The College of New Rochelle

Digital Commons @ CNR Faculty Publications

1989

Oxygen Electrode Mary Virginia Orna College of New Rochelle, [email protected]

Follow this and additional works at: http://digitalcommons.cnr.edu/facpubs Part of the Chemistry Commons Recommended Citation Orna, Mary Virginia. Oxygen Electrode. In Electrochemistry, Past and Present; Stock, J. T. and Orna, M.V., Eds.; American Chemical Society: Washington, DC, 1989; pp 196-210.

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Chapter 15 Oxygen Electrode History, Design, and Applications Mary Virginia Ornal Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 The measurement of dissolved oxygen levels in aqueous solutions is an important index in medicine, bio­ chemistry, molecular biology, and environmental, sanitation and industrial chemistry. This paper reviews the development of oxygen electrodes which work well under a variety of conditions.

The literature on ion-sensitive electrodes is vast and is growing at an enormous rate, so much so that the development of gas sensors and selective bioelectrode systems comprises only a small portion of this fast-developing field (1). However, the importance of gaseous oxygen and its measurement in numerous biologically-related systems has led to a large body of literature devoted to this very specific application. For example, in my laboratory, we have applied the use of the oxygen electrode to structure-activity correlations of known and potential carcinogens by measuring the kinetic parameters of enzymes reacting with these chemicals (2). This paper addresses the sub-field of oxygen sensors with respect to their early history, principles of measurement, their current design, and applications of the modern generation of these sensors. There is no doubt that Priestley discovered oxygen in August of 1774, although a work by Klaproth, as cited by Duckworth (22._, indicates that an oxygen-like component of the atmosphere was recognized by the Chinese as early as the eighth century. The confirmation of Priestley's discovery was related to the consumption of oxygen by a biological system. The oxygen-biology link has driven the development of methods for measuring oxygen production and consumption. Two distinct methods of measurement have arisen based on quite different principles, viz., manometry and electrochemistry. The Warburg manometer, the quantitative culmination of the "blood-gas manometer" of Barcroft and Haldane, has been a standard

1Current address: Department of Chemistry, College of New Rochelle, New Rochelle, NY 10801 0097-6156/89/0390-0196$06.00/0 o 1989 American Chemical Society

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ce of apparatus in biology and biochemistry laboratories for many P1 rs. The Warburg manometer is based upon measurement of the y ure or volume changes resulting from the evolution or sumption of oxygen by a system, and it has certain advantages over electrochemical technique in that: (1) it lends itself to ltiple simultaneous runs, (2) is insensitive to product cumulation, and (3) is able to cope with high endogenous levels of ygen (4) . However, the sensitivity of the method is fairly low, , the necessary apparatus is complex, fragile and expensive. In ddition, the sample flasks are relatively large and cumbersome mpared to the micro-chambers that are now available for mini-oxygen lectrodes, a technical advance that allows for rapid, accurate determination of oxygen even in severely sample-limited conditions. Tj,e three most serious limitations of manometric techniques are: (1) tjje inability to follow rapid changes in the gas phase; (2) the relatively long time required for equilibration and measurement; and (3) the inability to probe different regions of the same biological system (5_) • Because of the limitations listed above, manometry as the method of choice for measurement of oxygen tension has long since been superseded by the use of the oxygen electrode. The electrochemical technique is rapid and sensitive, provides continuous information about oxygen tension changes rather than just average values, lends itself to addition of reagents and substrates during a run, and can be used in a variety of settings in vitro, in vivo and in the field. Furthermore, the apparatus is simple, relatively inexpensive, and can easily be interfaced with digital data acquisition devices for purposes of data analysis.

History of Oxygen Measurement by Electrochemical Techniques The fundamental reaction that allows electrochemical measurement of oxygen tension is the 4-electron reduction of 0 (1) at a cathode maintained at a fixed potential sufficiently negative to reduce all of the oxygen that diffuses to its surface, essentially a polarographic technique. Although such an arrangement seems relatively simple, it was over 150 years in development from the first description of the phenomenon of polarization. Recognition of electrode problems, particularly the problem of electrode polarization (6_) , links the history of electrochemistry and bioelectric phenomena. In 1801, Gautherot described the existence of a residual current of brief duration that remained in charged silver and platinum wires even after removal of the source of charge. Later, Oersted recognized that this so-called "secondary current" could produce muscular contractions in frogs, but it was not until 1826 that the term "polarization" was applied to the phenomenon by de la Rive. During the course of the remainder of the 19th century, numerous workers wrestled with this phenomenon, particularly since it was a problem in dealing with the physiological effects of electricity. Becquerel, Peltier and Matteuci attributed polarization to the deposition of thin films of gas or foreign materials on the

