Anal. Chem. 1990, 62, 2429-2436
to faster surface diffusion of Au in the presence of chloride. A more detailed study is now under investigation. €&&try NO.Au, 7440-57-5;HC10~,7601-90-3;Cl-, 16887-00-6; HCl, 7647-01-0; AuOH, 12256-43-8;A u ~ O1303-58-8. ~,
LITERATURE CITED (1) Hubbard, A. T. Chem. Rev. 1988. 88, 633-656. (2) Ross, P. N.; Wagner, F. T. Advances In Electrochemistry and Electrochefnbl Engineering; Gerlscher, H., Ed.: Wlley: New York, 1984; VOl. 13, pp 69-112. (3) Kdb, D. M. Z . Phys. Chem. (Munlch) 1987, 154, 179-199. (4) Yeager, E. J . Electrochem. Soc. 1981, 128, 16OC-171C. (5) Sonnenfeld, R.; Hansma, P. K. Sclence 1988, 232, 211-213. (6) GewMh, A. A.; Bard, A. J. J . Phys. Chem. 1988, 92, 5563-5566. (7) Itaya. K.; Sugawara. S.; Hlgaki. K. J . Phys. Chem. 1988, 92, 6714-6718. (8) Wiechers, J.; Twomey, T.; Koib, D. M.; Behm, R. J. J . Electroanal. Chem. Interfacial Electrochem. 1988, 248, 451-460. (9) Trevor, D. J.; Chidsey, C. E. D.; Loiacono, D. N. Phys. Rev. Len. 1989, 82, 929-932. (10) Green, M. P.: Hanson, K. J.; Scherson, D. A.; Xing, X.; Richter, M.; Ross, P. N.: Can, R.; Lindau, I. J . Phys. Chem. 1989, 93, 218 1-2 184. (11) Itaya. K.; Sugawara, S.; Sashikata, K.; Furuya, N. J . Vac. Scl. Techn d . A 1990, 8 . 515-519. (12) Sonnenfetd, R.; Schneir, J.; Drake, 6.; Hansma, P. K.; Aspnes, D. E. A w l . Php. Len. 1987, 50, 1742-1744. (13) Tomita, E.; Matsuda, N.; Itaya, K. J . Vac. Scl. Techno/.,A 1990, 8 , 534-538. (14) Hallmark, V. M.; Chiang, S.; Rabok, J. F.; Swaien, J. D.; Wilson, R. J. Phys. Rev. Len. 1987, 59, 2879-2882.
2429
(15) SasMkata, K.; Honbo, H.; Furuya. N.; Itaya, K. Bull. Chem. Soc.Jpn., in press. . Interfacial (16) Clavilier, J.; Armand, D.; Wu, 6 . L. J . € k t f o ~ n a l Chem. Electrochem. 1982, 735, 153-166. (17) Motoo, S.; Furuya, N. J . Electroanal. Chem. IntetfaciaIElectrochem. 1984, 167, 309-315. (18) Motoo, S.; Furuya, N. Ber. Bunsen-Ges. Phys. Chem. 1987, 91. 457-461. (19) Cadle, S. H.; Bruckenstein. S. J . Efectroanal. Chem. InterfaclalEktrochem. 1973. 48, 325-331. (20) Rand, D. A. J.; Woods, R. J . Electroanal. Chem. InterfacialEfectrochem. 1972. 3 5 , 209-218. Bracker, C. E. Science 1988, (21) Hansma. P. K.; Eiings, V. B.; Marti, 0.; 242. 209-216. (22) Itaya, K.; Tomita, E. Surf. Sci. 1988, 207, L507-L512. (23) Schneir, J.; Sonnenfeld, R.; Marti, 0.; Hansma, P. K.; Demuth, J. E.; Hamers. R. J. J . Appl. Phys. 1988, 63, 717-721. (24) D'agostino, A. T.; Ross, P. N. Surf. Sci. 1987, 185, 88-104. (25) Angerstein-Kozbwska, H.; Conway, B. E.; Hamelln, H.;Stolcoviciu, L. J . Electroanal. Chem. Interfacial Electrochem. 1987, 228,429-453. (26) Hamelin, A. Modern Aspect of EIectrochem&fry; Bockris, J. O'M., Conway. B. E., Eds.; Butterworths: London, 1985; Vol. 16, pp 1-101. (27) Cadie, S. H.; Bruckenstein, S. Anal. Chem. 1974. 46, 16-20. (28) Jakievic, R. C.; Elie, L. Phys. Rev. Len. 1988. 60, 120-123.
RECEIVED for review May 14, 1990. Accepted July 18, 1990. This work was supported by Ministry of Education, Science and Culture, Grant-in-Aid for Research No. 63850160 and 480540125498, and a foundation of Nippon Soda Industrial Corporation.
Cyanide Detection Using a Substrate-Regenerating, Peroxidase-Based Biosensor Mark H. Smit and Anthony E. G. Cam* Centre for Biotechnology, Imperial College of Science, Technology, and Medicine, London SW7 2AZ, United Kingdom
An enzyme-based, dual working electrode system Is described for the sendng of cyanlde. Horseradish peroxidase (HRP) Is Incorporated as the senslng element. A contlnuous monltorln~d oxklattve actMty by the enzyme resuJts through the generatkn and rageneratbn of substrates at the electrode surfaces. Thus, HRP Is oxldlzed by hydrogen peroxide generated from dbolved oxygen, at the prbnary electrode, and then reduced through the secondary electrode by mediated electron transfer using ferrocene as a carrler. Ferrocene regeneratlon at thls electrode Is proportlonal to the lntrlnslc actlv#y of HRP. The dynamics of the system are Investigated by uslng a rotatlng rlng-dlsk electrode. The enzyme Is Immoblflzed to provlde better control over its catalytlc actlvlty and to Increase the llfetlme of the biosensor. Cyankle lnhlbIthof current can be modeled by reverdh blndng khetks. Detection of cyanlde is possible In submicromolar (ppb) concentrations, wlth a hall maxknal response at 2 wM. The response tkne for detectlon of Introduced cyanlde Is within 1 8. The sensor can be operated between 5 and 40 OC, and cyanide inhlbl#on Is unaffected by pH changes between 5 and 8. The sensor Is reprocludble for cyanlde determlnatlon and Is stabk for over 6 months.
