Methylene Blue Encapsulation in Cucurbit[7]uril: Laser Flash

May 27, 2009 - 140 μs (see inset of Figure 1), and its intersystem crossing quantum ... nitrogen, 0, 79.5, 140 .... The solutions were optically matc...
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Methylene Blue Encapsulation in Cucurbit[7]uril: Laser Flash Photolysis and Near-IR Luminescence Studies of the Interaction with Oxygen Marı´ a Gonzalez-Bejar,† Pedro Montes-Navajas,‡ Hermenegildo Garcı´ a,*,‡ and J. C. Scaiano*,† †

Department of Chemistry, Centre for Catalysis Research and Innovation, University of Ottawa, Ottawa, Ontario, Canada, K1N 6N5, and ‡Instituto de Tecnologı´a Quı´mica CSIC-UPV, Universidad Polit ecnica de Valencia, 46022 Valencia, Spain Received April 4, 2009. Revised Manuscript Received May 5, 2009 The effect of methylene blue (MB) encapsulation in cucurbit[7]uril (CB[7]) on triplet excited-state behavior and singlet oxygen (1O2) generation has been studied by using laser flash photolysis (LFP) and time-resolved near-IR luminescence spectroscopy. The lifetime of the triplet excited state of MB is longer in the CB[7] cavity (140 μs for MB-CB[7] vs 79.5 μs for aqueous MB). Cucurbituril also protects the dye triplets from quenching by oxygen, reducing the quenching rate constant [kq(O2)] from 2.6  109 M-1 s-1 to 0.2  109 M-1 s-1. The quantum yield of 1O2 production in the air-equilibrated D2O solutions is similar for free MB and for MB-CB[7], and the singlet oxygen lifetime is ∼70 μs, suggesting its decay occurs in the aqueous (D2O) phase. The generation of singlet oxygen is delayed by CB[7]; this is attributed to the time required for oxygen to access the CB[7] nanocavity and react with the MB triplet. Thus, the rate-limiting step for sensitization is the entry of oxygen into the CB[7] cavity. Encapsulation inside CB[7] increases the relative efficiency of photoinduced MB2+• dication-radical generation, for which a modest yield is observed.

Introduction There is considerable current interest in the properties of photosensitizers and their ability to generate singlet oxygen (type II mechanism) and/or other reactive oxygen species (type I mechanism), causing tumoral cell apoptosis and/or necrosis, a process that has applications in the area of photodynamic therapy (PDT).1,2 Most photosensitizer dyes aggregate easily and have poor solubility in aqueous solutions, thus leading to loss of photochemical activity and limited cell-penetrating properties.3 To overcome these limitations, there have been several approaches3,4 such as combination of photosensitizers (either conjugation or complexation) with polymers or macrocycles, such as liposomes, lipids, and oligosaccharides.5-10 Cucurbiturils (CBs), which are water-soluble cyclic oligomers of glycoluril units linked by methylene bridges (Scheme 1), have emerged as versatile *Corresponding author. E-mail: [email protected] (J.C.S.); [email protected] (H.G.). (1) Dougherty, T. J. Photochem. Photobiol. 1993, 58, 895–900. (2) Brown, S. B.; Brown, E. A.; Walker, I. Lancet Oncol. 2004, 5, 497–508. (3) Ideta, R.; Tasaka, F.; Jang, W.-D.; Nishiyama, N.; Zhang, G.-D.; Harada, A.; Yanagi, Y.; Tamaki, Y.; AIda, T.; Kataoka, K. Nano Lett. 2005, 5, 2426–2431. (4) Kim, S.; Ohulchanskyy, T. Y.; Pudavar, H. E.; Pandey, R. K.; Prasad, P. N. J. Am. Chem. Soc. 2007, 129, 2669–2675. (5) Baugh, S. D. P.; Leung, D. K.; Wilson, D. M.; Breslow, R. J. Am. Chem. Soc. 2001, 123, 12488–12494. (6) Dai, X.-H.; Dong, C.-M.; Fa, H.-B.; Yan, D.; Wei, Y. Biomacromolecules 2006, 7, 3527–3533. (7) Ristori, S.; Salvati, A.; Martini, G.; Spalla, O.; Pietrangeli, D.; Rosa, A.; Ricciardi, G. J. Am. Chem. Soc. 2007, 129, 2728–2729. (8) Jang, W.-D.; Nishiyama, N.; Zhang, D.; Harada, A.; Jiang, D.-L.; Kawauchi, S.; Morimoto, Y.; Kikuchi, M.; Koyama, H.; Aida, T.; Kataoka, K. Angew. Chem., Int. Ed. 2005, 44, 419–423. (9) Synytsya, A.; Kral, V.; Blechova, M.; Volka, K. J. Photochem. Photobiol., B: Biol. 2004, 74, 73–84. (10) Tang, W. X. H.; Park, E. J.; Philbert, M. A.; Kopelman, R. Biochem. Biophys. Res. Commun. 2008, 369, 579–583. (11) Lagona, J.; Mukhopadhyay, P.; Chakrabarti, S.; Isaacs, L. Angew. Chem., Int. Ed. 2005, 44, 4844–4870. (12) Lee, J. W.; Samal, S.; Selvapalam, N.; Kim, H. J.; Kim, K. Acc. Chem. Res. 2003, 36, 621–630. (13) . Mock, W. L. Cucurbituril. In Topics in Current Chemistry; Weber, E., Ed.; Springer-Verlag: Berlin, 1995; Vol. 175, p 1.

