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Anal. Chem. 1987, 59,736-739
(12) Johnson, D.C. J. Nectrochem. Soc. 1072, 779, 331-335. (13) Koch, W. F.; Stolz, J. W. Anal. Chem. 1982,54,340-342.
RECENEDfor review September 19,1986. Accepted November 3, 1986. Certain commercial equipment,, instruments, or
materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment is necessarily the best available for the purpose.
In Vitro Stability of an Oxygen Sensor Joseph Y. Lucisano, Jon C. Armour, and David A. Gough* Department of Applied Mechanics a n d Engineering Sciences, Bioengineering Group, University of California, Sun Diego, La Jolla, California 92093
Fabrication and operation of a three-electrode oxygen sensor that utilizes potentlosiatic instrumentationare described. The long-term stability of the sensor is demonstrated by continuous operation under quasi-physiologic conditions. With further development, this sensor may find application as an implant in the body or in other long-term monitoring situations.
The electrochemical oxygen sensor has been a powerful tool for revealing the role of oxygen in biological systems. Application of this sensor has been the main experimental methodology in many thousands of studies over more than 40 years. Virtually all, however, have been studies in which the sensor is used for a period of only a few days at most before recalibration is necessary. There is a need for a stable oxygen sensor that is suitable for continuous application in long-term monitoring situations without the need of frequent recalibration. Such a sensor would make possible certain important monitoring applications that are not now feasible ( I , 2). Electrochemical oxygen sensors are based on the electrocatalytic reduction of oxygen on a noble metal working electrode surface. When the electrode is appropriately polarized, the reaction that most likely occurs (3) is the following two-step process:
O2 + 2H+ + 2e-
-
Hz02+ 2H+ + 2e-
H202
-
2H20
The background medium is usually a thin layer of electrolyte held adjacent to the electrode surface by an oxygen-permeable polymer membrane. When the electrode is polarized within a certain cathodic range with respect to a potential reference electrode, the rate of these reactions is sufficiently rapid that the process becomes limited by mass transfer. This results in a "plateau", in which there is relatively little variation in current with applied potential. The electrode can be most easily operated as a sensor in this potential range. The membrane between the electrode surface and the sample medium serves several purposes. Most importantly, it minimizes access to the electrode surface of substances from the sample that may interfere with the oxygen-reduction process. In sensor designs where all electrodes are located on the same side of the membrane, the membrane is typically composed of a hydrophobic polymer, such as Teflon or polyethylene that is permeable to oxygen but virtually impermeable to polar solutes, and therefore is largely responsible for the selectivity of the sensor for oxygen. In another design commonly known as the monopolar sensor, the oxygen electrode is located behind a hydrophilic membrane and the 0003-2700/87/0359-0736$01 SO/O
second electrode is on the other side of the membrane in the sample medium. In this case, the membrane must allow electrolytic conduction between the two electrodes and minimize the access of large molecules to the electrode surface. The membrane, however, affords relatively little protection against the effects of diffusible polar molecules. In either design, the membrane may also provide a significant diffusional resistance to oxygen to maintain invariant mass-transfer conditions, an important requirement for reliable concentration determinations. Two approaches have been most commonly used to design a sensor based on this process. The so-called polarographic design incorporates a noble metal working electrode at which the oxygen-reduction process occurs and a silver/silver chloride anode. The electrolyte is typically phosphate buffer containing potassium chloride. A potential of approximately 500 mV is applied between the two electrodes to maintain the working electrode cathodic within the plateau potential region. The purpose of the silver/silver chloride electrode is twofold. First, the electrode must provide a stable potential that serves as a reference for polarizing the working electrode. Second, the electrode must also serve as a counter electrode through which the concentration-dependent current passes. These two functions can be performed by a single electrode when the current density involved is small enough to be accommodated without altering the reference potential. This is one reason for having a small working electrode. A well-known version of this type of sensor is the Clark oxygen sensor ( 4 ) , where both electrodes are mounted in a common housing on the same side of the membrane. Monopolar electrodes ( 5 , 6 )are another example. The second common approach to designing a sensor is the galvanic configuration (7). In this case, the working electrode is the same as in the previous design but the second electrode and electrolyte are composed of materials that give rise to a reference potential within the diffusion-limited plateau region. An example of these materials is a lead electrode in contact with a lead acetate electrolyte. A low resistance connection between the electrodes for purposes of current measurement brings the potential of the working electrode to the approximate potential of the second electrode, which is at the operating potential. Under these conditions, the process at the electrode is identical with that of the previous design, but there is no need to apply a potential between the two electrodes (8). Both types of sensors have been subject to electrochemical problems that reduce the stability of the signal. One such problem is the presence of undesired electrochemical processes at the working electrode. This includes poisoning of the electrode surface by apolar compounds from the sample (such as certain anesthetic agents) that are diffusible in the polymer 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
