Designing Stable Redox-Active Surfaces: Chemical Attachment of an

May 13, 2008 - The production of stable redox-active layers on electrode surfaces can lead to improvements in electronic device design. Enhanced stabi...
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Langmuir 2008, 24, 6351-6358

6351

Designing Stable Redox-Active Surfaces: Chemical Attachment of an Osmium Complex to Glassy Carbon Electrodes Prefunctionalized by Electrochemical Reduction of an In Situ-Generated Aryldiazonium Cation Susan Boland,† Fre´de´ric Barrie`re,‡ and Do´nal Leech*,† School of Chemistry, National UniVersity of Ireland, Galway, Ireland, and UMR CNRS No. 6226, Sciences Chimiques de Rennes, Equipe MACSE, UniVersite´ de Rennes 1, France ReceiVed October 15, 2007. ReVised Manuscript ReceiVed February 25, 2008 The production of stable redox-active layers on electrode surfaces can lead to improvements in electronic device design. Enhanced stability can be achieved by pretreatment of electrode surfaces to provide surface chemical functional groups for covalent tethering of redox complexes. Herein, we describe pretreatment of glassy carbon electrodes to provide surface carboxylic acid groups by electro-reduction of an in situ-generated aryl diazonium salt from 3-(4aminophenyl)propionic acid. This surface layer is characterized by attenuated total reflection infrared spectroscopy, atomic force microscopy, and electrochemical blocking studies. The surface carboxylic acid generated is then used to tether an osmium complex, [Os(2,2′-bipyridyl)2(4-aminomethylpyridine)Cl]PF6, to provide a covalently bound redox-active monolayer, E0′ of 0.29 V (vs Ag/AgCl in phosphate buffer, pH 7.4), on the pretreated glassy carbon electrode. The layer proves stable to pH, temperature, and storage conditions, retaining electroactivity for at least 6 months.

Introduction There has been a heightened interest lately in modifying the surfaces of conducting materials with specific chemical functionalities, by the electrochemical reduction of aryl diazonium salts.1–10 Reduction of these salts on carbon-based electrodes leads to elimination of dinitrogen and the formation of a strong C-C bond with the surface. This surface derivatization can thus be used to introduce a terminal functional group such as alkyl, nitro, alcohol, ester, or carboxyl groups5 to various surfaces, to which redox-active molecules can be attached11 and lead to potential applications in chemical sensing,12 biosensing,13 molecular electronics,14 and corrosion protection,15 to name a few. Up until now, the main method to introduce chemical functional groups on conducting substrates had been to use thiol chemistry on gold, forming well-ordered monolayers with relative ease.16 However, there are several drawbacks to using thiol * Corresponding author. † ‡

National University of Ireland. Universite´ de Rennes 1.

(1) Downard, A. J. Langmuir 2000, 16, 9680–9682. (2) Downard, A. J. Electroanal. 2000, 12, 1085–1096. (3) Downard, A. J.; Roddick, A. D. Electroanalysis 1995, 7, 376–378. (4) Delamar, M.; Desarmot, G.; Fagebaume, O.; Hitmi, R.; Pinson, J.; Saveant, J. M. Carbon 1997, 35, 801–807. (5) Allongue, P.; Delamar, M.; Desbat, B.; Fagebaume, O.; Hitmi, R.; Pinson, J.; Saveant, J. M. J. Am. Chem. Soc. 1997, 119, 201–207. (6) Kariuki, J. K.; McDermott, M. T. Langmuir 1999, 15, 6534–6540. (7) Kariuki, J. K.; McDermott, M. T. Langmuir 2001, 17, 5947–5951. (8) Liu, Y.-C.; McCreery, R. L. Anal. Chem. 1997, 69, 2091–2097. (9) Anariba, F.; DuVall, S. H.; McCreery, R. L. Anal. Chem. 2003, 75, 3837– 3844. (10) Itoh, T.; McCreery, R. L. J. Am. Chem. Soc. 2002, 124, 10894–10902. (11) Ricci, A.; Rolli, C.; Rothacher, S.; Baraldo, L.; Bonazzola, C.; Calvo, E.; Tongalli, N.; Fainstein, A. J. Solid State Electrochem. 2007, 11, 1511–1520. (12) Dubois, L.; Viel, P.; Bureau, C.; Palacin, S. J. Am. Chem. Soc. 2004, 126, 12194–12195. (13) Bath, B. D.; Martin, H. B.; Wightman, R. M.; Anderson, M. R. Langmuir 2001, 17, 7032–7039. (14) Cahen, D.; Hodes, G. AdV. Mater. 2002, 14, 789–798. (15) Viel, P.; Bureau, C.; Deniau, G.; Zalczer, G.; Lecayon, G. J. Electroanal. Chem. 1999, 470, 14–22. (16) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481–4483.

