Figure 7 represents current-concentration data for NADH in pyrophosphate buffer. The fall off in signal a t high NADH concentrations is believed to be due to electrode fouling. Enhancement of the NADH oxidation current and increased sensitivity is achievable by increasing the rotational speed of the electrode, which does not enhance the transport-independent background current. CONCLUSIONS The advantages of steady-state voltammetry have been compared with conventional scanning voltammetry. While the basic principles regarding steady-state voltammetry have been known to electrochemists for many years ( I ), the benefits of SSV as applied to solid electrodes have not been generally recognized. SSV has the distinct advantage of substantially lower background currents, which in turn permit the examination of very low concentrations of electroactive material. In addition, the low current requirements of SSV significantly decrease iR drop through the
cell, reducing the need for a more complex three-electrode system and supporting instrumentation. When applied to NADH, the use of SSV prevents fouling of the glassy carbon surface. In addition, SSV gives a current limited plateau on the current-voltage curve for NADH, not normally observable with conventional scanning techniques. SSV also shows considerable promise in the study of electrode surface reactions: a t very low current densities (below 100 nA/cm2), SSV may permit the most direct quantitative observations yet made of oxidative or reductive changes in the electrode surface that are caused by changes in applied potential.
RECEIVEDfor review April 30, 1974. Accepted August 1, 1974. This work was supported in part by j’unds from the Wisconsin Alumni Research Foundation (University of Wisconsin-Madison Graduate School Project No. 140323) and in part by the National Science Foundation (Grant No. 40694x1.
Improved Penicillin Selective Enzyme Electrode L. F. Cullen, J. F. Rusling, Arthur Schleifer, and G. J. Papariello Analytical and Physical Chemistry Section, Wyeth Laboratories, lnc., P.O. Box 8299, Philadelphia, Pa. 79 707
From a systematic evaluation of several basic penicillinase electrode configurations, a penicillin selective enzyme electrode has been developed with characteristics superior to an earlier design. In preparing this improved electrode configuration, penicillinase is immobilized by adsorption onto a fritted glass disc which is affixed to the end of a flat-surface pH glass electrode. The hydrogen ions produced by the penicillin-penicillinase enzymatic reaction are sensed by the glass electrode. The steady-state potentiometric response developed is proportional to the logarithm of the intact penicillin concentration. In designing this analytical system, cliemical and geometrical parameters related to the immobilized enzyme-substrate reaction were considered and optimized. Data collected on a variety of penicillin species demonstrate the versatility of the analytical technique. The procedure is applicable to the analysis of penicillins in the 3.5-1100 pg/ml range with a method precision of f3% at the 100 pg/ml level. In exemplifying the analytical utility of this electrode configuration, the analyses of penicillin capsule formulations and fermentation broths are described.
In a recent publication, this laboratory reported on the successful development of an electrode which is specific for the analysis of penicillins ( I ) . This electrode was based on the concept of an enzyme electrode first introduced by Clark and Lyons ( 2 ) . Since that time, a number of other immobilized enzyme electrodes have been developed; e g . , urea ( 3 ) ,amygdalin ( 4 ) , glucose ( 5 ) ,and L-amino acid (6) G. J. Papariello, A . K. Mukherji, and C. M. Shearer, Anal. Chern., 45, 790 (1973). L. C. Clark and C. Lyons, Ann. N.Y. Acad. Sci., 102, 29 (1962). G. G. Guilbault. G. Nagy. and S. S. Kuan. Anal. Cbirn. Acta., 67, 195 (1973).
