Covalently Anchored Carboxyphenyl Monolayer via Aryldiazonium Ion

May 29, 2014 - Covalently Anchored Carboxyphenyl Monolayer via Aryldiazonium Ion Grafting: A Well-Defined Reactive Tether Layer for On-Surface Chemist...
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Covalently Anchored Carboxyphenyl Monolayer via Aryldiazonium Ion Grafting: A Well-Defined Reactive Tether Layer for On-Surface Chemistry Lita Lee,†,‡ Haifeng Ma,†,§ Paula A. Brooksby,†,‡ Simon A. Brown,†,§ Yann R. Leroux,∥ Philippe Hapiot,∥ and Alison J. Downard*,†,‡ †

MacDiarmid Institute for Advanced Materials and Nanotechnology, Victoria University of Wellington, Post Office Box 600, Wellington 6140, New Zealand ‡ Department of Chemistry, and §Department of Physics and Astronomy, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand ∥ Institut des Sciences Chimiques de Rennes (Equipe MaCSE), CNRS, UMR 6226, Université de Rennes 1, Campus de Beaulieu, Bat 10C, 35042 Rennes Cedex, France S Supporting Information *

ABSTRACT: Electrografting of aryl films to electrode surfaces from diazonium ion solutions is a widely used method for preparation of modified electrodes. In the absence of deliberate measures to limit film growth, the usual film structure is a loosely packed multilayer. For some applications, monolayer films are advantageous; our interest is in preparing well-defined monolayers of reactive tethers for further onsurface chemistry. Here, we describe the synthesis of an aryl diazonium salt with a protected carboxylic acid substituent. After electrografting to glassy carbon electrodes and subsequent deprotection, the layer is reacted with amine derivatives. Electrochemistry and atomic force microscopy are used to monitor the grafting, deprotection, and subsequent coupling steps. Attempts to follow the same procedures on gold surfaces suggest that the grafted layer is not stable in these reaction conditions.

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protecting group in the para position.3 The protecting group prevents radical attack at already grafted phenyl groups and, after removal, leaves a reactive functionality. Monolayers or near monolayers of thiophenolate,3 benzaldehydes,4 aminomethylphenyl,5 and phenylethynylene groups (Ar-Eth-H)6 have been prepared by this protection−deprotection approach. Expanding the library of reactive tether monolayers increases the types of chemistries that can be used to couple target molecules to surfaces. Carboxylate-terminated tether layers are very useful for immobilizing a wide range of species. In particular, amide bond coupling with amine derivatives has been widely applied in the preparation of modified surfaces, most frequently for the immobilization of biomolecules. These methods have been demonstrated using multilayer carboxyterminated films grafted from the corresponding diazonium ion;5,7−17 however, there are no reports of well-characterized monolayer carboxy-terminated films from diazonium-based grafting. In this work, we describe a protection−deprotection strategy for preparing a monolayer of carboxyphenyl (Ar-

INTRODUCTION Since the first demonstration by Pinson, Savéant, and coworkers,1 aryldiazonium salts have been widely used for the preparation of covalently anchored, robust, nanoscale organic films on conducting, semiconducting, and non-conducting substrates.2 A key advantage of the surface modification strategy is the strong anchoring to many materials; however, a disadvantage is the difficulty of controlling the film structure. Grafting from aryldiazonium salts relies on generation of aryl radicals that attack the substrate surface but can also attack already grafted groups. Hence, the grafting method usually leads to disordered multilayers that are a far from ideal platform for building up well-defined surface layers through further coupling reactions. Furthermore, the multilayer films are usually poorly conducting and, thus, pose a significant barrier to electron transfer between the electrode and solution-based redox centers. This is a serious limitation for applications, such as electrochemical sensors. There is a clear need for methods that generate monolayer films from aryldiazonium salts and, in particular, for monolayers with reactive terminal groups for subsequent on-surface chemistry. Pedersen, Daasbjerg, and co-workers were the first to demonstrate the use of diazonium derivatives with a bulky © 2014 American Chemical Society

