2891
Anal. Chem. 1984, 56, 2891-2896 (9) Wong, R. C.; Burd, J. F.; Carrico, R. J.; Buckler, R. T.; Thoma, J.; Boguslaski, R. C. Clin. Chem. (Wlnston-Salem, N . c . ) 1979, 25,
666-691.
(14) The, T. H.; Feltkamp, T. R. Immunology 1970, 18, 865-873. (15) Kobayashi, Y.; Miyai, K; Tsubota, N.; Watanabe, F. SteroMs 1979, 3 4 ,
829-834.
1101 McGreaor. A. R.: Crookall-Greenina. J. 0.: Landon, J.; Smith, D. S. \
- I
C l i . ch/m. Acta 1970, 83, 16l-lb6. (11) Weber, G. Blochem. J . 1952, 57, 145-156. (12) Dendllker. W. 8.; Halbert, S.P.; Florlm, M. C.; Alonso, R.; Schapiro, H. C. J . EXP. Med. 1965, 122, 1029-1048. (13) Dandllker, W. 6.; Alonso, R.; Meyers, C. Y. Immunochemlstfy 1967, 4 . 295-302.
RECEIVED for review May 10,1984. Accepted July 16,1984. Support for this work was provided by a fellowship granted 'Onby the United States to J. *' vention, Inc. (Rockville, MD).
Alterations in Potentiometric Response of Glucose Oxidase Platinum Electrodes Resulting from Electrochemical or Thermal Pretreatments of a Metal Surface James F. Castner' and Lemuel B. Wingard, Jr.* Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsyluania 15261
A h e a r correlation has been observed between the log (glucose concenttatlon) and measured potentlals at bare Ptand at enzyme/Pt-Indicating electrodes. A comparison study of five dlfferent methods of electrode pretreatment was undertaken to evaluate the Nernstlan behavior of the sensors. The potentlostatlcally controlled double-layer and the platlnlc chloride-treated electrodes exhlblted the greatest dlfferences In potentlometrlc response between the metal and the enzyme/metal electrodes. Sensors fabricated with flame-oxldlzed and with potentlostatic-oxldlzed or reduced Pt surfaces demonstrated little dlfference In Nernstlan response between the two types of lndlcatlng electrodes. Varlatlons In pH, Ionic strength, and oxygen concentratlon had only minor effects. The mechanism of the enzyme electrode appears to be associated with the products of the blocatalytlc reaction; the electrochemlcal reaction of glucose at the bare metal electrode appears to be dependent on the Pt surface Chemistry.
The development of new types of biocatalytic surfaces coupled to potentiometric electrodes is a n area of analytical chemistry that has been extremely active during the past few years. Enzymes, microbial cells, special membranes, and immunoproteins have been incorporated into a variety of electrode configurations to impart selectivity for specific drugs, endogenous compound^, antibodies, or ions (1, 2). These electrodes respond to the selected materials potentiometrically. There is a need for obtaining continuous in vivo measurements of glucose levels for the clinical management of ambulatory insulin-dependent diabetic patients. The development of a small potentiometric enzyme electrode system has been proposed as one approach to meet this clinical need. Such an electrode should respond over a range of about 2.7-22.2 mM glucose (50-400 mg/100 mL) with a mean of roughly 6.6mM (120 mg/100 mL) for placement in the circulatory system. Alternatively, the electrode could be implanted in a nonvascular part of the body, so long as the glucose concentration responded quickly to changes in blood lPresent address: E. I. du P o n t de Nemours & Co., Inc., Biomedical Products, Wilmington, DE 19898. 0003-2700/84/0356-2891$01.50/0
glucose levels (3). Nonvascular placement would reduce the clotting complications caused by contact of blood with foreign materials. An electrode consisting of glucose oxidase immobilized on platinum ( 4 ) ,with or without added catalase (5),was shown in vitro to produce a potentiometric response to glucose. The proposed overall reaction sequence at this electrode was as follows. /3-D-glucose
+02
glucose oxidase
D-glucono-&lactone D-glucono-&lactone
+ H20
catalase
H202
-
+ H202 (1)
D-gluconic acid
H2O + l / 2 0 2
(2)
(3)
The hydrogen peroxide produced in reaction 1had a major influence on the magnitude of the observed Nernstian-like response (6, 7). It was recognized that a potentiometric response was generated when bare platinum metal was placed in solutions of different concentrations of glucose (6). However, this glucose-generated potentiometric response was enhanced markedly by the presence of glucose oxidase immobilized on the platinum surface (4-6). The difference in response between the enzyme electrodes and bare platinum electrodes was at least 10 times greater for glucose than for possible interfering endogenous materials at normal physiologic levels, with the exception of glycine (only 7 times greater for glucose) (5,8). In previous studies the platinum support was cleaned by soaking the metal in 20-50% nitric acid plus heating it until white-hot in a natural gas flame. Preliminary results showed that the potentiometric response was influenced by the type of pretreatment that the platinum surface was given (7). The characteristics of the oxide film present on the surface of the platinum metal may have been influenced by the surface pretreatment; however, the detailed electrochemistry of platinum oxide films is still not completely understood (9, 10). The present study was undertaken to examine the relationships between platinum surface pretreatments and potentiometric response elicited from (1)a glucose oxidase enzyme electrode and (2) a non-enzyme bare platinum indicator electrode for the andytes glucose and hydrogen peroxide. The 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