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electrodes. The concerted work of de la Rive, Fechner, Schroeder, Ohm, Lenz and Beets finally gave rise to the concept of what is now known as concentration polarization. Further developments in the field of electrochemistry as applied to biological systems were inextricably entwined with the interpretation and, often, the avoidance, of polarization effects. Around the turn of the century, the electrochemical reduction of oxygen was discovered by Salomon (_7) and von Danneel (8) working in Nernst's laboratory, and shortly thereafter by F. Cottrell (9) and Grassi (10). It was later observed that when oxygen was reduced at a platinum cathode, a current-voltage curve with a plateau current proportional to oxygen concentration could be obtained (5). However, 25 years were to elapse before Jaroslav Heyrovsky (11) clarified and developed (12) the basic principles and instrumentation of polarographic analysis, a feat for which he was awarded the Nobel prize in 1959 (13). Another 20 years passed before Heyrovsky"s work was translated into a reliable tool for oxygen analysis by Davies and Brink, who were the first to utilize a platinum cathode in tissue studies (14), and yet another 14 years went by before Clark's membrane-covered platinum electrode (15) enabled routine measurement of oxygen tension in a variety of sample types. Despite the fact that the measurement of oxygen tension is essentially a polarographic technique, the use of the dropping mercury electrode never became popular for biochemical measurements, although some instances of its use can be found in the literature (16). The platinum electrode has always been the electrode of choice for biochemical systems. An excellent historical review of the development of this so-called "oxygen cathode" is given by Davies (17). Basic Principles Measurement of oxygen availability in tissues or fluids, i.e., the oxygen tension of a system when expressed as pressure of 0 , is influenced by many factors that render absolute measurements extremely difficult. Attention must be paid to such variables as the diffusion coefficient of oxygen in different tissues, the degree of stirring and the possibility of poisoning the electrode surface by a variety of mechanisms (18). The different designs of these electrodes are all attempts to overcome these limitations, but for this very reason are not universally applicable. Basically, the oxygen cathode consists of a small area of platinum or gold that is polarized to approximately 0.6 V with respect to a reference anode, usually a calomel or a silver-silver chloride half cell; the accompanying circuitry is designed to measure the current passing through this cell. The reaction taking place at the cathode has been proposed to follow several different courses, viz., according to Laitinen and Kolthoff (19), a two-electron reduction,

2H+ + O

+ 2e~

^-

HO

or, according to Davies and Brink (14), a two-step process,

(2)

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Oxygen Electrode: History, Design, and Applications

2H 0 + 0 + 2e 2 2 H o + 2e 2 2

.. H o + 20H 2 2 J1r 20H •

199 (3) ( 4)

While the precise stoichiometry of the electrode reaction is unimportant, it is very important that the reaction be consistent for the duration of any one experiment. When a potential is initially applied to the oxygen cathode, the concentration of oxygen in its immediate vicinity decreases rapidly, since the rate of reaction at the electrode is much more rapid than the rate of diffusion through the medium. As soon as this concentration gradient has developed, the rate of oxygen reduction is diffusion-dependent, and in this particular situation, Fick's diffusion law can be applied. If we express the quantity of oxygen passing through the diffusion layer in unit time as d[02]/dt, then -D (dc/dx)

(5)

where D is the diffusion coefficient of oxygen in aqueous solution and (dc/dt) is the concentration gradient in the diffusion layer. since the measured current, i, is proportional to the amount of oxygen reaching and being reduced at the cathode, we can rewrite Equation 5 as i = kD (dc/dx) •

(6)

