H Sensors Based on Enzyme Entrapment in Ferrocene-Containing

Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West,. Montreal, Quebec, Canada H3G 1M8. NADH and ...
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Anal. Chem. 1998, 70, 4320-4325

NAD(P)H Sensors Based on Enzyme Entrapment in Ferrocene-Containing Polyacrylamide-Based Redox Gels Hai-Zhi Bu, Susan R. Mikkelsen,* and Ann M. English*

Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8

NADH and NADPH sensors were developed by entrapping lipoamide dehydrogenase (LD) and glutathione reductase (GR), respectively, in a redox gel formed by the copolymerization of vinylferrocene with acrylamide and N,N′-methylenebisacrylamide. Addition of LD or GR to the gel polymerization mixture resulted in a significant acceleration of free-radical copolymerization. The redox gels were secured on the surface of a carbon paste electrode with a dialysis membrane, and the resultant enzyme electrodes showed linear amperometric response to their substrates (NADH for LD and NADPH for GR) up to 3 mM when the immobilized ferrocene (Fc) and entrapped enzyme concentrations were ∼0.90 mM and 1.0 mg/mL, respectively. The substrate concentration over which the catalytic current was found to be linear depended on the concentrations of both the Fc mediator and the enzyme in the redox gels. The observed linearity indicates that the enzyme electrodes can be used as sensors to quantitate both NADH and NADPH in aqueous solutions. The pH-activity profiles of the enzyme electrodes as well as their storage and operational stabilities were examined. It was unexpectedly observed that both entrapped LD and GR exerted different effects on the electrochemical properties of the immobilized Fc/Fc+ redox couple, although they have similar structural and catalytic properties. In LD-containing gels, the redox couple is electrochemically irreversible (∆Ep ) 285 mV), while in GR-containing gels, the mediator exhibits the same quasi-reversible electrochemical behavior as in the absence of protein (∆Ep ) ∼95 mV). The electrocatalytic currents (ic) of the enzyme electrodes vs enzyme loading were investigated, and it was found that high LD loading (g1.5 mg/mL) reduced ic but high GR loading did not. It is concluded that the GR-containing redox gel electrode is highly suitable for use with dehydrogenases that require NADP+ as a cofactor. Electrical communication between enzyme redox centers and electrodes is essential in amperometric biosensors.1-4 However, (1) Janata, J.; Josowicz, M.; DeVaney, D. M. Anal. Chem. 1994, 66, 207R. (2) Scheller, F.; Schubert, F. Biosensors; Elsevier: Amsterdam, 1992. (3) Turner, A. P. F. Curr. Opin. Biotechnol. 1994, 5, 49. (4) Heller, A.; Maidan, R.; Wang, D. L. Sens. Actuators B 1993, 13, 180.