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cyclic hosts that can form strong inclusion complexes with positively charged organic guests.11-14 The remarkable properties of inclusion complexes with small organic molecules have been used to meet specific needs, such as a new “supramolecular dye laser”,15 probes for biomolecular systems,16 hydrolysis inhibition of potential drugs toward some enzymes,17 development of label-free continuous enzyme assays,18 and size-control and stabilization of gold nanoparticles.19 In particular, cucurbit[7]uril (CB[7]), with a cavity volume of 0.279 nm3, has been shown to display a variety of benefits with fluorescent dyes, which include fluorescence and brightness enhancement, protection toward fluorescence quenchers, photostabilization, solubilization, and deaggregation.15-24 We have recently examined the encapsulation of methylene blue (MB), a common singlet oxygen sensitizer, in CBs and reported that CB[7] forms a strong 1:1 host-guest complex with MB (Kb(MB-CB[7]) = (1.26 ( 0.28)  107 M-1 in water).20 MB exists in a monomer-dimer equilibrium in aqueous solutions, but CB[7] minimizes MB aggregation even at high dye concentration. Thus, monomeric MB is the main species present in solution, which is a prerequisite for its photosensitizer activity.9 It is well-known that photoexcitation of the dye produces triplet states (14) Rudkevich, D. A. Bull. Chem. Soc. Jpn. 2002, 75, 393–413. (15) Mohanty, J.; Pal, H.; Ray, A. K.; Kumar, S.; Nau, W. N. ChemPhysChem 2007, 8, 54–56. (16) Bhasikuttan, A. C.; Mohanty, J.; Nau, W. N.; Pal, H. Angew. Chem., Int. Ed. 2007, 46, 4120–4122. (17) Hennig, A.; Ghale, G.; Nau, W. N. Chem. Commun. 2007, 1614–1616. (18) Hennig, A.; Bakirci, H.; Nau, W. N. Nat. Methods 2007, 4, 629–632. (19) Corma, A.; Garcia, H.; Montes-Navajas, P.; Primo, A.; Calvino, J. J.; Trasobares, S. Chem. Eur. J. 2007, 13, 6359–6364. (20) Montes-Navajas, P.; Corma, A.; Garcia, H. ChemPhysChem 2008, 9, 713–720. (21) Koner, A. L.; Nau, W. M. Supramol. Chem. 2007, 19, 55–66. (22) Shaikh, M.; Mohanty, J.; Singh, P. K.; Nau, W. M.; Pal, H. Photochem. Photobiol. Sci. 2008, 7, 408–414. (23) Zhou, Y.; Yu, H.; Zhang, L.; Sun, J.; Wu, L.; Wang, L. J. Inclusion Phenom. Macrocyclic Chem. 2008, 61, 259–264. (24) Sueishi, Y.; Asano, K.; Yamaoka, M.; Yamamoto, S. Z. Phys. Chem. 2008, 222, 153–161.