electrodes. In this penicillin selective enzyme electrode, penicillin P-lactamase (penicillinase) is immobilized in a thin membrane of polyacrylamide gel molded around and in intimate contact with a hydrogen ion glass electrode. I t was reported that hydrogen ions produced from the hydrolysis of penicillin by B-lactamase would be selectively detected by the p H sensitive glass electrode w e d as the sensor. Unfortunately, subsequent work has demonstrated that the character of the glass electrode is changed in this enzyme electrode so that the glass sensing membrane is no longer sensitive only to hydrogen ions. Many monovalent cations could produce a change in potential with this electrode. A study to obtain an understanding of this electrode’s broad cation sensitivity led to the development of a new electrode configuration. In this improved electrode, penicillinase is immobilized by adsorption onto a fritted glass disc which is then affixed to the end of a flat-suyface pH glass electrode. This improved electrode is sensitive to changes in pencillin concentration and insensitive to cation concentration effects. Besides being more selective, this new electrode is easier to prepare than the previously reported electrode. I t is more Nernstian in behavior, more stable, more reproducible, more sensitive, and has a longer life than the previous electrode. EXPERIMENTAL Apparatus. Polyacrylamide Gel Penicillinase Electrode. A detailed description of the preparation of the polyacrylamide gel penicillinase electrode and of experimental condi1;ions for potential measurements used in this investigation are described by Papariello et ul. ( I ). Dialysis M e m b r a n e Electrode Configurations. [n the prepara(4) R. A . Llenado and G. A . Rechnitz, Anal. Chem., 43, I457 (1971) (5) S. J. Updike and G. P. Hicks, Nature (London), 214, 9136 (1967). (6) G. G. Guilbaul! and E. Hrabankova, Anal. Lett., 3, 53 ( 1970).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 13, NOVEMBER 1974
1955
NPII
i-
TIN I,
ri
L
Penicillinare rolutlon' 12 I
Id
'Oialpir
membranes
""llrm 3 " I I
Figure 1. Dialysis membrane electrode configurations
Figure 3. Glass disc penicillin selective enzyme electrode configuration
Figure 2. Schematic wiring diagram of voltage offset switch B1. 1.35-V dc. mercury battery; R I , I-Mohm resistor: R2-Rl0. resistors trimmed to 741 ohms: SI, single pole 10-position rotary switch: T-5 and T-6. recorder input terminals (1-mV full-scale sensitivity); J-303 and J-304, pH meter output terminals (10-mV full scale output)
tion of the dialysis membrane electrode geometries (Types I, I1 and 111), as shown in Figure 1, A. H. Thomas dialysis tubing with a 0.001-inch wall thickness was employed. This membrane has a molecular weight exclusion of 12,000 (7). Bacillus cereus 569 19-lactamase has a reported average molecular weight of 31,500 (8). Ruhber O-rings were used to anchor the dialysis membrane on the flatsurface pH electrode. Glass Disc Penicillinase Electrode. In the glass disc penicillinase electrode configuration, a Markson Science Inc. flat-surface p H electrode (Model No. 1205) was employed as the hydrogen ion sensor. In preparing this electrode configuration, a course porosity fritted glass disc (Kontes Glass Co., No. K-952050-0012, 14-mm o.d. X 2.3-mm thickness) was designed to completely cover the electrode sensing surface by shaping the disc to tightly fit a cylindrical polyethylene sleeve (11-mm i.d. X 5.5-mm height). Disc thickness was reduced to 1.3 f 0.1 mm. Disc reduction was carried out by hand sanding with 180 mesh silicon carbide paper with disc measurements made by a micrometer. The disc was immersed in water on an ultrasonic vibrator and vibrated for 5 min to remove adhering silicon carbide. I t was then dried 1 hr a t 105 "C and inserted into the base of the polyethylene sleeve. Six \'-shaped grooves were cut in the sleeve to enhance the adhering properties of the sleeve on the electrode. In activating the fritted glass disc, a solution containing 40 mg of Penicillinase A (B. cereus 569 0-lactamase, 5000 units/mg, from Riker Laboratories) per ml H20 was prepared. Under ambient conditions, 1 ml of the enzyme solution was added dropwise to the disc-sleeve assembly which was placed with the disc end down in a 2.0-ml capacity wide-mouth Teflon bottle cap. Thirty minutes after initial 0-lactamase addition and again a t the 1-hr and 1.5-hr (7) Dialyzer Tubing, Tech. Bull. No. DU-29-10M-8-71, A. H. Thomas Co., Philadelphia, Pa., 19105. (8) N. Citri, in "The Enzymes." 3rd ed., Vol. 4, P. D. Boyer, Ed., Academic Press, New York, N.Y., 1971, p 23.
