Anal. Chem. 1883, 65, 2067-2071
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Covalent Electropolymerization of Glucose Oxidase in Polypyrrole. Evaluation of Methods of Pyrrole Attachment to Glucose Oxidase on the Performance of Electropolymerized Glucose Sensors Bernadette F. Y. Yon-Hin, Maria Smolander,?Thomas Crompton, and Christopher R. Lowe. Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 lQT, U.K.
Methods for covalently immobilizing glucose oxidase in polypyrrole are investigated. The enzyme was chemically modified with pyrrole using one of three different reactive side chains found in the protein. The reactions involve carbodiimide coupling to either lysyl or carboxyl residues on the enzyme and Schiff base reaction of the carbohydrate moiety. Optimal coupling was achieved with the carbodiimide reaction, 15-20 mol of pyrrole/mol of enzyme compared with 6 mol of pyrrole/mol of enzyme for the Schiff base method. The pyrrole-substituted enzymes were electrochemically active, showing that the pyrrole moieties were oxidizable. Electropolymerized enzyme films deposited from solutions of free pyrrole and amounts of native or pyrrole-modified enzyme of equivalent activity resulted in covalently immobilized enzymes showing both higher enzyme activities and amperometric glucose responses than polypyrrole-entrapped native enzyme. The apparent Michaelis constant (KM') and pH optimum of the modifiedenzymes electrodes correlated with that of the native enzyme electrode. Enzyme films generated from carbodiimide-modified enzymes were 6-fold more stable to thermal denaturation than native enzyme electrode.
INTRODUCTION The use of electrochemically generated polymers as immobilizationmatrices for enzymeshas attracted considerable interest since we reported the incorporationof glucose oxidase (GOx) in polypyrrolefilms.lJ This new and simple technique allows the one-step deposition of enzymesa t electrodes under mild conditions. Sincethe immobilization process is restricted to the electrode surface, it is effective both for the selective deposition of different enzymesa t multielectrode devicesand for the well-defined localization of enzymes at microelectrodes.3 Subsequent studies have shown that other electropolymerized systems such as p~ly(N-methylpyrrole),~ t Current address: Vl",Biotechnical Laboratory, P.O. Box 202, SF02151 Espoo, Finland. (1)Foul&, N. C.: Lowe, C. R. J. Chem. SOC., Faraday Trans. 1 1986, 82, 1259-1264. (2) Foul&, N. C.; Lowe, C. R. Anal. Chem. 1988,60, 2473-2478. (3) Yon Hin. B. F. Y.: Sethi. R. 5.: Lowe. C. R. Sens. Actuators 1990, E1 ,.650-554. ' (4) Bartlett, P. N.; "hitaker, R. G. J. ElectroanaL Chem. 1987,224, 37-48,
0003-2700/93/0365-2067$04.00/0
polyaniline: poly (0-phenylenediamine)?and poly(pheny1ene oxide)' may also be used in a similar way to entrap GOx. Although the mechanism of enzyme immobilization in these polymers is uncertain, it is probably due to electrostatic interactions between the negatively charged protein and the growing cationic polymer resulting in the incorporation of GOx as the counter polyanion. However, while this concept is attractive for the construction of biosensors, there are potential problems with enzyme leakage from the polymer and limited immobilized activity for enzymes of low specific activity. In a more recent study, we reported a new deposition strategy involving the covalent attachment of GOx to the polymer and which combines the conceptsof spatial resolution provided by the electrochemical immobilization and the enhanced stability generally accompanying covalentbonding.8 Terminal amine groups on the surface lysyl residues of the enzyme were chemicallymodified with pyrrole moieties, and the pyrrole-modified GOx was copolymerizedwith pyrrole to produce conducting polymer films containing covalently immobilized GOx. The resulting enzyme electrodes showed increased activity and thermal stability compared to polypyrrole-immobilized GOx, in which the enzyme was physically entrapped as a counter-anion. In this paper, we investigate further the performance of covalently electropolymerized GOx electrodes. Pyrrole functionalities were chemically coupled to GOx by modifying different side chains on the enzyme (Figure 1). The modification reactions involved carbodiimide coupling (schemes A and B in Figure 1)of either carboxy derivatives of pyrrole to amine groups of lysyl residues of GOx or amine derivatives of pyrrole to carboxyl groups of GOx. Alternatively, amine derivatives of pyrrole are coupled to the carbohydrate moiety of GOx by Schiff base formations (scheme C in Figure 1).It is known that the chemicalmodification of proteins can alter their properties significantly.10Jl In this report, we compare the enzyme characteristics of the three pyrrole-modifiedGOx preparations with that of the native enzyme and their electrooxidationat glassy carbon electrodes. Enzymepolymer films were prepared by copolymerizationwith underivatized pyrrole and evaluated with respect to activity, stability, pH optimum, and amperometric response to glucose. (5) Shinohara, H.; Chiba, T.; Aizawa, M. Sens. Actuators 1988, 13, 79-86. ..