However, the magnitude of the concentration gradient is proportional to the amount of oxygen in the medium; therefore, the following generalization can be made (3£): (7) This equation is the fundamental basis for oxygen electrode measurements since the reduction current is directly proportional to oxygen concentration (strictly speaking, activity), which means, in practical applications, the amount of oxygen reaching the cathode. This equation assumes that the diffusion coefficient, D, is constant for a particular experiment and that the oxygen is transported to the electrode by the process of diffusion alone. If the applied voltage to the oxygen cathode is varied and the corresponding current produced measured, it is possible to construct current-voltage curves similar to those displayed in Figure 1 (21, �). The current-voltage relationship varies with the type of �­ electrode employed and the nature of the solution. A normal working applied voltage is that giving the least variation in current with voltage, i.e., a voltage in the plateau region of the curve where the current is limited by diffusion alone. For most systems, an applied voltage of -0.6 V fulfills this condition. The Design and Modification of Oxygen Electrodes Numerous designs for the oxygen electrode have been described in the literature. The variety in designs arises from the many applications

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ELECTROCHEMISTRY, PAST AND PRESENT

6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6

a: a:

3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 0.0

-0.10

-0.20

-0.30

-0.40

-0.50

-0.60

-0.70

-0.80

-0.90

VOLTS

Figure 1. Voltammograms for a typical Clark-type electrode indicating the nature of the plateau at (1) 95.0% oxygen and (2) air saturation.

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to which the basic electrode has been put, from homogeneous, controlled parameter experiments to heterogeneous biological media and in-the-field monitoring. Design considerations include: response time; sensitivity and its decrease over a period of time (electrode aging); linearity; stability; possible secondary effects of the consumption of.oxygen; production of hydroxide ions at the electrode surface; sensitivity to temperature; pH; mechanical mixing and movement; and possible reduction of sample constituents other than oxygen. In addition, probes to be inserted into tissues require special design (�). The basic design of oxygen electrodes falls into two main c ategories, open electrodes and coated electrodes. These, in turn, fall into several subcategories as listed in Table I. By far, the most important advance in the design of oxygen electrodes was the clark-type electrode (Figure 2) introduced by Clark in 1956 (15). Clark's idea was to isolate both platinum cathode and referen� anode, both bathed in a suitable electrolyte, from the body of the solution by a thin, nonconducting membrane that is readily permeable to oxygen (24,25). This design became and remains the design of choice in oxygen electrodes, and continuous improvements in both its specificity and size have made it ideal for most applications. The electrode is now commercially available from several suppliers and the ancillary instrumentation is both inexpensive and robust. In my laboratory, we have used a Teflon covered YSI-5331 platinum/silver electrode with a half-saturated KCl solution as electrolyte (Yellow Springs Instrument Co., Yellow Springs, OH) inserted into a 1.8 mL incubation chamber (Gilson Medical Electronics, Inc., Middleton, WI) in such a way that it is possible to complete a measurement in less than five minutes. Under these conditions, it is possible to make rapid, multiple determinations under the same experimental conditions, thereby enhancing reproducibility of the data. Mathieu (26) has given a complete description of the variables that�ffect the electrode system - effects of temperature on oxygen solubility and porosity of the Teflon membrane, effect of atmospheric pressure and of water salinity on the solubility of oxygen and effect of velocity of water passage through the membrane and how to compensate for them. Ferris and Kunz (27) have summarized the principal design considerations for""°oxygen-sensing electrodes, viz., membrane materials, thickness and performance, electrolyte nature and film thickness, electrode configuration, external pressure and temperature variation, and signal amplification requirements, and Ultman and co-workers (�) developed a spherical model that relates the sensitivity of the Clark electrode to cathode radius and electrolyte layer permeability and thickness for two different data sets, one obtained from polypropylene-covered and the other obtained from Teflon-covered Clark electrodes. The material from which the membrane is constructed has been the subject of much study (29-32) with respect to permeability, selectivity and hydrophobicity. Reports of numerous modifications of the Clark electrode have appeared in the literature since it was first devised, mainly because the traditional Clark cell must be miniaturized for many biochemical and biological applications. However, miniaturization introduces a

2. Clark

1. Platinum/Polymer

B. Coated Electrodes Coat

Robust; very stable; readily changeable membrane Insulation of electrode circuit from sample Anode & cathode behind same membrane

Decreased sensitivity; increased response time Reduction in aging; decreased movement artifact

Utilizes pulsed polarizing voltage Improved stability; insensitivity to convection Possible to obtain absolute 0 measurements

Diffusion current independent of vibration frequency above a minimum frequency Relatively small response time

b. Vibrating

3. Recessed

High sensitivity; relatively small response time Decreased sensitivity to external vibrations Very thin diffusion layer

Relatively large currents Rapid establishment of steady-state current

2. Moving

a. Rotating

Small current; rapid aging Requires little ancillary apparatus Sensitive to external vibrations

Characteristics

1. Stationary

A. Open Electrodes

Type

Table I. Types of Oxygen Electrodes

i

O

t/3

a71

o n

H

S

1

*

*

^

-J

;

J^

Lock

ma ,/

f.

