Anal. Chem. 1092, 6 4 , 11 12-1 117
1112
cantly, and this can be accomplished without an appreciable sacrifice in through-put rate if appropriately alternated stop-go timing is adapted in a suitably configured manifold. ACKNOWLEDGMENT We acknowledge valuable input from Don C. Olson, Shell Development Co., Houston, TX. This research was supported by an unrestricted research grant from the Shell Development Co. (Houston, TX). V.K. was partially supported by Nbright Foundation Grant 14358. Registry No. PVDF, 24937-79-9; HCN, 24937-79-9; H2S, 7783-06-4; sodium isonicotinate, 16887-79-9; 3-methyl-lphenyl-2-pyrazolin-5-one, 89-25-8; sodium N-chloro-4-methylbenzenesulfonamide, 127-65-1. REFERENCES (1) Lodge, J. P., Jr., Ed. Methods of AC Sampllng and Analysis, 3rd ed.; Intersociety ammmee, Lewis Publishers: CIWISW. MI, 1969; ~ o 808, pp 586-588.
(2) Cicerone, R. J.; Zellner. R. J . & w.h y_ s . Res. 1989. 88 (C15). . . 10669- 10696. (3) Manka, D. P. In Autometed Stream Analysls for Recess Control; Manka, D. P., Ed.; Academic Press: New York, 1984 Vol. 2, pp 169- 182. (4) Kublh, V.; Dasgupta, P. K.; Marx, J. N. Anal. Chem. 1982, 64, 36-43. ( 5 ) Kublh, V. Anal. Chlm. Acta 1992, 259, 45-52. (6) Dean, J. A., Ed. Lange's Handbook of Chemkrby, 12th ed.; M-awHill: New York, 1978; pp 5-15. (7) Internatha1 Crltlcal Tables of Numerical Deta. Vol. 3 , physics, ChemChemlsby and Technobgy; McGraw-Hill: New York, 1928; p 365. (8) Stumm. W.; Moraan. J. J. Auuatlc Chemlstw. 2nd ed.; Wiiev: New York, 1961; p 169. (9) Crank, J.; Park, G. S. Diffusion In Polymers; Academic Press: New York, 1968; Chapter 11. (10) Dasgupta, P. K.; Dong, S.; Hwang, H.; Yang, H.G.; Genfa, 2. Atmos. Environ. 1988. 22. 949-964. (11) Dasgupta, P. K. ACS A&. Chem. Ser., in press. '
d F~WWED
13, 1992.
for review November 4,1991. Accepted February
Enzyme-Based Bilayer Conducting Polymer Electrodes Consisting of Polymetallophthalocyanines and Polypyrrole-GI ucose Oxidase Thin FiIms Zhisheng Sun and Hiroyasu Tachikawa* Department of Chemistry, Jackson State University, Jackson, Mississippi 39217-0510
Bilayer conductlng polymer electrodes, which condst of a polymeiallopMhalocyanIne(PMePc) and potypyrrole Incorporating glucose oxldase (PPy-GOx), were prepared on the glassytarbon electrode by the successive electrochemlcal depodtlon of two different polymers. The bilayer fllm electrodes showed catalytlc behavlor, whlch Included an enhanced empenwnetrlc response current wkh the substrate and a slgnlflcantly shmed oxldatlon potentlal (-700 mV) of the response current. The bllayer electrodes also showed a fast response time and good staMllty wkh the substrate. A Mlayer mlcroekctrode, whkh was prepared by uslng both PCuPc and PPy-GOx polymer fllms, also showed a good amperometrlc response wHh the substrate.