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direct electron transfer between electrodes and redox proteins is, in most cases, prevented by the insulating protein shell around the active site. Use of freely diffusing or immobilized redox mediators provides a general means to facilitate electron transfer between redox enzymes and electrode surfaces and has allowed the development of various amperometric sensors for detecting a variety of biologically important molecules.5,6 Dehydrogenase enzymes requiring either NAD+ or NADP+ as specific cofactors have not been frequently exploited in enzyme electrodes, mainly due to the difficulty in regeneration of the cofactors. Direct electrochemical oxidation of NAD(P)H at electrodes has been studied, but dimerization to enzymatically inactive species and passivation of the electrode surface due NAD(P)+ absorption are observed; in addition, a large overpotential is required, so interference from other electroactive species is inevitable.7-9 Several redox-mediator-modified electrodes have proven to be successful in overcoming the overpotential constraint and in inducing specific electrochemical regeneration of NAD(P)+.10-14 Furthermore, co-immobilization of a dehydrogenase with the redox mediator on a base electrode has resulted in increased acceleration of NAD(P)H oxidation.15-26 Lipoamide dehydrogenase (LD), or diaphorase, is the most commonly (5) Byfield, M. P.; Abuknesha, R. A. Biosens. Bioelectron. 1994, 9, 373. (6) Usmani, A. M., Akmal, N., Eds. Diagnostic Biosensor Polymers; Maple Press: York, PA, 1994. (7) Day, R. J.; Kinsey, S. J.; Seo, E. T.; Weliky, N.; Silverman, H. P. Trans. N.Y. Acad. Sci. 1972, 34, 588. (8) Jaegfeldt, H. J. Electroanal. Chem. 1985, 110, 295. (9) Torstensson, A.; Gorton, L. J. Electroanal. Chem. 1981, 130, 199. (10) Willner, I.; Riklin, A. Anal. Chem. 1994, 66, 1535. (11) Laurinavicius, V.; Kurtinaitiene, B.; Gureviciene, V.; Boguslavsky, L. I.; Geng, L.; Skotheim, T. Anal. Chim. Acta 1996, 330, 159. (12) Sprules, S. D.; Hart, J. P.; Pittson, R.; Wring, S. A. Electroanalysis 1996, 8, 539. (13) Geng, L.; Boguslavsky, L. I.; Kovalev, I. P.; Sahni, S. K.; Kalash, H.; Skotheim, T. A. Biosens. Bioelectron. 1996, 11, 1267. (14) Skoog, M.; Johansson, G. Biosens. Bioelectron. 1991, 6, 407. (15) Tatsuma, T.; Watanable, T. J. Electroanal. Chem. 1991, 310, 149. (16) Chang, H.-C.; Ueno, A.; Yamada, H.; Matsue, T.; Uchida, I. Analyst 1991, 116, 793. (17) Matsue, T.; Kasai, N.; Narumi, M.; Nishizawa, M.; Yamada, H.; Uchida, I. J. Electroanal. Chem. 1991, 300, 111. (18) Chang, H.-C.; Osawa, M.; Matsue, T.; Uchida, I. J. Chem. Soc., Chem. Commun. 1991, 611. (19) Matsue, T.; Nishizawa, M.; Sawaguchi, T.; Uchida, I. J. Chem. Soc., Chem. Commun. 1991, 1029. (20) Tang, X.-J.; Johansson, G. Anal. Lett. 1995, 228, 2595. (21) Montagne, M.; Marty, J.-L. Anal. Chim. Acta 1995, 315, 297. (22) Cosnier, S.; Lous, K. L. Talanta 1996, 43, 331. S0003-2700(98)00287-X CCC: $15.00

© 1998 American Chemical Society Published on Web 09/17/1998

employed enzyme for catalyzing the two-electron oxidation of NAD(P)H.15-26 A co-immobilized mediator shuttles two electrons between the enzyme and the electrode surface, so that the following overall reactions take place:

NAD(P)H + oxidized mediator h NAD(P)+ + reduced mediator + H+ (1) reduced mediator f oxidized mediator + 2e(electrode) (2)

