Anal. Chem. 1995, 67, 2776-2780
A Polypyrrole/Three-Enzyme Electrode for Creatinine Detection Hitoshi Yamato,* Masaki Ohwa, and Wolfgang Wemet
International Research Laboratories, Ciba-Geigy (Japan) Ltd., 10-66Miyuki-cho, Takarazuka 665, Japan
To detect creatinine, an enzyme electrode was made by co-immobilizationof three enzymes, creatininase, creatinase, and sarcosine oxidase, in an active polypyrrole (PPy) matrix. Besides platinum, polypyrrole doped with a sulfated phenow resin (S-PHE) was used as a base electrode. The device was fabricated by electropolymerization of pyrrole in the presence of water soluble polyanions, enzymes, and a phosphate buffer, using the abovementioned base electrodes. At a potential of 400 mV vs Ag/AgCl, the sensors responded to creatinine with a sufFicient sensitivity. Under a nitrogen atmosphere, the response current was higher when PPy/S-PHE was used as a base electrode. As a main transduction pathway, a direct electron transfer from sarcosine oxidase to PPy chains is discussed. In addition, the influences of the different polyanionic dopants, the thickness of the active layer, and the concentration of sarcosine oxidase in preparation solutions on the sensor‘s performance were examined. Creatinine is known as an end product of the creatine metabolism, and its concentration in blood serum and urinary excretion is not very much affected by dietary changes.’ Consequently, the creatinine level is an important diagnostic index for renal, muscular, and thyroid function. The creatinine concentration is still often detected using a spectrometric method, based on the Jaffe reaction,2 in which creatinine forms an orange-red complex with picric acid. The results obtained in this way are seriously affected by other blood metabolites, such as a-oxo acids or amines. Therefore, an enzymatic method has been developed, which is, however, a time-consuming Because of the lack of a reliable and quick way to detect creatinine blood levels, electrochemical sensors based on three enzymes, creatininase (CRN), creatinase (CR), and sarcosine oxidase (SO), (Figure 1) have been As can be seen in eq 3 of Figure 1, oxygen is needed to reoxidize SO.”.” Using a Clark oxygen electrode, Nguyen et aL6 detected creatinine via the oxygen concentration change. This method, however, is seriously influenced by the oxygen concen(1) Bergmeyer. H. U.; Bergmeyer. J.; Grassl, M. Methods ofenzymatic analysis. Vol. WII, 3rd ed.; VCH Verlagsgesellschaft mbH: Weinheim, 1985; pp 488-
507. ( 2 ) Jaffe. M. 2. Physiol. Chem. 1886, 10, 391.
(3) Kinoshita. T.; Hiraga. Y. Chem. Pkarm. Bull. 1980, 28, 3501. (4) Fossati. P.: Prencipe. L.; Berti, G. Clin. Chem. 1983, 29, 1494. ( 5 ) Siedel, J.; Mollering. H.; Ziegenhorn, J. Clin. Chem. 1984, 30, 968. (6) Ngupen. V. K.; Wolff, C.-M.; Seris. J. L.; Schwing, J.-P.Anal. Ckem. 1991, 63, 611. (7) Tsuchida. T.: Yoda, K. Clin.Chem. 1983, 29, 51. (8) Jorns. M.S. Biochemistry 1985, 24, 3189. (9) K-Konishi. Y.;Suzuki. H. Biochim. Bioghys. Acta 1987, 915, 346.
