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An efficient poly(pyrrole–viologen)-nitrite reductase biosensor for the mediated detection of nitrite
Electrochemistry Communications 6 (2004) 404–408 www.elsevier.com/locate/elecom
An efficient poly(pyrrole–viologen)-nitrite reductase biosensor for the mediated detection of nitrite Serge Da Silva a, Serge Cosnier a
a,*
, M. Gabriela Almeida b, Jose J.G. Moura
b
Laboratoire d’Electrochimie Organique et de Photochime Redox, UMR CNRS 5630, Bat CHIMIE, Institut de chimie moleculaire de Grenoble, FR CNRS 2607, Universite Joseph Fourier Grenoble I, BP 53, 38041 Grenoble, Cedex 9, France b Departamento de Quımica, Faculdade de Ci^encias e Tecnologia, REQUIMTE, Centro de Tecnologia Quımica e Biotecnologia, Universidade Nova de Lisboa, 2825-516 Monte de Caparica, Portugal Received 21 January 2004; received in revised form 12 February 2004; accepted 13 February 2004
Published online:
Abstract A biosensor for nitrite analytical determination was developed using a cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 immobilized and electrically connected on a glassy carbon electrode by entrapment in an electrogenerated poly(pyrrole–viologen) matrix. The modified bioelectrode was studied by cyclic voltammetry and a catalytic current was observed in presence of nitrite. The linear range of the electrode response was 5.4–43.4 lM. The detection limit and the sensitivity were 5.4 lM and 1721 mA M1 cm2 , respectively. The K app M value determined from the Lineweaver–Burk plot was 86 lM. The biosensor fully maintained its electroenzymatic activity towards nitrite after four days. No catalytic response was observed in the presence of nitrate ions while interference from sulfites was considered negligible. Finally, the biosensor composition was optimized in term of monomer–enzyme ratio. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Biosensor; Polypyrrole; Nitrite reductase; Viologen; Enzyme wiring
1. Introduction Nitrates and nitrites are largely used as preservatives and fertilizing agents. However, a continuous exposition of these ions to humans can have serious health implications. Particularly, nitrites can react irreversibly with hemoglobin to produce methemoglobin, therefore reducing blood capacity to transport oxygen. The recognition of such a threat obliges, in most industrial countries, to nitrite detection, determination, and monitoring in environmental, food and physiological samples. The European Community have established the maximum admissible levels of nitrate and nitrite in drinking water at 50 and 0.1 mg/L, respectively. Nitrite determination is mainly carried out by spectrophotometry (Griess reaction), ionic chromatography, po*
Corresponding author. Tel.: +33-47-651-4998; fax: +33-47-6514267. E-mail address: [email protected] (S. Cosnier). 1388-2481/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2004.02.007
larography, capillary electrophoresis and fluorescence spectrophotometric methods [1]. However, these centralized and sophisticated analytical systems are representing delays in solving problems in emergency situations where the rapid diagnostic of diseases, food quality control or environmental pollution monitoring are issues of critical concern. As a consequence, there is, for three decades, a growing interest in the design of biosensors which combine intimately the recognition properties of biological macromolecules to the sensitivity of transducers; biosensors based on an electrochemical transduction constituting the main category. One of the most popular configurations of biosensor consists of enzyme immobilized within a polymer, which is generated over an electrode. The electrosynthesis of organic conducting polymers indeed allows the reproducible deposition of biological macromolecules with controlled spatial resolution [2]. In addition, the electropolymerization of polymers functionalized by redox groups is an attractive approach for the
S. Da Silva et al. / Electrochemistry Communications 6 (2004) 404–408
immobilization of biomolecules where the electron transport to enzymes is ensured by electron hopping between immobilized redox centers [3–5]. In that sense, nitrite reductases (NiRs) are natural candidates to build specific nitrite biosensors [6–8]. They may be classified by their reaction products (NO or NHþ 4 ) or their prosthetic groups (copper, hemes cd1; heme c and siroheme): both copper and heme cd1 containing NiRs are responsible for the reduction of nitrite to nitric oxide, in a two-electrons step, while siroheme and cytochrome c type NiRs catalyze the direct six-electrons reduction of nitrite to ammonia [9–11]. The development of electrochemical nitrite biosensors is, however, faced with the major obstacles of the poor stability of the nitrite reductase and the difficulty in establishing an electrical communication with the immobilized reductase. In this context, we report here the simultaneous electrical connection and immobilization of ccNiR from nitrate-grown cells of Desulfovibrio desulfuricans ATCC 27774 (a sulfate reducing organism) by an electrogenerated polypyrrole film N -substituted by viologen groups. This enzyme has a potential use in the biosensor technology as it performs one specific and welldefined chemical transformation. In addition, it is readily isolated in large amounts and is particularly stable [12,13]. ccNiR from D. desulfuricans is isolated as a mixture of heterooligomeric complexes of high molecular mass, in agreement to its high tendency to aggregate in solution. The enzyme complexes are composed by a periplasmic catalytic subunit of 61 kDa (NrfA) and a transmembrane subunit of 19 kDa (NrfH). The total in vitro stoichiometry is 2 NrfA:1 NrfH. NrfA contains five c-type hemes and NrfH contains four [13]. The resulting redox polypyrrole-nitrite reductase electrodes were, thus, characterized and their potentials for the electrochemical detection of nitrite will be hereby described.
