96
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
Enzyme Collagen Membrane for Electrochemical Determination of Glucose Daniel R. Th6venot" and Robert Sternberg Equipe de Bio6lectrochimie et d'Analyse du Milieu, Laboratoire d' Energetique Biochimique, Universitd Paris Val de Marne, Avenue du General de Gaulle, F940 70, Cr6teil C6dex, France
Pierre R. Coulet, Jean Laurent, and Danible C. Gautheron Laboratoire de Biologie et Technologie des Membranes du CNRS, Universite Claude Bernard, Lyon I , 43, boulevard du 1 1 novembre 19 18, F6962 7, Villeurbanne, France
A new trace glucose analyzer has been designed using electrochemical sensors. The differential device includes (a) a glucose sensor consisting of a modified gas electrode in which the pH detector was replaced by a platinum disk and the porous film by a collagen membrane on which 0-o-glucose oxidase has been covalently bound after an acyl-azide activation process; (b) a compensating electrode mounted with a nonenzymatic collagen membrane. After injection of a glucose containing sample into the reaction vessel, where the probes are immersed, an anodic current is detected at the enzyme working electrode. Current outputs of both electrodes are subtracted and twice differentiated; a steady state is reached and the stationary and dynamic responses are recorded. Both responses are proportional to glucose concentration in the 0.1 pM-2 mM range, and the reproductibility was found to be better than 2 % using these conditions. The extreme sensitivity exhibited by our system, Le., 10 nM, is better than previously reported data by 3 orders of magnitude, and is very favorable for trace glucose assays in food and biological samples.
T h e association of electrochemical sensors with enzymatic films, leading t o t h e so-called enzyme electrodes, is a rapidly expanding analytical method ( I , 2). In the presence of glucose oxidase (GOD), glucose is oxidized according t o t h e reaction: 0-D-glucose
+ 0,
-
GOD
gluconic acid
+ H,02
(1)
H202 formation is usually followed by reaction with a chromogenic precursor in the presence of peroxidase, including a necessary incubation time before measurement. An alternative method is the anodic oxidation of H202:
-
H202
O2 + 2e-
+ 2Hf
(2.1
leading t o t h e design of glucose sensors (3, 4 ) . Furthermore, e n t r a p m e n t of GOD in a n immobilized form avoids t h e drawbacks encountered when using a n enzyme solution ( I , 4). In t h e present work, reconstituted collagen membranes having undergone an acyl-azide activation process and bearing covalently bound GOD were associated to the amperometric detection of H z 0 2for t h e determination of traces of glucose ( 5 ,6 ) .
EXPERIMENTAL Instrumentation. The glucose sensor consisted of two electrodes; both electrodes contained a platinum disk and a collagen membrane maintained in close contact by a screwed cap (Figure 1). Common auxiliary electrodes (A) were platinum wires 0003-2700/79/0351-0096$01 O O / O
and references (R) were Ag/AgCl. Electrodes E, and E? were filled with 0.2 M acetate buffer, 0.1 M KCI. pH 5.6, and mounted with both a 6'-u-glucose oxidase (GOD, EC 1.1.3.4) collagen membrane and a nonenzgmatic one, respectively. The electrodes were either dipped into the buffer solution or mounted with flowthrough caps as shown on Figure 1. In the latter case, the flow rate was constant and equal to 0.7 mL/min. A potential difference of 650 mV was maintained between the platinum working electrodes W, and Wz, and a reference R, by a potentiostat. Current outputs of W 1and iV2 were first subtracted, and then twice differentiated with a time base of 1 s (Figure 2 ) . Thus, 4 different current vs. time