chemistry, particularly for subsequent attachment of redox-active molecules. Well-ordered, stable, redox-active monolayers can be prepared by functionalization of long-chain alkanethiols chemisorbed on gold. The use of such long-chain alkanethiols, however, results in a sharp decrease in the rate of electron transfer between the tethered redox-active molecule and the underlying electrode surface, thereby decreasing the electrochemical signal. In addition, alkanethiols can be desorbed from the electrode surface by means of oxidation or reduction or the use of high temperatures. Monolayer mobility due to the weakness of the gold-thiol, and the high mobility of the gold surface,17 are also a disadvantage for any possible applications, particularly when using short-chain alkanethiols. Redox-active monolayers of osmium complexes can be formed by self-assembly of the pyridine moiety of a dipyridyl ligand of the complex on platinum electrodes, providing well-ordered and relatively stable layers.18,19 The stability of these chemisorbed systems is less well studied than the thiol-gold system, but reports of a 10% decrease in surface coverage of the layers over a 15 min period19 coupled to the reliance on the nitrogen lonepair interaction for self-assembly would seem to indicate longterm instability of this layer. The electrochemical reduction of aryl diazonium salts is proven to be an excellent method to irreversibly attach molecules to carbon,2 semiconductors,14 metals,20 and silicon.21 Kariuki and McDermott6 have reported on the nucleation and growth of aryl diazonium-derived film in both two and three dimensions on carbon surfaces. This increase in the functionalized conducting surface area has advantages over other systems, mainly, the ability to covalently and stably attach molecules of interest in quantities that can exceed monolayer coverage of the electrode geometric (17) Ranganthan, S.; Steidal, I.; Anariba, F.; McCreery, R. L. Nano Lett. 2001, 1, 491–494. (18) Forster, R. J.; Faulkner, L. R. J. Am. Chem. Soc. 1994, 116, 5444–5452. (19) Acevedo, D.; Abruna, H. D. J. Phys. Chem. 1991, 95, 9590–95941. (20) Hurley, B. L.; McCreery, R. L. J. Electrochem. Soc. 2004, 151, 252–259. (21) Allongue, P.; de Villeneuve, C. H.; Cherouvier, G.; Cortes, R.; Bernard, M. C. J. Electroanal. Chem. 2003, 500, 161–174.

10.1021/la7031972 CCC: $40.75  2008 American Chemical Society Published on Web 05/13/2008

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area. The generation of a new array of hybrid materials based upon the incorporation of transition metal complexes into these diazonium systems is a rapidly expanding area of research. For example, Liu et al.22 have prepared gold and glassy carbon (GC) electrodes by reduction of a carboxylated aryl diazonium salt, andsubsequentlyattached,throughamidelinkage,ferrocenemethylamine to the surfaces. The previously reported slow decomposition of the oxidized ferrocenyl group may, however, impart instability to the redox activity of ferrocene-modified monolayers.23,24 Redox layers consisting of immobilized complexes of osmium may have advantages over iron and ruthenium-based systems, because of the favorable redox potential of the Os(II/III) transition and the relative stability of complexes in the Os(III) oxidation state.25 Danilowicz et al.25 first reported on the synthesis of osmium complexes with ligands bearing free aldehyde and carboxylic acid functional groups as building blocks for integrated chemical systems. In their studies, the complexes were covalently attached to a poly(allylamine) polymer that was subsequently coimmobilized with glucose oxidase, by adsorption and diepoxide crosslinking, on GC electrodes, resulting in a glucose sensing system. However, over time, the expected desorption of the polymer is observed, and after one day only 70% of the activity remains. Enhanced stability of osmium-based redox polymers on electrode surfaces may be achieved by tethering the film to a functionalized electrode surface, such as a self-assembled layer of cysteamine on gold.26 Recently, Ricci et al.11 prepared redox-active layers consisting of osmium complexes with ligands bearing functional groups (amine, aldehyde) that can be tethered to gold electrodes pretreated to introduce surface functional groups via thiol or diazonium salt chemistry. However, no information on the stability of these layers was provided. Stability, reproducibility, and robustness of modified electrodes are key factors for any potential future applications. Here we report on carbon surfaces modified using electrochemical reduction of in situ-generated aryl diazonium salt, and their characterization by atomic force microscopy (AFM), attenuated total reflection infrared spectroscopy (ATR-IR), and ability to block solution redox electrochemistry.22,27 We also report on the synthesis and characterization of an osmium complex with a 4-(aminomethyl)pyridine (4-AMP) ligand, resulting in a complex that has properties suitable for both redox catalysis and immobilization chemistry. The electrochemical behavior of this complex covalently attached to a GC electrode that is prefunctionalized by reduction of an aryl diazonium salt generated in situ from 3-(4-aminophenyl)propionic acid is reported, with the subsequent layer proving to be extremely durable.