^v /

Recorder or Computer

/ X

X

!>-»-,

c:

\r /

t Constant Temperature Bath Outlet

Magnetic - Stirring Bar

- O-Ring - Reaction Chamber — Membrane Enclosing KCI - KCI Solution (Half Saturated) - pt Cathode

Figure 2. Schematic drawing of a Clark-type oxygen electrode.

Constant Temperature Bath Inlet

_L

-0.6 to -0.8 V

Power Supply

\

S

S3'

I 9

I

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ELECTROCHEMISTRY, PAST AND PRESENT

number of additional problems, including different electrochemical behavior of microelectrodes and sensor lifetime limitations (33). Fatt (34) has described a modified Clark electrode that gives 99% of final response in 1.24 s for a 155 mmHg change of p[O_] in the gas phase and also an ultra-micro electrode which shows only a 2% difference in response between a vigorously stirred and an unstirred solution (35) . Friese (36) describes a new type of thermosterilization resistant Clark electrode in which the platinum cathode is insulated with glass; the electrode compartment between anode sjnd cathode is made of a porous sintered glass, and the electrode body is ground flat and covered with two layers of Teflon. This modification allows measurements to be made in systems where there is a great deal of mechanical and thermal stress. Other workers have reported improvements in the ancillary circuitry surrounding the electrode, in particular, stable, sensitive amplification (37), and multichannel recording permitting automatic reading and recording of oxygen consumption from as many as six cells simultaneously (38). Modifications for simultaneous spectrophotometric and polarographic measurements have also been described (39,40). Details for the construction of an oxygen polarograph designed for undergraduate use have also appeared in the literature (41). Mancy and co-workers (42) have described a galvanic cell consisting of a silver-lead couple for determination of oxygen concentrations in fluids. This cell has been used successfully as a completely portable instrument for oxygen monitoring in natural waters and wastes. Mackereth has found long term instability of sensitivity in this and similar devices, as well as a problem with the small current output (on the order of tens of microamperes). He has described an improved galvanic cell (43) which has a current output roughly ten times that of the conventional diffusioncontrolled electrode. It is most advantageous for measurements where there is a high degree of pollution; however, it is necessary to continuously renew the membrane surface by techniques such as air bubbling (44). A recent improvement in sample cell size has been offered by Yellow Springs Instrument Co. in their product literature. The YSI 5356 Micro Oxygen Chamber provides capability of handling samples as small as 600 microliters in either batch mode or in a flow-through mode. As applications in the use of oxygen electrodes continue to multiply, design modifications and adaptations of existing apparatus can be expected to keep pace. Performance Characteristics and Behavior Although the direct relationship between current output and oxygen concentration (activity) as given in Equation 7 holds for most systems under diffusion-controlled conditions, it is admittedly an oversimplification of the situation. Using a first order, one-dimensional two-layer model, and applying Fick's laws of diffusion, Mancy et al. (42) derived expressions for the transient current obtained on stepping the dissolved oxygen cell from open circuit to a potential where diffusion-controlled reduction of

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oxygen takes place at the cathode. They have shown that if one considers the fact that the diffusion coefficients of the electrolyte thin film and the membrane are different, and that the permeability coefficient of the membrane is a non-negligible parameter, the steady state current of the system is given by i = nFAP C/b m where i n F A P C b

(8)

= steady state current = number of electrons transferred = the Faraday = cathode area = membrane permeability coefficient = oxygen concentration of the test solution = membrane thickness.