Received: April 9, 2014 Revised: May 26, 2014 Published: May 29, 2014 7104

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in a small amount of ACN and reprecipitated by the addition of cold diethyl ether. The precipitate was collected under vacuum and stored in the freezer and in the dark. 1H NMR (500 MHz, CD3CN, ppm) δ: 8.57 (d, 2H, J = 9 Hz), 8.34 (d, 2H, J = 9 Hz), 7.90 (d, 2H, J = 7.5 Hz), 7.73 (d, 2H, J = 7.5 Hz), 7.48 (t, 2H, J = 7.5 Hz), 7.40 (t, 2H, J = 7.5 Hz), 4.81 (d, 2H, J = 6.5 Hz), 4.48 (t, 1H, J = 6.5 Hz). Electrochemistry, Surface Modification, and Coupling Reactions. All electrochemical measurements were performed using an Eco Chemie Autolab PGSTAT302 potentiostat/galvanostat. Glassy carbon (GC) working electrodes (3 mm diameter) were polished with a slurry of 1 μm alumina powder in water, followed by ultrasonication in water and drying with N2(g). PPF working electrodes were mounted horizontally between an insulated metal base plate and a glass solution cell. A Kalrez O-ring and four springs from the base plate of the glass cell sealed the solution above the PPF while maintaining a copper electrical contact to the PPF external to the solution.18 The auxiliary electrode was a Pt mesh, and the reference electrode was a saturated calomel electrode (SCE) for measurements in aqueous solutions and a calomel electrode with 1 M LiCl(aq) for non-aqueous solutions. Unless stated otherwise, Ar-COO-Fm groups were electrografted to GC and PPF from a solution of 5 mM N2+-Ar-COO-Fm and 0.1 M TBABF4 in ACN using 5 potential cycles between 0.80 and −0.75 V at a scan rate of 50 mV s−1. The modified electrode was rinsed with acetone, sonicated in ACN for 5 min, and dried with N2(g). The protecting Fm group was cleaved from the layer by immersing the modified electrode in a stirred solution of 20% piperidine in DMF for 40 min. Electrodes were rinsed with acetone and water after deprotection (preliminary experiments demonstrated that sonication was not required for this cleaning step). For experiments monitoring the response of redox probes at grafted electrodes, cyclic voltammograms were first obtained using the unmodified electrode in the redox probe solution; the electrode was rinsed with water (or acetone for the ferrocene redox probe) and dried with N2 gas between each scan and prior to modification. Ferrocenyl (Fc) and nitrophenyl (NP) groups were immobilized on Ar-COOH-modified surfaces by acid chloride coupling reactions.14 Carboxyphenyl-modified substrates were heated under reflux in anhydrous CH2Cl2 (6 mL) with oxalyl chloride (25 μL) and pyridine (8 μL) for 1 h under a N2 atmosphere. All volatiles were then removed under vacuum, followed by the introduction of NBAHCl or FcCH2NH2 (10 mg) in anhydrous CH2Cl2 (6 mL) with excess of triethylamine under a N2 atmosphere. The reaction was stirred for 5 min in an ice bath and then at room temperature overnight. The resulting modified electrodes were washed in vigorously stirred clean CH2Cl2 for 10 min, followed by sonication in ethanol (EtOH) for 2 min; the modified electrodes were dried with a stream of N2(g). Cyclic voltammograms of immobilized Fc and NP groups were obtained in 0.1 M LiClO4−EtOH and 0.1 M H2SO4 solutions, respectively. The surface concentration of immobilized Fc was determined by averaging the areas under the anodic and cathodic peaks from the third voltammetric cycle obtained at a scan rate of 200 mV s−1. The third scan was used because the background currents changed between the first and second and sometimes third cyclic voltammogram. The surface concentration of immobilized NP groups was estimated from the first cycle obtained at a scan rate of 100 mV s−1, using the NP reduction and the hydroxyaminophenyl oxidation peak areas and the number of electrons involved in each redox process.20 Voltammetric peak area analysis for NP and Fc redox processes was performed by correcting the baseline using a third-order polynomial and integrating the area under the peak. Uncertainties derived from the experimental data are indicated in the text. For sample sizes of >2, uncertainties are the standard deviation of the indicated number of samples (n). For a sample size of 2, the uncertainty indicates the range of values obtained. There are estimated additional uncertainties of 20 and 10% for absolute surface concentrations of NP and Fc, respectively, reflecting the difficulty of defining appropriate peak baselines. The geometric working electrode area was assumed for all surface concentration calculations.

