Electrochemical Behavior of Nitrate Reductase Immobilized in Self

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Electrochemical Behavior of Nitrate Reductase Immobilized in Self-Assembled Structures with Redox Polyviologen Nancy F. Ferreyra,† Liliane Coche-Gue´rente,‡ Pierre Labbe´,‡ Ernesto J. Calvo,§ and Velia M. Solı´s*,† INFIQC, Departamento de Fı´sico Quı´mica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´ rdoba, Pabello´ n Argentina, Ciudad Universitaria, 5000 Co´ rdoba, Argentina, Laboratoire d’Electrochimie Organique et de Photochimie Re´ dox, UMR 5630, Universite´ Joseph Fourier Grenoble 1-CNRS, BP 53, 38041 Grenoble Cedex 9, France, and INQUIMAE, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabello´ n 2, Ciudad Universitaria, AR-1428, Buenos Aires, Argentina Received November 13, 2002. In Final Form: February 15, 2003 We report on a novel bioelectrode based on self-assembled multilayers of nitrate reductase (NR) intercalated with a cationic viologen-functionalized polyvinylpyridinium (PV) polymer specially synthesized for this purpose. Different samples of polymer proved to have varying substitution ratios, according to NMR experiments. The electrostatic self-assemblies were built up on a thiol-modified gold surface using a strategy previously described by Hodak et al. The electrochemical behavior of PV and the response of the electrodes toward nitrate were followed by cyclic voltammetry. The catalytic currents obtained were proportional to the number of NR-immobilized layers, as well as to the polymer substitution degree, proving that the enzyme remained active under immobilization conditions and that it can be regenerated by PV redox moieties. Experiments with quartz crystal microbalance electroacoustic impedance were carried out to follow quantitatively the multilayer film formation. The kinetics of the adsorption processes and the influence of the ionic strength are investigated. The evaluation of water and ion exchange during the reduction of PV is also achieved from electroacoustic and electrochemical experiments. Results indicate an important water exchange associated with the polymer redox behavior.

Introduction Nitrate reductases (NRs) are a group of enzymes widely distributed in nature. They are related to two main metabolic routes, the assimilatory uptake and utilization of NO3- for biosynthetic purposes and the utilization of this anion as an oxidant in the respiration chain, under anaerobic conditions.1,2 NRs catalyze the reduction of nitrate to nitrite with a reduced pyridine nucleotide as the natural enzyme regenerator. The general properties of these enzymes are fully reviewed in refs 3-5. The enzymes are homodimers or homotetramers of subunits whose molecular weights are approximately 95-100 and 50 kDa, respectively.6 Each subunit contains FAD (the site for NAD(P)H+ oxidation), a b-type cytochrome, and a molybdenum-pterin group (the site for nitrate reduction) in a 1:1:1 stoichiometry. Each cofactor domain constitutes an autonomous structural element, and even isolated, it retains its partial activity. Thus, the molybdenum domain is responsible for nitrate reduction, being also operative in the presence of synthetic electron donors.7,8 A study of the kinetic properties of NRs * Corresponding author. Tel: +54-351-4334169/80. Fax: +54351-4334188. E-mail: [email protected]. † Universidad Nacional de Co ´ rdoba. ‡ Universite ´ Joseph Fourier Grenoble 1-CNRS. § Universidad de Buenos Aires. (1) Devorakova, A.; Demnerova, K.; Mackova, M.; Pazlarova, J.; Rauch, P.; Ferri, E.; Girotti, S. Chem. Listy 1998, 92, 126. (2) Richardson D. J.; Watmough, N. J. Curr. Opin. Chem. Biol. 1999, 3, 207. (3) Hille, R. Chem. Rev. 1996, 96, 2757. (4) Philippot, L.; Hojberg, O. Biochim. Biophys. Acta 1999, 1, 1446. (5) Moura, J.; Moura, J. J. G. Curr. Opin. Chem. Biol. 2001, 5, 168. (6) Steiner, F.; Downey, J. Biochim. Biophys. Acta 1982, 706, 203.

is presented in ref 9. In this paper, we employ NR from Aspergillus nidulans, whose enzymatic properties have been reported in the literature.10 Its molecular weight is about 190 kDa,11 and its isoelectric point is 6.12 ( 0.05 at 22 °C.12 Several studies involving enzymatic electrodes with NR as the recognizing element and redox colorants as artificial regenerators have been reported.13-15 In particular, methyl viologen (MV) has received a lot of attention.14,15 Other investigators have proposed the use of redox polymers electroformed on the electrode surface. Those polymers, besides providing an effective immobilization matrix, would serve as enzyme regenerators. In this sense, different viologen-derivatized polypyrroles have been used, the enzyme being incorporated on the electrode surface during the electropolymerization procedure.16-19 (7) Strehlitz, B.; Grundig, B.; Vorlop, K. D.; Bartholmes, P.; Kotte, H.; Stottmeister, U. Fresenius’ J. Anal. Chem. 1994, 349, 676. (8) Kirstein, D.; Kirstein, L.; Scheller, F.; Borcherding, H.; Ronnenberg, J.; Diekmann, S.; Steinrucke, P. J. Electroanal. Chem. 1999, 474, 438. (9) Campbell, W. H. Cell. Mol. Life Sci. 2001, 58, 194. (10) Kalaloutskii, K. L.; Ferna´ndez, E. Plant Sci. 1995, 105, 195. (11) McDonald, W. D.; Coddington, A. Eur. J. Biochem. 1974, 46, 169. (12) Steiner, F. X.; Donwey, R. J. Biochim. Biophys. Acta 1982, 706, 203. (13) Ferreyra, N. F. Doctoral Thesis, Universidad Nacional de Co´rdoba, Co´rdoba, Argentina, 2002. (14) Moretto, L. M.; Ugo, P.; Zanata, M.; Guerriero, P.; Martin, C. R. Anal. Chem. 1998, 70, 2163. (15) Ferreyra, N. F.; Solis, V. M. J. Electroanal. Chem. 2000, 486, 126. (16) Cosnier, S.; Le Lous, K. Talanta 1996, 43, 331. (17) Cosnier, S. Biosens. Bioelectron. 1999, 14, 443. (18) Cosnier, S.; Galland, B.; Innocent, Ch. J. Electroanal. Chem. 1997, 433, 113. (19) Cosnier, S.; Gondran, Ch. Analusis 1999, 27 (7), 558.

