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Electrocatalysis with monolayer modified highly organized macroporous electrodes
Electrochemistry Communications 5 (2003) 747–751 www.elsevier.com/locate/elecom
Electrocatalysis with monolayer modified highly organized macroporous electrodes Samia Ben-Ali a, David A. Cook b, Stuart A.G. Evans b, Anne Thienpont a, Philip N. Bartlett b,*, Alexander Kuhn a,* a
Laboratoire d’Analyse Chimique par Reconnaissance Mol eculaire, Ecole Nationale Sup erieure de Chimie et de Physique de Bordeaux 16 avenue Pey Berland, 33607 Pessac, France b Department of Chemistry, University of Southampton, Southampton SO17 1BJ, UK Received 11 June 2003; received in revised form 4 July 2003; accepted 8 July 2003 Published online: 12 August 2003
Abstract For the first time the inner surface of highly organized macroporous electrodes, obtained by electrodeposition of gold into the interstitial spaces of a self-assembled close packed array of latex beads, is modified with a monolayer of a catalyst. The combination of the increased specific surface area of these electrodes and the high catalytic rate constant of the redox mediator allows us to construct surfaces showing good electrocatalytic efficiency. As a model system the electrooxidation of NADH using nitrofluorenone mediators is studied. Ó 2003 Elsevier B.V. All rights reserved. Keywords: Macroporous electrodes; Modified electrodes; Electrocatalysis; NADH oxidation; Biosensors
1. Introduction Electrode modification with catalytically active molecules has been a very active field of research for several decades and original ideas to tailor the molecular character of the electrode interface continue to appear each year [1,2]. Most of the fundamental studies have been performed on classic flat electrode surfaces. However, for some applications, such as in vivo measurements, it is important to use very small electrodes (microelectrodes) with the result that the measured currents are quite small due to the limited active surface area. For other applications where a large amount of substrate has to be converted in a short period of time, as in the case of fuel cells, quite large electrodes can be used but it would be beneficial if they had a very high active surface area. In both cases porous electrodes might form part of the solution to the problem. Plati*
Corresponding authors. Tel.: +33-5-40-00-65-73; fax: +33-5-40-0027-17 (Alexander Kuhn). E-mail address: [email protected] (A. Kuhn). 1388-2481/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1388-2481(03)00175-9
num black and carbon felt electrodes are examples of quite pragmatic and popular ways to enhance the active area of an electrode with a given macroscopic size, but in both cases it is difficult to control the pore size distribution and to fine-tune the physico-chemical properties. In this communication we show that the adsorption of a monolayer of highly active redox catalyst [3] into the pores of well-organized macroporous electrodes [4] allows the rational design of electrodes with a controllable electrocatalytic activity. Macroporous metal phases were first prepared by chemical routes [5,6], and more recently the electrodeposition route has been explored with success [7–9]. To the best of our knowledge the electrochemistry of this type of substrates with a highly regular structure, modified or unmodified, has not been reported. In order to test our idea we use the electrochemical oxidation of nicotinamide adenine dinucleotide (NADH) to NADþ because it provides an attractive model system and recently a new family of redox mediators based on a nitrofluorenone skeleton has been developed in our group [3]. These molecules form
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studying the adsorbed mediator and at the negative end of the potential range for catalysis experiments. 2.3. Procedures
Scheme 1. Molecular structure of the nitrofluorenone derivative used in this study ((4-carboxy-2,5,7-trinitro-9-fluorenylidene)malononitrile).
monolayers on various electrode materials like glassy carbon, gold or platinum [10] and can therefore be easily adapted to the different macroporous electrodes that have been obtained with metals like platinum, palladium, cobalt and gold [4,9], or even with conducting polymers [11]. We have chosen the adsorption of (4-carboxy-2,5,7trinitro-9-fluorenylidene)malononitrile (Scheme 1) on organized macroporous gold in order to demonstrate that the pores are modified with the mediator monolayer throughout the film and that the total inner surface is accessible for the catalytic NADH oxidation.
