Anal. Chem. 1996, 68, 3951-3957
Modification of Ferrocene-Containing Redox Gel Sensor Performance by Copolymerization of Charged Monomers Hai-zhi Bu, Ann M. English,* and Susan R. Mikkelsen*
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8
In the field of amperometric oxidoreductase electrodes, much effort is currently being aimed at the immobilization of redox mediators that shuttle electrons between the enzyme’s redox centers and the electrode (or transducer).1-4 To date, ferrocene has been one of the most successful redox mediators due to its well-behaved electrochemical properties. For this reason, a variety of immobilization procedures for ferrocene species have been developed to fabricate reagentless amperometric biosensors. Okamoto and co-workers synthesized a series of ferrocenecontaining siloxane polymers to facilitate electron transfer between glucose oxidase (GOx) and electrode surfaces. A number of stable amperometric enzyme electrodes were fabricated by mixing GOx and the redox polymers with a carbon paste matrix which was packed into an electrode holder.5-10 Calvo and co-workers
prepared biosensors by cross-linking a ferrocene-containing acrylamide-acrylic acid copolymer11 or ferrocene-containing poly(allylamine)12 with GOx on the surface of a glassy carbon electrode. [(Ferrocenyl)amidopropyl]pyrrole and ([(ferrocenyl)amidopentyl]amidopropyl)pyrrole were synthesized by Foulds and Lowe, and enzyme electrodes were generated by the electrochemical codeposition of the redox monomers and GOx on the surface of platinum electrodes.13 Recently, Schuhmann entrapped ferrocene-modified GOx within electrochemically grown conducting polypyrrole layers.14 Iijima and co-workers prepared two water-soluble ferrocene-containing polymers by the reaction of ferrocenyl chloride with both a homopolymer and a copolymer of L-lysine, and glucose-sensing electrodes were constructed by trapping the polymeric mediators and GOx at glassy carbon electrodes with a semipermeable cellulose membrane.15 Using the ferrocene-containing L-lysine homopolymer, glucose concentrations up to 6 mM could be determined. Hendry and co-workers synthesized two redox polymers, poly(ferrocenylenemethylene) and methoxyphenyl-substituted polyferrocene, which act as good mediators following cross-linking and coimmobilization with periodate-modified GOx on carbon electrodes.16 An amperometric enzyme electrode employing horseradish peroxidase (HRP) covalently bound to a glassy carbon electrode and a ferrocenemodified polyaniline film deposited by electrochemical polymerization of N-(ferrocenylmethyl)aniline monomer was reported by Mulchandani and co-workers.17 Riklin and co-workers18 recently developed a novel approach involving reconstitution of apoGOx with a synthetic ferrocene-tethered FAD cofactor. Electrical contact between the electrode and the resulting enzyme in solution was greatly enhanced, and the authors concluded that reconstitution of flavoenzymes with synthetic cofactors should provide electroactive biocatalysts for possible use in amperometric biosensors.
(1) Janata, J.; Josowicz, M.; DeVaney, D. M. Anal. Chem. 1994, 66, 207R228R. (2) Turner, A. P. F. Curr. Opin. Biotechnol. 1994, 5, 49. (3) Davies, M. L.; Tighe, B. J. Sel. Electrode Rev. 1991, 13, 159-226. (4) Heller, A.; Maidan, R.; Wang, D. L. Sens. Actuators B 1993, 13, 180. (5) Hale, P. D.; Inagaki, T.; Karan, H. I.; Okamoto, Y.; Skotheim, T. A. J. Am. Chem. Soc. 1989, 111, 3482-3484. (6) Inagaki, T.; Lee, H. S.; Skotheim, T. A.; Okamoto, Y. J. Chem. Soc., Chem. Commun. 1989, 1181-1183. (7) Gorton, L.; Karan, H. I.; Hale, P. D.; Inagaki, T.; Okamoto, Y.; Skotheim, T. A. Anal. Chim. Acta 1990, 228, 23-30. (8) Hale, P. D.; Boguslavsky, L. I.; Inagaki, T.; Karan, H. I.; Lee, H. S.; Skotheim, T. A.; Okamoto, Y. Anal. Chem. 1991, 63, 677-682. (9) Hale, P. D.; Lee, H. S.; Okamoto, Y.; Skotheim, T. A. Anal. Lett. 1991, 24, 345-356.