Published on Web 05/27/2009

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Scheme 1. Structures of CB[7] and MB. The Glycouril Unit Has Been Highlighted in CB[7]

Scheme 2. Photosensitized Generation of Singlet Oxygen and Other Reactive Oxygen Species

Figure 1. Transient absorption spectra of a deaerated aqueous MB solution (λex 308 nm) (A) measured at 0.5 μs (O), 48 μs (b) and 101 μs (9) after the laser pulse; (B) in the absence (b) or in the presence (O) of CB[7] measured at 0.5 μs after the laser pulse (λex 308 nm, E = 10 mJ). The inset shows time profiles measured at 420 nm on the microsecond time scale for both of them (MB and MB-CB[7]).

following intersystem crossing. Thus, ground-state molecular oxygen (triplet oxygen, 3O2) can quench the dye triplet state by energy transfer to generate singlet oxygen 1Δg, as well as by electron transfer to produce reactive radicals.25 MB has very low fluorescence quantum yield (ca. 0.03), long triplet lifetime, high intersystem crossing quantum yield (ΦISC), high singlet oxygen sensitization (ΦΔ∼0.52) and low toxicity.26 Its structure and reactivity are shown in Schemes 1 and 2, respectively. In the context of therapeutic applications, MB can efficiently treat several types of cancer, viruses, and fungi infections, and, in contrast to other sensitizers such as photofrin, it can oxidize mitochondrial NAD(P)H.26-29 Among other techniques, near-infrared phosphorescence (NIRP) is commonly used as an analytical tool to measure singlet oxygen, which phosphoresces at ∼1270 nm. In a recent study, we have reported the singlet oxygen generation by aromatic aminoacids, proteins and immunoglobulins utilizing NIRP detection.30 Most of the work reported on cationic dyes-CB[7] host-guest complexes has focused on fluorescence,31 but laser flash photolysis (LFP) and NIRP studies of these supramolecular assemblies have not been reported. We have selected MB because of its application in PDT10,26 and its affinity for CB[7]. In this (25) Kochevar, I. E.; Redmond, R. W. Methods Enzymol. 2000, 319, 20–28. (26) Tardivo, J. P.; Del Glio, A.; de Oliveira, A.; Gabrielli, D. S.; Junqueira, H. C.; Tada, D. B.; Severino, D.; Turchiello, R. F.; Baptista, M. S. Photodiagn. Photodyn. Ther. 2005, 2, 175–191. (27) Danzinger, R. M.; Bar-Eli, K. H.; Weiss, K. J. Phys. Chem. 1967, 71, 2633–2640. (28) Junqueira, H. C.; Severino, D.; Dias, L. G.; Gugliotti, M.; Baptista, M. S. Phys. Chem. Chem. Phys. 2002, 4, 2320–2328. (29) Severino, D.; Junqueira, H. C.; Gabrielli, D. S.; Gugliotti, M.; Baptista, M. S. Photochem. Photobiol. 2003, 77, 459–468. (30) Chin, K. K.; Trevithick-Sutton, C. C.; McCallum, J.; Jockush, S.; Turro, N. J.; Scaiano, J. C.; Foote, C. S.; Garcia-Garibay, M. A. J. Am. Chem. Soc. 2008, 130, 6912–6913. (31) Nau, W. M.; Mohanty, J. Int. J. Photoenergy 2005, 7, 133–141.

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contribution, we have employed LFP and the direct detection of the time-resolved phosphorescence of singlet oxygen generated by MB in aqueous solutions and compared the results to those obtained in presence of CB[7]. To the best of our knowledge, this is the first study of the triplet excited state and singlet oxygen generation of a photosensitizer encapsulated in CB[7]; it is of interest because it prevents the MB π-stacking and could avoid reductive processes.