INTRODUCTION Entrapment of enzyme molecules into a conducting polymer has become an attractive method for the preparation of enzymebased electrodes as biosen~orsl-'~ because the conducting polymer offers good electrical conductivity, electrode surface activity, and an easy preparation procedure for the enzyme immobilization on the electrode surface. Foulds and Lowe1p2 and other authors*l0 have prepared enzyme-based electrodes by electrochemicallypolymerizing polypyrrole and substituted polypyrroles which are doped with small anions. Enzyme molecules are entrapped in the polypyrrole film matrix. Conducting polypprole-enzyme films can also be prepared in the enzyme solution without s m a l l anions,3Joand the doping behavior of the enzyme molecules in the PPy film has been *Towhom correspondence can be directed.
investigated. Simultaneous immobilizations of enzyme and mediators such as ferrocenecarboxylic acid, hydroquinonesulfonate ion, etc. to mediate the electron transfer from enzyme to electrode have been r e p ~ r t e d . ~InJ ~order to improve the performance of the PPy-GOx f i b electrode as a biosensor for the determination of glucose, there have been attempts to use a bilayer state including montmorillonite clay f i b and enzyme bovine serum albumin film.13 In this paper we describe a bilayer conducting polymer electrode which consists of a polymetallophthalocyanine(PMePc) redox polymer film and a conducting polypyrrole-glucose oxidase (PPy-GOx) film. The electrochemistry of phthalocyanines has received a great deal of attention due to good catalytic behavior.lkZ1 A greatly reduced oxidation potential has been observed for a variety of organic compounds a t the electrode incorporated with cobalt phthalocyanine.22-n Recently, thin fiims of poly metal phthalocyanines have been synthesized electrochemically by Li and The polymer thin film of metal phthalocyanine on the electrode has shown electrical conductivity in a wide potential range and may be used as a promising surface modification material in electroanalytical applications. The catalytic behavior for cysteine, mercaptosuccinic acid, glutathione, oxalic acid, hydroquinone, and catechol have been investigated by different electrochemical method^.^^^^^ To our knowledge, there has been no report of polymetallophthalocyanines being used for application in enzyme-based biosensors. In recent years the redox polymer used as an enzyme electrode matrix has received much interest for the preparation of the enzymebased biosens~r.~l-~ Enzymes have been immobilized in redox polymers to provide simple and sensitive
0003-2700/92/0364-1112$03.00/00 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
amperometric biosensors. In addition, the redox polymer can be used as a catalytic species which enhances the enzymesubstrate reaction by either directly mediating the electron transport from enzymes to the e l e ~ t r o d e ~orl -catalyzing ~ the reaction of the products and reactants of enzyme-substrate rea~tion.~’So far the redox polymers including the poly(vinylpyridine) (PVP) complex of Os(bpy),Cl, ruthenium penhammine or ferrocene derivatives,3l-= and polysiloxane with ferrocene as well as the polyporphyrin polymer,37 etc. have been used to make a glucose oxidasebased electrode. It is expected that the use of the redox polymer of polymetallophthalocyanine can provide a new biosensor matrix. We prepared the bilayer electrode by successive electrochemical polymerizations of two different polymers: catalyst polymer (PMePc) and enzyme polymer (PPy-GOx). A greatly enhanced response current was observed a t the GC/ PMePc/PPy-GOx bilayer film electrode compared with that observed