2.3.1. Preparation of the macroporous electrodes The working electrodes employed here were 0.5 mm diameter platinum discs sealed in glass with 1 cm diameter polished faces (0.05 lm alumina). The method for the self-assembly of the latex spheres consists basically of the slow evaporation of a small volume of dilute bead suspension (0.5–2 wt% in water) at the substrate surface. The templated samples are then put into the commercial gold plating bath (at 25 °C) (Technic Gold 25) 10 min prior to the reduction, in order to allow the solution to penetrate all the way throughout the template. Then a potential of )0.85 V vs. SCE is applied until the required charge has been passed. After carefully rinsing with water the electrodes are transferred into THF for 15 min to dissolve the latex spheres, leading to the desired regular macroporous structure with a thickness controllable by the charge used during the electrodeposition step. This thickness is expressed in terms of number of half-sphere layers (see Fig. 1).
2. Experimental 2.1. Reagents Latex bead solutions (500 nm diameter) were obtained from Duke scientific and gold-25 plating solution from Technic Inc. (4-carboxy-2,5,7-trinitro-9-fluorenylidene)malononitrile has been synthesized following the procedure reported in a former study [12]. Tris and calcium chloride dihydrate were purchased from Merck and Sigma, respectively, and used as received. Tris buffer was prepared by dissolving the required amount of compound and adjusting the pH to 8 by addition of HNO3 . The reduced form of b-nicotinamide adenine dinucleotide (NADH) was obtained as the disodium salt with 98% purity (Sigma). Solutions were prepared from ultra pure water that had been passed through a purification train (Milli-Q Plus 185, Millipore). 2.2. Apparatus Cyclic voltammetry (CV) was carried out in a conventional one compartment cell with an Autolab PGSTAT 30 potentiostat (Ecochemie) at ambient temperature (20 2 °C) in a solution that had been bubbled with nitrogen for at least 15 min. Potentials were measured with respect to a commercial Ag/AgCl (3 M KCl) reference electrode (BAS) and the counter electrode was a platinum wire. If not otherwise stated, scans were started at the positive end of the potential range for
Fig. 1. (A) Schematic drawing of the structure of a macroporous electrode with a total of 5/2 sphere layers. (B) Scanning electron micrograph (topview) of a macroporous gold film obtained by electrodeposition. White scale bar 1 lm.
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2.3.2. Preparation of a mediator monolayer A monolayer of (4-carboxy-2,5,7-trinitro-9-fluorenylidene)malononitrile was adsorbed on the macroporous gold surface by the following procedure. After cleaning and characterising the electrode surface by cycling in 0.5 M H2 SO4 a catalyst precursor layer is obtained by dipping the electrode for at least 2 h in a 5 mM solution of the nitro-compound in Tris buffer. This gives enough time for the solution to penetrate into the pores. Afterwards, the electrode is rinsed with ultrapure water leaving a layer of the organic molecule at the interface. Subsequently, the electrode is dipped into the pure Tris supporting electrolyte and the catalyst is activated by transforming two of the three nitro groups into hydroxylamine by choosing the adequate negative potential, )500 mV, during the first scan.
3. Results and discussion First of all we performed some basic experiments with the original electrode surfaces to make sure that all pores in such a device are electrochemically active. Fig. 2A shows that, despite the quite hydrophobic character of gold, an aqueous supporting electrolyte (0.5 M H2 SO4 ) seems to wet the whole inner surface of the electrode as the characteristic gold oxide reduction peak is increasing regularly when going from 1/2 to 3/2 and 5/2 sphere gold layers. Measurement of the charge under the stripping peak allows an estimation of the active surface area when assuming a specific charge of 386 lC/cm2 [13]: This gives areas of 0.25, 0.86 and 2.09 mm2 for the 1/2, 3/2 and 5/2 films, respectively. The first two values are in very good agreement with what would be expected from an increase of the number of half sphere layers by a factor of 3, whereas the 5/2 electrode shows a value, which slightly exceeds that expected for the specific surface area. The order of magnitude of these experimental results fits well to the theoretical value, obtained in a first order approximation for a hexagonal close packed half sphere layer with 96% packing density (0.35 mm2 ). One can therefore, conclude that the inner surface of the pores is rather smooth. In a second step it was interesting to see whether one could decorate the inner surface of the electrodes with a monolayer of a redox active molecule. In our particular model system the modification of these surfaces with the redox mediator reveals the same trend as the one observed for the gold oxide stripping peak. Fig. 2B has been obtained for the three types of electrodes when cycling in pure Tris supporting electrolyte after deposition of the nitrofluorenone layer. The peak is characteristic for the reversible two electron–two proton reaction of the R-NO/R-NHOH redox couple [3,14]. Actually it represents the convolution of two reacting
Fig. 2. (A) Cyclic voltammograms of (a) 1/2, (b) 3/2 and (c) 5/2 sphere layer gold electrodes in 0.5 M H2 SO4 at v ¼ 100 mV/s. (B) Cyclic voltammograms in 0.1 M Tris buffer pH 8 of the electrodes used in (A) after their modification with a monolayer of (4-carboxy-2,5,7-trinitro9-fluorenylidene)malononitrile (v ¼ 100 mV/s).
