹O₂ generation by CAP irradiation.^(15,35,36) In the present paper, we used CAPPJ with helium as a carrier gas to evaluate CAPPJ as a ¹O₂ generating method in water. ESR with a secondary amine probe was used for ¹O₂ detection and results were compared with other ¹O₂ generation methods.

Materials and Methods

Chemicals

Ultrapure helium gas (>99.999%) was obtained from Saisan Co., Ltd. (Saitama, Japan). 2,2,6,6-tetramethyl-4-piperidone (TEMPD), 2,2,6,6-tetramethyl-4-piperidinol (TEMP-OH), 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide (TPC) and carbamoyl-PROXYL were obtained from Sigma-Aldrich Co. Ltd. (St. Louis, MO). Histidine (His), sodium azide (NaN₃) and 1,4-diazabicyclo[2,2,2] octane (DABCO) were obtained from Fujifilm Wako Pure Chemical Industries Ltd. (Osaka, Japan). 4-Methyl-1,4-etheno-2,3-benzodioxin-1(4H)-propanoic acid (endoperoxide, EP) was obtained from WAKEN B TECH Co. Ltd. (Kyoto, Japan). All reagents were of the highest grade available and used without further purification.

Irradiation of water with CAPPJ

CAPPJ was generated using a plasma head (TPN-20; NU global, Nagoya, Japan) and an electric regulating unit (PN-110TPG; NU global) with helium as the carrier gas. The helium gas flow rate was regulated by a mass flow controller (CUBE GM2; Fcon Co., Ltd, Nankoku, Kochi, Japan), and set at 5 L/min except for the experiment of Fig. 3, where the flow rate of 3 L/min was used. The flow rate of 3 L/min resulted in slightly greater nitroxide radical formation (approximately 20% increase) than the 5 L/min condition (unpublished result). The 1 ml sample solution was placed in a glass container, inner diameter 2 cm and 2 cm in height, and irradiated with CAPPJ at a distance of 1.5 cm between the solution surface and plasma jet tip.

ESR measurement

The CAPPJ irradiated solution was immediately transferred to a quartz flat cell, and ESR measured. The X-band ESR spectra were recorded using a JES-FA100 ESR spectrometer (JEOL Ltd., Akishima, Tokyo, Japan). Mn²⁺ was used as the external standard. The signal intensity was calculated by dividing the signal height at the lowest magnetic field side of the nitroxide radical by the signal height of Mn²⁺. ESR spectra were analyzed with a WinRad ESR Data Analyzer (Radical Research Co., Ltd., Hino, Tokyo, Japan).

Generation of ¹O₂ from EP or rose bengal solution photoirradiation

The ¹O₂ generating reagent, EP, was heated at 40°C to generate ¹O₂ by thermal decomposition. Alternatively, ¹O₂ was generated by visible light irradiation of rose bengal solution using a Schott Megalight 100 with halogen lamp (Moritex Corporation, Yokohama, Japan) as the light source at a distance of 3 cm.

Results

Oxidation of ¹O₂ detector probes by CAPPJ irradiation of water

The sterically hindered secondary amines such as TEMP-OH, TEMPD and TPC are often used as ¹O₂ detector probes. TEMPD and TEMP-OH with a piperidine skeleton, and TPC with a pyrrolidine skeleton, react with ¹O₂ to form stable nitroxide radicals (Fig. 1) which are subsequently detected by ESR.

Fig. 1.

Chemical structure and reaction of ¹O₂ detection probes.

By irradiating a TEMPD solution (0.2 mM) with CAPPJ for 1 min, a three-line ESR signal, typical for nitroxide radicals, was observed (Fig. 2A). When a TEMP-OH solution (0.2 mM) was irradiated with CAPPJ, signal splitting in the ESR spectrum was observed (Fig. 2B), which may be due to oxidation of the hydroxy group at the 4’ position of the TEMP-OH piperidine skeleton by ^•OH.^(28,37) When a TPC solution was irradiated by CAPPJ, a similar three-line signal was detected as in the TEMPD solution (Fig. 2C). No nitroxide radical was observed for all three secondary amine probes in the presence of helium gas flow without discharge of the CAPPJ. Although nitroxide radicals were formed and detected by all three detection probes, TPC was superior to TEMP-OH and TEMPD, as TEMP-OH shows ESR signal splitting and TEMPD is easily oxidized by air and dissolved oxygen.⁽²⁸⁾ We therefore used TPC in subsequent experiments.

Fig. 2.

ESR spectra obtained after CAPPJ irradiation of secondary amine solutions. Each solution was irradiated by CAPPJ for 1 min at distance of 1.5 cm. The helium gas flow rate was set to 5 L/min. (A) 0.2 mM TEMPD solution, (B) 0.2 mM TEMP-OH solution, and (C) 1 mM TPC solution. MnO₂ signal (arrows) is shown as the external standard.

