Anel. Chem. 1992, 64, 1541-1545
1541
Covalent Electropolymerization of Glucose Oxidase in Polypyrrole Sorrel E. Wolowacz, Bernadette F. Y. Yon Hin,and Christopher R. Lowe* Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1 QT, U.K.
Glucoro oxidase (GOx) has been chemically derivatized by coupling K(2-carboxyethyl)pyrrole lo surface lysyl rerldues wilh a carbodlhnide-promotedreaction. The modtfledenzyme (30.2 f 0.5 mol of pyrrde/moi of GOx) displayed almost identkai properties lo the natlve enzyme except that the PI was sllghtly more acidic and the staMlty was &fold higher at pH 7.0 and 60 O C . The proleln-substitutedpyrroles were acce#dble to electrochemical oxidation and in the presence of free pyrrole generated copolymers whkh covalently incorporated Oox. Thls new procedure incorporated more 8iuymeactMtyintotheconducthgpdymcHthantheprevbusiy r m e d phydcalentrapmenttechnique at equivalentenzyme concentratlonr,in the polymerizatlon media. The covalently entrapped enzyme was 171-foldmore stable than the soluble native enzyme and was used to generate a giucotw-senrltlve electrode.
INTRODUCTION The capacity of glucose oxidase (GOx) (EC 1.1.3.4) to catalyze the oxidation of 8-D-glucose to 8-D-gluconolactone has been widely promoted as a model system for the design and fabrication of reliable, low-cost amperometric biosensora withhighstorage and operationalstability.12 The enzyme is readily available, highly active, and relatively stable and is expected to play a key role in the developmentof diagnostic devices to monitor glucose for diabetics.3-6 However, an important consideration in the future development of practical devices is how to develop new immobilization strategies which enhance electron exchange between redox enzymes and electrode surfaces. Conventionaltechniques such as physical adsorption? covalent attachment?#' and cross-linking with albuminsor collagen819often lead to significant loss of enzyme activity and are inappropriate for the construction of miniature sensors which require the precise immobilization of proteins on spatially discrete electrode areas. Electrochemicalimmobilizationin conductingprovides an elegant alternative for the one-step deposition of enzymes on small-area electrodes of defined geometry.'OJ1 Such immobilization provides a simple approach for controlling the amount and spatial distribution of enzyme in the polymer (1) Bartlett, P. N.; Whitaker, R. G. Biosensors 1987/88,3, 359. (2) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. 0.;Aston, W.
J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984,56, 667. (3) Turner, A. P. F.; Pickup, J. C. Biosensors 1986,1, 85. (4) Free, H. M.; Free, A. H. Anal. Chem. 1984,56,664. (5) Tatershall, R. B. Diabetologica 1979,16, 71. (6) Tran-Minh, C. Ion Sel. Elec. Rev. 1985, 7, 41. (7) Saeao, S. V.; Pierce, R. J.;Walla, R.; Yacynych, A. M. Anal. Chem. 1990,62,1111. (8) Barker,S.A. Biosensors: ~ndamentalandApplications;Turner,
A. P. F., Karube, I., Wilson, G. S.,Eds.; Oxford UniversityPress: Oxford, 1987; p 85. (9) Bowers, L. D. Anal. Chem. 1986,58,513. (10) Foulds, N. C.; Lowe, C. R. J. Chem. SOC.,Faraday Trans. 1986, 82, 1259. (11) Umana, M.; Waller, J. Anal. Chem. 1986,58, 2979. 0003-2700/92/0384-1541$03.00/0
and is especially applicable to the construction of multianalyte microelectronic sensors.12 Extensive studies have provided ample evidence that redox enzymes may be incorporated intopolypyrrole,10-21 polyaniline,n poly(N-methylpyrro1e)mSpolyindole,%and polyphenol' fiis electrochemically deposited on appropriate electrodes. More recently, mediators have been coentrapped with the redox enzymesin order to facilitate electron exchange with the base electrode'3J6J8Jg although, with the exception of one report,%direct electron transfer between GOx and the polypyrrole backbone has not been observed. The majority of studies to date have concentrated on the physical entrapment of enzymes in electrogenerated polypyrrole.'*21 While this presents few problems with enzymes such as GOx which exhibit relatively high specific activities or which can act as a strong counteranion to the cationic polypyrrole matrix, it could present serious difficulties with other systems, where the amount of enzyme physically entrapped within the pyrrole matrix is low and the steadystate oxidation currents are correspondingly small.12 This paper presents an alternative approach in which a pyrrole analogue is covalently attached to GOx and the modified enzyme is subsequently electrochemically polymerized with free pyrrole monomers to produce electrogenerated films containing covalently immobilized enzyme. It was envisaged that covalent entrapment might improve the stability of the immobilized enzyme and the amount and reproducibility of the activity deposited.