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tration in blood samples. Tsuchida et al.; developed a Pt-based three-enzyme electrode in which hydrogen peroxide (Figure 1, eq 3) is amperometrically detected. However, this electrode requires a working potential of 600 mV vs SCE. Under such conditions, blood metabolites such as ascorbic acid or uric acid are likely to be oxidized at Pt electrodes leading to incorrect results. Recently, conducting polymers have been paid much attention as an enzymeimmobilizing matix for biosensors.lO,ll When conducting polymers were used for the immobilization of oxidoreductase, in some cases a direct electron transfer from the enzyme to the electrode at lower potentials was discussed. Yabuki et a1.12reported the immobilization of glucose oxidase (GOx) and claimed a direct electron transfer to be responsible for the detected signal. Still, some ambiguity in the electron transfer mechanism remains;13J4however, the immobilization of enzymes in the polypyrrole (PPy) matrices is widely used because of the accurate control of the incorporated amount of enzyme and the high potential of this method to miniaturize the devices. Recently, Aizawa et reported the immobilization of three enzymes, CRN, CR, and SO, in the PPy/CI or PPy/toluenesulfonate matrixes. The resultant electrode responded to creatinine. This paper describes the preparation of an creatinine enzyme sensor in which three enzymes are incorporated into a PPy/ polyanion matrix and some factors influence the sensor performance. As base electrode materials, either Pt or PPy/sulfated phenoxy resin (SPHE)l6-I9is used, and the two are compared. The influences of the different polyanionic dopants for the PPy matrix, the “enzyme layer” thickness, and the concentration of SO in the preparation solutions on the performance of the devices are presented and discussed. The response of the sensors under an inert atmosphere provides evidence for a direct electron transfer mechanism from the enzyme to the active matrix. EXPERIMENTAL SECTION Chemicals. Sarcosine oxidase (EC 1.5.3.1,from Arthrobacter sp., 5.6 unitdmg) was obtained from Toyobo (Osaka, Japan). Creatininase (EC 3.5.2.10, from Microorganism, 8.8 units/mg) and (10) Deshpande, M. V.; Amalnerkar, D. P. Puog. Polym. Sci. 1993, 18, 623. (11) Bartlett. P. N.; Cooper, J. M. J. Electuoanal. Chem. 1993, 362, 1. (12) Yabuki, S.; Shinohara, H.; Aizawa, M.J. Chem. Soc., Chem. Commun. 1989, 945. (13) Belanger. D.; Nadreau, J.; Fortier, G.J , Electroanal. Chem. 1989,274, 143. (14) Cooper, J. M.; Bloor, D. Electroanalysis 1993, 5, 883. (15) Aizawa. M.; Khan, G. F.: Kobatake. E.; Ikariyama, Y.Proc. Int. Con5 SolidState Sens. Actuators, 7th 1993, 522. (16) U’ernet. W; Stoffer, J. U S . Patent 34.514, 1994. (17) Wernet. W. Syntk. Met. 1991, 4 1 . 843. (18) Wernet. W.; Yamato, H.; Kai. K.; Koshiba, T.: Ohwa, 11.;Rotzinger, B. Solid State Ionics 1992, 53-56, 1125. (19) Yamato. H.; Wernet, W.; Ohwa, 11.;Rotzinger, B. Synth. Met. 1993, 57, 3550. 0003-2700/95/0367-2776$9.00/0 0 1995
American Chemical Society
+ H20
Creatinine Creatine
+
H2O
Creaininase
-
Creatine
\
Crutinue
-
Sarcosine
+
Sarcosine oxidrre
