Anal. Chem. 1994,66, 510-515
Novel Membraneless Amperometric Peroxide Biosensor Based on a Tetrathiafulvalene-pTetracyanoquinodimethane Electrode Ulrlch Korell and Ursula E. Splchlger' Department of Organic Chemistty, Swiss Federal Institute of Technology (ETH), Universitatstrasse 16, CH-8092 Zurich, Switzerland
Amperometric biosensorsfor the detectionof hydrogen peroxide are prepared by adsorbing peroxidase (POD,EC 1.11.1.7, lipophilized with caprylic aldehyde) to TTF-TCNQ/silicone oil paste electrodes. This is the first time a reductaseis coupled to an organic conducting salt electrode. At -50 mV vs Ag/ AgCl and pH 6.0, the current vs concentrationfunction can be describedby the enzymekineticMichaelis-Menten formalism. Stable signals are obtained within 10 s. The detection limit is typically in the low nanomolar range for H2Oz. The enzyme stability under storage, standby, and various operation conditions is discussed. The coupling of immobilized redox enzymes to amperometric electrodes is a well-established technique for the preparation of biosensors.1,2 In comparison with other analytical techniques, such sensors combine the simplicity of amperometric assays with the substrate selectivity of the enzymes. In addition, they are cost-effective since a small amount of immobilized enzyme can be used for a large number of determinations. In this study, amperometric electrodes were prepared from lipophilized horseradish peroxidase (POD, EC 1.1 1.1.7), the organic conducting salt tetrathiafulvalene-p-tetracyanoquinodimethane (TTF-TCNQ), and silicone oil (Si oil). The enzyme catalyzes the reduction of hydrogen peroxide by a broad variety of electron donors, e.g., those released from TTF-TCNQ electrodes. A number of publications on amperometric POD electrodes already exist. Most of the biosensors described so far rely on an electron donor dissolved in the working buffer, e.g., ferro~yanide3-~or iodideS8v9 Recently, there have been reports on apparently direct electron transfer between surface-oxidized carbon electrodes and ~0~.1&14
(1) Mascini, M.; Guilbault, M. M. Biosensors 1986, 2, 147. (2) Kalcher, K. Electroanalysis 1990, 2, 419. (3) Yao, T.; Sato, M.; Kobayashi, Y.; Wasa, T.Anal. Biochem. 1985,149, 387. (4) Kojima, J.; Morita, N.; Takagi, M. Anal. Sci. 1988, 4, 497. (5) Wang, J.; Ozsoz, M. Electroanalysis 1990, 2, 647. (6) Yao, T.; Satomura, M.; Wasa, T.Anal. Chim. Acta 1992, 261, 161. (7) Schubert, F.;Saini, S.; Turner, A. P. F.; Scheller, F. S e w . Acruarors 1992, 8 7 , 408. (8) IvnitskkD. M.;Dzantiev,B. B.; Egor0v.A. M.;Kashkin,A. P. Prikl. Biokhim. Mikrobiol. 1985, 21, 821. (9) Ivnitskii, D. M.; Sitdikov, R. A.; Kurochkin, V. E. Anal. Chim. Acta 1992,
261, 45. (10) JBnsson, G.; Gorton, L. Electroanalysis 1989, 1, 465. (1 1) Wollenberger, U.; Bogdanovskaya,V.; Bobrin, S.; Scheller, F.; Tarasevich, M. Anal. Letr. 1990, 23, 1795. (12) Kulys, J.; Schmid, R. D. Bioelectrochem. Bioenerg. 1990, 24, 305. (1 3) Gorton, L.; JBnsson-Pettersson, G.;Csdregi, E.; Johansson, K.; Dominguez, E.; Marko-Varga, G.Analyst 1992, 117, 1235.