Time-dependent oxidation of TPC by CAPPJ irradiation

The ESR signal intensity increased linearly during the 1 min CAPPJ irradiation of the TPC solution (2 mM, 1 ml) (Fig. 3). The same tendency was observed in the irradiation of TEMPD and TEMP-OH solutions (data not shown). Therefore, it is plausible that the secondary amine moiety of the ¹O₂ detection probes was oxidized to form nitroxide radicals by ¹O₂.

Fig. 3.

Change in the ESR signal intensity of the nitroxide radical by CAPPJ irradiation time. TPC solution (2 mM, 1 ml) was irradiated with CAPPJ at a distance of 1.5 cm and irradiation time varied. The helium gas flow rate was 3 L/min. After irradiation, the solution was immediately transferred to a quartz flat cell, and ESR measured. Error bar shows SD (n = 3).

Effect of TPC concentration on ESR signal intensity

Varying concentrations of TPC solutions were irradiated with CAPPJ and ESR spectra were measured. When the TPC concentration increased, the ESR spectra intensity increased. This increase was not linear, however, and saturated at high TPC concentrations (Fig. 4A). A double reciprocal plot⁽³⁸⁾ (pseudo-Lineweaver-Burk plot with the reciprocal of signal intensity versus the reciprocal of TPC concentration) yielded a straight line (Fig. 4B and C). The value of the y-intercept was 0.0739 when the 60 s irradiation data was used. The inverse of this value (1/0.0739 = 13.5) corresponds to the quantity of nitroxide radicals produced over 60 s in the presence of a large excess of TPC. When the 30 s irradiation data was used, the y-intercept was 0.1479 and the inverse of this value was 1/0.1479 = 6.8. In other words, as there was sufficient TPC in the system, it is presumed that the TPC reacts with most of the ¹O₂ generated. Nitroxide radical concentrations were then estimated by a calibration curve, generated by relating the signal intensity and radical concentration for carbamoyl-PROXYL solutions of known concentrations. The amount of nitroxide radical detected was estimated to be 17.5 μM for 60 s of CAPPJ irradiation and 8.7 μM when irradiated for 30 s.

Fig. 4.

Change in ESR signal intensity by TPC solution concentration. TPC solution (1 ml) with varying concentrations (0.2–100 mM) was irradiated by CAPPJ at a distance of 1.5 cm. The helium gas flow rate was set to 5 L/min. After the irradiation, the solution was immediately transferred to a quartz flat cell, and ESR spectra measured. Error bar shows SD (n = 3). (A) The signal intensity is plotted against the TPC concentration. The irradiation time was 60 s. (B) Double reciprocal plot of the data used for the graph (A). The reciprocal of the signal intensity is plotted against the reciprocal of the TPC concentration. (C) Double reciprocal plot of the data of 30 s irradiation. The reciprocal of the signal intensity is plotted against the reciprocal of the TPC concentration.

Effect of ¹O₂ quenchers/scavenger on nitroxide radical production

To verify nitroxide radical formation due to ¹O₂, we added His as a ¹O₂ scavenger, or NaN₃ and DABCO as a ¹O₂ quencher (Fig. 5A). The ESR signal intensity was significantly suppressed by His, NaN₃, and DABCO addition. The suppression was 56 ± 1.4% by 2 mM His, 36 ± 0.8% by 2 mM NaN₃, and 26 ± 4.6% by 2 mM DABCO. Furthermore, NaN₃ and His inhibited signal intensity in a concentration dependent manner (Fig. 5B). The inhibition was 57 ± 4.5% in the presence of 4 mM NaN₃ and 74 ± 0.5% in the presence of 4 mM His. However, complete inhibition was not observed even in the presence of 4 mM quencher/scavenger.

Fig. 5.

Change in the ESR signal intensity by the addition of ¹O₂ quencher/scavenger. (A) The mixture (1 ml) of TPC solution (2 mM) and ¹O₂ scavenger (2 mM) was irradiated by CAPPJ for 60 s. The solution was irradiated at a distance of 1.5 cm. The helium gas flow rate was set to 5 L/min. Immediately after irradiation, the solution was transferred to a quartz flat cell and the ESR spectrum measured. Error bar shows SD (n = 3). *p<0.01 vs control by t test. (B) Effect of quencher/scavenger concentration on the signal intensity. The mixture (1 ml) of TPC solution (2 mM) and NaN₃ solution (1, 2, 4 mM) or His solution (1, 2, 4 mM) was irradiated with CAPPJ for 60 s. Error bar shows SD (n = 3).

Effect of mannitol on nitroxide radical production

To examine whether ^•OH is involved in the nitroxide radical formation, we added mannitol, a scavenger of ^•OH, to the solution. As shown in Fig. 6, the nitroxide radical ESR signal intensity was inhibited 10%, 25%, and 46% by 1 mM, 2 mM, and 4 mM mannitol, respectively.

