Anal. Chem. 1995, 67,1536-1540
A Continuous, Implantable Lactate Sensor Dale A. Baker and David A. Qough*
Department of Bioengineering, University of Califomia, San Diego, La Jolla, Califomia 92093-0412
An implantable sensor for continuous monitoring of
lactate in the bloodstream or tissues has been made. The sensor includes a lactate electrode based on immobilized L-lactate oxidase coupled to a potentiostatic oxygen electrode and an oxygen reference electrode to account for local oxygen. This design renders the response essentially independent of oxygen concentration. In vitro characterization demonstrated that the sensor can respond specifically to lactate over a broad concentration range. This response is stable for more than 1 week during continuous in vitro operation at body temperature. As an acute implant in the canine right atrium, the sensor produced a continuous recording of lactate that was in good agreement with conventional, discrete blood lactate assays. Lactate is one of several metabolites for which development of a continuously operating, implantable sensor would be advantageous. A sensor that can function in the tissues or bloodstream may be the optimal means of monitoring such metabolites that vary substantially over a period of a few minutes and can have important consequences for health. An implantable lactate sensor would have unique value in the diagnosis and study of acidosis, acute circulatory shock, and heart disease, as well as for continuous monitoring in surgery, exercise physiology studies, and The ideal sensor would respond continuously and specitically to lactate over a substantial range, function effectively under low-flow conditions and oxygen concentrations that often accompany lactate production, require only infrequent recalibration, and avoid an unacceptable biocompatibility response. For short-term clinical applications, the sensor need function for only 3-4 days. Previous lactate sensors have been of three t y p e ~ . ~ The - l ~ fist type, the majority of lactate sensors, detects lactate in discrete samples in vitro and is not intended for continuous use. The second type is designed for frequent blood lactate assay, but as (1) Rady, M. Y. Resuscitation 1992, 24, 55-60. (2) Toffaletti,J. G. Cn't. Rev. Clin. Lab Sci. 1991, 28, 253-268. (3) Vincent, J. L.; Dufaye, P.; Berre, J.; Leeman, M.; Degaute, J. P.; Kahn, R J. Cn't. Care Med. 1983, 11, 449-451. (4) Henning, R J.; Weil, M. H.; Weiner, F. Circ. Shock 1982, 9, 307-315. (5) Moret, P. R Lactate: Physiologic, Methodologic, and Pathological Approach; Springer-Verlag: New York, 1980. (6) Broder, G.; Weil, M. H. Science 1964, 143, 1457-1459. (7) Scheller, F.; Schubert, F. Biosensors; Elsevier: London, 1992. (8) Turner, A P. T.; Karube, I.; Wilson, G. Biosensors Fundamentals and Applications; Oxford University Press: Oxford, UK 1987. (9) Wang, D. L.; Heller, A Anal. Chem. 1993, 65, 1069-1073. (10) Campanella, L.; Tomassetti, M.; Mazzie, F. Biosens. Bioelectron. 1993, 8, 307-314. (11) Katakis, I.; Heller, A Anal. Chem. 1992, 64, 1008-1013. (12) Schneider, B. H.; Daroux, M. L.; Prohaska, 0. J. Sew. Actuators B 1990, 6, 565-570. (13) Clark, L. C.; Spokane, R B.; Homan, M. M.; Sudan, R; Miller, M. Trans. ASMO 1988, 34, 259-265. (14) Hu, Y.; Zhang, Y.; Wilson, G. S. Anal. Chim. Acta 1993, 281, 503-511.