In general, redox enzymes exhibit relatively low mediator selectivity. Hence, a variety of redox species has been exploited for electron mediation, including ferrocenes,16,17 ferricyanide,15,18,20,21 viologens,22-26 and anthraquinone-2-sulfonate,19 but only viologens have been co-immobilized with dehydrogenases on electrodes.22-26 In fact, most enzymatic NAD(P)+ regeneration systems have been coupled to a second NAD(P)+-dependent dehydrogenase reaction, and the resulting bienzyme electrodes have been used in the analysis10-16,20,21 and electroenzymatic synthesis23-26 of the cosubstrates. Glutathione reductase (GR) has been shown to catalyze the oxidation of NADPH using ferrocene monocarboxylic acid as a redox mediator free in solution.27 To the best of our knowledge, GR has not been used to date in a NADPH sensor. It was found to be inactive after immobilization in a viologen-acrylamide copolymer,28,29 and this was attributed to the lack of direct electrontransfer communication between the polymer and the buried active site of GR.28 We have recently developed a ferrocene (Fc)-containing polyacrylamide-based redox gel (designated PVAB) which can be formed in a one-step procedure on vinylferrocene (VF), acrylamide (AA), and N,N′-methylenebisacrylamide (BIS) copolymerization.30,31 The PVAB gel was successfully used to fabricate a glucose sensor by entrapment of glucose oxidase (GOx) in the redox gel. We now report the preparation and performance of NADH and NADPH sensors fabricated by entrapping LD and GR in the PVAB redox gels as a new application of these gels. The influence of the enzyme molecules on the rate of copolymerization, and the effects of the entrapped proteins on the electrochemical properties of the redox gel electrodes as well as on the electrocatalytic responses of the enzyme electrodes, were examined. Calibration curves (catalytic current vs substrate concentration), pH-activity profiles, and the storage and operational stabilities of the two sensors were determined. (23) Sobolov, S. B.; Leonida, M. D.; Bartoszko-Malik, A.; Voivodov, K. I.; McKinney, F.; Kim, J.; Fry, A. J. J. Org. Chem. 1996, 61, 2125. (24) Leonida, M. D.; Fry, A. J.; Sobolov, S. B.; Voivodov, K. I. Bioorg. Med. Chem. Lett. 1996, 6, 1663. (25) Viovodov, K. I.; Sobolov, S. B.; Leonida, M. D.; Fry, A. J. Bioorg. Med. Chem. Lett. 1995, 5, 681. (26) Fry, A. J.; Sobolov, S. B.; Leonida, M. D.; Voivodov, K. I. Tetrahedron Lett. 1994, 35, 5607. (27) Cass, A. E. G.; Davis, G.; Green, M. J.; Hill, H. A. O. J. Electroanal. Chem. 1985, 190, 117. (28) Willner, I.; Riklin, A.; Lapidot, N. J. Am. Chem. Soc. 1990, 112, 6438. (29) Willner, I.; Lapidot, N.; Riklin, A.; Kasher, R.; Zahavy, E. J. Am. Chem. Soc. 1994, 116, 1428. (30) Bu, H.-Z.; Mikkelsen, S. R.; English, A. M. Anal. Chem. 1995, 67, 4071. (31) Bu, H.-Z.; English, A. M.; Mikkelsen, S. R. Anal. Chem. 1996, 68, 3951.

