15725
J. Phys. Chem. 1995, 99, 15725-15727
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Detection of the O2(lAg) 02(3,) Transition in Aqueous Environments: A Fourier-Transform Near-Infrared Luminescence Study Jurina M. Wessels* and Michael A. J. Rodgers" Center for Photochemical Sciences, Bowling Green State University, Bowling Green, Ohio 43403 Received: June 20, 1995; In Final Form: August 30, 1995@
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Fourier-transformed near-infrared luminescence studies of the Oz('A,) Oz(3Zi)transition in H20 in the presence of various concentrations of the singlet oxygen quencher sodium azide were performed. In neat HzO (z 4 ps) the signal-to-noise ( S I N ) ratio obtained was 236. In the presence of M NaN3, where the lifetime of 02('A,) is reduced to approximately 180 ns, the 0z(lAg) phosphorescence could still be detected with an acceptable S I N ratio of 8. From these measurements a bimolecular rate constant of (5.8 f 1.1) x lo8 M-I s-l for NaN3 was obtained.
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Introduction Singlet molecular oxygen, Oz(lAg), is a metastable excited state of ground-state molecular oxygen, 0 2 ( 3 qwhich , is receiving increasing attention as a reactive intermediate in a variety of photochemical, photobiological, enzymatic, and environmental processes.' It is believed that Oz(lAg)is involved in oxidative processes causing cytotoxic damage in living systems2 Furthermore, Oz('A,) is generally accepted to be a key intermediate in photodynamic tumor therapy. The production of the metastable excited state of molecular oxygen via the type II pathway involves energy transfer from an electronically excited state of a sensitizer molecule S to Oz(3E,) (eqs 1 and 2).
The lifetime of O2(lAg) in condensed media ranges from to lo-' s, depending on the solvent, and is determined mainly by nonradiative dea~tivation.~ The short lifetime of O2(lAg) in water (t 4 p ~coupled ) ~ with a weak radiative rate constant makes the measurement of 0 2 ( IAg) quantum yields via its 1.27 pm phosphorescence particularly difficult. The phosphorescence quantum yield is further reduced in biological environments, where the local concentration of quenchers such as amino acids may be high. The lifetime of Oz('A,) in cells has been proposed to vary between 100 ns (in membra ne^)^ and 250 ns (in the cytoplasm).6 The only noninvasfve and probably most sensitive method for the detection of 02(lAg) is the observation of the nearinfrared emission at approximately 1270 nm. Several timeresolved measurements of photosensitized OZ(IA,) in the presence of biomolecules have been performed in deuterated ~ a t e r , where ~ - ~ the intrinsic lifetime of Oz('A,) is larger than in HzO by approximately a factor of 20. However, the timecorrelated single-photon-counting technique was successfully applied to the detection of 02(lAg) in H2Oe8-lo In HzO-based environments, such as cells, the rate of formation of photosensitized 0 2 ( 'A,) in air-saturated conditions is significantly lower than its rate of decay, and time-resolved methods are likely to fail to detect Oz(lA,) under such conditions." On the other
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Abstract published in Advance ACS Abstracts, October 1, 1995.
0022-3654l9512099-15725$09.00/0
hand, continuous-wavetechniques may be useful, and a steadystate setup has been successfully employed in the detection of Oz('Ag) in neat water with a good signal-to-noise ( S I N ) ratio." Parker reported 0z(lAg) emission during photodynamic treatment of tumors, although the poor SIN ratio presented probably precluded quantitative analysis.I2 Using steady-state excitation conditions, the use of FT techniques for the detection of OZ(IA,) has been demon~trated.'~.'~ It was shown recently that an interferometer-based near-IR spectrometer yields signal-tonoise ( S I N ) ratios that are a factor of 10-20 higher than those obtained from monochromator-based devices.I5 Additionally, the use of such an instrument for the detection of photosensitized O;?('A,)emission from cellular suspensions was demonstrated.16 In this study we report steady-state measurements of photosensitized 02(lAg) in aqueous suspensions of liposomes in the presence of varying concentrations of the 02(lAg) quencher NaN3 that reduce the O2(lAg) lifetime up to 180 ns. A Fouriertransform near-infrared luminescence (FTNIRL,) spectrometer was employed to ensure high sensitivity and optimum signalto-noise ratio. The influence of the S I N ratio on evaluation of the integrated area is discussed.