1536 Analytical Chemistty, Vol. 67, No. 9, May 1, 1995
part of an extracorporeal sampling system in which blood is withdrawn from body and diluted prior to exposure to the s e n ~ o r . ~ ~Sample - ~ O dilution is necessary to achieve sensitivity over a useful lactate range, to minimize the effects of interfering substances, and to provide sufficientoxygen for this type of sensor. There have been several notable reports describing this sensing approach.21-26These systems require a sample fluid handling system, usually based on a dual-lumen catheter, in which one lumen delivers an anticoagulant to maintain the blood fluidized, while the other lumen brings the anticoagulant/blood mixture to the sensor chamber. Extracorporeal sensors of this type need frequent recalibration, do not allow for continuous monitoring, and require additional fluid handling components for the calibrant solution. Mixing and pumping of the fluids also create time lags that add to the inherent response time of the sensor. The third type of lactate sensor is the implanted sens0r.2~This type of sensor does not require fluid handiing and can potentially achieve the ideal of continuous monitoring in specitic anatomical locations, provided that certain sensor design requirements not previously addressed are met. The implantable lactate sensor reported here is analogous to the oxygen-based enzyme electrode sensor we developed previously for g l u c o ~ e . ~The ~-~ sensor ~ is based on a reaction catalyzed by L-lactate oxidase: lactate
+ 0, - pyruvate + H20,
The enzyme is immobilized in a gel coupled to an oxygen-sensitive (15) Mitzutani, F.; Sasaki. R; Shimura, Y. Anal. Chem. 1983, 55, 35-38. (16) Kaisheva, A; Atanasov, P.; Gamburzev, S.; Dimcheva, N.; MeV, I. Sens. Actuators B 1992, 8, 53-57. (17) Adamowicz, E.; Burstein, C. Biosens. Bioelectron. 1988, 3, 27-43. (18) Weaver, M. R; Vadgama, P. M. Clin. Chim. Acta 1986, 155, 295-308. (19) Kambe, I.; Matsunaga, T.;Teraoka, N.; Suzuki, S. Anal. Chim. Acta 1980, 129,271-276. (20) Mascini, M.; Moscone, D.; Palleschi, P. Anal. Chim. Acta 1984, 157, 4551. (21) Hgkanson, H.; Kyrolinen, M.; Mattiasson, B. Biosens. Bioelectron. 1993, 8, 213-217. (22) Mascini, M.; Fortunati, S.; Moscone, D.; Palleschi, G.; Massi-Benedetti, M.; Fabietta, P. Clin. Chem. 1985, 31, 451-453. (23) Meyerhoff, C.; Bischof, F.; Mennel, F. J.; Stemberg, F.; Bican, J.; Pfeiffer, E. F. Biosens. Bioelectron. 1993, 8, 409-413. (24) Pfeiffer, D.; Setz, IC;Schulmeister, T.; Scheller, F. W.; Luck, H. B.; F'feiffer, D. Biosens. Bioelectron. 1992, 7, 661-671. (25) Shimojo, N.; Fujino, IC;Kitahashi. S.; Nakao, M.; Naka, IC;Okuda, IC Clin. Chem. 1991,37,1978-1980. (26) Palleschi, G.; Mascini, M.; Bemardi, L.; Zeppilli, P. Med. Biol. Eng. Comput. 1990,28,B25-B28. (27) Durliat, H.; Comtaf M. Anal. Chem. 1980, 52, 2105-2112. (28) Gough, D. A,; Leypoldt, J. IC;Armour. J. C. Diabetes Care 1982, 5, 190198. (29) Gough, D. A,; Lucisano, J. Y.; Tse, P. H. S. Anal. Chem. 1985,57, 23512357. (30) Gough, D. A; Armour, J. C.; Lucisano, J. Y.; McKean, B. D. Trans.ASAZO 1986, 32, 148-150. (31)Armour, J. C.; Lucisano, J. Y.; McKean, B. D.; Gough, D. A Diabetes 1990, 39, 1519-1526.
