Anal. Chem. 1988, 60,2028-2030
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Fiber-optic Fluorosensor for Oxygen and Carbon Dioxide Otto S. Wolfbeis* and Leonie J. Weis Analytical Division, Institute of Organic Chemistry, Karl-Franzens University, 8010 Graz, Austria
Marc J. P. Leiner and Werner E. Ziegler AVL-List GmbH, Kleist-St. 48, 8020 Graz, Austria
The capability of fiber-optic ilght guMes to transmit a varlety of optical signals simultaneously has been explolted to construct an optkal flber sensor for measurement of both oxygen and carbon dioxlde. The oxygen-sensitlve material (a Kieselgel-adsorbedfluorescent metal-organic complex) and the C0,-sensitlve materlal (an immobilized pH indicator in a buffer solution) are entrapped in a gas-permeabie polymer matrlx that is attached to the distal end of the fiber. Both indicators have the same excitation wavelength (in order to avoid energy transfer) but quite different emission maxlma. The two emlsdon bands can easily be separated with the help of interference fliters and give Independent signals. Oxygen can contlnuously be determined In the 0-200 Torr (0-26.6 kPa) range with flTorr accuracy and CO, in the 0-150 Torr (0-20 kPa) range with f l Torr. The accuracy is hlgher at low partial pressure, so that the detectlon limits are at -0.5 Torr In both cases.
It has frequently been stated that the high flux of information density is one of the advantages of fiber-optic cables over other types of information carriers. This, in fact, is nowaday extensively utilized in fiber-optic telecommunication systems with their extremely high bit rates. In the case of chemical sensors, another advantage of fibers may additionally be exploited, namely the simultaneous transmission of light of different color. This may pave the path for performing multiple analyses with one sensor. We report here on the first fiber-optic chemical sensor in which the feature of multiwavelength communication is exploited to sense two analytes with one fiber bundle. Several types of sensors for oxygen have been reported so far, some based on chemiluminescence (1) or thermoluminescence (2),but most on dynamic fluorescence quenching (3-8) including lifetime measurements (8). Optical determination of carbon dioxide has exclusively been performed by monitoring changes in the p H of a buffer solution via an added pH-sensitive dye (3,4, 7,9-11). To keep it in position and to prevent interferences by protons in liquid sample solution, the buffer plus dye solution is usually entrapped in a gas-permeable polymer such as silicone rubber. A proper choice of indicators with suitable spectral properties, together with a modified membrane composition has now led to the construction of a single sensor for two analytes which is described below. EXPERIMENTAL SECTION Chemicals. Oxygen and carbon dioxide, both of >99% purity, were taken from cylinders and mixed with nitrogen by using a RDM 280 mass flow controller (Air Liquide, Vienna) to give gases of defined percent O2and C 0 2 composition. The precision of the gas mixing device is specified to be within f l %of the actual value. Most of the experiments were performed at a barometric pressure of 740 Torr (98 kPa). Tris(2,2’-bipyridiyl)ruthenium(II)dichloride (RTDP, from Strem Chemicals, Newburyport, MA), 1-hydroxypyrene-3,6,8trisulfonate (Lambda Probes & Diagnostics, 8053 Graz, Austria),
one-component silicones of type E43 and E15 (Wacker Chemie, Burghausen, FRG) and Kieselgel (LiChroSpher Si 300, Merck, Darmstadt, FRG) were used as obtained. Sensing Membranes. Transparent 0.25 mm thick polyester membranes were used as solid supports, onto which the two indicator layers where deposited (Figure 1). Layer and support were then attached to the end of the fiber optic. The C02-sensitive layer (CSL) consists of HPTS covalently immobilized onto cellulose granules by analogy to the method published for the pH sensor (12)and embedded into Hydrogel. It was soaked with 15 mM bicarbonate buffer and then covered with a 80 wm thick layer of the oxygen-sensitive material (OSL) which essentially is a suspension of Kieselgel particles, dyed with RTDP, in E43 silicone rubber (13). The uppermost layer is the optical isolation (01) that prevents ambient light and sample fluorescence from entering the fiber. It consists of a 20-pm layer of red E15 silicone. A cross section of the membrane is shown in Figure 1. Optical Arrangement. Light from a 6-W 4.6-V halogen tungsten filament lamp was focused onto a 460-nm interference fiiter and launched into a bifurcated fiber-optic waveguide bundle (from Volpi AG, CH-8952 Schlieren, Switzerland), the common end of which (10 mm 0.d.) contained the sensing layer. Fluorescence (and scattered light) returns through the second half of the fiber bundle, passes either a 520-nmor 630-nm interference filter and is detected with a photodiode (BPW 21, from Siemens, D-8000 Munich, FRG). After passing a current-to-voltage converter (1 V/nA) and a frequency filter (0.3 Hz), the signal is amplified and displayed in an LCD display and recorded on an X/t recorder. Simultaneously, the data were stored in an lT3M-XT personal computer, which also governs the automatted measurement procedure including gas supply and sample flow-through.