EXPERIMENTAL SECTION Reagents. N-(2-Cyanoethyl)pyrrole, pyrrole, Na-HEPES [sodium 442hydroxyethy1)-1-piperazineethenesulfonate],DEC [1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride], diethyl ether, n-heptane,DMAB [p-(dimethylamin0)benzaldehyde],fluorescamine,and ferrocenemonocarboxylicacid were (12) Yon Hin, B. F. Y.; Sethi, R. S.;Lowe, C. R. S e w . Actuators 1990, E l , 550. (13) Foulds, N. C.; Lowe, C. R. Anal. Chem. 1988,60,2473. (14) Iwakura, C.; Kajiya, Y.; Yoneyhama, H. J. Chem. Soc., Chem.
Commun.1988,1019. (15) Mataue, T.; Kasai, N.; Narumi, M.; Niehizawa, M.; Yamada, H.; Uchida, I. J. Electroanal. Chem. Interfacial Electrochem. 1991,300,111. (16) Janda, P.; Weber, J. J. Electroanal. Chem. Interfacial Electro-
chem. 1991,300,119. (17) Slater, J. M.; Watt, E. J. Anal. R o c . 1989,26, 397. (18) Yabuki, S.; Shinohara, H.; Ikariyama, Y.; Aizawa, M. J. Electroanul. Chem. Interfacial Electrochem. 1990,18, 297. (19) Tamiya, E.;Karube,I.; Hattori,S.;Suzuki,M.; Yokama, K. Sens. Actuators 1989, 18, 297. (20) Kajiya, Y.; Sugai, H.; Iwakura, C.; Yoneyama, H. Anal. Chem. 1991, 63, 49. (21) BBlanger, D.; Nadreau, J.; Fortier, G. J. Electroanal. Chem. Interfacial Electrochem. 1990, 274, 143. (22) Shinohara, H.; Ohiba, T.; Aizawa, M. Sens. Actuators 1988,13, 79. ...
(23) Bartlett, P. N.; Whitaker,R. G.J.Electroanal. Chem. Interfacial Electrochem. 1987,224, 27. (24) Bartlett, P. N.; Whitaker,R. G. J. Electroanal. Chem. Interfacial Electrochem. 1987,224, 37. (25) Pandey, P. C. J. Chem. SOC.,Faraday Trans. 1988,84,2259. (26) Yabuki, S.; Shinohara,H.;Aizawa, M. J. Chem. SOC.,Chem. Commun. 1989,945.