Formaldehyde Sarcosine + 0 2 + H2O Figure 1. Reaction scheme for the enzymatic detection of creatinine creatinase (EC 3.5.3.3, from Bacillus sp., 7.0 units/mg) were obtained from Toyo Jozo (Tokyo, Japan). Creatinine from Wako (Osaka, Japan) was used without further purification. Pyrrole (Croda Japan, Osaka) was purified by distillation and stored under nitrogen at 4 "C prior to use. Poly(styrenesulfonate sodium salt) (PSSNa) (Aldrich, Milwaukee, WI) was used as received. Poly(sulfated hydroxyethyl methacrylate trimethylammoniumsalt) (S PHEMA-TA) and poly(sulfated vinyl alcohol-co-vinyl acetate trimethylammonium salt) (SVA-VAC-TA) were synthesized by the sulfation of alcohol groups of poly(hydroxyethy1 methacrylate) (Aldrich) and poly(viny1 alcohol-co-vinyl acetate) (Kuraray, Okayama, Japan) with sulfur trioxide/pyridine complex.16 Other chemicals were reagent grade and were used as received. Milli-Q water was used for all experiments. Preparation of PpY/SPHE Base Electrode. Electrochemical polymerization of PPy/SPHE film was carried out according to the previous r e ~ o r t s . ~ ~The - l ~films were grown using a charge of 5 C/cm2, leading to a film thickness of about 100 pm. After being peeled off from the electrode, PPy/SPHE was successively rinsed with propylene carbonate and ethanol for 5 h each and dried at 50 "C in vacuo overnight. The dried PPy/SPHE film was cut into rectangular pieces (5 x 25 mm2)with a scalpel. The active surface area was controlled by covering with a Teflon tape (Chukoh, Tokyo Japan; ASF-110). Cyclic Voltammetry of Pyrrole. The electrolyte solution used for cyclic voltammetry consisted of 20 mM phosphate buffer (PB) of pH 6.2, 0.2 M pyrrole, and 0.1 M PSSNa. The electrolyte was put into an electrochemical cell in which a Pt plate electrode (0.25 cm2) or a PPy/SPHE electrode (0.25 an2)was placed as a working electrode, and a Pt wire and Ag/AgCl electrode were placed as auxiliary and reference electrodes, respectively. The cyclic voltammetry was carried out with a potential sweep from 200 to lo00 mV at a rate of 20 mV/s for five cycles under a nitrogen atmosphere. Preparation of Enzymes Layer. For the preparation of the PPy/enzymes layer, a Pt plate electrode (0.25 cm2) or a PPy/S PHE electrode (0.25 cm2) was used as the base electrode. The electrolyte solution used for the preparation consisted of, unless otherwise specified, 20 mM PB (PH 6.2), 0.2 M pyrrole, 0.1 M PSSNa, 5 mg/mL CRN, 5 mg/mL CR and 2.5-20 mg/mL SO. Enzyme immobilization was typically performed by a potentiostatic method at 800 mV in a NTsaturated unstirred solution. The enzyme layer thickness was controlled by the amount of charge passing during the polymerization. The PPy/enzymes electrodes were immersed in a PB of pH 7.5 for 30 min after preparation to remove adsorbed enzymes and then thoroughly rinsed with a PB of pH 7.5, followed by air-drying. The dried PPy/enzymes electrodes were stored at -20 "C under an air atmosphere.
Steady-State Current Measurement for the Creatinine Assay. The determination of creatinine by PPy/enzymes electrodes was performed by an amperometric method. A PPy/ enzymes electrode was placed in a threeelectrode cell containing
Urea
+
Glycine +
H202
(3)
20 mM PB of pH 7.5 under nitrogen, unless otherwise noted. When a potential of 400 mV was applied, an anodic background current flowed and gradually decreased with time. Therefore, the electrode was polarized at 400 mV for 16 h to reach a steady state prior to the creatinine determination. After this conditioning process, a known amount of creatinine was injected into the solution under stirring. After 1 min of stirring, the current was monitored under the steady state for 6 min. From the results of the measurement using various creatinine concentration, a calibration curve was plotted.