curves were available and usually recorded after a glucose pulse. Electronics were supplied by Solea Tacussel Willeurbanne. France) and consisted of a Deltapol differential current amplifier, PRG5 potentiostat, a Minisis millivoltmeter, and a Derivol derivating amplifier. Recorders were Solea Tacussel EPL 2, with a T V 11 GD plug-in unit and three-traces Linear 395. Unless otherwise mentioned, the temperature of all solutions wa5 carefully thermostated at 30.0 f 0.1 "C (Colora cryothermostat IVK 5 DS). Solutions and Reagents. Insoluble films of highly polymerized reconstituted collagen (20 cm wide) were a gift of the Centre Technique du Cuir (Lyon, France). Their thickness is about 0.1 mm in dry state and from 0.3 to 0.5 mm when swollen. They do not need to be tanned and can be used several years without damage (7, 8). Lyophilized .I-D-glUcOSe oxidase (GOD, EC 1.1.3.4) grade I, peroxidase (POD, EC 1.11.1.7), beef liver catalase (EC 1.11.1.6), and 2,2'-azinodi(3-ethylbenzthiazoline sulfonate) (6) (ABTS) were supplied by Boehringer, France. Unless otherwise mentioned, all chemicals used were reagent grade. The stock solution of 0.1 M glucose was allowed to mutarotate at room temperature at least 3 h before use, and was stored a t 4 "C. Both electrodes were filled and dipped in 0.2 M acetate buffer-4.1 M KC1 solutions (pH 5.6). The stock solution of 0.1 M H2O2was frequently assayed by titration with 0.1 K Ce(SOJ2 using ferrous o-phenanthroline as indicator. Enzyme Assay for Free and Bound GOD. Two different methods were used to measure GOD activity in 0.1 M glucose solution. following gluconic acid or H 2 0 2formation. (ai Gluconic acid was titrated by use of a pH-stat in an unbuffered medium containing 0.9 mL of 0.5 M glucose, 0.1 mL of 1 mg/mL beef liver catalase, and 3.5 mL distilled water (regulated to pH 6 by addition of 10 mM KOH); the equipment consisted of a commercially available pH-stat device coupled to a microburet and a recorder: either Urectron 4,Electroburex buret and EPL2 recorder from Solea-Tacussel were used, or Metrohm E 300B and 473. Solea-Tacussel Electroburap buret and Sefram graphispot recorder. In routine experiments, 1 cm on the recording chart corresponded to 16 pL delivered by the microburet. (b) Hydrogen peroxide formation was followed using the POD-ARTS reagent: after addition of 0.2-mL samples into 0.8 mL of reagent (0.02mg/mL POD and 1.0 mg/mL ABTS) the blue coloration was followed at 420 nm. Procedure. Both electrodes El and E2 were allowed to equilibrate in the buffer solution during 15 to 30 min after stepping the potential of the working electrodes to +650 mV vs. Ag/AgCl C 1978
American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1 , J A N U A R Y 1979
97
-
dipped
electrodes
flaw-through
electrodes
1' ' N k ~ - N H ~
( 3 days,
room t e m p e r . )
(cgerniqht,
Figure 1. Scheme of the dtfferentlal glucose sensor with either stationary (dipped)or flow-through electrodes (W,) and (W,) platinum disk working (A) platinum wire auxiliary, and (R) Ag/AgCI reference electrodes
----
row tewer. )
+
-# '
mv-meter]
C
of SOmEglyc'ne-NaOH (EH9.l)
2 . 5 G~O O~ i ; . j m i
( 2 h, 4'C)
% 3
- $&:e!1:c
:
: -P
4
- SI0KCE.i
:
C.l
KC1
E
1OC
hH
-
GOD
Tin
a c e t a t e b u f f e r ( O H 4 . 0 ) , 4°C
Figure 3. Preparation and storage of GOD collagen membranes
Figure 2. Electronics used for testing the glucose sensor c
70/
reference. This potential corresponds t o the diffusion limited current for H 2 0 2oxidation. The stead>-state current at both electrodes was usually, in the absence of glucose, 30 60 11.4, after 30 minutes standby time, their difference ( I , I ? ) being negligible for usual glucose determinations.