405
2.2. Enzymatic activity assay The determination of ccNiR activity was based on the amount of nitrite consumed after 5 min during the following enzymatic reaction: 100 lL of 40 mM methyl viologen was added in a test tube with 100 lL of ccNiR sample in 700 lL of phosphate potassium buffer (286 mM, pH 7.5), containing NaNO2 (71.4 lM). The solution was thermostated at 37 °C and the catalytic reaction was initialized by adding 100 lL of a solution containing 8 mg/ mL of sodium hydrosulfite and 8 mg/mL of sodium bicarbonate. Sodium hydrosulfite solution was prepared just before assays to avoid compound degradation. A blue color appeared instantaneously due to the formation of viologen radical cation. After exactly 5 min, the reaction was stopped by agitation with a vortex until the solution color becomes translucent. For each assay, a control experiment without enzyme and a blank without nitrite were carried out. The remaining nitrite concentration was then determined by adding 0.5 mL of 1% (w/v) sulfanilamide in HCl (32% weight) and 0.5 mL of 0.02% (w/v) N -(1-naphtyl)-ethylendiamin dihydrochloride to each tube test. Nitrite concentrations were calculated from the resulting absorbances measured with a Carry 1 UV–Vis Varian spectrophotometer (k ¼ 540 nm). 2.3. Electrochemical apparatus All electrochemical studies were performed with a conventional three-electrode potentiostatic system. Working electrodes were in glassy carbon matter purchased from Radiometer analytical (3 and 5 mm of diameter). A Pt wire was used as the counter electrode and placed in a separated compartment containing the supporting electrolyte. A saturated calomel electrode (SCE) was used as reference. All measurements were conducted in a Metrohm cell thermostated at 30 °C containing solutions de-oxygenated by an argon flux. An Autolab 100 potentiostat was used to carry out the electrochemical experiments.
2. Experimental
2.4. Bioelectrode preparation
2.1. Reagents and biochemicals
The pyrrolic monomer was dispersed in de-ionized water by ultra-sonication into stable, optically transparent solutions (5 mM). Glassy carbon electrode surfaces were polished with 2 lm diamond paste, sonicated and rinsed with water. A solution containing ccNiR and dispersed monomer was spread on glassy carbon electrodes in order to obtain 0.5 lg/mm2 of ccNiR and 0.07–0.76 nmol/mm2 (according to experimental conditions) of monomer. Solvent was evaporated under vacuum and the resulting electrode was transferred into a cell containing an aqueous 0.1 M LiClO4 solution (2 mL). Polymerization of adsorbed monomer was carried out by controlledpotential electrolysis for 15 min at 0.8 V vs. SCE.
ccNiR from D. desulfuricans ATCC 27774 was purified in Faculdade de Ci^encias e Tecnologia, Universidade Nova de Lisboa following the procedure described in literature [13] and stored in 0.3 M phosphate buffer, pH 7.5 at )20 °C. N -Methyl-N 0 -(12-pyrrol-1-yldodecyl)4,40 -bipyridinium ditetrafluoroborate was synthesized as previously reported [14]. Sodium hydrosulfite (85%) and N -(1-naphtyl)-ethylendiamin dihydrochlorid (98%) were purchased from Aldrich and sulfanilamide from Fluka. All other chemical reagents were of analytical grade. Water was doubly distilled in a quartz apparatus.