(6) Malitesta, C.; Palmisano, F.; Torsi, L.; Zambonin, G. Anal. Chem. 1990,62, 2735-2740. (7) Bartlett, P. N.; Tebbutt, P.; Tyrrell, C. H. Anal. Chem. 1992,64, 138-142. (8) Wolowacz, S. E.; Yon Hin, B. F. Y.; Lowe, C. R. Anal. Chem. 1992, 64,1541-1545. (9) Yasuda, Y.; Takahaahi, N.; Murachi, T. Biochemistry 1971, 10, 2624-2630. (10)Plapp, B. V. J. Biol. Chem. 1970,245, 1727-1735. (11) Kaiser, E. T.: Lawrence, D. S.:Rokita, S. E. Annu. Reo. Biochem. 1985,54,56&595, 0 I993 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
\
0 GOx
4;
,c,
Flgure 1. Chemical reactlons Involved In the modlflcatton of O x wRh pyrrole. Carbodllmlde coupllngof pyrrole to (A) Qox lysyl residues and (B)Qox carboxyl residues or (C) Schlff base coupllng of pyrrole to GOx carbohydrate residues.
EXPERIMENTAL SECTION Chemicals. N-(2-Cyanoethyl)pyrrole,pyrrole, lithium aluminum hydride, n-pentane,DEC [1-[3-(dimethylamino)propyll3-ethylcarbodiimide hydrochloride], diethyl ether, n-heptane, and DMAB [p-(dimethy1amino)benzaldehydel were obtained from Aldrich Chemical Co., Ltd. (Gillingham, Dorset, U.K.). Glucose oxidase (GOx) (@-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) from Aspergillus niger; peroxidase (donor:HzO2 oxidoreductase, EC 1.11.1.7) from horseradish; and Sephadex G-15 were purchased from Sigma Chemical Co., Ltd. (Poole, Dorset, U.K.). All other reagents and solvents were supplied by BDH Chemicals Ltd. (Poole, Dorset, U.K.) or Fisons Scientific Equipment (Loughborough, Leicestershire, U.K.). Synthesis of Pyrrole Analogues. N-(3-Aminopropyl)pyrrole was synthesized by reduction of N-(2-cyanoethyl)pyrrole with LiAlH, in dry diethylether in a yield of 93.6% and identified by infrared spectrometry.2 N-(2-Carboxyethyl)pyrrolewas prepared by the hydrolysis of N-(2-cyanoethyl)pyrrole in aqueous KOH.lZ The product was obtained in a yield of 76% and was confiied by melting point (58-59 "C) and infrared spectrometry. Chemical Coupling of Pyrrole Analogues to Glucose Oxidase. Carbodiimide Coupling Reaction. GOx wax chemically derivatized with pyrrole functionalities by a carbodiimidepromoted reaction as described in a recent publication.* The lysyl amino groups were coupled to N-(2-carboxyethyl)pyrrole using DEC in Na-HEPES buffer, pH 7.3. The modified enzyme (lysyl-modified GOx) was separated from the reaction mixture by gel filtration chromatography on Sephadex G-15. In the present study, the carboxyl groups on GOx were also modified using the carbodiimide couplingprocedure. N-(3-Aminopropyl)pyrrole (1 mmol)waa dissolved in 3 mL of 0.1 M sodiumphosphate buffer, and the pH was adjusted to 5.5, followed by the addition of a GOx solution (80mg in 1mL of buffer) and 0.5 mmol of DEC. The reaction mixture was stirred gently in an ice bath for 15min and incubated overnight at 4 "C. The chemically modified enzyme (carboxyl-modifiedGOx) was separated from the reaction mixture by gel filtration chromatography on Sephadex G-15and stored at +4 "C. The protein and FAD concentration of the (12)B l u e , R. C.;Lindwall, H. G. J. Org. Chem. 1946,10, 255-258.