INTRODUCTION The catalytic activity of enzymes not only is very specific for their substrates but also can be very sensitive to the presence of specific inhibitors. The amplifying nature of 0003-2700/90/0362-2429$02.50/0
enzyme activity coupled to the sensitivity of amperometric devices provides us with the possibility of developing sensitive and specific biosensors for the detection of toxins at very low concentrations. The sensitivity of the device would increase with the toxicity of the substance. A variety of enzymebased amperometricsystems have resulted from recent developments in methods for monitoring enzyme activity by electrochemical means. Transduction methods for the determination of enzyme activity are usually based on the electrochemical measurement of substrates or producti, either natural mediators, like oxygen and hydrogen peroxide (I),or artificial mediators, such as the ferrocenes (2). The design of enzyme electrodes is such that the current or potential measured is proportional to the reaction of the substrate with the enzyme. The magnitude of the signal is controlled by the rate-determining step involved in this reaction (3). Many sensors have been described that rely on high enzyme loading; their response is dependent on the transport of substrate to the catalytic site and is thus sensitive to measuring variations in the concentration of substrate. Sensors based on enzyme inhibition, however, rely on the measured response being proportional to the amount of catalytically active enzyme present. These sensors are therefore sensitive to any perturbation of enzyme activity. The theory and application of enzyme inhibition for the detection of substances has recently been addressed (4). This present report describes the development of a sensitive enzyme-based amperometric sensor for the detection and determination of cyanide, using a very stable enzyme, horseradish peroxidase (HRP), and incorporating substrate-gen0 1990 American Chemical Society
2430
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
erating and -regeneratingfeatures, which allows for continuous monitoring of the integrity of the protein's enzymatic activity without the need for added reagents. For the development of an enzyme-electrode-based, continuous cyanide sensor, several criteria must be fulfilled. The sensor must be sensitive, providing detection in the low ppm range. The response rate of the sensor must be rapid to allow for appropriate evasive action. Specificity is important, to avoid false alarms. With respect to biosensors, the feature of enzyme stability and reproducibility is very important to address. Finally, the sensor should be easy to use and be self-supporting; that is, it should not require constant maintenance and replenishment. The need for cyanide sensors is apparent from this compound's high toxicity and ubiquity. Cyanide is a byproduct resulting from industrial processes such as electroplating, metal mining, and organic synthesis. Much effort has been expended in the development of methods for cyanide detection and determination (5).These methods range from sensitive colorimetric (6, 7) methods and electrochemical analysis (8) to techniques requiring sophisticated instrumentation such as atomic absorption spectroscopy and computer-assisted pattern recognition. In the detection and determination of cyanide there are certain inherent difficulties. Many procedures require the purification or volatilization of cyanide. Most methods suffer from a lack of specificity and the problems of interference. The application of physical-chemical techniques to a method of continuous monitoring is still being sought (9).A biochemical understanding of how cyanide exerts its toxicity and recent developments in biosensor technology provide us with an alternative method for its detection. Cyanide expresses its toxicity by binding to the terminal component in the electron transport chain in the mitochondria, cytochrome oxidase. It blocks an intramolecular electron transfer thus stopping electron flow in the respiratory chain (10).This interruption of current suggests an electrochemical means for the detection of cyanide. Ideally, the biosensor for the detection of a certain toxin should incorporate that component upon which the poison acts to exert its fatal effect. Such an "electrochemical canarynfor cyanide, sulfide, and azide has been described (4,ll).In that device, the reduction of oxygen though successive electron transfers from a modified electrode to cytochrome c and then to cytochrome oxidase was monitored. Inhibition of the current resulted from the presence of cyanide. As inhibition-based sensors must operate under conditions of an enzyme-limited signal rather than a transport-limited one, then they will be susceptible to the nonspecific denaturation of the enzyme as well as the specific inhibitors. In the example of the cytochrome oxidase based sensor, the signal from the device gradually decreased even in the absence of the inhibitor and, although the two causes of loss of activity could be distinguished kinetically, the electrode had a limited operating lifetime. In these studies, therefore, horseradish peroxidase (HRP) has been used as the enzyme, and an electrochemical system has been developed to both generate substrates and monitor enzyme activity. Horseradish peroxidase is a heme-containing glycoprotein that catalyses the oxidation of substrates by hydrogen peroxide. Several reviews exist on the structure and function of HRP (12-14). A variety of techniques have been employed to elucidate the mechanism of action of this enzyme. It is now understood that peroxides transfer their oxidizing equivalents to HRP, creating an intermediate known as compound I, which is two oxidation states above the resting state. The enzyme then gets reduced to compound I1 and subsequently to the resting state in two steps by a suitable donor. The
Figure 1. Representation of the monitoring of HRP activity with two working electrodes, one held at -1.0 V and the other at 0 V. H,Op and ferrocene are generated and regenerated at the primary and secondary electrodes, respectively. The current generated at the secondary electrode is a function of the catalytic state of the enzyme.
overall reaction is a modified ping-pong mechanism and is as follows:
H20z+ HRP compound I
+ D(red.)
compound I1
kl
k2
compound I
+ H20
compound I1
+ D(ox.)
+ D(red.1 -% HRP + D(ox.1 + H20
(1) (2)
(3)
In our development of an electrochemical sensor we chose an electroactive donor as the indicator substrate. Ferrocenes have been shown to act as electron donors in the reduction of compounds I and I1 (15). This function has led to the demonstration of their use as mediators in a hydrogen peroxide biosensor incorporating peroxidases as the sensing enzyme (16). Ferrocenes have a number of advantages as enzyme mediators. They are easily oxidized and reduced at electrodes. They are stable in the reduced form and are neither light nor pH sensitive. Perhaps most importantly, the structure of ferrocene allows for the synthesis of a largevariety of analogues that can be designed to fit a particular enzymatic application (17).