737
Table I. Summary of Reports on the Stability of Continuously Operated Intravascular Electrochemical Oxygen Sensors sensor type
test conditions
stated stability and longest continuous operation (if mentioned)
sensors operated in 5 patients from 1 to 25 days, drift ranged from 0.02 to 0.6 mmHg/h drift ranged from 0.1 to 0.2%/h after 12 h of in vitro (0.02 M phosphate buffer, pH 7.4, 38 "C) operation in vitro (heparinizedhuman blood) sensor output drifted 0.3%/h after 12 h of operation in vivo (human and animal, various arteries and veins) sensor output drifted 0.5%/h after the initial few hours 53 implants were made, maximum continuous in vivo (human, radial artery) Clark type operation was 8.5 days (av 3.2 days), output drifted sufficiently to warrant recalibration every 12 h in vivo (dog, femoral artery, systemically heparinized) drift was k5.6% over 8 h Clark type implants made in 4 patients with sensor operation in vivo (human, radial and femoral arteries) for more than 40 h in each case, drift ranged from 0.02 to 0.3%/h, authors state that errors in bench-top blood gas analyses may have artifactually contributed to drift in vivo (human, various arteries) 50 patients were continuously monitored for monopolar periods of 2 h to 15 days (av 73 h), drift sufficient to warrant recalibration every 8 h 10 electrodes each, of four different constructions monopolar in vitro (buffer, 37 O C , pOz 150 torr) (40 electrodes total) operated continuously for 40 h, average drift ranged from 0.9 to 1.7 mmHg/h drift was 0.18%/h monopolar in vitro (isotonic saline or buffer, 37 O C , air equilib) in vivo (human, various arteries and veins) 45 patients continuously monitored for 2-362 h (av 82 h), electrodes were periodically recalibrated, average drift was 3.2 mmHg/h authors state that electrodes have operatored with combination pC0, and in vitro (saline) Clark-type pOz outputs correct to k5 mmHg for up to 12 days in vivo (human, newborn infants, umbilical artery) 36 infants received implants, 32 electrodes Clark type operated continuously for 10-190 h (av 75 h), 22 electrodes needed no recalibration in use (av implant time 88 h), 4 electrodes required one recalibration, 6 electrodes failed after an average of 49 h Clark-type, surface heparinized monopolar
in vivo (human, radial and femoral arteries)
membrane (9). If this poisoning process is diffusion-limited, a reduced rate of signal decay is expected for electrodes of high surface roughness. The formation or destruction of surface oxides may also affect the reaction of oxygen a t the electrode surface (10). The rate a t which this occurs may depend on the electrochemical operating conditions and current density distribution on the surface of the electrode. Another example is the electrodeposition of cations from the anode onto the working electrode surface (9). This leads to inactivation of the working electrode when the anode is composed of lead or certain other metals, but may not cause a change in sensitivity to oxygen with a silver anode, since a silver surface can be comparable t o noble metal surfaces for oxygen reduction (11). Another electrochemical problem is anode failure. Corrosion may occur when metal is electrolytically removed from the anode and deposited on the cathode, causing the eventual disintegration of the anode. Depending on the current distribution, the removed material may also be deposited in the form of a dendritic contact between the two electrodes resulting in a short circuit (12). The time required for these processes to occur may depend on the current density. There are other categories of problems unrelated to the electrochemical processes (see ref 13). Few in vitro studies aimed at evaluating the long-term stability of potentially implantable sensors have been published. Certain investigators have, nevertheless, achieved a limited degree of signal stability from implanted sensors under prolonged use. These in vivo studies are considered here, with the recognition that the two types of studies may not be directly comparable due to the additional physiological effects present in vivo. The results of several notable studies are
ref
14 15 15 15 16
17 17
18
6 19
19 20 21
summarized in Table I. There is some difficulty in making quantitative comparisons among these various studies since the goals of the investigators were different, the experimental conditions were not consistent, and the criteria for stability were defined in various ways. In virtually all cases, however, sensors exhibited significant drift, to the extent that routine recalibration was necessary every few days. Without corresponding in vitro results for most of these sensors, it cannot be ascertained if the drift was due to the properties of the respective sensor itself or to its use in the physiologic environment. We describe here a three-electrode oxygen sensor based on the classical potentiostatic operating principle. We have evaluated the long-term stability of this sensor during continuous operation under well-defined in vitro conditions with the objective of providing a standard for comparison to subsequent implant results.