eluted fractions were measured by the Bradford assay and the absorbance at 445 nm, respectively.* Schiff Base Reaction. Periodate oxidation of GOx was performed as described by Yasuda et a1.lO The enzyme solution (80 mg in 4 mL of 0.05M sodium acetate buffer, pH 5.5) was incubated with 25 Kmol of sodium metaperiodate on an ice bath for 30 min. The oxidized protein was recovered by filtration on a Pharmacia PD-10 column with 0.1 M phosphate buffer, pH 7.0 and then incubated with 1mmol of N-(3-aminopropyl)pyrrole at +4 "C overnight to allow Schiff base formation. The resulting pyrrole-modified GOx was reduced by sodium borohydride (20 mg). The enzymevolumewas reduced by means of ultrafiltration through a membrane (AmiconPM30, MWCO 3000))and dialyzed against 0.1 M sodium phosphate buffer to remove the remaining traces of N-(3-aminopropyl)pyrrole. The modified GOx (carbohydrate-modified GOx) was stored at +4 "C. The protein and FAD content of the modified enzyme solution was estimated as described above. Characterization of Pyrrole-Functionalizd GOx. The extent of derivatization achieved by both enzyme modification procedures was determined by assaying the pyrrole content of the modified GOx with DMAB.lS The specific activities of the modified enzymes were measured spectrophotometrically by the peroxidase-catalyzed reaction of an oxidizable dye, 4-aminoantipyrine and phenol." This assay was also used to investigate the effect of modifying different groups on the enzyme on the Michaelis constant for glucose (KM).SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)was used to check that no cross-linkingof the enzyme occurred as a result of the carbodiimide coupling process. Changes in the isoelectric point of the derivatized enzymes were determined by isoelectric focusing using gels of pH gradient 4-6 with a PhastSystem from Pharmacia LKB Biotechnology. The thermal stability of the enzymes was studied by inactivation at 60 "C. Aliquots of the incubated enzymes were diluted with ice-cold buffer, and the remaining activity was assayed spectrophotometrically. Electrochemistry. Direct current (dc) cyclic voltammetry and chronoamperometric experiments were performed with an EG&G Princeton Applied Research Model 273 potentioetat/ galvanostat interfaced to a PC. A single-compartment waterjacketed Pyrex cell with a working volume of 1 mL was used. Temperature regulation was provided by a Julabo F10 water bath and circulator, and unless otherwise stated, all experiments were carried out at 25 "C. The reference electrodewas a saturated calomel electrode (SCE) and the counter electrode a long piece of platinum wire. Working electrodes were either a mini glassy carbon electrode (3-mm diameter) or a platinum disc electrode (3-mmdiameter)from Metrohm AG, Herisau, Switzerland. Prior to use, working electrodes were polished with an alumina slurry and thoroughly rinsed in distilled water. In addition, glassy carbon electrodes were activated by cycling between -1.7 and 1.7V vs SCE for 30 min. The electrolyte solution used in all experiments was a 0.1 M sodium phosphate buffer solution containing 0.1 M sodium chloride, pH 7.0. GOx Polymer Film Preparation and Characterization. The enzyme f i e were deposited at platinum disc electrodes by electrochemically oxidizing an electrolyte solution containing 0.1 M freshlydistilled pyrrole and known amounts of either native or pyrrole-modified enzyme. The oxidation potential was fixed at 0.7 V vs SCE until the amount of charge passed was 10 m C. The resulting enzyme polymer electrodeswere rinsed thoroughly in distilled water and incubated overnight in buffered electrolyte at 4 "C before use. The enzyme activity immobilized in the polymer films was measured spectrophotometrically by the 4-aminoantipyrine assay. The electrode was placed in a stirred assay mixture contained in a thermostated cell, and the absorbance was monitored continuouslyat 510nm using a flow-through cuvette linked to the assay mixture. The electrode response to glucosewaa monitoredamperometridyby measuring the steadystate current for H202 oxidation at 0.7 V vs SCE. The pH dependence of the immobilized enzymes was compared. The stability of the immobilized enzymes was investigated, as (13)Muhs, M. A.;Weka, F. T. Anal. Chem. 1968,30, 259-266. (14)Ling, G.I.; Ramstorp,M.;Mattiasaon, B. Anal. Chem. 1982,122, 26-32.