nitroso/hydroxylamine couples because in the very first scan we have chosen a potential sufficiently negative to activate two out of the three nitrogroups. The integration of this double peak, corresponding to the exchange of four electrons per molecule, leads to charges that again vary in good agreement with the number of halfsphere layers, 2.8 108 , 1.0 107 and 1.7 107 C, respectively. If one calculates the individual surface coverages by taking into account the four electrons per immobilized mediator, similar values are obtained in all three cases: 2.9 1011 (1/2, curve a), 3.0 1011 (3/2, curve b) and 2.1 1011 mol/cm2 (5/2, curve c). The order of magnitude of these values seems to indicate that actually a monolayer of the compound has been adsorbed. From these results we furthermore conclude that every pore, no matter its location compared to the outer electrode/ electrolyte interface, is accessible to the mediator
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solution and once the molecules are adsorbed they remain electrochemically addressable. This feature is due to the interconnection of the spherical voids by channels [11], allowing the soaking of the whole organized sponge-like structure with either a solution containing the organic molecule, or the supporting electrolyte. The adsorbed mediator layer is reasonably stable; after an initial loss during the first 20 potential cycles, the signal remains identical for more than 200 cycles. The remaining molecules are tightly attached to the gold surface through the nitrile functions as could be shown by reflectance IR-spectroscopy. In the next step the catalytic activity of the mediator covered electrodes has been tested with respect to the electrooxidation of NADH, an important cofactor for over 300 enzymes. Electrochemical oxidation of NADH needs a suitable redox mediator because direct oxidation at bare electrode surfaces often requires large overpotentials [15]. In the past different types of electrode materials have been modified with a variety of redox mediators and interesting results in terms of overpotential and rate constants have been obtained [16,17]. Fig. 3A illustrates the initial catalytic current obtained for a 1/2 sphere electrode in 3.2 mM NADH (curve b) compared to the background current (curve a). The oxidation of NADH occurs, as in the case of modified flat electrodes at a potential of )50 mV vs. Ag/AgCl [3], decreasing the overpotential by more than 500 mV compared to an unmodified surface. It is evident from Fig. 3B that the catalytic current, as well as the capacitive current observed for the three different electrodes, scales with the number of half-sphere layers. When comparing the 3/2 and the 5/2 to the 1/2 electrode in a more detailed analysis of the oxidation currents at a given potential of the cyclic voltammogram (E ¼ þ100 mV), a ratio of 3.4 and 6.8 is measured, respectively. This correlates with the theoretically expected increase and indicates not only that all the pores are electroactive but also that NADH can reach the whole inner surface by diffusion on the timescale of the experiment. Compared to a flat electrode of the same macroscopic size, the 5/2 sphere electrode thus allows an increase of the catalytic current by more than one order of magnitude. Another way to greatly enhance the catalytic current, which has been reported for several types of mediator modified electrode [18–23], is by addition of Ca2þ ions to the supporting electrolyte. In the present case a quite significant increase of the signal by more than 500% can be observed (Fig. 3A, curve c). We attribute this, as in the case of flat modified electrodes, to the temporary formation of a ternary complex between the carboxyl group of the mediator, the Ca2þ ions and the phosphate groups of the coenzyme [24]. This facilitates the charge transfer between both reactants due to a closer interaction, and in addition the partition coefficient of NADH between the more hydrophobic ‘‘pore phase’’ and the
Fig. 3. (A) Cyclic voltammogram of a mediator modified 1/2 sphere gold electrode in (a) pure 0.1 M Tris buffer pH 8, (b) in the presence of 3.2 mM NADH and (c) in the presence of 3.2 mM NADH and 0.2 M CaCl2 . (B) Comparison of the catalytic currents in the presence of 3.2 mM NADH for 1/2, 3/2 and 5/2 sphere electrodes (a, b and c, respectively). (C) As in (B) but in the presence of 3.2 mM NADH and 0.2 M CaCl2 . (v ¼ 10 mV/s for all voltamogramms)
outer side of the electrode might be enhanced analogous to what has been reported for conducting polymer modified surfaces [25]. Fig. 3C compares these calcium
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depositing more and more half-sphere layers and the reported findings might be generalized for many different kinds of redox catalysts. Work in this direction is in progress in our laboratories.