Experimental Materials and Reagents. All chemicals were, unless otherwise stated, purchased from Sigma-Aldrich and used as received. All solutions were made from Milli-Q (18.2 MΩ cm) water unless otherwise stated. For electrochemistry, Teflon-shrouded GC electrodes (3 mm diameter) were used in conjunction with a platinum wire counter electrode and either a Ag/AgCl (3 M KCl) or a Ag/ AgNO3 reference electrode (0.1 M AgNO3, 0.1 M LiClO4 in acetonitrile) for aqueous or nonaqueous electrochemistry, respectively (all sourced from IJ Cambria). (22) Liu, G.; Liu, J.; Bocking, T.; Eggers, P. K.; Gooding, J. J. Chem. Phys. 2005, 319, 136–146. (23) Ju, H.; Leech, D. Langmuir 1998, 14, 300–306. (24) Zhang, L.; Godınez, L. A.; Lu, T.; Gokel, G. W.; Kaifer, A. E. Angew. Chem., Int. Ed. Engl. 1995, 34, 235. (25) Danilowicz, C.; Corton, E.; Battaglini, F. J. Electroanal. Chem. 1998, 445, 89–94. (26) Kavanagh, P.; Leech, D. Anal. Chem. 2006, 78, 2710–2716. (27) Kuo, T. C.; McCreery, R. L. Anal. Chem. 1999, 71, 1553–1560.

Boland et al. cis-Os(2,2′-bipyridyl)2Cl2 was synthesized from (NH4)2OsCl6 according to literature methods.28 Ligand substitution of a chloride in this complex by 4-AMP was achieved according to literature methods28 by heating an ethylene glycol solution of the ligand and complex at reflux, with precipitation of the resulting [Os(2,2′bipyridyl)2(4-AMP)Cl]PF6 by addition of an aqueous NH4PF6 solution. Calculated for OsC26H24N6ClPF6: C, 39.47%; H, 3.06%; N, 10.62%. Found: C, 39.86%; H, 3.25%; N, 10.23%. Apparatus. The electrochemical measurements were performed using an Autolab, EcoChemie, PGSTAT12 potentiostat (Eco Chemie BV, The Netherlands). All experiments were carried out with a three-electrode system at room temperature, unless otherwise stated. All solutions were deaerated with argon for 15 min prior to use. AFM measurements were carried out using a Veeco Digital Instruments Dimension 3100 system. Veeco AFM antimony doped silicon tips were engaged in tapping mode. Images were captured with scan sizes of 1.00, 2.50, and 5.00 µm at a scan rate of 1.001 Hz, taking 256 samples in the defined area. The Z scale is 250 nm, and the surface roughness values were evaluated from the scanned surface topography. The IR spectra of the various samples were collected in the 650-4000 cm-1 range using a Perkin-Elmer Universal ATR-Sampling system. Methods. Prior to modification, GC electrodes were cleaned in a piranha solution (75% H2SO4/25% H2O2, CAUTION) for 2 h, polished with 1 µm of alumina slurry on microcloth pads (Buehler) followed by thorough rinsing with Milli-Q water, sonication in Milli-Q water for 15 min, and subsequent drying with an argon gas stream. Modification of the GC electrodes by introduction of carboxylic acid functional groups was achieved by electrochemical reduction of the diazonium cation generated in situ from the 3-(4-aminophenyl)propionic acid.29 Briefly, 8 mM of NaNO2 was added into a 10 mM acidic solution (0.5 M HCl) of 3-(4-aminophenyl)propionic acid to generate the diazonium cation. The solution was kept in complete darkness and in an ice bath and allowed to react for 5 min under argon and stirring. Surface derivatization was carried out by electrochemical reduction, in the diazonium cation-generating solution, by scanning from 0.6 V to -0.4 V vs Ag/AgCl at 20 mV/s for four cycles. The resulting modified electrodes were removed and rinsed with large volumes of acetonitrile and then water, followed by ultrasonication for 1 min to remove any loosely bound species. Electrodes were then rinsed again with copious amounts of water and dried under a stream of argon. Identical procedures were adopted for introduction of carboxylic acid functional groups to GC rod (Goodfellow) substrates for AFM imaging, and on gold substrates (Evaporated Metal Film, Inc., Ithaca, NY), for ATR-IR. The derivatized surfaces contain carboxylic acid functional groups that can provide an anchor for further coupling chemistry. In order to estimate the number of free carboxylic acid functional groups available at the GC-modified electrode, a method of coupling the carboxylic acid to a diamine, ethylenediamine, using benzotriazol1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) as a reagent, similar to that of Castro et al.30 and Felix and Bandaranayake31 was employed. The formation of the subsequent surface-bound amine can then be easily monitored with the colorimetric Kaiser test32,33 by measuring, at 570 nm, the absorbance of a solution into which the electrode is dipped, using an extinction coefficient of 1.5 × 104 M-1 cm-1 for the chromophore. This yields a quantitative estimate of the amine functional groups and thus an estimation of the carboxylic acid functional groups. The BOP coupling procedure is as follows: the modified electrode was placed in a cell with 20 mL of acetonitrile and 3 mM of ethylenediamine. Addition (28) Kober, E. M.; Caspar, J. V.; Sullivan, B. P.; Meyer, T. J. Inorg. Chem. 1988, 27, 4587–4598. (29) Baranton, S.; Belanger, D. J, Phys. Chem. B 2005, 109, 24401–24410. (30) Castro, B.; Dormoy, J. R.; Evin, J. G.; Selve, C. Tetrahedron Lett. 1975, 14, 1219–1222. (31) Felix, A. M.; Bandaranayake, R. M. J. Pept. Res. 2005, 65, 71–76. (32) Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal. Biochem. 1981, 117, 147–157. (33) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem. 1970, 34, 595–598.