ANALYTICAL CHEMISTRY, VOL. 05, NO. 15, AUGUST 1, lQQ3 2089
Table I. Characteristics of Native and Pyrrole-ModifiedGOx native GOx Lysyl-mod. GOx no. of pyrroles 0 15-20 sp act. (% of native GOx) 100 97 PI
KM for glucose, mM lst-order rate constant of deactivation at 60 O C , 8-1
4.2 17 2.4
X
lo-'
carboxyl-mod. GOx
3.9-4.1 16 1.2 x lo-'
described above for the soluble enzyme deactivation studies, by measuring the loss of activity at 60 O C .
RESULTS AND DISCUSSION Covalent Modification of GOx with Pyrrole Analogues. The protein fractions collected from all three preparations showed the presence of pyrrole; since no pyrrole was detected in the protein fractions when a control mixture of pyrrole and GOx containing no coupling reagent was used, it was concluded that the pyrrole found in the protein fractions were covalently bound to GOx. The efficiency of coupling was greater with the carbodiimide-promoted reaction, which modified 15-20 enzymic amines with N-(2-carboxyethy1)pyrrole and 16 enzymic carboxyl groups with N-(%aminopropy1)pyrrolewhereas the Schiff base reaction coupled only six pyrrole moieties to the glycoenzyme residues. Characterization of Pyrrole-Modified GOx. Table I summarizes the characteristics of the modified and native enzymes. The substitution of enzyme amine groups with uncharged pyrrole functions decreased the PI(3.9-4.1) of the enzyme due to an increase in net negative charge, whereas substitution of carboxylgroups on GOx with pyrrole moieties shifted the p l to higher values, since the removal of carboxyl functions decreased the net negative charge on the enzyme. A diffuse band was observed between pI4.4-5.0 compared to a tightly focused band at PI4.2 for the native enzyme. On the other hand, the p l for Schiff base-modified GOx was similar to that of the native enzyme. It was necessary, however, to determine whether any cross-linking of the enzyme occurred during the carbodiimide-promotedreaction, since both carboxyl and amine groups are available for modification on the enzyme. SDS-PAGE revealed a single band attributable to the GOx monomer (MW 75 kDa) for both carbodiimide-modified enzymes,indicating that no crosslinking of the modified enzyme took place. Furthermore, the specific activities of the modified enzymes were not significantly impaired. The pseudo-first-order deactivation constants calculated from the thermal stability data showed that carbodiimide coupling of GOx via both amine and carboxyl groups on the enzyme increased ita stability by 2 and 3-fold, respectively, whereas the modification of the enzyme carbohydrate had little effect on the enzyme stability. A thermal inactivation study of periodate-oxidized GOx gave similar resulta.16 Covalent coupling to functional groups of the enzyme may affect ita kinetics for substrate oxidation and result in a change in Michaelisconstant (KM). The K Mvalues for all the modified GOx preparations were determined and found to be similar to that of the native enzyme. Electrochemistry of Pyrrole-Functionalized GOx. The anodic oxidation of pyrrole and pyrrole-functionalized GOx was investigated in phosphate-buffered aqueous solutions by cyclic voltammetry to determine whether the pyrrole functionalities covalentlybound to the enzyme were accessible for oxidation. Figure 2 shows the first oxidationwave recorded for solutions containing pyrrole and lysyl-modified GOx a t a glassy carbon electrode. A single irreversible oxidation peak (15) Nakamura, S.;Hayashi,S.; Koga,K.Biochim.Biophys.Acta 1976,
445,294-308.