Acknowledgements The authors thank the French-British Alliance program (No 3058XB), the French Ministry of Research and the ENSCPB for financial support. D.A.C. thanks MediSense for a fully funded research studentship.
Fig. 4. Cyclic voltammogram of a mediator modified 5/2 sphere gold electrode dipping in 0.1 M Tris buffer pH 8 containing 15 mM NADþ , 0.2 M CaCl2 and 5 U/ml of GDH (thin line). After the addition of glucose (10 mM) a significant catalytic current is observed (thick line) (v ¼ 10 mV/s).
enhanced currents for all three electrodes and similar ratios as those obtained without Ca2þ are measured (3.5 and 6.4, respectively). As mentioned above the electrochemical reoxidation of NADH at low overpotentials is a very important topic in biosensor applications based on the use of enzymes belonging to the dehydrogenase family. We therefore had to make sure that the product of this electrochemical conversion of NADH is enzymatically active NADþ and Fig. 4 illustrates this point. The thin line corresponds to the background current of a 5/2 sphere electrode modified with mediator and dipping in a solution containing coenzyme, Ca2þ and glucose dehydrogenase. Upon addition of glucose a pronounced electrocatalytic current is recorded corresponding to the reoxidation of NADH formed during the enzymatic reaction (Fig. 4, thick line).
4. Conclusion We have demonstrated that macroporous gold electrodes can be modified over their entire inner surface with a redox mediator belonging to the nitrofluorenone family. The number of adsorbed molecules increases linearly with the number of pores and the nitrofluorenone derivative acts as a very efficient mediator for the electrocatalytic oxidation of NADH freely diffusing from the outside into the pores. The catalytic currents can be enhanced further by adding Ca2þ ions to the solution and the produced NADþ seems to be enzymatically active. The performance of this type of modified electrodes could be improved in a regular way by
References [1] R.W. Murray (Ed.), Molecular design of electrode surfaces, Techniques of Chemistry, vol. 22, Wiley, New York, 1992. [2] J.L. Anderson, L.A. Coury, J. Leddy, Anal. Chem. 72 (2000) 4497. [3] N. Mano, A. Kuhn, J. Electroanal. Chem. 477 (1999) 79. [4] P.N. Bartlett, J.J. Baumberg, P.R. Birkin, M.A. Ghanem, M.C. Nettl, Chem. Mater. 14 (2002) 2199. [5] P. Jiang, J. Cizeron, J.F. Bertone, V.L. Colvin, J. Am. Chem. Soc. 121 (1999) 7957. [6] O.D. Velev, P.M. Tessier, A.M. Lenhoff, E.W. Kaler, Nature 401 (1999) 548. [7] J.E.G.J. Wijnhoven, S.J.M. Zevenhuizen, M.A. Hendriks, D. Vanmaekelbergh, J.J. Kelly, W.L. Vos, Adv. Mater. 12 (2000) 888. [8] L. Xu, W.L. Zhou, C. Frommen, R.H. Baughman, A.A. Zakhidov, L. Malkinski, J.Q. Wang, J.B. Wiley, J. Chem. Soc. Chem. Commun. (2000) 997. [9] P. Bartlett, P. Birkin, M. Ghanem, Chem. Commun. (2000) 1671. [10] N. Mano, A. Thienpont, A. Kuhn, Electrochem. Commun. 3 (2001) 585. [11] P.N. Bartlett, P. Birkin, M.A. Ghanem, C.-S. Toh, J. Mater. Chem. 11 (2001) 849. [12] N. Mano, PhD Thesis, University of Bordeaux (2001). [13] R. Woods, in: A.J. Bard (Ed.), Electroanalytical Chemistry: A Series of Advances, vol. 9, Marcel Dekker, New York, 1980. [14] A. Darchen, C. Moinet, J. Electroanal. Chem. 78 (1977) 81. [15] E. Steckhan, Top. Curr. Chem. 170 (1994) 84. [16] I. Katakis, E. Dominguez, Mikrochim. Acta 126 (1997) 11. [17] L. Gorton, E. Dominguez, in: G.S. Wilson (Ed.), Encyclopedia of Electrochemistry, Bioelectrochemistry, vol. 9, Wiley, Europe, 2002. [18] E. Katz, T. Lotzbeyer, D.D. Schlereth, W. Schuhmann, H.L. Schmidt, J. Electroanal. Chem. 373 (1994) 189. [19] F. Tobalina, F. Pariente, L. Hernandez, H.D. Abruna, E. Lorenzo, Anal. Chim. Acta 358 (1998) 15. [20] F. Pariente, F. Tobalina, G. Moreno, L. Hernandez, E. Lorenzo, H.D. Abruna, Anal. Chem. 69 (1997) 4065. [21] C.X. Cai, L.H. Yin, K.H. Xue, J. Mol. Catal. A 152 (2000) 179. [22] L.T. Kubota, L. Gorton, J. Solid State Electrochem. 3 (1999) 370. [23] N. Mano, A. Kuhn, J. Electroanal. Chem. 498 (2001) 58. [24] N. Mano, A. Kuhn, S. Menu, E.J. Dufourc, Phys. Chem. Chem. Phys. 5 (2003) 2082. [25] C.S. Toh, P.N. Bartlett, N. Mano, F. Aussenac, A. Kuhn, E.J. Dufourc, Phys. Chem. Chem. Phys. 5 (2003) 588.
