Anal. Chem. 1990, 62, 2735-2740 (10) Lakowicz, J. R. Rinclples of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (11) Lentz, E. R.; Barenholz, Y.; Thompson, T. E. Biochemistry 1976, 75 (20),4521-4528. (12) Lentz, E. R.; Barenholz. Y.; Thompson, T. E. Biochemistry 1976, 75 (20),4529-4537. (13) Lakowicz, J. R.; Sheppard, J. R. Am. J . Human Genet. 1981, 3 3 , 155-165. (14)Sumbilla, C.; Lakowicz, J. R . J . Neurochem. 1982, 38 (6), 1699-1707. (15) Lakowicz, J. R.; Hogen, D. Biochemistry 1981, 20 (9, 1366-1373. (16) Lakowicz, J. R.; Cherek, H.; Bevan, D. R. The J . Biol. Chem. 1980, 255 (lo),4403-4406. (17) Parasassi, T.; Conti, F.; Gratton, E. Cell. Mol. Biol. 1986, '32 (l), 99-102. (18)Parasassi, T.; Conti, F.; Gratton, E. Ce//. Mol. Biol. 1968, 32 (l), 103-108. (19) Lakowicz, J. R.; Bevan, D. R.; Maliwal, B. P.; Cherek, H.; Baker, A. Biochemistry 1983, 22, 5714-5722. (20) Weber, G.; Farris, F. J. Biochemistry 1979, 78 (14),3075-3078. (21)Merlo, S.;Burgess, L. w.; Yager, P. Advanced Methods of Pharmacokinetic and Pharmacodynamic Systems Analysls : Proceedlngs of the 7990 Workshop of the Biomedlcel Simulations Resource; Plenum: New York,'in press. (22) Mwlo, S.: Burgess, L. W.; Yager, P. Sens. Actuators 1990, A27 -A23, 1150-1154.
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(23) Patent application #07/367,508. (24) Van Dijck, P. W. M.; De Kruijff, E.; Aarts, P. A. M. M.;Verkleij, A. J.; De Gier, J. Bimhem. Biophys. Acta 1978, 506, 183-191. (25)Olson, F.; Hunt, C. A.; Sroka, F. C.; Vail, W. J.; Papahadjopoulos, D. Biochim. Biophys. Acta 1979, 557, 9-23. (26) Blanck, T. J. J. Anesth. Ana@. 1981, 60 (6),435-436. (27) Schullery, S.E.; Schmidt, C .F.; Felgner, P.; Tillack, T. W.; Thompson, T. E. Biochemistry 1980, 79, 3919-3923. (28)Chang. E. L.; Gaber, B. P.; Sheridan, J. P. Biophys. J . 1982, 3 9 , 197-201. (29)Gaber, E. P.; Sheridan, J. P. Blochim. Biophys. Act8 1982, 685, 87-93. (30) Miller, R. D., Ed. Anesthesk?; Churchlll Livingstone: New York, 1981. (31)Hill, M. W. Blochim. Bbphys. Acta 1974, 356, 117-124. (32) Mabrey, A.; Sturtevant, J. M. Prm. M t l . Aced. Sci. U . S . A . 1976, 73 (1I ) , 3862-3866. (33) Abrams, S.;Merio, S.; Yager, P. Unpublished work.
RECEIVED for review February 2,1990. Accepted August 20, 1990. Support has been provided by the Center for Bioengineering,the Rotary Foundation, the Washington Technology Center, and UW-NIH Biomedical Research Support Grant PHS RR-07096.