This equation is based upon the assumption that only transport in the membrane is important and that the electrolyte layer between the membrane and the cathode is infinitely thin. Koudelka (33) , in treating the time interval between t=0 and attainment of the steady state, has pointed out that between 3 and 20 seconds after the potential step is applied, the sensor follows the Cottrell equation it = nFA(D/ 7Tt)1/2 C

(9)

where i = initial current D = membrane diffusion coefficient t = time. If this is the case, a plot of i vs. l/(t) is linear and i (t) /C is a constant. In practice, the behavior of amperometric oxygen sensors is considerably more complicated than implied by the simple diffusional model. For example, the time for a potentiostatic current to reach a steady value is often much longer than theory would predict, and the value of the steady state current finally achieved depends both on the thickness of the electrolyte layer and on the type of electrolyte in the cell. Hale and Hitchman (45) have examined examples of more complicated behavior, taking into account the transient current variation at switch-on, the effect of electrolyte layer thickness, and the pH of the electrolyte in the steady state situation. Additional studies on the behavior and performance characteristics of the oxygen electrode include: a three-dimensional mathematical modeling (46) ; the effect of the accumulation of cathodic reaction products on probe linearity; signal overshooting; tailing and hysteresis of transient characteristics for step changes in the concentration of oxygen for Clark-type probes (47) ; error produced by finite response time in rate determinations of oxygen consumption (48) ; and variations in responses of oxygen electrodes to variables in construction and use (49) . Obviously, this list of performance characteristics is not exhaustive, nor is the associated bibliography. It is merely the intent of this section of the paper to make the reader aware of the

206

ELECTROCHEMISTRY, PAST AND PRESENT

simplifying assumptions governing routine use of the oxygen electrode system and some attempts to address these problems. Calibration When it is necessary to obtain absolute values for oxygen uptake, the oxygen electrode must be calibrated. The simplest way to do this is to equilibrate the solution under study with air and make use of the solubility of oxygen in conjunction with its known partial pressure in air. The difficulty with this method is that it is limited to ' aqueous solutions and it is difficult to take such variables as pressure, temperature and ionic strength into account. In addition, any absolute measure of oxygen concentration must include corrections for the fact that activity, not concentration, of oxygen is the parameter being measured, and that the presence of both electrolytes and nonelectrolytes influences the solubility and the activity coefficient of oxygen in aqueous solutions. A number of calibration methods that address these problems are in the literature. The classic method of Winkler (50) is extremely tedious for routine use and is no longer used. The method that took its place, that of Estabrook and Mackler (51) , measures the oxygen consumed in the stoichiometric oxidation of a specified amount of the reduced form of nicotinamide adenine dinucleotide (NADH) as catalyzed by lysed mitochondria. The main drawback of this method is that it requires the preparation of mitochondria, a procedure which could take up to four hours. In the past two decades, several methods for calibration of the oxygen electrode, using readily available chemicals and relatively simple reactions, have been proposed. These will be described in the order in which they first appeared in the literature. The method of Robinson and Cooper (52) utilizes the reduction of N-methylphenazonium methosulfate (PMS) by NADH according to the following scheme: PMS + NADH + H+ -^+ 02 -^

PMSH

+ NAD+

(10)

PMS + H202

(11

catalase giving a net reaction of NADH + H+ + 1/2 0 -V-

NAD+ + H O

(13

This method has the advantage of directly determining the activity of oxygen, which is the species monitored, under the same physical conditions as the experiment, thus eliminating errors that can be as high as 25%. Billiar and co-workers (53) have proposed a calibration method based upon the stoichiometry of the xanthine oxidase reaction: Xanthine + H O + O

xanthine oxidase ^. . y uric acid + H? °2

,,., '14'

The difficulty with this reaction is the possibility of contamination

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207

of

xanthine oxidase with catalase or the catalytic oxidation of the product hydrogen peroxide to oxygen by trace metals present in the system, thus leading to spuriously low recorded amounts of oxygen consumption. The group reported that they did not observe any of the mechanisms of hydrogen peroxide breakdown operative in the system, but the method leaves other experimenters vulnerable to these problems unless care is taken to check for such contamination. Holtzman's method (54) circumvents the problems associated with the Billiar method by including catalase in the hypoxanthinexanthine oxidase reaction chain according to the following scheme: Hypoxanthine + 0

£.

xanthine oxidase ^_ >> uric acid + 2H 0 £,

£.