EXPERIMENTAL SECTION Materials. Acrylamide (AA), N,N′-methylenebisacrylamide (BIS), vinylferrocene (VF), hydroxypropyl-β-cyclodextrin (HPCD, average MW 1500), N,N,N′,N′-tetramethylethylenediamine (TEMED), H2O2, and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)1,2,4-triazine (ferrozine) were purchased from Aldrich. Lipoamide dehydrogenase (LD, EC 1.8.1.4) from torula yeast, glutathione reductase (GR, EC 1.6.4.2, type III) from baker’s yeast, NADH (disodium salt), flavin mononucleotide (FMN), trichloroacetic acid (TCA), and mineral oil (light white oil, d ) 0.84 g/mL) were obtained from Sigma. NADPH (tetrasodium salt) was supplied by Boehringer Mannheim. Mono- and dibasic sodium phosphate were 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). Enzyme Electrode Preparation. The LD- or GR-containing redox gels (LD- or GR-PVAB) and electrodes (LD- or GR-RGE) were prepared as described previously for GOx.30,31 Briefly, stock solutions of 400 mg/mL AA, 25 mg/mL BIS, and 10 mg/mL LD or GR in 0.1 M sodium phosphate buffer, pH 7.0 (PB), and 10 mM VF in 50 mM HPCD30 were prepared and stored in the dark at 4 °C. AA (48 mg) and BIS (7.5 mg) were mixed with VF and LD or GR where indicated; to catalyze the polymerization, FMN (30 nmol), H2O2 (1.76 µmol), and TEMED (3.30 µmol) were added, and the final volume was adjusted to 1.0 mL. Deoxygenation was performed by flowing water-saturated N2 over the surface of the solution for 10 min prior to irradiation with a 9-W UV lamp (Ultra-Violet Products). The polymerization was considered complete when a semirigid gel block was observed (2060 min). A LD- or GR-RGE was fabricated by placing a small piece (∼2 × 2 × 1 mm) of the LD- or GR-PVAB gel on the sensing surface of a carbon paste electrode (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 enzyme electrode was exhaustively washed with PB and subjected to cyclic voltammetry (0.0-0.5 V vs Ag/AgCl) until a constant profile was reached before recording the electrochemical measurements reported here.30 Electrochemical Measurements. Cyclic voltammetry was carried out using a Bioanalytical Systems BAS 100A potentiostat. A three-electrode single-compartment cell configuration was adopted with an enzyme electrode, an Ag/AgCl reference electrode (BAS), and a Pt wire (Fisher) auxiliary electrode. All measurements were performed at ambient temperatures (23 ( 1 °C) in solutions deaerated by purging with water-saturated N2. Catalytic currents were measured at 0.4 V vs Ag/AgCl by voltammetry at a scan rate of 0.01 V/s. This voltammetric method generates catalytic currents at enzyme electrodes that represent total active enzyme since, in the presence of excess substrate, all enzyme molecules in the gel are in the reduced form at the beginning of the anodic scan. This is not the case with amperometric measurements at fixed potential, where enzymes would be in the oxidized form throughout the gel phase, and the rate of substrate arrival would be measured. RESULTS AND DISCUSSION Enzyme Entrapment. Polymer entrapment is a simple and effective technique for protein immobilization. In our previous Analytical Chemistry, Vol. 70, No. 20, October 15, 1998

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studies,30,31 we developed a novel ternary catalyst system, consisting of 30 µM FMN, 1.76 mM H2O2, and 3.30 mM TEMED (FMNH2O2-TEMED), which was used to initiate the copolymerization of VF, AA, and BIS in the presence of GOx. It was found that GOx entrapped in the PVAB redox gels is a highly efficient catalyst,30,31 so the same polymer entrapment procedure was adopted in the present study for the immobilization of both LD and GR. The addition of LD or GR significantly accelerates copolymerization. For example, ∼85 min was required for the copolymerization of VF (1.00 mM), AA, and BIS in the absence of protein, while the copolymerization was complete in ∼30 min in the presence of 1.0 mg/mL LD or GR. Both LD and GR contain tightly bound FAD centers, and both are homodimers with a Mr of 106 kDa for the former32 and 105 kDa for the latter.33 LD and GR exhibit 33% sequence homology which extends to all domains, and they also have similar redox mechanisms.34 Hence, both enzymes affect the polymerization rates in the same way, presumably because of their similarities in structure and mechanism. In contrast, ∼200 min was required for the copolymerization to go to completion when 1.0 mg/mL GOx was added,30,31 which is surprising given that GOx also contains FAD centers and is a homodimer (Mr ∼155 kDa). Since neither LD nor GR is a glycoprotein, the retarding effect on polymerization observed only with GOx may be attributed to its carbohydrate shell.35,36 In freeradical polymerization, it is known that the rate of decomposition of initiators (or the rate of formation of primary radicals) is highly dependent on the reaction medium.37 To characterize the performance of an enzyme electrode, it is necessary to know the concentrations of immobilized enzyme and mediator. Following polymerization, the redox gels containing entrapped enzymes were anchored on CPE surfaces with a dialysis membrane. Since large species cannot diffuse through the dialysis membrane (e8000 MW cutoff), it is reasonable to assume that all the entrapped enzyme molecules remained in the covered gel layer. No measurable volume change occurred during polymerization of the redox hydrogels (which are >94% water with ∼5.5% w/w polymer matrix and 0.0035-0.030% w/w redox residues), presumably because hydration of the polymer is similar to that of the monomers. Therefore, the concentration of entrapped enzyme can be taken as that present in the polymerization mixture. The immobilized or copolymerized VF concentration ([VF]imm), which includes only VF residues that did not diffuse out of the gel phase on exhaustive washing,30,31 was determined spectrophotometrically as described previously.30 The procedure involves demetalation of the Fc groups by 5% TCA, followed by complexation of the free Fe2+ with ferrozine, a colorimetric reagent for iron.38 Thus, ignoring possible minute volume changes on polymerization, it was determined that 87-91% VF was im(32) Mattevi, A.; Obmolova, G.; Kalk, K. H.; Van Berkel, W. J.; Hol, W. G. J. Mol. Biol. 1993, 230, 1200. (33) Krauth-Siegel, R. L.; Blatterspiel, R.; Saleh, M.; Schiltz, E.; Schirmer, R. H.; Untucht- Grau, R. Eur. J. Biochem. 1982, 121, 259. (34) Williams, C. H., Jr. In Chemistry and Biochemistry of Flavoenzymes; Muller, F., Ed.; CRC Press: Boca Raton, 1992; Vol. III, pp 121-207. (35) Degani, Y.; Heller, A. J. Phys. Chem. 1987, 91, 1285. (36) Swoboda, B. E. P.; Massey, V. J. Biol. Chem. 1965, 240, 2209. (37) Seymour, R. B.; Carraher, C. E., Jr. Polymer Chemistry, An Introduction, 3rd ed.; Marcel Dekker: New York, 1992; pp 277-309. (38) Badia, A.; Thai, N. H. H.; English, A. M.; Mikkelsen, S. R.; Patterson, R. T. Anal. Chim. Acta 1992, 262, 87.