Electrocatalysis with monolayer modified highly organized macroporous electrodes Samia Ben-Ali a, David A. Cook b, Stuart A.G. Evans b, Anne Thienpont a, Philip N. Bartlett b,*, Alexander Kuhn a,* a
Laboratoire d’Analyse Chimique par Reconnaissance Mol eculaire, Ecole Nationale Sup erieure de Chimie et de Physique de Bordeaux 16 avenue Pey Berland, 33607 Pessac, France b Department of Chemistry, University of Southampton, Southampton SO17 1BJ, UK Received 11 June 2003; received in revised form 4 July 2003; accepted 8 July 2003 Published online: 12 August 2003
Abstract For the first time the inner surface of highly organized macroporous electrodes, obtained by electrodeposition of gold into the interstitial spaces of a self-assembled close packed array of latex beads, is modified with a monolayer of a catalyst. The combination of the increased specific surface area of these electrodes and the high catalytic rate constant of the redox mediator allows us to construct surfaces showing good electrocatalytic efficiency. As a model system the electrooxidation of NADH using nitrofluorenone mediators is studied. Ó 2003 Elsevier B.V. All rights reserved. Keywords: Macroporous electrodes; Modified electrodes; Electrocatalysis; NADH oxidation; Biosensors
1. Introduction Electrode modification with catalytically active molecules has been a very active field of research for several decades and original ideas to tailor the molecular character of the electrode interface continue to appear each year [1,2]. Most of the fundamental studies have been performed on classic flat electrode surfaces. However, for some applications, such as in vivo measurements, it is important to use very small electrodes (microelectrodes) with the result that the measured currents are quite small due to the limited active surface area. For other applications where a large amount of substrate has to be converted in a short period of time, as in the case of fuel cells, quite large electrodes can be used but it would be beneficial if they had a very high active surface area. In both cases porous electrodes might form part of the solution to the problem. Plati*
Corresponding authors. Tel.: +33-5-40-00-65-73; fax: +33-5-40-0027-17 (Alexander Kuhn). E-mail address: [email protected] (A. Kuhn). 1388-2481/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1388-2481(03)00175-9
num black and carbon felt electrodes are examples of quite pragmatic and popular ways to enhance the active area of an electrode with a given macroscopic size, but in both cases it is difficult to control the pore size distribution and to fine-tune the physico-chemical properties. In this communication we show that the adsorption of a monolayer of highly active redox catalyst [3] into the pores of well-organized macroporous electrodes [4] allows the rational design of electrodes with a controllable electrocatalytic activity. Macroporous metal phases were first prepared by chemical routes [5,6], and more recently the electrodeposition route has been explored with success [7–9]. To the best of our knowledge the electrochemistry of this type of substrates with a highly regular structure, modified or unmodified, has not been reported. In order to test our idea we use the electrochemical oxidation of nicotinamide adenine dinucleotide (NADH) to NADþ because it provides an attractive model system and recently a new family of redox mediators based on a nitrofluorenone skeleton has been developed in our group [3]. These molecules form
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studying the adsorbed mediator and at the negative end of the potential range for catalysis experiments. 2.3. Procedures
Scheme 1. Molecular structure of the nitrofluorenone derivative used in this study ((4-carboxy-2,5,7-trinitro-9-fluorenylidene)malononitrile).
