Anal. Chem. 1986, 58,220-222
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(8) Sepaniak, M. J.; Vargo, J. D.; Kettler, C. N.; Maskarinec, M. P. Anal. Chem. 1984, 54, 1252-1257. (9) Buffett, C. E.; Morris, M. D. Anal. Chem. 1982, 54, 1824-1825. (IO) Buffett, C. E.; Morris, M. D. Anal. Chem. 1983, 55, 376-378. (11) Morris, M. D. R o c . S f I E I n t . SOC. Opt. Eng. 1983, 426, 116-120. (12) Leach, R. A.; Harris, J. M. Anal. Chem. 1984, 56, 2801-2805. (13) Pang, T. J.; Morris, M. D. Appl. Spectrosc. 1985, 39, 90-93. (14) Nickoiaisen, S. L.; Blalkowski, S. E. Anal. Chem. 1985, 57,758-762. (15) Fang, H. L.; Swofford, R. L. I n “Ultrasensitive Laser Spectroscopy”; Kliger, D. S., Ed.; Academic Press: New York, 1983; pp 178-233. (16) Long, G. R.; Bialkowskl, S. E. Anal. Chem. 1984, 56, 2806-2811. (17) Mori, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1982, 54, 2034-2038. (18) Buffett, C. E.; Morris, M. D. Appl. Spectrosc. 1983, 37, 455-458. (19) Siebert, D. R.; Grabiner, F. R.; flynn, G. W. J . Chem. Phys. 1974, 60, 1564-1574.
(20) (21) (22) (23) (24)
Barker, J. R.; Rothem, T. Chem. Phys. 1982, 68, 331-339. Twarowskl, A. J.; Kliger. D. S. Chem. Phys. 1977, 20,253-258. Gigiio, M.; Vendramlni, A. Appl. fhys. Lett. 1974, 25,555-557. Welmer, W. A.; Dovlchi, N. J. Anal. Chem. 1985, 57, 2436-2441. Jackson, W. B.; Amer, N. M.; Boccara, A. C.; Fournier, D. Appl. Opt. 1981, 20, 1333-1343.
RECEIVED for review April 22, 1985. Resubmitted August 5, 1985. Accepted September 30, 1985. This research was supported in part by BRSG SO7 RR07093-17 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health.
Optical Sensor for Oxygen Based on Immobilized Hemoglobin Zhang Zhujun’ and W. Rudolf Seitz*
Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824
The oxygen sensor consists of a 0.5 mm thick layer of deoxyhemoglobin immobilized on catlon exchange resin and positioned on the common end of a bifurcated fiber optic bundle. An 0,-permeable TFE Teflon membrane separates the knmobiilzed reagent phase from the sample. The sensor Is based on the shlft in the Soret absorption band of hemoglobin upon association with 0,. The specific parameter measured Is the ratio of reflected intensities at 435 and 405 nm. This parameter may be used to measure 0, partlal pressure from 20 to 100 torr H a suitable cailbratlon curve is established. Reflected intensitles and Intensity ratlos decrease with the amount of Immobilized hemoglobin. For a reagent layer 0.5 mm thick, the optimum hemoglobin loading is 0.03 g/g catlon exchange resin. I t takes approximately 3 min to reach steady state when an 0, sample is introduced. The sensor must be stored in a reducing medium to prevent oxidation of hemoglobinto methemoglobin. I t Is stable for 2 days when stored at room temperature and for a week when stored at 4 OC.
