A Nitrite Biosensor Based on a Maltose Binding Protein Nitrite

A Nitrite Biosensor Based on a Maltose Binding. Protein Nitrite Reductase Fusion Immobilized on an Electropolymerized Film of a Pyrrole-Derived. Bipyr...
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Anal. Chem. 1997, 69, 4856-4863

A Nitrite Biosensor Based on a Maltose Binding Protein Nitrite Reductase Fusion Immobilized on an Electropolymerized Film of a Pyrrole-Derived Bipyridinium Q. Wu,† G. D. Storrier,† F. Pariente,‡ Y. Wang,§ J. P. Shapleigh,§ and H. D. Abrun˜a*,†

Department of Chemistry, Baker Laboratory, and Department of Microbiology, W216 Wing Hall, Cornell University, Ithaca, New York 14853, and Departamento de Quı´mica Analı´tica, Universidad Auto´ noma de Madrid, Canto Blanco 28049, Madrid, Spain

The preparation and electrochemical characterization of glassy carbon electrodes (GCEs) modified with electropolymerized films of the cation N-(3-pyrrol-1-yl-propyl)-4,4′bipyridine (PPB) are described. The behavior of a new biosensor, which exhibits a high catalytic activity for nitrite reduction and which consists of a maltose binding protein nitrite reductase fusion (MBP-Nir) immobilized on an electropolymerized film of PPB as an electrocatalyst, is also described. The insoluble perchlorate salt of the poly(benzyl viologen) dication was used to immobilize MBPNir onto an electrode previously modified with an electropolymerized film of PPB. The electropolymerized film of PPB on the GCE is redox active and exhibits special electron-transfer properties toward the MBP-Nir layer but not toward Nir (Nir without MBP fusion attached), suggesting an intimate interaction between the PPB film and the MBP-Nir layer. The kinetics of the catalytic reaction between the biosensor and nitrite anion were characterized using cyclic voltammetry and rotated disk electrode techniques, and a value of (4.6 ( 0.5) × 103 M-1 s-1 was obtained for the rate constant. Interest in the electrochemistry of redox enzymes and redox cofactors is driven both by an interest in basic aspects of electron transport in biological systems and by the desire to use biological electron transport systems in technologically relevant applications.1 The study of the electrochemical behavior of redox enzymes has received a great deal of attention, driven in many cases by the desire to construct practical, self-contained enzyme electrodes for biosensor applications. Various types of electroactive polymers have been used with redox enzymes. For example, a conducting polymer containing glucose oxidase immobilized in poly(pyrrole)2-5 and a redox polymer containing glucose oxidase immobilized in †

Department of Chemistry, Cornell University. ‡ Universidad de Quı´mica Analı´tica. § Department of Microbiology, Cornell University. (1) Lyons, M. E. G. Electroactive Polymer Electrochemistry; Plenum Press: New York, 1996. (2) Uman ˜a, M.; Waller, J. Anal. Chem. 1986, 58, 2979. (3) Dicks, J. M.; Hattori, S.; Karube I.; Turner, A. P. F.; Yokozawa, T. Ann. Biol. Clin. 1989, 47, 607. (4) Kajiya, Y.; Sugai, H.; Iwakura, C.; Yoneyama, H. Anal. Chem. 1991, 63, 49. (5) Bartlett, P. N.; Ali, Z.; Eastwick-Field, V. J. Chem. Soc., Faraday Trans. 1992, 88, 2677.

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poly(ferrocenylpyrrole) have been previously reported.6 Wellbehaved electrochemical responses of redox proteins7 as well as redox enzymes8 at electrodes modified with conducting polymers have been previously reported. In addition, there is evidence of adsorption of redox enzymes (e.g., horseradish peroxidase; HRP) onto the surfaces of conducting polymers such as polyaniline.8 Electron-transfer reactions play an important role in many biological systems, and numerous investigations have been performed to further understand the processes involved.9 Nitrite reductases (Nir’s) encompass a family of enzymes that carry out the reduction of nitrite ion (NO2-).10 The product of such processes can be either ammonia or nitric oxide. Ammonia production is either an assimilatory or a dissimilatory process whereas NO production is part of a dissimilatory pathway known as denitrification. Denitrification is a bacterially mediated process in which nitrate serves as an alternative electron acceptor. The overall pathway of denitrification is generally

ΝΟ3- f NO2- f NO f N2O f N2

One class of denitrifying enzymes has copper-containing sites that are responsible for reducing NO2-. The mechanism is quite complex and involves a delicate interplay between two types of copper: so-called type 1 and type 2. Because of the redox processes involved, there is a great deal of interest in the electrochemical behavior of nitrite reductases as well as in their application in sensor development. For example, Kohzuma and co-workers11 studied the direct electrochemistry of nitrite reductase using a di-4-pyridyl disulfidemodified gold electrode in the presence of Achromobacter cycloclastes apopseudoazurin, which is incorporated specifically to transfer electrons from the electrode surface to Nir. Kukimoto (6) Foulds, N. C.; Lowe, C. R. Anal. Chem. 1988, 60, 2473. (7) Bartlett, P. N.; Farington, J. J. Electroanal. Chem. 1989, 261, 471. (8) (a) Bartlett, P. N.; Cooper, J. M. J. Electroanal. Chem. 1993, 362, 1. (b) Bartlett, P. N.; Birkin, P. R.; Palmisano, F.; De Benedetto, G. J. Chem. Soc., Faraday Trans. 1996, 92, 3123. (9) King, R. B., Johnson, M. K., Kurtz, D. M., Kutal, C., Norton, M. L., Scott, R. A., Eds. Electron Transfer in Biology and the Solid State: Inorganic Compounds with Unusual Properties; Advances in Chemistry Series 226; American Chemical Society: Washington, DC, 1990. (10) Brittain, T.; Blackmore, R.; Greenwood, C.; Thompson, A. J. Eur. J. Biochem. 1992, 209, 793. (11) Kohzuma, T.; Shidara, S.; Suzuki, S. Bull. Chem. Soc. Jpn. 1994, 67, 138. S0003-2700(97)00595-7 CCC: $14.00