10.1021/la026841w CCC: $25.00 © 2003 American Chemical Society Published on Web 04/02/2003

Electrochemical Behavior of Nitrate Reductase

In general, the search for proper immobilization procedures for enzymes and other biopolymers has been receiving great attention since the performance of bioelectrodes is strongly dependent on the biopolymer microenvironment.20 In this sense, the incorporation of enzyme electrostatic self-assemblies (ESAs) including regenerators is very promising, since the amount and spatial distribution of the biocatalyst and mediators could be controlled.21-25 The ESA procedure, first introduced by Decher and Kunitake, is based on the alternating physisorption of oppositely charged macromolecules.26,27 The most remarkable feature of these thin polymer coatings is the possibility of controlling thickness, composition, and spatial structure at the molecular level. To start the selfassembling procedure, a stable surface charge excess is developed on the electrode surface by derivatization with a negatively charged species. Then, the stepwise electrostatic adsorption of polyelectrolytes from these species in solution, with surface charge reversal in every immersion step, brings the possibility of regulating adsorption by repulsion of soluble molecules of equal charge. In general, the resulting films are stable three-dimensional arrangements of layers joined mainly by electrostatic forces, although hydrophobic effects have also proved to have influence.28,29 In the case of enzyme electrodes, the enzyme is adsorbed at an appropriate pH considering the isoelectric point30-32 so that it carries an excess of positive or negative charge on its surface, and according to this, a polyanion or a polycation is used as the assembling partner. This procedure has been fully applied in the literature for the immobilization of a wide series of polyelectrolytes and biological molecules. Suitable materials and the structure and properties of self-assembled multilayers have been fully reviewed in ref 33. In previous publications, we used this methodology for the immobilization of different enzymes, among these, polyphenol oxidase intercalated with cationic polyallylamine built up on a thiol-modified gold surface.24 In addition, a complete study of the physical and chemical properties of this film, combining electrochemical, electroacoustic, and spectroscopic techniques has been recently performed.34 We report here on a novel bioelectrode based on selfassembled layers of NR intercalated with a redox cationic polymer, polyvinylpyridinium (PV), which we have syn(20) Bartlett, P. N.; Cooper, J. In Applications of Electroactive Polymers in Bioelectrochemistry and Bioelectronics: Electroactive Polymer Electrochemistry; Lyons, M. E. G., Ed.; Plenum Press: New York, 1996; Part 2, Chapter 9. (21) Rusling, J. F. In Protein Architecture; Lvov, Y., Mo¨hwald, H., Eds.; Marcel Dekker: New York, 2000; Chapter 13, p 337. (22) Hodak, J.; Etchenique, R.; Calvo, E. J.; Singhal, K.; Bartlett, P. N. Langmuir 1997, 13, 2708. (23) Yoon, H. C.; Hong, M.-Y.; Kim, H.-S. Anal. Chem. 2000, 72, 4420. (24) Forzani, E. S.; Solı´s, V. M.; Calvo, E. J. Anal. Chem. 2000, 72, 5300. (25) Calvo, E. J.; Battaglini, F.; Danilowicz, C.; Wolosiuk, A.; Otero, M. Faraday Discuss. 2000, 116, 47. (26) Decher, G. Science 1997, 277, 1232. (27) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (28) Lvov, Y. In Protein Architecture; Lvov, Y., Mo¨hwald, H., Eds.; Marcel Dekker: New York, 2000; Chapter 6, p 125. (29) Keller, S. W.; Kim, H.-N.; Mallouk, T. E. J. Am. Chem. Soc. 1994, 116, 8817. (30) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (31) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Biotechnol. Bioeng. 1996, 51, 163. (32) Hodak, J.; Etchenique, R.; Calvo, E. J.; Singhal, K.; Bartlett, P. N. Langmuir 1997, 13, 2708. (33) Bertrand, P.; Laschewsky, A.; Jonas, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319. (34) Forzani, E. S.; Lo´pez Teijelo, M.; Nart, F.; Calvo, E. J.; Solı´s, V. M. Biomacromolecules, submitted.

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thesized for this purpose. The redox polymer is also the enzyme regenerator. The film was built up on a thiolmodified gold surface, using an immobilization strategy described elsewhere.32 We used an electrochemical quartz crystal microbalance with electroacoustic impedance (EQCM) to follow quantitatively the multilayer film formation and also to estimate water and ion exchange during the reduction of PV. Voltammetric and amperometric measurements for the evaluation of the electrochemical properties of the redox polymers and catalytic response of the enzymatic electrodes were also employed. The effect of the redox-moiety content of the synthesized polyviologen on the electrochemical response, on the selfassembling capability, and on the enzyme performance is also analyzed. Experimental Section Preparation of the Viologen-Functionalized Polyvinylpyridinium. Four different samples of the viologen-functionalized polyvinylpyridinium polymer (that we called polyviologen, PV), namely, PVA, PVB, PVC, and PVD, were prepared as described in the Appendix. The degree of substitution in each one was evaluated by elemental composition analysis and 1H NMR in deuterated dimethyl sulfoxide using Brucker AC 250 MHz equipment, except for polymer B, for which a Brucker AC 500 MHz was employed due to the low solubility of this polymer. Electrochemical Measurements. All solutions were prepared with ultrapure water (18 MΩ cm-1) from a Millipore Milli Q system. Thiol solution, 2.0 × 10-3 M, was prepared with 3-mercapto-1-propane-sulfonic acid (Aldrich) in 1.6 × 10-3 M sulfuric acid (Merck). The enzyme used was NR (NAD[P]H) EC 1.6.6.2 from Aspergillus species (Sigma), dissolved in 0.10 M phosphate buffer (Merck) pH 7.5. The NR activity was measured as described elsewhere.13 1,1′-Dimethyl-4,4′-bipyridinium dichloride (methyl viologen, MV2+) from Sigma was used in some experiments. Cyclic voltammetry (CV) measurements at different potential sweep rates v were performed with an Autolab (Eco-Chemie, Utrecht, The Netherlands) equipped with a PGSTAT 30 potentiostat and the GPES 4.8 software package. Two identical 10 mL electrochemical cells (model VG-2, BAS) containing 0.100 M phosphate buffer pH 7.5 or NaNO3 prepared in the same solution were used consecutively. The same set of electrodes copiously rinsed with buffer was transferred from one cell to the other. In all cases, the solutions were carefully deoxygenated with nitrogen (99.99% purity), and the experiments were carried out under a nitrogen atmosphere, at room temperature. The reference electrode was Ag|AgCl|Cl- (3M), model RE-5B Mf 2052 BAS, and all potentials in the text are referred to this electrode. A platinum wire was the counter electrode. The working electrodes were gold sheets of (2.2 ( 0.10) cm2 geometric area. The cleaning procedure included polishing with 1200-grade emery paper followed by a careful sonication in deionized water for 1 min, immersion in “piranha” solution (1:3 H2O2/98% H2SO4) for 30 min, and rinsing with ultrapure water. Caution: piranha solution is very corrosive and must be handled with care at all times. The surfaces were then stabilized by cycling at 10 V s-1 in 0.5 M sulfuric acid between -0.25 and 1.50 V until a reproducible cyclic voltammogram corresponding to a clean surface was obtained. This cleaning procedure was repeated before each experiment. Prior to the preparation of the assemblies, we made two voltammograms at 0.100 V s-1 to check the surface conditions and to obtain information about the real surface area. It was measured by integration of the reduction charge of the oxide assuming 0.420 mC cm-2 real area.35 Preparation of PV-NR Assemblies. We employed the method of Hodak et al.32 The first step was the derivatization of the gold surface by soaking the electrode in thiol solution for 30 min, followed by a careful rinsing with deionized water. Thiol adsorption produces a negatively charged surface due to exposed (35) Wood, R. In Chemisorption at electrodes. Electroanalytical Chemistry; Bard, A. J., Ed.; Plenum Press: New York, 1974; Chapter 9.