Horseradish peroxidase was chosen because of its stability and availability as very pure preparations. Cyanide is a well-known inhibitor of peroxidase activity (18).It binds to the sixth coordination position of the iron (19).Its binding (20) and may thus inhibit is analogous to the binding of H202 the formation of compound I. The binding can be measured through the appearance of a distinct Soret peak at 422 nm (21)and is rapid with an association rate of 1.2 X 105 M-' 8'. Further kinetic data have shown that the dissociation rate constant is 0.2 S-I, giving an equilibrium dissociation constant around 2 pM (22). A dual working electrode system was designed to monitor HRP activity. This is shown schematically in Figure 1. The substrates are generated or regenerated at these electrode surfaces. Thus, the primary electrode is used to generate H202 from dissolved oxygen, and the rate of this reaction can be controlled by the applied potential. The secondary electrode is used to regenerate the reduced form of ferrocene, following its oxidation by HRP. Through the manipulation of the reactant concentrations, the enzyme concentration, and electrode potentials, conditions can be created in which the current generated at the secondary electrode by the reduction of ferrocenium is directly proportional to the HRP activity. Any perturbation of this activity by inhibitors would manifest itself as a decrease in secondary electrode current. The electrode configuration chosen to study this biosensor design was the rotating ring-disk electrode (RRDE). The transport of solution to the electrode is well understood (23) and can be controlled by the rotation rate. The hydrodynamics of these electrodes is such that fresh solution is transported up to the disk electrode and subsequently swept
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
I
H2O
FeCp2
eklrode. 2. Reactions occming at the &ace of the The glassy-carbon disk creates hydrogen peroxide, and the platinum ring detects and reduces ferrocenium.
out to the circumference. There is therefore a sequential transfer of components from the disk electrode to the ring electrode (Figure 2). Interestingly, a reducing current at the disk generates an oxidant that, through enzymatic catalysis, creates a reducing current at the ring. Because the potential applied to the disk is reductive, the only source of ferrocenium and thus cathodic ring current is through enzymatic oxidation. The net result of this catalytic system is the reduction of molecular oxygen to water using two electrons from the primary electrode and two electrons from the secondary electrode. The reactions taking place within this system are therefore as follows:
-+ + -
lo electrode (disk): O2 + 2e-
+
solution: H202 2H+ 2 O
+ 2FeCp2
electrode (ring): 2Fe+Cp2
+ 2H+
2H202 (4)
HRP
2H20
2Fe+Cp2 ( 5 )
2e-
2FeCp2 (6)
Further development of sensor design becomes possible once the fundamentals governing the creation and detection of catalytic currents are understood. In these studies we describe the components of this enzyme system, the coupling of enzyme to electrode, and its applications toward cyanide biosensing. EXPERIMENTAL SECTION Reagents. HRP was purchased as highly pure preparations (RZ > 3.0) from Biozyme. BSA (bovine serum albumin) was obtained from Sigma. (Hydroxymethy1)ferrocenewas obtained from Kodak. (Dimethylethano1amino)ferrocenewas a gift from MediSense, Inc. Ferrocenecarboxylate and -dicarboxylate and ((dimethylamino)methyl)ferrocenewere purchased from Aldrich, as was l-cyclohexyl-3-(2-morpholinoethyl)carbodiimidep methyltoluenesulfonate. NaCN and KCN were obtained from the British Drug House (BDH), as were most of the other laboratory chemicals including buffers and supporting electrolytes. ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) was bought from Boehringer Mannheim. HzOzwas purchased from BDH as 30% solution. All chemicals were of high purity (Analar or Aristar grade), and deionized water was used throughout. Apparatus. All the electrochemical instrumentation and electrodes were constructed in the Department of Chemistry, Imperial College. The platinum and glassy-carbon ring-disk electrodes were sealed in araldite or Kel-F and had the following dimensions: the disk had a diameter of 0.7 cm; the ring separated by a 0.05-cm spacer had an i.d. of 0.75 cm and an 0.d. of 0.8 cm. The electrodes were polished with a 0.3-pmaluminum oxide slurry and subsequently sonicated in a water bath. A four-electrode analogue potentiostat was typically used to control potentials at the working electrodes. Alternatively, a computer-controlled potentiostat built at Imperial College was used. Programs for this equipment were written by the Imperial College Chemistry Microprocessor Unit (ICCMU). The disk electrode was connected to a triangular wave generator for potential sweeps, and the ring electrode was supplied by a constant dc voltage source. The electrode rotation was controlled by a rotator from Ursar Scientific. All potentials are quoted with respect to a saturated calomel electrode (SCE). A 1-cm2platinum gauze served as the counter
2431
electrode. The electrochemical cells held volumes of 5-10 mL and were equipped with a water jackets for temperature regulation. Temperature was controlled by a water bath. Electrodes were positioned within the cell so as to minimize solution resistance. The collection efficiency was determined experimentally by using ferrocenecarboxylateto be 0.16, which agreed with the predicted value (23). The purity of HRP was determined by sodium dodecyl sulfate (SDS) gel electrophoresis, and by the RZ value (typically 3.2 or greater). The concentration of enzyme was measured from the absorbance at 403 nm by using an extinction coefficient of WOO0 M-l cm-’ (21). Ferrocenes were made up at the desired concentration on a weight basis. The dissolved oxygen concentration was determined by the Winkler method (24)to be 240 pM,which agreed with values obtained from a nomogram. All experiments were carried out at 22 OC. Spectrophotometric assays measuring the rate of ferrocene oxidations were carried out in the following manner: HRP, (hydroxymethyl)ferrocene, and hydrogen peroxide were mixed with the appropriate buffer, and the rate of change in absorbance was monitored at 330 nm with a Philips PU 8720 spectrophotometer. Measurement of Catalytic Currents. The electrocatalytic activity of HRP was typically assessed by immersing the eledrode in a buffered solution containing peroxidase and mediator. The electrode was rotated at a constant speed to control the transport of material to the disk electrode and subsequently out to the ring. A cathodic potential sweep was applied to the glassy-carbon disk. The platinum ring was held at constant potential, and the current at the ring as a function of the disk potential was measured. Before determination of the peroxidase-generated ring current, following electrode immersion, the ring current (background current)was allowed to stabilize (-5 min). A cathodic ring current resulting from the reduction of the enzymatically oxidized mediator was then measured at disk potentials that resulted in the partial reduction of molecular oxygen. The catalytic current was defined as the net reductive current occurring at disk potentials, resulting from the production of hydrogen peroxide, minus the ring current occurring at a disk potential of 0 V. The results of each analysis were traced on an XY recorder and usually expressed as the disk potential and ring current. Further experimental details are discussed in the main text. Immobilization of HRP onto the Electrode. The glassycarbon disk was activated by a combined chemical and electrochemical technique as described by Bourdillon (25). The disk was held at +2.2 V for 30 s, and during this time the platinum ring was held at -0.2 V. The electrode was transferred to a solution of 0.5 M NaKP04,pH 7.0, and the platinum ring was cleaned by cycling between -0.3 and +1.0 V for several hours, until a stable voltammogram resulted. The disk was then activated with carbodiimide (0.1 M for 1h in 0.1 M sodium acetate buffer, pH 5.0). Following washing with deionized water, the electrode was immersed in a solution of bovine serum albumin (BSA, 20 mg/mL in 0.1 M sodium acetate, pH 5.0, for 2 h). The glycoside moieties of HRP were oxidized with N d 0 4(8 mM, pH 8.3,O.l M NaHCOs, 2 h (26)),and the unreacted NaI04 was removed by adding an excess of ethanediol and passing the protein through a Sephadex G-25 column. The BSA-modified electrode was immersed in the oxidized peroxidase solution and rotated slowly for 2 h in a sodium bicarbonate buffer (0.1 M at pH 9.0). The resulting imine bond linking BSA to HRP was reduced with NaBH4 (100 pL of 5 mg/mL for 1 h and repeated once). The electrode was then rotated in 0.1 M NaKP04, and the ring was cycled between -0.3 and +1.0 V for 2 h to help remove any loosely adsorbed protein. Enzyme Inhibition Studies. All cyanide studies were performed in a fume hood, and all necessary precautions were taken to avoid contact and inhalation of the poison. A cyanide antidote was also kept available. NaCN and KCN were made up fresh in a 10 mM phosphate buffer, pH 7.5. The containers were kept tightly sealed, and the air in the containers was minimized to prevent the loss of HCN. The concentration of cyanide was determined by titration with silver nitrate using iodide as an end point (27). The cyanide was added to the electrochemical cell via a Hamilton syringe. The volumes introduced were typically 0.1%-0.5% of the total cell volume. Catalytic currents were measured, as described above, until a reproducible trace was