monolayers on various electrode materials like glassy carbon, gold or platinum [10] and can therefore be easily adapted to the different macroporous electrodes that have been obtained with metals like platinum, palladium, cobalt and gold [4,9], or even with conducting polymers [11]. We have chosen the adsorption of (4-carboxy-2,5,7trinitro-9-fluorenylidene)malononitrile (Scheme 1) on organized macroporous gold in order to demonstrate that the pores are modified with the mediator monolayer throughout the film and that the total inner surface is accessible for the catalytic NADH oxidation.
2.3.1. Preparation of the macroporous electrodes The working electrodes employed here were 0.5 mm diameter platinum discs sealed in glass with 1 cm diameter polished faces (0.05 lm alumina). The method for the self-assembly of the latex spheres consists basically of the slow evaporation of a small volume of dilute bead suspension (0.5–2 wt% in water) at the substrate surface. The templated samples are then put into the commercial gold plating bath (at 25 °C) (Technic Gold 25) 10 min prior to the reduction, in order to allow the solution to penetrate all the way throughout the template. Then a potential of )0.85 V vs. SCE is applied until the required charge has been passed. After carefully rinsing with water the electrodes are transferred into THF for 15 min to dissolve the latex spheres, leading to the desired regular macroporous structure with a thickness controllable by the charge used during the electrodeposition step. This thickness is expressed in terms of number of half-sphere layers (see Fig. 1).
2. Experimental 2.1. Reagents Latex bead solutions (500 nm diameter) were obtained from Duke scientific and gold-25 plating solution from Technic Inc. (4-carboxy-2,5,7-trinitro-9-fluorenylidene)malononitrile has been synthesized following the procedure reported in a former study [12]. Tris and calcium chloride dihydrate were purchased from Merck and Sigma, respectively, and used as received. Tris buffer was prepared by dissolving the required amount of compound and adjusting the pH to 8 by addition of HNO3 . The reduced form of b-nicotinamide adenine dinucleotide (NADH) was obtained as the disodium salt with 98% purity (Sigma). Solutions were prepared from ultra pure water that had been passed through a purification train (Milli-Q Plus 185, Millipore). 2.2. Apparatus Cyclic voltammetry (CV) was carried out in a conventional one compartment cell with an Autolab PGSTAT 30 potentiostat (Ecochemie) at ambient temperature (20 2 °C) in a solution that had been bubbled with nitrogen for at least 15 min. Potentials were measured with respect to a commercial Ag/AgCl (3 M KCl) reference electrode (BAS) and the counter electrode was a platinum wire. If not otherwise stated, scans were started at the positive end of the potential range for
Fig. 1. (A) Schematic drawing of the structure of a macroporous electrode with a total of 5/2 sphere layers. (B) Scanning electron micrograph (topview) of a macroporous gold film obtained by electrodeposition. White scale bar 1 lm.
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2.3.2. Preparation of a mediator monolayer A monolayer of (4-carboxy-2,5,7-trinitro-9-fluorenylidene)malononitrile was adsorbed on the macroporous gold surface by the following procedure. After cleaning and characterising the electrode surface by cycling in 0.5 M H2 SO4 a catalyst precursor layer is obtained by dipping the electrode for at least 2 h in a 5 mM solution of the nitro-compound in Tris buffer. This gives enough time for the solution to penetrate into the pores. Afterwards, the electrode is rinsed with ultrapure water leaving a layer of the organic molecule at the interface. Subsequently, the electrode is dipped into the pure Tris supporting electrolyte and the catalyst is activated by transforming two of the three nitro groups into hydroxylamine by choosing the adequate negative potential, )500 mV, during the first scan.