(10) Hale, P. D.; Lee, H. S.; Okamoto, Y. Anal. Lett. 1993, 26, 1-16. (11) Calvo, E. J.; Danilowicz, C.; Diaz, L. J. Chem. Soc., Faraday Trans. 1993, 89, 377-384. (12) Calvo, E. J.; Danilowicz, C.; Diaz, L. J. Electroanal. Chem. 1994, 369, 279282. (13) Foulds, N. C.; Lowe, C. R. Anal. Chem. 1988, 60, 2473-2478. (14) Schuhmann, W. Bisens. Bioelectron. 1995, 10, 181-193. (15) Iijima, S.; Mizutani, F.; Yabuki, S.; Tanaka, Y.; Asai, M.; Katsura, T.; Hosaka, S.; Ibonai, M. Anal. Chim. Acta 1993, 281, 483-487. (16) Hendry, S. P.; Cardosi, M. F.; Turner, A. P. F.; Neuse, E. W. Anal. Chim. Acta 1993, 281, 453-459. (17) Mulchandani, A.; Wang, C. L.; Weetall, H. H. Anal. Chem. 1995, 67, 94100. (18) Riklin, A.; Katz, E.; Willner, I.; Stocker, A.; Bu ¨ ckmann, A. F. Nature 1995, 376, 672-675.
Enzyme electrodes were fabricated by entrapping glucose oxidase (GOx) in charged ferrocene-containing redox gels formed by the copolymerization of vinylferrocenehydroxypropyl-β-cyclodextrin inclusion complex, acrylamide, and N,N ′-methylenebis(acrylamide), together with a positively or negatively charged monomer at pH 7, in the presence of GOx in aqueous solution. The one-step polymerization procedure was initiated by a ternary catalyst system consisting of flavin mononucleotide, H2O2, and N,N,N ′,N ′-tetramethylethylenediamine. The effects of varying the concentration and structure of the charged monomers on the electrochemical properties of the charged redox gels with and without GOx were investigated, and the pH-activity profiles of entrapped GOx were examined. The storage and operational stabilities of the sensors were established, and the influence of the interferents ascorbic acid, acetaminophen, and uric acid, which are present in body fluids, on the amperometric signals of the sensors was determined.
S0003-2700(96)00483-0 CCC: $12.00
© 1996 American Chemical Society
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Poly(vinylferrocene) (PVF) shows electrochemical behavior quite similar to that of free ferrocene19 and has been used as a polymeric mediator to fabricate glucose biosensors.20,21 Irie and Tanaka prepared a redox copolymer by copolymerization of N-isopropylacrylamide with vinylferrocene (VF),22 and the thermoshrinking property of the redox polymer was subsequently exploited in the construction of GOx electrodes by Tatsuma and co-workers.23 Poly(vinylferricenium) perchlorate (PVF+ClO4-), which shows interesting electrochemical properties when coated on platinum surfaces, was electroprecipitated by Gu¨lce and coworkers24 on a platinum electrode by electrooxidizing PVF, and immersion of the PVF+ClO4--coated platinum electrode in a GOx solution resulted in enzyme immobilization in the polymeric matrix due to ion association between the negatively charged GOx (pI ) 4.225) and PVF+. We recently reported a simple, one-step procedure for the preparation of neutral ferrocene-containing polyacrylamide-based redox gels.26 Photoinitiated free-radical copolymerization of VF with acrylamide (AA) and N,N ′-methylenebis(acrylamide) (BIS) was catalyzed in aqueous solution by a ternary catalyst system consisting of flavin mononucleotide, hydrogen peroxide, and N,N,N ′,N ′-tetramethylenediamine (FMN-H2O2-TEMED), and hydroxypropyl-β-cyclodextrin (HPCD) was used to convert VF into a water-soluble inclusion complex. GOx entrapment in the redox gels (termed PVAB to indicate their constituent components VF, AA, and BIS) was accomplished by polymerization from GOxcontaining solutions. We now report the preparation and properties of charged redox gels which result from the copolymerization of VF, AA, and BIS, together with a charged monomer, X. Redox gels, designated PVAB-X, where X represents one of four positively or negatively charged monomers, were prepared using the aforementioned catalyst system. The effects of varying the concentration and structure of X on the electrochemical responses of the redox gel electrodes were investigated. Reagentless glucose biosensors were fabricated by physically trapping the GOxPVAB-X gels at the surface of carbon paste electrodes with dialysis membranes, and the influence of X on pH-activity profiles of entrapped GOx was examined. The storage and operational stabilities of these sensors were also examined, and the influence of some commonly encountered interfering substances in body fluids27 (ascorbic acid, acetaminophen, and uric acid) on the amperometric signals of the sensors was studied. EXPERIMENTAL SECTION Materials. Acrylamide (AA), N,N ′-methylenebis(acrylamide) (BIS), vinylferrocene (VF), hydroxypropyl-β-cyclodextrin (HPCD, average MW 1500), N,N,N ′,N ′-tetramethylethylenediamine (TEMED), acrylic acid (ACA), 2-acrylamidoglycolic acid mono(19) Smith, T. W.; Kuder, J. E.; Wychick, D. J. Polym. Sci. 1976, 14, 24332448. (20) Chen, C. J.; Liu, C. C.; Savinell, R. F. J. Electroanal. Chem. 1993, 348, 317338. (21) Nguyen, A. L.; Luong, J. H. T. Appl. Biochem. Biotechnol. 1993, 43, 117132. (22) Irie, M.; Tanaka, T. Polym. Prepr., Jpn. 1991, 40, 461. (23) Tatsuma, T.; Saito, K.; Oyama, N. Anal. Chem. 1994, 66, 1002-1006. (24) Gu ¨ lce, H.; O ¨ zyo ¨ru ¨ k, H.; C¸ elebi, S. S.; Yildiz, A. J. Electroanal. Chem. 1995, 394, 63- 70. (25) Swobada, B. E. P.; Massey, V. J. Biol. Chem. 1965, 240, 2209-2215. (26) Bu, H. Z.; Mikkelsen, S. R.; English, A. M. Anal. Chem. 1995, 67, 40714076. (27) Cso¨regi, E.; Schmidtke, D. W.; Heller, A. Anal. Chem. 1995, 67, 12401244.