ANALYTICAL CHEMISTRY, VOL. 02, NO. 22, NOVEMBER 15, 1990
2432
-1200
-800
-400
0.5
-0.5
region, the current, which results from the reduction of ferrocenium at the ring, is not constrained by the rate of oxygen reduction a t the disk. The current is governed by solution reaction 5. The generation of ferrocenium, dominated by HRP enzyme kinetics, follows the following reaction kinetics (28)
l
-
a
4 I
c
g -1.0 0' -1.5
.1 .o
,,'\ /Oxygen Reduction at Disk (x
-1200
-SO0
.1.5
-400
Disk Potential (mV)
Figure 3. Currents resulting at the 1' and 2' electrodes as a result of disk potential sweeps wing a RRDE. Both currents were monitored concurrently. The ring was held at 0 V. Conditions: scan speed, 0.1 VIS; electrode rotation rate, 4 cyclesls; HMF, 0.3 mM; HRP, 1.25 pM; KCI, 0.1 M; KHPO,, 0.05 M, pH 7.0. Regions a, b, c, and d are described in the text.
achieved (-30 5). For the determination of the rate of inhibition, the disk was held at a constant potential to produce hydrogen peroxide, and current was monitored with time following the introduction of toxin. The testing of other inhibitors such as sodium azide and sodium sulfide was performed in an identical fashion.
RESULTS AND DISCUSSION Generation of Catalytic Current. The two-electrode system described here exploits the electron-transfer catalyzing properties of peroxidase by oxidizing the enzyme via the generation of hydrogen peroxide at the primary electrode and reducing it through the reduction of ferrocenium at the secondary electrode. The design of this system results in all the enzymatic activity taking place close to the electrode surfaces. The current at the secondary electrode (ring) results from the reduction of ferrocenium and reflects the ability of peroxidase to undergo its catalytic cycle. The actual profile of the reduction current seen at the ring is a function of the concentrations of the enzyme and substrates, as well as the rotation rate of the electrode, the sweep rate at the disk, and the potential of the ring. The magnitude of this current also depends on the fraction of enzymatically generated ferrocenium that actually reaches the ring. An example of a trace depicting the ring current as a function of the disk potential is shown in Figure 3. For illustrative purposes, the reduction of oxygen at the primary electrode (the disk) is superimposed on this trace. Four distinct regions of faradaic activity (a, b, c, d) result at the ring as the disk becomes more cathodic. Initially (beginning from the right), the horizontal nature of the trace, a, indicates that there is no ferrocene-mediated electroactivity at the ring. A t potentials more negative than about -0.3 V, b, there is an increase in cathodic ring current as the disk becomes more reducing. This increase is directly proportional to the production of hydrogen peroxide at the disk. The actual current produced is therefore initially controlled by reaction 4 and appears to be independent of the concentrations of enzyme and mediator. The reason for this is that there is an excess of enzyme over hydrogen peroxide; all of the peroxide is consumed as it is made. As the rate of peroxide generation increases, the magnitude of the ring current becomes controlled by the reaction rate of the enzyme. The third region of this modified voltammogram, c, occurs when the enzyme kinetics dominate the current profile and the reaction between the enzyme and substrate becomes the limiting factor in the generation of ring current. Thus, in this
where u is d(Fe+Cp2)/dtand the rate constants correspond to eqs 1-3. [HRPIo and [FeCp,], are the concentration of enzyme and mediator at the electrode surface, and [H2O2l0 is the concentration of electrogenerated hydrogen peroxide. This kinetic equation implies that the ring current generation shows saturation profiles for both substrates and is directly proportional to the enzyme concentration. This proportionality can been shown through current measurements (in region c ) at various enzyme concentrations; a linear relationship exists with increasing enzyme concentrations. The same proportionality manifests itself following enzyme inactivation through cyanide. The final region of the diagram shows a decrease in ring reduction current. This results from the disk electrode reducing oxygen directly to water. The details of the shape of this trace are important for biosensor construction, as, in our present application, optimum sensor performance occurs when current production is enzyme limited. Thus there is a range of potentials at which the primary electrode can be operated, which are determined by the concentration of enzyme and mediator and the rate of oxygen reduction to hydrogen peroxide. The potentials at which enzyme kinetics dominate the ring current can also be analyzed by monitoring the ring current as a function of disk current during a disk potential sweep. IR/IDis about 0.03 when H202production dominates the ring current. At disk potentials where excess hydrogen peroxide formation allows enzyme kinetics to dominate the ring current, this ratio decreases. This relatively low collection efficiency (3%) is not due to inefficient enzyme kinetics but rather due to the auto disk reduction (ADR) of ferrocenium. That is, the ferrocene that is enzymatically oxidized is reduced directly at the disk (see Figure 2). Typically, with these electrode dimensions, about 80% of the ferrocene undergoes ADR (the "disk-disk collection efficiency"). hesently, we are developing methods to decrease ADR. Catalytic Ring Current as a Function of Electrode Rotation Rate. With two-electrode systems, the transfer of material between electrodes is important. The rate at which substrates reach the disk and are transported laterally to the ring is controlled by the rotation rate of the electrode. Studies to determine the effect of rotation rate on ring current revealed that the ring current decreased with increasing rotation rate. Under the conditions described in Figure 4 the maximum current was achieved at a rotation rate of 2 Hz. We interpret the maximum in the rotation speed dependence as reflecting the balance between mass transport and enzyme reaction kinetics. At higher rotation speeds there is less time for the hydrogen peroxide to react with the HRP and generate ferrocenium before it is lost to bulk solution. Consistent with this interpretation is the observation that the maximum shifts to higher rotation speeds at greater HRP concentrations. Similar results have been discussed in more detail by Wilson and co-workers for a glucose oxidase rotating ring-disk electrode (29,30). Most of our experiments were done by using a rotation rate of 4 Hz, which gave both a good flux offerrocenium to the ring and a sufficient transit time for enzyme reaction in solution. Ferrocene as a Mediator. Ferrocenes have recently become one of the mediators of choice for amperometric enzyme assays (31). Ferrocenes typically display reversible redox
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
-
5-
-
4.-4 -
-
C
g 3-
0
ol
g
2-
0
5
10
20
15
25
30
35
Flgurs 4. Effect of the electrode rotation rate on the ring current. Conditions: (hydroxymethyl)ferrocene, 1 mM; HRP, 2.5 $4. The current was measured at a disk potential of -1.2 V, during a disk potential sweep of 0.1 VIS.