We have prepared an optical sensor for oxygen based on immobilized hemoglobin. The sensor exploits the shift in the Soret absorption band when deoxyhemoglobin combines with oxygen to form oxyhemoglobin. The ratio of the hemoglobin reflectances a t 405 and 435 nm serves as a measure of 0 2 partial pressure. One attractive feature of this approach is that it involves a true equilibrium. In contrast, the oxygen electrode requires steady-state mass transfer to 0, to the electrode surface and thus is subject to error if factors which affect 0, mass transfer are not properly controlled (1). The same is true for an 0 2 sensor based on chemiluminescence (2). Optical 0, sensors based on fluorescence quenching avoid this problem but are not true equilibrium sensors because response depends on the relative rates of fluorescence and nonradiative return to the ground state via interaction with 0, (3-6). Thus this type of sensor is sensitive to slight changes in medium, which affect fluorescence, and to the presence of other quenchers. Present address: Department of Chemistry, Shaansi Normal University, Sian, Shaansi, People’s Republic of China.
A more fundamental advantage of the approach described here is that it is based on a spectral shift. Because the oxygenated and deoxygenated forms each have distinct spectra, there is inherently more information available than there is from a fluorescence quenching based sensor where the measured parameter is a decrease in intensity. The most serious limitation of the O2sensor based on immobilized hemoglobin is that its useful lifetime is short because hemoglobin degrades. A second problem is that the measured response function is nonlinear with O2 partial pressure and is not readily described mathematically. EXPERIMENTAL SECTION Apparatus. The apparatus for the O2sensor is similar t o apparatus used earlier in our laboratory for pH and pC0, sensors (7,8). The output of a 250-W tungsten halogen lamp (Edmund Scientific) is passed through one of two dielectric interference filters with peak transmittances at 435 nm and 405 nm and bandwidths of 6.8 nm and 9.2 nm, respectively, at half-maximum transmittance. The filtered beam from the lamp is passed through one arm of a bifurcated fiber optic bundle (common end diameter 4.5 mm) to a thin layer of immobilized hemoglobin. The reflected intensity is monitored through the other branch of the bifurcated fiber optic bundle by an RCA 1P21 photomultiplier tube. The photocurrent is displayed on a Spex digital photometer (Model DPC 2) and recorded on a Heath SR-255B strip chart recorder. Figure 1shows a detailed view of the reagent phase and sample cell. A piece of Tygon tubing holds a 25 pm thick TFE Teflon membrane (American Durafilm Co., Inc.) on the end of a 7 mm i.d., 50 mm long piece of glass tubing. The membrane serves to separate the sample from the reagent phase, which consists of an immobilized suspension of deoxyhemoglobin in phosphate buffer. Samples are introduced or withdrawn by syringe. 0thenvise the cell is sealed to prevent ambient O2from entering. The sample cell is covered with A1 foil on the outside to enhance the reflected signal (a tactic that will only work if the sample is transparent at 405 and 435 nm). In operation, the entire sample cell assembly is covered with black cloth to exclude ambient light. Absorption spectra were obtained with a Shimadzu Model UV-200s double-beam spectrophotometer. A Matheson Model 7401T gas proportioner connected to nitrogen and oxygen cylinders was used to prepare standard oxygen solutions. The pH was measured with an Orion digital Ionanalyzer/SOl equipped with an Orion combination glass pH electrode. Reagents. Hemoglobin (substrate powder type 11), sodium dithionite and CM-Sephadex-C-50-120 were purchased from Sigma Chemical Co. Oxygen (99.6%) and nitrogen (99.7%) were
0003-2700/86/0358-0220$01.50/0 0 1985 American Chemical Society
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Flgure 1. Close-up vlew of common end of blfurcated fiber optic showing reagent phase and sample cell: a, glass tubing; b, TFE Teflon membrane; c, reagent phase (Immobilized hemoglobin suspended In buffer); d, sample cell; e, rubber stopper; f , stainless steel syrlnge needle; g, common end of bifurcated fiber optic bundle; h, short stainless steel syringe needle; i, O-ring seal; j, stlr bar; k, Tygon tubing.