COOH) groups from an aryldiazonium ion with a protected carboxylic acid group. We demonstrate that the deprotected layer forms a stable platform on glassy carbon for further onsurface chemistry.

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EXPERIMENTAL SECTION

All experimental details pertaining to modification and film characterization at Au electrodes are reported in the Supporting Information. Materials and Reagents. Potassium ferrocyanide [K4 Fe(CN)63H2O, Hopkin and Williams], ferrocene (Fc, Aldrich), 4nitrobenzylamine hydrochloride (NBAHCl, Aldrich), dopamine hydrochloride (Alfa Aesar), tetrahydrofuran (THF, Merck), tetrabutylammonium fluoride (TBAF, Acros Organic), N,N′-dimethylformamide (DMF, J.T. Baker), N,N′-dicyclohexylcarbodiimide (DCC, Alfa Aesar), di-tert-butyl dicarbonate (Boc2O, Aldrich), trifluoroacetic acid (TFA, Merck), 4-dimethylaminopyridine (DMAP, Merck), nitrosonium tetrafluoroborate (NOBF4, Aldrich), dioxane (Sigma-Aldrich), triethylamine (Merck), 9-fluorenylmethanol (FmOH, Aldrich), and acetonitrile [ACN, VWR BDH, high-performance liquid chromatography (HPLC) grade] were used as received. Millipore Milli-Q water (resistivity > 18 MΩ cm) was used for all aqueous solutions and washing. Tetrabutylammonium tetrafluoroborate (TBABF4),18 ferrocenemethylamine (FcCH2NH2),19 and pyrolyzed photoresist film18 (PPF) were prepared by literature methods. Synthesis of 9-H-Fluoren-9-ylmethyl 4-Aminobenzoate (NH2-Ar-COO-Fm). 4-Aminobenzoic acid (3.0 g, 0.022 mol) was dissolved in 75 mL of dioxane/water (2:1). Triethylamine (4.5 mL, 0.032 mol) was added to the mixture, followed by Boc2O (7.5 mL, 0.032 mol). The solution was stirred overnight at room temperature. Excess solvent was evaporated under vacuum, and 3 M HCl was added dropwise to the residue to obtain 4-{[(tert-butoxy)carbonyl]amino}benzoic acid (Boc-NH-Ar-COOH, 1) as a white precipitate, which was collected by filtration and washed with water (yield = 4.54 g, 88%). Proton nuclear magnetic resonance (1H NMR) (400 MHz, DMSO-d6, ppm) δ: 7.82 (d, 2H, J = 8.8 Hz), 7.54 (d, 2H, J = 8.8 Hz), 1.48 (s, 9H). Boc-NH-Ar-COOH (3.0 g, 0.013 mol), DMAP (80 mg), and FmOH (4.0 g, 0.020 mol) were dissolved in DMF (10 mL). The reaction mixture was stirred and cooled in an ice bath, followed by the addition of DCC (2.7 g, 0.013 mol). The solution was stirred for a further 5 min in the ice bath followed by 3 h at room temperature. Precipitated urea was removed by filtration, and the filtrate was evaporated under vacuum. The residue was then taken up in CH2Cl2 and washed twice with 0.5 M HCl, followed by saturated NaHCO3 solution, and dried over MgSO4. The solvent was removed under vacuum, and 9-H-fluoren-9-ylmethyl 4-{[(tert-butoxy)carbonyl]amino}benzoate (Boc-NH-Ar-COO-Fm) was obtained by column chromatography, eluted with CH2Cl2 (yield = 2.53 g, 48%). 1H NMR (400 MHz, CDCl3, ppm) δ: 8.03 (d, 2H, J = 8.4 Hz), 7.79 (d, 2H, J = 7.2 Hz), 7.65 (d, 2H, J = 7.6 Hz), 7.47 (d, 2H, J = 8.8 Hz), 7.41 (t, 2H, J = 7.2 Hz), 7.32 (t, 2H, J = 7.2 Hz), 4.59 (d, 2H, J = 7.2 Hz), 4.38 (t, 1H, J = 7.6 Hz), 1.26 (s, 9H). Boc-NH-Ar-COO-Fm (2.5 g, 0.006 mol) was dissolved in CH2Cl2 (15 mL) followed by the addition of TFA (10 mL), and the reaction was stirred for 1 h at room temperature. Excess reagent was removed under vacuum and sat. NaHCO3 was added dropwise to neutralize the solution. The white precipitate of NH2-Ar-COO-Fm was collected by filtration, and impurities were removed by dissolving in hexane (yield = 1.87 g, 95%). 1H NMR (400 MHz, CDCl3, ppm) δ: 7.93 (d, 2H, J = 8.4 Hz), 7.79 (d, 2H, J = 7.2 Hz), 7.66 (d, 2H, J = 7.2 Hz), 7.41 (t, 2H, J = 7.2 Hz), 7.32 (t, 2H, J = 7.6 Hz), 6.70 (d, 2H, J = 8 Hz), 4.55 (d, 2H, J = 7.2 Hz), 4.37 (t, 1H, J = 7.2 Hz). Synthesis of 4-[(9-H-Fluoren-9-ylmethoxy)carbonyl]benzene-1-diazonium Tetrafluoroborate (N2+-Ar-COO-Fm). The protected aryldiazonium salt was synthesized by dissolving NH2-Ar-COO-Fm (0.15 g, 0.45 mmol) in dry ACN. The reaction was stirred at −40 °C, followed by the addition of NOBF4 (65 mg, 0.55 mmol). The reaction was stirred for a further 2 h at −40 °C, and the solvent was evaporated under vacuum. The precipitate was redissolved 7105