at a GC/PPy-GOx single-layer film electrode. We examined two kinds of polymetallophthalocyanines including Co and Cu as the central metal for the catalyst polymer. The preparation condition of the electrode was studied in detail, and the analytical performance of the bilayer electrode was evaluated. The electrode showed good stability and reproducibility and a fast response time for the substrate. The bilayer enzyme f i b can also be prepared as an enzyme-based microelectrode. EXPERIMENTAL SECTION Chemicals. Cobalt(II)-4,4’,4’’,4’’’-tetraaminophthalocyanine (CoTAPc)and copper(II)-4,4’4’’,4~~’-tetraaminophthalocyanine (CuTAPc) were used as received from Midcentury Chemicals. Glucose oxidase (EC 1.1.3.4,Aspergillus niger, Type 11, 25 unita/mg) and fl-D(+)-glucosewere used as received from Sigma Chemical Co. Pyrrole (99% purity, Aldrich Chemical Co, Inc.) was purified by refluxing with zinc metal followed by the vacuum distillation. Tetraethylammonium perchlorate (TEAP) was received from Southwestern Analytical Chemicals, Inc., and dried at 120 “C for more than 24 h prior to use. Spectrophotometric grade Nfl-dimethylformamide (DMF) was used as received from Aldrich Chemical Co. Other chemicals were analytical reagenta and were used as received. Doubly distilled water was used as a solvent. Preparation of Bilayer Film Electrodes. The bilayer electrodes were prepared by successive electrochemical polymerizations of the PMePc and PPy-GOx. For example, a GC/ PMePc/PPy-GOx electrode was prepared by the electrochemical polymerization of the PMePc film on the glassy-carbon (GC) electrode followed by the electrochemical polymerization of the PPy-GOx film on top of the PMePc film, and the GC/PPyGOx/PMePc electrode was prepared by the reversed deposition order. The electropolymerization of the PMePc f i i was carried out in the DMF solution containing 10 mM MeTAPc monomer and 0.1 M TEAP by continuous scanning of the potential between 4.2 and +0.9 V. The thickness of the PMePc f i i was controlled by the number of potential cycles. The PPy-GOx film was formed by passing a constant oxidation current of 50 MAin the solution of 0.05 g/mL GOx and 0.35 M pyrrole without other supporting electrolytes. The thickness of the PPy-GOx f i i was controlled by the charge passed and was estimated to be lo00 A by passing 48 m C / ~ m ~ . ~ ~ Electrochemical Measurements. Electrochemical experimenta were carried out by an EG&G PARC Model 273 potentiostat/galvanostat and a Houston Instrument Model 2000 X-Y recorder. An electrochemical cell with three electrodes was used for both the electropolymerization and amperometric determination. An Ag/AgCl electrode was used as the reference electrode for the electrochemical polymerization of both polymetallophthalocyanines (PMePc) and polypyrrole-glucose oxidase (PPy-GOx) films, and a SCE was used for the amperometric determination of the glucose. A platinum wire with a large area was used as an auxiliary electrode, and a glassy-carbon (GC) electrode (BASMF2012,3.0-mmdiameter) was used as a working electrode. Prior to the electrochemical deposition of conducting
1113
7
0.4
0.8
1.2
POTENTIAL L V v s . SCE)
Figure 1. Amperometric response current and potential relationship at both GC/PMePc/PPy-GOx and GC/PPy-GOx film electrodes In PB (pH 7.4) with glucose (2.5 mM): (1) GC/PCoPc/PPy-GOx PCoPc layer (10.5 cycles between -0.2 and +0.9 V at 50 mV/s in DMF solution containing 10 mM CoTAPc and 0.1 M TEAP), PPy-GOx layer (5.0 mC); (2) GC/PCoPc/PPy-GOx PCoPc layer (10.5 cycles), PPy-GOx layer (1.0 mC); (3) GC/PCuPc/PPy-GOx PCuPc layer (10.5 cycles), PPy-GOx layer (1.0 mC); (4) GC/PPy-GOx, PPy-GOx layer (1.0 mC).