EXPERIMENTAL SECTION Sensor Design. As shown in Figure 1,the sensor incorporates a platinum working electrode, a silver/silver chloride reference electrode, and a platinum counter electrode, all mounted behind an oxygen-permeable hydrophobic membrane. The three electrodes are fine wires embedded in a glass or epoxy resin cylinder and connected to more substantial lead wires not shown. The electrodes are in electrolytic contact through an aqueous gel and encased by an outer layer of silicone rubber. The sensor is operated on the classical potentiostatic principle. As with other types of sensors, oxygen is electrochemically reduced on the platinum working electrode surface. The potential of the working electrode is specified by reference to the silver/silver chloride electrode. In this mode of operation however, the reference electrode is maintained electronically at a very high im-
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
200
0.06rnM 02
5 0
AgIApCI reference slecllwe
Figure 1. Oxygen sensor. The lead wires and glass housing were contained in a silicone rubber tube (not shown).
pedance to avoid significant current uptake. The main current passes to the inert platinum counter electrode, which is maintained at low impedance. This system provides separate electrodes for the two functions carried out by the anode in previous designs and has the advantage of directing very little current to the reference electrode, thereby prolonging its lifetime. It also makes possible the use of much larger ratios of working electrode to counter electrode area, thereby producing larger currents. This makes signal amplification and noise reduction less critical in sensors of small overall size. Sensor Fabrication. Electrodes were made by welding a small segment of 0.005-in.-diameter platinum or silver wire to one end of a long, insulated stainless steel wire. The welded regions of two such platinum electrodes and one silver electrode were then encapsulated in small beads of epoxy resin, cemented in a glass housing and fixed in the end of a silicone rubber tube in such a way that the stainless steel leads were inside the tube and the electrodes were exposed at the end. Electrodes were carefully bent to the parallel arrangement shown in Figure 1and trimmed to a length of 0.03 in. The working and counter electrodes were platinized (22) to a roughness factor of approximately 800, as estimated by anodic hydrogen stripping (23) The electrolyte gel was formed around the electrodes by dipping the end of the assembly in a solution of 20% poly(hydroxyethy1 methacrylate) (Polysciences,Inc.) in methanol, allowing the solvent to evaporate, and hydrating with electrolyte. The electrolyte was 0.01 M phosphate buffer, pH 7.3, containing 0.1 M KC1. The gel filled in the spaces between electrodes. An outer layer of silicone rubber was then made by dipping the end of the assembly in a solution of 25% silicone rubber (RTV 3140, Dow Corning) in toluene. The solvent was evaporated and the silicone rubber allowed to cure. This produced a layer approximately 10 pm thick. The gel could be dehydrated and rehydrated by exposure to an aqueous sample medium without loss of activity. The stainless steel wires were attached to an electrical connector at the opposite end. The sensors used here were approximately 2 mm in diameter and 1 mm in active length. This simple fabrication approach typically gave a high yield of sturdy, functional sensors. Testing, Sensors were checked for uniformity prior to longterm testing. The integrity of the silicone rubber coating was determined by measuring the resistance with respect to an external electrode by using a high impedance electrometer (Keithley Instruments Co., Model 616). Sensors with an apparent resistance of 1O'O R or greater could be used in complex media without interference from diffusible polar solutes and were considered to have an effective barrier. Sensors with significantly lower apparent resistance were recoated in silicone rubber. The background current in the absence of oxygen was found to be insignificant. Linearity of the response over a physiologic oxygen concentration range was verified by exposing sensors to oxygen concentrations ranging from 0.02 to 0.24 mM, made by equilibrating buffer solutions with analyzed gas mixtures of 2, 5 , 10, and 21% oxygen. Sensors typically required approximately 1min to return to steady state after a step change in oxygen concentration. Sensors were evaluated for stability in sealed, thermostated vessels a t 37 O C , containing 0.01 M phosphate buffer, pH 7.3. Solutions were maintained a t specified oxygen concentrations by equilibration with analyzed gas mixtures. The concentration was changed a t intervals of several days to demonstrate sensitivity.