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hydrate (AGA), 2-(dimethylamino)ethyl acrylate (DEA), [3-(methacryloylamino)propyl]trimethylammonium chloride (MTA), H2O2, R-D-glucose, and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4triazine (ferrozine) were purchased from Aldrich. Glucose oxidase (GOx, EC 1.1.3.4, grade II) from Aspergillus niger was obtained from Boehringer Mannheim. Flavin mononucleotide (FMN), trichloroacetic acid (TCA), L-ascorbic acid, uric acid, acetaminophen, and mineral oil (light white oil, d ) 0.84 g/mL) were received from Sigma. Mono- and dibasic sodium phosphate was from Fisher, and carbon powder (99.9995% purity) was from Johnson Matthey. All the chemicals were used as received, and solutions were prepared using nanopure water (Barnstead). Preparation of the PVAB-X Redox Gels. Stock solutions of 400 mg/mL AA, 25 mg/mL BIS, 100 mg/mL charged monomer, and 10 mg/mL GOx were prepared by dissolving each species in 0.1 M sodium phosphate buffer, pH 7.0 (PB). A stock solution of 10 mM VF was prepared by adding solid VF to a solution of 50 mM HPCD in PB, which was stirred until dissolution was complete. All solutions were stored in the dark at 4 °C. Aliquots of the stock solutions (AA + X ) 50 mg, BIS ) 7.5 mg) were mixed with VF and GOx where indicated; to catalyze the photopolymerization, FMN (30 nmol), H2O2 (1.76 µmol), and TEMED (3.30 µmol) were added,26 and the final volume was adjusted to 1.0 mL. Since oxygen inhibits the photoinitiated polymerization, the solution was deoxygenated by flowing watersaturated N2 over its surface for 10 min prior to polymerization. The container was then tightly sealed, and the mixture was irradiated with a 9-W UV lamp (Ultra-Violet Products) until a semirigid gel block was observed (30-320 min), at which time polymerization was considered complete. No measurable volume change occurred during polymerization. Working Electrode Preparation. A carbon paste electrode (CPE) was prepared as previously described.26 A redox gel electrode (RGE) or a RGE with entrapped GOx (GOx-RGE) was constructed by placing a small piece (2 mm × 2 mm × 1 mm) of the PVAB-X gel on the sensing surface of the CPE. The gel layer was covered and flattened to a thickness of ∼100-200 µm with a dialysis membrane (6000-8000 MW cutoff, Spectrum Medical Industries), which was anchored by a rubber O-ring. The resulting RGE or GOx-RGE was exhaustively washed with PB and subjected to cyclic voltammetry in PB from 0.0 to 0.6 V (vs Ag/ AgCl) at 10 mV/s until a constant profile was reached, typically after 3-5 cycles. Unless otherwise stated, sensors that had reached constant responses were used for all electrochemical measurements. Cyclic Voltammetry. A BAS-100A potentiostat was used for cyclic voltammetry. A standard three-electrode cell configuration was adopted with RGE or GOx-RGE, Ag/AgCl reference electrode (BAS, West Lafayette, IN), and Pt wire (Fisher) auxiliary electrode. PB (∼1.5 mL) was deaerated in the cell by sparging with watersaturated N2 for 10 min, and voltammetry was performed under a continuous N2 purge at room temperature (22-24 °C). Unless otherwise stated, voltammograms were recorded from 0.0 to 0.6 V (vs Ag/AgCl) at a scan rate of 0.01 V/s. This voltammetric method generates catalytic currents at GOx-RGEs that represent total active enzyme since, in the presence of excess glucose, all GOx in the gel is in the reduced form (FADH2) at the beginning of the anodic scan. This is not the case with amperometric measurements at fixed potential, where GOx would be in the