PREPARATION A N D PROPERTIES OF ENZYME COLLAGEN MEMBRANES Optimal Conditions for GOD Binding on Collagen Membranes. T h e mild general acyl-azide procedure for activation of collagen membranes was used (7, R ) , followed by a coupling of the enzyme (Figure 3). Carboxyl groups were first esterified by immersion of the crude nembranes into a methanol/0.2 M hydrochloride acid solution for at least 7 2 h, then were treated overnight with 1%hydrazine, and soaked for 3 min in 0.5 M NaN0,/0.3 M HC1 mixture a t 0-1 "Cjust before coupling. Thorough washings were performed a t each step and a t the end of the process, avoiding contact between reagents and enzyme solution. Optimization of the routine coupling process was applied to glucose oxidase binding, in order t o obtain a high surface activity. Several buffer solutions in the p H range 2.2 to 1 2 were used (Figure 4), 100 m M phosphate-citrate buffer from pH 2.2 to 7 , 50 m M triethanolamine-HC1 from p H 6 to 8, 49 m M veronal-HC1 from p H 5 to 9, 50 m M glycine-NaOH from p H 9 t o 11, and 50 m M boric acid-NaOH from p H 9 t o 1%. T h e highest retained activities were obtained for alkaline pH values of the enzyme coupling solution, ranging from 8 to 10. T h e activity of GOD collagen membranes was found equal t o 60 nmol.min~1.cm~2, when the coupling buffer was 50 m h l glycine-NaOH buffer a t p H 9.1; these conditions were selected for routine binding of GOD to collagen membranes (Figure 3). Properties of Enzyme Membranes. Enzyme membranes exhibited high mechanical resistance without any tanning, and no bacterial degradation was detected when the membranes were kept in buffer a t 4 "C for more than 2 years. Lyophilization of such membranes was conducted without significant loss of activity after rehydration. T h e activity of GOD collagen films was tested in the p H range 2.6 to 8.2 with a 0.2 pH step between each assay. No optimum p H shift was found compared to literature data ( 9 ) ;
2
4
6
8
1
0
1
2
PN Figure 4. Influence of buffer and pH on OOD coupling. (-D-) 50 mM glycine-NaOH, (- -0- -) 100 mM phosphate-citrate-Na', (-.-O---) 40 mM veronal-HCI, (-. .-A---) 50 mM triethancllamine-HCI, and (-.-.-V-...)
50 mM borate-Na' buffer solutions
Le.. a n optimum p H of 5.6-5.7 was obtained. T h e K , for glucose was equal to 3 m M after coupling. T h e essential property of such enzyme membranes is their resistance to denaturation either in storage or in use. Systematic tests were performed to determine the optimum storage conditions using different buffers and pH: 50 m M succinate ipH 4.01, 100 m M acetate ( p H L O ) , 50 m M cacodylate-HC1 (pH 6.0), 100 nihf phosphate (pH 7.01, and 50 mM Tris-HC1 (pH 8.0). A 35% decay of initial activity was observed after 50 days at 4 "C, except for succinate buffer where it was only half of this value. T h e influence of temperature on storage was determined using 100 mM acetate buffer, p H 4.5 (Figure 5). After a slight decrease during the first days, the activities of membranes stored either frozen a t 0 'C, or at 3 and 20 "C, reached a stable value of 96, 91, and 82% of initial activity, respectively. For membranes stored a t 30 "C, the residual activity was about ,554; after 45 days, showing
98
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
I 10+31 A
y
,
,
I
10-l
3
1
so
8
0
&
, 10
,
.