@ 1992 American Chemlcel Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
purchased from Aldrich Chemical Co., Ltd. (Gillingham,Dorset, UK). Glucoseoxidase (&Dglucose,oxygen 1-oxidoreductase,EC 1.1.3.4) (125 units mg-l type VII, Aspergillus nger), peroxidase (donor: hydrogen peroxide oxidoreductase,EC 1.11.1.7) (95units mgl type 1, horseradish), Sephadex G-15, and N-a-acetyl+ lysinewere obtained from Sigma Chemical Co., Ltd. (Poole,Dorset, UK). Nitrogen (Opfreegrade) was from BOC Ltd. (Guildford, Surrey, UK). All other reagents and solvents were supplied by BDH Chemicals Ltd. (Poole, Dorset, UK) or Fisons Scientific Equipment (Loughborough, Leicestershire, UK). Preparation of Pyrrole-Modified Enzyme. N42-cyanoethy1)pyrrole was hydrolyzed to N-(2-carboxyethyl)pyrroleby refluxing in aqueous KOH, and after acidification and ether extraction, a beige crystalline product was obtained from the etheral extrackn Recrystallization of the product from warm n-heptane produced white needle-likecrystals in a yield of 76 % The compound was confiimed to be N-(2-carboxyethyl)pyrrole by melting point (58-59 OC), infrared (IR), nuclear magnetic resonance (NMR), mass spectrometery (MS), and elemental analysis. Calcd for C7HsN02: C, 60.43; H, 6.47; N, 10.07. Found: C, 61.31; H, 6.47; N, 10.07. The covalent modification of GOx was performed by coupling N-(2-carboxyethyl)pyrrolewith enzymic amines using a modified method after Degani and Heller.= N-(2-Carboxyethyl)pyrrole (140 mg) was dissolved with gentle warming in 4 mL of 0.15 M Na-HEPES pH 7.3. DEC (100 mg) and 810 mg of urea were added, and the pH was readjusted to 7.2-7.3. The mixture was cooled to 0 OC, and 60 mg of GOx was added with stirring. The incubation wasstirred overnight at 4 "C (15h) and filtered through a 0.8 pM Millipore filter. The protein was separated from the reaction mixture by gel filtration chromatography using a Sephadex G-15column of 2-cm diameter and 20-cm height. The protein was eluted with 0.1 M sodium phosphate buffer pH 7.0, and the combined fractions were made 0.1% (w/v) in sodium azide and stored at 4 OC. The protein and FAD content of the eluted fractions was estimated using the Bradford assay and the absorbance at 445 nm, respectively. The degree of substitution of enzymic amines with N42-carboxyethy1)pyrrole was investigated by estimation of the pyrrole content and the reduction in amine content of the isolatedprotein. The pyrrole concentration was determined using a modified method after Muhs and Weiss.29 (Dimethy1amino)benzaldehyde (DMAB)[50pL,of 1% (w/v)]in orthophosphoricacid was added to400 pL of a sample containing 0-40 pg of pyrrole. After dilution to 1mL with glacial acetic acid and incubation for 2 h at 40 "C, the absorbance was read at 559 nm. Unmodified GOx was used as a no-sample blank, and pyrrole as the standard. The reduction in amine content was determined by estimation of the number of amines of the modified and unmodified protein by the fluorescamine assa-th N-a-acetyl-L-lysine as a standard. A time course for the modification of GOx by N-(2-carboxyethy1)pyrrole was performed. An incubation was prepared as described above and maintained at 18 OC. Samples (20pL) were taken at intervals into 3 mL of 0.1 M sodium phosphate buffer pH 8.0, and immediately assayed for amines using the fluorescamine assay.go The reaction of fluorescamine with amines is complete with milliseconds31and serves to quench the coupling reaction as well as determine the amine content. Characterization of the Modified Enzyme. The specific activity of the modified and unmodified enzyme was estimated spectrophotometrically using the horseradish peroxidase coupled assay for hydrogen peroxide detection.32 The extent of crosslinking of the protein due to the condensation of enzymic carboxylic acid and amine groups promoted by DECwas investigated by sodium dodecyl sulfakpolyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point was estimated by isoelectric focusing using an LKB Multiphore ii horizontal electrophore-
.
(27) Blume, R. C.; Lindwall, H. G. J. Org. Chem. 1945, 10, 255. (28) Degani, Y.; Heller, A. J. Phys. Chem. 1987,91, 1285. (29) Muhs, M. A.; Weiss, F. T. Anal. Chem. 1958, 30, 259. (30) Bohlen, P.; Stein, S.; Dairman, W.; Underfriend, S. Arch. Biochem. Biophys. 1973,155,213. (31) Ling, T. G. I.; Ramstorp,M.; Mattiasson, B. Anal. Biochem. 1982, 122, 26. (32) Undenfriend, S.; Stein, S.;Bdhlen, P.; Dairman, W.; Leimbruber, W.; Weigele, M. Science 1972, 178, 871.