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Chart 1

sulfonate groups. The following steps consisted of alternated immersions in the cationic polyelectrolyte and the anionic enzyme solutions for 20 min. As low ionic strengths favor the ESA procedure, PV solutions, 0.5 or 1.0 mg mL-1, were prepared in 0.01 M phosphate buffer pH ) 7.5, whereas a 0.10 M phosphate buffer of the same pH was used for NR solutions of different enzyme content. Polymer solutions were prepared just before use and preserved from light exposure. After each immersion step, the electrode was copiously rinsed with the corresponding phosphate buffer. The resulting electrodes were indicated as Au|thiol|(PV-NR)n, n being the number of bilayers developed. Electroacoustic Measurements. QCM measurements were performed using electroacoustic impedance. The components of a lumped element (LEM) Butterworth-Van Dyke equivalent circuit that represents the quartz crystal loaded with the viscoelastic film were obtained as previously described.25,36 ATcut quartz crystals (10 MHz) of 10-mm diameter (International Crystal Manufacturing Co. Inc., Oklahoma City, OK) with rugose gold electrodes (1 µm) of 0.196 cm2 active area were employed. They were mounted on the cell by means of O-ring seals, with only one face in contact with the electrolyte solution. The applicability of Sauerbrey conditions is clearly stated by means of a relatively simple analysis of quartz crystal electroacoustic impedance parameters, the film inductive impedance ∆XLf, and the film motional resistance ∆Rf. Thus, when ∆XLf . ∆Rf, the Sauerbrey equation is applicable as follows:37,38

∆m =

AxµQFQ 8πf02LQ

∆XLf

(1)

where LQ ≈ 7.5 mH for 10 MHz AT-cut quartz crystals. We carried out the electroacoustic impedance QCM measurements with NR self-assembled films under liquid in order to evaluate rheological changes after enzyme and polycation adsorption steps as well as to measure the corresponding mass increase in the layer-by-layer electrostatic buildup.

Results and Discussion Chemical Properties of the Viologen-Functionalized PV. The general formula for PV is shown in Chart 1, where the counterions in the bipyridinium cationic group are indicated as A-; this anionic species could be BF4- or I -. Differences in the synthesis procedure of PVA, PVB, PVC, and PVD (see the Appendix) may be responsible for differences in the redox moiety content in the polycation samples. From the analysis described in the Appendix, we can conclude that the degree of substitution and therefore the (36) Etchenique, R.; Calvo, E. J. J. Phys. Chem. B 1999, 103, 8944. (37) Rickert, J.; Brencht, A.; Go¨pel, W. Anal. Chem. 1997, 69, 1441. (38) Calvo, E. J.; Etchenique, R.; Bartlett, P. N.; Singhal, K.; Santamarı´a, C. Faraday Discuss. 1997, 107, 141.

Figure 1. Current-potential profiles as a function of v/V s-1 performed on Au|thiol|PVB in 0.10 M phosphate buffer pH 7.5, for the following v/V s-1 values: (solid line) 0.100; (dashed line) 0.150; (dotted line) 0.200; (O) 0.300; (b) 0.400; (0) 0.500. The polymer solution concentration was 1 mg mL-1, prepared in 0.10 M phosphate buffer. Figure inset: Ipc versus v for CV experiments of Figure 1.

charge density of the different polymer samples can be ordered according to the following sequence: PVB > PVD > PVC > PVA. Also, the molar masses of PVB and PVA were estimated as 94 and 72 kDa, respectively. Electrochemical Behavior of PV. In preliminary experiments, we analyzed the electrochemical response of polymer samples with the lowest and the highest substitution ratios, PVA and PVB, respectively, to check their redox properties. For these purposes, we employed a vitreous carbon electrode (VC) modified as follows: 5 µL of each PV sample solution 1.0 mg mL-1 in 0.01 M phosphate buffer was deposited on the surface, allowing the solvent to evaporate under nitrogen (CV|PV electrode). As expected from the chemical characterization described in the previous section, PVA gave a poor and sluggish electrochemical response, and therefore, samples of this polymer were not considered for further experiments. On the contrary, PVB gave reversible peak currents. The I-E profiles recorded in buffer solution presented a welldefined reversible peak with reduction peak potential Epc ) -0.480 V, which can be assigned to the first redox couple for the viologen moiety, V2+(PVB)/V•+ (PVB), not shown. To analyze the enzymatic response in the presence of PVB as the enzyme mediator, 5 µL of solution containing 4.2 mg mL-1 NR (enzymatic activity, 4.4 U mL-1) was deposited over the CV|PVB electrode and entrapped behind a dialysis membrane (cutoff, 12 000). Chronoamperometric experiments performed with this electrode at a potential value of -0.600 V, selected from the CV experiments, presented stationary currents which varied with the substrate concentration in a Michaelian way (not shown). These results put into evidence that PVB is electroactive on the electrode surface, and also that the redox moieties behave as a cosubstrate in the enzymatic reaction. After having verified the redox and the enzyme regeneration capability of PVB on the VC electrode, we analyzed the adsorption and the electrochemical response of PVB, PVC, and PVD on the gold thiolated surface. Figure 1 shows CV experiments at different potential sweep rates v for Au|thiol|PVB in phosphate buffer 0.10 M pH 7.5 in the potential interval of the first redox couple, which is the process of interest in the enzyme regeneration step. The good electrochemical response obtained with PVB is a clear indication that the polymer was successfully adsorbed on the thiolated gold surface and electrically “wired” to the electrode. Epc remained constant


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line, blank experiments) or on Au|thiol|(PVB-NR)1 (full line), prepared after 20 min of immersion in 1.0 mg mL-1 PVB solution and 9.1 mg mL-1 NR (enzymatic activity, 4 U mL-1). According to the current-potential profile without enzyme, no chemical reactions between nitrate and PVB took place. At 0.002 V s-1, Figure 2A, a clearly defined catalytic wave 5.4 µA higher than the Ipc value in the blank experiment was observed, proving that the enzyme can also be regenerated by PVB under these immobilization conditions. Residual dioxygen was responsible for the small wave observed at around -0.3 V. When the same experiments were performed at 0.100 V s-1, Figure 2B, almost no difference was observed between voltammograms recorded with and without enzyme. The very low v values necessary to obtain catalytic currents under CV conditions point to a sluggish enzymatic reaction. This fact was observed also for soluble mediators and was discussed elsewhere.13 The increasing negative currents at the end of the voltammetric profiles can be associated with hydrogen evolution, taking place before the viologen radical cation reduction. The enzymatic reactions responsible for the catalytic currents can be stated as follows. k1

kcat

NR(MoIV) + NO3- {\ } [NR(MoIV) - NO3-] 98 k -1

NR(MoVI) + NO2- (2)