Results and Discussion LFP and NIRP studies employed 308 nm pulsed laser excitation. The transient spectrum of MB has three maxima located at 420 nm, 520 nm, and in the 700-900 nm range (Figure 1A). The decays fit well to first-order kinetics. The bands at 420 and ∼820 nm have been assigned to monomeric MB triplets (3MB*) (τT ca. 60-90 μs), whereas the band at 520 nm has been assigned to the MB2+• dication-radical generated by electron photoejection from MB (τ ca. 190 μs), a relatively common process for MB under conditions of laser excitation.32 An alternative assignment of the 520 nm band to the triplet excited state of dimeric MB [3(MB)2*] appears unlikely in this case since, at the MB concentration used in the laser experiments (5.8  10-6 M), only about 2% of MB is present as dimer in aqueous solution, while, in the presence of CB[7], the dimer concentration is even lower. In the presence of excess CB[7], only monomers should be present.20 The MB transient spectrum recorded 0.5 μs after excitation in the presence of CB[7] showed an unchanged maxima (Figure 1B), but with some differences relative to the experiment in the absence of CB[7]. First, 3MB* was longer lived, ca. 140 μs (see inset of Figure 1), and its intersystem crossing quantum yield was estimated as 0.44, assuming the triplet extinction coefficient was not influenced by CB[7] inclusion. Second, the relative intensity of the peak at 520 nm, corresponding to MB2+•-CB [7] radicals immediately after the laser excitation, increases with (32) Kamat, P. V.; Lichtin, N. N. J. Phys. Chem. 1981, 85, 3864–3868.

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Gonz alez-B ejar et al. Table 1. 3MB* Lifetime (τT) under N2, Air, or O2 Aqueous Solutions in the Presence or Absence of CB[7] τT ( μs)

Figure 2. (A) Dependence of the ratio of the absorbance at 520 to 420 nm on the laser pulse energy. (B) Transient absorption spectra of a deaerated aqueous MB solution in the absence (5.8  10-6 M) (b) or in the presence ([MB] = 9.3  10-6 M) of CB[7] (9.3  10-5 M) measured at 0.5 μs after laser excitation (λex 308 nm, E = 30 mJ) under nitrogen.

atmosphere

[O2] (mM)

MB

MB-CB[7]

nitrogen air oxygen

0 0.27 1.27

79.5 1.8 0.3

140 16.0 3.8

Figure 3. Luminescence decay traces observed at 1270 nm after excitation of an air-saturated solution of MB in the absence or presence of CB[7] in D2O following 308 nm laser excitation; note the delay caused by CB[7] addition.

respect to that of 3MB*-CB[7], suggesting that encapsulation favors electron photoejection or reduces back reaction. A common test for solvated electron involves saturating the solution with N2O, an excellent electron scavenger.33,34 Under N2O saturation, we observe that the broadband in the 700-900 nm region is partially quenched, indicating the involvement of solvated electrons (result not shown). The reason for incomplete quenching is the absorption of the MB triplet in this region. Figure 2A shows that the ratio of the absorbance monitored at 520 to 420 nm depends on the laser power. Both in the absence and presence of CB[7] there is a dependence on power; however, this effect is far more pronounced when CB[7] is present, indicating that, in this case, the 520 nm includes a major component due to a two-photon process. Under our experimental conditions, there is no significant MB free in solution, and that in the CB[7] cavity will be present as a monomer.20 The fact that the intercept (Figure 2, top) does not go through the origin is due to the weak absorbance of the MB triplet at 520 nm. The absorbance of the species absorbing at 520 nm is much stronger when CB[7] is present in the MB solution. This suggests that CB[7] can assist two-photon processes that we propose involve photoionization, and probably decrease the probability of back electron transfer (or electron recapture). Thus the 520 nm signal is assigned to MB2+•, the product of MB photoionization.32 A small growth at 520 nm is frequently observed, and is likely due to electron capture by ground-state MB to give the corresponding radical. In the presence of air or N2O, this growth is not observed, since both N2O and O2 are electron scavengers (see Supporting Information, Figure S1). The signal intensity is also weaker under air, as expected, given that oxygen is a good triplet quencher (vide infra); the effect is smaller when CB[7] is present, reflecting the cavity’s protection of the triplet.