Table I. Slopes of Potential vs. Logarithm of Concentration for Electrodes in Glucose or Hydrogen Peroxide" slopes, mV/decade platinumenzyme,b in glucose
bare platinumb method double-layer oxidation reduction flamed platinic chloride
in hydrogen peroxide -13 f 3.7 (n = 3 ) -14 f 2.2 (n = 3) -18 f 2.8 (n = 3) -12 f 3.0 (n = 3) -29 f 8.0 (n = 3)
in glucose -22 -23 -43 -54 -60
4.6 (n = 4) *f 6.5 (n = 3) f 11 (n = 3) f 9.5 (n = 3) f 9.6 (n = 3)
-54 -29 -44 -43 -95
f 3.2 (n = 4) f 2.8 (n = 12) f 2.6 (n 14)
f 5.4 (n 15) f 17' (n = 3)
comparison of means, in glucose, P valued 0.0001
0.015 NS 0.011
0.038
'Determined from plot of change in potential (from base-line potential with buffer only) vs. logarithm of M glucose or M hydrogen peroxide concentration. Slopes obtained by linear least-squares fitting. Solution conditions at pH 7.4, 25 " C , and oxygen saturation. *Resultsshown as mean f std dev for n electrodes. 'No catalase was added. Comparisons via Student's t test; NS means not significant. influences of pH and ionic strength on the potentiometric response also were investigated. A mechanism is suggested to explain the observed potentiometric behavior of the electrode systems. EXPERIMENTAL SECTION Materials. Commercial-grade platinum foil 0.05 mm thick was purchased from Fisher Scientific (manufactured by Englehard). Platinum lead wires of 0.26 mm diameter were purchased from Medwire Corp. Glucose oxidase Type I1 (20% enzyme protein; 15000 units/g of solid; catalase impurity 0.46% of glucose oxidase activity), 25% aqueous glutaraldehyde Grade I, and pD-glucose were purchased from Sigma. Aqueous catalase (about 72 mg/mL or 4.1 X lo6 units/mL) was obtained from Worthington-Millipore Corp. All of the materials except glutaraldehyde were used as received. Only glutaraldehyde that had an absorbance ratio a t 235/280 nm of less than 0.5 was used. Material having a larger ratio was purified with activated carbon to remove polymeric glutaraldehyde (11). Low oxygen content (C0.5 ppm) nitrogen of 99.998% purity from Airco was bubbled through a solution of VOSO, and water prior to use (12). Instrumentation. Electrochemical pretreatment of the platinum foil was carried out with a Model RDE-3 potentiostat from Pine Instrument Co., Grove City, PA. Current-potential profiles were recorded with a Model 2000 X-Y recorder from Houston Instrument Co. Measurement of the potentiometric response from the enzyme and control electrodes was carried out with a Model 610C Keithly Electrometer (Cleveland, OH) connected to a Model 2000 Fisher Scientific strip chart recorder. All potentials were measured with respect to a saturated KC1 Ag/AgCl reference electrode. A platinum screen was used as the auxilliary electrode in the voltammetry experiments. This electrode was isolated from the electrolyte solution by a medium-pore frittedglass membrane. Platinum Pretreatment. Five methods were used to pretreat the platinum foil. Four of the methods were electrochemical (oxidation, reduction, double-layer, and platinic chloride). The other procedure was heating in a Bunsen burner flame. The electrochemical pretreatments were carried out under anaerobic conditions in 0.5 M sulfuric acid. For the oxidation pretreatment the platinum foil was poised at +1200 mV for about 6 min. The reduction pretreatment consisted of setting the platinum foil electrode at -200 mV for 6-15 min to obtain a steady current. The double-layer pretreatment began by potentiostati d l y controlling the foil at +400 mV for 3 min. Then the potential was stepped to +1300 mV and held 3 min, stepped to -300 mV and maintained 3 min, and finally stepped to +400 mV and held until a steady current was obtained. The objective of the double-layer pretreatment was to oxidize the surface and then reduce it so a8 to leave the metal surface in an electrochemicallynonactive state. In the platinic chloride procedure the metal foil was cleaned by electrolysis for 10 min a t -230 mV in 1 N HC1. This was followed by soaking the foil in a mixture of 5% chloroplatinic acid and 0.03% lead acetate. During this soaking pairs of platinum electrodes were galvanostatically connected. A current density of 100 mA/cm2 was imposed on the foil for six 1-min intervals with reversal of the anode and cathode after each minute (13). For the flamed pretreatment the platinum foil was held in the flame of a Bunsen burner until the foil glowed white-hot for about