carbohydrate-mod.GOx
16
6
85
75
4.4-5.0 17 0.7 X lo-'
4.3 18 2.4 X lo-'
I
loo
-
75
3
50
U
-
lysyl-modified GOx
-
r?
c.
c Q,
&
25-
0
0-
-blank 7
-25 ! -0.5
I
I
I
0.0
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az
-
1.5
(VI
J carbohydrate-modified GOx
C
c 25-
E
carboxyl-modified GOx
L
a 0
0-
-0.5
blank
0.0
0.5
Potential v s SCE
1 .o
1.5
(VI
Figure 2. Cyclic voltammograms for the oxidation of pyrrole, 5 mM; lysyl-modlfiedGOx,[GOx] = 0.1 mMand [pyrrole] = 1.5mM;carboxylmodlfied GOx, [GOx] = 0.050 mM and [pyrrole] = 0.85 mM; carbohydratsmodlfledGOx, [GOx] = 0.00 mM and [pyrrole] = 0.332 mM. Activated glassy carbon electrodes were used In a phosphate buffer containing 0.1 M NaCI, pH 7.0. Scan rate was 100 mV s-l.
was observed for pyrrole and lysyl-modifiedGOx at 0.91 and 1.1V, respectively, showing that pyrrole covalently bound to GOx was accessible to oxidation. The peak potential for the modified GOx was more positive than that of pyrrole, as expected for N-substituted pyrrole derivatives.16 The cyclic voltammogram for carboxyl-modifiedGOx gave an oxidation peak for GOx-bound pyrrole at 0.97 V. On the other hand, the carbohydrate-modified GOx did not give a well-defined oxidation peak, although an oxidation current for the bound pyrrole was recorded at potentials above 0.78 V. This is probably due to the reduced number of pyrrole groups on each GOx molecule. Evaluation of GOx Polymer Film Electrodes. Although the pyrrole-modified GOx solutions were electrochemically (16) Sundaresan,N.S.;Basak, S.;Pomerantz,M.;Reynol&,R.J. Chem.
SOC.,Chem. Commun. 1967,621422.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
60 50
1.5
I
40 30 20 10 0.0
0 native entrapped
Lysyl modified
Carboxyl modified
Carbohydrate modified
lmmobilised GOx Flgure 3. Correlation of immobiilzed enzyme activity with modified function on GOx. The enzyme polymer films were generated from electrolyte solution containing 50 units of natlve or pyrrolamodified GOx and 0.1 M pyrrole.
I
I
I
I
I
I
3
4
5
6
7
8
9
PH Figure5. pH dependenceof the current responseof the Wx electrodes in 10 mM glucose. (V)Native W x in soiutbn, (A)polypyrrdeentrapped GOx, (+) polypyrroie lysyl-modified W x , (0) polypyrroie carboxylpolypyrrole carbohydratamodified GOx. modifled Wx,and (0)
20
-aa
15
I
H I-
. 10
w
c
E
L
L
I 0
L 50
100
150
200
incubation time (min) 0
0
20
40
60
[glucose1 (mM) Flgure 4. Calibration curves for glucose at different enzyme polymer electrodes. (A)Polypyrrole-entrapped GOx, (e)polypyrrole lysylmodified Wx,(0) polypyrroie carboxyl-modified W x , and (0) polypyrrole carbohydrate-modified GOx, measured at 0.7 V vs SCE. Inset shows Hanes plot for determlnatlon of apparent KM'.