(15)

catalase with a net reaction of Hypoxanthine + O

>' uric acid

+ 2 HO

(17)

This method is highly advantageous in that it gives highly reproducible results, the reagents are readily available, inexpensive and stable for long durations. In addition, possible metal ion catalysis of hydrogen peroxide decomposition would not interfere with the overall stoichiometry of the system. Misra and Fridovich (55) have proposed a method of calibration that entirely avoids the use of enzymatic reactions. Their chemical system takes advantage of the fact that phenylhydrazine is rapidly oxidized to phenyldiimide by ferricyanide ion, and that the phenyldiimide formed reacts very rapidly with oxygen to produce phenol and nitrogen gas. The authors were able to demonstrate that the accumulated reaction products do not alter the sensitivity of the method. Finally, Wise and Naylor (56) reported a calibration procedure that involves the auto-oxidation of duroquinol at pH 9.0 to produce hydrogen peroxide, which subsequently disproportionates to form water and molecular oxygen. The regeneration of oxygen presents the same problem as in the method of Billiar, but the authors claim that fast electrode response times at higher temperatures allow the method to give good agreement with calculated values while at the same time allowing for highly accurate determination of the starting material, duroquinol, since it has a strong absorbance between 260 and 270 nm. This method has been shown to provide accurate values over the temperature range 5-45 C. While the choice of calibration method depends upon the reagents available and the specific application of each worker, it must be emphasized that calibration should not be overlooked since the monitor in itself is relative and must be related to actual oxygen concentrations (activities) in order to render any but the most qualitative oxygen consumption measurements meaningful. Applications Applications of the oxygen electrode are many and varied, and to cite all of the references related to these applications would comprise a

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ELECTROCHEMISTRY, PAST AND PRESENT

volume in itself. In addition to the many review articles already cited (4,5,13,17,18,20,21,26), there exist several additional reviews including general references on polarography (57-60). In addition, the Scientific Division of Yellow Springs Instrument Co. has published a booklet, "YSI Oxygen Electrode References," (61) which is available free of charge from the company. Although it is three years old, this is the most comprehensive bibliography available. It is divided into ten parts and covers Bacteria, Yeasts and Molds, Biochemical Assays and Techniques, Cell Fractions, Intact Organisms, Pathology and Toxicology, Pharmacological Studies, Radiation Research, Special Methods and Applications, Tissues and Cells and a list of Reviews. It is briefly annotated, which is a great advantage, but the list is limited to those applications using the YSI Model 53 (now 5300) Biological Oxygen Monitor or some modification thereof. In addition to the applications covered in (61), this author has found that kinetics research was done with the oxygen monitor as early as 1938 (62), and reaction mechanisms studies (63) are also susceptible of measurement. Stoichiometry (64-67), monitoring the levels of other gases (68-70), pollution control (71) and biochemical assays (72-76) are also areas of use. It is hoped that this brief review will help to situate the oxygen electrode among other electroanalytical methods as an important tool in biochemical research. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Arnold, M.A.; Meyerhoff, M.E. Anal. Chem. 1984, 56, 20R-48R. Orna, M.V.; Mason, R.P. In preparation. Duckworth, C.W. Chemical News, 1886, 53, 250. Trudgill, P.W. In CRC Handbook of Methods for Oxygen Radical Research; Greenwald, R.A., Ed.; CRC Press, Inc.: Boca Raton, FL, 1985; pp. 329-42. Lessler, M.A.; Brierley, G.P. In Methods of Biochemical Analysis; Click, D., Ed.; Wiley-Interscience: New York, 1969; Vol. 17, pp. 1-29. Feder, W. Ann. N.Y. Acad. Sci. 1968, 148, 3-4. Salomon, E. 2. Physik. Chem. 1897, 24, 55. von Danneel, H. Z. Elektrochem. 1897-98, _4, 227. Cottrell, F.G. Z. Physik. Chem. 1903, 42, 385. Grassi, U. 2. Physik. Chem. 1903, 44, 460. Heyrovsky, J. Trans. Far. Soc. 1923-24, 19, 785. Heyrovsky, J.; Shikata, M. Rec. Trav. Chim. 1925, 44, 496-8. Lubbers, D.W. Int. Anaesthes. Clinics 1966, £, 103-27. Davies, P.W.; Brink, Jr., F. Rev. Sci. Instr. 1942, 13, 524. Clark, L.C. Trans. Soc. for Art. Int. Organs 1956, £, 41-57. Petering, H.G.; Daniels, F. J. Am. Chem. Soc. 1938, 6£, 2796-2802. Davies, P.W. In Physical Techniques in Biological Research; Nastuk, W.H., Ed.; Academic Press: New York, 1962; Vol. 4, pp. 137-79. Silver, I.A. Int. Anaesthes. Clinics 1966, _4, 135-53. Laitinen, H.A.; Kolthoff, I.M. J. Phys. Chem. 1941, 45, 1061.