Steady-StateCurrent Measurement for the Oxidation of Formaldehyde and Hydrogen Peroxide. To prepare a PPy/ PSS layer on PPy/SPHE films, a solution of 20 mM PB, 0.2 M pyrrole, and 0.1 M PSSNa was used for the electropolymerization with a charge of 1.6 C/cm2. The responses of Pt, PPy/SPHE, and PPy/SPHE covered with PPy/PSS electrodes toward formaldehyde and hydrogen peroxide were detected at a potential of 400 mV vs Ag/AgCl in a 20 mM PB solution at pH 7.5. After conditioning at 400 mV for 30 min, the response current was monitored with injections of defined quantities of formaldehyde or hydrogen peroxide into the stirred buffer solution. Apparatus. All the electrochemical experiments were carried out with a HA-501 potentiostat/galvanostat and a HAB-151function generator (Hokuto Denko, Tokyo, Japan). The signal was recorded by a Compaq 386 computer with homemade software via an A/D converter. RESULTS AND DISCUSSION
PPy/S-PHE as a Base Electrode for the Creatinine Sensor. PPy/SPHE is known as one of the most stable conducting polymers. Its redox potential is around -200 mV vs Ag/AgCl, where it undergoes reversible doping-dedoping reactions in, e.g., propylene carbonate solutions. In the potential range between -200 and 800 mV, PPy/SPHE behavior is electrochemically inert in aqueous solutions even after extended use, and PPy/SPHE has excellentmechanical proper tie^.'^-^^ Therefore, PPyISPHE seems to be a suitable base electrode material for sensor devices. Furthermore, PPy/SPHE shows a unique selectivity toward the electrochemically active substances in aqueous solution. Cyclic voltammetry revealed the usual redox behavior of ferrocene compounds; however, &[Fe(CN)61 shows no significant electrochemical response. This is regarded as a consequence of the highly hydrophobic surface of PPyPSPHE. The electrochemistry of a growing PPy/PSS film on PPy/S PHE using cyclic voltammetry was studied in an aqueous PB solution at pH 6.2 and was compared to similar experimentsusing a Pt base electrode. Cyclic voltammograms for both electrodes are shown in Figure 2 for consecutive scans. During the first scan, the polymerization of PPy/PSS on PPy/SPHE started at 700 mV. The anodic current continued to increase after switching the scan direction at 1000 mV. In subsequent scans, the polymerization potential decreased to values below 600 mV. To Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
2777
Buffer injection Creatinine in' tion (final conc.:
%w
(500
w
(1
i
i
10 Time (sec)
Figure 3. Typical current-time curve of a steady-state current measurement for a creatinine assay. PPy/PSS/enzymes electrode (polymerization charge, 1.6 C/cm2) was polarized at 400 mV in 20 mM PB (pH 7.5).
n 200
4M)
600
800
Potential (mV)vs. Ag/AgCl
1200
40 E e 4
30
5
800
BM
600
er:
400
I
20
1000
10
200 0
200
400
800
1000
Potential (mV)vs. Ag/AgCI
Figure 2. Cyclic voltammograms for the oxidation of pyrrole on PPy/ S-PHE and Pt electrodes. Electrolyte: 20 mM PB (pH 6.2) containing 0.2 M pyrrole and 0.1 M PSS-Na. Scan rate, 20 mV/s.
generate a first layer of PF'y/PSS on PPy/SPHE, an overpotential similar to that of a Pt electrode is required. However, on Pt, the oxidation current is about three times higher. It was confirmed that a PpY/PSS layer can be electrochemically grown on PF'y/S PHE from an aqueous solution as well as on a Pt electrode. Creatinine sensors were made as described in the Experimental Section. The pH of the buffer solution was set to 6.2 as a compromise, taking the enzyme stabilitiesz0rz1and the effective conductivity of the PPy layers into account. At pH values above 6.0, the conductivity of PPy significantly drops,22 and above pH 7.0, the electropolymerization is almost completely inhibited.23 To measure the response of creatinine sensors, the devices were put in an electrochemical cell and polarized at 400 mV vs Ag/AgCl. The initially observed background current gradually (20) Toyobo Enzymes; Toyobo Co., Ltd.: Osaka, Japan, 1992. (21) Toyo Jozo Enzymes: Toyo Jozo Co., Ltd.: Tokyo, Japan, 1986. (22) Wernet, W.: Monkenbusch, M.: Wegner, G. Mol. Cyst. Liq. Cryst. 1985. 218, 193.