20
30
storage time
,
40 / days
lo-l=-
1
.-c
E
d
E
50
Figure 5. Influence of the temperature of storage on the maintenance of collagen bound GOD activity. Acetate buffer 0.1 M (pH 4.5) at ( 0 ) 0, (0)4 , (m) 20, and (0)30 O C
G
0i
10-5
10-6
- 7
10-3
10-4
1i-z 0
[glucose] / M Figure 7. Calibration curves with stationary electrodes. (-0-) steady-state and (- 0 -) dynamic responses to glucose pulses. (A) log (responses) vs. log (glucose). (6)Relative responses vs. log (glucose)
. A dipped electrodes
h?
101
l;Fi
E -lot 0 I -
/
5
0
I
1
time
2
3
/ rnin
Flgure 6. Response curves for a 10 pM glucose pulse with the stationary electrodes. (-) I , - I , vs. time: steady-state response. (- - -) I , vs. time: non-enzymatic response. (-.-.-) d(I, - I,)/dt vs. time: dynamic response. (-...-...) d2 ( I , - I,)/dt' vs. time: signal used in automated devices
t h a t t h e use of such GOD membranes, in solutions thermostated a t 30 "C for several weeks, is possible.
A SENSOR FOR THE DETERMINATION OF TRACESOFGLUCOSE T h e union of a GOD collagen membrane and an amperometric detector of H202,as described in Figure 1, yields a glucose sensor giving a response within 1 min and possessing a very high sensitivity (ca 10 nM). When glucose is added to t h e buffer solution into which both electrodes El a n d E2 are dipped, or when t h e glucose concentration increases in a flow-through solution, 4 different current vs. time curves may be recorded (Figure 6). Z2 remains very small showing the absence of any GOD activity of activated collagen on E2. ( I I- 1 2 ) reaches a steady-state value after 2 t o 3 min: this is t h e steady-state response of t h e sensor. d(Z, Z2)/dt is maximum after 30 to 50 s: the height of this peak is t h e d y n a m i c response of t h e sensor. d2(Il - I , ) / d t 2 may control a switch when reaching a null point, a n d be used for printing t h e dynamic response; this ~
0
1
20
C
t
2
: a
b
*
. I
3
-10
0
:
-20 0
5
10
15
assays Figure 8 . Determination of the precision of the glucose sensor by 13 successive assays. ( 0 )steady-state and (0)dynamic responses for successive (A) 20 p M and (6)50 p M glucose determinations
device was actually built and gave excellent results, although, because of electronic time-lag, the zero value of t h e second derivative is obtained ca. 15 s after the maximum of the first derivative. Since both responses are proportional t o glucose concentration over a very large range (Figures 7 and 8),successive glucose determinations are possible every 2 t o 3 min without washing the electrodes. If, however, a washing is necessary, only about 3 min are required for t h e currents t o return t o their background values. T h e lowest glucose concentration which can be detected by the sensor is less than 10 n M with the dipped electrodes and 100 nM with the flow-through ones: for these extremely dilute solutions, t h e precision of glucose determination ranges between 50 and 100%. T h e limit of detection depends on the noise level, which is somewhat less
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
for steady-state t h a n for dynamic responses, a n d for "dipped-in" than for flow-through electrodes. I n the latter case, the noise level was mainly related to the irregularity of flow-rates obtained with our peristaltic pump. T h e sensor responses become independent of glucose concentration a t concentrations higher than 10 m M (Figure 7A); the concentrations which can be determined range from 10 n M to 10 m M (Le., over 6 orders of magnitude). Linearity of the calibration curves, as tested by the concentration independence of the relative responses I / C and dZ/C-dt (Figure 7B), is observed over 4 to 5 concentration decades from ca 0.1 pM t o 2-5 mM.