The bimolecular rate constants for oxygen quenching of triplet MB and MB-CB[7] solutions were calculated from the plots of the reciprocal triplet lifetimes against the oxygen concentration for solutions under nitrogen, air, and oxygen; they are 2.6  109 M-1 s-1 and 0.2  109 M-1 s-1 for free and complexed MB, respectively (Table 1 and Figure S2). This is in good agreement with the value of 3  109 M-1 s-1 previously reported for MB.35 The quenching rate constant is 1 order of magnitude lower in the presence of CB[7] since CB[7] protects MB from external quenchers. The quenching rate constant for MB in D2O is in line with other reported values.36 Oxygen quenching of the excited triplet state leads to singlet oxygen formation by energy transfer. Its lifetime can be measured most easily in deuterated solvents, such as D2O, by direct observation of singlet oxygen phosphorescence at 1270 nm. The decay of singlet oxygen was monoexponential with a lifetime of 70 μs, a reasonable value for D2O as solvent (see Figure 3).36,37 The rate of singlet oxygen generation slows down dramatically when CB[7] is added to the solution of MB in D2O, i.e., the 3MB* quenching by ground state O2 is slower, but the 1O2 generated has the same lifetime. Thus, we propose that this delay in the formation of the emissive 1O2 species is due to the time required for oxygen to react with the MB triplet inside the nanocavity and fits well with the idea that 3MB* is protected inside the organic capsules. The quantum yield of singlet oxygen generation (ΦΔ) of the MB encapsulated in CB[7] was found to be 0.44 (determined using MB in D2O as a standard (ΦΔ = 0.52))26 (Figure S3). In order to investigate the influence of oxygen concentration on the singlet oxygen generation rate and lifetime in the presence of CB[7], we performed experiments at variable oxygen concentration by using mixtures of oxygen and nitrogen. The solutions were equilibrated with either air or oxygen-nitrogen mixtures ranging from 20% to 100% oxygen. As we increased the total oxygen concentration, the singlet oxygen growth was faster, and more singlet oxygen was generated. Figure 4A shows representative kinetic data acquired

(33) Janata, E.; Schuler, R. H. J. Phys. Chem. 1982, 86, 2078–2084. (34) Turro, N. J.; Ramamurthy, V.; Scaiano, J. C. Principles of Molecular Photochemistry: An Introduction; University Science Publishers: New York, 2008; p 493.

(35) Nilsson, R.; Merkel, P. B.; Kearns, D. R. Photochem. Photobiol. 1972, 16, 109. (36) Ogilby, P. R.; Foote, C. S. J. Am. Chem. Soc. 1983, 105, 3423–3430. (37) Schmidt, R. J. Am. Chem. Soc. 1989, 111, 6983–6987.

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from NIRP) agree well for both MB (free) or for the MB-CB[7] complex and are in accordance with the mechanism proposed above.

Conclusion

Figure 4. (A) Luminescence decay traces observed at 1270 nm after excitation of a solution of MB-CB[7] in D2O following 308 nm laser excitation at several oxygen concentrations. (B) Linear dependence of singlet oxygen photogeneration rate-constants of MB solutions in D2O in the absence (O) or in the presence (b) of CB[7] on overall oxygen concentration.

after excitation of the MB-CB[7] complex. The data was analyzed as a series of two first-order kinetic processes, the first one corresponding to the signal growth, and the second one to the decay, which, as already indicated, is consistent with the 1 O2 lifetime in D2O.36 Further, the growth rate constants (kg) showed perfect linear dependence on oxygen concentration, consistent with triplet-triplet energy transfer from MB to oxygen following a bimolecular mechanism (Figure 4B), with the entry of oxygen into the CB[7] cavity as the rate-determining step. In fact, there are two mechanistic proposals that would explain the slow down in singlet oxygen generation in the presence of CB[7]: (a) The rate-determining step could be the exit of 3MB* into the aqueous phase. In some cases, changes in dipole moment upon triplet excitation are known to lead to enhanced exit from supramolecular systems.38,39 (b) As we propose above, the rate determining step can be the entry of oxygen into the CB[7] cavity containing excited MB. In the case of proposal “a”, a quenching plot, such as that of Figure 4B, should not be linear, but rather, it should lead to a plateau as there is sufficient oxygen to trap all the exiting MB triplets. The rate at the plateau would then correspond to the exit of 3MB* from the CB[7] cavity. Clearly this is not the case, thus leaving explanation “b” as the preferred explanation. This linear correlation allowed us to calculate the rate constants for the singlet oxygen generation after oxygen quenching of MB and MB-CB[7] (2.1  109 M-1 s-1 and 0.2  109 M-1 s-1, respectively). The rate constants for triplet MB quenching by oxygen (from LFP of 3MB* decay) and the singlet oxygen generation rate constants (based on the growth of luminescence (38) Barra, M.; Bohne, C.; Scaiano, J. C. J. Am. Chem. Soc. 1990, 112, 8075–8079. (39) Liao, Y.; Frank, J.; Holzwarth, J. F.; Bohne, C. J. Chem. Soc., Chem. Commun. 1995, 199–200.