sis system in conjunction with a Multitemp ii thermostatic circulator and a Macrodrive 5 constant power supply. Twomillimeter polyacrylamide gels (T = 5 %, C = 3% ) containing Ampholine buffer (pH 3.5-10) were prefocused for 500 Volthours, loaded, and run using voltage, current, and power limits of 1500V, 50 mA, and 4 W, respectively,for 8000 Volt-hours. The PIwas estimated by comparison with LKBIEF protein standards. The pH optimum of the modified and unmodified enzyme was investigated between pH 4 and 9 pH by spectrophotometric assay of GOx activity at pH intervals using appropriate buffers (sodium acetate pH 4-6, sodium phosphate pH 6.5-7, and Trizma pH 8-9). The stability of the modified and unmodified enzymes at 60 OC was determined by removal of 20-pL samples of an enzyme solution incubated at 60 OC into 2 mL of ice-cold buffer and spectmphotometric estimation of remaining activity. The Michaelis constant for glucose with oxygen as the electron acceptor was determined by making initial rate measurements of assaye containing -40 pg mL-l GOx and 0-20 mM glucose. Electrochemical Measurements. dccyclicvoltammetry and chronoamperometric measurements were performed with an EG&GPrinceton Applied Research Model 273 potentiostat/galvanostat. The electrochemical cell was a three-electrode waterjacketed Pyrexcell with a workingvolume of 2 mL. Temperature regulation was provided by a Julabo VC1 water bath and circulator, and unless otherwise stated, all experiments were carried out at 25 OC. Output from the potentiostat was recorded on a Philips PM 8043 X-Y-t recorder. The reference and auxiliary electrodes were a sintered Ag/AgCl electrode (-45 mV saturated calomel electrode (SCE) and +lo7 mV VB SCE in 0.1 M sodium phosphate in presence of 10 mM NaClOd and a 10cm-long platinum wire, respectively. The working electrode comprised either a mini glassy carbon electrode (3-mmdiameter) or a disposable graphite disk (5-mm diameter) cut from a sheet of graphite foil (1-mm thickness) supplied by Johnson Matthey (Royston, He&, UK). The graphite disk was inserted into a Teflon electrode holder designed in-house providing an exposed electrode surface of 3-mm diameter. The supportingelectrolyte was 0.1 M sodium phosphate pH 7.0, containing 10 mM sodium perchlorate in all experiments, unless otherwise stated. The ferriciniumion and its derivativeshave been widelystudied as mediators for amperometric glucose sensors to circumvent the potential problems of oxygen limitation and interference by ascorbic acid. It is therefore important that the modified COX is ammenable to reoxidation by mediators such as ferrocene for its use in a "second generation" type sensor. The second-order rate constant for the reoxidation of the reduced form of modified GOx by ferrocenemonocarboxylicacid was determined electrochemically using the analysis of Nicholson and ShahbgCyclic voltammetry was performed in a nitrogen-purged quiescent electrolyte containing 50 mM glucose and 0.5 mM ferrocenemonocarboxylic acid. A series of voltammograms were recorded for a set of scan speeds betwen 5 and 100 mV 8-l in the presence of 5-30 pM modified GOx. The pseudo-firsborder rate constants at each enzyme concentration were derived from the ratio of kinetic and diffusion-limited currents and scan speed making use of a working curve (Figure 14 of ref 33). The second-order rate constant was derived from the slope of a plot of the pseudofist-order rate constant against enzyme concentration. Preparation and Evaluation of Enzyme Electrodeo. Modified enzyme copolymer films were electrodeposited at graphite disk electrodes by oxidizing unstirred deaerated aqueous electrolyte solutions containing 0.1 M pyrrole and known amounta of enzyme-modified pyrrole. The optimum potential for the immobilization of modified GOx was investigated and observed to be around 0.7 V vs Ag/AgCl. Consequently, the enzyme f i i s were generated at a fixed potential of 0.7 V vs Ag/AgCl until a charge of 10mC had accumulated, rinsed thoroughly, and stored overnight in fresh buffered electrolyte at 4 "C before use. The enzymeactivityimmobilizedin the polymer f i i was determined by the chromogenic-4-aminoantipyrineassay.31 The electrode was placed in a stirred assay mixture contained in a thermostated cell, and the absorbance monitored continuously by circulating the assay medium through a flow through cuvette. The stability of the immobilized modified GOx was investigated (33) Nicholson, R. S.; Shain, I. Anal. Chem. 1964, 36, 706-723.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
0.8
__o,
-
Table I. Enzyme Characteristics of Modified and Unmodified Glucose Oxidase
FAD pyrrole
modified
1
PI pH optimum
E
%
4
0.2
l-1
0.0 0
120 160 Elution VOI,ml
40
80
"-
I uv
-0
0
- 24
80
U
60
- 16 -8
t
0
0 0
5
10
15 t,
20
25
hours
Figure 2. Time course for the modification of GOx with N(2car-
boxyethyibyrrole at
GOx 4.2
5.5-6.5
5.5-6.5
0
Flgure 1. Isolation of chemically modified W x by gel filtration.
