Photochemistry and Photobiology Vol. 45, No. 4, pp. 4591164, 1987 Printed in Great Britain

003 1 -8655/87 $03 .OO + 0.M Pergamon Journals Ltc

STEADY-STATE NEAR-INFRARED DETECTION OF SINGLET MOLECULAR OXYGEN: A STERN-VOLMER

QUENCHING EXPERIMENT WITH SODIUM AZIDE

ROBERT D. HALL* and COLIN F. CHIGNELL Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, PO Box

12233, Research Triangle Park, NC 27709, USA

(Received 30 June 1986; accepted 10 September 1986)

Abstract-A sensitive near-infrared detection system incorporating improvements to existing method- ologies has been used to characterize the sodium azide quenching of the steady-state luminescence of singlet molecular oxygen at 1270 nm. Stern-Volmer plots which were linear up to 80% quenching of the '0, generated by rose bengal and eosin Y yielded a rate constant of 5.8 2 0.1 x lox M- ' s-I for the quenching of '0, in water, while the rate constants obtained in deuterium oxide with the same sensitizers were 6.28 X lox M-' s-' and 6.91 x lox M-' s-' , respectively. A flow system minimized the effects of photobleaching of the rose bengal. With a mercury arc light source, the instrument can be used in photosensitization experiments to detect low levels of '0, production in aqueous media.

INTRODUCTION

In the last decade, the direct spectroscopic obser- vation of the primary (1270 nm) emission of lo2

has emerged as an important tool for the detection of this active oxygen species in chemical and bio- logical systems. Initially Krasnovsky (1976, 1979) used red-sensitive photomultiplier tubes to detect the emission at 1270 nm. Subsequently Khan and Kasha (1979) and Khan (1980) showed that a cooled lead sulfide detector greatly increased instrument sensitivity; Khan (1981) also demonstrated that even greater sensitivity could be obtained with ger- manium diodes. Several other groups have employed similar detectors for the detection of emission (Hurst et af., 1982; Parker and Stanbro, 1982; Ogilby and Foote, 1982; Lengfelder er af., 1983; Torinuki and Miura, 1983). Pulse laser tech- nology has made it possible to study the lifetime of '0, under a variety of conditions (Hurst and Schus- ter, 1983; Rodgers, 1983). At the same time, steady- state instrumentation has been used to determine whether peroxidase enzymes can produce lo2 under conditions of biological importance (Kanofsky , 1983, 1984; Khan, 1983, 1984; Khan et af., 1983).

The possibility that lo2 is an important causative agent in the inflammatory response elicited by cer- tain photosensitizers to which humans are exposed led us to investigate the applicability of spec- troscopic detection of '02 to such problems. However, despite the recent advances in lo2 detec- tion, direct spectroscopic observation of lo2 in bio- logical milieux remains extremely difficult. In aque- ous systems, including those containing amphipathic molecules, the quantum yield of '02 phos- phorescence is lo-' at best (Krasnovsky, 1981a,b), and the cell is an ocean of potentially reactive mol-

*To whom correspondence should be addressed.

ecules which most certainly reduces the quantum yield even further. Therefore, our initial work has been limited to in vitro systems (Hall et af., 1986).

Previous studies have utilized indirect methods of '02 detection to demonstrate that the azide anion rapidly quenches lo2 in hydroxylic solvents (Foote el al., 1972; Hasty et al., 1972; Foote, 1979; Lindig and Rodgers, 1981). However, Chou and Khan (1983) have successfully used steady-state instru- mentation to determine the Stern-Volmer behavior of ascorbic acid quenching of lo2 in water. Con- tinuous irradiation of chrysene sulfonate was used to generate lo2. Ascorbic acid apparently did not quench the fluorescence of chrysene sulfonate but did quench '0, by a chemical pathway.

In this paper, we describe a direct measurement of the Stern-Volmer quenching constant for the quenching of photochemically generated '02 by azide anion in water and deuterium oxide. The following equations represent the principal pro- cesses occurring in our experimental system:

hv P- IP*

3p* + 3 0 2 - lo2 + P (3) k ,

(4) N, + 302 photochemical products

where k,,, is the rate of intersystem crossing between the singlet and triplet excited states of the pho- tosensitizer, P ; and k l is the rate of quenching of the triplet excited state of the sensitizer by ground-

459

460 ROBERT D. HALL and COLIN F. CHIGNELL

state oxygen. The '0, sensitized by P is quenched at a rate dependent upon the concentration of the azide anion and the rate constant, k,. The photo- sensitizer can also react with '0, and with the azide anion through either of its excited states. We have used a flow system to minimize the effects of these processes on the observed '0, emission intensities. In any case, intersystem crossing is so rapid for the photosensitizers we have used (Lambert and Neckers, 1985) that singlet-state reactions should be of secondary importance. The triplet state of eosin Y is known to react slowly with the azide anion (Kraljic and Lindqvist, 1974) and the lower triplet state energy of rose bengal makes it likely that its triplet state also reacts slowly with the azide anion (Gollnick et al. , 1972).