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Scheme 1. Reaction Steps for the Preparation of the Protected Carboxylate Diazonium Salt, N2+-Ar-COO-Fm

Scheme 2. Strategy for Preparation of the Ar-COOH Monolayer and for Coupling Fc and NP Groups to the Layer

Atomic Force Microscopy (AFM) Measurements. AFM measurements were made with a Dimension 3100 and Nanoscope IIIa controller (Digital Instruments, Veeco, Santa Barbara, CA). Film thicknesses were measured on modified PPF working electrodes by removing a small section of film by scratching with an AFM tip and then recording average line profiles across the film and scratch.18 Three scratches were made on each sample, and at least eight transverse cross-sections were chosen from the corresponding images, yielding at least eight average line profiles. Each profile gave two film thicknesses: one from the step on the right side of the section and the other from the step on the left side of the section. Thus, the film thickness reported for each sample is the mean of at least 16 values, and the uncertainty is the standard deviation of the mean. Molecular dimensions were calculated using the freeware Avogadro 1.1.1.21 Computational Methods. The conformation of Ar-COO-Fm groups was optimized using density functional theory (DFT) calculations at the B3LYP/6-31G* level. All calculations were performed using the Gaussian 03 package with the default parameters.22 The xyz coordinates of the optimized geometry are given in Table S1 of the Supporting Information.

Figure 1. Five consecutive scans at a bare GC electrode in a solution of 5 mM N2+-Ar-COO-Fm in 0.1 M TBABF4-ACN. Scan rate = 50 mV s−1.