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S. Da Silva et al. / Electrochemistry Communications 6 (2004) 404–408
3. Results and discussion 0.5 lg/mm2 of ccNiR was directly co-adsorbed with 0.5 nmol/mm2 of amphiphilic viologen derivative on a glassy carbon surface. The subsequent electropolymerization of the adsorbed monomers induced the physical entrapment of the adsorbed ccNiR in the in situ generated polypyrrole film. The electrochemical behavior of this modified electrode was investigated by cyclic voltammetry in 0.1 M KCl solution containing 0.05 M Tris–HCl buffer (pH 7.5). As expected, in the absence of nitrite, the cyclic voltammogram displays a reversible peak system at E1=2 ¼ 0:42 vs. SCE assigned to the one-electron reduction of the polymerized viologen groups (Fig. 1(a)). In the presence of nitrite (1 mM), a marked increase in the cathodic current is observed while the anodic one decreased (Fig. 1(b)). This electrocatalytic phenomenon reflects the efficient electrochemical wiring of the immobilized nitrite reductase to the electrode surface likely via a hopping mechanism between the polymerized redox mediators. Viologen dications were reduced into viologen radical cations at the electrode surface and re-oxidized by ccNiR, which in turn catalyzes the reduction of nitrites into ammonium ions. In addition, control experiments were performed in similar conditions with an electrode covered by a pure poly(viologen) film. Without ccNiR, no catalytic current was observed after addition of nitrites. Such phenomenon unambiguously indicated the electroenzymatic reduction of nitrite by the bioelectrode. The catalytic current (I cat ) values were derived from cyclic voltammograms subtracting the base line (current measured at the same potential from the cyclic voltammogram performed without nitrites) from the current measured at )0.70 V vs. SCE in the presence of nitrites. As shown in Fig. 2, the catalytic current magnitude increased linearly with nitrite concentration up to 43.4 lM, with a sensitivity of 1721 mA M1 cm2 . The detection limit was 5.4 lM. At high nitrite concentrations the current response
Fig. 1. Cyclic voltammetry of poly(pyrrole–viologen)-ccNiR electrode performed in de-oxygenated 0.05 M Tris–HCl buffer (pH 7.5) containing 0.1 M KCl in the absence (a) and in presence (b) of 1 mM NaNO2 ; scan rate 20 mV s1 .
Fig. 2. Catalytic current response of the biosensor obtained by cyclic voltammetry as a function of nitrite concentration (currents are reported in absolute values); same electrochemical conditions than in Fig. 1.
deviated from linearity and approached a constant value reflecting the maximum rate of the enzymatic reaction under nitrite saturating conditions. Upon plotting the catalytic current vs. substrate concentration (Fig. 2) a typical Michaelis–Menten profile was observed. The K app M value as determined from the Lineweaver–Burk plot (1/I cat vs. 1/[NO 2 ]) was 86 lM. This electroenzymatic system constitutes the first example of a nitrite biosensor based on ccNiR immobilization in a poly(pyrrole–viologen) matrix. The analytical characteristics of the poly(pyrrole–viologen)ccNiR electrode differed markedly from those previously reported for other ccNiR-based biosensors. In particular, the bioelectrode exhibited a lower K app M value and higher nitrite sensitivity [7,8]. Most probably, the membranar nature of D. desulfuricans ccNiR and the presence of an hydrophobic sub-unit facilitate its interaction with the hydrophobic microenvironment provided by the organic polymer surrounding the immobilized enzyme. This may enhance the protein structure stability and hence counterbalance the matrix denaturation effects. In addition, the improvement in biosensor performance also may be ascribed to both high specific activity and stability of ccNiR from D. desulfuricans ATCC 27774. The poly(viologen)-ccNiR electrodes were also examined for their storage stability by recording the catalytic current response to nitrite (1 mM) once a day at the same time; in between, the bioelectrodes were stored at +4 °C in 0.05 M Tris–HCl buffer, pH 7.5. Fig. 3 shows the polymer electroactivity and catalytic current evolutions as a function of time. No loss of activity was observed after four days reflecting the absence of enzyme release as well as the stability of the polymeric coating. Afterward, the signal of the polymerized viologen decreased successively by 19%, 22% and 29% while the catalytic current dropped by 53%, 72% and 92% for five, six and seven days, respectively. Thus, it appears that the catalytic current (i.e., the enzyme activity) decreased in a faster way than the electroactivity of the
S. Da Silva et al. / Electrochemistry Communications 6 (2004) 404–408
Fig. 3. Time influence on the peak current of the one-electron reduction of the polymerized viologen groups (grey) in absence of nitrite and on the catalytic current response of the biosensor (black) in presence of 1 mM NaNO2 (currents are reported in absolute values); same electrochemical conditions than in Fig. 1.