purchased from Welders Supply Co. Deoxygenated water was prepared fresh daily by boiling deionized water vigorously for about 20 min and then cooling under a nitrogen atmosphere. Nitrogen was then bubbled through this water to eliminate the remaining oxygen. Standard oxygen solutions were prepared by dissolving known mixtures of oxygen and nitrogen in the deoxygenated water using the gas proportioner. Phosphate buffers (0.020 M) were prepared by dissolving 1.37 g of potassium dihydrogen phosphate in 500 mL of water and adjusting to the desired pH with 2 M potassium hydroxide. Unless otherwise indicated, the pH was 7.20 in all experiments. Procedures. Hemoglobin was immobilized by electrostatic attraction to preswollen CM-Sephadex C-50-120 ion exchange resin by a published procedure (9). Unless otherwise indicated, the loading was 0.030 g/g of resin. Absorption spectra were measured on suspensions of immobilized hemoglobin in 0.020 M phosphate buffer at pH 7.20 after allowing time for the suspension to settle. Swollen resin suspended in buffer was placed in the reference beam. To get spectra of deoxyhemoglobin, 5 mg of sodium dithionite was added to reduce oxyhemoglobin. The sensor is prepared in a nitrogen-filled glovebag. Immobilized hemoglobin on 0.5 g of resin is reacted with sodium dithionite to reduce all the hemoglobin to the deoxy form. After several washes with 0.020 M phosphate buffer at pH 7.20 to remove excess sodium dithionite, the resin is placed in the glass tubing of the sample cell, covered with TFE Teflon membrane, and positioned in the sample cell. The sample cell is filled with 5.0 mL of deoxygenated water. At this point the glass tubing is removed from the glovebag. A 3-min interval is allowed after adding solution before making a measurement to let the sensor reach equilibrium.
RESULTS AND DISCUSSION Absorption Spectra. Figure 2 shows absorption spectra for oxy- and deoxyhemoglobin immobilized on cation exchange resin. For comparison, solution spectra are included. Immobilization on a cation exchanger causes the Soret band to shift to longer wavelength by about 5 nm but does not otherwise affect the spectrum. In particular, it does not cause band broadening, which would make it more difficult to resolve oxy- and deoxyhemoglobin spectrally. The wavelengths chosen for quantitative measurements were 405 and 435 nm, which are selective for oxy- and deoxyhemoglobin, respectively. Response Time. It takes about 3 min to reach constant response at room temperature with a 0.5 mm thick hemoglobin layer. Presumably the main component of the response time is the time required for mass transfer of O2 into the immobilized hemoglobin phase. Reducing the thickness of the hemoglobin layer and increasing the temperature have the expected effect of shortening the response time. After an 02-containing sample is removed, it takes about 5 min for the signal to return to its original value. With use, the fall time becomes longer. Stability. Oxyhemoglobin oxidizes to methemoglobin in a matter of hours (IO),interfering with the ability of sensor to respond to oxygen. To minimize the extent of this process
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the sensor should be kept in an oxygen-free environment when not in use. Covalent immobilization of the hemoglobin was found to reduce the rate of oxidation. Methemoglobin once formed can be reduced back to deoxyhemoglobin by treatment with dithionite. This allows for continued use of the sensor once the excess dithionite is washed away. Immobilized hemoglobin also irreversibly degrades with time. Hemoglobin immobilized on the cation exchanger degraded after 2 days a t room temperature and 7 days when stored at 4 "C. The instability problem is the most serious limitation of the sensor based on immobilized hemoglobin. Other immobilization methods may lead to improved stability; however, if they cause spectral broadening they may adversely affect response to O2 If the degradation process produces observable changes in the spectrum of immobilized hemoglobin, then the onset of degradation can be determined by monitoring the intensity at a third wavelength. This is possible if degradation involves oxidation to methemoglobin since the absorption spectrum of methemoglobin differs significantly from the spectra of oxy- and deoxyhemoglobin (10). It would have to be determined experimentally whether other degradation
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
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processes gave rise to observable spectral changes. Response to Oxygen. Figure 3 shows reflected intensities at 405 nm and 435 nm as a function of oxygen partial pressure. As expected, increasing levels of O2causes the reflected intensity to increase a t 435 nm and decrease at 405 nm. Response to O2 is sigmoid in character. In part this may be due to the well-known cooperativity of hemoglobin (IO). However, the degree of cooperativity is reduced by immobilization.