RESULTS AND DISCUSSION The sensing method is based on the simultaneous excitation of two fluorescent indicators with well-separated emission bands. Figure 2 shows the excitation and emission spectra of HPTS and RTDP; the arrows indicate the analytical wavelengths. Since the two layers are spatially separated, the optical density of the COz-sensitive layer has to be kept low in order to allow sufficient light to shine onto the oxygensensitive layer. The thickness of the C02-sensitivelayer can be kept small because HPTS has a fairly high absorbance and very high quantum yield (close to l.O), whereas RTDP has low absorbance and low quantum yield (ca. 0.1). Unlike in other work on oxygen (5) and COz sensors (10) we prefer to use silicone, which is an almost ideal material. It lends itself to easy manufacturing of thin layers, is available in a number of modifications, and, most important, has the best permeability for gases among all polymers (14, 15). The sensing schemes for oxygen and COz are entirely different in that oxygen acts as a dynamic quencher of the fluorescence of RTDP, thereby interacting with the first excited singlet state of the indicator. Carbon dioxide, on the other hand, changes the pH of a 15 mM bicarbonate buffer solution according to GO2 + H 2 0 HC03- + H+. This is a ground-state reaction resulting in a shift in an acid-base equilibrium of a fluorescent pH indicator that is “seen” by the fiber. Typical response curves are shown in Figures 3 and 4. The total signal change in going from 2.8% to 15.0% COPand from
0003-2700/88/0360-2028$01.50/0 @ 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 1988
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Flgure 1. Cross section of the membrane used for simultaneously sensing oxygen and carbon dioxide: S, solid support; C.S.L., carbon dioxide sensitive layer containing cellulose granules with immobilized pH indicator and soaked with bicarbonate buffer; O.S.L., oxygen-sensitlve layer with entrapped Kieselgel beads containing the oxygen indlcator; 0.I., 'optical Isolation.
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Figure 2. Absorption and emission spectra of RTDP [trls(2,2'-bipyrldyl)ruthenium(II ) dichloride] (A and A') and HPTS [ l-hydroxypyrene-3,6,8-trlsulfonate] (B and 9') In a sensor with internal buffer of pH 8. The arrows indicate the analytical wavelengths. The absorption spectrum of RTDP strongly changes at concentrations above 5 mM.
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Figure 3. Typical response of the double sensor toward oxygen admixed to nitrogen (left) and carbon dioxide (right) and electrical signal intensity changes obtained (in mV) by measuring fluorescence at 630 nm.