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Analytical Chemistry, Vol. 66,No. 4, February 15, 1994
Organic conducting salts are unique in that they are intrinsically conducting at room temperature. Thus, electrodes can be prepared from TTF-TCNQ and Si oil without any further conducting additive (as, for example, graphite powder in carbon paste electrodes). Moreover, crystalline TTFTCNQ is insoluble in water but releases more or less soluble electroactive species (TTF+, TTFO, TCNQO, TCNQ-, or TCNQ2-) upon destruction of thecrystal latticeat the electrode surface by either cathodic or anodic processes. These socalled mediators can shuttle electrons between redox enzymes and the electrode surface. The rate of release of each mediator species and the efficiencyof the catalytic cycle can be controlled by choosing the appropriate applied potential, buffer concentration, pH and stirring rate.15J6 Hence, biosensors based on TTF-TCNQ are very versatile in that these parameters can be optimized for each analytical application. It was shown recently that the electron transfer through these mediators is so efficient that TTF-TCNQ/enzyme (sulfite oxidase,17 xanthine oxidase1*J9) electrodes can be operated in airsaturated solutions since molecular oxygen is not competitive as an electron acceptor. In the past few years, a number of redox enzymes have been coupled to organic conducting salt electrodes.17-*2 However, all enzymes employed so far are oxidases or dehydrogenases, where the mediator transfers electrons from the reduced form of the enzyme to the electrode surface (Figure 1a). In this study, we report on the first coupling of a reductase to an organic conducting salt electrode. In this novel type of biosensor, the mediator transfers electrons in the opposite direction, Le., from the electrode surface to the oxidized form of the enzyme (Figure lb). The POD was lipophilized by reaction with caprylic aldehyde and then immobilized on a TTF-TCNQ/Si oil paste electrode by adsorption. This technique avoids diffusion problems associated with membranes commonly used for enzyme immobilization. EXPERIMENTAL SECTION Reagents. Acetonitrile (puriss. p.a.), caprylic aldehyde (purum), hydrochloric acid (fuming, puriss. p.a.), peroxidase (14) CsBregi, E.; Gorton, L.; Marko-Verga, G. Anal. Chim. Acta 1993, 273, 59. (15) Korell, U.; Lennox, R. B. Anal. Chim. 1992,64, 147. (16) Zhao,S.; Korell, U.;Cuccia, L.;Lennox, R. B. J . Phys. Chem. 1992,96,5641. (17) Korell, U.; Lennox, R. B. J . Electroanal. Chem. 1993, 351. 137. (18) Korell, U.; Spichiger, U.E. Electroanalysis 1993, 5, 869. (19) Korcll, U.; Spichiger, U. E. ElectroaMlysis, in press. (20) Albery, W. J.; Bartlett, P. N.; Cass, A. E. G.Phil. Trans. R . Soc. London B 1987, 316, 107. (21) McKenna, K.; Brajter-Toth, A. Anal. Chem. 1987, 59, 954. (22) Zhao, S.;Lennox, R. B. Anal. Chem. 1991,63, 1175.
0003-2700/94/036605 10$04.50/0
0 1994 American Chemical Society
4 electrons per hypoxanthine
a
t mediator (reduced form)
xanthine oxidase (oxidized form)
mediator (oxidized form)
xanthine oxidase (reduced form)
0
OH
hypoxanthine
uric acid 2 electrons
Per H202
b
mediator (reduced form)
mediator (oxidized form)
peroxidase (oxidized form)
peroxidase (reduced form)
OH-
H202
Flgure 1. Flow of electrons in mediated amperometric enzyme electrodes. (a, top) Typical oxidase (xanthine oxidase, EC 1.2.3.2);(b, bottom) horseradish peroxidase.