© 1997 American Chemical Society

and co-workers12 established a model of the electron-transfer pathway between Nir and pseudoazurin which involves electron transfer from the type 1 copper of pseudoazurin to the type 1 copper of Nir, on to the type 2 copper of Nir, and subsequently to nitrite. The electrocatalytic reduction of nitrite has been achieved with iron porphyrins13,14 and related complexes such as cobalt15 and manganese porphyrins.16 Co(III)-1,4,8,11-tetraazacyclotetradecane17 was demonstrated to act as a catalyst for nitrite reduction at a potential of ∼-1.3 V (vs SCE). Vicente and co-workers18 reported on a Nafion-(Bu4N)2[MoO2CC(S)(C6H5)2)2]-modified electrode at which nitrite produced an electrocatalytic current at -0.8 V (vs Ag/AgCl). Strehlitz and co-workers19 developed nitrite sensors by using different electron mediators such as methyl viologen, phenazines (phenosafranin, safranin T, N-methylphenazinium, 1-methoxy-N-methylphenazinium), and triarylmethane redox dyes (bromphenols blue and red). We have been involved in the development of analytical strategies for the determination of NO (the product of Nir) and related species using chemically modified electrodes. In particular, we previously demonstrated that electrodes modified with an electropolymerized film of [Cr(v-tpy)2]3+ exhibit a very high activity toward the reduction of NO.20 As part of our continued interest in denitrification processes and in the development of analytical methodologies, we have developed an amperometric biosensor for the determination of nitrite based on the immobilization of a maltose binding protein nitrite reductase (MBP-Nir) with poly(benzyl viologen) (PBV) onto a glassy carbon electrode (GCE) previously modified with an electropolymerized film of the N-(3-pyrrol-1-yl-propyl)-4,4′bipyridinium cation. In this paper, we describe the electropolymerization of PPB and the electrochemical characteristics of the resulting electroactive films as well the preparation and characterization of a nitrite biosensor. In addition, we have studied the kinetics of the reaction between the biosensor and nitrite in solution by cyclic voltammetry and rotated disk electrode techniques. EXPERIMENTAL SECTION Reagents. 2,5-Dimethoxytetrahydrofuran (98%), 3-bromopropylamine hydrobromide (98%), 4,4′-dipyridyl (98%), and ammonium hexaflorophosphate (99.99%) were obtained from Aldrich Chemical Co., Ltd. and were used as received. The bromide salt of poly(benzyl viologen) was synthesized according to a published procedure which yields polymers with a molecular weight of ∼11 000.21 N-(3-Bromopropyl)pyrrole and N-(3-pyrrol-1-yl-propyl)4,4′-bipyridinium hexafluorophosphate (PPB) were prepared by (12) Kukimoto, M.; Nishiyama, M.; Murphy, M. E. P.; Turley, S.; Adman, E. T.; Horinouchi, S.; Beppu, T. Biochemistry 1994, 33, 5246. (13) Barley, M. H.; Rhodes, M. R.; Meyer, T. J. Inorg. Chem. 1987, 26, 1746. (14) Younathan, J. N.; Wood, K. S.; Meyer, T. J. Inorg. Chem. 1992, 31, 3280. (15) Cheng, S. H.; Su, Y. O. Inorg. Chem. 1994, 33, 5847. (16) Yu, C. H.; Su, Y. O. J. Electroanal. Chem. 1994, 368, 323. (17) Taniguchi, I.; Nakashima, N.; Matsushita, K.; Yasukouchi, K. J. Electroanal. Chem. 1987, 224, 199. (18) Vicente, F.; Carcia-Jaren ˜o, J. J.; Tamarit, R.; Cervilla, A.; Domenech, A. Electrochimica Acta 1995, 40, 1121. (19) Strehlitz, B.; Gru ¨ ndig, B.; Schumacher, W.; Kroneck, P. M. H.; Vorlop, K.D.; Kotte, H. Anal. Chem. 1996, 68, 807. (20) Maskus, M.; Pariente, F.; Wu, Q.; Toffanin, A.; Shapleigh, J. P.; Abrun ˜a, H. D. Anal. Chem. 1996, 68, 3128. (21) Factor, A.; Heinshon, G. E. J. Polym. Sci., Polym. Lett. Ed. 1971, 9, 289.