BASIC PROPERTIES OF THE GLUCOSE SENSOR Reproducibility. For definite values of the geometrical parameters of the system (electrodes-collagen membranes ), t h e removal and remounting of the membranes on the platinum anode may shift t h e steady-state and dynamic glucose responses by 4 and 8%, respectively. Precision. T h e precision of glucose determinations has been tested during a set of experiments by successive additions of glucose concentrations into the same medium. T h e electrodes were either dipped into the solution (Figure 8A) or the solution was allowed to flow through them, in sequence with a pure buffer solution (Figure PB). Both the steady-state and dynamic responses seem independent of the number of assays run within ca. 1 h, and also independent of the number of H2O2determinations. For 13 successive assays of a 20 and a 50 pM glucose, the standard deviation from mean is usually lower than 2% and 4% using the stationary (dipped) and flow-through electrodes. respectively. Selectivity. T h e selectivity depends on both the enzyme and the electrochemical detection method. GOD, itself, is very specific for 8-D-glucose; so electrode El (Figure 2) presents a high selectivity for glucose compared to usual sugars: the selectivity coefficient is smaller than E1.10-~for glucose over fructose, lactose, and sucrose. A 1m M addition of these three sugars, either separately or mixed, gives a steady-state response lower than 1 nA, whereas a further addition of 1 m M glucose gave a 3 . 5 - p A steady-state response. Since species other than enzymatically generated H202may diffuse through the collagen membranes and be oxidized on platinum at +0.65 V vs. AgjAgCl (Le., 0.94 V/NHE), the use of a compensating nonenzymatic electrode (E,) eliminates the possible interferences of species such as H202,ascorbate, urate, and t>Tosine. For example, an addition of 3.9 ptM H202 will give a steady-state response a t each electrode of about 35 nA whereas the differential response (II- I,) is, after 2 min, lower than 0.05 nA. Compared to the usual values of glucose steady-state responses (1to 3 mA.M-'), the selectivity coefficient for glucose over nonenzymatically generated H 2 0 2ranges between 4 x and 1.25 X depending upon experimental parameters (balance of the compensating electrode and GOD activity of the film). Influence of GOD Activity. T o study the relationship between the glucose responses of the sensor and the GOD membrane activity, membranes were prepared with various concentrations of GOD coupling solutions, leading to surface All these activities ranging from 4 to 80 nmol.rnin-',cm-'. membranes were mounted on the same electrode ( E i ) , and the calibration curves were determined in each case. When plotting the slopes of these calibration curves ( I / C ) vs. the exhibited GOD activity, as shown on Figure 9, a linear relationship appears. This demonstrates t h a t the rate limiting factor of the sensor is the GOD surface activity of the collagen membrane and t h a t the best sensitivity is obtained with the most active enzyme membrane.
5,
99
1
4-
3. 21-
4
0 0
20
40
GOD activity/
60
8'0
1
100
nmoie . rnin-'.crn- 2
Figure 9. Influence of GOD activity of the collagen films on the slope of the Calibration curve of the glucose sensor. Steady-state responses, 30 "C
Influence of Temperature. T h e sensor responses to glucose are as dependent on temperature as on t h e GOD activity of the collagen films. Because this dependence reaches 4-5%/"C a t 30 "C and 7-8%j0C at 20 "C, it is necessary to carefully thermostat t h e solutions in contact with both electrodes. Nevertheless, the sensor may be used over a large temperature range: 15 t o 40 "C. Storage. Besides the above mentioned high resistance of the GOD collagen films t o bacterial degradation and GOD denaturation, we have observed excellent storage patterns for the glucose sensor, itself. For example, a given glucose electrode mounted with the same GOD membrane exhibited a decrease of less than 6% of the slopes of the calibration curves of the steady-state responses (Z/C), when stored a t room temperature for more than 8 weeks, and checked a t 30 "C for more than 60 h (cumulative time), totaling 310 glucose assays either in stock solutions or in output flow of bioreactors containing starch derivatives. CONCLUSION In a previous work, a generally useful mild coupling method for enzyme on collagen membranes using acyl-azide activation has been reported ( 7 , 8 ) .T h e excellent performances of such covalent binding allowed us to use these membranes in solid phase enzymology ( I O . l l ) ,in bioreactors (12, 131, and as an active part of glucose sensors ( 5 , 6). T h e proteinaceous environment provided by the collagen, itself, ensures a noticeable stability of the bound enzyme, either stored a t 4 "C for years, or mounted on an electrode a t room temperature for months. No microbial attack was detected in our conditions of storage and utilization. Even with a low amount of bound enzyme (50-100 m u j m e m b r a n e of 1-cm diameter), the device was found able to detect LO n M glucose solutions, the linearity of calibration curves obtained being from 100 nM to 2 mM. T h e differential device, either with immersed or flow-through electrodes, can be used for the determination of traces of glucose in whole blood or plasma (6). T h e monitoring of glucose production in the output flow of bioreactors using starch derivatives as substrate and specific immobilized hydrolases is under investigation. T h e potential of the described device, using either different oxidases or co-immobilized enzymes yielding H202,is very promising. In the latter case, glucose must be the product of the first enzymatic steps and GOD the final enzyme, ensuring the enzymatic oxidation of glucose and the production of H202. In all cases, the use of enzymes in an immobilized form, together with amperometric detection, avoids single-use expensive reagents, and leads to a low cost analytical device.