(POD, EC 1.11.1.7, lyophilized, 854 unit/mg, from horseradish), p-tetracyanoquinodimethane(TCNQ, purum), tetrasodium pyrophosphate decahydrate (MicroSelect), and tetrathiafulvalene (TTF, puriss.) were obtained from Fluka (Buchs, Switzerland). Silicone oil (Si oil), high temperature
( d = 1.050 g c m 9 , was purchased from Aldrich (Steinheim, Germany). Aqueous solutions were prepared with doubly quartzdistilled water. All experiments were carried out using 100 mM sodium pyrophosphate buffer, adjusted to pH 6.00 with HC1. The buffer was stored at room temperature for up to 2 weeks. Hydrogen peroxide stock solution (8.8 mM) was prepared from 30% H202 (p.a., Merck, Darmstadt, Germany), stored at room temperature protected from light, and used within 24 h. Tetrathiafulvalene-p-tetracyancquinodimethane (TTFTCNQ) was synthesized according to the procedure described by Ferraris et al.23 and was not recrystallized. Elemental analysis of the material employed in these studies confirmed the 1.OO:l.OO stoichiometry of the salt. TTF-TCNQ/Si oil paste was prepared from TTF-TCNQ and Si oil (1 .OO:1.60 w/w) by mixing using a stainless steel spatula. Native enzyme suspension was made from 5.0 mg of POD lyophilizate and 100 pL of sodium pyrophosphate buffer. It was stored at 4 OC and used within 4 days. Lipophilized enzyme suspension was prepared as follows: 5.0 mg of POD lyophilizate was mixed with 100 pL of caprylic aldehyde and spread on a glass plate. Water from the reaction and excess aldehyde were removed by keeping the mixture in a desiccator over calcium chloride at > 11 mbar for 10 h. The lipophilized enzyme was stored at 4 OC. Immediately before preparing the first electrode, the dry product was mixed with 100 NL of sodium pyrophosphate buffer, and the suspension was stored at 4 OC. Enzyme Electrode Preparation. Rotating disk electrodes were prepared from Kel-F tubing of 4.00-mm i.d. A Teflon disk with a hole (diameter 1.0 mm) in the center was pressfitted into the tubing close to one end, leaving a cavity of approximately 3-mm depth. The other end of the tubing was press-fitted into a brass connector, which was in electrical contact with the rotator. Electrical contact between the brass connector and the cavity was made by a Pt wire (diameter 1.O mm), which was held in pIace by the Teflon disk. Immediately before using the electrode, the cavity was filled with TTFTCNQ/Si oil paste so that the end of the Pt wire in the cavity was completely covered by the paste. The surface of the paste was smoothed with a glass slide, carefully wiping off any excess material from the Kel-F tubing. The resulting disk electrode had a diameter of exactly 4.00 mm and a geometric area of 12.57 mm2. Enzyme electrodes were prepared by placing 10 p L of the respective POD suspension on the electrode surface. The electrode was dried at room temperature, which took ca. 1-1.5 h and then rotated in sodium pyrophosphate buffer at 25 Hz for 15 min to remove any enzyme not firmly adsorbed. The wash buffer was then discarded. Instrumentation and Procedure. An Ag/AgCl electrode (Moller, Zurich, Switzerland), filled with saturated aqueous KC1 solution, served as a reference electrode. The counterelectrode consisted of a 200-mm-long Pt wire of 0.30-mm diameter. The electrode system was controlled by a potentiostat Model OE PP2 (Oxford Electrodes, Oxford, England). The working (23) Ferraris, J.; Cowan, D. 0.;Walatka, V.; Perlstein, J. H. J . Am. Chem. Sm.
1~3.95.948.
Analytical Chemistry, Vol. 66, No. 4, February 15, 1994
511
j 50 nA I
I
I
+
4
1
Figure 2. Typical response of the electrode based on lipophillzed POD. The trace starts at the left where 100.0 mL of pyrophosphate buffer is present. Each arrow indicates the addition of 10.0 pL of 8.8 mM H202,at constant rotation rate of 25.00 Hz. The time span between the additions was 120 s.
electrode was generally poised at -50.0 mV vs AgfAgCl and rotated by an Oxford Electrodes rotator. The current was recorded on 2 chart recorder (Model 314, W+W, Basel, Switzerland). Connections were made with shielded cables, but no further means against electric noise pickup were employed. The measuring cell consisted of a cylindrical glass beaker of 70-mm height and 50-mm i.d. and was covered with a polyethylene disk with appropriately drilled holes for the electrodes. The cell was kept under water-saturated nitrogen and protected from light. All experiments were carried out at 25.0 f 0.2 OC. The baseline (Le., the background current) was established in 100.0 mL of HzO2-free sodium pyrophosphate buffer. It was considered stable when the drift was 10.2 nA/min. In experiments performed at constant rotation rate (Le., 25.00 Hz), one increment of 8.8 mM H202 solution (10.0 or 50.0 pL) was added every 120 s by means of a motor buret (Dosimat 655, Metrohm, Switzerland). Between runs, the electrodes were kept in peroxide-free sodium pyrophosphate buffer under operating conditions. When varying the rotation rate, it was changed every 120 s in the sequence 25.00,20.25, 16.00, 12.25,9.00,6.25, and 25.00 Hz again. After this, one 50-pL increment of 8.8 mM hydrogen peroxide solution solution was added, and the rotation rate sequence was restarted. The program was controlled by a laboratory-made electronic
0
.loo
. 2
4
I
.zoo
3 2
.300
1
0
10
20
30
40
50
60
70
[ H z W 1 PM
Figure 3. Typical set of four calibration curves (1-4), recorded with the same electrode, based on ilpophilized POD. Freshly prepared electrode (e, 1); 2 (H, 2), 18 (A,3), and 20 (+, 4) h after the first run. Each run required 60 min; between runs, the sensor was operated in peroxide-free buffer. For comparison: new TTF-TCNQ/Si oil electrode, coated with native peroxidase (0). The baseline current (Le., at [H202] = 0) was subtracted.