modification of the syntheses of Carpio et al.22 for the preparation of N-(2-bromoethyl)pyrrole and N-(2-pyrrol-1-ylethyl)-4,4′-bipyridinium hexafluorophosphate. Standard nitrite solutions were prepared daily. Electrochemical measurements were performed in water (purified by passage through a Milli-Q purification system) and acetonitrile (AN, Burdick and Jackson distilled in glass, dried over 4-Å molecular sieves). Tetra-n-butylammonium perchlorate (TBAP; G. F. Smith) was recrystallized three times from ethyl acetate and dried under vacuum at 90 °C for 72 h. In aqueous media, potassium phosphate buffers (Fisher) containing NaClO4 were employed as supporting electrolytes. All other reagents were of at least reagent grade quality and were used without further purification. Solutions were deoxygenated by purging with prepurified nitrogen gas for 15 min. Enzyme. The copper-containing nitrite reductase from Rhodobacter sphaeroides strain 2.4.3 was used as a source of nitrite reductase in these experiments. The gene, designated nirK, encoding this protein has been sequenced and found to have >60% identity with copper-containing nitrite reductases from other denitifiers.23 Nir from the closely related R. sphaeroides f. sp. denitrificans has been purified and shown to contain a type 1 and a type 2 center, with the type 2 center being the apparent site of nitrite binding and reduction.24 To permit the rapid, large-scale isolation of the 2.4.3 Nir, we have attempted to heterologously express the protein in Escherichia coli. This has been done using two different expression systems. To facilitate cloning of nirK into both expression systems, a SalI restriction site was introduced by site-directed mutagenesis by mutating a single residue at position 120 of the open-reading frame. Modification at this site permits cloning of SalI fragments that retain 90% of the Nir apoprotein sequence, including all the residues required for ligation to the copper centers. The SalI fragment was initially cloned into the vector pMALC2 (New England Biolabs). This created an in-frame fusion between the malE open-reading frame in the vector and the nirK open-reading frame. malE encodes a high-affinity maltose binding protein. The fusion of nirK to malE creates a MalE-nirK chimera (designated MBP-Nir) which can be readily purified on the basis of the affinity of MalE for maltose. The malE-nirK fusion plasmid was transformed into the E. coli strain DH5R. The strain was cultured at 37 °C in LB medium containing 150 µM CuSO4 and 100 µg/mL ampicillin. Once the cells had reached an absorbance at 600 nm of 0.5, 0.3 mM isopropyl β-D-thiogalactopyranoside (IPTG) was added to induce expression of the fusion and the cells were grown an additional 2 h. Cells were then harvested by centrifugation, resuspended in 50 mM phosphate buffer (pH 7.4), and lysed by passage through a French pressure cell. The MBPNir was affinity purified by passage over an amylose column (New England Biolabs; as per instructions). Elution of the bound protein was achieved by addition of phosphate buffer plus 100 mM maltose. SDS/polyacrylamide gel electrophoresis indicated samples eluting from the amylose column had a single band of ∼80 kDa, the expected size of the MBP-Nir fusion. Absorbance and EPR analysis of the purified protein indicated the presence of type 1 (22) Carpio, H.; Galeazzi, E.; Greenhouse, R.; Guzma´n, A.; Velarde, E.; Antonio, Y.; Franco, F.; Leon, A.; Pe´rez, V.; Salas, R.; Valde´s, D.; Ackrell, J.; Cho, D.; Gallegra, P.; Halpern, O.; Koehler, R.; Maddox, M. L.; Muchowski, J. M.; Prince, A.; Tegg, D.; Thurber,T. C.; VanHorn, A. R.; Wren, D. Can. J. Chem. 1982, 60, 2295. (23) Tosques, I. E.; Kwiatkowski A. V.; Shi, J.; Shapleigh, J. P. J. Bacteriol. 1997, 179, 1090. (24) Michalski, W. P.; Nicholas, D. J. D. Biochim. Biophys. Acta 1985, 828,130.

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and type 2 copper centers nearly identical to the copper centers in the enzyme purified from R. sphaeroides f. sp. denitrificans.25 This indicates the MalE protein has no obvious effect on assembly of the copper centers. The fusion was active in assays in which nitrite reduction was measured using either methyl viologen or cytochrome c as electron donors.25 The SalI nirK fragment used to construct the MalE expression system was also cloned into the vector pET-17b (Novagen) to permit expression of a form of Nir that lacks MalE. The expected size of the product of the gene fusion in the pET clone is 39.9 kDa. This is in comparison to the 40.3 kDa of the Nir apoprotein. The recombinant Nir expressed from the pET vector has 32 amino acids at its N-terminus that are not present in wild-type Nir. The purification and characterization of the pET-derived Nir will be presented elsewhere.26 However, the procedure is very similar to the purification protocol developed for purification of Nir from R. sphaeroides f. sp. denitrificans.24 The visible and EPR spectra of the pET-derived Nir were nearly identical to the spectra obtained from the MBP-Nir.26 The rates of Nir activity were also similar in both forms of the enzyme. Instrumentation. Electrochemical experiments were carried out with either an IBM EC-225 voltammetric analyzer or an EG&G Parc 173 potentiostat and a 175 universal programmer. Data were recorded on a Soltec VP-64236 X-Y recorder or collected on a Nicolet 4094 digital oscilloscope. Three-compartment electrochemical cells (separated by medium-porosity sintered glass disks) were employed. All joints were standard taper so that all compartments could be hermetically sealed with Teflon adapters. This is important to ensure that there is no gas leakage to/from the electrochemical cell. A GCE (geometric area 0.071 cm2) was used as a working electrode. It was polished prior to use with 1-µm diamond paste (Buehler) and rinsed thoroughly with water and acetone. A large-area platinum wire coil was used as a counter electrode. For rotated disk electrode experiments, a Pine Instruments rotator and RDE-3 bipotentiostat were employed. A glassy carbon rotated disk electrode from Pine (geometric area 0.283 cm2) was used as the working electrode. All potentials are referenced to a sodium saturated calomel electrode (SSCE) without regard for the liquid junction potential. Synthesis. N-(3-Bromopropyl)pyrrole. 2,5-Dimethoxytetrahydrofuran (6.6 g, 0.05 mol), 3-bromopropylamine hydrobromide (11.2 g, 0.05 mol), sodium acetate (22 g, 0.26 mol), and glacial acetic acid (25 mL, 0.44 mol) in dioxane (50 mL) were heated to 140 °C and distilled for 1 h. After cooling, the remaining oil was poured into saturated aqueous NaCl solution (150 mL) and aqueous Na2CO3 was added until the solution was basic. The solution was extracted with CH2Cl2 and then the organic extract washed with saturated aqueous NaCl, saturated aqueous Na2CO3, and saturated aqueous NaCl solutions. The organic layer was dried with MgSO4 and concentrated in vacuo. The resulting oil was purified by column chromatography (silica with CH2Cl2 as eluent), and the first fraction was collected and concentrated to give N-(3-bromopropyl)pyrrole as a clear liquid (1.5 g, 17%): 1H NMR (CDCl3) δ 6.82 (d, 2H, J ) 2.0 Hz, pyrr(2,5)), 6.32 (d, 2H, J ) 2.0 Hz, pyrr(3,4)), 4.18 (t, 2H, J ) 6.3 Hz, CH2Br), 3.42 (t, 2H, J ) 6.2 Hz, NCH2), 2.36 (q, 2H, J ) 6.2 Hz, CH2). (25) Toffanin, A.; Scholes C. P.; Shapleigh, J. P., unpublished data. (26) Olesen, K.; Veselov, A.; Zhao, Y.; Wang, Y.; Scholes, C. P.; Shapleigh, J. P. Biochemistry, submitted.