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Figure 1. Cyclic voltammograms of a redox gel electrode (RGE) containing 0.92 mM immobilized VF in the absence and presence of NAD(P)H in PB at a scan rate of 0.01 V/s.

mobilized from polymerization solutions containing 1.0 mM VF and 0.5-2.5 mg/mL LD or GR. The yield of VF immobilization was relatively insensitive to the concentration of enzyme added, and here, as elsewhere,30,31 we have assumed that 100% of the AA and BIS monomers were copolymerized into the gels. Characterization of Biosensor Performance. Matsue and co-workers reported that NADH can be electrocatalytically oxidized by ferrocene derivatives on a glassy carbon electrode in buffered solution (pH 7.0).39 Large catalytic effects were observed for (ferrocenylmethyl)trimethylammonium perchlorate and (ferrocenylmethyl)dimethylamine, and a moderate effect was observed for ferrocenecarboxylic acid.39 In the present study, no catalytic oxidation of NAD(P)H at a PVAB redox gel electrode (RGE) was observed (Figure 1), indicating that the immobilized ferricenium cation (Fc+) in the gel phase is not an effective electron acceptor from NAD(P)H. However, characteristic catalytic currents from the enzymatic oxidation of NADH and NADPH by LD-containing (LD-RGE) and GR-containing (GR-RGE) redox gel electrodes, respectively, were clearly observed (Figure 2), indicating that the immobilized ferrocene residues are efficient mediators of electron transfer from reduced LD or GR to the electrode surface. The proposed mechanism for electron transfer from NAD(P)H to the electrode surface in both LD-RGE and GRRGE is shown in Figure 3. The entrapment of GR in the PVAB redox gel is, to the best of our knowledge, the first time active GR was immobilized to construct an amperometric NADPH biosensor. Moreover, the cyclic voltammogram of the GR-RGE in PB at a scan rate of 0.01 V/s in the absence of NADPH shows a peak potential separation (∆Ep) of ∼95 mV [Figure 2b(I)], which is very close to the ∆Ep (∼97 mV) for the RGE in PB (Figure 1), indicating quasi-reversible electrochemical behavior.40 However, the LD-RGE shows irreversible electrochemical characteristics (∆Ep ≈ 285 mV) in PB without NADH at 0.01 V/s [Figure 2a(I)]. In our previous study,30 we found that the GOx-RGE also exhibits irreversible electrochemical behavior in PB, suggesting that both GOx and LD may hinder heterogeneous electron transfer between the redox sites and the surface of the CPE and/or physical motion of the redox centers, resulting in decreased electron self-exchange between neighboring centers. In any event, LD and GR, which possess (39) Matsue, T.; Suda, M.; Uchida, I. J. Electroanal. Chem. 1987, 234, 163. (40) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, Fundamentals and Applications; John Wiley and Sons: New York, 1980; pp 230-231.