Table 1. Structures and pKa Values of the Monomers symbol
MW
AGA ACA DEA MTA AA VF
163.1 72.1 143.2 220.7 71.1 212.1
structure
pKa
CH2dCHCONHCH(OH)COOH 3.1 CH2dCHCOOH 4.6 CH2dCHCONHCH2CH2N+H(CH3)2 8.5 CH2dC(CH3)CONHCH2CH2N+(CH3)3 >12 CH2dCHCONH2 CH2dCH(C5H4)Fe(C5H5)
ref 29 28 30 31
oxidized form (FAD) throughout the gel phase, and the rate of glucose arrival would be measured. RESULTS AND DISCUSSION Characterization of Copolymerization. Acrylic acid (ACA),28 2-acrylamidoglycolic acid (AGA),29 2-(dimethylamino)ethyl acrylate (DEA),30 and [3-(methacryloylamino)propyl]trimethylammonium chloride (MTA)31 (Table 1) readily copolymerize with AA and BIS. At pH 7.0, ACA and AGA are negatively charged, while DEA and MTA are positively charged (Table 1). In our previous report,26 we demonstrated conclusively that VF, although it behaves as a retarder, copolymerized with AA and BIS in aqueous solution. The optimal catalyst system was found to consist of 30 µM FMN, 1.76 mM H2O2, and 3.30 mM TEMED (FMN-H2O2-TEMED),26 and this catalyst system was used in the present work to effectively initiate the copolymerization of VF, AA, BIS, and a charged monomer, X. In the presence of 0.20-2.00 mM VF, 30-260 min is required for the copolymerization of VF, AA, and BIS to go to completion. Addition of X to the polymerization mixture significantly altered the polymerization rates; substitution of 4-20% (w/w) of AA with ACA, DEA, and MTA accelerated the copolymerization by 1030%, whereas substitution with AGA resulted in 20-50% deceleration. The mechanism(s) for acceleration and deceleration of this free-radical copolymerization are not clear; possibly, MTA, DEA, and ACA form more reactive free radicals than AA and hence increase efficiency of chain propagation, while AGA may form a more stable radical, leading to reduced polymerization efficiency. The polymerization time was also sensitive to the concentration of GOx in the polymerization solution. For example, the addition of 1.0 mg/mL GOx increased the polymerization time 2-3-fold at VF concentrations between 0.2 and 1.6 mM, and even longer polymerization times were observed at higher GOx concentrations, suggesting that the large GOx molecules (MW ∼155 kDa32) may act as a barrier to polymerization. The addition of X did not alter the pH dependence of the polymerization rates, which are relatively constant between pH 5 and 14.26 Polymerization in the pH range 5-8 is recommended for GOx entrapment, since GOx is most stable in solution around pH 5, but below pH 2 or above pH 8, catalytic activity is rapidly lost.33,34 (28) Mosbach, R.; Koch-Schmidt, A. C.; Mosbach, K. In Methods in Enzymology, Immobilized Enzymes and Cells; Mosbach, K., Ed.; Academic Press: New York, 1976; pp 53-66. (29) Righetti, P. G. Immobilized pH Gradients: Theory and Methodology; Elsevier: Amsterdam, 1990; pp 5-51. (30) Baade, W.; Hunkeler, D.; Hamielec, A. E. J. Appl. Polym. Sci. 1989, 38, 185-201. (31) Watterson, A. C.; Chin, D. N.; Salamone, J. C.; Clough, S. B. Polym. Prepr., Am. Chem. Soc. Div. Polym. Chem. 1989, 30, 254-255. (32) O’Malley, J. J.; Weaver, J. L. Biochemistry 1972, 11, 3527-3532. (33) Coulthard, C. E.; Michaelis, R.; Short, W. F.; Skrimshire, G. E. H.; Standfast, A. F. B.; Birkinshaw, J. H.; Raistrick, H. Biochem. J. 1945, 39, 24-36.