Figure 2. Voltammperograms performed at 0.002 V s-1 (A) and 0.100 V s-1 (B) in NaNO3 2.5 × 10-2 M + 0.10 M phosphate buffer pH 7.5, for (dashed line) Au|thiol|PVB and (solid line) Au|thiol|(PVB-NR)1 electrodes, after an adsorption time of 20 min in the following solutions: PV 1.0 mg mL-1 and NR 9.1 mg/mL with 4.0 U/mL of enzymatic activity, prepared in 0.01 and 0.10 M phosphate buffer, respectively. Experiments were carried out under anaerobic conditions at room temperature.

s-1

in the interval 0.100 e v e 0.500 V , whereas the corresponding peak current (Ipc) varied linearly with v (see figure inset). This behavior indicates a reversible electron transfer involving adsorbed electrochemical species. Current-potential profiles obtained for Au|thiol|PVC and Au|thiol|PVD showed also electroreduction peaks at Ep ) 0.500 V and ∆Ep ) 0.040 V (not shown). The corresponding reduction charges in µC cm-2 real area were as follows: PVB ) 2.42, PVC ) 1.74, and PVD ) 2.18. These values correlated qualitatively with the substitution ratios determined from NMR spectra (2.42, 1, and 2, respectively; see the Appendix) in the sense that the reduction charge is increasing with the substitution ratio of polymer samples. A better correlation between NMR and electrochemical data would not be expected, since the reduction charge is proportional to the number of viologen groups electrically wired to the surface, and this is not necessarily equal to the number of adsorbed moieties due to steric factors. Furthermore, the number of polymer moles adsorbed on the surface has to be dependent on the hydrophobicity of each polymer, which in turn is dependent on the substitution degree. According to the points discussed above, a simple rationalization of the differences observed between the NMR and the electrochemistry experiments is not easy. To verify the enzyme regeneration capability of the immobilized polymer, the same type of experiment was performed on the Au|thiol|PVB|NR electrode. Thus, Figure 2 shows CV profiles obtained in 2.50 × 10-2 M NaNO3 in phosphate buffer pH 7.5 either on Au|thiol|PVB (dotted

k1 and k-1 stand for the formation and dissociation constants for the enzyme-substrate complex, and kcat is the complex dissociation constant to give nitrite as the reaction product. If we consider that each viologen redox moiety undergoes a one-electron redox step, V2+ + e a V•+, and that the regeneration of NR(MoIV) involves two electrons per mole of substrate, the following stoichiometry relation holds: k

NR(MoVI) + 2V•+ 98 NR(MoIV) + 2V2+

(3)

where k is the NR/PV•+ regeneration constant. The following global reaction takes into account the stoichiometry relationship between the substrate and the redox group in PV:

NO3- + 2V•+ + 2H+ f NO2- + 2V2+ + H2O (4) Although PVC and PVD gave clear electroreduction signals, they proved to have very poor enzyme regeneration capability. Small and irreproducible catalytic currents, even at potential sweep rates lower than 0.002 V s-1, were observed in both cases, and accordingly, these polymer samples were not considered for preparing enzymatic electrodes. After verifying the successful electrochemical and catalytic response of the Au|thiol|(PVB-NR) electrode, we extended our analysis to electrodes with self-assembled multilayers. Only polymer B was used, and the electrodes were referred to as Au|thiol|(PVB-NR)n, n being the number of bilayers involved. The electrochemical behavior observed was similar to that of Figure 1, but the peak currents were dependent on n. Figure 3 shows Ipc at v ) 0.100 V s-1 measured at Epc ) -0.470 V as a function of n, PVB being the outermost layer. Voltammograms were recorded in buffer solution in the absence of nitrate, after the adsorption of each PVB layer. A linear relationship is observed for the first four bilayers, this being a clear indication that similar amounts of PVB were incorporated in each adsorption cycle and

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Figure 3. Ipc at v ) 0.100 V s-1 versus the number of bilayers for self-assembled structures on a thiol-modified gold electrode performed in 0.10 M phosphate buffer pH 7.5 in the absence of substrate. Numbers indicate the PV-terminated layer number. Adsorption time: 20 min. Adsorption solutions: PVB 1.0 mg mL-1 and NR 4.0 mg mL-1 with 0.7 U mL-1 of enzymatic activity. Experiments were carried out under anaerobic conditions at room temperature. The arrow indicates the Ipc value obtained after overnight immersion of the electrode in PV solution.

also that the electrochemical connection between the electrode and the polymer redox moieties was maintained throughout the film. Although a significant lower Ipc was obtained for the fifth bilayer, it was increased up to the corresponding linear value after overnight immersion in the PVB solution (see the arrow). This points to the presence of possible slow adsorption and/or reorganization processes, which would favor the film electrochemical conductivity. This issue will be discussed later. For a good performance of the electrode, not only a suitable electrochemical polymer response is necessary. Also, the enzyme has to be active in the film environment. The catalytic response of Au|thiol|(PVB-NR)n as a function of n was analyzed by CV experiments performed at 0.002 V s-1 in 0.10 M phosphate buffer + 1.0 × 10-2 M NaNO3 (not shown). Four similar gold electrodes were modified with two, four, six, and eight self-assembled bilayers, respectively. The corresponding catalytic current densities at -0.480 V after subtraction of the blank peak current values in µA cm-2 were as follows: 0.25, 0.60, 1.25, and 1.56. In these experiments, although the gold geometric areas were similar and all the electrodes were treated in the same way, the rugosity of each surface would not be the same. Therefore, the gold oxide reduction charge recorded by CV at 0.100 V s-1 in 0.5 M sulfuric acid was measured in order to have a rough estimation of the real surface area of each electrode. It was assumed that an oxide reduction charge of 420 µC is equivalent to 1 cm2 of real surface area.35 This real area was taken into account to perform a normalization of the catalytical current obtained for different electrodes. Although for the catalytic currents the linear correlation is not very good, a steady increase of the catalytic response after the incorporation of each new bilayer was clearly evident. To infer the extent of enzyme-polymer “wiring”, CV experiments were performed in buffer solution also containing soluble MV2+. Figure 4A shows voltammograms recorded at 0.100 V s-1 on Au|thiol|PVB in 0.10 M phosphate buffer + 2.0 × 10-4 M MV2+ (dotted line), in buffer solution without MV2+ (full line), and on Au|thiol in 0.10 M phosphate buffer + 2.0 × 10-4 M MV2+ (dashed line). The comparison of these