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Scheme 1. Proposed Reaction Scheme for the In Situ Generation of an Aryl Diazonium Cation Followed by Electrochemical Reduction of the Generated Aryl Diazonium Cation to Effect Carbon-Carbon Coupling

of 1.5 mM BOP with stirring at room temperature was followed by the addition of triethylamine (5 mM). The reaction was stirred for 30 min, after which 5 mL of a saturated NaCl solution was added to quench the reaction. Covalent attachment of the [Os(bpy)2(4-AMP)Cl]PF6 complex to the carboxylic acid-terminated GC modified electrode was carried out by following the procedures of Liu et al.34 Briefly, the modified surfaces were incubated with aqueous solutions of 10 mM Nhydroxysuccimide (NHS) and 40 mM 1-ethyl-3(3-diethylaminopropyl)carbodiimide hydrochloride (EDC) for 1 h. After the activation, the electrodes were rinsed with water and incubated in a solution of 5 mM [Os(bpy)2(4-AMP)Cl]PF6 in phosphate buffer (pH 6) for 24 h.

Results and Discussion Introduction of Carboxylic Acid Functional Groups at Carbon Surfaces. Aryl-diazonium salts were generated in situ in acidic solutions containing sodium nitrite and an appropriate aryl-amine, which in this case is 3-(4-aminophenyl)propionic acid.29 Electrochemical reduction of the subsequent aryldiazonium salt on GC electrodes generates aryl radicals that can couple, through carbon-carbon bond formation, to the carbon surface, as suggested in Scheme 1. The first reduction scan of a cyclic voltammogram for this process, presented in Figure 1, shows one reduction peak centered at -0.1 V, corresponding to the reduction of the aryl diazonium salt, generating the radical. During the second cycle, the redox peak disappears and the cyclic voltammogram exhibits only a small reduction current. Subsequent cycles show no change in electrochemistry from the second, indicating the presence of the coupled layer.29 Surface coverages of grafted aryl moieties, Γ, as estimated from integration of the charge passed under the reduction peak on the first scan, reach 2 ((0.82) × 10-9 mol/cm2 of the geometric area of the electrode. Downard35 calculated surface concentrations of nitrophenyl groups covalently grafted to pyrolyzed photoresist films of 1.4-2.3 × 10-9 mol/cm2. While it is difficult to directly correlate these results because of differences in the materials and diazonium salts used and differences in electrode preparation procedure, in both instances the surface coverages are close to the surface coverage estimated by assuming monolayer coverage of aryl moieties, 1.35 × 10-9 mol/cm2 of the geometric area of the electrode.36 However, a highly ordered aryl-monolayer is not (34) Liu, J.; Gooding, J. J.; Paddon-Row, M. N. Chem. Commun. 2005, 631– 633. (35) Yu, S. S. C.; Tan, E. S. Q.; Jane, R. T.; Downard, A. J. Langmuir 2007, 23, 11074–11082. (36) Liu, G.; Bocking, T.; Gooding, J. J. Electroanal. Chem. 2007, 600, 335– 344.

Figure 1. Cyclic voltammograms for the electrochemical grafting of the aryl diazonium cation generated in situ from 3-(4-aminophenyl)propionic acid to GC in Ar saturated solution containing 8 mM NaNO2, 10 mM 3-(4-aminophenyl) propionic acid, and 0.5 M HCl. Scan rate of 20 mV/s. The first and fourth scans are shown.