The immobilized VF concentration ([VF]imm), which includes only those VF species that cannot diffuse out of the gel matrix, was determined spectrophotometrically as previously described.26 The procedure involves demetalation of the ferrocene residues by 5% TCA, followed by complexation of the free Fe2+ with ferrozine, a colorimetric reagent for iron.35 For the four charged redox gels, it was found that ∼93% VF was immobilized when VF concentrations in the polymerization mixtures ([VF]sol) were in the range 0.2-0.8 mM but decreased to ∼80% at [VF]sol ) 2.0 mM. These results are essentially identical to those reported for the neutral gel,26 indicating that the concentration of charged monomer had no obvious effect on the VF immobilization yield. The addition of GOx resulted in a slight reduction (2-6%) in [VF]imm, which was sensitive to the concentration of enzyme in the polymerization solution. Here and elsewhere, we are assuming that 100% of the charged monomers were incorporated into the gels. Characterization of Charge Transfer. The diffusion coefficient for charge transfer (Dct) can be determined from cyclic voltammograms by varying the scan rate (ν), assuming that charge transfer to and from the redox centers is under diffusion control.26,36 The relationship between the peak current (ip) and ν is given by the Randles-Sevcˇik equation:36
ip ) (2.69 × 105)ADct1/2C*ν1/2
(1)
where A is the electrode surface area and C* the concentration of redox centers (VF residues) in the gel film. Hence, at given VF and monomer concentrations, Dct values were obtained from the slopes of plots of ip vs ν1/2. Since the charged gels contain fixed redox-active VF residues, fixed electroinactive anions or cations, and mobile electroinactive counterions, the measured Dct values could reflect rates of electron hopping between VF residues and/or diffusion of the mobile counterions. Given the low concentration of VF residues (0.93 mM) in the gels, it is anticipated that electron hopping would be largely controlled by segmental motion of the gel matrix.37 From Figure 1, it can be seen that RGEs prepared using the two negatively charged gels, PVAB-AGA and PVAB-ACA, exhibit anodic peak currents (ipa) and Dct values 20-50% higher than those of the RGE prepared from a neutral redox gel under the same conditions. The PVAB-AGA and PVAB-ACA gels contain 13-65 and 28-140 mM immobilized anionic COO- groups, respectively. The positively charged redox gels, PVAB-MTA and PVABDEA, which contain 9-45 and 14-67 mM immobilized cationic groups, respectively, exhibit ipa and Dct values that are within 20% of those observed for the neutral gel. To preserve electroneutrality within the gels, diffusion of mobile counterions must be coupled to electron transfer. For the anionic gels, cation migration out of the gels rather than anion (HPO42-, H2PO4-) migration into the gels would maintain electroneutrality as the VF moieties are oxidized, but for the positively charged gels, migration of phosphate counterions into the gels is most likely required. (34) Keilin, D.; Hartree, E. F. Biochem. J. 1948, 42, 221-229. (35) Badia, A.; Thai, N. H. H.; English, A. M.; Mikkelsen, S. R.; Patterson, R. T. Anal. Chim. Acta 1992, 262, 87. (36) Murray, R. W. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13, pp 191-368. (37) Inzelt, G. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1994; Vol. 18, pp 138-157.
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Table 2. Diffusion Coefficients for Charge Transfer in Redox Gels Containing 0.93 and 1.61 mM [VF]imm Dct × 108, cm2/sa redox gel
[VF]imm ) 0.93 mMb
[VF]imm ) 1.61 mMb
ratio
PVAB-DEA PVAB PVAB-MTA PVAB-ACA PVAB-AGA
6.1 7.2 8.4 9.5 12
22 28 30 34 44
3.6 3.8 3.6 3.6 3.7
a Cyclic voltammetry was performed in N -saturated PB from 0.00 2 to 0.60 V (vs Ag/AgCl) at scan rates of 0.01, 0.05, 0.10, 0.20, and 0.40 V/s, and Dct values were calculated using eq 1. The charged monomer concentration in the redox gels was 4 mg/mL, corresponding to 28 mM DEA, 18 mM MTA, 56 mM ACA, and 25 mM AGA. b Concentration of VF residues immobilized in the gels (see text).
Characterization of Biosensor Performance. Four sets of charged redox gels (GOx-PVAB-X, X ) AGA, ACA, MTA and DEA) containing the optimal concentrations of entrapped GOx (1.0 mg/mL) and immobilized VF (0.91 mM) determined previously26 were prepared with 2-10 mg/mL X. GOx-RGEs were fabricated using the gels, and catalytic currents (ic) were measured in N2-saturated PB containing saturating (40 mM) glucose at 0.45 V vs Ag/AgCl by voltammetry at a scan rate of 10 mV/s. The electrocatalysis of glucose oxidation can be written in terms of the following scheme:26
GOx-FAD + glucose f GOx-FADH2 + gluconolactone (2) GOx-FADH2 + 2VF+ f GOx-FAD + 2VF + 2H+ (3) VF f VF+ + e-
Figure 1. (a) Steady state anodic peak currents at 10 mV/s and (b) apparent charge transfer diffusion coefficients of the PVAB-X gels vs charged monomer concentration in gels containing 0.93 mM immobilized VF. X ) AGA (b), ACA (O), MTA (2), and DEA (4). Cyclic voltammetry was carried out in N2-saturated PB from 0.00 to 0.60 V vs Ag/AgCl; 10 mg/mL corresponds to 63 mM AGA, 140 mM ACA, 45 mM MTA, and 70 mM DEA.