'=
unmodified
GOx 3.9-4.1
pseudo-fit-order rate constant of 3.02 X 1W s-l 18.3 X 1V 8-l enzyme deactivation at 60 OC K, (Michaelis constant) 11.1mM 12.2 mM K,(second-orderrate constant for 1.8X l@ 1.8 X 1@ enzyme mediation with ferroL mol-' 8-1 L mol-' 8-1 cenecarboxylic acid)
v)
45
1541
18 "C.
by measuring the rate of loss of activity at 60 OC and compared with that of an electrode containingpolypyrrole-entrappedGOx. Finally,the electrochemicalresponse of a modiied GOx electrode to glucose was determined using ferrocenemonocarboxylic acid as soluble mediator.
RESULTS AND DISCUSSION Covalent Modification of GOx with N-(2-Carboxyethy1)pyrrole. The covalent modification of GOx was performed by carbodiimide coupling of N-(2-carboxyethyl)pyrrolewith enzymic amines using a modified method after Degani and Heller.28 These authors suggested that the presence of the carbohydrate moiety of GOx conceals the exterior of the protein from chemical modification of this kind and that a limited concentration of urea (3.4 M) is required to 'open" the structure slightly. Initial attempts to modify GOx in the presence of 3.4 M urea yielded an enzyme with some degree of substitution (5-6 mol of pyrrole/mol of GOx); however, 4 0 4 0 % of the activity was lost. The elution profile of gel filtrations of such incubations showed two FAD peaks: the f i t was coincident with protein elution, while the second contained no protein, indicating that a substantial percentage of the prosthetic group was dissociating from the apoenzyme. Attempts to conduct the modification under conditionswhich stabilizethe apoenzymeand reconstitute the modified protein with FAD produced no improvement in activity. However, when modificationwas performed in the absence of urea the specific activity of the isolated enzyme was identical within the experimental error to that of the unmodified enzyme, and a single FAD peak was observed concurrent with the protein peak (Figure 1). The small pyrrole peak coincident with the protein peak is attributable to covalently bound pyrrole and cannot be accounted for by adsorption to the protein since an equivalent peak was not observed during the elution of a control incubation containing no carbodiimide. The time course of the reaction (Figure 2) showed that 85% of enzymic amines were modified after 1h and the reaction
had reached 95 % completion after 22 h, 30.2 k 0.5of a possible 32 amines being modified. Characteristics of the Chemically Modified Enzyme. Table I compares the properties of the modified and native GOx. The pH optimum, Michaelis constant (K,)for glucose and the second-orderrate constant (k,)for enzyme mediation with ferrocenemonocarboxylicacid were essentially identical. In principle, the substitution of amino groups with uncharged pyrrole moieties will result in an increase in net negative charge and a shift of pI to a lower value. Heterogeneityin the degree of substitution will cause a variation in PI throughout the population of enzyme molecules. As expected, isoelectric focusing of the modified enzyme revealed a diffuse band at a slightly more acidic pH than the PI of the native enzyme, 4.2.% SDS-PAGE revealed a single band attributable to the GOx monomer for both modified and unmodified GOx with a migration consistent with a molecular weight of 75 kDa. No higher molecular weight bands indicative of cross-linking of the modified protein were observed. In addition, the pseudofist-order rate constants for the thermal denaturation of modified and unmodified enzyme at pH 7.0 and 60 "C showed that the modified enzyme was 6-fold more stable than the native GOx. Electrochemistry of Pyrrole-Modified GOx. The accessibility of the enzyme-substituted pyrroles to electrochemical oxidation was investigated by cyclic voltammetry. Figure 3a shows the first oxidation wave recorded for solutions containing pyrrole and unmodified and modified enzyme. A single irreversible anodic peak was observed at 0.88 V