30 s. The treated electrode was cooled in air or by plunging it into cold water. Preparation of Immobilized Enzyme Electrodes. The enzyme electrodes consisted of a matrix of bovine serum albumin, glucose oxidase, and catalase that was held together by crosslinking with glutaraldehyde. The electrode fabrication method is described in greater detail elsewhere (14). Briefly, the matrix was formed in a 1.43-cm-diameter by 0.38-cm-deep well in a poly(methy1 methacrylate) mold. A 1.30-cm-diameter piece of pretreated platinum foil, with attached lead wire, was placed in the bottom of the well; then the enzyme matrix solution was added and allowed to cross-link. This solution consisted of 42 mg/mL albumin, 18 mg/mL catalase, 0.014 mg/mL glucose oxidase, and 7.5 mg/mL glutaraldehyde in 0.1 M phosphate buffer, p.H 7.4. After the cross-linking reaction was completed the platinumenzyme matrix unit was removed from the well and washed for 1-2 days with 0.1 M sodium phosphate buffer, pH 7.4, to remove loosely attached enzyme. Potentiometric Measurements. The enzyme electrodes, or pretreated platinum non-enzyme control electrodes, plus a Ag/ AgCl reference electrode were placed in 0.1 M phosphate buffer, pH 7.4, in a jacketed flask maintained at 25 "C. Water-saturated oxygen was bubbled through the buffer to maintain an oxygensaturated solution. Agitation of the solution was maintained by a magnetic stirrer. When the potential between the test and reference electrodes reached a steady-state value defined as the base-line potential, sufficient buffered @-D-glucosesolution was M (5 added to obtain a glucose concentration of 2.77 X mg/100 mL). When the potential again reached a steady-state reading, another incremental glucose addition was made. This sequence was repeated several times until the final glucose conM (400 mg/100 mL) was reached. The centration of 2.22 X potentiometric results were expressed as the change in potential with respect to the base-line potential (Le., buffer only). Similar potentiometric measurements were made with bare platinumpretreated control electrodes in glucose and again with the glucose replaced by hydrogen peroxide, over a peroxide concentration range of from to M. For hysteresis experiments the concentration of glucose was changed, first in increasing and then decreasing order. RESULTS A N D DISCUSSION Platinum Pretreatment. Several albumin-enzyme matrix electrodes were made with the platinum support pretreated by each of the five methods. The electrodes were tested for their potentiometric response to glucose in oxygen-saturated solutions in 0.1 M phosphate buffer at p H 7.4 and 25 "C. When the potentials were plotted against the logarithm of the glucose concentration, the linearity was excellent over the range of 2.77 x to 2.22 x M glucose. The slopes, calculated by linear least-squares fits, are shown in Table I. For every slope the correlation coefficient was 0.98 or better. Similar potentiometric measurements were made with pretreated bare platinum electrodes in oxygen-saturated, p H 7.4, solutions containing either glucose or hydrogen peroxide. The slopes for the hydrogen peroxide results were fitted by using the data over the full range of concentrations tested (10-5-10-2 M). The slopes from the pretreated bare platinum electrode
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
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Table 11. Base-Line Potentials of Enzyme Platinum and Bare Platinum Electrodes in Buffer"
method double-layer oxidation
reduction flamed
bare platinum
electrodes, mV platinum-enzyme
362 f 35 (n = 7) 307 f 40 (n = 6) 384 f 52 (n = 6) 305 f 34 (n = 6) 375 f 17 (n = 6)
P values NS NS NS 0.003 0.005
394 f 9 (n = 4) 326 f 19 (n = 12) 374 f 24 (n = 14) 376 f 17 (n = 15) 420 f 13 (n = 3)
platinic chloride ODetermined in oxygen-saturated solution of 0.1 M sodium phosphate buffer at pH 7.4 and 25
data also are listed in Table I. The correlation coefficients ranged from 0.99, for most of the results, to 0.94. The magnitudes of the slopes ranged considerably between the three different modes of testing as well as between the different methods of pretreatment. The range of results suggested that several different electron-exchangereactions were taking place, with the overall reaction probably dependent on the method of platinum pretreatment. With the enzyme matrix present, four of the pretreatment methods gave slopes typical of one to two electron-transfer reactions. The fifth method of pretreatment, Le., platinic chloride, showed a super-Nernstian response. The reason for this is not known; perhaps mixed redox reactions or kinetic effects were present. The slopes in Table I also served as a basis for comparing the five methods of platinum surface pretreatment for enhancement of the enzyme electrode response as compared to the bare platinum response to glucose. The double-layer and platinic chloride methods of pretreatment showed the greatest differences in slopes between the enzyme and bare-metal responses t o glucose. All but the electrochemically reduced platinum gave statistically significant differences between the mean slopes for the enzyme and bare platinum electrodes. These differences in slopes probably were influenced by (1) the