1956
intervals, this Penicillinase A solution is recycled through the fritted glass disc in the same manner as previously described. After standing an additional 2 hr, the disc-sleeve assembly was thoroughly washed with water and affixed onto the flat-surface p H electrode. The electrode was then equilibrated in a 0.7% phosphate buffer ( p H 6.9 i 0.1) a t 5 "C for a period of not less than 24 hours prior to its initial use. When not in use, the electrode was stored a t 5 "C in the 0.7% phosphate buffer to preserve enzyme activity. Potential and p H measurements were made using a Corning Model 12 Expanded Scale p H meter. A standard, combination p H electrode (A. H. Thomas, Model No. 4094-L15) was used t o make the p H measurements. The enzyme electrode and p H combination electrode were connected to the p H meter using a Beckman Electrode Switch accessory (Model No. 97200). The potentiometric output of the enzyme electrode was continuously displayed on a Beckman 10-inch Linear Potentiometric Recorder (Model No. 100500). The pH meter and recorder were interfaced through an offsetting voltage switch device, as shown in Figure 2. This device significantly enhances method precision by amplifying the 40.5-mV resolution characteristics of the Corning Model 1 2 pH meter to a f O . l mV/0.25 cm resolution capability on the recorder. A Beckman Permaprobe Solid-state electrode (Model No. 39406) served as the reference electrode for the penicillinase electrode. In preparing a sample cell, the stem of a Sherwnnd type 5.5mm polypropylene funnel was removed to permit the insertion of the solid-state reference electrode. With the reference electrode inverted, the funnel is secured to the electrode with Teflon tape, forming a sample cup which can accommodate a maximum solution volume of 12 ml. A small motor-driven stirring rod (2500 rpm) and glass disc penicillinase electrode are immersed in the penicillin sample solution. The complete disc-enzyme electrode configuration is illustrated in Figure 3. Reagents. Stock solutions (0.1M) of sodium ampicillin, potassium cyclacillin (WY-4508),potassium penicillin G (potassium benzylpenicillin), potassium penicillin V ( potassium phenoxymethyl penicillin), sodium dicloxacillin monohydrate, potassium phenethicillin, and benzylpenicilloic acid were prepared. These stock solutions were prepared on the day of use. T o obtain the solution with the penicillin concentration desired, these stock solutions were diluted with water or 0.01M KC1 solution in the appropriate manner. Stock solutions (0.1M) of Analytical Reagent grade NaC1, KC1, KBr, NHdC1, (NH4)&04, CaS04, MgC12, and CaC12 were prepared and diluted to the appropriate concentration in water. In studying the polyacrylamide gel penicillinase electrode and dialysis membrane electrode geometries, stock solutions were diluted with water and adjusted to a p H of 6.4 by use of dilute hydrochloric acid or dilute sodium hydroxide as described hy Papariello et al. ( I ) . In using the glass disc electrode. the penicillin solu-
ANALYTICAL CHEMISTRY, VOL. 46, NO. 13, NOVEMBER 1974
-\ I
I 1 I
1M)-
-
-
120-
E f
W Reccr Cer
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p? Cleter
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Electrwe Shlltch Accessor)
w
80
Enzyme
-
Electrode Configuration 40 -
Figure 4. Enzyme electrode analytical system
tions were diluted to the desired concentration with a 0.01M KC1 solution and adjusted t o a pH of 6.9. P r o c e d u r e f o r Glass Disc Penicillinase Electrode. A diagram of the enzyme electrode analytical system is shown in Figure 4. The pH combinaticln electrode is inserted into a beaker containing 50-ml of sample solution, which contains penicillin, ideally, in the concentration range of to 2.5 X 10-4M. The solution is magnetically stirred and adjusted to p H 6.900 f 0.005 with dilute sodium hydroxide or dilute hydrochloric acid using the p H meter in the expanded scale mode. A 3.0-ml aliquot of this p H adjusted sample solution is pipetted into the sample cell containing the enzyme electrode. Switching the pH meter into the mV mode, potential measurements are obtained until a steady-state potential is recorded. T h e steady-state potential is read, and the penicillin concentration is determiced from a calibration curve of enzyme electrode potential response L ' S . log of penicillin concentration from reference standard solutions measured in the same manner as the sample solution. On aging of the enzyme electrode, the slope from the penicillin calibration curve gradually decreases. When this slope decreases to approximately 43-46 mV per decade, a fresh penicillinase activated glass disc must be substituted for the used disc. After sample measurement, the sample solution is aspirated to waste and the cell flushed 3 times with a 0.01M KC1 wash solution, previously adjusted to p H 6.9 f 0.01 with dilute sodium hydroxide. The enzyme electrode is washed free of residual penicilloic acid by immersing the electrode in 3-ml portions of the 0.01M KC1 solution in the sample cell. T h e electrode is washed until the monitored potential returns t o a millivolt response equivalent t o the 0.01M KCI wash so1ut.ion. Washout time is less than 6 minutes a t penicillin concentrations of 2.5 X 10-4M or below. All measurements were made a t ambient room temperature with stirring, except when otherwise noted.