vs Ag/ AgCl for the pyrrole-modified enzyme, showing that at least some of the pyrrole monomers on the enzyme were available for electrochemical oxidation. The peak potential was more positive than that of pyrrole as expected for N-substituted pyrrole derivative@ and coincident with that of N42-carboxyethy1)pyrrole. Electrochemicaloxidation of supporting electrolyte containing modified GOx alone produced no observable film on the electrode surface presumably because of steric hindrance involved in the dimerisation of pyrrole radical cations covalently attached to enzyme molecules. Therefore the formation of copolymers between free and enzyme-bound pyrrole was investigated. Cyclic voltammetry of mixtures of pyrrole and pyrrole-modified GOx (Figure 3b) showed a single sharp oxdiation peak rather than two distinct peaks or a broad peak characteristic of a mixture of two independent redox species. Furthermore, the peak potential shifted from that of pyrrole-modified GOx to that of pyrrole as the ratio of free to substituted pyrrole was increased. This behavior is consistent with the cooxidation of two species to form random ~opolymers'3,3~ and provides evidence for the concomitantcovalent immobilization of GOx during copolymer formation. The sharpness of the peak indicates that the population of contributing groups are oxidized at a very similar potential and rate and the (34)Pazur, J. H.;Kleppe, K. Biochemistry 1964,3, 578. (35)Sundaresan, N.S.;Basalt, S.; Pomerantz, M.; Reynolds, J. R. J.
Chem. Soc., Chem. Commun. 1987,621.
ANALYTICAL CHEMISTRY, VOL. 64,NO. 14,JULY 15, 1992
1544
100
0 I
I
I
I
I
I
0.0
0,ii
I .2
0.0
0.6
I .2
--
50 I.
3
400
t , min
Cycllc voltammograms of mlxtures of pyrrole and Gox-modlfledpyrrole under the same condttions as a. ,Ep,.values wsre at 0.81 and 0.84 V for 1O:l and 6:l pyrole/QOx-modlfiedpyrrole, respectively. E
300
Flguro 5. Dotmination of pseudo-firstorder rate mtant of deneturation of (0)unmodlfled and (0)modified QOx lmmobillzd In potypyrrok film at 60 O C , pH 7.0.
Figure 3. (a)Cyclic voltammograms of untwdifled Gox (42pM), pyrrole (0.87mM), and Gox-modlfiedpyrrole ([py] = 0.86 mM, [Gox] = 46 pM) at an activated glassy carbon electrode at a scan rate of 100 mV s-' in a Nrpurged 0.1 M sodlum phosphate soiutlon containing 10 mM NaC104. The oxidation peak potentlal, &, for pyrrole was observed at 0.7 V and that of Gox-modHied pyrrole at 0.68 V. (b)
(u
200
'
0
0
4
3
$
i
4
1
0
10
31
20
[Glucose], mM F@m 6. Calibration curve for the amperomtrlc response of glucose at a polypyrrobmodlfledO x ekctrode at 26 OC, pH 7.0. Steadystate currents were measured at 0.3 V wlth 0.5 mM forrocenemonocarboxyik acid as mediator. 0: 0
I
10
I
1
I
20
30
40
Electrolytic [GOx], U/ml
Dependence of immoblllzed enzyme actMty on the concentration of Gox (0)unmodified and (0)pyrrole modified, present In the polymerization solution. Flgure 4.
coincidence of this potential with that of N-(2-carboxyethyl)pyrrole may indicate that the environment on the protein surface does not inhibit the oxidation significantly,and large overpotentials are unnecessary. Growth of Copolymer Films of Modified GOx with Pyrrole. Smoothblack adherent f i i were grown at graphite disk electrodes by the cooxidation of mixtures of pyrrole and pyrrole-modified GOx. The apparent amount of enzyme activity immobilized at the electrodes was determined by the coupled spectrophotometric assay. Previous studies on the physical entrapment of GOx in polypyrrole films has shown a correlation between the enzyme concentration in the electropolymerization medium and the resultant enzymatic activity incorporated on the electrode surface.lO A comparison of the apparent immobilized activity of modified as opposed to unmodified GOx is shown in Figure 4 as a function of the concentration of enzyme present in the electrolyte during polymerization. An increase in electrodeimmobilizedenzyme activity was observed for both modified and native enzymes.