30 nA/pM at low concentrations, the detection limit of this system (i.e., the analyte concentration giving rise to a current increase of 3 SD) can be estimated as 0.010 pM H202. A typical set of four current versus concentration curves (1-4) measured with an electrode based on Iipophilized POD is given in Figure 3. The data of curve 1 was obtained with the freshly prepared biosensor; those of curves 2-4 were obtained after 2, 18, and 20 h of operation, respectively. Between assays, the electrode was operated in HzOz-free buffer. Curve 5 was recorded using a new POD electrode freshly prepared from native enzyme and is given for comparison purposes. Evidently, lipophilization of the enzyme with caprylic aldehyde enhances the response behavior significantly. To evaluate the kineticsof the enzyme-catalyzed reduction of H202, the data of Figure 3 were plotted as Hanes graphs (Figure 4). These plots are linear if the data fit the Hanes equation (adapted from ref 25):
RESULTS AND DISCUSSION A typical response curve of the peroxidase biosensor is given in Figure 2. After the addition of H202 stock solution, the reductive current rises steeply to reach a stablevalue within ca. 10 s. Mixing of the solution is effected exclusively by the rotation of the working electrode at 25.00 Hz. The time required for homogeneously distributing the 10.0-pL stock solution added to the buffer (100.0 mL) is about 3-5 s as estimated from experiments with a colored test solution. Hence, the sensor is expected to exhibit a response time of considerably less than 10 s when operated in smaller cells. The width of the current trace is normally 0.3-0.5 nA. Using a standard deviation of fO.l nA and a typical slope of
where [SI is the concentration of substrate (Le., HzOz), i,,, is the maximum current (here, the most negative current), and K M E is the apparent Michaelis constant (i.e., the substrate concentration at which 50% of ,i is measured). Given that
(24) Korell, U. Ph.D. Thesis, ETH Zurich, 1993.
( 2 5 ) Segel, I. H. Enzyme Kinefics, 1st ed.;Wiley: New York, 1975.
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Analytical Chemistry, Vole 66,No. 4, February 15, 1994
is] f i =
fim,,
+ KME/imax
(1)
4.8
-20
'
"
-10
i
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I
0
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I
10
'
20
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'
I
30
.
40
I
.
50
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(
60
70
Flgure 4. Data of Figure 3, plotted as Hanes graphs. The same symbols were used. Table 1. Enzyme Klnetlc Parameters of Data Presented In Flgures 3 and 4 operation , i 3 of Km r20f enzyme type time (h) (A)initial, z (pM) Hanes plot
lipophilized
native
0.0
2.0 18.0 20.0 0.0
-441.8 -370.8 -283.9 -180.2 -106.1
100.0 83.9 64.3 40.8
14.2 17.4 17.8 18.3 19.5
0.999 0.995 0.999 0.999 0.998
the stoichiometry of the enzyme-catalyzed reduction of H202 is exactly defined (one molecule accepts two electrons), i and i,,, are proportional to the turnover rate u and the maximum turnover ratev,,,, respectively. The Hanes equation is directly related to the Michaelis-Menten f o r m a l i ~ m : ~ ~
v=
[Slumax/([Sl+
0
5
10
15
20
25
operation time / h
[ H A 1 I PM
KM)
(2)
Hence, data yielding linear Hanes plots will equally satisfy the Michaelis-Menten equation.26 Since the linearity of the Hanes plots is generally very good, the data were analyzed according to the MichaelisMenten formalism. The enzyme kinetic parameters calculated from the data presented in Figures 3 and 4 are given in Table 1. Lipophilization of POD with caprylic aldehyde resulted in a 4-fold increase in i,,,. This is not surprising since the immobilization of enzyme on the lipophilic electrode surface is exclusively based on hydrophobic interaction. The K M Eof the sensor with lipophilized POD is even lower than the one of the sensor with native enzyme, indicating that the lipophilization procedure does not generate an additional diffusion barrier around the enzyme molecule. Furthermore, the former sensor had a significantly better stability than the latter one, with which in the second run (not shown in Figure 3) virtually no catalytic activity was measured. The loss of relative enzyme activity between the second and the third runs (i.e., within 16 h), expressed in percentage of the initial imaX(Table l ) , is of similar magnitude as that between the first and the second or between the third and the fourth runs (Le., within 2 h each). This indicates that the procedure of changing the cell solution (including the exposition of the electrode to open circuit conditions) causes (26) Inthiscontext, [S],KD(e,andi,generallyreferto thesubstratebeingreduced during the enzyme-catalyzed reaction, Le., H1O2. i-and KMeare "apparent" values determined from Hanes plots.