Figure 4. Current-potential profiles performed in 0.10 M phosphate buffer pH 7.5 in self-assembled structures. Adsorption solutions: PVB 0.5 mg mL-1 and NR 3.9 mg mL-1 with 3.3 U mL-1 of enzymatic activity prepared in 0.01 and 0.10 M phosphate buffer, respectively. Adsorption time: 20 min. Experiments were carried out under anaerobic conditions at room temperature. (A) v ) 0.100 V s-1 under the following conditions: (dotted line) Au|thiol electrode after the addition of 2.0 × 10-4 M MV2+, (solid line) Au|thiol|PVB electrode in buffer solution, and (dashed line) Au|thiol|PVB electrode after the addition of 2.0 × 10-4 M MV2+. (B) Reduction scan at v ) 0.002 V s-1 on the Au|thiol|(PVB-NR)1 electrode in the following solutions: (solid line) 1.0 × 10-2 M NaNO3; (dashed line) 1.0 × 10-2 M NaNO3 + 2.0 × 10-4 M MV2+ in 0.10 M phosphate buffer.

voltammograms has allowed us to assign the first peak system at -0.480 V and the second one at -0.650 V to the V2+(PVB)/V•+(PVB) and MV2+/MV•+ redox couples, respectively. Figure 4B shows the cathodic voltammetric responses at 0.002 V s-1 on Au|thiol|(PVB-NR)1 in 0.10 M phosphate buffer + 1.0 × 10-2 M NO3- with (dotted line) and without (full line) the addition of 2.0 × 10-4 M MV2+. According to previous results,39 this mediator concentration is high enough to reach a stationary current independent of mediator concentration. In the absence of MV2+, one catalytic wave with E1/2(1) ) -0.400 V was observed with a plateau current of 8.8 µA; when the soluble mediator was added, an additional wave at E1/2(2) ) -0.600 V was also evident and the plateau current increases up to 37 µA. According to Figure 4A, the catalytic waves at E1/2(1) and E1/2(2) in Figure 4B have to be assigned to the enzymatic reaction mediated by the reduced forms of PVB and MV2+, respectively. The reduction processes of moiety V2+(PVB) and MV2+ take place at well-separated potentials, allowing us to assign the catalytic current at -0.500 V to the mediation of V•+(PVB) ( eq 3) and that observed at (39) Ferreyra, N. F.; Solis, V. M. Anal. Chim. Acta, submitted.


-0.650 V to the mediation of both V•+(PVB) and MV•+, whose formation takes place simultaneously at this potential. Equation 3, describing the NR regeneration with the viologen reduced moiety, is in fact a multistep reaction during which two electrons are exchanged between the oxidized form of the enzyme, NR(MoVI), and two redox moieties, V•+(PVB) or two molecules of MV•+. To perform a direct comparison for both catalytic currents at -0.500 and -0.650 V, it is necessary to consider several parameters that can potentially act as rate limiting in the global process. (i) At -0.500 V, V•+(PVB) is electrogenerated at the electrode surface and the charge transport is assumed to occur through the (PVB-NR)1 layer by an electron hopping mechanism. (ii) At -0.650 V, both V•+(PVB) and MV•+ are electrogenerated at the electrode surface. Charge transport within the (PVB-NR)1 assembly can occur by electron hopping along the PVB chain, by diffusion of the MV•+ through the assembly, and by electron transfer from MV•+ to V2+(PVB). (iii) Charge transport is also expected to depend on the concentration of redox centers in the (PVB-NR)1 layer. An estimation of the film thickness can be obtained from averaged apparent density values informed for other systems (2.1 ( 0.3) g cm-3.34,40 If the mass increments measured from QCM experiments are considered, the thickness of the assembly can be estimated to be around 10 nm. The charge needed to reduce the (PVB-NR)1 layer (Figure 4) allowed us to evaluate the concentration of V•+(PVB). This value was thus estimated to be about 0.4 mol L-1, 3 orders of magnitude higher than the concentration of diffusing MV•+, 2.0 × 10-4 mol L-1. Taking into account the high reversibility of the V2+(PVB)/V•+(PVB) redox couple (see Figure 1) and the very slow v values employed in our experiments to obtain a significant catalytic current (Figure 4B), it appears reasonable to assume that charge transport does not constitute a limiting step at -0.500 V. This assumption is reinforced by the high local concentration of V2+(PVB) redox centers in the (PVB-NR)1 layer. Several reasons could be the origin of the significant increase of the catalytic current recorded at -0.650 V. First, some molecules of NR should not be accessible to the V•+(PVB) as a consequence of some steric constraints in contrast to the much more mobile MV•+. A second explanation could find its origin in the driving force of the electron-transfer step between the redox mediator and the Mo(VI) active site of NR. Indeed, the MV•+ mediator should be more rapid in the regeneration step than V•+(PVB) as a consequence of the advantage of a 0.180 eV driving force (see Figure 4A). Finally, owing to the high increase of catalytic current in regard to the low concentration of MV•+, we believe that the difference in contribution for the regeneration step is a consequence of balance between the high mobility of diffusing MV•+ molecules and the expected rigidity of the PVB polymer backbone. The formation of the encounter complex between the redox mediator and the enzyme active site is indeed expected to be much more rapid with the small size of MV•+ than with the polymer chain linked to V•+(PVB). Despite the limitation in the regeneration process that the polymer presents in comparison with the mediator in solution, the catalytic current can be increased by assembling several layers, as was previously discussed. (40) McGeachie, J.; Summers, L. A. Z. Naturforsch., B 1986, 41b (10), 1255.

Langmuir, Vol. 19, No. 9, 2003 3869

Figure 5. Variations of film electroacoustic parameters during polyelectrolyte adsorption: (solid line) inductive impedance ∆XLf; (dashed line) motional resistance ∆Rf. The numbers stand for consecutive polyelectrolyte adsorption steps. (A) After the injection of 200 µL of 1.0 mg mL-1 PVB to 200 µL 0.01 M phosphate buffer. (B) After the injection of 200 µL of NR 7.8 mg mL-1 with 5.5 U mL-1 enzymatic activity to 200 µL of 0.1 M phosphate buffer.

Quartz Microbalance Study of PV and NR Adsorption in Self-Assembled Structures. We have used QCM experiments in order to monitor the electrostatic adsorption of PV onto the thiolated gold surface as well as the alternated incorporation of NR and PV layers. The experimental procedure employed has been used before, and it is described in ref 32. Figure 5 shows the time dependence of quartz crystal electroacoustic impedance parameters for the alternated adsorption of successive layers of cationic PVB, Figure 5A, and anionic NR, Figure 5B. The solution compositions were 0.5 mg mL-1 PVB + 0.01 M phosphate buffer and 3.9 mg mL-1 NR + 0.10 M phosphate buffer, respectively. In both cases, no significant variations in the motional damping resistance ∆R were observed, with values lower than 4% of ∆XLf after 5 PVB layers (Figure 5A) and around 10% in the case of NR after six adsorption steps (Figure 5B). This is a clear indication that the incidence of viscoelastic effects due to the rheological properties of the polycation solution can be considered as negligible.25,36 On the other hand, the reactive impedance ∆XLf, assigned to mass changes, evidenced the mass increase occurring after each adsorption step, which can be evaluated by applying eq 1.25,36 The fact that both the enzyme and the redox polymer behave according to the Sauerbrey equation allows the mass monitoring of both adsorption steps. Figure 6 shows the corresponding mass increments observed after the adsorption of each polyelectrolyte (PV and NR) as a