However for modified GOx the maximum activity achieved was 30% higher than that of the unmodified enzyme, and the activity reached a saturation level at a lower concentration of enzyme in the supporting electrolyte. This improvement in loading efficiency could be advantageous for the immobilization of expensive or scarce enzymes or those with low specific activities. Characterization of Enzyme Polymer Films. Good enzyme stability is critical for the development of successful enzyme biosensors. The rate of loss of enzyme activity a t 60 "C was determined for both modified and unmodified GO. entrapped in polypyrrole films. Figure 5 shows the semilogarithmic plot for the determination of the pseudo-first-order rate constant for the denaturation of unmodified and modified GOx. Both immobilized enzymes showed a first-order rate of decay in activity, with pseudo-first-order rate c o ~ t a n t e for unmodified and modified GOx, calculated from the dopes of Figure 5, of (6.7 & 0.05) X and (1.07 0.005) X s-l,respectively. The stability of the covalently immobilized enzyme was (6.2 f 0.3)-fold higher than that of the p h y e i d y entrapped enzyme, 28-fold higher than the modified enzyme in solution, and 171-fold higher than the native soluble enzyme. The electrochemical response to glucose of electrodee generated by the covalent immobilization of modified GO. in polypyrrole films was investigated using ferrocenemonocarboxylic acid as mediator. Steady-state current measurementa in the absence of oxygen were recorded at 0.3 V vs
*
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, l9g2
AgIAgC1 in the presence of glucose. Figure 6 represents a typical calibration curve for glucose at an immobilized modified GOx electrode. The enzyme reaponse was consistent with Michaelis Menten kinetics, giving an apparent Michaelis constant (Km')of 11.9mM, virtually indistinguishablefrom the soluble native and pyrrole-modified enzymes. Since there is no change in the apparent Kmf,we can conclude that the apparent enzyme activity measured at the copolymer electrodes is indicative of the electrode active enzyme loading.
CONCLUSIONS Despite the obvious elegance of the concept of electrodeposition of enzymes in conducting polymers,lOJ1 a number of persistent problems remain to be resolved before a more widespread application of the technique could be envisaged. Many of these difficulties arise because the morphology, mechanicalproperties, and conductivityof electrochemicallypolymerized conducting polymers are critically dependent on a number of facets of the system, including the electrochemical parameters, solvent, nature of the counterion, and the presence or absence of protein.lO~BJ7 Scanning electron micrographs of polypyrrole show an extremely porous structure, with typical pore sizes up to a few micrometers, which change to a more fibrillar structure in the presence of increasing amounts of GOx.l0 It is also likely that relatively (36)Reynolds, J. R.J. Mol. Electron. 1986,2, 1. (37) Din, A. F.;Hall, B. ZBM J. Res. Deu. 1983,27,342.
1645
acidic enzymes such as GOx would nonspecifically adsorb to the morphologically complex polypyrrole films and thereby complicate the entrapment proce~s.'~Consequently, controlled and reproducible deposition of enzymes by physical entrapment in electrogenerated polymers, especially in the presence of other coentrapped reagents such as mediators14-19 or coenzymes,18 would be difficult to achieve. On the other hand, covalent electrochemical deposition of the proteins in the conductingpolymer has a number of distinct advantages: it allowsthe controlled and reproducible deposition of proteins irrespective of their size or overall charge, the stabilization of the deposited proteins by covalent interactions with the matrix backbone, the deposition of high activities from lower concentrationsof enzyme in the polymerization mixture, and the prospect of covalent co-incorporationof a variety of other reagents, mediators, and coenzymes, concurrent with the enzyme deposition, while still retaining all the advantages of a single one-step deposition procedure.
ACKNOWLEDGMENT The financial support of the Science and Engineering Research Council is gratefully acknowledged.
RECEIVED for review January 27, 1992. Accepted April 10, 1992.