The Stern-Volmer description of dynamic quenching (Jameson, 1984) given in equation 5 was used to find the quenching constant, K,,,:

r. = 1 + K s Y [ Q ] I

where [Q] is the concentration of the quencher and I J I is the ratio of the emission intensity of '02 in the absence and presence of quencher, respectively. The quenching rate constant can be derived from K,, by using the appropriate excited-state lifetime of lo2, T, and the relation:

K,, = K, T

The rate constants for the quenching process have been derived using the lifetimes of '02 in water and deuterium oxide determined by Rodgers (1983).

We have used two different photosensitizers, rose bengal and eosin Y, to generate lo2. We also describe some improvements in '02 detection that have increased the overall signal-to-noise capability of the instrument and extended its applicability to photosensitization experiments.

MATERIALS AND METHODS

The deuterium oxide (99.8 atom %D) and the rose bengal (certified grade) were from Aldrich Chemical Co. (Milwaukee, WI). The concentration of rose bengal in all the experiments was 20 K M (Asdxnm = 1.1). The eosin Y. a certified grade from Hartman-Reddon Company. was used at a concentration of 11 p M (A,,,,,,, = 1.0). The sodium azide was recrystallized from ethanol.

emission detector was constructed from com- ponents following the general descriptions of similar instruments given by Khan (1981) and Kanofsky (1983). The light source liquid filter, sample holder and the mono- chromator and driver were all purchased from Kratos Instrument Co. (Westwood, NJ). Light from a 150 W Xe lamp or a 100 W Hg arc lamp passes through a shutter assembly, a 10-cm pathlength liquid filter and through selected additional filters. The lamps were chosen for their convenient size and small arc image. Water was circulated through the liquid filter. In order to generate IO, with rose bengal, a KV418 filter and KG2 and KG3 filters (Schott Optical Glass Co; Duryea, PA) were used to eliminate short wavelength radiation and any residual near-infrared radiation. Schott neutraI density filters (NGI to NG12) were added to vary the intensity of the excitation

The

beam. The sample holder accommodates a 10 mm: cuvette. The fluorescence flow cell was from Precision Cells, Inc. (Hicksville, NY); it has a chamber with a 0.5 me volume and a 5 mm excitation light path. At a righl angle to the excitation beam, light is collected by a lens system and passes through another filter, usually a Corion Corp. (Holliston, MA) LL950 cut-off filter to remove scattered light in the UV-visible wavelength range. A monochromator (Model GM252) is used to isolate the desired wavelengths, and the light then passes through E light-chopper set at 68 Hz (Model 125A, Princeton Applied Research; Princeton, NJ). Finally, a lens focusser the light onto the 25 mmz detector of a germanium detec. tion system (Model 403 L, Applied Detector Corp.. Fresno, CA) containing the detector. a liquid-nitrogen dewar and a pre-amplifier. The light-chopper, lens anc germanium detection system are all enclosed in a black plexiglass box with a light-tight removable top. A lock-in amplifier (Model numbers 4110 and 4114 from Evan5 Associates; Berkeley, CA) isolates the appropriate elec- tronic signal and an interface (Interactive Structures. 1°C. : Bala Cynwyd, PA) digitalizes and inputs the signal to an Apple 11+ computer (Apple Computer Inc.). A modified version of an SLM-AMINCO (Urbana, IL) computer pro. gram is used to drive the monochromator and to acquire. store and analyze data.

All absorption and filter transmittance measurement5 were made with a Gilford Response spectrophotometer. Measurements were made of the irradiance of the Xenon and mercury arc lamps at the cuvette holder with a Yellon Springs Model 65A Radiometer and, where necessary. screen filters from Hitachi Corp. The peristaltic pump was from the LKB Corp. (Model 2120).