Overall, the Dct values fall within the narrow range of (6-12) × 10-8 cm2/s for the neutral, positively, and negatively charged redox gels (Table 2), and this, coupled with the known 94% (w/w) water content of the gels, suggests that mobile counterion diffusion does not limit the rate of electron transfer. [VF]imm in the redox gels has a stronger influence on Dct values than the charged monomer concentration. When [VF]imm is increased 1.7 from 0.93 to 1.61 mM, all the Dct values increase by a factor of ∼3.7 (Table 2). This uniform increase in Dct suggests that charge transfer occurs by the same mechanism in all the gels, and the sensitivity to [VF]imm is indicative of rate-limiting electron hopping. The smaller variation in Dct values with charged monomer concentration (Figure 1b) could be due to the effects of a changing electrostatic environment within the gel on the segmental motion of the gel matrix, which is expected to control electron hopping rates.37 3954
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(electrode)
(4)
where FAD and FADH2 represent the oxidized and reduced forms, respectively, of the flavin adenine dinucleotide bound to the active site of GOx, and VF represents an immobilized ferrocene residue in the gel. GOx-FADH2 is reoxidized by the ferricenium residues (VF+) generated by electrooxidation (eq 4). Since the VF species are regenerated very rapidly by reaction 3, the electrocatalytic oxidation wave in the voltammogram is very much enhanced. However, no reduction wave is observed because all the VF+ species are consumed by GOx-FADH2 oxidation (eq 3).26 From Figure 2, it can be seen that the magnitude of ic follows the same trend as ipa in the absence of GOx (Figure 1a), with signals increasing in the order GOx-PVAB-DEA < GOx-PVAB < GOxPVAB-MTA < GOx-PVAB-ACA < GOx-PVAB-AGA. Plots of ic vs negatively charged monomer concentration level off at ∼4 mg/ mL charged monomer, corresponding to 25 mM AGA and 56 mM ACA. Enhanced ic values could arise from enhanced enzyme activity and/or charge mobility in the gels on addition of the negatively charged monomers. However, since ic (Figure 2) and ipa (Figure 1a) exhibit essentially identical trends with both ACA and AGA concentration, it is likely that enhanced charge mobility increases ic. Our previous work26 has shown that enzyme activity does not limit ic at a concentration of 1 mg/mL entrapped GOx. The catalytic current response of a GOx-RGE containing 4.0 mg/mL AGA to 0-50 mM glucose was measured in air- and N2saturated PB. As shown in Figure 3, the GOx-RGE displays typical Michaelis-Menten behavior toward glucose, and the reduced ic
Figure 2. Catalytic currents (ic) at 0.45 V (vs Ag/AgCl) vs charged monomer concentration in the GOx-PVAB-X gels. X ) AGA (b), ACA (O), MTA (2), and DEA (4). Voltammetry was performed with enzyme electrodes containing 1.0 mg/mL entrapped GOx and 0.91 mM immobilized VF in N2-saturated PB containing 40 mM glucose at a scan rate of 10 mV/s.
Figure 4. (a) Storage stability vs time of GOx-containing redox gels. (b) Stability vs use of enzyme electrodes. The gels and electrodes contained 1.0 mg/mL entrapped GOx, 0.91 mM immobilized VF, and 4.0 mg/mL AGA (b), ACA (O), or 0 mg/mL charged monomer ([, neutral gel). Data points represent catalytic currents at 0.45 V (vs Ag/AgCl) measured in N2-saturated PB with 40 mM glucose by voltammetry at a scan rate of 10 mV/s.
Figure 3. Catalytic currents (ic) at 0.45 V (vs Ag/AgCl) vs glucose concentration. Voltammetry was performed with a GOx-PVAB-AGA enzyme electrode containing 1.0 mg/mL entrapped GOx, 0.91 mM immobilized VF, and 4.0 mg/mL (25 mM) AGA in PB under N2 (b) and air (O) at a scan rate of 10 mV/s.
