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IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 38, NO. 5, MAY 1991

Design and Validation of the Transparent Oxygen Sensor Array Bradley J. Sargent and David A . Gough

Abstract-A new oxygen monitoring system has been developed that consists of an array of transparent, independentlyconnected oxygen sensors mounted on a glass plate. This system is intended for continuous monitoring of oxygen at multiple locations on the surface of exteriorized tissues, while simultaneously visualizing microvascular activity. The sensor array is fabricated by techniques similar to those employed for semiconductors, which facilitate customization of sensor size and pattern to best monitor oxygen in a given physiologic preparation. Methods are described for characterizing the performance of the sensor array in vitro, including studies on the oxygen sensitivity, stability of the signal, and detection of oxygen gradients. An example of in vivo application of the sensor array is also presented.

INTRODUCTION N a previous paper [ 13, we described a novel oxygen sensor system consisting of an array of oxygen microelectrodes fabricated on a transparent glass substrate. The sensor system was composed of an array of oxygen electrodes in an arbitrary two-dimensional pattern, a highimpedance reference electrode, and a common counter electrode, all mounted on a glass plate. The array was operated by a multichannel potentiostat instrumentation system. The oxygen electrodes were independently connected to simultaneously indicate local oxygen concentration in the immediate vicinity of each sensor. This transparent sensor array system was designed as an experimental tool for the study of the dynamic distribution of oxygen near the surface of exteriorized organs and tissues. The transparent oxygen sensor array would be placed in contact with the surface of surgically exteriorized tissues and oriented over the microvascular network with the aid of a microscope. This would provide the capability of continuously monitoring oxygen concentration at many locations on the tissue surface while simultaneously visualizing the microvascular activity in the underlying tissue. The system is a means of studying the relationship between the dynamic oxygen distribution in tissue and microvascular architecture and function, and

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Manuscript received November 2. 1989; revised July 20, 1990. This work was supported by a grant from the National Institutes of Health. B . J. Sargent was with the Department of Applied Mechanics and Engineering Sciences, University of California, San Diego, La Jolla. CA 92093. He is now with the Institute de Microtechnique, University of Neuchitel, Neuchltel, Switzerland. D. A . Gough is with the Department of Applied Mechanics and Engineering Sciences, University of California, San Diego, La Jolla, CA 92093. IEEE Log Number 9144699.

may lead to a new understanding of local control of oxygen transport in tissues. This system provides an alternative to individual oxygen microelectrodes and previous nontransparent sensor arrays. Microelectrodes that are inserted into tissues allow recording of oxygen concentration at only one location at a time [2], [3], making it difficult to define the dynamic oxygen distribution. Nontransparent oxygen sensor arrays have previously been used to monitor oxygen concentration of the surface of tissues and organs [4], [5J,but these sensor arrays do not allow visualization of the tissue and results must therefore be analyzed without knowledge of the proximity of the sensors to microvascular structures. The oxygen sensor array concept is shown schematically in Fig. 1. From the top, a series of thin-film metal strips emanates from the multipin connector on the right and runs parallel across the transparent plate and converges to narrow, closely spaced ends on the left. In the expanded view, the ends form a sensor pattern where the active electrode areas are defined by openings in the insulation layer. There are eight similar platinum working electrodes at which electrochemical oxygen reduction occurs, a common silver/silver chloride reference electrode to which the potential of the working electrodes is compared, and a common platinum counter electrode to which the current passes. From the side view, the various layers including the substrate, thin-film metal layer, insulation, hydrophilic gel and outer silicone rubber membrane, and connector contact structure can be seen. The purpose of these structures will be described later. The sensor array was made using semiconductor fabrication technology. These fabrication techniques allowed the sensor size, number, and distribution on the plate to be individualized to obtain maximal information from a particular tissue preparation. Other investigators have used similar methods for production of other types of sensors, including sensor arrays for recording intracortical potentials in brain tissue [6], for monitoring the activity of nerve [7] and cardiac cells [8], and for auditory prostheses [9]. These applications also benefitted from the precise pattern control offered by semiconductor processing techniques. We previously gave a preliminary description of methods of fabrication of the sensor array and models of oxygen distribution in front of individual electrodes [ 11. The present paper describes improved methods of array fab-