ratio of glucose oxidase to catalase, (2) the amount of immobilized glucose oxidase, and (3) the thickness of the enzyme membrane. Potentiometric testing of the platinic chloride electrodes was carried out by using glucose oxidase albumin matrices that were prepared without the addition of catalase. Although the platinic chloride method of pretreatment showed an enhanced response with the enzyme platinum electrode, as compared to the bare platinum electrode, the platinum black method of pretreatment was not studied extensively. This was due to the difficulty in making reproducible electrode surfaces. The relative slopes shown in Table I for the flamed method of pretreatment were somewhat surprising. In earlier studies (5) in which the platinum had been pretreated by flaming plus soaking 24 h in 20% nitric acid at room temperature, the platinum-enzyme electrodes had shown markedly greater slopes than had the bare platinum electrodes when tested in glucose. Subsequent tests showed that this difference in slopes between the enzyme and bare platinum electrodes varied between suppliers of the same grade of platinum foil. One explanation attributes the differences to changes in the surface contamination of the platinum. Recent ESCA studies (15)showed that impurities, such as silicon, in commercial-grade platinum migrate to the surface when the platinum is held a t white-hot temperature in a natural gas flame. The platinum used in the earlier studies reportedly had a typical Si level of 1-3 ppm (16). This was lower than the Si level of 6-12 ppm reported as typical for the presently used commercial-grade platinum (17). The effect of a coating of silicon oxide on the platinum surface would be a reduction in the area available for platinum oxide formation. The role of platinum oxide in the suggested mechanism of the potentiometric response is discussed later.
comparison of means,b
OC.
bStudent's t test; NS means not
The slopes for the bare platinum electrodes tested in glucose can be divided into two statistically separate groups: (1) double-layer, oxidation (mean 22 mV/decade) and (2) reduction, flamed, platinic chloride (mean 52 mV/decade). The mean slopes of the two groups differed a t the P < 0.5 level, as determined by the Newman-Keds multiple range test. The mean slopes with the enzyme present were different a t the P < 0.01 level among the double-layer, oxidation, and platinic chloride methods of pretreatment. In the present study four additional electrodes were pretreated by chemical oxidation of the platinum foils, e.g., 1.5 h in 1%aqueous hydrogen peroxide. This test was carried out because the enzyme electrodes all get exposed to hydrogen peroxide as a result of the glucose oxidase-catalyzed reaction. Therefore, it was necessary to see if hydrogen peroxide oxidation had the same influence on the potentiometric response as did oxidation by other methods. The bare platinum electrodes were tested in buffered glucose solutions at pH 7.4, 25 "C, saturated with oxygen. The mean slope of the potentiometric response vs. logarithm of the glucose concentration for the hydrogen peroxide pretreatment was -39 (f8.2) mV/decade of concentration. This was different at P < 0.05 from the slope of -23 (*6.5) mV/decade for the electrochemically oxidized platinum pretreatment (Table I). Both methods of pretreatment presumedly produced an oxidized platinum surface, but perhaps the oxide film had a different composition or different physical nature with the two pretreatment methods. The base-line potentials (Le,, the potential measured in buffer without added analyte) are listed in Table I1 for both the enzyme-platinum and bare platinum electrodes. Two statistically different groups were observed with the bare platinum: (1) oxidation, flamed and (2) double-layer, reduction, platinic chloride. All of the base-line potentials, except for the reduction pretreatment, were increased by addition of the enzyme matrix to the platinum. The increases were statistically significant only for the flamed and platinic chloride pretreatments. Whether these increases were due to modified junction potentials or to some other factor is not known. Influence of Method of Enzyme Immobilization. In an alternative method of enzyme immobilization, allylamine was coupled to electrochemically pretreated (double-layer) platinum. The terminal amine was activated by using glutaraldehyde; glucose oxidase was coupled to the aldehyde, as described elsewhere (18). For a control electrode albumin was substituted for the glucose oxidase. These electrodes were tested for their potentiometric response to buffered, oxyensaturated 0.27-22 mM glucose solutions at pH 7.4 and 25 "C. The slope with the platinum-allylamine-enzyme electrode was -45 mV/decade, similar to the mean slope listed in Table I for the electrochemical double-layer pretreatment and an enzyme matrix. The platinum-allylamine-albumin electrode results gave a slope of essentially 0. The allylamine-albumin apparently protected the platinum surface from interaction with glucose.