(23) Qian, R.: Pei, Q.; Huang, Z. Mukromol. Chem. 1991,192, 1263.
2778 Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
0 0
1
2
3
4
5
6
Creatinine Conc. (mM)
Figure 4. Calibration curve for creatinine obtained with PPy/PSS/ enzymes electrode prepared on PPy/S-PHE (0) and Pt (0).The electrode was potentiostatically prepared at 800 mV in PB (pH 6.2) containing 0.2 M pyrrole, 0.1 M PSS-Na, 10 mg/mL CRN, 10 mg/mL CR, and 10 mg/mL SO. The polymerization charge was 0.8 C/cm2. The creatinine assay was carried out at a potential of 400 mV in 20 mM PB (pH 7.5) under N2.
decreased, and the period to become constant was in the range of 30 min to several hours, depending on the thickness of the PPylenzymes layer. Therefore, all the sensors were conditioned overnight (-16 h). Before creatinine detection, the blind value was measured by injection of buffer solution. No change was observed. After each injection, the solution was stirred vigorously for 1min, and then the stirring was stopped to record the steadystate current. A typical current-time curve is shown in Figure 3. The steady-state currents for creatinine concentrations from 200 pM to 5 mM are plotted to make calibration curves, the calibration curves for Pt-and PPy/SPHE-based creatinine sensors are displayed in Figure 4. In the considered concentration range, the responses of both sensors were almost linear with the concentration without the use of any diffusion-controlling cover membrane. The absolute response current was typically 53%
Table 1. Oxidation Current bA/cm2) of Formaldehyde and Hydrogen Peroxide at Various Electrodes in a SteadpState Current Measurement.
10 mM HCHO 10 mM HzOz
Pt
electrode PPy/SPHE PPy/SPHE/PF$/PSS
32 323
no response no response
no response 80
The electrodes were constantly polarized at 400 mV in 20 mM PB (PH 7.5) upon injections of substances.
H,O,
HCHO
soy
I
I
I
cnz I Cnz I
Cnz
cn3
OH
PSS
I Cnz I
oso3. S-PHEMA
Table 2. Creatinine Response Current @A/cm2)of PPy/Polyanion/Enzymes Elecrodes.
H201
PSS
response current for 5 mM creatinineb pt
PFylS-PHE
S-VA-VAC
Figure 6. Chemical structure of dopants.
800
polyanion SPHEMA 120
SVA-VAC 256
PpYlS-PHWPpYlPSS
Figde 5. Scheme for the electrochemical reaction of hydrogen peroxide and formaldehyde at various electrodes. P, oxidative product of hydrogen peroxide; Q, oxidative product of formaldehyde.
higher at 5 mM creatinine for PPy/SPHE-based sensors. The reason for this finding is still not clarified,but it is possibly related to a higher surface area of PPyISPHE base electrodes. Moreover, another advantage of PPy/SPHE is the strong adhesion to the PPy/PSS enzyme layer. It was observed that the PPy/PSS enzyme layer was easily peeled off from the Pt electrode in the drying process after measurement. On the other hand, the PPy/ PSS enzyme layer strongly adhered to the PPy/SPHE electrode in the same process. Mechanism of Electron Transfer. According to Figure 1, formaldehyde 0 and hydrogen peroxide (ID are reaction products of sarcosine. Hydrogen peroxide as well as formaldehyde can be amperometrically detected at the electrodes. A third pathway to transfer electrons to the electrode is a direct electron transfer (III) from SO to the electrode. To evaluate the efficiency of I and 11, the electrochemical responses of Pt, PPy/SPHE, and PPy/ SPHEIPPyIPSS electrodes (without enzymes) to formaldehyde and hydrogen peroxide were measured. The electrodes were polarized at 400 mV, and the steady-state current was monitored. Formaldehyde was oxidized only at the bare Pt electrode (Table 1). On PPy/SPHE and PPy/SPHE/PPy/PSS electrodes, no oxidation current was observed. Toward hydrogen peroxide, both R-based and PPy/SPHE/PPy/PSS electrodes responded. No signal was obtained from the PPy/SPHE electrode alone. The results are schematically summarized in Figure 5. Therefore, a Pt-based sensor should basically show a higher response current against creatinine than