Figure 4 shows how the amount of immobilized hemoglobin affects the reflected intensities and the intensity ratio. As amount increases, reflected intensities decrease, ultimately approaching a limiting value. The intensity ratio also decreases with increasing amount. As would be expected, the slope of the plot of reflected intensity vs. amount is very large for small amounts. Similar results were obtained in an experiment in which the loading of immobilized hemoglobin was held constant at 0.030 g/g of resin and the thickness of the reagent phase was varied. As the thickness is increased from 0.1 to 2.0 mm, the reflected intensity decreases and the response time is longer. We chose to work with a thickness of 0.5 mm because this was the smallest practical value using ow procedures. With a more sophisticated arrangement, it would be better to work at smaller thicknesses and higher hemoglobin loadings. This would lead to shorter response times with otherwise similar response characteristics. Factors Affecting Response. The affinity of hemoglobin for oxygen is a function of pH and ionic strength (IO). Experimentally it was found that reflected intensity for an oxygen partial pressure of 100 torr was a function of both pH and the buffer concentration. The conditions chosen in this study were designed to maximize response for po, = 100 torr. Within experimental error response to oxygen was independent of temperature from 30 to 40 "C although the response time was shorter at the higher temperature. The relative standard deviation of replicate measurements on the same solution was 2-4%. It presumably would be improved if the instrument were designed to automatically measure intensity ratios rather than requiring consecutive measurements of intensities at each wavelength. The question of interferences was not addressed experimentally. However, they can be predicted from known properties of hemoglobin. Carbon monoxide would be a serious interference because it binds very strongly with hemoglobin. Gases that would oxidize hemoglobin would also interfere. Gases that act as acids or bases will interfere if the amount of gas that transfers across the membrane is sufficient to change the pH of the phosphate buffer in the reagent phase. However, this was not observed as a problem in the course of this study. Registry No. 02, 7782-44-7; Teflon, 9002-84-0.
LITERATURE CITED (1) Hitchman, M. L. I n "Chemical Analysis"; Elving, P. J., Winefordner, J. D., Kolthoff, I. M., Eds.; Wlley: New York, 1978; Vol. 49. (2) Freeman, T. M.; Seitz, W. R. Anal. Chem. 1981, 53, 98-102. (3) Peterson, J. I.; Fitzgerald, R. V.; Buckhold, D. K. Anal. Chem. 1984, 56, 62-67. (4) Luebbers, D. W.; Opitz, N. Sens. Actuators 1983, 4 , 641-654. (5) Kronels, H. W.; Marsoner, H. J. Sens. Actuators 1983, 4 , 587-592. (6) Wolfbeis, 0.S.; Offenbacher, H.; Kroneis, H.; Marsoner, H. Mikrochim. Acta 1984, 153-158. (7) Saari, L. A.; Seitz, W. R. Anal. Chem. 1982, 5 4 , 821-823. (8) Zhujun, 2.; Seitz, W. R. Anal. Chim. Acta 1984, 160, 305-309. (9) Antonini, E.; Fanelli, M. R. R. I n "Methods in Enzymology"; Mosbach, K., Ed.; Academic Press: New York, 1978; Vol. 44, pp 538-543. (IO) Fairbanks, V. F. I n "Fundamentals of Clinical Chemistry", 2nd ed.; Tietz, N. W., Ed.; W. 6 . Saunders: Philadelphia, PA, 1976; Chapter 6. pp 401-454.
RECEIVED for review January 22, 1985. Resubmitted June 7, 1985. Accepted August 22,1985. Instrumentation Laboratory, Inc., provided financial support for this research.