Figure 2. (a) Cyclic voltammograms of a LD-redox gel electrode (LD-RGE) containing 0.91 mM immobilized VF and 1.0 mg/mL entrapped LD. (I) No NADH. (II) 5.0 mM NADH. (b) Cyclic voltammograms of a GR-redox gel electrode (GR-RGE) containing 0.90 mM immobilized VF and 1.0 mg/mL entrapped GR. (I) No NADPH. (II) 5.0 mM NADPH. Cyclic voltammetry was performed at 0.01 V/s in PB.

Figure 4. Catalytic current vs NADH (b) and NADPH (O) concentration. Measurements were taken with a LD-RGE containing 0.91 mM immobilized VF and 1.0 mg/mL entrapped LD (b), and a GRRGE containing 0.90 mM VF and 1.0 mg/mL entrapped GR (O) in N2-saturated PB at 0.4 V vs Ag/AgCl.

Figure 3. Mechanism for the electrocatalytic oxidation of NAD(P)H by lipoamide dehydrogenase or glutathione reductase (reduced form, Ered; oxidized form, Eox).

similar structures and catalytic mechanisms, exert different effects upon the immobilized Fc/Fc+ redox couple, whereas LD and GOx, which have quite different structures and catalytic mechanisms, have similar insulating effects on the redox couple. Typical calibration curves for NADH and NADPH obtained at a LD-RGE and a GR-RGE, respectively, are shown in Figure 4. Both enzyme electrodes show a linear response to substrate up to ∼3.0 mM, but with different slopes or sensitivities, these being 0.67 µA/mM for the LD-RGE and 1.05 µA/mM for the GR-RGE. At each substrate concentration, the GR-RGE generates a larger catalytic current than the LD-RGE, which may be a consequence of higher catalytic activity of entrapped GR and/or the weaker matrix effect of GR on electron transport, as suggested above. Conversion of the catalytic currents (ic) to current densities gives a value of 15.8 µA/cm2 for the LD-RGE and 24.2 µA/cm2 for the GR-RGE in 3.0 mM substrate. These are surprisingly high current densities, considering the extremely low VF loading (∼0.9 mM) of the redox gels. The catalytic oxidation of NADH and NADPH by Fc+ involves reactions I and II (Figure 3). The overall redox process is controlled by reaction II at low [NAD(P)H], and reaction I is ratelimiting at high [NAD(P)H], as revealed by the deviation of plots of ic vs [NAD(P)H] from linearity above 3 mM NAD(P)H (Figure 4). The linear response range of the sensors increased to 4 mM NAD(P)H upon increasing [VF]imm from 0.90 to 1.46 mM at an

Figure 5. Catalytic current vs concentration of entrapped LD (b) and GR (O). The enzyme electrodes were prepared using redox gels containing 0.90 mM immobilized VF, and measurements were taken by voltammetry at 0.4 V vs Ag/AgCl in PB with 5.0 mM NAD(P)H at 0.01 V/s.