100
ANALYTICAL CHEMISTRY, VOL. 51. NO. 1, JANUARY 1979
LITERATURE CITED
(10) P. R. Coulet, C. Godinot, and D. C. Gautheron, Biochim. Biophys. Acta, 391, 272 (1975). (1 1) J. M. Engasser, P. R. Coulet, and D. C. Gautheron. J. Bioi. Chem., 252, 7919 (1977). (12) J. M. Brillouet, P. R . Coulet, and D. C. Gautheron, Biotechnol. Bioeng., 16, 1821 (1976). (13) J. M. Brillouet, P. R. Coulet, and D. C. Gautheron, Biotechnol. Bioeng., 19, 125 (1977).
(1) G. G. Guilbault, in "Immobilized Enzymes, Antigens, Antibodies and Peptides"; H. H. Weetali, Ed., M. Dekker New York. 1975, p 293. (21 , . K . Camman. in "Das Arbeiten mit Ionenselectiven Electroden". Sorinaer Veriag, New York, 1977, and Fresenius Z.Anal. Chem., 287, 1 {19?7), (3) L. C. Clark and C. Lyons, Ann. N .Y Acad. Sci., 102, 29 (1962). (4) G. G. Guilbault and G. J. Lubrano, Anal. Chim. Acta, 64, 439 (1973). (5) D. R. ThBvenot, P. R. Coulet, R. Sternberg, and D. C. Gautheron, in "Enzyme Engineering" Vol. 4 , Plenum, New York. 1978, p 219. (6) D. R . Thevenot, P. R . Coulet, R . Sternbera. and D. C. Gautheron, Bioelectrochem. Bioenerg., 5, 541 (1978) (7) P. R. Coulet, J. H. Julliard, and D. C. Gautheron, French patent (ANVAR) 73-23-283, publ. number 2.235, 133 (1973). (8) P. R . Couiet. J. H. Julliard, and D. C. Gautheron, Biotechnol. Bioeng., 16, 1055 (1974). (9) T. E. Barman, in "Enzyme Handbook", VOI. 1 Springer-verlag, New York, 1969, p 112.
RECEIVED for review July 25, 1978. Accepted October 11, 1978. This investigation was supported in p a r t by "Dglggation GBngrale la Recherche Scientifique e t Technique" G r a n t 76.7.0920.