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
Table IV. Effect of Ionic Strength on Potentiomatic Response of Double-Layer Pretreated Bare Platinum Electrodes" ionic strength
slope,b mvldecade
0.23 0.37 0.72 0.87
v)
-34 -34 -40 -16 -25 -20
1.22
W
a
1.72 -80 I
I
I
I
r
1 1 1 1 1
I
,
,
1
,
,
1
2 x ~ ~ - 4
I
162
GLUCOSE
(M)
0.10
0.14 0.23 0.29
-17 -16 (6) -11 (3) -18 (7)
-28 -26 (7) -19 (5) -29 (9)
-38 -38 (9) -30 (7) -40 (11)
-50 -51 (8) -41 (6) -49 (14)
'In 0.1 M sodium phosphate buffer but without added salts to maintain a constant ionic strength, oxygen-saturated, 25 "C. bPotentials shown as mean and (standard deviation) with n = 3; exceDt where n = 2. no standard deviation is shown. Direction of Change of Glucose Concentration. The slopes shown in Table I were determined by increasing the glucose concentration of the test solution from the lowest to the highest value. Several tests also were run with decreasing as well as increasing glucose concentrations, to see if the curves were superimposable. The results in Figure 1show possible hysteresis occurring for the flamed pretreated platinumenzyme electrode tested in glucose. The data points in Figure 1deviate by as much as 7 mV from the linear least-squaresfitted line. Potentiometric results with individual enzyme electrodes have shown a standard deviation of f8 mV a t 5.5 X lod3M glucose (19). Thus, it is questionable whether the trends in the Figure 1results were true deviations from the fitted line or were merely due to experimental variation. Less hysteresis was observed with the double-layer pretreated bare platinum; however, no hysteresis experiments were carried out with the double-layer pretreated enzyme electrodes. pH, Ionic Strength, a n d Oxygen Effects of Potentiometric Response. Electrochemically pretreated double-layer bare platinum electrodes were tested in glucose solutions containing 0.1 M sodium phosphate buffer at several pH values. Although a pH effect was expected, none was observed (Table 111). The measured potentials were independent of pH over the range of pH 5.4-8.4 at all four of the glucose concentrations that were tested. For the Table I11 data, the ionic strengths of the 0.1 M phosphate buffers were allowed to vary with the pH. Therefore, subsequent measurements were carried out to determine the influence of ionic strength on the potentiometric response. The ionic strength of 0.1 M phosphate buffer, p H 7.4, was varied by addition of sodium chloride. The results (Table IV) were obtained with platinum
13.5
(n = 2)
(n 3)
11.3
(n = 2) (n = 3) (n = 3)
h7.1 16.4
.
Table 111. Measured Potentials vs. pH at Constant Glucose Concentration for Double-Layer Pretreated Bare Platinum Electrodes
5.4 6.4 7.4 8.4
(n = 3)
"In 0.1 M sodium phosphate buffer with added sodium chloride. Slope of potentiometric response vs. logarithm of glucose concentration; correlation coefficients 0.98-0.99 except one experiment at 1.72 ionic strength where r = 0.86.
Figure 1. Hysteresls test for enzyme electrode in 0.1 M phosphate buffer, pH 7.4, glucose solutions at 25 O C . Sequence of glucose concentrations in dlrection of arrows: (0)ascending and (A)descending concentrations. Platinum pretreated by flaming. Enzyme matrix 0.16 c m thick. Line fit by linear least squares; r = 0.98.
potentials,bmv, at the following glucose concns ionic pH" strength 2.77 mM 5.55 mM 11.1mM 22.2 mM
std dev
\ Pt
t
0,
aluconlc
I /
I:E\ glucuronlc acld
Figure 2. Summary of postulated sources of potentiometric response to glucose and hydrogen peroxide at pH 7.4. G stands for glucose and GO for glucose oxidase. See text for explanation.