polymers, the GC electrode was polished with 0.05-pm alumina powder (Buehler Ltd.)for 5 min, sonicated for 5 min, and then was washed thoroughly with distilled water. The amperometric determination of glucose at the bilayer electrode was carried out in phosphate buffer (PB) solution. The response time was obtained from current-timawes of amperometric response current which were recorded under stirring condition by a Houston Instrument Model 2000 X-Y recorder equipped with a time-base module. RESULTS AND DISCUSSIONS Amperometric Response to Glucose. Figure 1shows the relationships of the amperometric currents from the bilayer GC/PMePc/PPy-GOx electrodes and the potential when glucose was added to the solution. An apparent catalytic enhancement was observed for the GC electrode modified by the bilayer of PMePc/PPy-GOx film compared to the GC electrode modified only by the PPy-GOx film. For the PPy-GOx single-layer electrode, the catalytic oxidation current of glucose was observed when the potential of 0.9 V or higher was applied to the electrode. However, the catalytic oxidation current was observed by applying less than 0.2 V to the GC/PCoPc/PPy-GOx bilayer electrode. The potential of the GC/PCoPC/PPy-GOx electrode for the amperometric determination of glucose shifted toward a less positive potential by almost 700 mV, and the current level also increased compared with the catalytic response at the GC/PPy-GOx film electrode. The response current level at the GC/ PCoPc/PPy-GOx electrode with the glucose increased when the potential was moved from 0.2 to 0.4 V and then decreased when the potential was further moved toward the positive direction. The maximum response current was observed around 0.4-0.5 V for the GC/PCoPc/PPy-GOx electrode. A similar relationship between the amperometric current and the potential was observed for the bilayer electrode of GC/ PCuPc/PPy-GOx, and the maximum current was observed a t -0.6 V. We consider that the enhanced response current at the bilayer GC/PMePc/PPy-GOx electrode for g l u m oxidation is caused by the excellent catalytic behavior of the PMePc film. On the basis of the structural feature of the bilayer electrode, two possible catalytic mechanisms may be considered. Mechanism I. The PMePc polymer behaves as a mediator for the electron transport from the enzyme redox center to the electrode, that is
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
1114
Chemical reaction between polymer and substrate in solution:
glucose(s) + PMePc(fXox)
-
gluconic acid(s)
20
t -
I
PMePc(fXred)
Electrode reaction: PMePdfXred) PMePc(fXox) + ewhere s means solution and f means film. Mechanism II. The enzyme catalyzes the oxidation of the substrate in the presence of oxygen, and the PMePc f i i catalyzes the oxidation of H20z,which is a product of enzymesubstrate reaction. The reactions are expressed by the following: Chemical reaction: glucose(s) + 02(s) PMePc(D(ox) + H202(f)
t
-
GOx(D
gluconic acid(s) + H202(f')
0.4
1.2
0.8
POTENTIAL ( V v s . SCE)
Figuro 2. Amperometrlc response current and poteat41 relationship at QCIPMePc fllm electrodes In P8 (pH 7.4) with H,O, (0.5 mM): (1) QC/PCoPc, 15.5 cycles between -0.2 and 0.9 V; (2) QC/PCuPc, 10.5 cycles; (3) bare (3C electrode.
2H' + O2 + PMePc(fXred1
-
I
Electrode reaction: PMePdfXred) PMePc(fXox) + eIn the two mechanisms the electrode reaction involves the oxidation of the PMePc fii. However, the main difference between mechanism I and mechanism I1 is that oxygen is involved in the enzyme reaction of mechanism I1 and the reaction takes place only in the presence of 02. On the other hand, mechanism I will not be affected by oxygen. In mechanism I1 the PMePc film should have a good catalytic behavior toward HzOp Figure 2 shows the amperometric response currents for hydrogen peroxide from the GC/PMePc thin-film electrode and bare GC electrode at different potentials. It can be seen that H202can be oxidized only at -0.9 V or higher at a bare GC electrode. However, H202can be oxidized at around 0.1 V at the GC/PCoPc film electrode and 0.2 V at the GC/ PCuPc film electrode. The oxidation potential of H202shifted negatively almost 700 mV for the PCuPc f i i and 800 mV for the PCoPc film, compared to that at the GC electrode. The results suggested that the PMePc film is a very good electrochemical catalyst for the oxidation of HzOP Figure 3 shows the effect of oxygen on the catalytic