Potentiometric Enzyme Electrode for Lactate Toshio Shinbo"' and Masaaki Sugiura National Chemical Laboratory for Industry, Hiratsuka Branch, Hiratsuka, Japan
Naoki Kamo Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
A potentiometric enzyme electrode for lactate is developed. The enzyme electrode is constructed by coating the sensor membrane of the redox electrode with a film of enzyme-gelatin gel layer. The enzyme used is lactate dehydrogenase which catalyzes the oxidation of lactate by ferricyanide. The change in the concentration ratio of ferricyanide to ferrocyanide is monitored by the redox electrode which is a membrane electrode sensitive to redox potential of the solution. A plot of the potential of enzyme electrode vs. !og [lactate] gives an S-shaped curve which depends on enzyme quantity and ferricyanide concentration. A relation between the enzyme electrode poteniial and substrate concentration is derived semitheoretically. The equation explains the results successfully.
Enzymes have been used widely in t h e field of analytical chemistry owing to their substrate specificity ( I ) . T h e enzyme electrode, one of m a n y useful analytic a1 devices. is a n electrochemical sensor made by combination of a n enzyme reaction with an electrode. T h e enzymc electrode enables us t o determine t h e concentration of a specific organic species continuously a n d easily. Many enzymc electrodes have been reported in the past decade. which are classified in two groups; (1) T h e change in concentration of a charged substance produced by a n enzyme reaction is measured potentiometrically with use of t h e ion-selective electrode (2-6). (2) T h e a m o u n t of current caused by oxidation or reduction of a product or a reactant of t h e reaction is measured polarographically ( 7 -13). For enzymes catalvzing t h e oxidationreduction reaction. the polarographic method has mainly been employed (7-13). In t h e present paper. we report a new potentiometric enzyme electrode with use of t h e eii7yme catalyzing t h e I Present address, National Chemical I aboratory for Industry. Hiratsuka Branch. 1-3-4 Nishiyawata. Hira rqukashi, Kanagawaken, .Japan.
0003
'00/79~0351-0100$01O O / G
oxidation-reduction reaction. For monitoring t h e progress of such reactions, a new membrane electrode sensitive to redox potential (redox electrode) was developed. T h e sensoimembrane of t h e redox electrode is a plasticized poly(vinylchloride) membrane containing dibutylferrocene (PVC-Fc membrane). I t responds to the redox potential of the solution. T h e enzyme electrode was constructed by coating t h e sensor membrane of the redox electrode with a film of enzymegelatin gel. T h e enzyme used is lactate dehydrogenase (cytochrome b2, EC 1.1.2.3) which catalyzes t h e oxidation of lactate in t h e presence of a n electron acceptor such as ferricyanide;
pyruvate
+ 2Fe(CN):-
+ 2H+
T h e change in t h e concentration ratio of ferricyanide to ferrocyanide in t h e enzyme gel layer caused by t h e enzyme reaction is detected by t h e redox electrode.. T h e equation relating the potential of the enzyme electrode t o the substrate concentration is derived semitheoretically and it explains the results obtained successfully.
EXPERIMENTAL Materials. Lactate dehydrogenase (cytochrome bp,EC 1.1.2.3; prepared from yeast) and catalase (EC 1.11.1.6) were purchased from Sigma Chemical Co. L-Lactate was purchased from Sigma Chemical Co. and used after neutralization with NaOH. Dibutylferrocene and dioctylphthalate were obtained from Tokyo Kasei Co. (Tokyo, Japan). Gelatin was obtained from Nippi Co. (Tokyo, Japan) and purified by electrodialysis a t room temperature for 30 h. Preparation of PVC-Fc Membrane a n d Redox Electrode. In 10 mL of tetrahydrofuran (THF) were dissolved 250 mg of poly(vinylch1oride) (PVC),500 mg of dioctylphthalate (DOP),and 300 mg of dibutylferrocene (Fc). This solution was poured into a Petri dish (60 cm2 in area) and evaporated slowly a t room temperature to form a thin membrane (0.14.15 mm in thickness). A piece of the PVC-Fc membrane was peeled off and glued to a PVC tube with THF. An AglAgC1 electrode was used as the inner reference electrode which consisted of a silver wire mounted in a glass pipet filled with an agar gel containing 3 M KCI. The CC 1978 American Chemical Society