Glucose Fast-Response Amperometric Sensor Based on Glucose Oxidase Immobilized in an Electropolymerized Poly(o =phenylenediamine)Film Cosimino Malitesta, Francesco Palmisano,* Luisa Torsi, and Pier Giorgio Zambonin
Laboratorio di Chimica Analitica, Dipartimento di Chimica, Uniuersitci degli Studi di Bari, 4, Trau.200 Re David-70126 Bari, Italy
o -Phenylenedlamlne has been used for glucose oxidase (GOx) lmmobillzatlon on Pt electrodes by electrochemical polymerlzatlon at +0.65 V vs SCE. By thls approach the enzyme Is entrapped In a strongly adherent, highly reproduclble thln membrane, whose thickness Is around 10 nm. This one-step procedure produces a glucose sensor wlth a response time less than 1 s, an active enzyme loading higher than 3 units/cm2 of electrode surface, a high sensitlvity, and a sufflclently wlde llnear range. The glucose response shows an apparent Mkhaells-Menten constant, K', = 14.2mM, and a limltlng current density, of 181 pA/cm2. The product &D of partltion and dlffusion coefficients of glucose in the polymer film is on the order of lo-'' cm2/s. Due to permselectlvlty characteristics of the membrane, the access of ascorbate, a common Interfering specles, to the electrode surface Is blocked. To our knowledge, thls represents the first report of a membrane capable, at the same time, of immobilizing GOx and rejecting ascorbate. The Interesting electrode behavlor can be rationallzed by using an existing model predlctlng the amperometrlc response of an Immobliized GOx system.
INTRODUCTION Analytical devices combining the specificity of biological systems with the advantages of electrochemical transduction have gained an increased popularity in the last decade ( I ) . Enzymatic redox reactions are particularly amenable to be interfaced with electrochemical transducers, since electron exchange is a key step in their natural cycle. A particular
effort in this field has been directed to the realization of an amperometric sensor for the clinically significant substrate glucose (2-9) using the archetypal redox enzyme glucose oxidase (GOx). Among the biosensors designed for this purpose, "immobilized-enzyme electrodes" play a key role. The advantages of immobilized-enzyme electrodes include a minimum pretreatment of the analyzing matrix, the need of a small sample volume, and the possible recovery of the enzyme for repeated use. However, the usefulness of sensors based on immobilized enzymes depends on several factors such as the immobilization method, thickness and stability of the entrapping membrane, the activity and stability of the entrapped enzyme, the response time, and the storage conditions required. In conventional amperometric enzyme electrodes, up to three membranes are employed to overcome problems associated with enzyme immobilization, electroactive interferences, and electrode fouling. Cellulose acetate casting (10) of Pt electrodes is a common way to eliminate the effects of protein adsorption onto electrode surface and to reduce interferences due to electroactive species other than HzO2. However the use of both cast and discrete membrane films are essentially limited to two-dimensional electrode surfaces because of the practical difficulties in controlling thickness, reproducibility, and uniformity of the polymer film on electrode surfaces with more complex geometry (e.g. reticulated vitreous carbon). Very recently, Sasso et al. (11)demonstrated that in such a case electropolymerization possesses definite advantages over conventional casting. Poly(l,2-diaminobenzene)was electrochemically coated on a platinized reticulated vitreous carbon with covalently bound GOx, producing a sensor virtually free from
0003-2700/90/0362-2735$02.50/00 1990 American Chemical Society
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interference and fouling problems. Recent improvements in the enzyme entrapment technology include the use of chemically modified electrodes and, in particular, of electrodes modified by a layer of electrodeposited conducting organic polymer. By this approach the spatial distribution of the immobilized enzyme can be readily controlled and the thickness of the enzyme film easily varied. Furthermore, a multilayer structure using different enzymes and/or different polymers, could, in theory, be built up. Poly(pyrro1e) (PPy) modified electrodes, obtained by electrochemical polymerization of pyrrole are, by far, the most studied systems (12-15). PPy electrodes however appear to be degraded (13)in the presence of H202at the potential value normally used for amperometric detection of this species. An indirect strategy (13)based on the determination of hydrogen peroxide via Mo(V1)-catalyzed iodine reduction of +0.2 V vs Ag JAgCl has been adopted to overcome the above problem. Recently (16),GOx has been incorporated into a poly(indo1e) film electrochemically deposited onto a platinum surface. A working potential of +1.6 V vs Ag/AgCl was required to detect HzOz. This very high potential, however, would certainly preclude the practical use of this sensor because of unavoidable interferences caused by electroactive endogenous components of the matrix. Contrary to the nonelectrochemical approach to membrane enzyme entrapment (in which the enzyme loading and membrane thickness can be independently varied), the electrochemical method permits, once the optimum entrapment conditions are established, an increase of the amount of the entrapped enzyme to be obtained only by increasing the film thickness. However a thicker film implies a slowdown in the response time (ranging, in the most favorable cases, between 20 and 40 s), a lowering of electrode sensitivity, and, at the best, a widening of the response linear range (a desirable feature). An interference-free sensor having a high enzyme content immobilized in a thin polymer film, a low response time, a high sensitivity, and a sufficiently large linear range appears highly desirable. In this paper an electrochemically synthetized poly(ophenylenediamine) (PPD) film has been used for GOx immobilization. A very thin, stable, and strongly adherent film was obtained capable of incorporating more than 3 units/cm2 of GOx. The one-step procedure for sensor preparation took typically 15 min only. An enzyme electrode device with a response time less than 1 s can be obtained. The permselectivity character of the membrane allowed the removal of ascorbate interference.