R E S U L T S AND DISCUSSION Polyacrylamide Gel Penicillinase Electrode. A major consideration in the design of an immobilized enzyme electrode from an analytical viewpoint is substrate-specificity. The published polyacrylamide gel penicillinase electrode system was reported to selectively sense the hydrogen ions produced a t the membrane by the enzyme catalyzed hydrolysis of the penicillin to the corresponding penicilloic acid. The electrode potential ( E )is described as being logarithmically related to the penicillin concentration in the following manner: nm
Equation 1 predicts a slope of 59 mV per decade change a t 25 "C in penicillin concentration when log (Penicillin) is plotted us. the observed potential. In studying the geometry and configuration of this electrode in an attempt to improve its reproducibility, the electrode was found to have significant sensitivity to changes in concentration of monovalent cations. A polyacrylamide gel electrode prepared without penicillinase present, however, did not show sensitivity to changes in salt concentration. The potentiometric response of the published electrode with penicillinase present to Na+, K+, Ca2+, and sodium
01
I 10
I 10'
I
I
I
103
104
10 =
108
10'
Cation Concentration M
Figure 5. Response of the polyacrylamide gel penicillinase electrode to cations
+--+
HCI, 0- - -0 Sodium ampicillin, A--A NaCI, 0- - -0 KBr, - -7 CaCI2, A- - -A HCI In system without penicillinase
B--B KCI, 7in gel layer
200
-
-- 160-
f
w 120-
80
-
Figure 6. Response of Type I dialysis membrane electrode configuration to cations
0--0 Sodium ampicillin in 10-'M NaCl solution, B- - -B Sodium ampicillin in lO-'M NaCl solution, 0--0 Sodium ampicillin in HzO, A- - -A NaCI, 0- - - 0 HCI
ampicillin under the prescribed experimental conditions is presented in Figure 5 . Ammonium ions produced a response similar to the above monovalent cations while Mg*+ behaved in a manner similar to Ca2+. Responses to hydrogen ion concentrations by the polyacrylamide electrode systems prepared with and without penicillinase in the gel layer are also shown. Figure 5 reveals that the potential responses and resulting slope for sodium ampicillin (slope: 54 mvldecade) are only slightly greater than those obtained for Na+ (slope: 48 mvldecade) over an equivalent molar concentration range. This indicates that the electrode response for the sodium ampicillin is due mainly to the presence of Na+ ion rather than as a result of the enzymatic hydrolysis of the penicillin. Further, there is a drastic decrease in the hydrogen ion sensitivity of the polyacrylamide gel electrode system with penicillinase in the immobilizing layer. A change in electrode potential of 59 mV would be expected (Equation 1) per decade change in hydrogen ion concentration a t the membrane-solution interface. However, starting a t the prescribed initial p H of 6.4, one obtains a non-Nernstian re-
ANALYTICAL CHEMISTRY, VOL. 46, NO. 13, NOVEMBER 1974
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1957
150
*.
I
I
I
I
I
150
Figure 7. Response of Type II dialysis membrane electrode configuration to cations
-+
6- HCI (slope: 59 mV/decade), A--A Sodium ampicillin in 10-'M NaCl solution (slope: 53 mvldecade), 0 - - - a Sodium ampicillin in H20 (slope: 46 mV/decade), .--U NaCl (slope: -2 mV/decade)
-10'
(9)H. Nilsson, A. C. Akerlund. and K. Mosbach, Biochim. Biophys. Acta., 320,529 (1973).
1958
'
I
1
10
10 Cation Concentratlo" M
I
I
10 '
10
Figure 8. Response of Type 111 dialysis membrane electrode system
to cations, penicillinase, units/25 rnl in brackets and slope, mV/decade in parentheses
.---.