electrodes cut from the same sheet of platinum foil used in the Table I11 experiments. The Table IV data show that the slopes, of the potential vs. logarithm of glucose concentration plots, were slightly less at high ionic strength. Chloride ion has been shown to influence the adsorption of glucose on platinum (20). Therefore, if adsorption of glucose on the platinum surface was a variable in the present potentiometric effect, then the Table IV results may have been influenced some by the different chloride levels. The significance of the pH results in regard to the proposed mechanism of the potentiometric effect is discussed later. The electrochemically oxidized bare platinum electrodes of Table I also were tested for their potentiometric response to hydrogen peroxide and glucose under a nitrogen atmosphere. With the electrodes immersed in buffer, purifiedwater-saturated nitrogen was bubbled through the buffer to reduce the level of dissolved oxygen. Then, deoxygenated buffered solutions of hydrogen peroxide or glucose were added, and the potential was noted. With hydrogen peroxide solutions the mean slope changed slightly from -14 f 2.2 mV/ decade under air-equilibrated conditions to -7 4.2 mV/ decade under nitrogen-saturated conditions. In glucose solutions the mean slopes were -23 f 6.5 mV/decade with air and -39 f 16 mV/decade with nitrogen. Both the glucose and hydrogen peroxide slopes were not significantly different, with and without oxygen, as judged by the t test. Previous results for the enzyme electrode showed only minor changes in potentiometric response due to a reduction in gaseous oxygen from 100% to 12% (5). Postulated Mechanism. A summary of the proposed mechanism for generation of the potentiometric response is shown in Figure 2. Three possible sources are suggested to account for the observed results. These sources are (1)hydrogen peroxide/oxygen/water redox reactions, (2)surface
*
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
reactions between hydrogen peroxide, oxygen, and platinum metal, and (3) the catalytic oxidation of glucose and corresponding reduction of the oxidized platinum surface. The potentiometric response of bare platinum immersed in aerobic hydrogen peroxide solution at pH 7.4 became more negative as the peroxide concentration was increased (see Table I and ref 7 ) . This suggests that a reductive reaction was taking place at the indicating electrode. In addition, four of the pretreatment methods gave slopes of -12 to -18 mV/decade for bare platinum exposed to hydrogen peroxide (Table I) in the absence of glucose. As a first approximation of the expected magnitude of the slope, we can consider a Nernstian system. For such a system a slope of about -15 suggests a four-electron transfer. The reduction or oxidation of hydrogen peroxide is known to undergo a two-electron transfer (see eq 4 and 5) (21). It should also be noted that HzOz =
O2+ 2H+ + 2e- E"
= -0.68 V
+
H202 2H+ + 2e- = 2Hz0 Eo = +1.77 V
(4) (5)
both reactions are pH dependent. Since the observed results indicate a possible four-electron-transfer reaction, which is pH independent, the source of the observed potential, therefore, does not seem to be simply a hydrogen peroxide bulk solution redox reaction. The results suggest that the observed potentials came from platinum surface reactions, rather than from reactions in the bulk solution. Platinum metal is known to be a catalyst for the decomposition of hydrogen peroxide to oxygen and water, possibly by a combination of eq 4 and 5. Additional hydrogen peroxide likely would act on the platinum surface to form one or more platinum oxide species (the platinum-oxygen and platinumoxide surface chemistry is discussed later). Soon after insertion of the bare platinum electrode into the solution, the concentration of hydrogen peroxide and other species at or on the platinum surface should reach a quasi steady state. The resulting potential of the platinum electrode would be due to the net effects of the several half-cell reactions. When glucose oxidase was attached to the platinum surface and the resulting electrodes were tested in aerobic glucose solution, the observed potentials again became more negative with increasing glucose levels. This was not surprising since glucose oxidase catalyzes the formation of hydrogen peroxide. Therefore, similar rections presumedly were present for both the enzyme platinum electrode tested in aerobic glucose solution and the bare platinum electrode tested in aerobic hydrogen peroxide solution. However, the predominant reaction may have been different in these two situations, as evidence by comparison of the slopes in Table I. The chemistry of the interaction of platinum metal surfaces with oxygen species in aqueous solution is a complex process that still is not well understood (9,10,22).PtOz, PtO, Pt-0, and PtOH are the major species that are thought to be involved. Pt-0 represents oxygen dissolved in the platinum lattice (22). The process of platinum oxide formation has been described (9) as initially an oxidation in which OH groups reversibly attach to the surface layer of platinum atoms. At or near monolayer coverage the outer surface atoms of platinum and OH undergo an irreversible rearrangement in which the OH groups migrate into the platinum atomic lattice. One of the oxide species could undergo reduction, driven by hydrogen peroxide, to give the observed potentiometric response. For example, the reactions listed in eq 6 or 7 (21) could be coupled with eq 4 and still give a positive value for Eo. The Pt(OH)2
+ 2H+ + 2e- = Pt + 2 H z 0 Pt2++ 2e- = Pt