407
monomer, the electropolymerization yield being 7%. In contrast, the evolution of the biosensor response depicted a bell shape, the maximum value being reached for 0.253 nmol/mm2 of deposited monomer. Keeping in mind that the immobilized amount of enzyme being constant, the ascendant part of the bell curve reflects an improvement of the electrical wiring of ccNiR due to the density increase of the electrogenerated Vþ sites surrounding the protein shell. Then, the decrease in biosensor response for increasing monomer amount may be more likely ascribed to the increase in film thickness. The latter, indeed, induced a large increase in diffusional constraints towards the permeation of nitrite and ammonium.
4. Conclusions viologen groups highlighting a continuous ccNiR denaturation, in spite of its hydrophobic feature. The influence of sulfites, known to have some reactivity with ccNiR [15], and nitrates which are frequently present in natural nitrite samples, as potential interferents on the biosensor sensitivity was examined. No appreciable change in the biosensor response was observed for nitrates (1 mM) while sulfites (1 mM) induced a very slight signal increase, namely 1.7%. A biosensor optimization study was also performed by varying the amount of monomer spread on the electrode surface, the amount of deposited ccNiR being constant (0.5 lg/mm2 ). After each electropolymerization, the enzymatic activity of electrolyte solutions was examined indicating a quasi absence of enzyme release (only 5%) for all the configurations. Fig. 4 presents the electroactivity of the redox polymer and the biosensor response to nitrite as a function of the amount of deposited monomer. As expected, the electroactivity of the polymer monotonously increased with the deposited amount of
ccNiR from D. desulfuricans ATCC 27774 was successfully immobilized and electrically connected on a glassy carbon electrode by co-adsorption and electropolymerization of an amphiphilic pyrrole–viologen derivative. Thanks to the specific properties of ccNiR, a catalytic current was observed in presence of nitrite ions in spite of the hydrophobic feature of the polymeric matrix. To our knowledge, this is the first time that a nitrite reductase was connected to a poly(pyrrole–viologen) to perform a third generation electrochemical biosensor. Indeed, all the previous attempts to construct nitrite reductase based electrochemical biosensors required the presence of mediator species for analyte determination. Sensor performances matched with those encountered in literature and even seem slightly better thanks to the good specific activity, high stability and hydrophobic feature of ccNiR. On the other hand, very few examples of bioelectrodes modified with a combination of functionalized redox polymers and reductases, exhibited such a fast enzyme response as exemplified by the electrocatalytic currents reported in this work.
Acknowledgements The authors thank the Commission of the European Communities Research Directorate for their support under the PEBCAT project contract No. EVK1-CT2000-00069, FSE and the Fundacß~ao para a Ci^encia e Tecnologia, through the PhD Grant No. PRAXIS XXI/ BD/11349/97 (MGA) Fig. 4. Electroactivity of the poly(pyrrole–viologen) ðsÞ and catalytic current to nitrite ðjÞ (1 mM) as a function of the amount of monomer deposited on the electrode surface (currents are reported in absolute values). The amount of electroactive polymerized monomer was determined by integration of the charge recorded under the reduction wave of V2þ=þ system in the absence of nitrite. Each point was calculated from at least three independent experiments. Same electrochemical conditions than in Fig. 1.
References [1] M.J. Moorcroft, J. Davis, R.G. Compton, Talanta 54 (5) (2001) 785. [2] S. Cosnier, Anal. Bioanal. Chem. 377 (2003) 507. [3] W. Schuhmann, Mikrochim. Acta. 121 (1995) 1. [4] S. Cosnier, Biosens. Bioelectron. 14 (1999) 442.
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[5] F. Palmisano, P.G. Zambonin, D. Centonze, Fresenius J. Anal. Chem. 366 (2000) 586. [6] M. Scharf, C. Moreno, C. Costa, C. Vandijk, W.J. Payne, J. LeGall, I. Moura, J.J.G. Moura, Biochem. Biophys. Res. Commun. 209 (3) (1995) 1018. [7] B. Strehlitz, G. Gr€ undig, W. Schumacher, P.M.H. Kroneck, K.-D. Vorlop, H. Kotte, Anal. Chem. 68 (1996) 807. [8] Q. Wu, D. Storrier, F. Pariente, Y. Wang, J.P. Shapleigh, H. Abruna, Anal. Chem. 69 (1997) 4856. [9] I. Moura, S. Bursakov, C. Costa, J.J.G. Moura, Anaerobe 3 (1997) 279.