0003-2700/95/0367-1536$9.00/0 0 1995 American Chemical Society
Lactate
Oxygen
\ n
Oxygen
n
Electrode
Oxygen Reference
,Electrode
Fill
Figure I. Schematic of the implantable lactate sensor. The sensor is 2 mm in diameter and 20 cm in length, with a 3-mm-long active
region. electrode. Lactate and oxygen from the body react withii the enzyme gel and unconsumed oxygen is detected by the electrode, producing a lactatemodulatedoxygendependent current, ihwThe ambient oxygen concentrationis indicated by a second electrode within the enzyme, the oxygen reference electrode, which produces an oxygen-dependent current, io. The currents are subtracted to give the signal of interest, the lactate-dependent difference current, i ~which , can be related to lactate concentration when certain conditions are met. The complete lactate sensor is therefore composed of a lactate electrode, an oxygen reference electrode, and an appropriate means of current subtraction. This principle, together with the catalytic specificity of lactate oxidase, makes possible a sensor that is specific for lactate and potentially transparent to ambient oxygen.28 A separate, additional requirement is the need to maintain a stoichiometric excess of oxygen relative to lactate within the enzyme gel to assure that the reaction/diffusion process is limited by lactate, rather than by oxygen. The design must compensate for the “oxygen deficit”28that prevails physiologically, in which there is a relative shortage of oxygen compared to lactate. These conditions are due to a combination of physical and biological factors, such as the low solubility of oxygen in biological fluids, metabolic processes that consume oxygen, high lactate production under anoxic conditions, and pathologic interruption of local circulation. As a result, lactate concentrations of interest may range to 25 mM, whereas biological oxygen concentrations are at most -0.15 mM (equivalent to 100 mmHg partial pressure) in arterial blood and can be much lower locally in tissues (e.g., 0.01 mM or 7 mmHg) under conditions of The oxygen deficit must be addressed by design of the lactate electrode and cannot be remedied by use of the oxygen reference electrode. SENSOR DESIGN
The sensor is shown schematically in Figure 1. The lactate electrode and oxygen reference electrode are located in series within a single silicone rubber tube.30 The lactate electrode contains the immobilized enzyme in a cylindric, cross-linked albumin gel located at the end of the hydrophobic silicone rubber tube. The potentiostatic oxygen electrodes that are the basis of the lactate electrode and oxygen reference electrode each consist of three small wire electrodes, two platinum and one silver, embedded in an epoxy stub. The wires are maintained in electrical contact by a layer of electrolyte (not shown) and are (32) Isselbacher, IC J., et al., Eds. Harrison‘s Principles oflntemal Medicine, 13th ed.; McGraw-Hill New York, 1994.
coated with a thin layer of porefree silicone rubber to prevent electrochemical interference and electrode poisoning by polar biochemicals, which are impermeable in silicone rubber. The conductive leads from the lactate electrode pass through a region filled with silicone rubber and the epoxy stub of the oxygen reference electrode. The leads from both electrodes are attached to electrical connectors at the distal end (not shown). In some cases, the lactate and oxygen reference electrodes were also fabricated in separate tubes rather than in a single tube as described above. The principle of the potentiostatic oxygen sensoS3 and associated instrumentation3 have been described previously. The lactate electrode is based on the two-dimensional sensor design described previously for the implantable glucose sensor.29 The outside is a tube of silicone rubber, which is impermeable to lactate but highly permeable to oxygen. Both lactate and oxygen can diffuse into the gel layer through the exposed annular end parallel to the axis of the oxygen sensor, but only oxygen can diffuse radially to gel through silicone rubber surface. This provides a twedimensional supply of oxygen to the enzyme region (both radial and axial) but only a one-dimensional supply of lactate (axially only). It has been shown29 that this simple design feature can eliminate an oxygen deficit without introducing an unacceptable response lag.35 In the present lactate sensor design, the oxygen reference electrode is based on radial oxygen diffusion. This implantable lactate sensor builds on our previous develop ment of an implantable glucose sensor. The glucose sensor has been described extensively in models of the in vitro steady state and transient response^,^^^^ which models can be employed with kinetic and transport parameters specific to lactate. The oxygen sensor used here is a threeelectrode, potentiostatic design that has been shown to be highly stable during continuous, long-term in vitro 0peration.3~An implantable dual-potentiostatand telemetry instrumentation system for testing the sensor in vivo has been adapted The glucose sensor has proven to be effective as an intravascular implant in dogs for continuous monitoring of blood glucose for periods of over 100 days without limitations due to biological incompatibility or need for calibration.31 If immobilized lactate oxidase is sufficiently stable, a lactate sensor with similar analytical capabilities can be envisioned. We report here fabrication of the implantable lactate sensor, in vitro characterization, and in vivo application for continuous short-term monitoring of blood lactate in a dog heart preparation. EXPERIMENTAL SECTION
Sensor Fabrication. The two oxygen electrode systems consisted of short segments of 100;umdiameter platinum and silver electrode wires, welded to insulated stainless steel lead wires and encapsulated in a parallel arrangement in a bead of epoxy resin. The resulting electrode fixtures were each -1 mm in diameter with a 1-mm exposed active length. The fixtures were dip coated with a hydrophilic polymer solution containing electrolyte, dried, coated with a thin layer of silicon rubber, and cemented in series into the end of a silicone rubber tube. The lead wires attached to connectors. The annular region in the oxygen reference electrode was filled with silicone rubber. Slight (33) Lucisano, J. Y.; h o u r , J. C.; Gough, D.A Anal. Chem. 1987, 59, 736739. (34) McKean, B. D.; Gough, D.A IEEE Trans. Biomed. Eng. 1988,35, 526532. (35) Lucisano, J. Y.; Gough, D.A Anal. Chem. 1988, 60, 1272-1281.