expected to form on GC electrodes that have a microscopically heterogeneous surface,6,37 and there may thus be local multilayer (overlap of monolayer) formation. The slight discrepancy in Γ, as estimated from integration of the charge passed under the reduction peak on the first scan, and that for a close-packed monolayer of aryl moieties possibly indicates the presence of several layers, or incomplete reaction of all of the electrogenerated aryl radicals.29 Pinson and Podvorica have, for example, concluded that integration of charge under voltammetric peaks provides monolayer coverages or below, but such an approach does not necessarily indicate that complete monolayer coverage is obtained.38 It is reported6,7,9 that deposition of layers of grafted aryl moieties, under conditions similar to those used here, may proceed by nucleation and growth, yielding roughened surfaces with “mushroom”-like features of several nanometers thickness, even though monolayer coverages or less were estimated from voltammetric procedures. To further investigate this, a quantitative colorimetric Kaiser test31,32 can be used to estimate free NH2 functional groups at surfaces. In order to use this test, conversion of the carboxylic acid-terminated surface to an amine-terminated surface was achieved by BOP-mediated coupling of ethylenediamine.30The Kaiser test31,32 then allows estimation of a total free NH2 functional group coverage of 2.3 × 10-10 mol/ cm2 of the geometric area of the electrode, providing an estimation of just over one-tenth of a monolayer of carboxylic acid functional groups on the geometric surface of the electrode. The 10-fold difference in the estimates for surface coverage of carboxylic acid groups by this method, and by integration of the charge for reduction of the diazonium salt, may be attributed to several factors. The discrepancy may be attributed to the fact that some of the electro-generated aryl moieties do not couple to the surface. For example, Liu and McCreery8 estimated that only half of the electrogenerated radicals from a 4-nitrophenyl diazonium salt actually coupled to the GC surface. In addition, incomplete reaction of the BOP coupling procedure or coupling of some of the diamine to two carboxylic acid surface groups should also be considered. The presence of several layers, inferred from AFM data and as reported on by others,6,7,9 could also sterically hinder and block access of the BOP reagent to functional groups that are buried. AFM measurements were performed on the bare and modified GC surfaces. AFM images (Figure 2a) show the surface of the bare GC to be roughened, with disordered polygraphitic domains, (37) Liu, Y. C.; McCreery, R. L. J. Am. Chem. Soc. 1995, 117, 11254–11259. (38) Pinson, J.; Podvorica, F. Chem. Soc. ReV. 2005, 34, 429–439.

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Figure 2. AFM characterization and cross-sectional analysis of bare GC (a) and of GC modified by grafting of the aryl diazonium salt (b).

associated grain boundaries from electrode polishing procedures, and also surface metamorphosis during pretreatments. It has been reported that nucleation of the aryl diazonium salt electroreduction reaction on a highly ordered pyrolytic graphite (HOPG) basal plane occurs at distributed atomic-scale defect sites, which extends across the surface even with three-dimensional layer growth.6 A comparison of images in Figure 2a,b provides evidence of the nucleation and growth of layers. In figure 2b, the basal plane appears relatively unchanged, compared to 2a, while the surface topography now consists of several “mushrooms” corresponding to the initial reduction sites and consequent growth of layers.6,9 A polymerization reaction (Scheme 2), where electrogenerated radicals can attack grafted moieties to give a multilayer network, is postulated to occur under the deposition conditions used, facilitating the formation of three-dimensional layers.6,39 Kariuki and McDermott6,7 demonstrated the formation of multilayers on GC electrodes via the reduction of diazonium salts and that aryl radicals can be generated through a 20 nm thick film. From AFM images, the surface roughness for several scan sizes was determined. The surface roughness value, averaged from images taken with different scan sizes, increases from 14.9 nm for an unmodified GC to 38.3 nm for the diazonium-derivatized surface. Further evidence that electrografting has occurred on the surface can be provided from examination of the solution voltammetric behavior of ferricyanide before and after modification of the electrode (Figure 3). Before modification, the peak-to-peak separation, ∆Ep, of the cyclic voltammograms at 100 mV/s in pH 7.4 buffer is 170 mV (vs Ag/AgCl), while after modification by reduction of the diazonium salt, generated in situ from 3-(4aminophenyl)propionic acid, no peaks are seen in the potential (39) Laforgue, A.; Addou, T.; Belanger, D. Langmuir 2005, 21, 6855–6865.

Scheme 2. Scheme Depicting the Postulated Route for Multilayer Formation during the Electrochemical Reduction of the Aryl Diazonium Salt

Designing Stable Redox-ActiVe Surfaces

Figure 3. Cyclic voltammograms at a scan rate of 100 mV/s for a 10 mM Fe(CN)63- (pH 7.4, 0.2 M phosphate buffer) solution before (top) and after (bottom) grafting of the aryl diazonium salt. Middle scan represents a voltammogram in pH 2, 0.2 M phosphate buffer, 10 mM Fe(CN)63- at the grafted surface.