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N-(3-Pyrrol-1-yl-propyl)-4,4′-bipyridinium Hexafluorophosphate. To a solution of 4,4′-bipyridine (0.41 g, 2.8 mmol) in acetonitrile (4 mL), N-(3-bromopropyl)pyrrole (0.36 g, 1.9 mmol) was added at room temperature. The solution was heated to 80 °C, stirred for 20 h, and then allowed to cool to room temperature. N-(3Pyrrol-1-yl-propyl)-4,4′-bipyridinium hexafluorophosphate was obtained by dropwise addition of the reaction solution containing the bromide salt into a stirring aqueous solution containing excess ammonium hexafluorophosphate. The resulting precipitate was filtered, washed with water, and then recrystallized from acetonitrile/water to afford a yellow/brown crystalline solid (0.62 g, 80%): 1H NMR ((CD3)2SO) δ 9.15 (d, 2H, J ) 6.4 Hz, bpy(2, 6)), 8.90 (d, 2H, J ) 5.4 Hz, bpy(2′, 6′)), 8.61 (d, 2H, J ) 6.3 Hz, bpy(3, 5)), 8.05 (d, 2H, J ) 5.5 Hz, bpy(3′, 5′)), 6.76 (t, 2H, J ) 1.9 Hz, pyrr(2, 5)), 5.99 (t, 2H, J ) 2.0 Hz, pyrr(3, 4)), 4.65 (t, 2H, J ) 7.2 Hz, N+CH2), 4.05 (t, 2H, J ) 7.0 Hz, pyrr-CH2), 2.50 (overlap DMSO peak, CH2); 13C NMR ((CD3)2SO) δ 152.38, 151.20, 145.55, 141.03, 125.41, 122.05, 120.63, 108.14, 58.67, 45.87, 31.90. Preparation of Biosensor and Modified Electrodes. Electrodes were modified with electropolymerized films of PPB by scanning the potential (at 100 mV/s) between +0.90 and -0.35 V for a prescribed length of time (depending on the desired coverage) in a thoroughly degassed 1 mM solution of PPB in acetonitrile (0.1 M TBAP). Following the polymerization step, the coated electrodes were rinsed thoroughly with acetone and water. The surface coverage of the polymer film, Γ (in mol/cm2), was determined, in clean acetonitrile (0.1 M TBAP) or in H2O (0.1 M NaClO4) with no dissolved PPB, from the integrated charge under the cyclic voltammetric wave centered at ∼-0.20 V at slow sweep rates. Average coverage values of (1.0-1.5) × 10-9 mol/ cm2 were obtained which represent approximately three to five equivalent monolayers, respectively. A mixture of 4 µL of enzyme (25 mg/mL) and 5 µL of 0.5 g/mL PBV solution was added onto the PPB-modified electrode surface and the solvent allowed to dry in air. Then, 10 µL of 0.01 M NaClO4 was added on top of the electrode surface. The biosensor could be used after drying in air for 30 min. When not in use, the biosensor was stored frozen in order to retain the activity of the enzyme. Electrochemical Measurements and Biosensor Response. Cyclic voltammetric studies of nitrite reduction at the biosensor were carried out at varying sweep rates. Aqueous electrochemical experiments were conducted at pH 5.6, where the enzyme exhibits maximal activity. The measurement of the heterogeneous chargetransfer rate constant for the electrodes modified with electropolymerized films of PPB was carried out using the method of Laviron.27 Cyclic voltammograms for the modified electrodes were obtained at varying sweep rates from 0.05 to 200 V/s, recorded on a digital oscilloscope, and transferred to a personal computer for further analysis. The rate constant for the reaction of the biosensor and nitrite was determined by both cyclic voltammetry and rotated disk electrode (RDE) experiments. In the RDE experiments, the potential was swept at 5 mV/s from 0 to -0.5 V. The PPB-PBV-MBP-Nir biosensor was placed in buffer solution for 15 min prior to use to ensure solvent equilibration. The biosensor response was assayed in 0.1 M phosphate buffer containing 0.001 M NaClO4 solution. (The perchlorate was added to prevent dissolution of the PBV layer.) The biosensor was (27) Laviron, E. J. Electroanal. Chem. 1979, 101, 19.