inhibition of electron transfer to diazonium ion in solution, consistent with the formation of a non-conducting grafted layer. After grafting an Ar-COO-Fm film, deprotection of the carboxylate groups in a stirred solution of 20% piperidine in DMF24 (step ii of Scheme 2) was investigated. The effect of immersion in the deprotection solution was monitored at 20 min intervals by transferring the modified electrode to a solution of 1 mM dopamine in 0.1 M H2SO4 and recording cyclic voltammograms at the modified electrode. Dopamine was chosen for this study because of the sensitivity of its response to the presence of specific surface structures.25,26 Fast kinetics for the solution-based dopamine redox couple relies on catalysis by either adsorbed dopamine or surface oxygen functionalities, probably involving hydrogen bonding between dopamine and the surface species.25,26 Figure 2 shows that, before deprotection, there is no evidence of dopamine redox chemistry at the modified electrode, consistent with a lack of accessible catalytic surface sites. After 20 min in the deprotection solution, well-defined peaks are observed but with current approximately half that at the bare GC surface. After 40 min, the peak currents are close to those for the bare surface and a further 20 min in the deprotection solution gives little further change. The reappearance of relatively fast dopamine redox chemistry indicates that accessible catalytic sites are generated by

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RESULTS AND DISCUSSION The protected aryl diazonium salt, N2+-Ar-COO-Fm, was synthesized in 4 steps from 4-aminobenzoic acid (Scheme 1). In the first step, the amine group of 4-aminobenzoic acid was protected with tert-butyloxycarbonyl (Boc). A Steglich esterification23 of the Boc-protected aminobenzoic acid with FmOH gave the 9-fluorenylmethyl (Fm)-protected compound. The Boc group was then removed using TFA to yield the Fmprotected aniline derivative, NH2-Ar-COO-Fm. Finally, desired N2+-Ar-COO-Fm was obtained by reaction with NOBF4 under a N2(g) atmosphere at −40 °C. Ar-COO-Fm films were electrografted to GC and PPF in ACN using the isolated protected diazonium salt (step i of Scheme 2). Cyclic voltammograms obtained in the grafting solution (Figure 1) show reduction of the diazonium ion in the first scan but only featureless voltammograms with relatively low current in subsequent scans. This behavior is typical of that seen when grafting from aryldiazonium salts. The disappearance of the well-defined reduction process after 1 cycle indicates 7106

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Figure 2. Cyclic voltammetry in a solution of 1 mM dopamine in 0.1 M H2SO4 at a scan rate of 100 mV s−1: (black line) bare GC, (red line) GC modified with N2+-Ar-COO-Fm before deprotection, and GC modified with N2+-Ar-COO-Fm after deprotection in 20% piperidine/ DMF for (blue line) 20 min, (green line) 40 min, and (brown line) 60 min.

Figure 3. Cyclic voltammograms obtained in a solution of (a) 1 mM ferrocene in 0.1 M TBABF4-ACN, (b) 1 mM dopamine in 0.1 M H2SO4, (c) 1 mM ferrocyanide in 0.1 M HCl, and (d) 1 mM ferrocyanide in 0.1 M PB (pH 6.9) at a scan rate of 100 mV s−1: (black line) bare GC and GC modified with N2+-Ar-COO-Fm (red line) before deprotection, (green line) after immersion in DMF for 40 min, and (blue line) after deprotection in 20% piperidine/DMF for 40 min.

treatment in the deprotection solution. This is consistent with the expected removal of the large Fm groups, which may expose several types of catalytic sites: dopamine physisorbed on the GC surface within the sparsely packed monolayer, native carboxylic acid groups on the GC surface, and terminal carboxylic acid groups of the grafted monolayer. Hence, it appears that, after 40 min of immersion in a stirred solution of 20% piperidine in DMF, removal of Fm groups is essentially complete. For further experiments, 40 min was adopted as the standard deprotection time. The deprotection reaction and properties of the deprotected grafted layer were further examined using ferrocene and ferrocyanide as additional redox probes. Cyclic voltammograms were obtained before deprotection, after immersion of the ArCOO-Fm modified electrode in stirred DMF solution in the absence of piperidine, and after subsequent immersion in the deprotection solution. The red lines in Figure 3 show that, for all redox probes, the Ar-COO-Fm layer exerts a strong blocking effect with only very low currents obtained over the potential range where the redox reactions occur at a bare GC electrode (black lines). After immersion of the modified electrode in DMF, the layer becomes significantly less blocking toward the redox reactions of ferrocene, dopamine, and ferrocyanide (in 0.1 M HCl) (green lines). For the latter redox probe, the cyclic voltammogram (green line in Figure 3c) has a distinctly sigmoidal shape, consistent with permeation of the redox probe through a porous film.27 Subsequently, the modified electrodes were immersed in 20% piperidine/DMF solution for 40 min, after which the response (blue line) to the redox probes (with the exception of ferrocyanide at pH 6.9) was close to those at the bare GC electrodes. The changes in the voltammograms described above confirm that there is a relatively thick and non-porous layer prior to immersion in DMF, which significantly slows the electron transfer rate for redox probes in solution. After immersion of the modified electrodes in DMF solution, the films become more porous, indicating swelling of the film, and/or removal of physisorbed impurities. Immersion in the deprotection solution has a much stronger effect on the barrier properties of the films: the films have little influence on the redox response, suggesting that the films are now very thin and/or highly porous. Comparing the voltammograms of the ferro-/ferricyanide