window because of the blocking properties of the modified electrodes. This may be rationalized to be due to electrostatic repulsion between the negative charges of the surface carboxylate groups (the pKa of the carboxylic group in 3-(4-aminophenyl)propionic acid is 4.6) and those of the redox couple. Increasing the pH of the electrolyte to more alkaline conditions does not result in any increased blocking by the layer, indicating that, at pH 7.4, effectively all the surface carboxylic acid groups are deprotonated. In acidic solutions, the neutral surface, resulting from protonation of the surface carboxylate, is not as efficient at blocking the redox cycling of the ferricyanide probe, with ∆Ep of 247 mV and anodic and cathodic currents decreased by 45% and 4%, respectively, compared to those for ferricyanide redox cycling at a bare electrode (Figure 3). However, in the context of application of these modified electrodes as supports for immobilization of redox catalysts, it is essential that the modified surface does not unduly impede electron transfer between the redox catalyst and the electrode. To investigate whether this is the case, experiments such as those shown in Figure 3 and discussed above were undertaken using ferrocenemethanol, a redox probe that is neutral in the Fe(II) redox state. In contrast to ferricyanide, the ∆Ep for the

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ferrocenemethanol0/+ redox transition was identical, at approximately 130 mV, before and after the modification protocol, in pH 7.4 buffer. The carboxylic acid-functionalized carbon electrode exhibits only a slightly diminished current response, a 17% decrease in anodic and cathodic peak currents, compared to those at the bare electrode, toward the ferrocenemethanol0/+ redox couple at this pH. Recent studies have shown that aryl diazonium salts can be grafted to metal and semiconductor surfaces other than carbon,22 allowing use of gold slides as a support for examination of spectroscopic and microscopic properties of these grafted layers. Thus, in order to confirm surface attachment to electrodes, ATRIR spectra of gold slides modified using reduction of the aryl diazonium salt were measured, as well as the spectrum of the (4-aminophenyl)propionic acid starting material (Figure 4). All spectra share a broad absorbance peak at about 2960 cm-1 attributed to a C-H stretching and a peak at 1400 cm-1 attributed to C-H scissoring and bending.40 An O-H stretching frequency is observed at 2960-2850 cm-1, and a sharp CdO stretching frequency is seen at 1700 cm-1; a broad peak seen at 1150 cm-1 can also be attributed to this stretching. A sharp peak at 1500 cm-1 is characteristic of aromatic rings, while phenyl ring substitution bands can be seen in a sharp peak at 830 cm-1. The absence of any significant bands in the 2300-2130 cm-1 range or the 3300-3000 cm-1 range, where the NdN stretching and -NH2 transitions occur, for the gold slide modified by reduction of the aryl diazonium cation is indicative of surface attachment.40 Osmium-Modified Electrodes. A widely explored range of redox catalysts and mediators are based on polypyridyl complexes of osmium and ruthenium, because of the relative ease of synthetic variation of their structures, and hence properties, and the relative stabilities of the complexes in both oxidation states (II/III), with osmium complexes providing increased stability compared to analogous ruthenium complexes.28 Exploration of the possibility of attachment of such redox complexes was undertaken by the synthesis and characterization of a redox complex, [Os(bpy)2(4AMP)Cl]PF6, that has properties suitable for both redox catalysis and also possesses a free amine functional group amenable to immobilization chemistry. Synthesis of this complex, via the well-established route of prior preparation of an Os(bpy)2Cl2 precursor, was performed in ethylene glycol, as described by others11,28 in order to speed up the reaction compared to water/ ethanol mixtures. Characterization of the resulting complex was

Figure 4. ATR-IR spectra of starting material, 3-(4-aminophenyl)propionic acid (black), and of a gold-coated slide modified by grafting of aryl diazonium salt (red).

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Figure 5. Cyclic voltammogram of [Os(bpy)2(4-AMP)Cl]PF6 in acetonitrile containing 0.1 M LiClO4 at a scan rate of 0.1 V/s (from inner to outer). Scheme 3. Scheme Depicting Chemical Attachment of [Os(bpy)2(4-AMP)Cl]PF6 to the Functionalized Surface via Classical Carbodiimide Coupling

undertaken using microanalysis, UV-visible spectrophotometry, ATR-IR spectroscopy, and cyclic voltammetry (CV), confirming the success of the synthetic procedure. A cyclic voltammogram of a solution of the complex in acetonitrile using a carbon working electrode, Figure 5, yields a reversible couple centered around a redox potential of 0.17 V vs Ag/AgNO3. This reversible couple is associated with the Os(II/III) signal and the value corresponds well to a value of 0.13 V predicted for this complex using the method established by Lever41 accounting for ligand contributions to the redox potential of a series of metal complexes in organic media. A redox potential of 0.29 V versus Ag/AgCl for the Os(II//III) transition of the complex can be estimated from CV of the complex in aqueous phosphate buffer, pH 7.4, solutions. Covalent attachment of the [Os(bpy)2(4-AMP)Cl]PF6 to the carboxylic acid-terminated carbon electrodes, previously prepared by reduction of the aryl diazonium salt generated in situ from 3-(4-aminophenyl)propionic acid, was carried out through carbodiimide coupling following the procedures of Lui et al.34 (scheme 3). The CV of the resulting osmium-modified surface exhibits well-defined oxidation and reduction peaks corre(40) Ricci, A.; Bonazzola, C.; Calvo, E. J. Phys. Chem. Chem Phys. 2006, 8, 4297–4299. (41) Lever, A. P. B. Inorg. Chem. 1990, 29, 1271–1285.