EXPERIMENTAL SECTION Preparationof Enzyme Electrodes. The base electrode used for the preparation of the PPD/GOx electrodes consisted, unless otherwise specified, of a Pt disk (1-mm diameter) sealed in Pyrex glass. The electrode surface was polished with diamond paper, washed, and electrochemically pretreated by potential cycling between -0.21 and +1.19 V vs SCE in 0.5 M H&301 until a steady state was reached (17). PPD films were electrochemically grown from a fresh solution containing 5 mmol/L of o-phenylenediamine(1,2-diaminobenzene) in an acetate buffer (pH 5.2, I = 0.2). Enzyme immobilization was performed by adding 500 units/mL of GOx to the ophenylenediamine (0-PD) solution prior to immobilization. Films were grown for 15 min at a constant applied potential of +0.65 V vs SCE from deaerated unstirred solution. Polypyrrole enzyme electrodes (Pt/PPy/GOx) were prepared at +0.75 V vs SCE for 20 min by employing a phosphate buffer solution at pH 6.8 containing 0.2 mol/L of pyrrole (Py) and 0.13 pmol/L of GOx (12). The enzyme electrodes were thoroughly washed after preparation and stored in a phosphate buffer pH 7.0 ( I = 0.2) at +4 "C when not in use.
Chemicals. GOx (type VI1 from Aspergillus Niger; 137000 units/g) and @-D-ghCOSewere obtained from Sigma Chemical Co. (St. Louis, MO). Stock glucose solutions (freshly prepared, in a phosphate buffer, pH 7.0, every 2 weeks) were allowed to mutarotate at room temperature overnight before use. o-PD and Py were obtained from Aldrich (Steinheim, FRG) and purified just before use by vacuum sublimation at 90 "C and vacuum distillation at 62 "C, respectively. All the other chemicals were analytical grade. Nitrogen used to obtain a controlled atmosphere in the electrochemical cell was of high purity grade. Apparatus. All the electrochemical experiments were carried out by a PAR 174 A polarographic analyzer (EG&G Princeton Applied Research) coupled to a Hewlett-Packard Model 1070 X-Y-t recorder (Hewlett-Packard, Palo Alto, CA) and a conventional three-electrode system with a Pt foil as counter electrode and a saturated calomel electrode (SCE) as reference. When necessary, solutions were stirred by a magnetic stirrer (Lab-Line Instruments Inc, IL). RDE (rotating-disk-electrode)experiments were performed by using an EDI-Controvit system (Tacussel, Villeurbaine, France), using a 3 mm diameter Pt-disk electrode embedded in a PTFE (polytetrafluoroethylene) body. ESCA (electron spectroscopy for chemical analysis) measurements (see ref 18 for details)were performed by a Leybold LHSlO spectrometer equipped with a sample holder suitable for angledependent measurements. Estimation of the Active Entrapped Enzyme. The enzymatic activity present on each enzyme electrode was estimated by an amperometric method. A Pt electrode was potentiostated at +0.7 V vs SCE in a stirred solution containing 0.1 M glucose in a phosphate buffer (pH 7.0, I = 0.2). When the background current diminished to a nearly constant value, a known amount of GOx solution in a phosphate buffer was injected and the "i vs t" curve for the oxidation of the resultant hydrogen peroxide was recorded. A calibration curve could be constructed by plotting the initial rate of production of HzOz (Ailat) vs [GOx]. The amount of enzyme activity present on each electrode, expressed as equivalent enzyme activity in solution, was estimated by repeating the above measurements with the test electrodes (of course not wired) in place of the soluble enzyme. Evaluation of PPD Film Thickness. The thickness of the PPD film was evaluated by using the intercept (19) of a Koutecky-Levich plot of (limitingcurrent)-' vs (rotation for the reduction of hydrogen ion at a rotating PPD film electrode in a 0.2 M trifluoroacetate buffer (pH 3.0). A k D value of 3.5 X cmz/s (19) for the product of partition ( k ) and diffusion (D) coefficient of H+ in PPD films was used in the calculation. Thickness was also evaluated by angle-dependent ESCA measurements (20)of the Pt 4f signal coming from the underlying Pt sheet on which a PPD film was grown. An exponential decay of signal intensity with the sine of electron take-off angle was used for thickness calculation (20). The escape depth value derived according to ref 21 ranged between 2.5 and 4 nm for a film density ranging between 1.5 and 1 g/cm3. As far as we know the PPD polymer density has not been reported. However the above assumption appeared reasonable since literature density values for different kinds of polymers (22) lie in the above range. RESULTS AND DISCUSSION Growth of Polymer Films. Figure 1shows a typical cyclic voltammogram for the oxidation of o-PD in a phosphate buffer (pH 7.0) solution. The oxidation wave appears completely irreversible, and on successive scans, the peak current dropped significantly with each scan until ultimately no current flowed. This behavior is indicative of a (polymeric) film coating the electrode and blocking the access of the monomer to the electrode surface. This finding is indirect, but clear, evidence of formation of a very compact and insulating film substantially free from holes. o-PD forms polymeric films on electrochemical oxidation at nearly all pH values and on a variety of electrode materials (23-25), but studies on polymer structure are substantially lacking. Recently, Chiba et al. (23) polymerized o-PD from sulfuric acid solution onto a basal plane pyrolytic graphite electrode and obtained a film with
ANALYTICAL CHEMISTRY, VOL. 62, NO. 24, DECEMBER 15, 1990
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c z Y
U U 3
0
c z W
I
0.0
POTENTIAL V v a Ag/AgCl
I p!
3
0
Flgure 1. Cyclic voltammograms for the oxidation of 5 mM ophenylenediamine at a platinum electrode in a deaerated phosphate buffer (pH 7.0, I = 0.2). Scan rate: 50 mVls. Curves 1-3 and 6 refer to the first, second, third, and sixth cycles, respectively.
a thickness of 2.5 l m (0.1 C/cm2 of charge involved) and a conductivity (dry state) of S cm-'. The same authors hypothesized for the polymeric film a ladder structure containing phenazine rings. Extrapolation of these results to the present case appeared however doubtful, since charge involved, as well as film thickness, are considerably lower here. A Pt sheet covered with PPD fii, when analyzed by ESCA, showed the Pt 4f signal, indicating the presence of a very thin film. Angle-dependent ESCA measurements gave, in fact, a thickness value ranging from 5 to 8 nm depending on the assumed polymer density (vide ante). Such a low thickness is hardly measurable by other conventional techniques, since it is even below the limit afforded by an interference microscope. An independent estimation of film thickness could be obtained, however, by RDE measurements on the reduction wave of hydrogen ions which are known to permeate through the film (19). From the intercept of a Koutecky-Levich plot of (limiting current)-' vs (rotation rate)-'I2 a thickness around 10 nm could be estimated. The permeability toward organic molecules is, of course, of great concern when attempted use of a polymeric membrane is for enzyme entrapment aimed at the realization of an amperometric sensor (for instance a glucose sensor based on glucose oxidase). In this case matrix endogenous species such as ascorbate, which are more easily oxidized than H202(the amperometrically detected species), represent unavoidable interferents. Such a kind of interference is usually removed by an additional membrane with a defined cutoff in terms of molecular weight. A single membrane capable of entrapping the enzyme and rejecting the above interferent species is highly desirable but not yet available. Figure 2 shows cyclic voltammograms obtained, in a phosphate buffer, pH 7, containing 4 mM of ascorbic acid, at a Pt bare electrode (solid line) and a PPD-covered Pt electrode (dashed line). As can be seen, the electrochemistry of ascorbic acid is nearly completely suppressed at the polymer-modified electrode. In the context outlined above these findings appear of considerable interest and peculiar to the studied system. It should be considered, in fact, that for a poly(pyrro1e)-modified electrode (the archetype of electrosynthetized polymer for glucose oxidase entrapment) just the opposite situation is verified in fact ascorbate, as well as other anionic species, showed an enhanced electrochemistry due to the electrostatic interaction with the polymer matrix containing fixed cationic sites (26). Preliminary experiments on the permselectivity of PPD films toward substrates other than ascorbic acid (e.g. uric acid, dopamine, dihydroxyphenylacetic acid, hydroquinone, and (3,4-dihydroxyphenyl)aceticacid) seem to indicate that the access of redox couples to the
P O T E N T I A L V v s Ag/AgCI Flgure 2. Cyclic voltammograms for the oxidation of 4 mM ascorbic acid in a deaerated phosphate buffer (pH 7.0, I = 0.2) at a Pt bare electrode (solid line) and PPD-covered Pt electrode (dashed line). Initial potential: -0.3 V. Scan rate: 50 mV/s.