oxidizable, it was not possible to generate visible polymer films solely from them. This is not surprising since the covalentattachment of pyrrole to a protein will result in steric hindrance to a-a' coupling of pyrrole radical cations and, hence, impede the formation of polymeric enzyme films on the electrode. Instead, copolymer filmswere electrodeposited from solutions containing known amounts of modified GOx and 0.1 M pyrrole. The amount of polymer grown was fixed by controlling the amount of charge passed to 10m C. Figure 3 compares the apparent immobilized enzyme activity, measured spectrophotometrically, of enzyme electrodes generated from covalently electropolymerized GOx to that of polypyrroleentrapped native GOx. The covalently electropolymerized GOx f i i s showed higher enzymeactivity than the entrapped native enzyme for polymerization solutions containing equivalent amounts of soluble enzyme activity. Enzyme films prepared from carbodiimide-coupled GOx
Flgure 6. Pseudo-firstorder plot of deactivation of immobilized (A) native,(+) lysyknodlfied, (0) carboxyl-modifled, and (0) carbohydratemodlfied GOx in polypyrrole at 00 O C , pH 7.0.
exhibited a 4.4-4.8-fold increase in activity while those from Schiff base-modified GOx were 2-fold higher in activity compared to native GOx. The results indicate that a higher immobilized activity was achievedwith GOx of higher pyrrole content. This correlation between enhanced enzyme incorporation and pyrrole content of the enzyme combined with observations of electrooxidation of GOx-bound pyrrole suggests that the protein-modified pyrrole species are copolymerizing with pyrrole under the conditions the polymer films are grown. Figure 4 shows typical calibration curves for the steadystate oxidation of HzOz as a function of glucose concentration. The observed amperometric response seemed to correlate with the measured immobilized activity. The apparent Michaelis constant (KM')for both native and pyrrole-modified enzymes, using data from Figure 4, were determined using the Hanes plot for Michaelis-Menten kinetics (eq 1).
[sl/i, = [sl/i- + KM'/i(1) where [SI is the glucose concentration, i, is the steady-state current, and, i is the maximum current under saturating substrate conditions. The KM' for polypyrrole-entrapped native GOx electrode was 19 mM while those of electrode-
ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
immobilized lysyl-modified, carboxyl-modified, and carbohydrate-modifiedGOx were 18,15, and 15 mM respectively. These values agree well with the KM value of the soluble enzyme, indicating that the electrodes are under the control of enzyme kinetics. The pH dependence of the immobilized enzymes was investigated by measuring the steady-state amperometric response to 10 mM glucose. Figure 5 shows that irrespective of the enzyme modification method, the electrodes exhibiteda broad pH optimum of 5.0-6.5 compared to a pH optimum of 5.5-6.5 for soluble enzyme.2 The lowering of the pH optimum may be due to the positively charged polypyrrole matrix excluding protons so that the enzyme pH environment is higher than the bulk solution pH. The thermal stability of the i m m o b W enzymes, determinedas a function of activity loss at 60 OC, is depicted in Figure 6 as a pseudofirst-order deactivation plot. The maximum increase in stability was obtained with the carbodiimide-coupledGOx; 3-fold compared to the polymer-entrapped native enzyme.
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with retention of enzyme activity. Since one of the aims of using pyrrole is to produce electropolymerized enzyme fibs, the number of pyrrole units bound to each GOx molecule is critical in determining the enzyme loading of the f h s . Our results clearly demonstrate that covalently immobilized GOx films obtained from the enzyme modified by carbodiimide pyrrole coupling exhibited improved electrodeactive enzyme loading and stability compared to physically entrapped GOx films. These characteristics are promising for the development of miniature biosensors and for application to more labile enzymes.
ACKNOWLEDGMENT The financial support of the Science and Engineering Research Council is gratefully acknowledged. Olvi-Foundation and the Foundation for Biotechnical and Industrial Fermentation Research are thanked for personal grants to M. Smolander.
CONCLUSIONS We have shown that it is possible to modify GOx with pyrrole functionalitiesby different covalent couplingmethods
RECEIVEDfor review December 18, 1992. Accepted April 9, 1993.