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Figure 6. Cyclic voltammograms, 0.1 V/s, illustrating the “break-in” period at GC electrodes modified by conjugation of the osmium complex to the surface carboxylic groups introduced via grafting using the aryl diazonium salt. Initial scan (top) and scan after 200 repetitive scans (bottom) are shown.

sponding to the Os(II/III) redox couple, with a redox potential estimated as 0.29 V (vs Ag/AgCl). This redox potential corresponds closely to that observed for the solution complex dissolved in phosphate buffer, indicating that immobilization has little effect on the thermodynamics of the redox transition. The coupling procedure results in the formation of chemisorbed, surface attached, and physisorbed osmium complex, as evident from results of repetitive scanning CV of the modified electrodes in buffer (Figures 6 and 7). Stable voltammograms are obtained only upon cycling repeatedly in buffer for ∼200 cycles at a scan rate of 100 mV/s. Further confirmation of the presence of physisorbed complexes is observed when the coupling procedure is repeated in the absence of the coupling reagents, EDC/NHS. Redox peaks centered at 0.30 V are observed upon transfer of such modified electrodes to an electrochemical cell and recording of cyclic voltammmograms in phosphate buffer, pH 7.4, which, upon repeated scanning, quickly decrease. The redox peak currents for the surface attached osmium complex scale linearly with scan rate, indicating a surface confined electrochemical response, expected for such films.42 An estimate of total osmium surface coverage (ΓOs) can be evaluated from the charge passed (Q), by integrating the area under the voltammetric peaks at these low scan rates, using the equation

ΓOs ) Q/nFA

(1)

where n is the number of electrons transferred, F is Faraday’s constant, and A is the electrode area. The osmium surface coverage estimate can provide important details on the packing density and layer thickness for the complex covalently attached to the derivatized surface. From data on related osmium complexes, a surface coverage of 1 × 10-10 mol/cm2 can be estimated for monolayer coverage.18,19,43,44 Taking the geometric area of the GC electrode (0.0707 cm2), the surface coverage of osmium is estimated to be 2.9 × 10-10 mol/cm2, roughly 3 times the monolayer coverage of the complex. A surface coverage value exceeding one monolayer can be explained by the microscopic roughness of the GC electrode, and also the formation of (42) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; Wiley: New York, 2001. (43) Casero, E.; Darder, M.; Takada, K.; Abruna, H. D. Langmuir 1999, 15, 127–134. (44) Russ, J. C. Computer-Assisted Microscopy. The Measurement and Analysis of Images; Plenum: New York, 1992, p 450.

Designing Stable Redox-ActiVe Surfaces

Figure 7. (a) Overlaid cyclic voltammograms, 0.1 V/s, of GC electrodes modified by conjugation of the osmium complex to the surface carboxylic groups introduced via grafting using the aryl diazonium salt. The first and 200th scans are shown after the initial “break-in” period (see Figure 6). (b) Stability of the oxidation peak current of the osmium layer, estimated from cyclic voltammograms at 0.1 V/s in phosphate buffer, pH 7.4 (see Figure 8a), with storage of the electrode in phosphate buffer, pH 7.4 at 4 °C between scans.

multilayers at defect sites on the electrode surface.6 The osmium surface coverage evaluated corresponds well with the carboxylic acid surface group availability for coupling, estimated from the Kaiser test results following BOP-mediated coupling of a diamine to the carboxylic acid derivatized surface (Vide supra). Vijaikanth et al.45 reported surface concentrations of 3 times the monolayer coverage for an iron complex grafted to a GC surface modified utilizing an aryl diazonium salt. Ghodbane et al.46 reported the attachment of a ferrocene complex to a bromophenyl-modified GC electrode, achieved using aryl diazonium salt derivatization, yielding a surface coverage of 3.5 × 10-10 mol/cm2, while Liu et al.36 reported on attachment of a copper complex to an aryl diazonium-modified gold electrode with a surface coverage of 2.19 × 10-10 mol/cm2. Where there are no lateral interactions between surface confined redox centers and a rapid equilibrium is established with the electrode (ideal reversible system), a zero value for peak-topeak splitting (∆Ep) and a full width at half-maximum (fwhm) of 90.6 mV are expected for a one-electron transfer.42 The CV of the bound complex at pH 7.4 shows nonideal peak separation (∆Ep ∼ 90 mV) at slow scan rates, and fwhm greater than the ideal at ∼160 mV. This nonideality may be attributed to a combination of lateral interactions between the charged redox complexes and lateral diffusion of electrons and charge at these rough surfaces. (45) Vijaikanth, V.; Capon, J.-F.; Gloaguen, F.; Schollhammer, P.; Talarmin, J. Electrochem. Commun. 2005, 7, 427–430. (46) Ghodbane, O.; Chamoulaud, G.; Be´langer, D. Electrochem. Commun. 2004, 6, 254–258.