3. Results and discussion First of all we performed some basic experiments with the original electrode surfaces to make sure that all pores in such a device are electrochemically active. Fig. 2A shows that, despite the quite hydrophobic character of gold, an aqueous supporting electrolyte (0.5 M H2 SO4 ) seems to wet the whole inner surface of the electrode as the characteristic gold oxide reduction peak is increasing regularly when going from 1/2 to 3/2 and 5/2 sphere gold layers. Measurement of the charge under the stripping peak allows an estimation of the active surface area when assuming a specific charge of 386 lC/cm2 [13]: This gives areas of 0.25, 0.86 and 2.09 mm2 for the 1/2, 3/2 and 5/2 films, respectively. The first two values are in very good agreement with what would be expected from an increase of the number of half sphere layers by a factor of 3, whereas the 5/2 electrode shows a value, which slightly exceeds that expected for the specific surface area. The order of magnitude of these experimental results fits well to the theoretical value, obtained in a first order approximation for a hexagonal close packed half sphere layer with 96% packing density (0.35 mm2 ). One can therefore, conclude that the inner surface of the pores is rather smooth. In a second step it was interesting to see whether one could decorate the inner surface of the electrodes with a monolayer of a redox active molecule. In our particular model system the modification of these surfaces with the redox mediator reveals the same trend as the one observed for the gold oxide stripping peak. Fig. 2B has been obtained for the three types of electrodes when cycling in pure Tris supporting electrolyte after deposition of the nitrofluorenone layer. The peak is characteristic for the reversible two electron–two proton reaction of the R-NO/R-NHOH redox couple [3,14]. Actually it represents the convolution of two reacting
Fig. 2. (A) Cyclic voltammograms of (a) 1/2, (b) 3/2 and (c) 5/2 sphere layer gold electrodes in 0.5 M H2 SO4 at v ¼ 100 mV/s. (B) Cyclic voltammograms in 0.1 M Tris buffer pH 8 of the electrodes used in (A) after their modification with a monolayer of (4-carboxy-2,5,7-trinitro9-fluorenylidene)malononitrile (v ¼ 100 mV/s).
nitroso/hydroxylamine couples because in the very first scan we have chosen a potential sufficiently negative to activate two out of the three nitrogroups. The integration of this double peak, corresponding to the exchange of four electrons per molecule, leads to charges that again vary in good agreement with the number of halfsphere layers, 2.8 108 , 1.0 107 and 1.7 107 C, respectively. If one calculates the individual surface coverages by taking into account the four electrons per immobilized mediator, similar values are obtained in all three cases: 2.9 1011 (1/2, curve a), 3.0 1011 (3/2, curve b) and 2.1 1011 mol/cm2 (5/2, curve c). The order of magnitude of these values seems to indicate that actually a monolayer of the compound has been adsorbed. From these results we furthermore conclude that every pore, no matter its location compared to the outer electrode/ electrolyte interface, is accessible to the mediator
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solution and once the molecules are adsorbed they remain electrochemically addressable. This feature is due to the interconnection of the spherical voids by channels [11], allowing the soaking of the whole organized sponge-like structure with either a solution containing the organic molecule, or the supporting electrolyte. The adsorbed mediator layer is reasonably stable; after an initial loss during the first 20 potential cycles, the signal remains identical for more than 200 cycles. The remaining molecules are tightly attached to the gold surface through the nitrile functions as could be shown by reflectance IR-spectroscopy. In the next step the catalytic activity of the mediator covered electrodes has been tested with respect to the electrooxidation of NADH, an important cofactor for over 300 enzymes. Electrochemical oxidation of NADH needs a suitable redox mediator because direct oxidation at bare electrode surfaces often requires large overpotentials [15]. In the past different types of electrode materials have been modified with a variety of redox mediators and interesting results in terms of overpotential and rate constants have been obtained [16,17]. Fig. 3A illustrates the initial catalytic current obtained for a 1/2 sphere electrode in 3.2 mM NADH (curve b) compared to the background current (curve a). The oxidation of NADH