enzyme loading of 1.0 mg/mL (data not shown). Thus, the LDRGE and GR-RGE can be used for the quantitative analysis of NAD(P)H in the low millimolar range in aqueous solutions. To examine the effects of protein loading on the observed catalytic currents, a series of PVAB gels were prepared from solutions containing a fixed concentration (1.0 mM) of VF ([VF]imm ) 0.90 mM) and varying LD or GR concentrations between 0.3 and 2.5 mg/mL. Plots of ic at 0.4 V vs entrapped enzyme concentration in PB with 5.0 mM NAD(P)H are shown in Figure 5. The ic values initially rise sharply upon increasing the entrapped enzyme concentration, but above 1.5 mg/mL a gradual decline in ic is observed for the LD-RGEs, while ic increases more slowly with GR concentration. Generation of catalytic current by an enzyme electrode is controlled by enzyme activity and mediator “activity”, which is taken here to include the electron self-exchange rate of the mediator (factors that influence the self-exchange rates of the immobilized Fc mediators are discussed in ref 41), the electron-transfer rate between the mediator and the enzyme’s Analytical Chemistry, Vol. 70, No. 20, October 15, 1998

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Figure 6. Catalytic currents vs pH of a LD-RGE (b) containing 0.91 mM immobilized VF and 1.0 mg/mL LD, and a GR-RGE (O) containing 0.90 mM immobilized VF and 1.0 mg/mL GR. Buffers: pH 3-5, 0.1 M citric acid-sodium citrate; pH 6-8, 0.1 M NaH2PO4-Na2HPO4; pH 9-10, 0.1 M H3BO3-NaH2BO3. Catalytic currents were measured by voltammetry at 0.4 V vs Ag/AgCl in the presence of 5.0 mM substrate (NADH or NADPH) at 0.01 V/s. Each point is the average value obtained with three freshly prepared sensors.

active site (reaction I, Figure 3), and the heterogeneous electrontransfer rate at the electrode surface. In the initial steeply rising portions of the ic vs [E] curves (Figure 5), ic is limited by enzyme activity (reaction II, Figure 3) in the gels. The decrease in ic at higher [LD] suggests that the response of the LD-RGE becomes limited by mediator activity. Similar behavior was observed for the GOx-RGE30 and also reported by Ohara and co-workers on varying the GOx concentrations in their polymer films containing complexed Os(4,4′-dimethylbipyridine)2Cl.42 In contrast, ic continues to increase with [GR] for the GR-RGE, but the attenuation of ic at higher [GR] indicates that the electrode response is limited by both enzyme and mediator activities. As can be seen from Figure 5, the optimal concentration of entrapped LD is ∼1.5 mg/mL, while >2 mg/mL would appear to be optimal for entrapped GR. In practice, 1.0 mg/mL is the preferred concentration for both enzymes, since the polymerization rates for these redox gels are relatively fast at this enzyme concentration, and the signals obtained with the sensors constructed from these gels are more reproducible (data not shown). In addition, the avoidance of high enzyme loading is beneficial in maximizing mass and charge transfer within the redox gel layer. The pH dependence of ic for the LD-RGE and GR-RGE was examined in 0.1 M buffer over the pH range 3-10. As shown in Figure 6, the optimal pH range was found to be ∼6-8 for both sensors. The ic vs pH profile of an enzyme electrode is determined by pH dependence of both the mediator and enzyme used, but in our previous work, the electrochemical response of the immobilized Fc residues was found to be independent of pH over the range 2-10.30 Therefore, it can be concluded that the oberved ic vs pH profiles for the LD-RGE and GR-RGE represent the pH-activity profiles of the entrapped enzymes. The storage stabilities of the LD-PVAB and GR-PVAB redox gels as well as the operational stabilities of LD-RGEs and GR(41) Bu, H.-Z.; English, A. M.; Mikkelsen, S. R. J. Phys. Chem. B 1997, 101, 9593. (42) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1994, 66, 2451.

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Figure 7. Stability vs use of a LD-RGE (b) containing 0.91 mM immobilized VF and 1.0 mg/mL LD, and a GR-RGE (O) containing 0.90 mM immobilized VF and 1.0 mg/mL GR. Data points represent catalytic currents measured by voltammetry at 0.4 V vs Ag/AgCl in the presence of 5.0 mM substrate (NADH or NADPH) at 0.01 V/s.