amperometric current at the GC/PCoPc/PPy-GOx f i i electrode. When the solution was saturated with either air or 02,the catalytic current was observed. However, no significant current was observed when the solution was saturated with N2. No significant difference was observed between the air-saturated solution and the solution saturated with 02. From the above experiments, the behavior of the bilayer GC/PMePc/PPyGOx film electrode was consistent with mechanism 11. A bilayer film electrode was also made by reversing the order of polymerization, first depositing a PPy-GOx film on the GC electrode followed by the depoeition of a PMePc fh. However, no response current was observed from the GC/ PPy-GOx/PMePc electrodes when the glucose was added to the solution. The above results suggested that the reaction between the enzyme and the substrate occurred at the inM a c e of the PF'y-GOx film and the solution and the catalytic oxidation of Hz02occurred at the interface of PMePc and Ppy-Gox (seeFigure 4). It is likely that the H20zproduced by the enzymatic reaction in the PPy-GOx film diffused through the PPy-GOx film and reached the PMePc film where it was oxidized. For the investigation of the effects of adsorbed enzyme molecules on the catalytic response current, the PMePc f i i electrode was immersed in 0.05 g/mL g l u m oxidase solution for several minutes (using the same time period needed for
TIME
Flguro 3. Effect of oxygen on the catalytic amperometrlc current at
the GC/pcopc/PPydox electrode: Pcopc layer, 10.5 cycles between -0.2 and +0.9 V; PPy-Gox layer; 1.3 mC. Applled potentlal: 0.5 V. Solutions: (1) N, saturated PB (pH 7.4) solution with 2.5 mM glucose and then bubbling succegsivety wlth 0, and N,; (2) N, satveted P 8 @H 7.4) solution (glucose absent at the beglnnlng); (3) air-saturated PB solution (glucose absent at the beglnnlng).
4
I
M (Red) PC
'
""& -w I
o2
GOX I ^
4 A
I
L
I
I
I
I
I F'MePc f i l m
PPy-GOx f i l m
solution
F I p w 4. Schema* desaiption of the catalytic reactlon at the bkyer electrode. G: glucose.
polymerization of PPy-GOx) before examining the presence of a catalytic response current. The GOx-treated GC/PMePc electrode showed an amperometric response current with glucose at 0.5 V, but the current level was substantially lower than that observed at the bilayer electrode (0.2 p A at the GOx-treated PCuPc film electrode and 0.08 p A at the GOxtreated PCoPc film electrode). The resulta suggested that the amount of enzyme incorporated in the bilayer electrode was much higher thanthat adsorbed on the surface of PMePc film. Effects of Film Thickness on Response Current. Figure 5 shows the thickness effect of PMePc and PPy-GOx films on the catalytic current of the bilayer electrode for the oxidation of glucose. The film thickness was controlled by the number of scanning8 during the continuous cyclic voltammetric polymerization of the metal phthalocyanine film. It can be seen that the response current showed the maximum values at certain film thicknesses of the PMePc. For the
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
1115
Table I. Optimum Conditions for the Preparation of the Bilayer GC/PMePc/PPy-GOx Electrode and Determination of Glucose GC/PCoPc/PPy-GOx
GC/PCuPc/PPy-GOx
scan between -0.2 and +0.9 V (8-20 cycles) scan between -0.2 and +0.9 V (10-20 cvclesl in in DMF solution containing 10 mMDMF' solution containing 10 mM CuTAPc and CoTAPc and 0.1 M TEAP 0.1 M TEAP PPy-GOx film formation pass 1.0-mC charge (50-pA oxidation current) pass 0.2-0.3-mC charge (50-pA oxidation current) in aqueous solution containing 0.05 g/mL in aqueous solution containing 0.05 g/mL GOx GOx and 0.35 M Py and 0.35 M Py amperometric determination of glucose in PB (pH 5-8) at 0.4-0.5 V in PB (pH 7-8.5) at 0.45-0.7 V PMePc film formation
4
-
1.0
c
z W
E E
a
0
0.5
-.-I R
I
-1
I
I
I
:c 2
PPY-GOX FILM THICKNSS (CHARGE, mc)
0
10
20
PWPC FILU THICKNSS ( MllBER
OF CYCLES )
Flgure 5. Effect of thlckness of polymer fllms on the amperometric response current wtth glucose (2.5 mM) In PB (pH 7.4) solutlon: (A) QC/PCoPc/PPydox pcopC layer (10.5 cycles between -0.2 and + O S V), PPy-QOx layer (thickness varied); (B) QC/PCoPc/PPy-Wx PCoPc layer (thlckness varled), PPy-Gox layer (1.0 mC); (C) GC/PCuPc/ PPy-QOx WuPc layer (10.5 cycles), P P y - W x layer (thlckness varied); (D)Gc/PcuPc/PPy-oox PCUPC layer (tMdvlessveried), PPy-oox layer (0.25 mC). Applied potentlal: 0.5 V.