Flgure 5. Current vs operation time graphs of two electrodes, based on iipophilired POD. One electrode was operated in a buffer containing 18 pM H202 (O),the other one was operated in a buffer contalnlng 62 pM H202(0).The respectivecurrent measuredwlth freshly prepared electrodes was defined as 100% .
a decrease in enzyme activity that is even larger than that within 16 h of continuous operation in peroxide-free buffer. On the other hand, the decrease within 16 h seems to be relatively small if open circuit conditions are avoided, e.g., if the sensor is used in a flow-through system. The effect of the H202 concentration on the sensor lifetime was investigated by continuously operating two electrodes in buffers of different H202 concentrations (Figure 5 ) , one with 18 pM H202 and the other with 62 pM H202. These concentrations correspond to approximately 1 and 3.5 K M E , respectively. The respective current measured with the freshly prepared electrodes was defined as Comparing these results with those of Figure 3, it can be concluded that the relative decrease with time is considerably larger in the buffer containing H202 than with no peroxide. Moreover, the sensor stability is much lower if the concentration is increased from 1 to 3.5 KME.However, given the low detection limit (typically 0.010 pM H202 as estimated above), even a large decrease of relative enzyme activity does not necessarily mean that the sensor is of no use any more. To obtain information about the stability of the suspension of lipophilized POD in pyrophosphate buffer, seven electrodes were prepared after the enzyme suspension had been stored at 4 O C for the time indicated on the abscissa of Figure 6. For each electrode, Hanes plots were prepared from 20 data points between038 and 17.6pM H2Oz (corresponding to the addition of 10 p L each of the H202 stock solution). Obviously, the specific activity of the enzyme suspension does not decrease within 22 days at 4 "C; after some days of storage, it is even higher than directly after preparation. On the other hand; the amount of activity immobilized on an electrode has to be determined for each sensor separately since it cannot be predicted. This scattering may be due to inhomogeneities of the enzyme suspension or to irregular deposition of the enzyme during the drying-on procedure. The sensor-to-sensor reproducibility can possibly be improved by s i m u l t a n e o u s l y preparing a large number of enzyme electrodes under identical conditions. (27) The loss of hydrogen peroxide due to enzyme-catalyzed reduction of HtO2 by the amperometric sensor can be calculated from the current and the time of operation. The resulting decrease in [H202] in the sample solution over 24 h is too small to be relevant in this context. Hence, the decrease in current can be regarded as being proportional to the loss of enzyme activity at the electrode surface.
Analytical Chemistry, Vol. 66, No. 4, February 15, 1994
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electroactive species to the electrode surface:28 j d = 0.62nF~D~/~v-'/~w'/~
.zoo0
0
10
5
15
20
25
enzyme storage time / days 24 i
I
I
I
:
10
20
15
25
enzyme storage time I days
Figure 6. Enzyme kinetic parameters, calculated from Hanes plots (20 data polnts each), obtained wlth seven electrodes, prepared from llpophllized POD suspension that had been stored at 4 O C for the time Indicated. The error bars (1 SD) of the enzyme klnetlc parameters are generally smaller than the symbols used.
i
I
-ILU"T
4
12
20
28
rotation rate / Hz
Flgure 7. Current vs rotation rate w for a new electrode (based on 4.4 (W): 8.8 (0);13.2 llpophilized POD) at dlfferent [H202]: 0.0 (0); (*); and 17.6 pM (A).