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differences in electrode size and oxygen flux were compensated electronically prior to signal subtraction. The enzyme was immobilized in a gel formed in the annular end cavity between the silicone rubber coating around the electrodes and the outer tubing. The gel contained 20 wt % dog serum albumin (Sigma Chemical Co., fraction V) and 5 wt % lactate oxidase from Pediococcus, dissolved in 0.01 M phosphate buffer, pH 7.3, containing 0.1 N KCl, and was cross-linked by adding a small amount of glutaraldehyde (Sigma Chemical Co., grade I). The representative size of the lactate sensor was 2 mm in diameter by 20 cm in length, with the lactate and oxygen reference electrodes occupying the final 3 mm. The aspect ratio,B or ratio of annular radius to length of the immobilized enzyme gel, was -0.25. The sensor described here is intended for intravascular application, but may be adaptable for use in tissues. In vitro Characterization. An automated testing apparatus% or a well-stirred,thermostated vessel containing phosphate buffer solution (PH 7.3, 37 "C) was used to demonstrate the in vitro sensor response to step and other changes in lactate and oxygen concentration. Solutions were maintained at specified oxygen concentrations by equilibration with analyzed gas mixtures. Oxygen concentrations and partial pressures were calculated with the aid of standard solubility tables.37 Controlled additions of a lactate standard solution to give the desired lactate concentration challenge were achieved by pumping or metered injection. The sensors were connected to individual potentiostats, similar to those described and up to 10 sensors were tested simultaneously. The analog signals from the sensors were multiplexed, digitized with a 12-bit analog-to-digital converter, and routed to a computer for display, analysis, and storage. The sensors generated -10 nA/mM lactate at oxygen concentrationsrepresentative of venous blood. Sensor Implantation. The lactate sensor was inserted into the jugular vein of an anesthetized dog and advanced through the superior vena cava so that the tip was located in the right atrium of the heart. The sensor was connected via percutaneous leads to external instrumentation. The sensor continuously recorded blood lactate during a 4 h period. Discrete blood samples were withdrawn through a catheter placed nearby in the atrium and assayed by standard methods for blood gas and lactate concentration with, respectively, a Radiometer blood gas analyzer (Model ABL3, Copenhagen, Denmark) and Yellow Springs Instruments lactate analyzer (Model 1500, Yellow Springs, OH). The implanted lactate sensor operated continuously during experimental studies of regional ischemia and cardiac biomechanics. The objective was to record lactate in venous blood returning from the heart itself during an episode of experimentally induced cardiac ischemia. Most of the venous blood from the cardiac circulation returns directly to the right atrium via the coronary sinus but mixes in the right atrium with venous blood returned from the body via the vena cava. The right atrium is therefore a reasonable location for detecting lactate in blood perfusing cardiac tissues, although quantitative interpretation of this information in terms of cardiac tissue metabolism may require additional measurements such as systemic and cardiac blood flow. Lactate monitoring in the right atrium is an essential step to demonstrate sensor effectiveness and reliability in blood prior to attempting (36)Baker, D.A;Gough. D. A.Biosens. Bioelectmn. 1993,8, 433-441. (37)Perry, R H.,e t al., Eds. Peny's Chemical Engineen' Handbook, 6th ed.; McGraw-Hill: New York, 1984. 1538 Analytical Chemistry, Vol. 67, No. 9,May
I, 1995
OB .-O 0.6 1
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Figure 2. In vitro sensitivity to lactate. Normalized lactatedependent difference current is plotted as a function of the ratio of the bulk lactate to oxygen concentration for various physiologic oxygen concentrations.