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Laviron has derived general expressions for the linear sweep voltammetric response for the case of surface-confined electroactive species.47,48 A heterogeneous electron transfer rate constant, k, can thus be estimated for the Os(II/III) transition (R ) 0.5 is assumed) by measuring the variation in the peak potential with scan rate. Plots of ∆E (defined as Epeak - E°) versus log scan rate, where ∆E is independent of log scan rate at low scan rates and proportional to log scan rate at higher scan rates, are used to extrapolate an average k of 90 s-1 for the osmium complex covalently attached to the derivatized surface. This value compares well to that estimated, 98 s-1, for similar complexes attached via conjugation to a 4-carboxyphenyl-modified gold electrode. This rate is, however, higher than that determined by Liu et al.34 for ferrocenemethylamine immobilized by covalent attachment to a 4-carboxyphenyl-derivatized GC surface. These authors claim a 10-fold decrease in rates for ferrocenemethylamine attached via this functional group to GC versus the same attachment on gold. Layer Stability. The osmium-modified electrode proved to be stable to repeated scanning in buffer. Initially there is a loss in surface coverage, but this can be attributed to the loss of any physisorbed molecules (Vide supra). Once the voltammetric signal had stabilized, there was no change upon repeated scanning (Figure 7a), over 800 cycles. Over a 3-week period, the electrode showed no leaching of the complex, although there was a slight variation in peak currents, possibly due to the swelling of the film or variations in room temperature (Figure 7b). When the electrode was not in use it was stored in phosphate buffer (pH 7.4) at 4 °C. No decrease in the electrochemical signal for the electrode was observed over a subsequent 6-month period, when the electrode was stored dry, demonstrating the remarkable stability of the layer. The pH of the buffer solution was varied to prove that the complex was not electrostatically bound. Although there was a variation in the shape of the voltammogram over the pH range 2-9.5, the CV signal for complex remains stable after repeated scanning in each buffer and remains stable in all the rinsing steps. The voltammetric behavior of the modified electrodes was slightly affected by the pH of the buffer solution. The shape of the voltammogram and the peak potentials were constant over the pH range 2-4.5. At pH above the pKa of the carboxylic acid (4.6), ∆Ep decreases from 124 mV (pH 2) to 87 mV (pH 7.4). While this could be indicative of an enhanced interaction between any remaining, unconjugated, carboxylate surface functional groups and the cationic osmium complex, the reasons for this change remain to be elucidated. To further probe the robustness of the modified electrode, the electrodes were subjected to a range of temperatures (5-40 °C), and ∆Ep was determined as a function of temperature. The electrodes were immersed in pH 7.4 buffer in a temperaturecontrolled heating bath, and the temperature was slowly increased, taking a CV at temperature intervals. In these experiments, the charge under the peaks remains constant both before and after heating, indicating the thermal stability of these modified electrodes throughout these experiments.

Conclusion GC electrodes have been modified with a carboxylic acidterminated layer by electroreduction of an in situ-generated diazonium salt of 3-(4-aminophenyl)propionic acid. This surface (47) Laviron, E. J. Electroanal. Chem. 1979, 101, 19–28. (48) Laviron, E. J. Electroanal. Chem. 1979, 100, 263–270.

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has been characterized by electrochemical, ATR-IR, and AFM methods, indicating strong evidence of nucleated growth of the layer on the surface. Chemical coupling of the redox molecule [Os(bpy)2(4-AMP)Cl]PF6 to the prefunctionalized surface can then be undertaken, to provide approximately 3 times the monolayer coverage of the redox-active species. Electrochemistry of the grafted osmium layer has been characterized in detail, and the electrode stability has been examined as a function of time and temperature. Rapid electron kinetics have been observed for the Os(II/III) redox couple that are in excellent agreement with those determined by

Boland et al.

Ricci et al.11 for this complex attached to a gold electrode bearing a similar diazonium film. Acknowledgment. Useful discussions with A. Downard on the use of aryl diazonium salts for grafting to carbon, and with C. Innocent, S. Tingry, and M. Cretin on the use of the Kaiser test are acknowledged. S.B. gratefully acknowledges support from an NUI, Galway College of Science Fellowship. This research is partly financed by the BIO-MEDNANO EU STREP, contract NMP4-CT-2006-017350. LA7031972