Figure 2. Plot of (E°′ - Ep) vs log sweep rate (V/s) (Laviron plot) for a glassy carbon electrode modified with an electropolymerized film of PPB in 0.1 M pH 5.6 phosphate buffer solution. Figure 1. (A) Consecutive cyclic voltammograms at a sweep rate of 0.10 V/s, depicting electropolymerization for a glassy carbon electrode in contact with an AN (0.1 M TBAP) solution containing 1 mM PPB. (B) Cyclic voltammogram in AN (0.1 M TBAP) (a) and in 0.1 M pH 5.6 phosphate buffer (b) at a sweep rate of 0.10 V/s for a glassy carbon electrode modified with an electropolymerized film of PPB.

placed in 3.0 mL of buffer solution at an applied potential of -0.35 V. After the potential was scanned for two or three cycles from 0 to -0.50 V, the background current decayed to a steady value and aliquots of a fresh solution of nitrite were added. The solution was stirred for 30 s and allowed to stand for 30 s for equilibration, and the catalytic current due to nitrite reduction was subsequently measured. RESULTS AND DISCUSSION Electrode Modification and Electrochemical Characterization of the Polymerized Film. PPB readily undergoes electrooxidatively initiated polymerization to give rise to electrodes modified with an electroactive film of PPB. Figure 1A shows a series of consecutive cyclic voltammograms for an electrode in contact with a 1 mM solution of PPB in AN containing 0.1 M TBAP. As can be ascertained, there is a reversible wave with a formal potential of -0.24 V which we ascribe to a bipyridiniumlocalized process, akin to a viologen. There is, in addition, an irreversible oxidation at ∼+0.90 V which we ascribe to a pyrrolelocalized process. Not only is this value consistent with a substituted pyrrole but also, upon consecutive scanning, there is an increase in the current associated with the bipyridiniumlocalized reduction, indicative of electropolymerization. When such an electrode is removed from the polymerization solution, rinsed with acetone, and placed in an AN (0.10 M TBAP) solution containing no dissolved PPB, the cyclic voltammetric response (Figure 1B-a) exhibits a redox process at -0.23 V, a potential that is virtually identical to that observed for the PPB in solution. In addition, the voltammetric wave is sharp and symmetric as would be anticipated for a surface confined redox process. The ∆Ep values are typically small, 20 mV at a sweep rate of 20 mV/s, and the peak currents are directly proportional to the sweep rate for values of up to 500 mV/s. At higher sweep rates, the waves take on a more “diffusional shape” and there is a concomitant increase in ∆Ep. Such deviations were more notable at higher coverages.

The films are also electroactive in aqueous solution and, again, the redox response is that anticipated for a surface-immobilized redox couple, as can be seen in Figure 1B-b, which shows a cyclic voltammogram for a GCE modified with an electropolymerized film of PPB in a pH 5.6 phosphate buffer solution. In this case, however, the formal potential was -0.34 V, a value that is negatively shifted by ∼100 mV from that obtained in nonaqueous solvents such as acetonitrile. This potential shift might reflect differences in solvation between aqueous and nonaqueous solvents. In order to determine the charge-transfer rate constant of PPB-modified electrodes, we made use of the approach developed by Laviron, which consists of measuring the variations of the peak potentials with scan rate and plotting the same as ∆Ep vs log v (where ∆Ep ) E°′ - Ep; Ep is the peak potential value for the anodic or cathodic wave, E°′ is the formal potential, and v is the sweep rate in V/s). From the slopes of the linear parts of such a plot (at high sweep rates) and from the extrapolated values where such lines cut the log v axis, the values of ks and R can be determined. Figure 2 shows a plot of ∆Ep vs log V (a so-called Laviron plot) for a PPB-modified electrode in pH 5.6 phosphate buffer. Using the procedure described by Laviron, the rate constant (ks) was determined to be (3.0 ( 0.3) × 103 s-1 and R was found to be 0.5. Thus, the electron-transfer process for PPB films in aqueous media appears to be moderately fast. Biosensor Description. In previous work,28 we had studied the electrochemical behavior of PBV deposited on a Pt electrode. In contrast to the soluble Br- salt, the ClO4- form of the PBV cation is insoluble in aqueous solutions. In order to immobilize a layer of PBV onto the electrode surface, an aqueous solution containing the Br- salt of PBV was added to a PPB-modified electrode. After the solvent had evaporated, an aliquot of aqueous KClO4 solution was added to the electrode surface, resulting in the deposition of the PBV film as the insoluble perchlorate salt. In the construction of the biosensor, the enzyme MBP-Nir was dissolved in the PBV-containing solution and the material deposited as described above for PBV. The biosensor is schematically depicted in Figure 3. It consists of two layers, the first being an electropolymerized film of PPB (28) Abrun ˜a, H. D.; Bard, A. J. J. Am. Chem. Soc. 1981, 103, 6898.

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Figure 3. Schematic depiction and working principle of biosensor.