Table I. Ring Current as a Function of Ferrocene Structure and Redox Potentiala
(hydroxymethy1)ferrocene
Ep,l2, current, re1 % m'$ mA current
(1,l'-dimethyl-3-
210 160
3.8 2.2
100 58
((dimethy1amino)methyl)ferrocene ferrocene-1,l'-dicarboxylate
295 490 420
0.64 0.22 0.10
17 6 3
ethano1amino)ferrocene ferrocenecarboxylate
0.12 0.10
0.08 0.06
gE
3
'
'I
I
I
I
I
I
I
I
2
3
4
5
6
7
0
9
0.00
PH
CPS
ferrocene derivative (0.5 mM)
0.14
- 0.04 - 0.02
1-
0
2433
'HRP 2.5 GM; currents taken at a disk potential of -1.2 V. Further details as in Figure 3.
kinetics, are easily oxidized at low potentials, and can be structurally modified to fit a particular biological function (17). In addition to their use as mediators in biosensor applications, they can be used in protein chemistry studies by measuring variations in kinetic rate constants as a function of their structural modification. These types of studies can help elucidate the nature of the enzyme active site where electron transfer occurs. The ring-disk setup used here provides a quick method of determining the interaction of a variety of ferrocenes with HRP. Table I displays several ferrocenes tested for their ability to act as mediators for HRP reduction. This enzyme interaction is shown in the resulting ring current. Care was taken to ensure that ring current reflected enzyme-dominated limiting catalytic current. The oxidizing potentials of both compound I and compound I1 are close to 1.0 V (32);thus the differences in reactivity of the various ferrocenes reside in both their accessibility to the active site and their redox potential. HRP may have a relatively nonpolar active site, and most isozymes carry a net p i t i v e charge at neutral pH. These features may help explain the differences between the various derivatives. The introduction of a permanently charged amino group, as with the (dimethylamino)methyl derivative dramatically reduces the catalytic rate. Although the negative charge on the carboxylate derivatives may favor binding, they are not as effective for mediation, possibly due to their polarity as well as their increased redox potential. The uncharged hydroxymethyl derivative gave the maximum current among those tested. Studies are currently being carried out to determine kinetic parameters such as Km and k, to separate the electrontransfer step from the preceding complex formation step. Effect of pH on HRP-Generated Ring Current. Since the system uses protons for the heterogeneous reduction of water, it wa8 important to determine the effect of pH on the HRP-generated current. It was also of interest to know the
Figure 5. Measurement of enzymatic oxidation of ferrocene by amperometric means and by spectrophotometric means. Condltlons for 0.3 amperometric analysis: HRP, 1.25 pM (hydroxymethyI)ne, mM. The spectrophotometric assay was performed as discussed In the text. Conditions: HRP, 1.25 pM, (hydroxymethyi)ferrocene, 0.5 mM; H202, 10 mM. Buffers (50 mM): pH 3-5.5 acetate, pH 5-7.5 potassium phosphate, pH 7-9 Tris.
optimal pH at which the sensor generated current. Figure 5 shows the effect of pH on ring current. Interestingly, the system shows a maximal response around pH 4.5. Spectrophotometric assays to measure the oxidation of ferrocene c o n f i i the stimulation of ferrocene oxidation at higher proton concentrations, implying protonation of an important group near the active site. With the spectrophotometric assay, however, there is no decrease below pH 4.5 but rather a plateau of activity around pH 3.5. A similar pH dependence has been seen for the oxidation of ferrocyanide (33)by HRP. The decrease in activity measured electrochemically below pH 4.5 may reflect some limitation in a nonenzymatic component of the system. Conveniently, the ring current shows little fluctuations for p H s around 7.0, allowing the sensor to be operated at a pH that enhances enzyme stability.
INHIBITION STUDIES Inhibition of Catalytic Current by Cyanide. The inhibitory effect of cyanide on HRP-generated ring current was measured under steady-state conditions. Cyanide was added from stock solutions to the electrochemical system, and binding was allowed to reach equilibrium (-30 s). The ring current generated under conditions of substrate-oxidationlimited kinetics was determined by cycling the disk between 0 and -1.2 V. The net ring current resulting from the catalytic activity of HRP in the region of HzOz production was determined by subtracting the ring current occurring a t 0 V. Thus, the ring current is a direct measure of the ability of HRP to oxidize ferrocene. Figure 6 shows the effect that cyanide has on the catalytic ring current. The inhibition of ring current can be standardized by expressing it as a percent inhibition of total current. These results can by analyzed in the first instance by using steady-state binding kinetics. If we assume that cyanide binds to HRP on a one to one basis
+
H R P CN- + HRP-CN (8) and the complex formgtion is directly proportional to the inhibition of current (which occurs under the appropriate conditions), then
where [CN] is the free inhibitor concentration ([CN] = [CNIT - [HRP-CN], [CN]Tis the total cyanide added) and [HRPCN] = [HRPIT X (% inhibition). Figure 7 shows a Scatchard analysis of a typical set of data. KI is the apparent inhibition constant for cyanide. From inhibition curves the KIwas
2434
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990 1 .o
14-
0.8
12-
c
-5, 1 0 -
E
j 0.6 0
-m
8-
’=6 0 . 4
Y-
6-
E
iL
40.2
2-
I
I
0
20
1 40
-1400
I
I
I
60
80
100
[Cyanide] (hM)
Flgure 6. Fractbnal ring current remaining fdlowing the introduction 0.3 mM, of potassium cyanide. CondWons. (hydro-&trrm, HRP, 2.5 pM. The ring current was measured at a disk potential of -1.2 V and was held at 0 V; the dlsk was swept at 0.1 V I S . The current was measured after 1 min equilibration following the addition of cyanide.