0018-9294/91/0500-0476$01.00 O 1991 IEEE

S A R G E N T A N D GOUGH: T R A N S P A R E N T O X Y G E N SENSOR A R R A Y

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rication and methods for characterizing the performance of the sensor array in vitro, including studies on the oxygen sensitivity, stability of the signal, and detection of oxygen gradients. A preliminary example of in vivo application of the sensor array is also presented. FABRICATION Fig. 2 outlines the techniques of sensor array fabrication showing, as an example, the construction of an array having two working electrodes, a reference electrode and a counter electrode. Glass was used as a support structure. A 20 pm polyimide layer (DuPont PI 2702D) that served as the substrate for thin-film metal deposition was applied to the glass support by spin coating and cured by exposure to ultraviolet light and high temperature ( > 38OOC). Care was taken to avoid subsequent surface contamination. A thin film of platinum was then deposited by vacuum evaporation, as shown at the top of Fig. 2. A theoretical es$ mate [lo] of the thickness of the metal layer is 370 A . Metalization was purposely heavy in order to ensure against failure due to insufficient metal thickness. The electrode pattern was then produced by a photolithographic procedure. A photosensitive polymer (Shipley 1400-33 positive photoresist) was spin-coated onto the metal surface as shown in step three. Residual solvent was removed by baking in a convection oven at 85°C for 30 min. The electrode pattern was transferred to the polymer layer by means of a lithographic mask, as shown in step four. The mask was placed over the polymer film and the part was exposed to ultraviolet light at 760 nm. In a positive resist system shown here, the UV light caused exposed polymer regions to decompose and become soluble in a developer solution. After the exposed polymer regions were removed in the developer solution,

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the polymer remained only in the desired electrode pattern, as shown in step five. The polymer was baked again at 110°C for 30 min to achieve a full cure, imparting acid

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IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 38, NO. 5 . MAY 1991

resistance. The part was then immersed in an acid bath to etch away the exposed metal. After rinsing with distilled water, the cured polymer was removed with organic solvents, rinsed again and dried, leaving only the metal tracings in the desired pattern, as in step six. A two-stage patterning process was necessary because the reference electrode was a different metal than the other electrodes. The technique for reference electrode deposition was based on photolithography. After acid etching the platinum and before removal of the cured polymer, silver for the reference electrode was deposited by vacuum evaporation. A photosensitive polymer was spun onto the part covering the cured polymer and silver and then a second lithographic mask with the reference electrode pattern was aligned over the part. After UV exposure, the second image was developed. A second acid etching was done, after which both patterns of polymer were removed leaving only the metal tracings as in step seven. This technique produced precise definition of the reference electrode. An alternative technique for reference electrode deposition based on physical masking was also used in some sensor arrays. Photolithography was used to pattern a negative polymer image on the substrate, followed by metal deposition over the entire surface. The underlying polymer served as a separation layer, allowing the unwanted metal to be removed when the polymer was dissolved. This method has the advantage of requiring fewer processing steps and eliminates the need for acid etching (a potential problem), but may leave a ragged edge, a feature that restricts the ability to define small structures. This “lift-off” procedure can also be used for materials other than metal, such as insulation materials. An insulation layer was deposited and patterned with openings aligned over the metal tracings to define the electrochemically active electrode area as shown at the bottom of Fig. 2. The insulation layer must be precisely pattemable, impermeable to water vapor, and have good adhesion to both the substrate and metal. The polyimide used previously as a platinum deposition substrate is photosensitive and was used as the insulation material. The resulting insulation layer was 20 pm in thickness and, depending on curing temperature, ranges from light yellow to brown in color. In the final step, two polymeric membrane layers were applied. The first, providing ionic contact between the electrodes, was a thin hydrophilic gel layer containing a buffer solution. The gel, composed of poly(hydroxyethylmethacrylate) (or p-HEMA), was dissolved in methanol and deposited by spin-coating to produce a 10 pm layer, which was then soaked in the electrolyte solution. The second outer hydrophobic membrane was composed of poly(dimethylsi1oxane) (or silicone rubber). This material allows passage of oxygen and water vapor but excludes polar molecules. The membrane prevents electrode poisoning and provides a consistent oxygen mass transfer boundary layer in front of the working electrodes. This polymer was formed from a monomer and catalyst (RTV

Fig. 3 . Photograph of the sensor array. The sensor array region is at the right and the electrical leads run right to left to the connector assembly. The scale is in centimeters.