its use in less accessible regions of the heart, such as directly in the coronary venous circulation. Cardiac ischemia was caused by graded occlusion of a dissected segment of the left anterior descending coronary artery using a clamp device in the openchest canine heart preparation. The heart was instrumented for pressure and regional blood flow measurements. Placement of the lactate sensor in the right atrium was validated by palpation and cinkradiographic (X-ray) recordings. Systemic anticoagulation was not used in this study. Other details of this experimental protocol have been reported
RESULTS AND DISCUSSION
Steady State in vitro Response to Lactate. The steady state response to lactate at different physiologic oxygen concentrations is shown in Figure 2. Values for the lactatedependent difference current normalized by the oxygen reference current, il/i0, are plotted as a function of the ratio of bulk lactate to oxygen concentration, Q/C,B. The experiments were carried out by recording the response to increasing lactate concentration at each oxygen concentration shown. The value of the abscissa also indicates the oxygen deficit. The symbols correspond to different oxygen concentrationsover the range of 0.02-0.21 mM (or 15150 mmHg oxygen partial pressure). Plotting the results in this fashion, suggested by previous modeling and experimental studies on the glucose sensor,29 greatly simplifies the interpretation, as the response to lactate is independent of oxygen concentration. This approach is also readily amenable to automatic computation in the following way. The oxygen reference electrode signal is io,from which C,,B is determined. The lactate electrode signal, ih,, is substracted from ioto give il. Therefore, a given current ratio il/iocorresponds to a specific concentration ratio Q / C ~ B with C ~ Bspecified, from which can be calculated, irrespective of oxygen concentration. These calculations can be made in real time using a simple algorithm. This universal calibration curve for a given sensor changes only with enzyme inactivation,Bwhich does not occur during the period of these studies, as shown later. These results indicate that this particular sensor can overcome an oxygen deficit of greater than 400,allowing detection to at least 8.0 mM lactate concentration (38)McCulloch, A D.;Omens, J. H.J. Biomech. 1991,24, 539-548. (39)Hashima. A R;Young, A A; McCulloch, A D.; Waldman, L. IC]. Biomech. 1993,26, 19-35. (40) May-Newman, K; Omens, J. H.; Pavelec, R S.; McCulloch, A D. Circ. Res. 1994,74,1166-1178.
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in the worst case of lowest oxygen concentration shown here (0.02 mM or 15 mmHg oxygen partial pressure) and up to -25.0 mM at 0.06 mM oxygen (or 45 mmHg partial pressure), equivalent to the highest expected values in tissues or venous blood. This approach to the determination of lactate is particularly powerful because it is transparent to oxygen and covers a broad lactate concentration range. In Vitro Response to Lactate Steps. Figure 3 is an example of the response of a sensor to lactate steps. The lactate difference current is recorded with time in response to successive steps in lactate concentration. Testing was performed at a constant oxygen concentration of 0.06 mM (or 45 mmHg oxygen partial pressure), verified by the oxygen electrode and discrete standard oxygen assays. The continuous tracing is the lactate sensor response, and the arrows indicate step challenges of specifled concentrations. Discrete samples were assayed for lactate and oxygen concentration with standard analyzers to venfy steady state concentrations. The transient response, typical of -30 sensors tested, was complete in -5 min. Retention of Sensitivity during Continuous In Vitro Operation. Figure 4 shows the retention of in vitro sensitivity to lactate of a representative sensor over a 1-week period of continuous in vitro operation at body temperature. This operational period was chosen as being adequate for experimentaland potential short-term clinical applications. The current ratio k/i, is plotted as a function of lactate concentration at a k e d physiological oxygen concentration. The sensor was operated continuously and the sensitivity evaluated on days 3, 5, and 7. The sensor retained a stable sensitivity to lactate up to -25 mM under these conditions, although the sensor began to lose