I
Scatchard Analysis 0.4-
E 0.3% z
-. I
8 0.2-
0.0-
I
0.2
I
I
0.4 0.6 0.8 Fractional Inhibition of Current
F W s 7. Scatchard analysis of data obtained as described for F w r e 4, using the equation: % I I [ C N ] = - % I / K , 4- I,,,,JKl. Points represent the means of five d e t e r ” f the standard deviation. fhe K , represents the free concendratkn of cyanide required for 50% inhibition.
calculated by both linear and nonlinear regression methods to be about 2 p M . This value agrees well with binding constants reported in the literature (22). This concentration corresponds to 52 X lo4 mg/mL or 52 ppb and is well within the desired detection limit. Cyanide Inhibition as a Function of [H202]. As emphasized earlier, sensors based on enzyme inhibition are most sensitive when the measured response (secondary electrode current in the present example) is directly related to the functional state of the enzyme. This point is illustrated by measuring the KI for cyanide under various rates of peroxide generation. The inhibition of the enzyme-catalyzed ring current was determined at various disk potentials. Figure 8 displays the apparent KI values for cyanide as a function of disk potential. As the disk potential becomes more negative, the KI for cyani9 decreases. Under conditions of low HzOz production the rihg current is proportional to the disk current and an excess of enzyme exists. Thus, the ring current is determined by the peroxide produced, and current inhibition is not proportional to enzyme inhibition. Below a disk potential of -0.8 V the inhibition of HRP by cyanide reaches a maximal efficiency. Cyanide Inhibition and Peroxidase Concentration. Equation 9 implies that the KI is independent of HRP concentration. Inhibition studies have shown that under enzyme-limited conditions the concentration of HRP does not
-1200
-1000
-800
-600
Disk Potential (mV)
F&gwe8. Inhibition constant for cyanide determined at different rates of H202production as determined by the dlsk potential. C ” s : 0.3 mM. Fwther Wak are HRP, 1.25 pM; (hydroxymthyl), as in Figures 3 and 4. The sensitivity of the system increases as the rate of peroxide generation increases, to obtain enryme-llmited ring currents.
affect the K I of cyanide. Thus, by standardizing the enzyme-generated current and its inhibition, the relative magnitude of inhibition is governed by the affinity of cyanide for HRP, and therefore the total concentration of cyanide can be determined, provided the free concentration of cyanide is known. The free concentrationcan be calculated if the enzyme concentration is known. Variations in sensor manufacture would therefore not be important. It should be noted, however that the total cyanide required to inhibit 50% of the current increases with protein concentration due to depletion of the free cyanide concentration. Cyanide Inhibition and Mediator. The inhibition of current by cyanide, when expressed as KI,was found to be independent of ferrocene concentration, as long as the system was operating under enzyme-limited conditions. Increasing the mediator concentration (with enzyme concentrations in the low micromolar range), however, increased the disk potentials required to achieve enzyme-limited kinetics. In our system, the enzyme is therefore not operating at its maximal velocity; rather the ring current is governed by the degree of saturation of the enzyme by the ferrocene. Increasing the mediator concentration produces larger currents but again restricts the disk potentials at which enzymelimited kinetics can be achieved; the actual concentration used would depend on the amount of enzyme present and on the sensitivity of the detecting electrode. The K I for cyanide was not affected by the structure of the mediator. Mediators may therefore be chosen for their stability, interaction with the enzyme, and redox potential. Cyanide Inhibition and pH. The effect of the pH of the electrolyte buffer on cyanide’s inhibition of HRP-generated catalytic current was determined. In contrast to the effect of pH on enzyme activity, the binding of cyanide was found to be unaffected by H+ concentration between pH 5.0 and 8.0. This is in agreement with studies by Dunford et al. (N), which suggest that although cyanide binding is dependent on the protonation state of the enzyme, it only decreases below pH 5.0. These results show that pH 7.0 is a suitable pH value for operation of this sensor, which as mentioned earlier is most suitable for enzyme stability. Enzyme Immobilization. Studies similar to those just described were performed by using an immobilized catalytic system. Enzyme immobilization is desirable for a variety of reasons. The stability of HRP increases following covalent coupling to supports (35). Immobilization of HRP to the carbon disk prevents protein fouling at the secondary (platinum) electrode and therefore prevents any decrease in the
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
sensitivity of current measurements. Furthermore, by restricting the enzyme to a specific site, the catalytic activity is easier to control and mathematically model and therefore optimize (29,30,36,37). The immobilization also increases the efficiency of the enzyme electrode (36)and would therefore support the possibilities for miniaturization. The enzyme immobilization was realized (as described in the Experimental Section) by using a BSA linker such as that described by Kamin and Wilson (30). BSA was coupled to an oxidized carbon electrode by means of carbodiimide activation. HRP was coupled to BSA through carbohydrate moieties that had previously been oxidized with NaI04, in a manner which has been very successful in the labeling of antibodies for enzyme immunoassays (26). There are several advantages of carbohydrate attachment. There is decreased risk of immobilization in a manner that restrids the diffusion of substrate to active site. Avoiding direct amino acid attachment may decrease enzyme denaturation at the the electrode surface. In addition, the large carbohydrate composition of HRP allows for multiple sites of immobilization, which may deliver a more secure and stable modified electrode product. The incorporation of a BSA linker was done for several reasons. It provided a layer of amine groups that could react with the aldehyde groups created from carbohydrate oxidation. The creation of a protein cushion protects HRP from the reductive potentials at the disk. It also provides a buffering microenvironment for the electrochemistry occurring at the electrode surface (such as pH changes). The spacer may also result in less ferrocenium being reduced directly at the disk. The effects of this spacer on ring collection efficiency are unknown. The enzyme-modifiedelectrode was analyzed for peroxidase activity through a colorimetric method (38). The electrode was immersed in an assay mixture and rotated at a speed that overcame any diffusion limitations (>4 cps). Absorbance changes were monitored by means of a spectrophotometer at a single wavelength. The actual enzyme activity was then determined from a standard curve. In this way, the electrodes were checked for activity rather than for the amount of protein that was immobilized. The amount of enzyme immobilized based on its activity was equivalent to approximately 2 X lW3 mol cm-2, which is similar to concentrations achieved through other immobilization