Experimental Section 5,10,15,20-Tetrakis( 1-dodecyl-3-pyridyl)-2 1H,23H-porphine tetrabr~mide'~ was incorporated into L-a-phosphatidylcholine dimyristoyl (DMPC, Sigma) liposomes at a molar ratio of sensitizernipid = 1/200 and was used for photosensitization of Oz('A,). Increasing concentrations of NaN3 were employed to systematically reduce the lifetime of Oz('A,). All measurements were carried out at room temperature (20 f 1 "C) in air-saturated solutions. The liposomal suspension had an absorbance (A) = 1.12/cm at the excitation wavelength. The 514 nm line of an Ar+ laser was used as an excitation source and brought to the cuvette via a 200 p m optical fiber. The laser power incident on the sample was 300 mW. The emission was detected at right angles via an AR-coated silicon metal filter using a NIR-FT-Raman accessory (FRA106, Bruker) coupled to a FT-IR spectrometer (IFS66, Bruker). The detector was a liquid nitrogen-cooled germanium diodelamplifier (Applied Detector Corp., Model 403G, Fresno, CA). Spectra were recorded at a resolution of 10 cm-', and 800 scans were averaged over a spectral range of 15 OOO cm-I. The diameter of the aperture was 12 mm. These conditions were set to sacrifice spectral resolution in order to gain signal intensity. 0 1995 American Chemical Society
Letters
15726 J. Phys. Chem., Vol. 99, No. 43, 1995
TABLE 1: Integrated Area and S/N Ratio of Oz('A,) Emission in I320 in the Presence of Varying Concentrations of NaN3 after Subtraction of the Backmound Luminescence S/N ratio CNaN3 (M) integral area (au)
0.O040 1
0.0035
A
0.0030
-3
0.0025
6
0.0020
'-
0.0015
v
I
0.0010
10-2
I
I
I
7600
7800
8000
8200
I 1
0.0025
0.0020 ?
3
v
,$' 0.0015 .*
1
1
O.oo00
7400
7600
7800
8000
8200
8000
8200
wavenumter (an'') 0.0030
0.0025
0.0020 ?
3
v
,$' 0.0015
I
.* 0.0010
0.0005
7400
7600
7800
84 19 8
Figure 1 shows emission spectra of the 0-0 emission band of the 02('A,) 0 2 ( 3 qtransition ) in the absence and in the presence of varying concentrations of NaN3 (details in caption). No 02(lAg) phosphorescence emission could be detected at lo-' M NaN3. This last spectrum was used for subtraction of background luminescence prior to evaluation of signal-to-noise (S/N)ratio and integral area (Table 1). The lifetime of 0 2 ('Ag) in the presence of NaN3 as a quencher can be evaluated according to
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0.0030 I
0.0005
263
Results and Discussion
wavenumber (an.')
0.0010
0.018 0.004 0.002
5 x 10-2
0.0005
7400
0.05
0 10-3
wavenumber (an.')
Figure 1. Phosphorescence spectra of the 0-0 transition of photosensitized 02('Ag) in HzO and in the presence of varying concentrations of NaN3 prior to subtraction of background phosphorescence (top) and after subtraction of the background phosphorescence (middle). The spectra correspond to the following concentrations of NaN3: (1) 0 M; (2) M; (3) 0.5 x M; (4) M; (5) lo-' M. (bottom) 02(lAg) phosphorescence spectrum in the presence of low2M NaN3 normalized to the signal intensity obtained in neat water.
l/r = kx
+ kQ[Q]
(3)
with the first-order rate constant kx = 2.5 x lo5 s-l and the bimolecular rate constant for quenching by NaN3 kQ = 5.6 x lo8 M-' s-1.19,20 Using these rate constants the lifetime t of M NaN3 is approximately 180 02(lAg) in the presence of ns, which is within the order of magnitude of the proposed lifetime of 02(lAg) in the cellular cytoplasm.6 Under these experimental conditions, the 0-0 transition of the 02(lAg) phosphorescence can still be detected (Figure 1) with an acceptable S/N ratio of 8 and be used for evaluation of the integrated area (Table 1). For evaluation of the S/N ratio, the peak-to-peak amplitude of the noise was measured at the baseline on the red side of the emission band. For evaluation of the quantum yield 4, the frequency range over which the emission was integrated was determined by the noise level of the weakest 02(lAg) emission detected. The integration limits were defined by the first minima of the signal on both sides of the emission peak as indicated in Figure 1, bottom. Integration over the signal was performed after defining the line connecting to the two points where the signal crosses the integration borders as the new baseline for each 02('Ag) emission signal. By defining the baseline as well as the integration limits in such manner, it is possible to restrict the error of the integrated value of the emission signal introduced by noise in the baseline region. Nevertheless, evaluation of integral areas for determination of rate constants from signals exhibiting small S/Nratios is subject to great uncertainty. By varying the integration limits by k 1 0 cm-I, an upper estimate for this uncertainty was obtained (see below). The areas under the 02(lAg) emission peak obtained as described above between 7910 and 7780 cm-' were evaluated for several NaN3 concentrations and plotted in a Stem-Volmer manner. The plot is shown in Figure 2. The slope provides a kQ value of (5.8 f 1.1) x lo8 M-' s-I, which agrees with literature value^.'^^^^ The error in k~ is derived from the uncertainty in the integrated areas, which itself strongly depends on the S/ N ratio of the 02(lAg) emission signal and the defition of the integration borders with respect to the noise on the signal. The possibility of evaluating the area under the 02('As) 02(3ZJemission peak is a prerequisite for the establishment of a quantitative relationship between the 0 2 ( A,) quantum yield and the yield of photooxidative damage. Gorman and Rodgers" have discussed advantages and disadvantages of time-resolved and steady-state detection of photosensitized 02('Ag) and concluded that as a result of the kinetic situation in biological environments, steady-state methodology is the method of
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J. Phys. Chem., Vol. 99,No. 43, 1995 15727
Letters
sheim, Germany, and The Center for Photochemical Sciences at Bowling Green State University. J.M.W. thanks the Alexander von Humboldt Foundation for support within the FeodorLynen Program.