7% to 20% oxygen (i.e., the clinically most interesting range) are 88 and 173 mV, respectively. The signal-to-noise ratio is >500. Obviously, there is no cross sensitivity between the two sensors and gases, as can be seen from the identity of the signals for oxygen and COP in different carrier gases. The spectral overlap of the two indicators and the efficiency of energy transfer must also be negligible at the analytical
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Figure 5. Relative fluorescence of the oxygen-sensitive layer in the double sensor and corresponding Stern-Volmer plot at a total gas pressure of 98 kPa.
wavelengths (520 and 630 nm) because otherwise there would be some interference to be observed. Figure 5 shows a plot of relative signal of the oxygen sensor versus the fraction of oxygen in the gas, together with a Stern-Volmer-type plot. The Stern-Volmer equation predicts the relation between optical signal intensity I and quencher concentration [O,] to be
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where Io is the fluorescence intensity in the absence of oxygen (i.e., maximum). Z is the fluorescence in the presence of a quencher in concentration [O,],and k,, is the quenching constant that is specific for each indicator/quencher combination and also depends on solvent and temperature. The linear graph in Figure 5 was obtained by a modified SternVolmer equation that accounts for contributions of stray light and unquenchable fluorescence (I,)
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It fits the experimental data with very good precision. 12, is found to be 0.08%-'. Given the slope of the Stern-Volmer plot, a 173-mV signal change over the analytically useful range, and a signal resolution of f0.2 mV, an accuracy of f l Torr (133 Pa) in the oxygen assay can be calculated. The change in optical signal versus C 0 2 partial pressure is shown in Figure 4. The signal drops with increasing COP partial pressure because of the lowering of the pH of the internal buffer. The relative signal change is most expressed a t low COz levels and becomes smaller as the COP pressure is raised. The clinically useful range is from 2.8% to 15% COP
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 1988
Given the signal change (88 mV) over this range and a signal-to-noise ratio of >500 and in view of the slope of the calibration curve in the range of interest, one can calculate the accuracy to be on the order of at least f l Torr again. At low levels of both oxygen and C 0 2the slopes of the response curves are distinctly steeper than in the usual range, which means that less than 1 Torr of the respective gases can be detected. In fact, from both the estimations and the experiments we find the detection limits to be in the order of 0.5 Torr. The response times strongly differ from each other, as can be seen from Figures 3 and 4. It is well known that oxygen sensors are faster than C02sensors because of both diffusional and chemical processes involved in the second case. Moreover, C02sensors tend to become destabilized once they are exposed to low C02 levels for some while and that they need hours for regeneration. Typical response times until the total signal change has occurred are 40 s for the oxygen sensor, but 3.8-5.2 min for the C 0 2 sensor. Note in Figure 4 that the response time is faster in going from lower to higher levels of C 0 2 than reverse. The longer response of the C 0 2 sensor is, of course, partially also due to the fact that the C02-sensing layer is covered by the oxygen-sensitive layer, which may prolong the time for equilibration. Sensing two analytes with one sensor requires two indicators having no cross sensitivity. At least three kinds of cross sensitivity in fluorescence-based multiple sensors have to be discerned: (a) a strong overlap of the fluorescence emission bands of the indicators, so that a analyte-specific wavelength with sufficient signal intensity cannot be found; (b) the indicator is not specific for a given analyte (oxygen, for instance, is a notorious and almost ubiquitous quencher of luminescence); (c) the emission band of one dye overlaps with the absorption band of the other, giving rise to energy transfer between analyte-sensitive and analyte-insensitive dye. Such a situation, which has been exploited in a pH-sensor (16),has to be avoided in this case. The other kind of energy transfer (the dipole-dipole interaction) is easily avoided in this case by spatial separation of oxygen- and C02-sensitivelayers, since resonance energy transfer inversely depends on the 6th power of distance. As a result of these considerations, the two indicators required for simultaneously sensing oxygen and C 0 2 (a) have to be specific for oxygen and COO,respectively, (b) have rather similar absorption maxima (and, therefore, excitation wavelength), and (c) have entirely different emission maxima. Suitable indicators were found in tris(2,2’-bipyridiyl)ruthenium(I1) chloride (RTDP, for oxygen) and in l-hydroxypyrene-3,6,8-trisulfonatetrisodium salt (HPTS, for carbon dioxide). RTDP is not affected by C 0 2 at levels up to 200 ‘I’orr (26 kPa) and has a pH-independent fluorescence in the pH 6-8 range. The fluorescence of HPTS is quenched by -3.5% only in going from pure nitrogen to pure oxygen, which is negligible.