and the second a mixture of enzyme and PBV. The PPB cationic layer is reduced to neutral PPB. This species transfers electrons to the enzyme layer reducing Nir to Nirred, which in turn reduces nitrite to nitric oxide. To demonstrate that Nir not only accepts electrons from the PPB layer but also reduces nitrite, we compared the currents of the redox process in the absence and in the presence of enzyme and found that no catalytic current due to nitrite reduction was generated in the absence of enzyme. In addition, in the absence of a polymerized film of PPB and with only a mixture of enzyme and PBV present on the electrode, no catalytic current of nitrite was observed at the above potential (∼0.35 V). These experiments suggest that the electron pathway is electrode surface f (PPB) f enzyme f nitrite. The working principle of the biosensor is depicted in Figure 3. Comparison of Electrocatalytic Activities of Surface-Immobilized MBP-Nir and Nir. As part of our investigations, we were interested in ascertaining whether different Nir’s would retain their catalytic activity upon immobilization since it is often found that immobilization gives rise to loss of activity. In this case, we compared the behavior of MBP-Nir with Nir where the former has a maltose-binding domain whereas the latter does not. The catalytic activities of the enzymes, Nir and MBP-Nir, for the reduction of nitrite in aqueous media were evaluated by comparing the voltammetric responses of the enzymes in solution and immobilized on an electrode surface using methyl viologen and a PPB-modified electrode as mediators in solution and on the surface, respectively. First, electrodes modified with PPB and one of the two nitrite reductases, Nir or MBP-Nir, in the presence and absence of nitrite were examined. Figure 4 shows cyclic voltammograms in 0.1 M phosphate buffer (pH 5.6) containing 0.001 M NaClO4 for GC electrodes modified with PPB-PBV-MBP-Nir (A) and PPBPBV-Nir (B), respectively, in the absence (a) and presence (b) of 133 µM nitrite. In the absence of nitrite (Figure 4A-(a), the response for the PPB-PBV-MBP-Nir biosensor was that previously described with a well-defined voltammetric wave centered at ∼-0.34 V. Upon addition of nitrite (Figure 4A-b, the cyclic 4860 Analytical Chemistry, Vol. 69, No. 23, December 1, 1997

Figure 4. Cyclic voltammograms in 0.1 M phosphate buffer (pH 5.6) containing 0.001 M NaClO4 at a sweep rate 5 mV/s for a glassy carbon electrode modified with (A) PPB-PBV-MBP-Nir and (B) PPB-PBV-Nir in the absence of nitrite (a) and the presence of 133 µM nitrite (b).

voltammogram exhibits a dramatic enhancement of the cathodic peak current with virtually no current on the reverse sweep typical of an ECcat process. It should be mentioned that the potential (-0.34 V) at which this catalytic reaction takes place is significantly less negative than that when methyl viologen, a commonly used mediator, is employed (see below). From calibration curves (shown in the subsequent section), the cathodic peak current increased linearly with nitrite concentration over the range from 1 to 160 µM. This behavior is to be contrasted with that obtained for a PPB-PBV-Nir electrode. In this case (Figure 4B), there is virtually no change in the voltammetric profile in the presence (b) or absence (a) of nitrite. This suggests that whereas the immobilized MBP-Nir retains its enzymatic activity, the immobilized Nir does not. This would suggest that the presence of the MBP domain plays an important role in helping the enzyme retain its activity upon immobilization. Given the above mentioned behavior, we were interested in determining whether there was a specific interaction between the PPB layer on the electrode and the MBP domain on the MBPNir. In order to test for this possibility, electrodes modified with a PPB film were exposed, for 30 min, to solutions of MBP-Nir or Nir at equal enzyme concentrations and subsequently tested for their reactivity toward the reduction of nitrite. The results are presented in Figure 5A and B for PPB-modified electrodes exposed to MBP-Nir and Nir, respectively. The PPB-modified electrodes (A-a, B-a) exhibited the response previously described with a well-defined voltammetric wave centered at -0.35 V. After immersion of the electrodes in the different enzyme solutions, the one exposed to MBP-Nir exhibited a slight decrease in peak current (A-b) whereas the one exposed to Nir exhibited a significant decrease, and in addition, the wave was shifted to more negative potentials and was also significantly broadened. Upon the addition of nitrite (1 mM), the electrode exposed to MBP-

Figure 5. Cyclic voltammograms in 0.1 M phosphate buffer (pH 5.6) at a sweep rate 10 mV/s for a glassy carbon electrode modified with PPB (A) (a) buffer only; (b) with 30 µL of MBP-Nir; (c) with 30 µL of MBP-Nir and 1 mM nitrite. (B) (a) buffer only; (b) with 30 µL of Nir; (c) with 30 µL of Nir and 1 mM nitrite.

Figure 6. Cyclic voltammograms in 0.1 M phosphate buffer (pH 5.6) containing 0.1 M KCl and 1 mM methyl viologen at a sweep rate of 10 mV/s for a glassy carbon electrode in the presence of (A) MBPNir and (B) Nir. (a) Without enzyme; (b) with 30 µL of enzyme in 3 mL of solution; (c) with 30 µL of enzyme in 3 mL of solution and 1 mM nitrite.

Nir exhibited a large current enhancement, whereas the one exposed to Nir did not. This is a most important observation since it suggests that the MBP domain in MBP-Nir appears to bind specifically to the PPB layer with retention of activity whereas the Nir appears to lose its activity. In addition, the fact that the response of the PPB layer is significantly degraded upon exposure to Nir would suggest that the enzyme is adsorbing onto the electrode surface. As a final point, we compared the activities of Nir and MBPNir in solution using methyl viologen as a soluble mediator. In this case, results dramatically different from those described above for PPB-modified electrodes were obtained. Figure 6 shows the responses for GC electrodes in the presence of 1 mM methyl viologen (A-a, B-a), in the presence of viologen and MBP-Nir (A-b) or Nir (B-b), and upon the addition of nitrite (A-c, B-c). As can be ascertained, in this case both enzymes exhibited comparable activity. These results suggest that electropolymerized films of PPB have a special/specific affinity for MBP-Nir. Not only does the