Eo N
Eo = 0.98 (6)
1.2
(7)
irreversible rearrangement step could result in possible hys-
2895
teresis between platinum oxide formation and reduction processes. This in turn could account for the possible hysteresis between an increasing or decreasing glucose concentration (9). Rearrangement of the platinum atomic lattice also has been shown to occur when oxygen dissolves in the metal. The presence of dissolved oxygen has been observed to a depth of 360 A; the lattice holes have remained after the dissolved oxygen has diffused out (22). Whether or not dissolved oxygen plays any role in the mechanism of the potentiometric response observed in the present studies is not known. However, with hydrogen peroxide present the concentration of oxygen should be elevated a t the platinum surface, due to the platinum-catalyzed disproportionation of the hydrogen peroxide. These conditions would provide a driving force for diffusion of oxygen into the metal. The overall scheme for the interaction of hydrogen peroxide with the platinum surface also is summarized in Figure 2. The potentiometric response with bare platinum in glucose solution at pH 7.4 appears to occur via adsorption and oxidation of glucose (23, 24) with concurrent reduction of a platinum oxide species. The reduction step is needed to account for the more negative response in potential with increasing concentration of glucose. Heyns and Paulsen (23) described the platinum-catalyzed oxidation of glucose in alkaline solution to form the gluconate salt. When they blocked the oxidizable group a t carbon-1, then the carbon-6 position was oxidized to the corresponding glucuronic acid when placed in contact with a platinum catalyst at 60 O C (23). Others (24) showed that the carbon-1 position could be oxidized in phosphate buffer at pH 7.4 with gluconic acid as the only product. In the present work the potentiometric response of flamed bare platinum was tested in gluconic acid and again in glucuronic acid at pH 7.4 in the absence of glucose. Over the range of glucuronic and gluconic acid concentrations of to 2.22 X M, the slopes for the potentiometric 2.77 X response vs. the logarithm of concentration were -18 (h5.9) ( n = 4) mV/decade for the first acid and -22 ( n = 2) mV/ decade for the second acid. These slopes were ess$ntially the same for the two acids but were markedly different from the value of -54 mV/decade for flamed bare platinum in glucose (Table I). However, if either gluconic or glucuronic acid was formed with bare platinum and glucose, then the potentiometric response should be pH dependent. The Table I11 data do not show a pH effect, suggesting that these acids are not involved in the production of the observed potential. The involvement of these acids is shown in Figure 2 only as a distant possibility. Two other reports have cited the adsorption of glucose (25) or phosphoric acid (26)on platinum and have suggested that the adsorbed material hindered the oxidation of the platinum surface. In summary, the postulated mechanism whereby hydrogen peroxide interacts with the platinum surface to reduce one or more oxide species appears to be consistent with our data and with the results cited earlier of others. The type and amount of platinum surface oxides may vary with the method of platinum pretreatment, thus providing a rationale to explain the gross differences in potentiometric response among the methods of pretreatment. The oxidation-reduction of the platinum oxide also incorporates an irreversible step that can serve as the basis for a small degree of hysteresis observed between increasing and decreasing changes in the glucose concentration. However, our present knowledge of the platinum oxide surface chemistry, and specifically which oxide species may be involved in the potentiometric response, is too uncertain to utilize this principle for development of a practical glucose sensor. If on the other hand an electrochemical method can be developed for periodic in situ regeneration of the
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platinum surface, then a potentiometric platinum-enzyme glucose sensor might become a reality. ACKNOWLEDGMENT We appreciate the skillful assistance of L. C. Cantin in carrying out the experimental part of this work. Registry No. Glucose, 50-99-7;hydrogen peroxide, 7722-84-1; platinum, 7440-06-4;glucose oxidase, 9001-37-0;platinic chloride, 16941-12-1. LITERATURE C I T E D Solsky, R. L. CRC Crlt. Rev. Anal. Chem. 1083, 14, 1-52. Ryan, M. D.; Wilson, G. S. Anal. Chem. 1082, 54, 20R-27R. Wingard, L. B., Jr. Fed. Proc., Fed. Am. SOC. Exp. 1083, 42, 288-291. Wingard, L. B., Jr.; Ellls, D.; Yao, S. J.; Schiller, J. G.; Liu, C. C.; Wolfson, S. K., Jr.; Drash, A. L. J. Solid-Phase Blochem. 1070, 4, 253-262. Wlngard, L. B., Jr.; Schlller, J. G.; Wolfson, S. K., Jr.; Liu, C. C.; Drash, A. L.; Yao, S. J. J. Biomed. Mater. Res. 1070, 13, 921-935. Llu, C. C.; Wingard, L. B., Jr.; Wolfson, S.K., Jr.; Yao, S. J.; Drash, A. L.; Schiller, J. 0. J . Nectroanal. Chem. 1070, 104, 19-26. Wlngard, L. B., Jr.; Castner, J. F.; Yao, S. J.; Wolfson, S. K., Jr.; Drash, A. L.; Liu, C. C. Appl. Biochem. Biotechnol. 1084, 9, 95-104. Wingard, L. B., Jr.; Llu, C. C.; Wolfson, S. K., Jr.; Yao, S. J.; Drash, A. L. Diabetes Care 1082. 5 . 199-202. Hoare, J. P. I n "Encyclopedia of Electrochemistry of the Elements"; Bard, A. J., Ed.; Marcel Dekker: New York, 1974; Vol. 2, pp 2 10-238. Angersteln-Koziowska, H.; Conway, B. E.; Sharp, W. B. A. Electroanal. Chem. Interfacial Electrochem. 1073, 43, 9-36. Rasmussen, K. E.; Albrechtsen, J. Histochemistry 1074, 38, 19-26.