procedures (37). The enzyme electrodes were analyzed for the ability to generate a catalytic ring current as described previously in this report. Higher concentrations of ferrocene (1mM range) were required to generate currents greater than 100 nA. Cyanide was found to inhibit this ring current in a manner analogous to the inhibition of HRP in solution. The apparent KI values for cyanide increased to 20 pM in this system. This is still in the low ppm range and therefore provides a suitable sensor design. This modified electrode was found to remain stable for more than 6 months when stored in 1M phosphate buffer at pH 7.0 and analyzed for activity and sensitivity at least once per week. Long-term continuous operation is currently under investigation. Rate of Response of System, Temperature Studies, and Selectivity. The rate of response of the sensor to the introduction of cyanide is governed by the rate of binding of the poison. In our system and with our instrumentation, an inhibition of current was seen as soon as cyanide was added. With a concentration of 2 pM cyanide, the inhibition reached equilibrium within 10 s. By transferring the electrode to a fresh solution of ferrocene, it was found that the this inhibition was rapidly reversible. Temperature studies revealed that the sensor could be operated between 5 and 40 "C. For inhibition studies, attainment of equilibrium is slower at lower tem-
2435
peratures. The KI at 5 "C decreases to about 1.5 pM. The current produced at various temperatures by the uninhibited system was analyzed by means of the Arrhenius equation, and the energy of activation (E.) of the rate-limiting step was found to be around 18 kJ mol-'. Selectivity studies have begun in this laboratory, and preliminary results show that typical HRP binding anions such as F and N; do not inhibit ring current up to concentrations of 10 mM. NazS has a KI of about 100 p M further examination of this inhibition revealed that Sz was serving as a good reductant for compounds I and I1 and thus competing with ferrocene for oxidizing equivalents. The inhibition by this substance decreased with enzyme turnover, due to its depletion through oxidation. It should be noted that a universal sensor for toxin gases is also desirable, and the ability for HRP to recognize more toxins is being studied.
CONCLUSIONS The dual working enzyme electrode provides an alternative method for sensing the presence of cyanide. Both the detection and determination of cyanide can be accomplished by analyzing the inhibition of current by steady-state or kinetic methods. The system operates by creating a constant catalytic current that is sensitive to the presence of poisons; this current is diagnostic of the catalytic state of the enzyme. A sensitive and stable enzyme, HRP, has been incorporated, and the primary and secondary electrodes both generate and regenerate substrates. The constant measurement of the difference between ring current at 0 and -1.2 V (for example) creates conditions where the enzyme activity is the only generator of ferrocenium; thus false signals are avoided. The introduction of electroactive or electropassive interferents would be noted by a change in the baseline (disk at 0 V) ring current. With any enzyme electrode under continuous operation, the feature of substrate depletion must be addressed. For this sensor, the balance between oxygen depletion through peroxide formation and its replacement by diffusion back into solution is one such consideration. It is possible to calculate or simulate both effects, and the results would depend on the electrodes' area and the overall sensor geometry. Although long-term operations with the rotating ring-disk system (by no means the optimal configuration) are still awaiting completion, we have found the depletion of oxygen through bulk electrolysis to not be a problem within at least 48 h of operation. Of equal importance is the depletion of ferrocene through its oxidation. This depletion would depend on its rate of oxidation and on the collection efficiency. We have developed methods for 100% collection of oxidized ferrocene (manuscript in preparation), which solves this problem. The model system described here reacts rapidly (within seconds) upon the introduction of cyanide and is reversible. The sensor is sensitive for the detection of cyanide into the submicromolar range. A rough calculation using Henry's law shows that for a gaseous concentration of 1 ppm a solution concentration of 10 pM is achieved at equilibrium (39). HRP, the biological component, is commercially provided in high purity and is very stable once immobilized. Temperature and pH fluctuations do not greatly affect the sensor's performance. The system regenerates the substrates by reducing ferrocenium; the oxygen availability is determined by the diffusion of oxygen from the atmosphere into solution, thus providing a continual source of hydrogen peroxide, provided the system is well buffered. The rate-limiting factor for the detection of cyanide would be its diffusion from the gaseous phase into the aqueous phase. Present developments in membrane technology allow for ways to minimize this barrier. In addition current developments in microelectrode technology, especially interdigitated electrodes (40)and microband electrodes (41),provide the pos-
2436
ANALYTICAL CHEMISTRY, VOL. 62, NO. 22, NOVEMBER 15, 1990
sibility of miniaturization. The constant current produced by interdigitated electrodes could be an analogous sytem to replace the ring-disk system. It is possible to measure peroxidase activity by using only a single rotating disk electrode, as well as inhibition of that activity. The disk current would be the sum of oxygen reduction and ferrocenium reduction (ADR) currents, with the latter contributing much less than the former under the desired conditions of low enzyme loading. We have found the inhibition of this ADR current to be an inaccurate method for cyanide detection because of its minor contribution to total current. With a secondary electrode potentiostated at 0 V, the background current is small and constant, thus allowing for accurate cyanide detection. We have attempted to replace the ring-disk system by a single static electrode using a potential pulse sequence, where a potential of -1 V is applied in the presence of oxygen, HRP, and mediator, to generate hydrogen peroxide. The electrode is then left on open circuit for a second period to allow the buildup of ferrocenium ions by the enzyme-catalyzedreaction. Finally, the ferrocenium ions are detected by their reduction at 0 V. Although our results are very preliminary, the currents observed during the application of 0 V are indistinguishable from the background. This is probably due to the ferrocenium being generated and rapidly reduced during the -1 V pulse, effectively destroying the peroxide during this period. Although further research is being carried out to simplify the system, we believe a two-working-electrode system provides the greates sensitivity and reproducibility. In summary, the incorporation of a biological component into a toxin-sensing system creates an "electrochemical canary", which, through its mimicry, may provide a more sensitive and specific device for the detection of poisons. The basis for its sensing is on the biological action of a toxin rather than on its molecular structure.