couple in 0.1 M HCl (Figure 3c) and in phosphate buffer (PB) at pH 6.9 (Figure 3d) confirms that deprotection generates a layer with the expected acid−base properties. After deprotection, the cyclic voltammogram recorded in 0.1 M HCl is indistinguishable from that obtained at the bare GC electrode; however, in the pH 6.9 solution, although there is an increase in the current for the redox couple after deprotection, the electron transfer rate clearly remains low (ΔEp = 279 mV) relative to that at the polished surface (ΔEp = 65 mV). The pKa of a multilayered Ar-COOH film is in the range of 2.8−3.3,28 and hence, the deprotected film will be protonated (neutral) in 0.1 M HCl (pH ∼ 1) and deprotonated (negatively charged) at pH 6.9. Although deprotection decreases the film thickness, which accelerates the electron transfer rate, the negative surface charge at pH 6.9 slows the electron transfer rate compared to that at the neutral deprotected surface, because of electrostatic repulsions with the negatively charged redox probe. The net effect is a smaller increase in the electron transfer rate after deprotection, for the ferri-/ferrocyanide couple at pH 6.9. The cyclic voltammetry of redox probes described above is qualitatively consistent with the changes expected on deprotection of the grafted layers; however, evidence for generation of a monolayer film is best obtained by direct measurement of the thickness of films grafted to PPF. PPF is a glassy carbon-like material with low surface roughness (rms roughness ≤ 0.5 nm) and allows for measurement of surface layers with low nanometer thickness.29,30 The thickness of films grafted to PPF was measured by removing small sections of grafted film by scratching with an AFM tip, followed by profiling across the scratch. Figure 4a shows a topographic image of a scratch in an Ar-COO-Fm film, and Figure 4b is the corresponding depth profile, which shows an average film thickness of 3.1 ± 0.3 nm. The calculated height of an ArCOO-Fm group oriented perpendicularly on a flat surface is ∼1.4 nm, and hence, the Ar-COO-Fm film is multilayered with 7107

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voltammogram matches those obtained for NP films grafted directly from 4-nitrobenzenediazonium ion,20 showing the irreversible reduction of NP groups to aminophenyl and hydroxaminophenyl groups at ∼ −0.5 V and the chemically reversible hydroxyaminophenyl/nitrosophenyl couple centered at ∼0.25 V. From the peak areas in the first cyclic scan of five modified electrodes, the estimated surface concentration of immobilized NP groups was (4.1 ± 0.7) × 10−10 mol cm−2. Figure 6a shows cyclic voltammograms of the modified

Figure 4. (a and c) AFM topography images and (b and d) depth profiles of Ar-COO-Fm-modified PPF (a and b) before and (c and d) after deprotection.