RESULTS

In the early stages of our work with pho- tosensitizers, we used a 150 W Xe lamp in the '02 emission detector. However, we found that residual scattered light from the Xenon emission line at 1260 nm can confound spectral analysis at low emission intensity levels. Moreover, the output of a 200 W Hg lamp at the wavelengths of Hg line emission greatly enhances the excitation rate of compounds which absorb at those wavelengths. In the case of rose bengal, the Hg line at 546 nm overlaps closely with the wavelength of maximum absorption of the photosensitizer (548 nm). Therefore, we used the Hg arc lamp for the Stern-Volmer experiment described in this paper.

Rose bengal photobleached under strong illumi- nation of the type used in these experiments so that the emission intensity of '0, steadily decreased in an unstirred solution during the signal acquisition time. Stirring a small volume of the solution in a 10 mmz cuvette was inconvenient and did not improve the reproducibility of the experimental results very much. Therefore, we developed a flow system using a peristaltic pump, a flow cell and teflon and fluororubber tubing. The peristaltic pump draws the solution through the flow cell directly from the stock solution. Flow rates of 1.5 me min-' and 2.0 me min-' were used in the quen- ching experiments with water and deuterium oxide, respectively; these were the slowest rates a t which minimal changes occur in singlet oxygen production

Singlet oxygen 461

0 0.4 0.0 1.2 1.6 2.0

Flow Rote ( m h n i n )

Figure 1. The emission intensity of '0, at 1270 nm and the absorbance of rose bengal in aqueous buffer following irradiation as a function of Row rate of solution through a spectroscopic flow cell. 1" is the emission intensity at the highest flow rate (2 mUmin) and A. is the absorbance at

548 nm before irradiation.

A

I I 1210 1230 1250 1270 l2bU 1310 1330 1350

W4VELENGTH ( n m l

Figure 2. Singlet oxygen emission spectrum sensitized by 21 FM rose bengal in aqueous 25 mM sodium phosphate buffer, pH 7.0 without (m), and with 0.34 mM (0) and

20 mM ( x ) sodium azide.

(see Fig. 1). The same flow rates were used with solutions of eosin Y, even though '02 production and the eosin Y absorbance showed less degradation under equivalent conditions (results not shown). The photobleaching of neither sensitizer was affec- ted by the presence of sodium azide. A stable and reproducible emission intensity reading at 1270 nm could be obtained while 4 to 5 me of solution flowed through the flow cell without recirculation.

Solutions for the Stern-Volmer plot were pre- pared by appropriate dilution of aliquots from stock aqueous solutions of rose bengal, eosin Y and sodium azide. Both the aqueous and deuterium oxide solutions contained 25 mM sodium phosphate at pH 7.0. In the case of the deuterium oxide buffers, we have assumed that pD = pH + 0.41 (Covington et al., 1968). All solutions were fully aerated.

Complete quenching of the '02 signal with sodium azide left a residual emission which we ascribe to the photosensitizer fluorescence (Fig. 2). Therefore, we used solutions of photosensitizer con-

taining 20 mM sodium azide in aqueous experiments and 5 mM sodium azide in experiments with deu- terium oxide in order to perform background sub- tractions. These azide concentrations did not sig- nificantly affect the emission of the photosensitizer measured outside the '02 spectral region. Maximum intensity and a baseline intensity were recorded with solutions containing no azide and the highest con- centration of azide respectively. After flowing buffer through the cell to guarantee clearance of the azide solution, an average emission intensity was recorded for each of the other solutions, followed by another determination of the intensities of unquen- ched and completely quenched '02 emission. Usually, there was a small lag time before a previous solution was completely displaced from the chamber by the next solution. Therefore, average values for emission intensity were recorded only after an inten- sity plateau had been reached. Each of the points in Figure 3 represents an average of three separate experiments. Figure 3A displays the Stern-Volmer plot obtained for aerated azide solutions using eosin Y as the sensitizer. It can be seen that a straight line quite adequately describes the quenching up to at least 80% of the initial intensity. Unfortunately, the '02 emission obtained by irradiating eosin Y was too weak to obtain reliable data at higher con- centrations of sodium azide. Photosensitization with rose bengal gave a five-fold greater '02 emission and, consequently, more reproducible results. The Stern-Volmer behavior for azide quenching in Fig. 3B is also linear up to at least 2.5 mM sodium azide. Companion experiments using oxygen-saturated sol- utions gave the same quenching curves within error (results not shown). It is not clear whether the apparent curvature at the highest concentrations of sodium azide is real or not. However, it is important to realize that the background value determined in each experiment becomes most critical in the region of highest quenching. Therefore, a least squares fit of the data points representing the four lowest concentrations of sodium azide in each plot was used to obtain the Stern-Volmer constants.