A
-200
OO
-400
-800
-600
Potential (mV vs. Ag/AgCi Electrode)
Figure 2. Current-potential relationship. The current is plotted as a function of applied potential at the working electrode with respect to the internal silverlsilver chloride electrode. Points represent steadystate current at 5 min with and without oxygen. 200 1 180 160
,
1
1
I
Three Continuousiy Operated Oxygen Sensors 0.06mM O,,3 7 T pH = 7.3, buffer
140 -
3 -
120-
. = 80:
j100060 -
. ,.% o - c
-0
-
& - f0i g
A & 0~ 0
-I
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
sensor output. Sensors were involved in other experiments during periods in which there are few or no data points. The reference oxygen current was used for normalization to allow comparison of sensors of slightly different electrode size and membrane thickness. The reference current was taken at 2 days because of the stabilization period that typically occurs during the initial hours of sensor operation, during which the current asymptotically approaches a steady value. In each case, after this stabilization period the current shows no systematic variations with time over periods of several months. The maximal random variation in current was within *lo% for periods of greater than 100 days. Approximately 80% of over 20 sensors tested have shown comparable results. The other 20% failed due to initially undetected flaws in fabrication. Several of these sensors have operated with the same degree of stability for significantly longer periods. The long-lived sensors eventually failed in one of two characteristic modes. In most cases, the current rose abruptly and returned to the original value several times over the period of a few hours before finally remaining at a high, off-scale value. In other sensors, the current suddenly began to drift downward and fell over a period of several weeks. Microscopic Examination of Oxygen Sensors. Certain sensors were dissembled after a period of operation for detailed microscopic examination. Scanning electron microscopy revealed that the reference electrode had partially dissolved and had become porous in some sensors. The working electrode acquired a granular surface structure with use, which, according to X-ray elemental analysis, was the deposition of a thin layer of silver. In cases of gradual sensor failure the original signal could be restored by appropriate polarization treatment or by replatinization of the working electrode. In cases of abrupt sensor failure, a small dendritic silver structure had formed a contact between the working and reference electrodes, apparently growing from the working electrode. The factors that contribute to these modes of failure remain to be determined. In all cases, the counter electrode retained its original surface composition and microstructure. The microscopic examination indicates that various processes may occur during operation. The working electrode gradually became coated with silver as a result of being cathodically polarized with respect to the reference electrode. Thus, the oxygen-reduction process was no longer occuring on a platinum surface. This is a common observation for conventional polarographic oxygen electrodes. The deposition of silver apparently did not affect the signal as long as no dendritic contact with the other electrodes was made. the transfer of silver from the reference electrode was, however, somewhat surprising because the reference electrode was maintained at very high impedance by the potentiostat circuit (>10l2R based on the input impedance of the amplifier). Calculations show that a leakage current of approximately A between the two electrodes over the operational period is sufficient in some cases to account for the small amount of material transferred. Transient local capacitive currents as a result of inadequate shielding of the leads may also have played a role. By comparison however, the rate of silver deposition during potentiostatic operation is likely to be much lower than that of a conventional silver-platinum two-electrode configuration of the same working electrode area, where all of the current passes through the silver/silver chloride electrode. These observations suggest that the reference electrode, although operating at very high impedance, was not truly reversible. This conclusion is consistent with previous experiments on bare and chlorided silver electrodes under similar conditions (24). Micropolarization studies with carefully prepared electrodes showed that the current is distinctly
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nonlinear and irreversible with very small applied potentials (