(12) Gordon, A. J.; Ford, R. A. "The Chemist's Companion"; Wlley: New York. 1972: 438. -(13) Marincic, L.; Soeldner, J. S.; Coiton, C. K.; Giner, J.; Morris, $. J . Nectrochem Soc 1070, 726, 43-49. (14) Wingard, L. B., Jr.; Cantin, L. A.; Castner, J. F. Blochim. Blophys. Acta 1983, 748. 21-27. (15) Proctor, A.; Castner. J. F.; Wlngard, L. B., Jr.; Hercules, D. M. Anal. Chem., submitted. (16) Rowe, M., Johnson, Matthey Co., Oct 1983, personal communicatlon. ( 1 7) Zysk, E., Engelhard Industrles, Sept 1983, personal communication. (18) Castner, J. F.; Wlngard, L. E., Jr. Blochemistry 1084, 23, 2203-2210. (19) Wingard, L. B., Jr.; Castner, J. F. I n "Enzyme Engineering"; Ghlbata, I., Fukui, S., Wingard, L. B., Jr., Eds.; Plenum Press: New York, 1982; VQI. 6,pp 415-416. (20) Giner, J.; Malachesky, P. Proceedings of the Artiflclal Heart Program CQnference, June 9-13, 1969, Washington, DC. (21) Dobos, D. "Electrochemical Data"; Elsevier: Amsterdam, 1975; pp 259-260. (22) Hoare, J. P. Nectrochim. Acta 1081, 2 6 , 225-232. (23) Heyns, K.; Paulsen, H. I n "Newer Methods of Preparative Organlc Chemistry"; Forest, W., Ed.; Academic Press: New York, 1963: Vol. 2, pp 303-335. (24) Rao, M. L. 8.; Drake, R. F. J. Nectrochem. SOC. 1060, 116,
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(25) deMeie, M. F. L.; Videia, H. A.; Arvia, A. J. Bloelectrochem. Bloenerg. 1082, 9 , 469-487. (26) Hsueh, K. L.; Gonzaiez, E. R.; Srinivasan, S. J. Electrochem. SOC. 1084, 131, 823-828.
RECEIVED for
review January 3,1984. Accepted July 31,1984. This work was supported by Grant lROl AM26370 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases of NIH.
Model of a Two-Substrate Enzyme Electrode for Glucose J o h n K. Leypoldt' a n d David A. Gough*
Department of Applied Mechanics and Engineering Sciences, Bioengineering Group, University of California, S a n Diego, La Jolla, Californiq 92093
A mathematlcai model of a two-substrate enzyme electrode Is presented. The electrode is a glucose-specific sensor In which enzymes are lmmobiilzed wlthln a membrane attached to an electrochernlcal sensor for oxygen! the cosubstrate of the enzyme reaction. The model presented here focuses on the reaction and dlffuslon phenomena that occur wlthln the enzyme membrane. I t Is shown that when the membrane has the approprlate permeability to both substrates and the enzyme reaction Is dlffuslon-llmited, the difference In the enzyme electrode current from that of an oxygen-sensitive reference electrode Is proportional to the bulk glucose concentration, irrespective of the ratlo of substrate concentrations In the bulk solutlon. Moreover, when the bulk oxygen concentration Is low, It may be advantageous to immobilize Ilmited amounts of enzyme In the membrane in order to control the range of glucose detectablllty. These predictions differ notably from those of a one-substrate model. The modellng results are summarlred concisely in three simple figures and verified by comparison with experimental observations. A method for identlfying the llmitlng substrate Is described and guldellnes are glven for sensor design.
"Enzyme electrodes" show promise for a variety of novel analytical applications (1-3). Broadly defined, these are Current address: Department of Medicine, V.A. Medical Center, San Diego, CA 92161.
chemical-specific sensors in which biological catalysts such as immobilized enzymes, immobilized cells, or layers of tissue are coupled to electrodes that are sensitive to a product or cosubstrate of the enzyme reaction. Early investigations with the type of enzyme electrode that requires only a single substrate ( 4 , 5 ) have demonstrated that this type of sensor can be used in some cases to analyze chemicals with little or no sample preparation, a feature that is not only convenient, but particularly advantageous for monitoring directly in living tissues. Many enzymes, however, require a cofactor or cosubstrate in order to be catalytically active. One approach to providing this reactant is to simply add it to the sample in sufficient amounts. Unfortunately, this eliminates the advantage of no sample preparation. In other situations where the cosubstrate is endogenous to the sample, an alternative is to design the sensor for aperation under existing cosubstrate concentrations. This may require, for example, specifying the immobilized enzyme content or tailoring the membrane permeability to the respective substrates. In order to proceed rationally with this approach, it is necessary to have a working model of the two-substrate sensor. Mathematical models of one-substrate enzyme electrodes proposed by a number of investigators (6-9) have been reviewed elsewhere ( I , IO) and have some relevance to twosubstrate enzyme electrodes. These models show that imposing diffusional limitations on the enzyme reaction can lead to improvements in sensor performance in various ways, such as, increasing the sensitivity to the substrate ( 6 ) ,extending the range of linearity (7,8), decreasing the sensitivity to en-
0003-2700/84/0356-2896$01.50/0 0 1984 American Chemical Society