[10] T. Brittain, R. Blackmore, C. Greenwood, A.J. Thomson, Eur. J. Biochem. 209 (1992) 792. [11] W.G. Zumft, Microbiol. Mol. Biol. Rev. 61 (4) (1997) 533. [12] M.C. Liu, C. Costa, I. Moura, Methods Enzymol. 243 (1994) 303. [13] M.G. Almeida, S. Macieira, L.L. Goncßalves, R. Huber, C.A. Cunha, M.J. Rom~ao, C. Costa, J. Lampreia, J.J.G. Moura, I. Moura, Eur. J. Biochem. 270 (2003) 3904. [14] S. Cosnier, B. Galland, C. Innocent, J. Electroanal. Chem. 433 (1997) 113. [15] I.C. Pereira, I.A. Abreu, A.V.M. Xavier, J. LeGall, M. Teixeira, Biochem. Biophys. Res. Commun. 224 (1996) 611.
An efficient poly(pyrrole–viologen)-nitrite reductase biosensor for the mediated detection of nitrite Serge Da Silva a, Serge Cosnier a
a,*
, M. Gabriela Almeida b, Jose J.G. Moura
b
Laboratoire d’Electrochimie Organique et de Photochime Redox, UMR CNRS 5630, Bat CHIMIE, Institut de chimie moleculaire de Grenoble, FR CNRS 2607, Universite Joseph Fourier Grenoble I, BP 53, 38041 Grenoble, Cedex 9, France b Departamento de Quımica, Faculdade de Ci^encias e Tecnologia, REQUIMTE, Centro de Tecnologia Quımica e Biotecnologia, Universidade Nova de Lisboa, 2825-516 Monte de Caparica, Portugal Received 21 January 2004; received in revised form 12 February 2004; accepted 13 February 2004
Published online:
Abstract A biosensor for nitrite analytical determination was developed using a cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 immobilized and electrically connected on a glassy carbon electrode by entrapment in an electrogenerated poly(pyrrole–viologen) matrix. The modified bioelectrode was studied by cyclic voltammetry and a catalytic current was observed in presence of nitrite. The linear range of the electrode response was 5.4–43.4 lM. The detection limit and the sensitivity were 5.4 lM and 1721 mA M1 cm2 , respectively. The K app M value determined from the Lineweaver–Burk plot was 86 lM. The biosensor fully maintained its electroenzymatic activity towards nitrite after four days. No catalytic response was observed in the presence of nitrate ions while interference from sulfites was considered negligible. Finally, the biosensor composition was optimized in term of monomer–enzyme ratio. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Biosensor; Polypyrrole; Nitrite reductase; Viologen; Enzyme wiring
1. Introduction Nitrates and nitrites are largely used as preservatives and fertilizing agents. However, a continuous exposition of these ions to humans can have serious health implications. Particularly, nitrites can react irreversibly with hemoglobin to produce methemoglobin, therefore reducing blood capacity to transport oxygen. The recognition of such a threat obliges, in most industrial countries, to nitrite detection, determination, and monitoring in environmental, food and physiological samples. The European Community have established the maximum admissible levels of nitrate and nitrite in drinking water at 50 and 0.1 mg/L, respectively. Nitrite determination is mainly carried out by spectrophotometry (Griess reaction), ionic chromatography, po*
Corresponding author. Tel.: +33-47-651-4998; fax: +33-47-6514267. E-mail address: [email protected] (S. Cosnier). 1388-2481/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2004.02.007
larography, capillary electrophoresis and fluorescence spectrophotometric methods [1]. However, these centralized and sophisticated analytical systems are representing delays in solving problems in emergency situations where the rapid diagnostic of diseases, food quality control or environmental pollution monitoring are issues of critical concern. As a consequence, there is, for three decades, a growing interest in the design of biosensors which combine intimately the recognition properties of biological macromolecules to the sensitivity of transducers; biosensors based on an electrochemical transduction constituting the main category. One of the most popular configurations of biosensor consists of enzyme immobilized within a polymer, which is generated over an electrode. The electrosynthesis of organic conducting polymers indeed allows the reproducible deposition of biological macromolecules with controlled spatial resolution [2]. In addition, the electropolymerization of polymers functionalized by redox groups is an attractive approach for the