0--0
sponse to hydrogen ions as seen in Figure 3. Thus, the potentiometric response measured by this electrode is due only in part to the hydrogen ions from the hydrolysis product with a significant contribution from the sodium cation of the penicillin salt. Dialysis Membrane Electrode Configurations (Types I, 11, a n d 111). T o obtain a further understanding of the effects of immobilized penicillinase a t the surface of the glass electrode, basic penicillinase electrode assemblies (Figure 1) were designed for systematic evaluation. The Type I electrode configuration behaved in essentially the same manner (Figure 6) as the published polyacryiamide gel electrode (Figure 5 ) ; that is, it was sensitive to all monovalent cations. When one tests the behavior of a Type I configuration without penicillinase present, one finds that it exhibits characteristic glass electrode Nernstian response for hydrogen ions and no response with other cations. The Type I electrode configuration is similar in design to the liquid enzyme layer penicillinase electrode recently described by Nilsson et al. (9). This electrode configuration was also sensitive to monovalent cation concentration effects. Millivolt responses with the Type I1 electrode configuration for Na+, H+, and sodium ampicillin concentrations are presented in Figure 7 . Unlike the two previously described electrode systems, this configuration with the dialysis membrane between the enzyme phase and glass electrode shows no response to Na+ and a high sensitivity to H+ between pH 6.4-3.4 (slope: 59 mV/decade). Further, a nearNernstian response (slope: 53 mvldecade) is obtained for sodium ampicillin solutions prepared in 0.1M NaC1. Prohibitively long response and washout times, however, preclude the use of this liquid enzyme layer electrode as an effective analytical device. niently study the effect of changes in penicillinase concentration on the properties of the p H glass electrode. The response of a Type I11 electrode to sodium ions a t various concentration levels of penicillinase is shown in Figure 8. At penicillinase concentrations of 10 X lo4 units and 15 x IO4 units/25 ml, a significant sensitivity to sodium ion is observed, whereas a t a low level of penicillinase concentration (2.5 X lo4 units/25 ml), there is little or no sodium ion sensitivity. A t this low enzyme concentration, the near
I 10
+-
Sodium ampicillin [2.5X lo4] (54), - - 6 NaCl [15 X lo4] (42). NaCl [lo X IO4] (38),A- - -A NaCl [2.5X lo4] (4)
Nernstian response (Le., slope: 54 mV/decade) to sodium ampicillin over the range of to 2.5 X IO-ZM can be attributed to enzymatic hydrolysis. The data collected with these three electrode configurations have helped to explain the behavior of the polyacrylamide gel penicillinase electrode. The data clearly demonstrate that the penicillinase concentration on the sensor surface of the pH glass electrode decreases its sensitivity toward hydrogen ions in the p H range 6.4-4.4 and drastically affects the electrode behavior to monovalent cations. Such an effect may be due to the adsorption of the penicillinase onto the glass surface. I t has been reported in the literature that penicillinase can be adsorbed on fine-mesh glass powder and that this property is used in the purification of this enzyme (10, 111. In all probability, this penicillinase adsorption onto the glass electrode sensing surface takes place during polymerization of the enzyme-gel solution or by leaching of the penicillinase from the polyacrylamide membrane during its initial equilibration period. This increased cation sensitivity and decreased hydrogen ion sensitivity was observed by Ling in studying the behavior of an oxidized collodion coated-glass electrode (12, 13). Ling found that the selectivity of the p H glass electrode is altered by a coating of oxidized collodion. This coated electrode was sensitive to monovalent cations (&., Li+, Na+, NH4+,Rb+, Cs+) but insensitive to divalent cations such as Mg2+ and Ca2+.Ling describes this change in electrochemical sensor properties through a mechanism of cation adsorption to fixed negative charges (carboxylate groups) due to the oxidized collodion on the glass membrane surface. The electrode potential, E, can be related to the concentration, C,+, of monovalent cations by the equation:
E = Eo
+
TRITn [ F K i ( C i + ) l 1'
L
,
t=t
-I
where K i is the adsorption constant of the i t h monovalent cation (of m total species) on the surface negative sites. Bacillus cereus 569 P-lactamase, with an isoelectric p H of 4.9, also contains a t a pH of 6.4 a significant level of fixed negative charges from ionized glutamic acid and as(10)N. Citri, Biochim. Biophys. Acfa., 27, 277 (1958). (11) M. Kogut. M. R . Pollock, and E. J. Tridgell, Biochem. J., 62,391 (1956). (12)G. N. Ling, in "Glass Electrodes for Hydrogen and Other Cations," G. Eisenman, Ed., Marcel Dekker, New York, N.Y., 1967,p 284. (13)G. N. Ling, J. Gen. Physiol. Suppl., 43, 149 (1960).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 13, NOVEMBER 1974