Figure 6. Cyclic voltammograms obtained in 0.1 M LiClO4/EtOH of a GC electrode modified with an Ar-COOH monolayer and coupled with Fc: (a) repeat scans at scan rates from 0.01 to 1 V s−1 and (b) plot of peak currents against the scan rate.

a minimum of 2−3 layers. After deprotection (panels c and d of Figure 4), the average film thickness is 0.4 ± 0.3 nm, consistent with a sparsely packed Ar-COOH monolayer (the calculated height of a perpendicularly oriented Ar-COOH group is 0.6 nm). Multilayer films form during grafting when aryl radicals attack modifiers already attached to the surface. For a grafted Ar-COO-Fm layer, the bulky Fm moieties sterically protect the phenyl groups but are themselves highly accessible to radical attack. The presence of a monolayer film after removal of the Fm-protecting groups confirms that the multilayer film is built up through radical attack at the Fm substituents rather than at the phenyl rings. An Ar-COOH monolayer is expected to be a useful tether for surface immobilization of amine derivatives via amide coupling. The reactivity of the Ar-COOH monolayer was tested by coupling with NBAHCl and FcCH2NH2 to give surfaces modified with NP and Fc groups, respectively. Reactions were carried out in DCM solution using oxalyl chloride to convert the carboxylate functionality to the more reactive acyl chloride functionality (step iii of Scheme 2). After the coupling step, modified electrodes were rinsed with DCM, sonicated in EtOH, and then transferred to blank electrolyte solution. Figure 5 shows a cyclic voltammogram obtained in 0.1 M H2SO4 for an Ar-COOH film after reaction with NBAHCl. The

electrodes after coupling FcCH2NH2 to the Ar-COOH monolayer by the same general method. The redox response at E1/2 = 0.34 V is assigned to the expected ferrocene/ ferrocenium couple. The cyclic voltammograms of the Fcterminated film obtained at scan rates of 0.01−1 V s−1 (Figure 6a) show a linear relationship between the peak current and scan rate (Figure 6b), indicting that there are no kinetic limitations under these conditions. The average surface concentration of Fc groups calculated from cyclic voltammograms of two modified surfaces was (4.1 ± 0.4) × 10−10 mol cm−2, matching the surface concentration of NP groups coupled by the same method. A freshly polished GC surface exposed to air typically has an O/C ratio of 0.07−0.2.31 Surface O is attributed to various functional groups, including carboxylic acids,31 hence raising the possibility that, under our conditions, coupling reactions occur directly to GC in addition to the grafted monolayer. To investigate the importance of direct coupling reactions, GC electrodes were subjected to the same conditions and series of steps as the grafted electrodes, except that no diazonium salt was present in the “grafting” step. After reaction with NBAHCl and FcCH2NH2, cyclic voltammetry (not shown) of the treated electrodes gave surface concentrations of 1.8 × 10−10 and (1.3 ± 0.6) × 10−10 mol cm−2 (n = 2) for NP and Fc groups, respectively. Hence coupling of the amine derivatives directly to activated GC may contribute to the measured surface concentrations of NP and Fc groups at the grafted ArCOOH electrodes, although the number of accessible surface carboxylic acid groups is presumably lower in the presence of the monolayer. A further blank experiment was undertaken to confirm that oxalyl chloride has the expected role in the coupling of the amine derivative to the Ar-COOH monolayer. For this experiment, all standard steps for preparation of the Ar-COOH monolayer and coupling NBAHCl to the monolayer were carried out but in the absence of oxalyl chloride. Cyclic voltammetry of the modified electrode (not shown) revealed a very small response attributed to NP groups, corresponding to

Figure 5. Repeat cyclic voltammograms obtained in 0.1 M H2SO4 at a scan rate of 100 mV s−1 of a GC electrode modified with an ArCOOH monolayer and coupled with NBAHCl. 7108

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a surface concentration of (0.4 ± 0.1) × 10−10 mol cm−2 (n = 2). Clearly, oxalyl chloride is necessary for a high yield coupling reaction, providing strong evidence that, in the presence of oxalyl chloride, the reaction proceeds with the formation of an amide bond, as shown in step iii of Scheme 2. The small amount of immobilized NP groups detected in the blank experiment is attributed to electrostatic interactions between NBAH+ and the Ar-COOH monolayer or chemisorption of the amine on the GC surface.32 The correspondence of the surface concentrations of NP and Fc groups coupled to the Ar-COOH-modified surfaces (4.1 × 10−10 mol cm−2) strongly suggests that the coupling reaction proceeds quantitatively and that the surface concentration of carboxylic acid groups is ∼4 × 10−10 mol cm−2. This value includes an unknown contribution (< ∼1.8 × 10−10 mol cm−2) from carboxylic acid groups on the GC surface. The theoretical maximum surface concentration of Ar-COOH groups grafted to a flat surface by this approach can be estimated by assuming that the density of grafted groups is governed by the size of the Fm-protecting group. Assuming that the protected aryl moieties are arranged in a hexagonal closest packing lattice (the highest density arrangement of disks of diameter d), the surface concentration, Γ, in mol cm−2 can be estimated using eq 1, where d is in Å.