S. Da Silva et al. / Electrochemistry Communications 6 (2004) 404–408
immobilization of biomolecules where the electron transport to enzymes is ensured by electron hopping between immobilized redox centers [3–5]. In that sense, nitrite reductases (NiRs) are natural candidates to build specific nitrite biosensors [6–8]. They may be classified by their reaction products (NO or NHþ 4 ) or their prosthetic groups (copper, hemes cd1; heme c and siroheme): both copper and heme cd1 containing NiRs are responsible for the reduction of nitrite to nitric oxide, in a two-electrons step, while siroheme and cytochrome c type NiRs catalyze the direct six-electrons reduction of nitrite to ammonia [9–11]. The development of electrochemical nitrite biosensors is, however, faced with the major obstacles of the poor stability of the nitrite reductase and the difficulty in establishing an electrical communication with the immobilized reductase. In this context, we report here the simultaneous electrical connection and immobilization of ccNiR from nitrate-grown cells of Desulfovibrio desulfuricans ATCC 27774 (a sulfate reducing organism) by an electrogenerated polypyrrole film N -substituted by viologen groups. This enzyme has a potential use in the biosensor technology as it performs one specific and welldefined chemical transformation. In addition, it is readily isolated in large amounts and is particularly stable [12,13]. ccNiR from D. desulfuricans is isolated as a mixture of heterooligomeric complexes of high molecular mass, in agreement to its high tendency to aggregate in solution. The enzyme complexes are composed by a periplasmic catalytic subunit of 61 kDa (NrfA) and a transmembrane subunit of 19 kDa (NrfH). The total in vitro stoichiometry is 2 NrfA:1 NrfH. NrfA contains five c-type hemes and NrfH contains four [13]. The resulting redox polypyrrole-nitrite reductase electrodes were, thus, characterized and their potentials for the electrochemical detection of nitrite will be hereby described.
405
2.2. Enzymatic activity assay The determination of ccNiR activity was based on the amount of nitrite consumed after 5 min during the following enzymatic reaction: 100 lL of 40 mM methyl viologen was added in a test tube with 100 lL of ccNiR sample in 700 lL of phosphate potassium buffer (286 mM, pH 7.5), containing NaNO2 (71.4 lM). The solution was thermostated at 37 °C and the catalytic reaction was initialized by adding 100 lL of a solution containing 8 mg/ mL of sodium hydrosulfite and 8 mg/mL of sodium bicarbonate. Sodium hydrosulfite solution was prepared just before assays to avoid compound degradation. A blue color appeared instantaneously due to the formation of viologen radical cation. After exactly 5 min, the reaction was stopped by agitation with a vortex until the solution color becomes translucent. For each assay, a control experiment without enzyme and a blank without nitrite were carried out. The remaining nitrite concentration was then determined by adding 0.5 mL of 1% (w/v) sulfanilamide in HCl (32% weight) and 0.5 mL of 0.02% (w/v) N -(1-naphtyl)-ethylendiamin dihydrochloride to each tube test. Nitrite concentrations were calculated from the resulting absorbances measured with a Carry 1 UV–Vis Varian spectrophotometer (k ¼ 540 nm). 2.3. Electrochemical apparatus All electrochemical studies were performed with a conventional three-electrode potentiostatic system. Working electrodes were in glassy carbon matter purchased from Radiometer analytical (3 and 5 mm of diameter). A Pt wire was used as the counter electrode and placed in a separated compartment containing the supporting electrolyte. A saturated calomel electrode (SCE) was used as reference. All measurements were conducted in a Metrohm cell thermostated at 30 °C containing solutions de-oxygenated by an argon flux. An Autolab 100 potentiostat was used to carry out the electrochemical experiments.