Rather than using UV-excitable dyes (which have the disadvantage of requiring expensive UV light sources and fused silica fibers), we decided to use dyes excitable by conventional light sources. No lasers have to be applied in our case. In addition, the signal from the sensors is intense enough to be detected with a photodiode rather than with a more expensive photomultiplier tube. All these facta contribute to keep the costs for optical sensors at a minimum. It is probably informative to cite from a paper of Lubbers and Opitz ( 4 ) who stated that “with a different set of filters the optical part of the sensor can be used for measuring different substances.” This seems to be a first hint for the possibility of sensing two analytes simultaneously, although the indicators used a t that time (pyrenebutyric acid and 4methylumbelliferone) required UV excitation and had poor spectral separation. Miller et al. (7)have developed a triple sensor for blood pH, pC02, and p 0 2 that consists of three 100-rm fibers with appropriate chemistries a t their ends. Although the whole sensing needle is only 1 mm thick, the size of the sensor is still a limiting factor. We think the new sensor type will present a considerable improvement in blood gas sensing since it can reduce the size of the sensing heads for two analytes by half. This is a result of the unique properties of optical fibers and may result in new types of invasive catheters. There is not obstacle in sight that would prevent the extension of this principle to a triple one-fiber sensor for oxygen, carbon dioxide, and pH. Registry No. RTDP,14323-06-9;02, 7782-44-7;COz, 124-38-9. LITERATURE C I T E D Freeman, T. M.; Seitz, W. R. Anal. Chem. lQ81,5 3 , 98. Hendricks, H. D. US Patent 3 709 663, 1973. Wolfbeis, 0. S. “Fiberoptic Sensors in Analytical and Cllnlcal Chemistry; Molecular Luminescence Spectroscopy: Methods and Appllcations; Schulman, S. G., Ed.; Wiley: New York, 1988; Chapter 3. Lubbers D. W.; Opitz, N. Sens. Actuators 1083,4 , 641. Peterson, J. I.; Fitzgerald, R. V.; Buckhold, D. K. Anal. Chem. 1084, 56, 62. Kroneis, H. W.; Marsoner, H. J. Sens. Acfuators 1083,4 , 587. Miller, W. W.; Yafuso, M.; Yan, Ch. F.; Hui, H. K.; Arick, S. Clin. Chem. (Winsfon-Salem, N . C . ) 1087,33. 1538. Lippksch, M.; Leiner, M. J. P.; Pusterhofer, J.; Wolfbeis, 0. S. Anal. Chim. Acta 1988,205, 1. Zhujun, 2 . : Seitz, W. R., Anal. Chim. Acts 1984, 160, 305. Vurek. G. G.; Peterson, J. I.; Goldstein, S. W.; Severinghaus, J. W. Fed. proc., Fed. Am. SOC.Exp. Blol. 1982,4 1 , 1484. Munkholm, Ch.; Walt, D. R.; Mllanovich, F. P. T8lenta 1988,35, 109. Offenbacher, H.; Wolfbeis, 0. S.; Furlinger, E. Sens, Actuators 1986, 9 , 73. Wolfbeis, 0. S.; Leiner, M. J. P.; Posch, H. E. Mikrochlm. Acta 1987, III, 359. Cox, M. E.; Dunn, 6. J . Pol)”. Sci. 1986,24. 621 and 2395. Brandrup, j.; Immergut, E. H. The Polymer Handbook; Wlley: New York, 1975; p 111-232 ff. Jordan, D. M.; Walt. D. R.; Milanovich, F. P. Anal. Chem. lW7, 59, 437.
RECEIVED for review February 3, 1988. Accepted April 25, 1988.