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Ferreyra et al. Table 2. Ionic Strength Effect on the PV Adsorption electroacoustic parameters/Ω PV adsorption in 0.01 M phosphate buffer

Figure 6. Plots of QCM areal mass versus bilayer numbers for PV-NR self-assembled multilayers after the adsorption of PVB and after the adsorption of NR. Data are from Figure 5. Table 1. Electroacoustic Parameters for PV2+(B) Solutions of Different Concentrations 0.5 mg/mL PV solution PV adsorption

NR adsorption

1.0 mg/mL PV solution PV adsorption

NR adsorption

∆R/Ω ∆XLf/Ω ∆R/Ω ∆XLf/Ω ∆R/Ω ∆XLf/Ω ∆R/Ω ∆XLf/Ω 2 2 2 3 3

15 30 68 105 105 116

2 4 8 10 9 9

27 48 74 78 91 87

2 6 17 22 29 37

19 37 70 101 88 96

6 6 7 12 17 20

24 38 54 52 51 76

function of the number of bilayers. It is clearly seen that from the third and following layers, a linear relationship holds for both PVB and NR adsorption, with very similar slope values. According to these results, similar mass increments after the adsorption of either the polycation or the enzyme were observed. It is important to note that the rheological behavior described in Figure 6 is observed for adsorption solutions of low polycation concentration. When the PVB concentration is doubled, keeping the rest of the experimental conditions unchanged, important changes in the electroacoustic impedance parameters, ∆R and ∆XLf, were observed during the adsorption of both the polycation and the enzyme. Table 1 summarizes the obtained results. While the mass incorporated from the 0.5 mg mL-1 PVB solution behaved like an acoustic thin film, layers incorporated from the 1.0 mg mL-1 solution showed significant viscoelastic losses. Under these conditions, the decrease of f0 observed after each adsorption step gave no direct information related to mass increments. Changes in the solution viscosity and the deposit of a less ordered polymer layer would be responsible for the observed behavior. Influence of the Ionic Strength on the Adsorption Processes. The influence of the ionic strength (I) was

PV adsorption in 0.01 M phosphate + 0.025 M NaCl

layer no.

∆R

∆XLf

∆R

∆XLf

1 2 3

2 2 2

15 30 68

4 8 10

72 89 128

analyzed after the comparison of the QCM parameters determined for the adsorption from electrolytic solutions containing 0.5 mg mL-1 PVB in 0.01 M phosphate buffer with or without 0.025 M NaCl. The enzyme was deposited from a solution containing 3.9 mg mL-1 NR in 0.10 M phosphate buffer. Each buffer solution was also employed for washing after each adsorption step. The corresponding values of ∆R and ∆XLf obtained for the deposition of each one of three bilayers in both electrolytic solutions are listed in Table 2. Although both electroacoustic parameters were modified by variations of I, ∆R values remained below 10% of ∆XLf in both cases, and accordingly, the Sauerbrey equation was applied. Differences in ∆XLf point to a greater adsorbed mass for the more concentrated ionic solution. It has been stated in the literature that the most important variable for determining multilayer thickness (besides the number of bilayers) is the salt concentration of the solution used for deposition. An approximately linear dependence of layer thickness on salt concentration has been noticed. This may be the consequence of the overcompensation of surface charge done by the salt ions.41 CV experiments in solutions containing NaCl were performed in order to determine the effect of the ionic strength on the PV charge-transfer process. Although QCM experiments clearly indicated that adsorption was more important in the concentrated ionic solution, the potentiodynamic current-potential profiles obtained with two different Au|thiol|(PVB-NR)1 electrodes, adsorbed from electrolytic solutions of different I, presented almost the same current density (differences below 1%). These results would be an indication of a poor connection between the electrode and the polymer redox residues present in the thicker layer, with no advantages in the sensitivity of the electrode response. Kinetic Analysis of the Adsorption Processes. Kinetic information about the adsorption process involving PV and NR was obtained from the analysis of the time variation of the Sauerbrey mass according to a double exponential kinetic law, eq 5.42,43

∆m ) A1(1 - e-k1t) + A2(1 - e-k2t)

(5)

where A1 and A2 correspond to the maximum ∆m values for each adsorption step, and k1 and k2 stand for the time constants of two different kinetic steps in the adsorption process. A single-exponential law, corresponding to a simple Langmuir type adsorption, gave very poor fits. These double-exponential binding curves have been explained considering nonspecific adsorption, conformational differences or configurational changes, the presence of two or more populations of binding sites, or steric hindrance and diffusion (either in the surface of the sensor or through the molecular layer).37,43 (41) Schlenoff, J.; Dubas, S. T. Macromolecules 2001, 34, 592. (42) Purvis, D.; Pollard-Knight, D.; Lowe, P. In Commercial Biosensors: Applications to Clinical, Bioprocess, and environmental Samples; Ramsay, G., Ed.; J. Wiley & Sons: New York, 1998; Chapter 5. (43) Barbero, C.; Calvo, E. J.; Etchenique, R.; Morales, G. M.; Otero, M. Electrochim. Acta 2000, 45, 3895.


Langmuir, Vol. 19, No. 9, 2003 3871 Table 3. Comparison of the Electroacoustic Parameters for Different PV Samples electroacoustic parameters (Ω) per layer PV2+(B)

Figure 7. (A) Time variation of the NR adsorption mass on Au|thiol|(PVB-NR)n: (0) first layer; (O) fifth layer; (solid line) theoretical values obtained with eq 5. Adsorption solutions: PVB 0.5 mg mL-1 and NR 3.9 mg mL-1 with 5.5 U mL-1 of enzymatic activity prepared in 0.01 and 0.10 M phosphate buffer, respectively. Adsorption time: 20 min. (B) Evolution of the adsorption constant values for NR on PV layers evaluated from eq 5, as a function of bilayer number. (I) (b) A1; (O) A2. (II) (b) k1; (O) k2.

Figure 7A shows the QCM mass transients for the first and the fifth NR adsorption layers, using 0.5 mg mL-1 PVB and 3.9 mg mL-1 NR solutions. As also shown in Figure 5, the adsorption rate is higher for the first layers, decreasing steadily for successive adsorption steps. The corresponding fits according to eq 5 are also included (full line). The very low residuals obtained between the experimental and theoretical values pointed to reasonable good fits which were independent of the initial values used for the fitting procedure. Values of A1, k1, A2, and k2 evaluated from the ∆m versus time plots for the adsorption of successive NR-capped layers similar to those of Figure 7A are shown in Figure 7B. As the number of layers is increased, A1 presents an increase with a hyperbolic decrease in k1. The parameter

layer no.