220,
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1
-
a180
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-
.-
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Flgure 9. Typical calibration curves for different penicillin species, slope, mV/decade in parentheses A- - -A Potassium cyclacillin (56), 0--0 Sodium dicloxacillin monohydrate (57), 0- - -0 Potassium phenethicillin (56),A--A Sodium ampicillin (57), 0- - -0 Potassium penicillin V (58), W--M Potassium penicillin G (58), NaCl ( as an Analytical Technique. In the field of pharmaceutical analysis, an important consideration is the development of a stability-indicating procedure which measures the level of intact drug in the presence of its decomposition products. As described by Papariello et al., the specificity of the electrode system is a result of the inherent selectivity of penicillinase for the penicillin substrate ( I ) . Penicilloic acid present in the sample prior to analysis has no effect on the enzyme electrode voltage measured. T o demonstrate this experimentally, a solution was prepared to contain benzylpenicilloic acid and potassium henzylpenicillin (penicillin G ) both a t a concentration of 2.5 X 10-4M. The observed potential did not differ from that obtained with a 2.5 X 10-4M solution of potassium benzylpenicillin. I t is well documented that the major route of penicillin decomposition is through the formation of penicilloic acid (23).Thus, the enzyme electrode is applicable to stability studies where partially degraded systems are measured. As described previously (Figure 9), the electrode system can be applied to the analysis of penicillin solutions containing 3.5 to 1100 pg per ml. Consequently, the method offers a tenfold increase in sensitivity over the two most widely used chemical techniques for penicillin analysisuiz., the iodometric titration method and the hydroxamic acid colorimetric procedure. T o demonstrate the analytical utility of this electrode configuration, the technique was used to analyze successfully commercial penicillin capsule formulations and filtered fermentation broths. In penicillin broth analysis, comparative data between the established iodometric titration procedure and penicillin enzyme electrode technique (21) W. J. Blaedei, T. R. Kissel, and R. C. Boguslaski, Anal. Chem., 44, 2030 (1972). (22) Penicillinase A, Tech. Buii. No. PAPI-V5, Riker Laboratories, Inc., Northridge, Calif. 91324. (23) M. A . Schwartz and F . H. Buckwalter, J. Pharm. Sci., 51, 11 19 (1962).
Table IV. Results of Analysis of 100 pg/ml Sodium Ampicillin Solution Using the Glass Disc Penicillinase Electrode Found, iig/ml
Day
Electrode 1
Electrode 2
Electrode 3
1
103 102 99 98 104 96 102 95 106 102 99 100 103 99 97 102 96 103 106 101
103 97 101
99 100 104
103 96 101
96 103 101
104 100 96
103 103 97
100 101 102
106 97 102
96 98 102 100
98 98 100 100
13.27
i.2.77
~3.07
2
3
4
5
Average Relative standard deviation
agreed within 2%. For a 250-mg ampicillin capsule formulation, both the precision and accuracy of this technique were determined by adding known quantities of ampicillin to the capsule excipient mixture and measuring the percentages recovered. Ten replicate assays produced a relative standard deviation of f3.2% with a recovery of 100%of the theoretical amount present. In demonstrating the precision of the electrode over a five-day period, three different electrodes were prepared and used to assay a 100 pg/ml solution of sodium ampicillin. Calibration curves for all three electrodes were prepared on a daily basis. The results of this study are presented in Table IV. The data illustrate the analytical usefulness of this electrode system. T o establish stability of the enzyme tlectrode, slopes were determined a t appropriate intervals over a four-week observation period. During this time, only a slight decrease in slope (i.e., 2-3 mV/decade) was found. This slight change does not affect the practical analytical value of the electrode. Further developmental studies in this laboratory are now being directed toward extending the useful lifetime of the enzyme electrode. The covalent coupling of' penicillinase t o the fritted glass disc via functional groups of the enzyme protein that are not essential to enzyme activity may offer an even more stable enzyme layer for this electrode. RECEIVEDfor review April 10, 1974. Accepted July 22, 1974.
ANALYTICAL CHEMISTRY, VOL. 46, NO. 13, NOVEMBER 1974
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