LITERATURE CITED (1) Wilson. G. S.; Thevenot, D. In Eiosensors: A Practical Approach; Cess, A. E. G., Ed.; Oxford University Press: Oxford, U.K., 1990; p 1-16. (2) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hili, H. A. 0.; Aston. W. J.; Higgins, I. J.; Plotkin, E. 8.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem . 1984, 56 667-67 1. (3) Eddowes, M. J. In Biosenscfs: A PracticaIApproech; Cess,A. E. G., Ed.; Oxford University Press: Oxford, U.K., 1990 p 21 1-262. (4) Aibery, W. J.; Cass, A. E. G.; Shu, 2 . X. Eiosens. Bioelecf.1990, 5 , 367-378. (5) Singh, H. B.; Wasi, N.; Mehra, M. C. Int. J. Environ. Anal. Chem. 1986, 2 6 , 115-136. (6) Aklridge, W. N. Analyst 1945, 7 0 , 474-475.
OUdbeUn. G. G.; Kramer, D. N. Anel. (3".1966, 38,834-636. Clark, W. J . chem.Soc. 1926, 749-775. Bentby, A. E.; Alder, J. F. A M I . Chkn. Acta 1689, 222, 63-73. Ntcholls. P. Trends Blochsm. Scl. 1983, 8, 353. Albery, W. J.; Cass. A. E. G.; Hubbard, J. A. M.; Shu, 2. X. Bkchem. SOC. Trans. 1988. 74, 1212-1213. (12) Dunford, H. B.; Stiliman, J. S. Coord. Chem. Rev. 1978, 79, 167-251. (13) Dunford, H. B. Adv. Inorg. Bioahem. 1982, 4 , 41-68. (14) Frew, J. E.; Jones, P. Adv. Inorg. 8Mnwg. Mech. 1984, 175-212. (15) Epton. R.; Hobson, M. E.; Marr, G. J . Organom&. Chem. 1678, 749, 231-244. (16) Frew, J. E.; Harmer, M. A.; Hili, H. A. P.; Libor, S. I. J . Electfaanal. Chem. Interfacial Electrochem. i988, 207, 1-10. (17) Hill, H. A. 0.; Sanghera, G. S. In Bkrpensors: A PracticalAppraech; Cess, A. E. G., Oxford University Press: Oxford, U.K., 1990; p 19-45. (16) Keiiin, D.; Mann. D. R o c . R. Soc. 0 1937. 122, 119-137. (19) Blumberg. W. E.; Peisach, J.; Wittenberg, B. A.; Wittenberg, J. B. J . Bioi. Chem. 1968, 243, 1654-1662. (20) Dolman, D.; Newell, G. A.; Thurlow, M. D.; Dunford, H. B. Can. J . Blochem. 1975, 53,495-501. (21) Keilin, D.; Hertree, E. F. Biochem. J . 1951, 49, 66-104. (22) Cotton, M. L.; Dunford, H. B.; Raycheba. J. M. T. Can. J . Biochem. 1973, 57,627-631. (23) Ak"y. W. J.; Hitchman, M. L. Rlng-Lxfc €kfrodes; Ciarendon Press: Oxford, U.K., 1971. (24) Hitchman, M. L. Measurement of Dissdved Oxygen; John Wiiey and Sons: New York, 1976. (25) Laval, J.-M.; Bourdibn. C.; Moiroux, J. J . Am. Chem. SOC. 1984, 106, 4701-4706. (26) Tijssen, P.; Kurstak, E. AMI. Blochem. 1984, 736,451-457. (27) Vogei, A. I. Quantltat/ve Inorgenic Anaiysk Inckrdyng Elementary InStrw"eta1AnaIyS16, 3rd ed.;Longmans, Green and Co., Ltd.: Essex, U.K.. 1961. (26) Cormier, M.J.; Richard, P. M. J. M I . Chem. 1968, 243, 4706-4714. (29) Shu. F. R.; Wilson, G. S. Anal. Chem. 1978. 48. 1679-1686. (30) Kamin, R. A.; Wilson, G. S. Anal. Chem. 1980, 52, 1198-1205. (31) Cess, A. E. G.; Davis, G.; Green, M. J.; Hill, H. A. 0. J. Electmanal. Chem. Interfacial Electrochem. 1965. 190, 117-127. (32) Hayashi, Y.; Yamazaki, I. J . Eiol. Chem. 1979. 254, 9101-9106. (33) Cotton, M. L.; Dunford, H. B. Can. J . Chem. 1973, 57, 582-587. (34) Dunford, H. B.; Hewson, W. D.; Stelner, H. Can. J . Chem. 1978, 56, 2844-2852. (35) DAnguiro, L.; Gailani, S.; Cremonesi, P. I n 0loc8tal)rsls h Orgbnk M d a ; Laane, C., Tramper, J., Lilly, M. D.; Elsevier Press: Amsterdam, 1987. (36) Laval, J.-M.; Bourdlllon, C.; Moiroux, J. J . Am. Chem. Soc. 1984, 706. 4701-4706. (37) Bowdilion, C.; Beley, M. Now. J . Chim. 1983, 7 , 521-526. (36) Putter, J.; Backer, R. Enzymes 7: Oxoredwtases, Transferases; Methods Of Enzymatic Analysis, Vol. 111; Burgmeyer, H. U., Ed.; Verlag Chemle Weinheim: Basel, 1983; pp 286-293. (39) International CritEcal Tables of Numerical mta, phvsics. Chemkby and Techolcgy; Washburn, E. W., Ed.; Mc&aw-HIII Book Co., Inc.: New York, 1928; p 261. (40) Seddon, 8. J.; oirautt, H. H.; Eddowes, M. J. J . Electraanal. Chem. 1989, 266, 227-238. Interfacial € k t r " . (41) Hiii. H. A. P.; Klein, N. A.; Psalti, I. S. M.; Walton, N. J. Anal. Chem. 1989, 67, 2200-2206. (7) (8) (9) (10) (11)
RECEIVED for review April 23,1990. Accepted August 2,1990. This work was supported by the Ministry of Defense. M.H.S. is a recipient of the Overseas Research Student (ORs)Award.