occurs, as in the case of modified flat electrodes at a potential of )50 mV vs. Ag/AgCl [3], decreasing the overpotential by more than 500 mV compared to an unmodified surface. It is evident from Fig. 3B that the catalytic current, as well as the capacitive current observed for the three different electrodes, scales with the number of half-sphere layers. When comparing the 3/2 and the 5/2 to the 1/2 electrode in a more detailed analysis of the oxidation currents at a given potential of the cyclic voltammogram (E ¼ þ100 mV), a ratio of 3.4 and 6.8 is measured, respectively. This correlates with the theoretically expected increase and indicates not only that all the pores are electroactive but also that NADH can reach the whole inner surface by diffusion on the timescale of the experiment. Compared to a flat electrode of the same macroscopic size, the 5/2 sphere electrode thus allows an increase of the catalytic current by more than one order of magnitude. Another way to greatly enhance the catalytic current, which has been reported for several types of mediator modified electrode [18–23], is by addition of Ca2þ ions to the supporting electrolyte. In the present case a quite significant increase of the signal by more than 500% can be observed (Fig. 3A, curve c). We attribute this, as in the case of flat modified electrodes, to the temporary formation of a ternary complex between the carboxyl group of the mediator, the Ca2þ ions and the phosphate groups of the coenzyme [24]. This facilitates the charge transfer between both reactants due to a closer interaction, and in addition the partition coefficient of NADH between the more hydrophobic ‘‘pore phase’’ and the
Fig. 3. (A) Cyclic voltammogram of a mediator modified 1/2 sphere gold electrode in (a) pure 0.1 M Tris buffer pH 8, (b) in the presence of 3.2 mM NADH and (c) in the presence of 3.2 mM NADH and 0.2 M CaCl2 . (B) Comparison of the catalytic currents in the presence of 3.2 mM NADH for 1/2, 3/2 and 5/2 sphere electrodes (a, b and c, respectively). (C) As in (B) but in the presence of 3.2 mM NADH and 0.2 M CaCl2 . (v ¼ 10 mV/s for all voltamogramms)
outer side of the electrode might be enhanced analogous to what has been reported for conducting polymer modified surfaces [25]. Fig. 3C compares these calcium
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depositing more and more half-sphere layers and the reported findings might be generalized for many different kinds of redox catalysts. Work in this direction is in progress in our laboratories.
Acknowledgements The authors thank the French-British Alliance program (No 3058XB), the French Ministry of Research and the ENSCPB for financial support. D.A.C. thanks MediSense for a fully funded research studentship.
Fig. 4. Cyclic voltammogram of a mediator modified 5/2 sphere gold electrode dipping in 0.1 M Tris buffer pH 8 containing 15 mM NADþ , 0.2 M CaCl2 and 5 U/ml of GDH (thin line). After the addition of glucose (10 mM) a significant catalytic current is observed (thick line) (v ¼ 10 mV/s).
enhanced currents for all three electrodes and similar ratios as those obtained without Ca2þ are measured (3.5 and 6.4, respectively). As mentioned above the electrochemical reoxidation of NADH at low overpotentials is a very important topic in biosensor applications based on the use of enzymes belonging to the dehydrogenase family. We therefore had to make sure that the product of this electrochemical conversion of NADH is enzymatically active NADþ and Fig. 4 illustrates this point. The thin line corresponds to the background current of a 5/2 sphere electrode modified with mediator and dipping in a solution containing coenzyme, Ca2þ and glucose dehydrogenase. Upon addition of glucose a pronounced electrocatalytic current is recorded corresponding to the reoxidation of NADH formed during the enzymatic reaction (Fig. 4, thick line).
4. Conclusion We have demonstrated that macroporous gold electrodes can be modified over their entire inner surface with a redox mediator belonging to the nitrofluorenone family. The number of adsorbed molecules increases linearly with the number of pores and the nitrofluorenone derivative acts as a very efficient mediator for the electrocatalytic oxidation of NADH freely diffusing from the outside into the pores. The catalytic currents can be enhanced further by adding Ca2þ ions to the solution and the produced NADþ seems to be enzymatically active. The performance of this type of modified electrodes could be improved in a regular way by
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