the PPy/SPHE-based sensor, if the reaction products (hydrogene peroxide, formaldehyde) contribute to the current signal. After the evaluation of the electrochemicalbehavior of different electrodes, the response of a creatinine sensor in oxygen- and nitrogen-saturated PB solutions was measured using the PPy/S PHE base electrode. If oxygen exclusively participates in the electron transfer process, a lower signal is expected under inert conditions. Contrary to this expectation, the response current in a nitrogen-saturated buffer solution was found to be 32%higher than that in an oxygen-saturated buffer at the creatinine concentration of 5 mM. This result suggests at least the partial involvement of a direct electron transfer from SO to conductive PPy chains. Taking into account that PPy/SPHE electrodes do
a The electrodes were potentiostatically prepared at 800 or 1000 mV in PB (PH6.2) containing 0.2 M pyrrole, 0.1 M polyanion, 5 mg/mL CRN, 5 mg/mL CR, and 5. mg/mL SO. The polymerization charge was 0.8 C/cm2. The creabnine assay was carried out at a potential of 400 mV in 20 mM PB (PH7.5) under N2.
not respond to H202 and that PPy/PSS shows less efficiency compared to Pt (see Table l), a major part of the electrons seems to be transferred directly from SO to the electrode when using a PPy/SPHE base electrode in an inert environment. Comparison of DitTerent PpY Dopants Used in Active (Enzyme-Containing) Layers. Besides PSS, partially sulfated polyfiydroxyethyl methacrylate) (SPHEMA) and partially sulfated copolymers of vinyl alcohol and vinyl acetate (SVA-VAC) (Figure 6) have been used as a dopant in active PPy layers on Pt base electrodes. The response current of the resultant electrodes against 5 mM creatinine is exhibited in Table 2. Among the three polyanions, PSS showed the highest response current; SVA-VAC and especially SPHEMA led to a significantly lower sensitivity, about one-seventh that of PSS. Structural differences between the active PPy layers and possibly different diffusion coefficients of the substrates and products of the enzyme reactions may be the reason for these findings. Effect of the Thickness of the Active Layer. The thickness of the active layer defines the magnitude of the current response. It seems reasonable to assume that the concentration of enzymes in the active layer is uniform and independent of the thickness. Two contrary effects have to be optimized: the total amount of immobilized enzyme and a growing diffusion problem of the substrate with increasing layer thickness. To optimize the device, active layers using a polymerization charge between 0.8 and 40 C/cm2 have been synthesized and measured. Calibration curves were obtained in the same shape with different magnitudes of the response current at various polymerization charge. Figure 7 shows the response current for 5 mM creatinine as a function of the polymerization charge. Up to a charge of 16 C/cm2, the response current is increasing, and then it slightly decreases and reaches about the same level at 40 as at 10 C/cm2. However, the noise level increased with the thickness of PPy/PSS layer; then the optimum thickness is approximately equal to the polymerization charge of 10 C/cm2. Marchesiello and Geniesz4developed a theoretical model based on a PPy/GOx sensor in which they considered the thickness (24) Marchesiello, M.; Genies, E. /. Electyoanal. Chem. 1993,358, 35.
Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
2779
3.0
'
0
0
10
20
30
40
0.0
0
5
10
15
20
Concentration of SO (mg/ml)
Polymerization charge (C/cm2)
Figure 7. Effect of the polymerization charge on the steady-state response of the PPy/PSS/enzymes electrode on PPy/S-PHE. The electrode was potentiostatically prepared at 800 mV in PB (pH 6.2) containing 0.2 M pyrrole, 0.1 M PSS-Na, 5 mg/mL CRN, 5 mg/mL CR, and 10 mg/mL SO. The creatinine assay was carried out at a potential of 400 mV in 20 mM PB (pH 7.5) under NP.