10−10 mol cm−2) is consistent with the presence of a monolayer of Ar-COOH groups grafted from the protected diazonium ion. The grafting, deprotection, and coupling steps were briefly investigated on Au surfaces, using the same conditions as for GC electrodes. Additionally, on Au electrodes, the amide coupling reaction was also attempted using milder reaction conditions (see the Supporting Information for experimental details and results obtained at Au). Although cyclic voltammograms of redox probes at the initially grafted and deprotected surfaces were consistent with the presence of the expected grafted and deprotected layers, only trace amounts of Fc and NP groups were detected electrochemically after coupling reactions of the corresponding amine derivatives with the deprotected layer. Ultrahigh vacuum scanning tunneling microscopy and non-contact mode AFM investigations showed that, after the initial grafting step, a continuous film covers the Au surface; however, we were unable to obtain clear evidence for a deprotected monolayer. It appears that the grafted film is substantially removed from the gold surface during deprotection. AuI−piperidine complexes are well-known,34 and hence, attack of the Au surface by piperidine may account for this observation.

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CONCLUSION Grafting and subsequent deprotection of a Fm-protected ArCOOH diazonium derivative give a monolayer film of ArCOOH groups on GC. Through amide-bond-forming reactions with amine derivatives, NP and Fc groups are coupled to the monolayer with a surface concentration of ∼4 × 10−10 mol cm−2. Our results suggest that the coupling reactions are close to quantitative, and thus, the surface concentrations of NP and Fc groups reflect the concentration of the Ar-COOH tether layer, albeit with a small contribution from species directly coupled to carboxylic acid groups on the GC surface. The ArCOOH monolayer thus provides a convenient base for building stably attached layers on the GC surface through subsequent on-surface chemistry. In contrast, on Au, the grafted layer appears to be largely stripped from the surface during the deprotection step. Extending the general approach to Au surfaces will require investigation of alternative deprotection reagents.

−8

Γ∼

1.92 × 10 d2

(1)

The diameter d of the disk representing the protecting group is evaluated by computing the conformation of the molecule and assuming free rotation of the protecting group along the aryl amine axis. The conformation of the molecule (Figure 7) was

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ASSOCIATED CONTENT

S Supporting Information *

Table of the xyz coordinates of the optimized geometry of ArCOO-Fm, experimental details of the grafting, deprotection, and coupling steps at Au electrodes, and the corresponding cyclic voltammograms and scanning probe microscopy images. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 7. Optimized structure of Ar-COO-Fm from DFT calculations at the B3LYP/6-31G* level.

AUTHOR INFORMATION

Corresponding Author

*Telephone: +64-3-364-2501. Fax: +64-3-364-2110. E-mail: [email protected].

optimized using DFT calculations at the B3LYP/6-31G* level. Using this methodology, we obtained d = 10.74 Å for a freely rotating molecule of Ar-COO-Fm, giving the theoretical maximum surface concentration on an ideal flat surface, Γtheo = 1.66 × 10−10 mol cm−2. When the typical roughness factor of polished GC (1.5−2.533) and the contribution from NP and Fc coupled directly to activated carboxylic acid groups on the GC surface are taken into account, our experimentally determined surface concentration of coupled NP and Fc groups (∼4 ×

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the MacDiarmid Institute for Advanced Materials and Nanotechnology. Lita Lee thanks the MacDiarmid Institute for a doctoral scholarship. The authors 7109

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thank Dr. Matthew Polson and Professor Richard McCreery for helpful discussions and Dr. J. S. Loring for use of Linkfit software.

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