enzyme appear to bind to this polymeric film, but also it retains a high level of activity. We speculate that the nonquaternized pyridine nitrogen (in the PPB film) might serve a dual role of binding site and promoter whereas the pyridinium group likely serves as redox mediator. On the other hand, no such specific interaction was present for Nir, and in fact, immobilization of Nir gives rise to a complete loss of activity. Since the only difference between Nir and MBP-Nir is that MBP-Nir has a maltose binding site, we assume that this gives rise to special electrostatic and/or steric interactions with PPB films. In fact, as shown above, it appears that the PPB layer is capable of binding MBP-Nir so that the layer of PBV is not strictly required. The PBV layer was employed, in part, so that the activity of both MBP-Nir and Nir immobilized on the electrode surface could be compared. In addition, when immobilized with PBV, the MBP-Nir layer appears to be more stable. That the MBP-Nir appears to “recognize” and bind to the PPB layer on the electrode while retaining high activity is, in our view, a most interesting and important observation since it suggests that the use of PPB-modified surfaces could be employed as a general strategy for enzyme immobilization by attaching an MBP domain to an enzyme. Kinetics of Nitrite Reduction. In order to assess the catalytic activity of PPB-PBV-MBP-Nir biosensors toward the reduction of nitrite, we employed both RDE and cyclic voltammetric methods. In the first case, the catalytic current for nitrite reduction at a known (50 µM) solution concentration was obtained as a function of the rate of rotation. A Levich plot of ilim vs ω1/2 deviated from linearity at high rates of rotation, suggesting kinetic rather than transport control. On the other hand, a KouteckyLevich plot29 of 1/ilim vs 1/ω1/2 was linear (as would be anticipated for a system that is kinetically controlled and assuming first-order kinetics) over the entire range of rates of rotation employed (up to 700 rpm). From the value of the intercept, kf was determined to be 4.7 × 103 M-1 s-1. In the second case, using cyclic voltammetry, the catalytic current for nitrite reduction was obtained at a given nitrite (133 µM) concentration as a function of sweep rate. Using the approach developed by Andrieux and Saveant,30 the rate constant was determined to be 4.4 × 103 M-1 s-1, which is virtually identical to the value determined from rotated disk experiments. These values are indicative of a reaction that is moderately fast. Effect of pH on the Biosensor Response. pH is known to affect enzymatic activity, in general, and in addition in the present case, the overall reaction of nitrite to nitric oxide involves a oneelectron/two-proton process, as shown in eq 1. Thus, one would

NO2- + 2H+ + e- f NO + H2O

(1)

anticipate pH effects in the biosensor response. In order to determine the optimum conditions for the biosensor, experiments were carried out over the pH range of 4-9. As anticipated, it was found that in the absence of nitrite the peak currents for the PPB-modified electrode were virtually independent of pH over the range of 4-9. On the other hand, the response of the biosensor in the presence of nitrite was strongly pH dependent. The (29) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 1980. (30) Andrieux, C. P., Saveant, J. M. J. Electroanal. Chem. 1978, 93, 163.

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biosensor exhibited virtually no response for the reduction of nitrite above pH 8.5 and a marked increase in response below pH 8.0 with a maximal response obtained around pH 5.6. The activity decreased again at lower pH values. A very similar pH dependency in activity has been reported for nitrite reductase from Bacillus halodenitrificans.31 In that particular case, the pH of maximal activity was estimated to be ∼6, which is in very good agreement with our measurements. Optimal Conditions for Catalytic Reduction of Nitrite at the PPB-PBV-MBP-Nir Biosensor. Since one of the objectives of the present work was the development of a biosensor for the determination of nitrite, we were interested in ascertaining the optimal conditions for its operation. Variables investigated included the surface coverage of the PPB layer, nature of the supporting electrolyte (buffer), amount of enzyme and PBV immobilized on the electrode surface, and concentration of perchlorate. It should be restated that although the PBV layer was not strictly necessary, the biosensor was more stable when the MBP-Nir was coimmobilized with PBV. In general, a relatively thin layer of PPB, in the range of 0.5 × 10-9 to 1 × 10-9 mol/cm2 gave the best results. These values represent a compromise between the magnitude of the response and possible transport limitations. From the various supporting electrolyte (buffer) systems employed, which included Tris, phosphate, N-(2hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), N,Nbis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), and 3-(Nmorpholino)propanesulfonic acid (MOPS), phosphate buffers gave the best results. As previously mentioned, perchlorate anions are necessary to prevent dissolution of the PBV layer. In general, the biosensor response was found to be virtually independent of the perchlorate concentration and a value of 1 mM was chosen as appropriate, although the choice of this specific concentration was somewhat arbitrary. We also studied the effect of enzyme loading on the biosensor. When increasing amounts of MBPNir (0, 25, 75, 100, and 125 µg) were immobilized, it was found that for up to 100 µg there was a concomitant increase in the catalytic current but for levels above this value the response became independent of enzyme loading. Hence, 4 µL of enzyme (at a concentration of 25 mg/mL) was used in all further experiments. Finally, the optimal amount of PBV used to immobilize the enzyme on the electrode surface (see comments above) surface was determined to be 5 µL of 0.5 mg/mL solution. Biosensors made by use of these conditions showed good stability and reproducibility. Stability and Reproducibility of Biosensors and Interference Effects. The reproducibility and stability of biosensors are particularly important, and experiments were conducted in order to investigate these aspects. In terms of reproducibility, it was found that the relative standard deviation of the catalytic current of nitrite for six replicate determinations was 3%. After this, the biosensor was used for 2 h (not continuously) and subsequently stored (frozen) for 1 and 2 days. The catalytic currents for nitrite reduction were measured after 1 and 2 days of storage and the values obtained were 93.8 and 85%, respectively, of that obtained on the first. Hence, this biosensor shows good reproducibility and stability, with enzyme activity lasting several days. It is especially important to investigate the reproducibility of several biosensors since it would be difficult to obtain identical (31) Denariaz, G.; Payne, W. J.; LeGall, J. Biochim. Biophys. Acta 1991, 1056, 225.