observed in air-saturated PB is due to competition between the VF+ centers and O2 for GOx-FADH2. The current density produced by the GOx-RGE (electrode surface area, 0.13 cm2) in 10 mM glucose in N2-saturated PB was calculated to be ∼17 µA/ cm2, which is 26% higher than that (13.5 µA/cm2) produced by the neutral gel electrode26 under the same conditions. As mentioned previously,26 these current densities are surprisingly high, considering the extremely low VF loading of the GOx-PVAB and GOx-PVAB-AGA gels (∼0.3 wt % of the dry polymer matrix). Figure 4a compares the long-term storage stabilities of the neutral and negatively charged redox gels containing entrapped
GOx. The gels were stored in sealed vials at 4 °C under nonsterile conditions, and each point in Figure 4a was obtained with a sensor freshly prepared from the stored gels. In addition to providing the largest signals, the AGA-containing gel possesses the best storage stability, with 88% signal retention after 2 months, while the ACA-containing and neutral gels exhibit 74 and 47% signal retention, respectively, after the same storage period. The stabilities of individual enzyme electrodes prepared from these gels are shown in Figure 4b; the sensors were used only to record the data points shown in Figure 4b and were stored in PB at 4 °C under nonsterile conditions when not in use. It can be seen that the AGA-containing, ACA-containing, and neutral sensors retained 87, 82, and 54% of their initial signals, respectively, after a week of periodic use. Hence, the two negatively charged gels and their sensors possess much greater long-term stabilities than the neutral gels and sensors, suggesting that GOx is more stable in the negatively charged gels. Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
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Table 3. Response of GOx-RGEs to Glucose (G) in the Presence of Physiologically Relevant Concentrations of Interferents (I)27 ic (µA) a
GOx-RGE
G
G + I1
G + I2a
G + I3a
GOx-neutral gel GOx-PVAB-ACA GOx-PVAB-AGA GOx-PVAB-MTA GOx-PVAB-DEA
1.84 ( 0.04 2.07 ( 0.04 2.32 ( 0.05 1.71 ( 0.05 1.38 ( 0.04
2.13 ( 0.04 2.09 ( 0.03 2.33 ( 0.03 2.18 ( 0.06 1.67 ( 0.05
1.99 ( 0.03 1.99 ( 0.05 2.32 ( 0.05 1.99 ( 0.04 1.61 ( 0.06
2.09 ( 0.05 2.05 ( 0.05 2.29 ( 0.04 2.05 ( 0.05 1.62 ( 0.04
a I , 0.1 mM ascorbic acid; I , 0.17 mM acetaminophen; and I , 0.48 1 2 3 mM uric acid. The sensors contained 1.0 mg/mL entrapped GOx, 0.91 mM immobilized VF, and 6.0 mg/mL charged monomer where applicable. Voltammetry was performed in N2-saturated PB containing interferent I and 10 mM glucose at a scan rate of 10 mV/ml, and ic values were measured at 0.5 V vs Ag/AgCl. Each ic value represents the average of three repeated measurements.
Sensitivity to Interferents. Glucose biosensors often suffer interference from electrooxidizable species such as ascorbic acid, acetaminophen, and uric acid. In recent years, different protective procedures, mainly involving the use of selective membranes, have been employed to effectively reduce signals resulting from interfering substances in glucose determination.27,38-40 We tested the effects of ascorbic acid, acetaminophen, and uric acid at relevant physiological concentrations27 on the glucose biosensors, as shown in Table 3. With the neutral gel sensor, the addition of 0.1 mM ascorbic acid, 0.17 mM acetaminophen, or 0.48 mM uric acid caused a 15.8, 8.2, or 13.6% increase in signal, respectively, compared with that observed in the absence of the interferent. Under identical conditions, no change in signal (within experimental error) was observed using either of the two negatively charged sensors on addition of the three interferents; thus, the AGA- or ACA-containing glucose sensors are less sensitive to negatively charged interferents than the neutral gel sensor. Interference was also tested using the positively charged sensors under the same conditions, and both the MTA- and DEAcontaining glucose sensors are more sensitive (16-28% increase in signal) to the interferents than the neutral gel sensor. Clearly, enhancement or elimination of interference can be controlled by controlling the nature of the electrostatic interaction between the gel phase on the electrode surface and the interferents. pH Effects on Redox Gel Electrodes and Sensors. The pH dependence of the ipa values obtained with PVAB-X redox gel electrodes and the ic values of GOx-PVAB-X enzyme electrodes was examined in 0.1 M buffer salts over the pH range 2-13. For comparison, pH profiles of the ipa values of homogeneous solutions of the ethylferrocene inclusion complex (EF-HPCD), and of the ic values for GOx using EF-HPCD as a mediator, were determined at a CPE, as shown in Figure 5. EF was used as a mediator in homogeneous solution because of its structural similarity to the copolymerized VF residues. From Figure 5a, it can be seen that the ipa values measured for homogeneous solutions of EF-HPCD showed no pH dependence in the range 2-13, but the cathodic peak currents (ipc) decreased by ∼20% between pH 10 and 13 (data not shown). Loss of EF+ above pH 10 is attributed to nucleophilic attack of OH- on the ferricenium ions.41 (38) Lumley-Woodyear, T.; Rocca, P.; Lindsay, J.; Dror, Y.; Freeman, A.; Heller, A. Anal. Chem. 1995, 67, 1332-1338. (39) Zhang, Y.; Hu, Y.; Wilson, G. S. Anal. Chem. 1994, 66, 1183-1188. (40) Vaidya, R.; Wilkins, E. Med. Eng. Phys. 1994, 16, 417-421.