sensitivity to the highest lactate concentrations during the second week of continuous operation (not shown). This effect is a result of the slow inactivation of immobilized lactate oxidase. These data suggest that the stability of immobilized lactate oxidase in this form is not a limitation for short-term continuous monitoring applications. In Vi0 Monitoring of Lactate. Figure 5 shows the results of application of the sensor as an implant. Blood lactate concentration was monitored continuously for a period of -4 h during operation of the sensor in the right atrium of an anesthetized dog. Lactate concentration, indicated by the sensor in the solid line and by standard discrete assay in the triangles, is shown as a function of time in minutes. Venous oxygen concentration (not shown) was also recorded by the oxygen reference electrode and by standard assay of discrete blood samples. Oxygen remained relatively constant at about 40-45 mmHg partial pressure throughout the experiment. The response to lactate was based on a priori, in vitro sensitivity, with no in vivo calibration or adjustment. Although there were fluctuations of relatively small amplitude in the sensor signal reflecting experimental perturbations, the concentration indicated by the sensor showed reasonable agreement with values determined by the standard assay. The surgical procedure was likely responsible for the initial elevation of the lactate concentration from typical resting values near 1.0 mM lactate. From approximately 53 to 63 min and from 100 to 105 min, the left ventricle was manipulated during placement of instrumentation and direct biomechanical measurements. These procedures may account for fluctuations in the sensor signal during these intervals, although it remains to be determined whether all fluctuations were a result of small lactate changes or reflections of changes in flow or other physical phenomena. The left anterior descending coronary artery was occluded in a graded fashion starting at 150 min, as indicated by the arrow in the figure. Incremental reduction in cardiac blood flow caused by the graded occlusion gave rise to the slow increase in venous blood lactate measured in the right atrium. Biocompatibility was an important aspect of sensor performance. The flexible sensor was free-floating in the right atrium, where fluid dynamics maintained the sensor tip away from the heart wall, pointed in the direction of flow and exposed to wellmixed blood flow. This site may minimize clotting and discourage encapsulation of the sensor in comparison to sites with higher shear flow conditions, such as intra-arterial or other intravenous Analytical Chemistry, Vol. 67, No. 9, May 1, 1995
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sites.4l Also, the materials in contact with the blood (silicone rubber and cross-linked albumin) have been reported to be relatively n o n t h r o m b o g e n i ~ Although . ~ ~ ~ ~ ~ systemic anticoagulation was not used in this open chest preparation, there was no indication of clot formation on the sensor at explantation and no signs of signal modifcation ascribable to clotting during the experiment. The pre-implant and postexplant determination of sensitivity to lactate were nearly identical. An implantable sensor for continuous blood lactate measurement as described in this report may be potentially useful in monitoring dynamic changes in the heart and in other sites of reduced blood flow and oxygen levels in the body. CONCLUSIONS The lactate sensor described here is effective for monitoring lactate over a broad concentration range at oxygen concentrations (41) Caro, C. G.; Pedley, T.J.; Schroter, R C.; Seed, W. k rite Mechanin offhe Circulation; Oxford University Press: Odord, UK, 1978. (42) Mchtire, L. V. Guidelinesfor Blood-Materials Interactions; NIH Publication 852185; US. Govt. Printing Office: Washington, DC, 1985. (43) Van Noort, R; Black, M. M. Biocompatibility of Clinical Implant Materiaki; Williams, D. F., Ed.; CRC: Boca Raton, FL, 1981; Vol. 2, pp 79-98.
1540 Analytical Chemistry, Vol. 67,No. 9,May 1, 1995
that prevail in poorly oxygenated tissues and venous blood. The sensor retains this broad sensitivity to lactate during continuous in vitro use at physiologic temperature for at least 1week, a degree of stability that is more than adequate for short-term clinical applications. Preliminary studies have shown that the sensor is biocompatible and can meet many requirements for continuous in vivo lactate monitoring in experimental studies. The sensor shows promise for use in a variety of biomedical applications. ACKNOWLEDQMENT We acknowledge the major role of Andrew McCulloch, James Covell, and Richard Pavelec in the animal studies. This work was supported in part by Grant HM7089 from the National Institutes of Health. Received
for
review October 6, 1994. Accepted February
7, 1995.@ AC940988D Abstract published in Advance ACS Abstracts, March 15, 1995.