141, Rh6ne-Poulenic) and was deposited by spin-coating to a thickness of 30-100 pm. Unless noted otherwise, all sensor arrays had membranes. Connection to the external instrumentation was made by a 10-pin strip connector. Silver epoxy was used to link the pins to individually exposed areas of the metal strips. The silver epoxy connection was finally covered with nonconductive epoxy for insulation and support. There are several differences in this procedure from that we reported previously [ 11. A major improvement is the use of the transparent polyimide as both a metalization substrate and the insulation layer. This allows the thin metal film to be sandwiched between a common polymeric material, giving rise to improved insulation. A second advantage of the use of polyimide is that the glass no longer functions as a substrate for metal adhesion but as a support structure for processing and handling. This suggests the possibility of developing a flexible, conformal sensor array by removing the polyimide-insulated array structure from the glass support. Other advantages include better control over the reference electrode deposition and patterning, which permits smaller and more complicated sensor array patterns to be fabricated. Fig. 3 is a photograph of the complete sensor array. The sensor array portion is at the right edge with the long straight insulated leads joining to the strip connector on the left. The scale shown is centimeters. Fig. 4 is a closeup photograph of the sensors. The eight working electrodes are the small round circles, the counter is the two rectangular squares in the center, and the reference is the large square near the top. The rounded edges of the insulation layer can be seen around the openings that define the active electrode areas. The thin ridges are the metalized strips covered by insulation that act as electrical leads. The circular working electrodes have a diameter of approximately 100 pm with a 200 pm center-to-center separation. CHARACTERIZATION Sensitivity to Oxygen The in vitro testing apparatus is shown schematically in Fig. 5 . Sensor array characterization was carried out in phosphate buffer in a thermostated vessel at 37°C. The

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oxygen partial pressure was maintained by equilibrating the buffer solution with mixed gases, resulting in partial pressures of 0-106 mm Hg, which brackets the physiologic range. The sensor array was connected by cable to the multichannel potentiostat [ 111 that operates each sensor simultaneously and independently of each other. The potentiostat output was sampled at specified rates by computer through an analog-to-digital interface and data were stored in digital form on disk and displayed on screen and on the printer. Sensors were operated in vitro continuously for 2 to 2 1 days with periodic changes in oxygen partial pressure. Fig. 6(a) and (b) shows typical recordings of the signals from sensor arrays with and without membranes during a testing cycle. The individual sensor currents are recorded as a function of time and the oxygen partial pressure is indicated. In Fig. 6(a), a sensor array without membranes is initially exposed to 35 mm Hg oxygen partial pressure, followed by a step change to 0 and then to 14 mm Hg oxygen partial pressure. The individual sensor signals indicate the range of variation among sensors and the temporal variation due to stirring effects. The time to reach the new steady signal is characteristic of equilibration of the buffer solution to the new oxygen partial pressure, not of the sensor response time, which is much faster. The current remaining after the change to zero oxygen is largely a result of residual oxygen in the test vessel and eventually falls to less than 1 nA for all sensor arrays after

Fig. 6 . Sensor array oxygen sensitivity. (a) A sensor array without membranes and (b) a sensor array with membranes were exposed to changes in oxygen partial pressure in virro in a thermostated vessel in buffer at 37°C.

oxygen has been totally removed. The individual currents are linear with oxygen partial pressure, at approximately 0.70 nA/mm Hg. In Fig. 6(b), a sensor array with membranes is exposed to steps in oxygen partial pressure of 0, 14, 35, 71, and 35. The initial overshoot as oxygen is changed from 0 to 14 mm Hg is an artifact resulting from residual room air (149 mm Hg oxygen) being flushed from the gas delivery line. The signals from the sensor array with membranes show much less fluctuation with time due to fluid agitation, suggesting that the oxygen gradients in front of individual sensors may be largely confined within the membrane. The steady-state currents of the membrane-covered sensors are also typically linear with oxygen partial pressure over the tested range, but the current for a given oxygen partial pressure is approximately half that for sensors with membranes, or about 0.35 nA/mm Hg.