GC/PMePc/PPy-GOx bilayer film electrodes, the maximum currents were normally observed from the films made by 10-15 cycles of potential scan (see Figures 5B and 5D). The effect of PPy-GOx film thickness on the catalytic behavior of the bilayer electrode for the glucose oxidation was very similar to that of a single-layer GC/PPy-GOx film electrode.10 For the GC/PCoPc/PPy-GOx film electrode, the maximum response current was observed when a 1.0-mC charge was passed for the polymerization of the PPy-GOx film (see Figure 5A). The PPy-GOx layer, which was deposited by passing 0.25 mC of the charge, provided the maximum response current for the GC/PCuPc/PPy-GOx film (see Figure 5C). The observed thickness effects for both the PMePc and PPy-GOx films on the response current may be caused by two factors: (1)diffusion process of H202in the PPy4.30~ film and (2) the amount of the enzyme immobilized in the PPy-GOx film. When the PPy-GOx film is too thick, the diffusion rate of HzOz in the film may be low, and therefore, the response current level drops. On the other hand, if the PPy-GOx film is too thin, the amount of GOx available for the reaction with the glucose must be very small. Characteristics of the Bilayer PMePc/PPy-GOx Electrode. From the results obtained above, the optimum condition for the preparation of the most sensitive bilayer PMePc/PPy-GOx electrodes for the determination of the glucose are shown in Table I. The characteristics of electrodes are as follows.
5
10
GLUCOSE CONCENTRATION ( m M )
Flgure 6. Callbratlon curves for the amperometric response current at GC/PMePc/PPy-GOx electrodes: (1) GC/PCoPc/PPy-GOx PCoPc (15.5 cycles between -0.2 and +0.9 V), PPy-GOx (1.0 mC); (2) GC/PCuPc/PPy-GOx PCuPc (10.5 cycles between -0.2 and +0.9 V), PPy-Wx (0.25 mC). The amperometric response was measured at 0.5 V after successbe addttlon of the 2.5 mM glucose solutlon.
Calibration Curve for the Determinution of Glucose. Figure 6 shows the amperometric response currents of both the GC/PCuPc/PPy-GOx and GC/PCoPc/PPy-GOx film electrodes with successive additions of the glucose into the PB buffer solution. The catalytic response currents of the bilayer electrodes showed a linear relationship at the low glucose concentration, but the catalytic response deviated from the linear relationship at a high glucose concentration. The apparent Michaelis-Menten constanta have been calculated on the basis of the EadieHofstee form of the Michaelis-Menten e q ~ a t i o n ; ~ ~12* and ~ ~ ,8.6 ~ ~mM , ~ are obtained for GC/ PCuPc/PPy-GOx and GC/PCoPc/PPy-GOx electrodes, respectively. Detection limits which were calculated in terms of signalto-noise (a signal level which is 3 times larger than the noise level can be detected) are 0.5 m M for GC/PCoPc/PPy-GOx and 0.25 mM for GC/PCuPc/PPy-GOx electrodes, respectively. The above results along with the results shown in Figure 6 indicated that the bilayer electrode can be used for the determination of glucose in the range from 0.5 to at least 10 mM. Response Time. Improvement of the response time of the enzyme electrode is an important topic in the field of the construction of biosensors. Much work has been done for the immobilization of the enzymes onto the electrode surface to improve the selectivity of electrochemical measurements. However, very few enzyme-based electrodes have shown a fast response time. The response time of enzymebad electrodes, which are prepared by chemical covalent and adsorptive couplings of the enzymes to the carbon-paste electrode as well as the entrapment of the enzyme into the carbon-paste mixture, is normally in the order of several minutes.40 Platinized microelectrodes utilizing surface-adsorbed enzymes have been reported to have a response time of several seconds,ll or leas.'2
1118
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
l1-24 TIME
Flgurs 7. Response time of the GC/PCuPc/PPy-Ox electrode with glucose: PCuPc layer, 10.5 cycles between -0.2 and +0.9 V. PPyO x layer 0.30 mC of charge passed. Condklons: glucose concentration (1) 1.0 mM, (2) 2.5 mM; measured at 0.5 V in PB (pH 7.4) solution.