The effect of changes in the electrode rotation rate was investigated to obtain additional information about the electrode mechanism (Figure 7 ) . To control the stability of the sensor at the respective H 2 0 2concentration, the current was measured twice at 25.00 Hz, i.e., at the beginning of the rotation rate sequence and at the end (i.e., 12 min later). As expected from previous experiments (e.g., Figure 5), the difference in current between the first and the second signal increases with increasing peroxide concentration. The catalytic current increases with the rotation rate o at all concentrations tested (Figure 7), indicating that the supply of substrate to the enzyme is critical. The slope of the i vs rotation rate graphs decreases with increasing H202 concentrations, since the sensitivity of the reaction rate to changes in the substrate concentration decreases according to the Michaelis-Menten formalism. When the data of Figure 7 at [H202] = 0 pM are plotted in Levich graphs (i vs w1I2, not shown), a good linear fit (r2 = 0.984) is obtained. The Levich equation describes systems where the current is entirely controlled by the transport of 574
Analytical Chemlstty, Vol. 66,No. 4, February 15, 1994
(3)
In eq 3, jd (A cm-2) denotes the steady-state current density under diffusion control, n (dimensionless) is the number of electrons exchanged, F (A s mol-') is the Faraday constant, c (mol cm-3) is the bulk concentration of the electroactive species in the solution; D (cm2s-l) is the diffusion coefficient, v (cm2s-l) is the kinematicviscosity, and w (Hz) is the rotation rate of the disk. In the absence of substrate, the electrode displays "inversen Levich behavior, meaning that the current is controlled by the diffusion of electroactive species away from the electrode surface. Faster rotation generally leads to a greater loss of mediator species to the bulk solution. In the presence of substrate, however, the linearity of the Levich plots is poorer (r2I0.96),indicating that the current is not solely controlled by mass transport. In this case, the enzymatic reaction limits thecurrent significantly. This mixed control of diffusion and catalytic conversion of the substrate is accounted for by the Koutecky-Levich formalism (i-* vs w-1I2 plots).29 This model, however, can only be applied if the background current (i.e., current measured at zero substrate concentration) is zero or negligible in comparison to the catalyticcurrent. Under theconditionsin this study, however, the background currents are of an order of magnitude similar to that of the catalytic ones (Figure 7), and high catalytic current densities (at high substrate concentrations) are not accessible due to the low sensor stability under substrate saturation conditions.
CONCLUSIONS Horseradish peroxidase (POD)is suspended in TTFTCNQ/silicone oil paste electrodes. The amount of immobilized enzyme activity can be increased considerably by lipophilizing the enzyme with the alkylating agent caprylic aldehyde. Stable signals are obtained within 10 s after the addition of H202, including the time required for the homogeneous distribution of the added stock solution within a 100-mL cell. The detection limit is typically in the low nanomolar range. At -50.0 mV vs Ag/AgCl and pH 6.00,the current vs concentration graphs can be described using the enzyme kinetic Michaelis-Menten formalism with very good correlation. Under the conditions investigated, the reductive current generally increases with increasing electrode rotation rate, indicating that the supply of substrate to the enzyme is at least partially signal limiting. Suspension of lipophilized POD in pyrophosphate buffer (pH 6.00) has been stored at 4 O C , and no loss in activity is observed within 22 days. The operational stability of the sensor decreases with increasing H202 concentration. Furthermore, exposing the enzyme electrode to open circuit conditions changes its calibration function. Therefore, it can be optimally employed in a flow-injection system where the electrode is continuously poised at the operation potential and flushed with peroxide-free buffer when not in use. (28) Filinovsky, V. Yu.; Pleskov, Yu. V. In Comprehensive Treafiseof Elecfrochemistry; Plenum Press: New York, 1984; Vol. 9. (29) Gorton, L.; Torstensson, A,;Jaegfeld, H.;Johansson, G. J . Electroam/. Chem. 1984, 161, 103.
In this study, it is demonstrated for the first time that a reductase can be coupled to organic conducting salt electrodes, yielding biosensors of favorable analytical performance. This probably opens up a number of new analytical applications. In particular, the POD Sensor can be used as an indicator electrode for enzymatic reactions where H202is consumed or generated.
ACKNOWLEDGMENT This work was partly supported by the Swiss National Science FoundationReceived for review August 279 1993. Accepted November 1% 1993.' e
Abstract published in Advance ACS Absrracrs, January 1, 1994.
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