Figure 8. Effect of SO concentration in the deposition solution on the steady-state response of the PPylPSSienzymes electrode on PPyi S-PHE. The electrode was potentiostatically prepared at 800 mV in PB (pH 6.2) containing 0.2 M pyrrole, 0.1 M PSS-Na, 5 mg/mL CRN, 5 mg/mL CR, and 2.5-20 mg/mL SO. The polymerization charge was 1.6 C/cmz. The creatinine assay was carried out at a potential of 400 mV in 20 mM PB (pH 7.5) under NP.
and the quality (active, inactive) of the enzyme-containing matrix. In active layers, the response is said to increase up to a certain thickness and then become constant. In inactive layers, the response current increases also up to a certain thickness but then sharply decreases due to a growing distance to the active electrode and finally reaches a much lower level. From our experiments and based on the predictions above, the PPyIPSS layer is an active matrix. Effect of the SO Concentration, Fortier et aL2j suggested that enzymes are physically entrapped in PPy layers rather than incorporated as dopants, because no effect of the pH was found during the immobilization of GOx in PPy. On the basis of this result, three enzymes, CRN, CR, and SO, in the present creatinine sensor were regarded to be physically entrapped during the electropolymerization of the PPy/PSS layer. Because three enzymes may compete for priority incorporation, the influence of the SO concentration in the preparation solution was studied with the fixed concentration (5 mg/mL) of the other two enzymes, respectively. Figure 8 shows the 5 mM creatinine response current as a function of the SO concentration in the polymerization solution. As can be seen, a concentration of 5 mg/ mL in the preparation solution is an optimized value. Below this value, an insufficient amount of SO may cause the current drop, and above it, SO may displace CRN and CR to be sufficiently incorporated.
Ag/AgCl. The reason for this rather unexpected result is not yet clarified but may be related to the total surface area, which is higher for PPy/SPHE electrodes. On the basis of measurements of PPy/SPHEs electrochemical activity toward formaldehyde and hydrogen peroxide, as well as experiments of biosensor performance made under nitrogen and oxygen atmosphere, a direct electron transfer mechanism was found to be involved in generating the output current. Among the polyanionic dopants used in the experiments, PSS is superior to SPHEMA or SVA-VAC. More detailed experimental work will be necessary to understand the differences. From the variation of the enzyme layer thickness, it can be concluded that PPy is an electrochemically active matrix and that the optimum thickness is reached using a polymerization charge of about 10 C/cm2. The magnitude of the response current is furthermore affected by the concentration of SO in the preparation solution, and 5 mg/mL SO is regarded as an optimum concentration when the concentration of CRN and CR is 5 mg/mL each. The drawbacks of hydrophilic PPy/PSS enzyme-containing layers in sensor devices are the long conditioning times in static measurements and problems with the long-term stability of the electrical properties of the active PPyIPSS matrix. More detailed data on this latter subject will soon be available. ACKNOWLEDGMENT
CONCLUSIONS It is possible to make a three-enzyme creatinine sensor by
physically entrapping the enzymes in an electrochemically active PPy/PSS matrix. A PPy/SPHE base electrode to prepare sensor devices leads to a higher response current as compared to that of a platinum base electrode at a detection potential of 400 mV vs
We thank Dr. G. F. Khan for his useful suggestions and discussions. Received for review March 2, 1995. 1995.B
Accepted
AC9502209 (25) Fortier. G . ; Brassard, E.; Belanger, D. Biosens. Bioelectron. 1990,5 . 473.
2780 Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
Abstract published in Aduance ACS Abstracts, July 1. 1995
May 25,
