Anal. Chem. 1995, 67, 1505- 1509
Measurement of Dissolved Oxygen in Water Using Glass-Encapsulated Myoglobin Kwang E. Chung,t Esther H. Lan,* Michael S. Davidrron,* Bruce S. Dunn,* Joan Sehrerstone Valentine,*pt and Jeffrey I. Zinkt Departments of Chemistry and Biochemistry and Materials Science and Engineering, University of Califomia, Los Angeles,
405 Hilgard Avenue, Los Angeles, Califomia 90095 Myoglobin (Mb) encapsulated in a glass matrix by the solgel method was examined as a sensing element for measurement of dissolved oxygen (DO) in water using electronic absorption spectroscopy in the visible region. The Mb-containinggel was porous and transparent, and the encapsulated Mb exhibited the same chemical and spectroscopic properties in the gel as in solution upon reduction of metMb with dithionite to give deoxyMb or absorption of DO to give oxyMb. The absorbance of a deoxyMb-containinggel changed linearly with time upon exposure to DO for the first 8 min, or longer, at three selected wavelengths, 418, 431.5, and 436 nm. The linear absorbance change rate 'was established in a few minutes, 8 min, as shown in Figure 3, the change rates were determined from data obtained within 5 min. The absorbance change rate for each water sample was constant with a correlation index greater than 0.98. The constant rates revealed that the amounts of DO depleted from water samples due to the absorption by deoxyMb were relatively very small, and nearly constant amounts of DO were available for the absorption by deoxyMb. Thus, the amount of Mb in the gel was withii the right range of concentration. As observed with Figure 3, the absorbance decreased faster with a higher DO concentration, suggesting that the rate of absorbance change is proportional to the DO concentration. The rates of the absorbance change are plotted versus the concentration of DO for three different wavelengths (418,431.5, and 436 nm) in Figure 5. In these plots, the absolute values of absorbance change rates were used for convenience whether the absorbance increased or decreased. Each data point represents the average value of triplicate determinations. Errors were 16% in Figure 5 4 and 12%in Figure 5B and C. Figure 5A reveals that the absorbance change rate at 418 nm can be divided into two parts. In a low concentration range, ~ 2 5 % air saturation, the absorbance change is extremely small and is almost insensitive to the DO concentration. In the higher concentrationrange, >25% air saturation, the absorbance change rate increased with the DO concentration. The correlation index was 0.961. This high correlation index indicates that the insensitivity at the lower concentration range may be due to the tendency of oxyMb to dissociate to form deoxyMb and oxygen at low oxygen concentrations,12since the absorbance change at 418 nm is mostly due to oxyMb. The absorbance changes at 431.5 and 436 nm are affected much less by the formation of oxyMb. Plots B and C of Figure 5 show that the linear relationships between the rate of the absorbaiice change and DO concentration at 431.5 and 436 nm extend closer to the origin. In the leastsquares fitting of the data, the origin, i.e., zero absorbance change rate with 0%air-saturatedwater, was included as an experimental point, based on the absorption behavior of Mb in solution.12 The correlation indexes are high, 0.972 at 431.5 nm and 0.984 at 436 nm. The better correlation appears to be due to much larger absorbance changes at these wavelengths than at 418 nm as well as a smaller contribution from oxyMb to the absorbance change. These correlation data reveal that the rate of absorbance change is directly related to the DO concentration. The correlation is apparently unique to the glassencapsulated Mb. The deoxyMb binds DO, resulting in the absorbance change, but the rate of the absorbance change depends on the transport of DO through the porous network of the gel matrix. The absorbance change rate represents the transport rate of DO as well as the disappearance of deoxyMb that absorbs DO to form oxyMb. The transport of DO is expected to be rate limiting since deoxyMb binds DO rather rapidly within milliseconds in solution.1QHowAnalytical Chemistry, Vol. 67,No. 9,May l , 1995
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Figure 2. Absorbance change versus time after exposure of deoxyMb gel to air-saturated water. 30
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Figure 3. Absorbance change at 431.5 nm after exposing deoxyMb gel to different DO concentrations (air-saturated water at room temperature contains -8 ppm of DO).
ever, the constant rates of absorbance change that we observed (Figures 3 and 4) are not what is expected on the basis of the theory, i.e., Fick’s first and second laws of diffusion in a porous solid.20s21It would predict that the disappearance of deoxyMb or the transport of DO to a central location in the rectangular gel that was monitored in our experiments would initially be small, increase gradually, reach a maximum, and then decrease gradually to zero. If the DO transport is not rate limiting, i.e., fast relative to the absorption rate of DO by deoxyMb, the disappearance of deoxyMb would occur within seconds. The constant rate of disappearance of deoxyMb for a relatively long time, i.e., until the disappearance of more than 70% deoxyMb (e.g., estimated from Figures 2 and 3), is unusual and indicates that the transport rate of DO is constant for the same period. This observation is currently under further examination for a possible relationship with the transport role of myoglobin in tissues.I3 Nevertheless, the excellent correlation reveals not only that Mb is homoge (19) Gibson, Q. H.; Roughton, F. J. W. J. Physiol. 1957,136, 507-526. (20) Cussler, E. L. DzBsion: Mass transfer influid systems;Cambridge University Press: New York, 1984. (21) Crank, J. The Mathematics ofDifision; Oxford University Press: New York, 1975.