Fig. 6.

Change in the ESR signal intensity by mannitol addition, a hydroxyl radical scavenger. The mixture (1 ml) of TPC solution (2 mM) and mannitol solution (1–4 mM) was irradiated with CAPPJ for 60 s. The solution was irradiated at 1.5 cm. The helium gas flow rate was set to 5 L/min. Immediately after irradiation, the solution was transferred to a quartz flat cell and the ESR was measured. Error bar shows SD (n = 3).

Comparison of nitroxide radical formation by CAPPJ to other ¹O₂ generation methods

We compared CAPPJ ¹O₂ generation to that by rose bengal photoirradiation and EP thermal decomposition (Fig. 7). The ESR signal intensity observed from CAPPJ irradiation was inhibited by addition of a large amount of NaN₃ (100 mM). The inhibition was 28 ± 2.8% at 30 s and 35 ± 3.0% at 60 s. The ESR signal intensity observed in rose bengal photoirradiation (0.2 mM) for 30 s and 60 s was similar as that observed for 30 and 60 s CAPPJ irradiation. Addition of a large amount of NaN₃ (100 mM) to the rose bengal system was accompanied by decreased signal intensity. The signal inhibition was 55 ± 5.7% at 30 s and 65 ± 5.2% at 60 s. For the thermal decomposition of EP, unlike the CAPPJ irradiation and the photoirradiation of rose bengal, the ESR signal could not be detected within a few minutes, despite a higher EP concentration (the SOAC assay used 0.4–0.8 mM, while we used 2 mM) and higher temperature (the SOAC assay used 35°C, while we used 40°C).^(25,26,39) Increasing the reaction time to 60 min, enabled ESR signal detection, although the intensity was very low. The addition of 100 mM NaN₃ resulted in 77 ± 5.2% inhibition on the ESR signal intensity at 60 min.

Fig. 7.

Comparison of ¹O₂ generation by CAPPJ with other ¹O₂ generation methods in the presence and absence of NaN₃. TPC concentration was 75 mM and NaN₃ concentration was 100 mM. TPC solution was irradiated with CAPPJ at a distance of 1.5 cm for 30 or 60 s, with or without NaN₃. The helium gas flow rate was set to 5 L/min. The mixed solutions of TPC and rose bengal (0.2 mM) were irradiated with visible light from a distance of 3 cm for 30 or 60 s, with or without NaN₃. The mixed solutions of TPC and EP (3 mM) were incubated at 40°C for 60 min, with or without NaN₃. Immediately after irradiation, the solution was transferred to a quartz flat cell and the ESR was measured. Error bar shows SD (n = 3).

Discussion

Of the several ¹O₂ measurement methods, ESR with sterically hindered secondary amine probes is often used.^(27,29,38) The secondary amine probe is oxidized to a stable nitroxide radical, which is detected by ESR. Of the several secondary amine probes,^{(29,38,40–42)} we compared three, TEMPD, TEMP-OH, and TPC, and found that TPC was superior (Fig. 2) and therefore, TPC was chosen for the present work.

The ESR signal intensity increased linearly during the 1 min CAPPJ irradiation of the TPC solution, indicating ¹O₂ formation. The concentration of the resultant nitroxide radical is related to the concentration of generated oxidant. We estimated the amount of ¹O₂ generated by CAPPJ irradiation by assuming 100% ¹O₂ reaction with TPC. By increasing the TPC concentration, the reaction between the oxidant and TPC increases. As shown in Fig. 4A, the ESR signal intensity saturated at higher TPC concentrations, and the double reciprocal plot yielded a straight line. The intercept of the plot y-axis shows the inverse of the signal intensity at an infinite TPC concentration. This signal intensity was then converted to nitroxide radical concentration using the calibration curve. The nitroxide radical concentrations were thus estimated to be 17.5 μM and 8.7 μM for the 60 s and 30 s sample irradiations, respectively. However, these estimations are not equal to the ¹O₂ concentration generated as the oxidation reaction efficiency by ¹O₂ is not 100% and oxidation species other than ¹O₂ contribute to the signal increase. Although the secondary amine probes we used are frequently used for ¹O₂ detection, probe reaction with other ROS is also possible. Indeed, Jablonowski et al. described nitroxide radical formation by the reaction of secondary amine probes with O₃ and ^•O.⁽⁴³⁾

We estimated the contribution of ¹O₂ in nitroxide radical production among the total oxidizing species formed by CAPPJ irradiation. NaN₃, DABCO, and histidine, which scavenge/quench ¹O₂, significantly inhibited the nitroxide radical signal intensity induced by CAPPJ irradiation (Fig. 5). This observation confirms the involvement of oxidizing species other than ¹O₂ in nitroxide radical formation. The ESR signal intensity was also inhibited by the presence of mannitol (Fig. 6), implicating ^•OH as an oxidizing species. Considering that nitroxide radical formation inhibition by NaN₃ and histidine was about 60–70% and mannitol inhibition was about 45%, ^•OH is clearly another major oxidizing species produced during CAPPJ irradiation that contributes to nitroxide radical formation. Indeed, we previously reported ^•OH generation by CAPPJ irradiation under the same conditions as the present study.⁽¹⁷⁾ The concentration of ^•OH generated by 60 s irradiation was high, approximately 270 μM (unpublished result).