In order to further characterize the quenching of lo2 by the azide anion, we carried out the same quenching study with deuterium oxide buffer. The sensitizer concentrations were the same as in the aqueous solutions. Although the plots displayed in Fig. 4 are the result of single experiments, they appear to be remarkably linear to over 90% quen- ching of the original '02 intensity.

A Stern-Volmer plot of quenching by the aque- ous buffer showed less than 2% quenching by 25 mM buffer (results not shown) and the emission enhancements we have found suggest that phos- phate buffer does not significantly quench '02 in deuterium oxide. Therefore, we have assumed that the lifetimes given by Rodgers (1983) for loz in water (4.2 ps) and deuterium oxide (55.0 ps) are applicable to our system. The solutions of rose

462 ROBERT D. HALL and COLIN F. CHICNELL

0.8 1.6 2.4 3.2 [Sodium Azide] (mM)

I

r

I I I I I I I I I 08 16 24 3 2

[Sodium Azide] (mM)

Figure 3 . Stern-Volmer quenching results obtained with aerated solutions of eosin Y(A) and rose bengal (B) in aqueous sodium phosphate buffer monitoring the emission intensity at 1270 nm. The lines are least squares fit of the points corresponding the four lowest concentrations of sodium azide.

0 80 160 240 320 400

[Sodium Aride] ( p M )

Figure 4. Stern-Volmer quenching results with aerated deuterium oxide solutions of rose bengal (0) and eosin Y (O), buffered with 25 mM sodium phosphate, pH 7.0. The

lines are linear least-squares fits of the data points.

bengal in deuterium oxide contained 3% water by volume while the solutions of Eosin Y in deuterium oxide contained 1.5% water by volume. Accord- ingly, we have estimated the lo2 lifetimes in those solutions by calculating the weighted averages of the bimolecular rate constants given by Rodgers (1983). These values (Table 1) were then used to calculate the rate constants for azide anion quen- ching of '02 in deuterium oxide. The Stern-Volmer constants and the derived rate constants are given in Table 1.

3

Table 1. Quenching effects of azide anion on singlet oxy- gen emission in water and deuterium oxide

Eosin Y Rose Bengal

In water: Ksv ( M - ' ) 2420 2450 T ( l o ? ) (PS) 4.2* 4.2" k, (M-I s - l ) 5.76 x lox 5.83 x lox

In deuterium oxide: Ksv (M-'1 31 806 25 070

k, (m- s-I) 6.91 x lox 6.28 x lox

Signal enhancement

T(lo*) 46.0; 39.9t

in deuterium oxide: 21-fold 18-fold

*Singlet oxygen lifetimes from Rodgers ?Slight contamination with water reduces singlet oxygen

lifetime from value in pure deuterium oxide (55 ps). See text for details.

(1983).

DISCUSSION

The azide anion is commonly used as a diagnostic agent for lo2. Several studies have demonstrated that the azide anion efficiently quenches '02 through a combination of physical and chemical mechanisms. Hasty et al. (1972) published a rate constant in methanol (2.2 x lo8 M-' s-' ) and Foote (1979) calculated a value for methanol-water (8:1, vol/vol) solution (2.8 x lo8 M - ' s - ' ) based on data in Foote et al. (1972).

More recently, Lindig and Rodgers (1981) used a laser flash technique to obtain a rate constant equal to 5.1 x lo8 M - ' s - ' for the quenching by sodium azide of lo2 sensitized by methylene blue in deuterium oxide. This last value is close to the rate constant we have calculated for the quenching of '0, sensitized by rose benaal in deuterium oxide.

Singlet oxygen 463

There is also very good agreement between the rate constants for sodium azide quenching in water and in deuterium oxide calculated according to the life- time data given by Rodgers (1983).

The dependence of the derived rate constant on the photosensitizer used in deuterium oxide may only reflect inaccuracies in the estimation of '02 lifetimes in solutions of deuterium oxide containing small amounts of water. It may also be the result of successful competition by rose bengal for '02 at the lower concentrations of sodium azide used in that solvent. Indeed, the photobleaching of rose bengal increased in deuterium oxide. It is not likely that self-quenching contributed to the quenching of 'Or in deuterium oxide since the intensity of 'Or is linear with photosensitizer excitation over the entire range of emission intensity observed in the present experiments (not shown). The 20-fold enhancement of the '02 signal that we obtained with the deu- terium oxide samples can be explained by noting that, in addition to the difference in the lifetime of '02 in water and deuterium oxide, part of the emis- sion of lo2 is re-absorbed by the water through which the signal travels before it leaves the cuvette. The transmittance of a 2 mm pathlength of water at 1270 nm is 70% while the transmittance of deu- terium oxide is close to 100% (unpublished result). This is consistent with the apparent enhancement obtained in the quenching experiments.