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Figure 4. Absorbance changes at 431.5 nm during 5 min of exposure of deoxyMb gels to 40% air-saturated water (0),(B) 80% air-saturated water (0),and (C)100% air-saturated water (0).
neously distributed, but also that the pore structure is uniform throughout the glass gel. The experimental data demonstratethat the rate of absorbance change in sol-gel encapsulated deoxyMb is directly proportional to the concentration of DO. Our results indicate that deoxyMb gels may be used as the basis for an accurate and relatively simple oxygen sensor. The fact that the correlation may be determined at only one wavelength greatly simplifies the instrumentalrequirements and data analysis in the practical application of the Mb gel to the measurement of DO. (It is, of course, possible to use two wavelengths in this method in order to eliminate or minimize possible interferences in certain water samples.) Although we used 5 min in these feasibility experiments, the analysis time required to establish an absorbance change rate is even shorter, Le., less than 2 min. Also, the amount of sample water can be reduced to 100 pL instead of the 3.5 mL used in our experiments, depending on the DO concentration. Furthermore, we have found that Mb gels prepared and stored under identical conditions give the same kinetic behavior when exposed to DO. Therefore, it is only necessary to test a few representative samples of a particular batch in order to confirm that all the samples of that batch, prepared at the same
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This new method for determination of DO using sol-gel glassencapsulated Mb provides important alternatives and capabilities over the MPOD method in analytical equipment and pr0~edure.l~ The new method has an advantage over the MPOD method in that does not require the stirring of the water sample; only a brief gentle shaking is necessary. Moreover, the sample is not exposed to air or a gas during the measurement. Thus, we believe that the new method in an optimized form has the potential to be more accurate and reproducible than the MPOD method. The Mb gel method may not prove to be as convenient as the MPOD method for continuous monitoring of DO in natural waters or process streams. Nevertheless, it could be automated for a batch wise continuous operation.
Concentration, 8 Air-saturation
Figure 5. Absorbance change rate at (A) 418 (0),(6)431.5 (0), and (C) 436 nm (0)versus DO Concentration.
time and stored under identical conditions, follow the linear correlation previously determined for a particular concentration of Mb and temperature. Regeneration of Used Gels and Brief Comparison with Other Methods. After a deoxyMb gel is exposed to DO, it is converted to an oxyMb gel and must be reconverted to a deoxyMb gel if it is to be reused. The regeneration of used gels was successfully accomplished by the same method earlier employed to reduce metMb gels to deoxyMb gels, i.e., treatment with a dithionite solution, which in this case reduces oxyMb gels to deoxyMb gels. It is likely that other chemical reduction methods can be found as well. Thus, the used gels may be chemically reduced and reused for subsequent determinations. A second method that we evaluated, applying vacuum to used gels immersed in degassed water at ambient temperatures to remove 02 and therefore to convert oxyMb to deoxyMb, was not effective in returning the gels fully to the deoxyMb state. An optical sensor for DO based on Hb immobilized on a cation exchange resin was previously reported.14 It differs substantially from our sol-gel encapsulated Mb DO sensor because its useful lifetime is short due to Hb degradation. By contrast, the Mb gels appear to have very long lifetimes and to be reusable. In addition, the simple linear relationship between the rate of change of the absorbance and the DO concentration and the short time required for a determination are found only in the case of the sol-gel encapsulated Mb method.
CONCLUSIONS Sol-gel glassencapsulated deoxyMb has characteristics that make it an excellent sensor for dissolved oxygen. These properties arise from a combination of the oxygen-binding property of deoxyMb and the porous structure and optical transparency of the sol-gel matrix. DeoxyMb gels have been shown to exhibit three characteristics necessary for a practical sensing element for DO detection: (1) an optical response that is sensitive to DO concentration; (2) an optical response that is established rapidly (in this case, the rate of absorbance change, which is established withii 2 min); (3) relatively simple analytical equipment and procedures. Furthermore, the method is expected to produce only negligible changes in the DO concentration in the water sample during the measurement, and stirring is not required. In addition, the volume of the sample can be as small as 100 pL, depending on the DO concentration. Thus, the encapsulated Mb sol-gel method has high potential as a new method for accurate and reproducible determination of dissolved oxygen in water. ACKNOWLEDGMENT This work was supported by NSF Grant DMR90-03080 and by Los Alamos-UC Campuses Collaborative Research Initiatives Grant UC-944-A-216. Received for review December 5, 1994. February 9, 1995.@
Accepted
AC9411785 *Abstract published in Advance ACS Abstracts, March 15, 1995.
Analytical Chemistry, Vol. 67,No. 9, May 7, 7995
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