mn
-?
200'
la
k9 W
q
100.
1
0
5
10
[GOx] (mU/mL) Flgure 3. Rate of production of H,O, AilAt as a function of the glucose oxidase concentration (see text for explanation).
electrode surface is controlled, as in the case of other nonconducting films such as poly(diviny1benzene) (27), by a mechanism of selective partitioning and diffusion within the film. Enzyme Immobilization. Glucose oxidase can be incorporated into PPD film by electrochemical codeposition of the enzyme and polymer onto the electrode surface. A pH value of 5.2 was chosen for the electrodeposition step since enzyme immobilizations performed at lower (e.g. 4.5) and higher (e.g. 6.9) pH values, both produced worse results in terms of electrode performances. The mechanism by which the enzyme is entrapped is uncertain; besides mechanisms usually invoked (.e.g mechanical entrapment and polymer charge balance by enzyme acting as counterion), adsorption of GOx at the platinum surface, prior to electropolynerization, could play a certain role (28). Under the above specified conditions, about 25-30 mU (U = units) (i.e. 3.2-3.8 U/cm2 of electrode surface) equivalent to 2.5-3.0 mU/mL of dissolved glucose oxidase could be incorporated into the film. The amount of oxidase activity incorporated in the PPD film was estimated from the rate of production of H202with the aid of the calibration curve in Figure 3. Obviously, this approach assumes that the solution enzyme
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 24, DECEMBER 15, 1990 1OL
b * b
I
-
Pt f P PD/GO
b
I-LA
I
02
04
06
08
P t 1P P y /GO
E(Voltvs S C E )
-
Figure 4. Normalized voltammogram for the oxidation of hydrogen peroxide at a R/PPD electrode in a phosphate buffer (pH 7.0, I = 0.2) containing 1 mM HO , .,
kinetics is identical with the immobilized enzyme kinetics, which could not be true, particularly when mass-transfer limitations through the polymer film are involved. Nonetheless the measured quantity can be used for comparison purposes. The amount of active enzyme entrapped in PPD film is more than 1 order of magnitude higher than that measured through a similar approach (and reported as solution equivalent) using poly(pyrro1e) as entrapping polymer (12). Electrode Characteristics. The reaction between GOx and glucose with oxygen as electron acceptor can be described by the following equations: GOx(ox) 8-D-glucose = GOx(red) D-gluconic acid
+
+
(1) GOx(red) + O2 = GOx(ox) + H202 (2) The production of H202can be easily monitored amperometrically, and the oxidation current (under constant oxygen content) can be related to the glucose concentration in the sample. The electrochemical behavior of H202on the Pt/PPD electrode (whose detailed description is beyond the scope of this paper) was found in general agreement with the findings of Gorton (29). The anodic branch of the current-potential profile a t a P t / P P D electrode in a 1 mM HzOz solution in phosphate buffer (pH 7.0) is shown in Figure 4. For both bare and modified Pt electrodes a plateau was reached nearly at the same potential (around 0.6 V vs SCE). On this basis, a working potential of +0.7 V was chosen for probing the electrode response. A typical response of a Pt/PPD/GOx electrode (potentiostated at +0.7 V in a 10 mL of stirred phosphate buffer pH 7.0) to the injection of 10 mmol/L of glucose is shown in Figure 5 . In the same figure the response obtained on a poly(pyrrole)-modified electrode with immobilized GOx (Pt/PPy/ GOx) is also shown. Two main features are readily apparent, namely a considerably faster response time (