3956 Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
Figure 5. (a) Steady state anodic peak currents vs pH of PVAB-X redox-gel electrodes. (b) Catalytic currents vs pH of GOx-PVAB-X enzyme electrodes. X ) AGA (b), MTA (O), and no X ([, neutral gel). The data points (9) in (a) were obtained in buffers containing 0.2 mM ethylferrocene (EF) and 1.0 mM HPCD at a carbon paste electrode (CPE), and those (9) in (b) were obtained in buffers containing 0.5 mg/mL GOx, 0.2 mM EF, and 1.0 mM HPCD at a CPE. The PVAB-X gels contained 0.93 mM immobilized VF, and 6.0 mg/ mL charged monomer where applicable, and the GOx-PVAB-X gels contained 1.0 mg/mL entrapped GOx, 0.91 mM immobilized VF, and 6.0 mg/mL charged monomer where applicable. Buffers: pH 2-5, 0.1 M citric acid-Na2HPO4; pH 6-8, 0.1 M NaH2PO4-Na2HPO4; pH 9-10, 0.1 M H3BO3-NaH2BO3; and pH 11-13, Na2HPO4-Na3PO4. Anodic peak currents were measured by scanning from 0.00 to 0.60 V, and catalytic currents were measured at 0.45 V (vs Ag/AgCl) in the presence of 10 mM glucose by voltammetry at a scan rate of 10 mV/s in N2-saturated buffers. Each point is the average value obtained with three freshly prepared RGEs (a) and GOx-RGEs (b).
The ipa values for the PVAB-X (X ) AGA, MTA) and neutral redox gel electrodes were insensitive to pH over the range 2-10, but decreased significantly above pH 10 (Figure 5a). Above pH 10, VF+ residues decompose, resulting in a lower concentration of electroactive species in the gel layer close to the electrode (41) Prins, R.; Korswagen, A. R.; Kortbeek, A. G. T. G. J. Organomet. Chem. 1972, 39, 335.
surface. These residues cannot be replaced, resulting in decreased electron transport to the electrode and smaller ipa values. In the homogeneous EF-HPCD system, decomposed EF+ would be insignificant with respect to the bulk EF concentration, so ipa remains essentially constant above pH 10, but ipc decreases because of the immediate decomposition at high pH of some of the EF+ produced during each potential scan period. Using EF-HPCD as a mediator, GOx exhibits high catalytic activity in the pH range 7-10 (Figure 5b). It has previously been reported that ferrocenes and a number of other electron acceptors from GOx give rise to pH optima around 7.5, unlike dioxygen, which has an optimum at ∼pH 5.5.42 Thus, EF-HPCD as an electron acceptor resembles other ferrocenes in that optimal GOx activity is observed at alkaline pH. The GOx-PVAB-AGA sensor exhibits the least sensitivity to pH, since its optimal range falls between pH 6 and 10. In comparison, the neutral GOx-PVAB sensor has an optimal pH range of 7-10, while the positively charged GOx-PVAB-MTA sensor has an optimal pH range of 6-9 (Figure 5b). It can also be observed from Figure 5b that the catalytic activities of the enzyme electrodes decrease more slowly with pH than does that of free GOx with EF-HPCD as a mediator, which is consistent with other reports.16,20 This suggests that the effective pH in the gels may be different from that in the bulk solution. It should also be noted that the decrease in catalytic activities of the enzyme electrodes above pH 10 is due not only to decreased GOx activity but also to decomposition of the VF+ residues (Figure 5a). If this latter contribution is taken into consideration, it is clear that entrapment of GOx in the gels, especially the negatively charged PVAB-AGA gel, considerably extends the pH range over which the enzyme is optimally active (Figure 5b). (42) Wilson, R.; Turner, A. P. F. Biosens. Bioelectron. 1992, 7, 165-185.
CONCLUSIONS Novel, ferrocene-containing, charged redox gels were prepared by incorporating charged monomers into polyacrylamide-based redox gels.26 The incorporation of negatively charged residues markedly improves the performance of glucose sensors constructed by entrapping GOx in the charged gel matrices, compared to the performance of neutral gel glucose sensors.26 The observed improvements are as follows: (1) the catalytic currents are enhanced, with sensors fabricated from AGA-containing gels showing the greatest enhancement; (2) both AGA- and ACAcontaining gels and their sensors possess much better storage and operational stabilities than the neutral gels and sensors, indicating that entrapped GOx is more stable in the negatively charged gels; (3) the negatively charged redox gel sensors are less sensitive to commonly encountered interferents in body fluids (ascorbic acid, acetaminophen, and uric acid) than the neutral gel sensor; and (4) the AGA-containing glucose sensor exhibits the least sensitivity to pH, since its optimal operating range falls between pH 6 and 10, while the neutral sensor has an optimal range of pH 7-10. ACKNOWLEDGMENT This work was financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), and H.Z.B gratefully acknowledges receipt of a Concordia University Graduate Fellowship.
Received for review May 16, 1996. Accepted August 23, 1996.X AC960483I X
Abstract published in Advance ACS Abstracts, October 1, 1996.
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