RGEs were examined. It was found that the LD-PVAB and GRPVAB gels stored in sealed vials at 4 °C under nonsterile conditions exhibit ∼90% activity after 1 month and ∼73% activity after 2 months. The operational stabilities for individual enzyme electrodes are shown in Figure 7. The sensors were used only to record the data points shown in Figure 7 and were stored in PB at 4 °C under nonsterile conditions when not in use. The LDRGEs and GR-RGEs retained 72 and 67% of their initial signals, respectively, after periodic use over 5 days, and 47 and 49%, respectively, after a 10-day period of use (Figure 7). Both sensors exhibit operational stabilities similar to those observed for the GOx-RGE.30 Incorporation of negatively charged monomers, such as 2-acrylamidoglycolic acid and acrylic acid, into the neutral PVAB redox gel led to significant improvements in the performance of the resultant glucose sensors containing entrapped GOx.31 However, no distinct improvement was observed for the NAD(P)H sensors constructed by entrapping LD and GR in negatively charged redox gels (data not shown). This reveals that the effects of charged residues in the gel on the performance of enzyme electrodes depend on the properties of the enzyme immobilized. CONCLUSIONS We have demonstrated that ferrocene-containing polyacrylamide-based redox gels (PVAB) provide an excellent matrix for the immobilization and mediation of LD and GR and represent the first example of GR immobilization with retention of NADPHoxidizing activity. The accelerating effect of both proteins on gel copolymerization, in contrast to the deceleration observed in the presence of GOx, facilitates preparation of the LD-PVAB and GRPVAB gels. The catalytic response of the LD-RGE and GR-RGE to substrate is controlled by enzyme activity and mediator activity; ic is limited by the former at low enzyme loading but the latter at high enzyme loading. Mediator activity appears to be controlled by the effects of the entrapped protein at high loading, resulting in an optimal gel loading for enzymes such as LD and GOx30 at which the sensors yield the highest catalytic currents. Understanding the relationship between the structure, catalytic activity

and matrix effects of an entrapped enzyme is important in designing new enzyme electrodes. The ideal enzyme would be one that showed high enzymic activity but exerted no inhibitory effects on mediator activity. GR resembles such an enzyme since it exhibits high enzymic activity even at high enzyme loading in the PVAB gels. The successful application of the PVAB redox gels to the preparation of NADH and NADPH sensors demonstrates the versatility of these gels. No doubt, they can be used to entrap other redox enzymes to fabricate the corresponding enzyme electrodes without appreciable difficulty. In fact, it is anticipated that many enzyme electrodes can now be developed by coentrapping one of the 200 or more NAD+- or NADP+-dependent dehydrogenases16 with LD or GR in the PVAB gels. In these sensors, the LD-PVAB or GR-PVAB system would enzymatically regenerate the essential NAD+ or NADP+ cofactor for the entrapped dehydrogenase, allowing the latter to respond to its substrate (alcohol, lactate, malate, etc.) via a cycling amplification mechanism. Finally, the PVAB gel entrapment method offers several advantages over other commonly used immobilization procedures

for the regeneration of NAD(P)H.15-26 These include (1) simple and rapid gel and sensor preparation; (2) very high catalytic current densities considering the extremely low loading of ferrocene residues (∼0.3% of the dry weight of PVAB);41 (3) high retention of enzyme activity; and (4) anticipated wide applicability, given that the gels are formed in neutral aqueous solutions and possess high water content (∼94%), a friendly environment for enzymes. ACKNOWLEDGMENT The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support. H.Z.B gratefully acknowledges receipt of a Doctoral Fellowship from Fonds pour la Formation de Chercheurs et l’Aide a` la Recherche (FCAR) and a Concordia University Graduate Fellowship.

Received for review March 12, 1998. Accepted July 4, 1998. AC9802877

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