As TPC also reacts with oxidizing species other than ¹O₂, the signal intensity inhibited by NaN₃ can be regarded as the nitroxide radical generated by only ¹O₂.⁽¹⁵⁾ Here, we estimated the concentration of ¹O₂ generated by CAPPJ irradiation in the presence of 75 mM TPC and 100 mM NaN₃, the same concentrations as previously used ⁽¹⁵⁾ and compare with results obtained by rose bengal photo-irradiation and EP thermal-decomposition. For EP thermal decomposition, the ESR signal was very small, suggesting limited ¹O₂ generation. As the SOAC measurement is performed over a long time, the EP decomposition forming ¹O₂ is slow. In the case of CAPPJ irradiation and rose bengal photoirradiation, nitroxide radical formation was greater than that from EP decomposition. The nitroxide radical formation amount was CAPPJ > rose bengal > EP. On the other hand, inhibition by NaN₃ was 35% for CAPPJ, 65% for rose bengal, and 77% for EP, which indicates EP thermal decomposition generates relatively pure ¹O₂. Considering total amounts of the nitroxide formed and inhibition by NaN₃, the order of ¹O₂ generation was estimated as rose bengal ≥ CAPPJ > EP. Since the rose bengal photoirradiation uses visible right, the energy of the light is quenched when a colored compound is used. Therefore, the rose begal photoirradiation is not applicable to the sample with colors. In contrast, the color of the sample does not affect the¹O₂ generation by CAPPJ irradiation.

It is reported that not only ¹O₂ but also ^•OH is generated in rose bengal photoirradiation.⁽²⁸⁾ Therefore, ^•OH may be an active oxidizing species in the rose bengal photoirradiation. We reported ^•OH, ^•H, and H₂O₂ formation by CAPPJ irradiation.⁽¹⁷⁾ Contribution of O₃ in nitroxide radical formation was also reported.^(34,43) In our approach, the contribution of O₃ cannot be ruled out as we detected a slight ozone-like smell during CAPPJ irradiation, however the O₃ contribution is likely small given the large estimated ^•OH contribution, at around 45% of total signal intensity.

Only 30–50% of the values obtained in Fig. 4B and C are generated by ¹O₂. From this, the total nitroxide concentration, 17.5 μM estimated at an infinite TPC concentration, converts to around 5–8 μM of ¹O₂ produced by 60 s CAPPJ irradiation. This value is the same order of magnitude as the amount of ¹O₂ produced reported by Takamatsu et al.⁽¹⁵⁾ We have values of about 270 μM for ^•OH generation (unpublished data) and about 60 μM for H₂O₂ generation⁽¹⁷⁾ by CAPPJ irradiation under the same conditions as the present study. The amount of these ROS produced were more than one order of magnitude larger than the amount of ¹O₂ produced estimated in this study. To increase ¹O₂ production by CAPPJ irradiation, various parameters can be tuned. Changing the carrier gas is one method to increase ¹O₂ production. Indeed, carrier gas selection is reported to change the amount of ¹O₂ generated.⁽¹⁵⁾ When O₂ and CO₂ are used as carrier gases, ¹O₂ generation was 63 μM and 90 μM, respectively.

In conclusion, ¹O₂ generation by CAPPJ irradiation under these conditions was confirmed. CAPPJ irradiation, however, generates reactive species other than ¹O₂ and the amount of ¹O₂ generated is rather low at a few μM. Therefore, CAPPJ with helium as the carrier gas under our reaction conditions does not generate ¹O₂ specifically. However, the system is easy to operate and superior to rose bengal photoirradiation for CAPPJ irradiation. The CAPPJ can be used as an oxidative stress inducing system with multiple kinds of ROS.

Author Contributions

TT contributed to the study design, data acquisition, and manuscript drafting. HN and KZ collected data. KT and AO contributed to critical revision of the manuscript. KA supervised the study, data interpretation and critical revision of the manuscript.

Conflict of Interest

We have not received any financial support or other benefits from commercial sources for the work reported in the manuscript. None of the authors have financial interests that could create a potential conflict of interest or the appearance of a conflict of interest with regard to this work.