2. Experimental
2.4. Bioelectrode preparation
2.1. Reagents and biochemicals
The pyrrolic monomer was dispersed in de-ionized water by ultra-sonication into stable, optically transparent solutions (5 mM). Glassy carbon electrode surfaces were polished with 2 lm diamond paste, sonicated and rinsed with water. A solution containing ccNiR and dispersed monomer was spread on glassy carbon electrodes in order to obtain 0.5 lg/mm2 of ccNiR and 0.07–0.76 nmol/mm2 (according to experimental conditions) of monomer. Solvent was evaporated under vacuum and the resulting electrode was transferred into a cell containing an aqueous 0.1 M LiClO4 solution (2 mL). Polymerization of adsorbed monomer was carried out by controlledpotential electrolysis for 15 min at 0.8 V vs. SCE.
ccNiR from D. desulfuricans ATCC 27774 was purified in Faculdade de Ci^encias e Tecnologia, Universidade Nova de Lisboa following the procedure described in literature [13] and stored in 0.3 M phosphate buffer, pH 7.5 at )20 °C. N -Methyl-N 0 -(12-pyrrol-1-yldodecyl)4,40 -bipyridinium ditetrafluoroborate was synthesized as previously reported [14]. Sodium hydrosulfite (85%) and N -(1-naphtyl)-ethylendiamin dihydrochlorid (98%) were purchased from Aldrich and sulfanilamide from Fluka. All other chemical reagents were of analytical grade. Water was doubly distilled in a quartz apparatus.
406
S. Da Silva et al. / Electrochemistry Communications 6 (2004) 404–408
3. Results and discussion 0.5 lg/mm2 of ccNiR was directly co-adsorbed with 0.5 nmol/mm2 of amphiphilic viologen derivative on a glassy carbon surface. The subsequent electropolymerization of the adsorbed monomers induced the physical entrapment of the adsorbed ccNiR in the in situ generated polypyrrole film. The electrochemical behavior of this modified electrode was investigated by cyclic voltammetry in 0.1 M KCl solution containing 0.05 M Tris–HCl buffer (pH 7.5). As expected, in the absence of nitrite, the cyclic voltammogram displays a reversible peak system at E1=2 ¼ 0:42 vs. SCE assigned to the one-electron reduction of the polymerized viologen groups (Fig. 1(a)). In the presence of nitrite (1 mM), a marked increase in the cathodic current is observed while the anodic one decreased (Fig. 1(b)). This electrocatalytic phenomenon reflects the efficient electrochemical wiring of the immobilized nitrite reductase to the electrode surface likely via a hopping mechanism between the polymerized redox mediators. Viologen dications were reduced into viologen radical cations at the electrode surface and re-oxidized by ccNiR, which in turn catalyzes the reduction of nitrites into ammonium ions. In addition, control experiments were performed in similar conditions with an electrode covered by a pure poly(viologen) film. Without ccNiR, no catalytic current was observed after addition of nitrites. Such phenomenon unambiguously indicated the electroenzymatic reduction of nitrite by the bioelectrode. The catalytic current (I cat ) values were derived from cyclic voltammograms subtracting the base line (current measured at the same potential from the cyclic voltammogram performed without nitrites) from the current measured at )0.70 V vs. SCE in the presence of nitrites. As shown in Fig. 2, the catalytic current magnitude increased linearly with nitrite concentration up to 43.4 lM, with a sensitivity of 1721 mA M1 cm2 . The detection limit was 5.4 lM. At high nitrite concentrations the current response
Fig. 1. Cyclic voltammetry of poly(pyrrole–viologen)-ccNiR electrode performed in de-oxygenated 0.05 M Tris–HCl buffer (pH 7.5) containing 0.1 M KCl in the absence (a) and in presence (b) of 1 mM NaNO2 ; scan rate 20 mV s1 .
Fig. 2. Catalytic current response of the biosensor obtained by cyclic voltammetry as a function of nitrite concentration (currents are reported in absolute values); same electrochemical conditions than in Fig. 1.