10
I A I
0 2
10
6
PH
Flgurs 8. Effect of pH on the amperometrlc response currents at GC/PMePc/PPy-GOx and GC/PMePc electrodes: (A) GC/PMePc/ PPy-Gox with 2.5 mM glucose; (B) GC/PMePc with 0.5 mM H202;(1) GC/pcd)c/PPydox Pcopc layer (15.5 cydes between -0.2 and + O S V), PPy-QOx (1 mC of charge passed); (2) GC/PCuPc/PPy-Gox PCuPc (10.5 cycles), PPy-Ox (0.25 mC). Applied potentlal: 0.5 V.
Pautano et alqUreported a fast response time of an enzymebased modified carbon-fiber microelectrode in which a biotin/avidin system was used to immobilize the enzyme. Figure 7 shows the response time of the GC/PCuPc/PPy-GOx electrode to the glucose. It can be seen that the response times (time required for the signal to change from 5% to 95% of limiting current) are 1 s for 1 mM glucose and 1.5 s for 2.5 mM glucose, respectively. This type of bilayer sensor, which does not possess protective membranes, is expected to have faster response time than those with protective membranes.40 This fast response behavior will meet the analytical requirement for monitoring the dynamic processes in many applications of biosensors. pH Effects. Figure 8A shows the effect of pH on the response current of bilayer GC/PCuPc/PPy-GOx and GC/ PCoPc/PPy-GOx film electrodes to 2.5 mM glucose. When the pH of the solution was either very low or very high, the level of the response current was low. The maximum response current was observed in the pH range 4-6 for the GC/ PCoPc/PPy-GOx film electrode and in the pH range 7-8.5 for the GC/PCuPc/PPy-GOx film electrode. Besides the effects of the pH on the GOx-glucosereaction in the PPy-GOx f i i , l o the catalytic behavior of the PMePc film to HzOzmay
I
50
5
c1
I
T I ME
Flgwe 9. Reproducibility and stability of the (3C/PCuPc/PPy-GOx fllm electrode. Amperometric response currents with glucose (2.5 mM) at 0.5 V in PB (pH 7.4) solution: (A) repetitive measurements; (B) single-response current witti tlme.