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Figure 7. Coverage-normalized catalytic current vs [NO2-] for four GC PPB-PBV-MBP-Nir biosensors prepared with different surface coverages. Inset: cyclic voltammetric response at 5 mV/s for a biosensor (6.6 × 10-10 mol/cm2) in 0.1 M phosphate buffer (pH 5.6) containing 0.001 M NaClO4 as a function of nitrite concentration [(a) 0, (b) 33.2, (c) 66.4, (d) 99.6, and (e) 132.8 µM].

surface coverages for multiple electrodes, a situation that would require a calibration curve for each biosensor. We compared the results of four different biosensors prepared under similar conditions but with somewhat different surface coverages. By constructing a calibration curve normalized to the surface coverage of PPB (icat/Γ vs [NO2-]), all four biosensors gave virtually the same coverage-normalized response so that the above plot essentially represents a universal calibration curve (Figure 7). This is clearly a very valuable aspect of this approach since it indicates that a universal calibration curve may be constructed. We tested the effects of oxygen and nitrate as potential interferences. Figure 8A shows the response of a biosensor in the absence (a) and in the presence (b) of 1 mM nitrate. As can be ascertained, the responses are virtually identical, indicating that nitrate does not interfere in the catalytic reduction of nitrite. On the other hand, the presence of oxygen gives rise to a significant interference effect. As can be seen in Figure 8B, the presence of oxygen gives rise to an enhancement of the current at -0.24 V. Thus, it is clear that oxygen interferes in the determination of nitrite with the biosensor. This not an unexpected result since, in general, Nir’s are capable of reducing O2. However, given that in our studies of nitrite electrocatalysis the solutions were thoroughly purged with N2 prior to the addition of nitrite and that Teflon joints were employed, we believe that any effects due to traces of O2 are negligible. Analytical Determination of Nitrite Using Biosensor. One of the objectives of the present work was the development of a biosensor for the determination of nitrite. As was mentioned earlier, in the presence of nitrite the biosensor exhibits an enhancement in the current response for the wave centered at -0.35 V. As shown in Figure 9, the amplitude of the catalytic current (in a cyclic voltammogram at 5 mV/s), defined as the difference of the peak currents in the presence and absence of nitrite, is proportional to the solution concentration of nitrite

Figure 8. Cyclic voltammograms in 3 mL of 0.1 M phosphate buffer (pH 5.6) solution containing 0.001 M NaClO4 at a sweep rate 10 mV/s for a PPB-PBV-MBP-Nir biosensor. (A) (a) without nitrate; (b) with 1 mM nitrate. (B) (a) buffer only; (b) after addition of 50 µL of air; (c) after addition of 200 µL of air.

Figure 9. Catalytic current vs nitrite concentration. Inset: low concentration range. Sweep rate 5 mV/s; 0.1 M pH 5.6 phosphate buffer containing 0.001 M NaClO4.

for values of up to 160 µM. In these studies, the optimized parameters previously established were employed. However, at higher concentration, the response deviates from linearity and appears to approach a concentration-independent value. Such behavior is typical of enzymatic processes where the reaction is

eventually limited by the activity of the enzyme. From inspection of the data in Figure 9 one can estimate the value of the apparent Michaelis-Menten constant to be ∼150 µM. This value is significantly larger than that of Nir26 in solution, which has been determined to be ∼15 µM. The origin of this significant difference is not clear at this time but it is likely that immobilization of the enzyme is responsible, at least in part. The limit of detection of nitrite was estimated by measuring the increase in current upon the addition of known amounts of a nitrite standard solution. Even at a 1 µM nitrite concentration, a clear increase in current was noted and we believe that the limit of detection is likely around this value. From the linear part of the calibration plot (Figure 9 inset), the sensitivity was determined to be 56 nA/µM. Thus, the results obtained indicate that glassy carbon electrodes modified with an immobilized film of PPBPBV-MBP-Nir can be used as biosensor for the nitrite reduction with very high sensitivity. CONCLUSIONS PPB can be electropolymerized on glassy carbon electrode surfaces, and the resulting modified electrodes retain their redox activity in aqueous and nonaqueous solutions. Further modification with MBP-Nir gave rise to electrodes that exhibited high activity for the reduction of nitrite. However, PPB electrodes modified with Nir did not exhibit such activity. This suggests the presence of specific interactions between the PPB layer and MBP-Nir but not with Nir. Although MBP-Nir would bind to PPB modified electrodes, more stable films were obtained when the MBP-Nir was coimmobilized with a film of PBV. A PPBPBV-MBP-Nir biosensor was shown to exhibit high activity toward the catalytic reduction of nitrite in solution at a potential of -0.35 V vs SSCE. The catalytic response exhibits a linear dependency on the concentration of nitrite in solution, with a limit of detection of ∼1 µM. Rate constant values of 4.7 × 103 and 4.4 × 103 M-1 s-1 for the reaction with nitrite were determined via rotated disk and cyclic voltammetric techniques. ACKNOWLEDGMENT This work was supported by the National Science Foundation, the Office of Naval Research, and the U.S. Department of Agriculture. F.P. acknowledges support by a NATO Fellowship. We thank Adam Borah, Joe Shipman, and Annita Toffanin for technical assistance in the early stages of the project.

Received for review June 9, 1997. Accepted September 22, 1997.X AC970595P X

Abstract published in Advance ACS Abstracts, November 1, 1997.

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