∆R

1 2 3 4 5 7

2 2 2 3 3

1 2 3 4 5 6

2 4 5 7.5 10 9,5

∆XLf

PV2+(C) ∆R

PV Adsorption 15 3 30 5 70 7 105 9 104 15 44 NR Adsorption 27 5 48 5 73 7 78 12 91 12 87 40

∆XLf 21 41 102 101 104 123

23 59 62 73 58,5 70

PV2+(D) ∆R

∆XLf

2 5 3

10 10 25

2 5

20 35

A2 and k2 remained almost constant in the entire interval studied. The empirical nature of eq 5 discards precise physical interpretations of the above results, although it is clear that the protein adsorption is a complex process, involving at least two kinds of contributions. Some are independent of the number of layers, as suggested by the constancy of A2 and k2. Other processes having an influence on A1 and k1 were slowed by the previously adsorbed layers. Time constants for the successive adsorption of PVB layers showed the same tendency as NR layers. Adsorption of PVB from a 1.0 mg mL-1 solution was not followed due to the inapplicability of the Sauerbrey equation. Adsorption Behavior of the Different Polymer Samples. Table 3 summarizes the electroacoustic parameters for electrodes modified with an increasing number of self-assembled bilayers, adsorbed from 0.5 mg mL-1 solutions of PVB, PVC, and PVD and a 3.9 mg mL-1 NR solution. No significant differences were observed between PVB and PVC. On the other hand, PVD only presented a weak tendency to get adsorbed on the electrode surface. According to these results, it is not easy to establish a correlation between polymer structure, rheological properties, electrochemical behavior, and catalytic activity of the polymers, the analysis remaining empirical. Synthesis procedures may have a strong influence not only on the degree of substitution but also on the water content of each sample, which must also influence the polymer behavior. Evaluation of Water and Ion Exchange during the Reduction of PVB from Electroacoustic and Electrochemical Experiments. The estimation of the electrochemical activity of PVB on multilayer assemblies, considering the water and ion uptake, can be performed by the simultaneous measurement of the electroacoustic impedance parameters ∆R and ∆XLf and currentpotential profiles. These experiments allowed the characterization of mass changes and viscoelastic phenomena taking place in the adsorbed film in parallel with the charge-transfer processes.43 As previously stated, the elasticity of the PVB layer made it possible to evaluate mass changes from frequency shifts, which in this case could be associated with the ion and water exchange that follows the electrochemical reaction. Figure 8 shows the QCM response in parallel with a CV experiment on an Au|thiol|(PVB-NR)6 electrode at 0.002

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Ferreyra et al.

Conclusions

Figure 8. EQCM experiment for the Au|thiol|(PVB-NR)6 PVB electrode. (A) Current-potential profile at 0.002 V s-1 in 0.10 M phosphate buffer pH 7.5. (B) Electroacoustic parameters as a function of potential: (solid line) inductive impedance ∆XLf; (dashed line) motional resistance ∆Rf. (C) Electroreduction charge as a function of potential. (D) QCM areal mass as a function of the reduction charge. Adsorption solutions: PVB 0.5 mg mL-1 and NR 3.9 mg mL-1 with 5.5 U mL-1 enzymatic activity prepared in 0.01 and 0.10 M phosphate buffer, respectively. Adsorption time: 20 min.

V s-1 in deoxygenated 0.10 M phosphate buffer solution.In this case, the geometric area of the electrode was considered. In Figure 8A, the second voltammetric cycle is shown. As previously mentioned, the peak at -0.450 V corresponds to the electrochemical reduction of the polymer bipyridinium groups, involving a total amount of 224 µC cm-2. As before, we consider the presence of 69 redox centers per PVB molecule and the transfer of one electron per redox center. Therefore, 3.42 × 10-11 mol cm-2 (3.2 × 10-6 g cm-2) was the total amount of electroactive PVB present in the seven layers self-assembled on the electrode. As the reduction proceeded, the decrease in positive charge in the film was compensated by the release of counteranions and the associated water molecules from the film to the solution, according to the electroneutrality condition. Therefore, a decrease in ∆XL was observed, Figure 8B, without appreciable changes in ∆R. The opposite behavior was evident for the positive potential sweep, when the polymer was oxidized. In Figure 8C, the charge involved during the negative sweep, evaluated by integration of the corresponding i-E profile, is plotted as a function of E. These charge values were correlated to the corresponding mass changes as shown in Figure 8D. The linear response points to the applicability of Faraday’s law, and the negative slope is a clear indication of the mass loss taking place during the polymer electrochemical reduction. From this slope, the mass change was evaluated as 1022 g/Faraday, which amounts to 2.4 × 10-6 g cm-2. This mass can be considered as the sum of the masses of water and anions exchanged during the redox process. Taking into account the molar mass for the corresponding anion (96 g mol-1), the mass of water interchanged for each mole of reduced viologen corresponds to 51 mol. Such high ion-water mass interchanged has also been reported for other systems.43

For the first time, NR has been incorporated by the ESA procedure. For this purpose, polymers of different degrees of substitution were prepared, the composition being determined by NMR. The electrochemical behavior of the polymer sample with the highest content of redox viologen moieties (12.1% of substitution) evidenced a reversible redox couple with a lower reduction potential than other soluble regenerators, such as methyl viologen or phenosafranin. This is an obvious advantage from the analytical point of view. Nevertheless, the regeneration capability of PV in relation to soluble methyl viologen is lower, probably due to steric factors, a lower driving force for the redox process, and a poor mobility of the polymer in relation to that of PV. Both the enzyme and the polymer (in diluted solutions) presented an elastic behavior in electroacoustic impedance experiments, and accordingly, it was possible to follow the areal mass increments after each adsorption step for both polyions. As expected, the ionic strength of the solution had a positive effect on the amount of polyelectrolyte incorporated in each layer, although this did not correlate with the electrochemical response. QCM studies allowed us to conclude that immobilization of NR in self-assembled layers using cationic polyviologen is an excellent procedure in order to control the amount of the biocatalyst, this being proportional to the number of bilayers. The electrochemical response also confirms the linear correlation between catalytic activity and enzyme surface concentration. Acknowledgment. Financial support from the Consejo Nacional Investigaciones Cientı´ficas y Tecnolo´gicas (CONICET), Ecosud-France/Argentina, Agencia Co´rdoba Ciencia Sociedad del Estado, ANPCyt, Universidad de Buenos Aires, FONCYT, and Universidad Nacional de Co´rdoba is gratefully acknowledged. Appendix Synthesis of Polyviologen Samples. Step a. 1-(2-Bromoethyl)-4,4′-bipyridinium bromide was prepared according to ref 40 by the following procedure: a given amount of 4,4′-bipyridine (from Riedel de Haen) was refluxed with a proper volume of 1,2-dibromoethane (Aldrich) at 80 °C for 3 h. The brown precipitate was collected and exhaustively extracted with boiling ethanol. The mixture was filtered to remove an undesired insoluble polymer of viologen. The desired product was obtained as a yellow precipitate which was separated from the filtrate, washed with ether (yield around 50%), and used without further purification. 1H NMR (δ; D2O): 3.98 (2H, CH2Br); 5.05 (2H, CH2N); 7.85-8.89 (H, A). Step b. N-1-(2-Bromoethyl)-N′-methyl-4,4′-bipyridinium was synthesized by refluxing 1-(2-bromoethyl)-4,4′-bipyridinium bromide with a 10-fold molar excess of methyl iodide in dimethylformamide (DMF) at 76 °C for 24 h. The resulting orangered precipitate was washed with ether in order to eliminate the nonreacted methyl iodide. 1H NMR (δ; D2O): 3.77 (2H, CH2Br); 4.44 (3H, CH3N); 5.02 (2H, CH2N); 8.45-9.09 (H, A). To increase the polymer solubility in DMF, halide ions were interchanged by BF4- passing product b through an ion exchange column (Amberlite IRA-93) in BF4- form. Nevertheless, taking into account the strong affinity of iodide to the pyridinium group,33 it has to be expected that some iodide ions remain as counterions after the ion exchange procedure. This fact was verified for the case of two polymer samples, PVA and PVB, by means of an elemental analysis of iodine ions, finding a weight percentage of 10.36 and 6.77 for each sample. Step c. The preparation of the viologen-functionalized redox polymer was performed from an adapted procedure described in