Stability In vitro stability of the steady-state signal was evaluated by calculating the signal drift during periods of exposure to constant oxygen. Typically, the signal was initially erratic, but stabilized by the end of the first day of operation to a drift of within 5 % / h . This degree of stability could be maintained for several hundred hours and is comparable to stability of other potentiostatic oxygen sensors [ 111. Oxygen Gradient Detection Tests were done in which an oxygen gradient was imposed across the sensor array to demonstrate the ability to

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IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 38, N O . 5 . MAY 1991

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detect spatial concentration differences. The experimental apparatus is shown in Fig. 7(a). The apparatus consists of a silicone rubber slab placed over the sensor array with two 250 pm diameter silicone rubber catheters embedded in the silicone rubber slab near each edge of the sensor array. A Saran layer was placed on top of the silicone rubber slab to prevent oxygen access from the atmosphere. Gases of different oxygen contents were passed through each tube to establish an oxygen gradient. The silicone rubber layer was thin compared to the distance between catheters. Typically, one tube was maintained at 71 mm Hg oxygen and the other at zero. The results for the two sensor arrays are shown in Fig. 7(b). The recorded steady-state oxygen partial pressure is plotted as a function of normalized distance between the parallel catheters. Sensors in the array nearer the catheter with the higher oxygen partial pressure have higher readings indicating that the array does detect an oxygen gradient. The broken line represents a simple linear gradient between the catheters. The nonlinear response, shown by the second-order polynomial fit to the data, is most likely due to oxygen consumption by the electrodes as the oxygen diffuses across the array surface.

Response to an Oxygen Step A simple experiment was done to measure the signal transient in response to a step change in oxygen partial pressure. The sensor array was placed in a thermostated vessel of stirred buffer solution at 37°C. The solution was equilibrated to 0 mm Hg by argon gas sparging. To create the concentration step, sparging was stopped and a specific amount of buffer that had been previously equilibrated to 7 10 mm Hg oxygen partial pressure was rapidly injected into the stirred vessel to bring the oxygen partial pressure to 35 mm Hg. The results from a variety of sensor arrays indicate that 30 s or less is required for sensors of this size and membrane thickness to reach 90% of the final value. I N VIVO STUDIES Preliminary in vivo experiments were performed to demonstrate the sensor array performance under conditions of intended use. The objective was to use the sensor array in living tissues and show an example of the type of results that can be obtained. An extensive analysis of the in vivo response will be the subject of later communications. The cremaster muscle of an anesthetized rat was surgically exposed leaving the vasculature attached and circulation intact [ 121. This muscle is thin and transparent and has been used previously in studies of microcirculation. The muscle was carefully draped over a microscope stage with minimal disturbance of the local intravascular blood flow. The sensor array was placed face down on the top of the thin muscle over the area of study with the aid of a micropositioning device forming a seal to the atmosphere. Peripheral regions of tissue that were not covered by the sensor array were covered with Saran film to provide isolation from atmospheric oxygen. The femoral vein was catheterized to facilitate collection of venous blood for comparative oxygen assay. After a period of stabilization, the anesthetized rat was respirated for specified periods with different mixtures of inspired gas ranging from 0 to 710 mm Hg oxygen partial pressure. Typical results taken 2 h after placement of the sensor array are shown in Fig. 8. The normalized current, or current of a given sensor divided by the sensor in vitro calibration current at 35 mm Hg oxygen partial pressure, is recorded as a function of time. Different broken or solid lines correspond as indicated to the different sensors. The diamonds indicate venous blood oxygen partial pressure in the scale on the right from blood samples assayed with a standard blood gas analyzer. The labeled bars on the abscissa indicate periods during which the inspired gas was other than atmospheric. These data show several interesting characteristics. First, there is substantial short-term fluctuation in the signal from individual sensors. This may correspond to local variations in perfusion or oxygen variation. Second, there are differences in signal depending on the location of individual sensors. Sensors 1 through 4, which were located close to microvessels, gave a substantially higher signal

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than sensors 5 through 8 , which were more distant. Third, perturbations in inspired oxygen gave rise to substantial changes in both venous blood oxygen partial pressure and local tissue oxygenation, with sensors located near microvascular structures being affected more dramatically. Finally, when oxygen delivery to the tissue was interrupted completely the response of all sensors dropped to zero. These local oxygen partial pressure recordings need to be correlated with the microscopically observed tissue function in order to obtain a deeper understanding of the role of oxygen in living tissues. These studies demonstrate that the transparent oxygen sensor array can provide new, and more extensive information about superficial tissue oxygen distribution. THE PROCESS OF SENSOR A R R A Y DESIGN Determination of the sensor array configuration that yields maximal useful information in a given monitoring situation will be an iterative process that includes in vivo experimentation. The ideal sensor number, size, spacing, orientation, and membrane thickness may be different for various applications because the microvascular architecture and metabolic demands of tissues can differ greatly. Moreover, important information such as the dimensional scale for oxygen distribution and the mass transfer resistance in layers near the tissue surface may not be known prior to the initial in vivo experiments. Refinements in the array configuration based on information from exploratory experiments and models of oxygen distribution may be required. It is therefore, difficult at present to define the ideal sensor array configuration. This process of specification by itself may improve understanding about the physiologic role and distribution of oxygen. The important advantage of the fabrication techniques described here is that the sensor array configuration can be readily mod-

ified without significant changes in the fabrication process.