also be affected by the pH, especially with a different central metal for the phthalocyanines. As shown in Figure 8B, this possibility was confirmed by the relationship between the catalytic currents of the PMePc films of a different central metal and the pH for the oxidation of H202 The observed pH effects of the PMePc films for the oxidation of H202are consistent with those of the bilayer electrodes for the determination of the glucose. Stability. The stability of the bilayer GC/PMePc/PPyGOx film electrode was studied by adding the same concentration of glucose. As shown in Figure 9, the electrode showed good reproducibility. For the GC/PCuPc/PPy-GOx electrode, the relative standard deviations of four repetitive measurements were 5.8%,5.1% ,and 5.7% for the solutions of 1, 2.5, and 5.0 mM glucose, respectively. The response current which was recorded under a controlled potential of 0.5 V showed little change (less than 5%) in 5 min (see Figure 9B). The lifetime of the electrode was measured by keeping it in the pH 7.4 PB solution. The results showed that 80% of the activity of a new electrode was maintained after 1week, and 50% activity remained after 2 weeks. After 1 month, the electrode still showed a significant response to the glucose. Selectivity. As an amperometric glucose electrode, the bilayer GC/PMePc/PPy-GOx electrode may be affected by other electrooxidizablespecies such as ascorbic acid, uric acid, acetaminophen, etc. However, the low operating potential of the bilayer electrode would reduce the effects of the intarferanta which can be oxidized at a higher oxidation potential. We compared the amperometric behaviors of both a singlelayer GC/PPy-GOx electrode (amperometric measurement was operated at 1.0 V) and a bilayer GC/PCuPc/PPy-GOx electrode (operated at 0.5 V) in the solutions of interferants including ascorbic acid, uric acid, acetaminophen, desipramine, and chlorpromazine. The results showed that medical compounds such as desipramine and chlorpromazine produced severe interference for the determination of glucose by using the single-layer GC/PPy-GOx electrode, but almost no interference could be observed at the bilayer GC/PCuPc/ PPy-GOx electrode. Both ascorbic acid and acetaminophen showed less interference for the measurement of glucose at the bilayer GC/PCuPc/PPy-GOx electrode than the singlelayer GC/PPy-GOx electrode. However, the amperometric response current for uric acid at the bilayer GC/PCuPc/ PPy-GOx electrode was more than 2 times higher than that at the single-layer electrode. The results suggested that the oxidation of the uric acid was catalyzed by the catalyst polymer (PCuPc).
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
CONCLUSION The bilayer conducting polymer electrodes consisting of catalytic polymer film and enzyme polymer f i can be prepared on a GC electrode by successive electrochemical depositions of PMePc and PPy-GOx films. The bilayer electrodes showed much improved response behaviors as a glucose sensor compared with a single-layer PPy-GOx f i electrode: an enhanced amperometric response current with glucose and a significantly shifted oxidation potential of the response current. The response current at the bilayer electrode with glucose is high enough that it can be used as a bilayer microelectrode: a PCuPc/PPy-GOx bilayer film prepared on a platinum microelectrode (100-Mm diameter) showed a good amperometric response toward glucose (0.25-12.5 mM concentration). The type of central metal in the PMePc film showed significant effecta on the behavior of the bilayer f i electrodes. A bilayer film electrode consisting of PCoPc and PPy-GOx (GC/PCoPc/PPy-GOx) showed more significant catalytic effects with the glucose by shifting the operating potential to leas positive potential compared with those observed at the GC/PCuPc/PPqhGOx electrode. The GC/PCoPc/PPy-GOx electrode also oxidized HzOzat much less positive potential than the GC/PCuPc/PPy-GOx electrode. The pH effects on the catalytic response currents at these two different bilayer eledrodes are much different (the maximumresponse currents were observed in the pH range 4-6 for the GC/PCoPc/ PPy-GOx electrode and in the pH range 7-8.5 for the GC/ PCuPc/PPy-GOx electrode, respectively). These experimental results suggested that the type of central metal in the PMePc film plays an important role for the observed catalytic behavior of bilayer conducting polymer electrodes. Besides these two PMePc films, the use of other phthalocyanines as well as their analogues with different metal centers may lead to the development of more sensitive biosensors. ACKNOWLEDGMENT This work was supported in part by the Office of Naval Research and the National Institutes of Health (Grant S06GM08047). REFERENCES Foulds, N. C.; Lowe, C. R. J . Chem. Soc., Faracley Trans 1 1988, 82, 1259. Foulds, N. C.; Lowe. C. R. Anal. Chem. 1988, 60, 2473. Pandey, P. C. J . Chem. Soc., Faracley Trans 1 1988, 84, 2259. Tomiya, E.; Karube, I.Sens. Actuators 1989. 18, 297. Iwakura, C.; Kajiya, Y.; Yoneyama, H. J. Chem. Soc..Chem. Common. 1988, 1019.
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RECEIVED for review October 28, 1991. Revised manuscript received February 18, 1992. Accepted February 21, 1992.