30
References and Notes
20
% 10
O.Oo0
0.005
0.010
concentrationof NaN, (M)
Figure
2. Stem-Volmer plot of the integral area of the emission as a function of time.
02(IAg)
choice. Kanofsky et al. have obtained excellent results from time-resolved measurements of 02(‘Ag) in DzO-based solutions in the presence of biomolecules and cell The work reported here has demonstrated that a FTNIRL steady-state setup demonstrates the possibility of detecting 02(‘Ag)under nearbiological conditions (HzO-based) in a quantitative way.
Acknowledgment. The authors gratefully thank Louis Wang from Applied Detector Corp. for loaning the Bruker FT-IRRaman spectrometer complete with one of his detectors, and Dr. W. E. Ford for providing 5,10,15,20-tetrakis(l-dodecyl-3pyridyl)-21H,23H-porphinetetrabromide. Support for this work was obtained from the NIH (Grant GM24235), from the GSFForschungszentrum fiir Umwelt und Gesundheit, Oberschleis-
(1) Kanofsky, J. R. Chem. Bid. Interact. 1989, 70, 1. (2) Weishaupt, K. R.; Gomer, C. J.; Dougherty, T. J. Cancer Res. 1976, 36, 2326. (3) Ogilby, P. R.; Foote, C. S. J . Am. Chem. SOC.1983, 105, 3423. (4) Rodgers, M. A. J.; Snowden, P. T. J . Am. Chem. SOC. 1982, 104, 5541. (5) Kanofsky, J. R. Photochem. Photobiol. 1991, 53, 93. (6) Baker, A.; Kanofsky, J. R. Photochem. Photobiol. 1992, 55, 523. (7) Baker, A.; Kanofsky, J. R. Photochem. Photobiol. 1993, 4, 720. (8) Egorov, S. Yu.;Kamalov, V. F.; Koroteev, N. I.; Krasnovsky, Jr., A. A.; Toleutaev, B. N.; Zinukov, S. V. Chem. Phys. Lett. 1989,163,421. (9) Zinukov, S. V.; Kamalov, V. F.; Koroteev, N. I.; Krasnovsky, Jr., A. A. Opt. Spektrosk. 1991, 70, 790. (10) Krasnovsky Jr., A. A. SPIE Proc. 1993, 1887-44, 177. (11) Gorman, A. A.; Rodgers, M. A. J. J . Photochem. Photobiol. 1992, 14, 159. (12) Parker, J. G. IEEE Circuits Devices Mag. 1987, January, 10. (13) Macpherson, A. N.; Truscott, T. G.; Tumer, P. H. J. Chem. SOC., Faraday Trans. 1994, 90,1065. (14) Macpherson, A. N.; Tumer, P. H.; Truscott, T. G. Appl. Spectrosc. 1994, 48, 539. (15) Wessels, J. M.; Charlesworth, P.; Rodgers, M. A. J. Photochem. Photobiol. 1995, 61, 350. (16) Bohm, F.; Marston, G.; Truscott, T. G., Wayne, R. P. J. Chem. Soc., Faraday Trans. 1994, 90, 2453. (17) 5,10,15,20-Tetrakis(l-dodecyl-3-pyridyl)-2 lH,23H-porphine tetrabromide was synthesized by quatemization of 5,10,15,20-tetra-3-pyridyl21H,23H-porphine (Midcentury) with a 40 molar e x e s of 1-bromodecane (Aldrich) in refluxing N,N-dimethylformamide for 2 h.I8 The compound was purified by gel permeation chromatography through a column of Sephadex LH-20 with methanol as a solvent. UV-vis (methanol) (Amu(nm), 6 (M-I cm-I): 418, 3.32 x lo5; 514, 1.95 x 104; 548, 3.90 x lo3; 580, 7.41 x lo3; 640, 1.56 x lo3. (18) Okuno, Y.,Ford, W. E., Calvin, M. Synthesis 1980, 537. (19) Lindig, B. A.; Rodgers, M. A. J. Phorochem. Photobiol. 1981, 33, 672. (20) Hall, R. D.; Chignell, C. F. Photochem. Photobiol. 1987.45, 459. Jp9517460