CONCLUSIONS A new research tool has been developed and characterized in vitro. The in vitro studies on sensitivity, signal stability, response dynamics, and spatial oxygen resolution provide a background for the application of the transparent oxygen sensor array in physiologic studies. It is hoped that this sensor array will become a useful tool for understanding the role of oxygen in tissues. REFERENCES D. A. Gough, B. J. Sargent. and P. H . S . Tse, “A transparent oxygen sensor array,” Ann. Biomed. Eng.. vol. 14. pp. 153-159, 1986. I. A . Silver, “The oxygen micro-electrode,” Med. Electron. Biol. E n g . , vol. 3, p. 377. 1965. W. J . Whalen, J . Riley, and P. Nair, “A microelectrode for measuring intracellular PO,.” J . Appl. Physiol., vol. 23, no. 5 , pp. 798801, 1967. D. W. Lubbers. “Methods of measuring oxygen tensions of blood and organ surfaces,” f n t . Anesrhesiol. Clin.. v o l . 4 , pp. 103-127, 1966. M. Kessler and W. Grunewald. “Possibilities of measuring oxygen pressure fields in tissue by multiwire platinum electrodes,” Progr. Resp. Res.. vol. 3. pp. 147-152, 1969. 0. Prohaska, F. Olcaytug. K. Womastek. and H . Petsche, “A multielectrode for intracortical recordings produced by thin-film technology,” Electroenceph. Clin. Neurophys., vol. 42, pp. 421-422. 1977. J . L. Novak and B. C. Wheeler. “Recording from the Aplysia abdominal ganglion with a planar microelectrode array,” f E E E Trans. Biomed. Eng., vol. BME-33, no. 2 , pp. 196-202, 1986. D. A. Israel, W. H . Barry. D. J . Edell, and R. G . Mark, “An array of microelectrodes to stimulate and record from cardiac cells in culture,” Amrr. .I. Physiol., vol. 27, pp. 669-647, 1983. H . S. Lusted, “ f n I?IY) electrical stimulation using multichannel photolithographic electrode arrays,“ f E E E Trans. Biomed. E n g . , vol. BME-33, no. 8, pp. 800-803, 1986. D. H. Kay, Techniques f o r Electron Microscopy. Oxford. England: Blackwell. 1965. J . Y . Lucisano, J. C. Armour. and D. A. Gough. “ f n virro stability of an oxygen sensor,” Anal. Chrm., vol. 59, pp. 736-739, 1987.

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[12] S . Baez, “Anope cremaster muscle preparation for the study of blood vessels by in vir microscopy,” Microvas. Res., vol. 5, pp. 384394, 1973.

Bradley J. Sargent received the Ph.D. degree in bioengineering from the University of California, San Diego, in 1989. He has served as a Postdoctoral Fellow and Instructor at the University of California, San Diego. He is currently at the Institut de Microtechnique, University of Neuchitel, Switzerland, His research interests include the development of oxygen and glucose sensors for biological studies and computer modeling of sensor function.

David A. Gough received the Ph.D. degree in materials science and engineering in 1974 from the University of Utah, Salt Lake City. From 1974 to 1976, he was an N.I.H. Postdoctoral Fellow at Harvard Medical School and had appointments at the Joslin Clinic and Peter Bent Brigham Hospital. He then moved to the University of California. San Diego, where he is presently a Professor of Bioengineering and ViceChair of the Department of Applied Mechanics and Engineering Sciences. He spent the 19861987 academic year at the Massachusetts Institute of Technology, Cambridge. His main reseiuch interests are the implantable glucose sensor, oxygen transport in tisswes, and biotechnology.

Design and validation of the transparent oxygen sensor array.

A new oxygen monitoring system has been developed that consists of an array of transparent, independently-connected oxygen sensors mounted on a glass ...
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