deviated from linearity and approached a constant value reflecting the maximum rate of the enzymatic reaction under nitrite saturating conditions. Upon plotting the catalytic current vs. substrate concentration (Fig. 2) a typical Michaelis–Menten profile was observed. The K app M value as determined from the Lineweaver–Burk plot (1/I cat vs. 1/[NO 2 ]) was 86 lM. This electroenzymatic system constitutes the first example of a nitrite biosensor based on ccNiR immobilization in a poly(pyrrole–viologen) matrix. The analytical characteristics of the poly(pyrrole–viologen)ccNiR electrode differed markedly from those previously reported for other ccNiR-based biosensors. In particular, the bioelectrode exhibited a lower K app M value and higher nitrite sensitivity [7,8]. Most probably, the membranar nature of D. desulfuricans ccNiR and the presence of an hydrophobic sub-unit facilitate its interaction with the hydrophobic microenvironment provided by the organic polymer surrounding the immobilized enzyme. This may enhance the protein structure stability and hence counterbalance the matrix denaturation effects. In addition, the improvement in biosensor performance also may be ascribed to both high specific activity and stability of ccNiR from D. desulfuricans ATCC 27774. The poly(viologen)-ccNiR electrodes were also examined for their storage stability by recording the catalytic current response to nitrite (1 mM) once a day at the same time; in between, the bioelectrodes were stored at +4 °C in 0.05 M Tris–HCl buffer, pH 7.5. Fig. 3 shows the polymer electroactivity and catalytic current evolutions as a function of time. No loss of activity was observed after four days reflecting the absence of enzyme release as well as the stability of the polymeric coating. Afterward, the signal of the polymerized viologen decreased successively by 19%, 22% and 29% while the catalytic current dropped by 53%, 72% and 92% for five, six and seven days, respectively. Thus, it appears that the catalytic current (i.e., the enzyme activity) decreased in a faster way than the electroactivity of the
S. Da Silva et al. / Electrochemistry Communications 6 (2004) 404–408
Fig. 3. Time influence on the peak current of the one-electron reduction of the polymerized viologen groups (grey) in absence of nitrite and on the catalytic current response of the biosensor (black) in presence of 1 mM NaNO2 (currents are reported in absolute values); same electrochemical conditions than in Fig. 1.
407
monomer, the electropolymerization yield being 7%. In contrast, the evolution of the biosensor response depicted a bell shape, the maximum value being reached for 0.253 nmol/mm2 of deposited monomer. Keeping in mind that the immobilized amount of enzyme being constant, the ascendant part of the bell curve reflects an improvement of the electrical wiring of ccNiR due to the density increase of the electrogenerated Vþ sites surrounding the protein shell. Then, the decrease in biosensor response for increasing monomer amount may be more likely ascribed to the increase in film thickness. The latter, indeed, induced a large increase in diffusional constraints towards the permeation of nitrite and ammonium.
4. Conclusions viologen groups highlighting a continuous ccNiR denaturation, in spite of its hydrophobic feature. The influence of sulfites, known to have some reactivity with ccNiR [15], and nitrates which are frequently present in natural nitrite samples, as potential interferents on the biosensor sensitivity was examined. No appreciable change in the biosensor response was observed for nitrates (1 mM) while sulfites (1 mM) induced a very slight signal increase, namely 1.7%. A biosensor optimization study was also performed by varying the amount of monomer spread on the electrode surface, the amount of deposited ccNiR being constant (0.5 lg/mm2 ). After each electropolymerization, the enzymatic activity of electrolyte solutions was examined indicating a quasi absence of enzyme release (only 5%) for all the configurations. Fig. 4 presents the electroactivity of the redox polymer and the biosensor response to nitrite as a function of the amount of deposited monomer. As expected, the electroactivity of the polymer monotonously increased with the deposited amount of
ccNiR from D. desulfuricans ATCC 27774 was successfully immobilized and electrically connected on a glassy carbon electrode by co-adsorption and electropolymerization of an amphiphilic pyrrole–viologen derivative. Thanks to the specific properties of ccNiR, a catalytic current was observed in presence of nitrite ions in spite of the hydrophobic feature of the polymeric matrix. To our knowledge, this is the first time that a nitrite reductase was connected to a poly(pyrrole–viologen) to perform a third generation electrochemical biosensor. Indeed, all the previous attempts to construct nitrite reductase based electrochemical biosensors required the presence of mediator species for analyte determination. Sensor performances matched with those encountered in literature and even seem slightly better thanks to the good specific activity, high stability and hydrophobic feature of ccNiR. On the other hand, very few examples of bioelectrodes modified with a combination of functionalized redox polymers and reductases, exhibited such a fast enzyme response as exemplified by the electrocatalytic currents reported in this work.
Acknowledgements The authors thank the Commission of the European Communities Research Directorate for their support under the PEBCAT project contract No. EVK1-CT2000-00069, FSE and the Fundacß~ao para a Ci^encia e Tecnologia, through the PhD Grant No. PRAXIS XXI/ BD/11349/97 (MGA) Fig. 4. Electroactivity of the poly(pyrrole–viologen) ðsÞ and catalytic current to nitrite ðjÞ (1 mM) as a function of the amount of monomer deposited on the electrode surface (currents are reported in absolute values). The amount of electroactive polymerized monomer was determined by integration of the charge recorded under the reduction wave of V2þ=þ system in the absence of nitrite. Each point was calculated from at least three independent experiments. Same electrochemical conditions than in Fig. 1.
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