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From the present work, we conclude that photoexcitation of MB encapsulated in CB[7] leads to the MB triplet state that can generate singlet oxygen by energy transfer. In the interaction of oxygen with the MB triplet, oxygen needs to enter the CB[7] nanocavity in order to quench 3MB*, and this is the rate-limiting step for the generation of singlet oxygen. This process can be readily followed spectroscopically by LFP (triplet MB) and NIRP (1O2), providing the first determination of the rate constant (2  108 M-1 s-1) for the penetration of oxygen into the CB[7] cavity, where MB affords significant protection from oxygen. The effect is almost entirely kinetic, since the reduction of the rate of sensitization by an order of magnitude causes only a minor decrease in the quantum yield of 1O2 generation, i.e., 0.44 in the complex versus 0.52 for free MB. Laser excitation also leads to some MB photoionization; however, this is a two-photon process and is not expected to play a significant role under conditions of lamp excitation.

Experimental Section Materials. MB chloride and CB[7] were commercial samples (Aldrich) and used as received. For the LFP and NIRP studies, milli-Q water (18.2 Ω) or deuterium oxide (99.99%) from Aldrich were used, respectively. All chemicals and solvents were used without further purification. Experimental Procedure. The solutions were optically matched (0.1) at 308 nm for the LFP and NIRP variable oxygen concentration studies. MB was 5.8  10-6 M when free, and the complex MB-CB[7] was prepared using a ratio of 1:10 ([MB] = 9.3  10-6 M and [CB[7]] = 9.3  10-5 M). Spectroscopy Instrumentation. All the absorbance spectra were run using a Cary-50-Bio UV-visible spectrophotometer. LFP measurements were performed after excitation with a Lumonics Pulse Master excimer laser (308 nm, 10 ns, ∼10 mJ/pulse), using fused silica cells with a path length of 1.0 cm. The data were acquired and analyzed with a customized Luzchem Research mLFP-111 apparatus with an orthogonal pump/probe configuration. The probe source was a ceramic xenon lamp coupled to quartz fiber-optical cables. The laser pulse and the mLFP-111 system were synchronized with a Tektronix TDS 2012 digitizer, operating in pretrigger mode. The signals from a compact Hamamatsu photomultiplier were initially captured by the Tektronix digitizer and transferred to a computer for data analysis and archival. The singlet oxygen phosphorescence decay traces were measured as follows: The solutions were placed in a 1  1 cm2 fused silica cuvettes capped with septa and irradiated with 308 nm laser pulses from a Lumonics Pulse Master or HD-500 excimer laser after purging with the corresponding gas mixture for at least 15 min. NIR emission studies40 were carried out using a Peltiercooled (-62.8 °C) Hamamatsu NIR detector (Model H10330-75) operating at 900 V coupled with a computer-controlled grating monochromator. A long pass filter, type FEL1150 filter from Thorlabs was placed in front of the monocromator. The photocurrent from the PMT was stored on a digital oscilloscope (Tektronix TDS 2012). Signal rise times as short as 50 ns were measured using an SR-445 amplifier from Stanford Research. Luzchem LFP software was used to acquire and process the data. In both systems (LFP and NIR singlet oxygen phosphorescence), the data were transferred to a personal computer operating (40) Cojocaru, B.; Laferriere, M.; Carbonell, E.; Parvulescu, V.; Garcı´ a, H.; Scaiano, J. C. Langmuir 2008, 24, 4478–4481.

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LuzChem LFPv3 software. The solutions for the experiments in the absence of oxygen were deoxygenated by bubbling with pure nitrogen, and the experiments at variable oxygen concentration were carried out by using mixtures of oxygen and nitrogen (ranging to 20% to 100% oxygen) prepared with a flow gas controller-mixer system. The concentration of oxygen was calculated using Henry’s law: ½O2  ¼ KH0 3 PO2

ð1Þ

(λex) would be