Figure 9.

Langmuir, Vol. 19, No. 9, 2003 3873

1

H NMR spectroscopic analysis, using deuterated dimethyl sulfoxide as the solvent.

the literature.44 The viologen resulting from step b was dissolved in DMF together with poly(4-vinyl pyridine), PVP, MW ) 60 000, with a mean polymerization degree of 571 (Aldrich). The ratios of the two reagents corresponded to a molar excess of 2.5 of viologen per equivalent vinyl pyridine mole number. The mixture was stirred at X °C for Y hours, depending on the polymer sample: for PVA, X ) 45 °C, Y ) 72 h; for PVB, X ) 80 °C, Y ) 48 h; for PVC, X ) 45 °C, Y ) 96 h; for PVD, X ) 45 °C, Y ) 120 h. The resulting redox polymer was precipitated from the reaction mixture by addition of acetone under stirring. Differences in the degree of substitution of each polymer sample should be expected. Step d. The products of step c were dissolved in water and then dialyzed. For PVA and PVB, this procedure was done against water in order to eliminate unreacted viologen molecules (b), while the PVC and PVD were dialyzed first against KCl 0.5 M for 72 h and then against distilled water. A dialysis membrane from Spectrum Laboratories (MW cutoff, 12 000-14 000) was used. The dialysis procedure was stopped when the conductivity of the dialysis medium reached the value of the distilled water. These different dialysis procedures determined the polymer counterion composition. It is assumed that almost all Br- and BF4- ions in PVC and PVD were interchanged by Cl-. Finally, all polymer samples were lyophilized. Estimation of the Structure and Substitution Ratio. This was performed from 1H NMR spectroscopic analysis. The proposed structure is shown in Chart 1, with different substitution ratios depending on the polymer sample. Small letters indicate the different aromatic hydrogen atoms considered in the 1H NMR spectroscopic analysis, from which the structure and the substitution ratio were estimated. According to Chart 1, in each macromolecule of polyviologen, there are eight kinds of aromatic protons, labeled Ha, Hb, Hc, Hd, He, Hf, Hg, and Hh. Thus if N is (44) Eng, L. H.; Elmgren, M.; Komlos, P.; Nordling, M.; Lindquist, S. E.; Neujahr, H. Y. J. Phys. Chem. 1994, 98, 7068.

the number of monomer units composing the macromolecules and if x is the number of substituted pyridine groups, we can consider that there are 2xHa, 2xHb, and 2(N - x) of each kind of Hc, Hd, He, Hf, Hg, and Hh. To assign the NMR signals, the NMR spectra of the following compounds have been recorded in DMSO: polyvinyl pyridine, bromoethyleviologene, and N-ethylpolyvinylpyridinium (obtained by full quaternization of polyvinyl pyridine using bromoethane). Comparison of the NMR spectra of these compounds with those of the polyviologen samples allowed us to concluded that Ha, Hb, Hc, Hd, He, Hf, Hg, and Hh would give NMR signals nearby the following chemical shifts: 2xHa, ∼6.57 ppm; 2xHb, ∼8.24 ppm; 2(N - x)He, ∼9.3 ppm; 2(N - x)Hh, ∼9.3 ppm; 2(N - x)Hf, ∼8.8 ppm; 2(N - x)Hg, ∼8.8 ppm; 2(N - x)Hd, ∼8.93 ppm; and 2(N x)Hc, ∼7.99 ppm. As an example, Figure 9 shows the spectrum for PVB. The integral signal at 6.6 ppm (area A1) corresponding to 2xHa of the pyridine ring has to be proportional to the number of unsubstituted pyridine moieties, x. On the other hand, the multiplet signal centered at about 9 ppm, recorded at a chemical shift higher than that of Hb, can be assigned to the 10(N - x) protons He, Hh, Hf, Hg, and Hd. From these, 8(N - x) correspond to the bipyridinium ring and 2(N - x) to the Hd of the substituted pendant pyridine ring of the polymer backbone. Since the integrated area is proportional to the number of protons, we can conclude that 2x ) kA1 and 10(N - x) ) kA2, with k as the proportionality coefficient. The substitution ratio is therefore (N - x)/N ) [1 + 5(A1/A2)]-1. The resulting (%) values were as follows: PVA ) 3.9, PVB ) 12.1, PVC ) 5.0, and PVD ) 10.0. From the substitution ratio of each polymer sample, the mean molecular mass can be evaluated provided the corresponding counterion composition is known. Taking into account the stoichiometry, the algorithms A1 and A2 were employed for PVB. In this calculation, it was assumed that the counteranions A-

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Ferreyra et al.

were BF4- or I-. The mass content of this last anion, as weightby-weight percentage (m(I)% w/w) was determined by elemental composition analysis.

m(I)% w/w ) [(N - x)(1 - y)2MI]/MPV

(A1)

MPV ) xMU + (N - x)MS + 2(N - x)yMBF4 + 2(N - x)(1 - y)MI (A2) y being the fraction of bipyridinium groups that have BF4- as the counterion considering that the rest, (1 - y), are balanced by iodide. MPVB, MI, MBF4, MU, and MS are the molar masses of PVB, I-, BF4-, the unsubstituted monomer, and the substituted

monomer, respectively. In the evaluation of MU and MS, the counterion masses were not considered. According to eqs A1 and A2, y ) 0.44 and MPVB ) 94 kDa. The molar mass for the polymer PVA was calculated in the same way; the values obtained were y ) 0.18 and MPVB ) 72 kDa. The chloride content in PVC and PVD determined by elemental analysis was 0.33% and 6.45%, respectively, at variance with the values evaluated from the substitution ratio, which are 4.3% and 7.41%, respectively. This clearly points to the fact that the counterion composition may include undetermined amounts of iodide or BF4-. Therefore, it was not possible to calculate the corresponding molar masses.

LA026841W

Electrochemical Behavior of Nitrate Reductase Immobilized in Self

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