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Beechey, R.B.; Ribbons, D.W. In Methods in Microbiology; Morris, J.R.; Ribbons, D.W., Eds.; Academic Press, New York, 1972; Vol. 68, pp. 25-53. 21. Connelly, C.M. Fed. Proc. 1957, 16, 681-84. 22. Hagihara, B. Biochim. Biophys. Acta 1961, 46, 134-42. 23. Connelly, C.M.; Bronk, D.W.; Brink, Jr., F. Rev. Sci. Instr. 1953, 2±, 683-95. 24. Clark, Jr., L.C.; Wolf, R.; Granger, D.; Taylor, Z. J. Appl. Physiol. 1953, j>, 189-93. 25. Clark, Jr., L.C.; Lyons, C. Ann. N.Y. Acad. Sci. 1962, 102, 29-45. 26. Mathieu, J.L. Eau et Industrie 1980, 48, 99-112. 27. Ferris, C.D.; Kunz, D.N. Biomed. Sci. Instrum. 1981, r/, 103-8. 28. Ultman, J.S.; Firouztale, E.; Skerpon, M.J. J. Electroanal. Chem. 1981, 127, 59-66. 29. Severinghaus, J.W.; Bradley, A.F. J. Appl. Physiol. 1958, _13, 515-20. 30. Refojo, M.F.; Yasuda, H. J. Appl. Poly. Sci. 1965, ^, 2425-35. 31. Rattner, B.D.; Miller, I.F. J. Biomed. Mat. Res. 1973, ]_, 353-67. 32. Michaels, A. J. Poly. Sci. 1961, 50, 393-412. 33. Koudelka, M. Sensors and Actuators 1986, JJ, 249-58. 34. Fatt, I. J. Appl. Physiol. 1964, 19, 550-53. 35. Fatt, I. J. Appl. Physiol. 1964, 19, 326-29. 36. Friese, P. J. Electroanal. Chem. Interfacial Electrochem. 1980, 106, 409-12. 37. Carr, L.J.; Hiebert, R.D.; Currie, W.D.;Gregg, C.T. Anal. Biochem. 1971, _41, 492-502. 38. Carr, L.J.; Larkins, J.H.; Gregg, C.T. Anal. Biochem. 1971, 41, 503-509. 39. Ribbons, D.W.; Hewett, A.J.W.; Smith, F.A. In Biotechnology and Bioengineering; Gaden, Jr., E.L., Ed.; John Wiley and Sons, New York, 1968; Vol. X, pp. 238-42. 40. Trudgill, P.W. Anal. Biochem. 1974, 58, 183-89. 41. Reed, K.C. Anal. Biochem. 1972, 50, 206-212. 42. Mancy, K.H.; Okun, D.A.; Reilley, C.N. J. Electroanal. Chem. 1962, ^, 65-92. 43. Mackereth, F.J.H. J. Sci. Instr. 1964, 41, 38-41. 44. Fujimoto, E.; Iwahori, K. Environ. Technol. Lett. 1983, _4, 397-404. 45. Hale, J.M.; Hitchman, M.L. J. Electroanal. Chem. 1980, 107, 281-94. 46. Lemke, K. Internationales Wissenschaftliches Kolloquium Technische Hochschule Ilmenau 1985, 30, 165-8. 47. Linek, B.; Sinkule, J.; Vacek, V. J.~Electroanal. Chem. 1985, 187, 1-30. 48. Ito, S.; Yamamoto, T. Anal. Biochem. 1982, 124, 440-45. 49. Carey, F.G.; Teal, J.M. J. Appl. Physiol. 1965, 20, 1074-77. 50. Winkler, L.W. Z. Anal. Chem. 1914, 53, 665. 51. Estabrook, R.W.; Mackler, B. J. Biol. Chem. 1957, 229, 1091-1103. 52. Robinson, J.; Cooper, J.M. Anal. Biochem. 1970, 33, 390-99. Q>

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