The rate constant for the quenching of eosin Y by azide anion ( k , = 1 x lo6 M-' s-') determined by Kraljic and Lindqvist (1974) is two orders of magnitude below the rate of quenching of 'OZ deter- mined in the present experiments. Our results also suggest that the azide anion cannot compete with oxygen as a quencher of the triplet state of rose bengal. The linear quenching plots (up to at least 80% quenching of lo2) and quenching rate con- stants which are relatively unaffected by the nature of the sensitizer suggest that the derived quenching constant represents the dynamic quenching of '02 by azide anion.

We have estimated the sensitivity of the instru- ment using the results obtained in the quenching experiments. The spectral irradiance of the exciting light after passing through 10 cm of water and the Schott KV418, KG2 and KG3 filters was 1.58 X lo4 W m-* at the cuvette. The diameter of the beam cross-section was about 3 mm. Thus, the radiant flux was 0.1 W. Taking into account the wavelength dependence of the photon energy and converting the irradiance to photons/sec, the rate of photon absorbance, Q, by rose bengal can be estimated by using Eq. 7 ,

Q = 5.65 x 10"z AfF (A) {l-lO-A(A)}. (7) Tf (A) AA (photons/s)

where fE (A) is the fraction of radiant power, A (A) is the absorbance of the sensitizer, Tf (A) is the combined transmittance of the lenses and optical

filters in the excitation light path at wavelength A and AA is the wavelength interval. The quantities, fE (A), were calculated from a typical profile of spectral irradiance provided by Kratos Instrument Co. for the 200 W Hg lamp. T1 (A) and A (A) were measured with a spectrophotometer. The sum- mation was carried out with a computer using a 5 nm wavelength interval and absorbance values for the most dilute solution of rose bengal (1.0 F M ) for which an unambiguous spectrum of lo2 could be obtained at 1270 nm. Under these conditions, pho- ton absorption was 4.0 x 10Ih photonsls. If the quantum yield of 'Or production by rose bengal is 0.76 (Lamberts and Neckers, 1985), then the instrument can detect '02 in an aqueous medium when the rate of '02 production is 50 nanomolis. Since the quantum yield of '02 phosphorescence has been reported to be about in water (Kras- novsky, 1981a,b), this translates to a rate of 'Or emission of 50 fmol/s. The performance of our instru- ment is comparable to that of good commercial fluorometers (Jameson, 1984) when the quantum yield of '02 in water is taken into account.

The very low quantum yield of 'Or in aqueous media and the small illuminated volume require that '02 production occur at very high rates (1 mM s-') in terms of concentration in order to allow the detection of its luminescence. Furthermore, rose bengal is an ideal photosensitizing agent because it has a high absorptivity and its intersystem crossing is efficient. Photosensitizers which have lower absorptivities or which produce 'Or less efficiently than rose bengal must be studied at proportionately high concentrations. Ultimately, therefore, a com- bination of stronger excitation power and higher levels of diode sensitivity will be needed in order to routinely monitor in vivo production of '02 by most photosensitizing agents.

Chou, P.-T. and A. U. Kahn (1983) L-Ascorbic acid quenching of singlet delta molecular oxygen in aqueous media: Generalized antioxidant property of vitamin C . Biochem. Biophys. Res. Commun. 115, 932-937.

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Foote, C . S. (1979) Quenching of singlet oxygen. In Singlet Oxygen (Edited by H. H. Wasserman and R. W. Murray), pp. 139-171. Academic Press, New York.

Foote, C. S. , T. T. Fujimoto and Y. C . Chang (1972) Chemistry of singlet oxygen. XV. Irrelevance of azide trapping to mechanism of ene reaction. Tetra- hedron Left. 1, 45-48.

Gollnick, K . , T. Franken, G . Schade and G. Doer- hoeffer (1972) Photosensitized oxygenation as a function of the triplet energy of sensitizers. Ann. N.Y.

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Hasty, N., P. B. Merkel, P. Radlick and D. R.

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