pubs.acs.org/Langmuir © 2010 American Chemical Society
Electrochemically Controlled Adsorption of Fc-Functionalized Polymers on β-CD-Modified Self-Assembled Monolayers Galina V. Dubacheva,*,† Angeline Van Der Heyden† Pascal Dumy,† Oznur Kaftan,‡ Rachel Auzely-Velty,‡ Liliane Coche-Guerente,*,† and Pierre Labbe† † D epartement de Chimie Mol eculaire, UMR CNRS-UJF 5250, ICMG FR-2607, Universit e Joseph Fourier, BP 53, 38041 Grenoble, Cedex 9, France, and ‡Centre de Recherches sur les Macromol ecules V eg etales (CNRS), Universit e Joseph Fourier, BP 53, 38041 Grenoble, Cedex 9, France
Received May 19, 2010. Revised Manuscript Received July 20, 2010 This work presents an in situ study of the adsorption/desorption behavior of ferrocene(Fc)-functionalized linear polymers on a gold surface covered with β-cyclodextrin(β-CD)-modified self-assembled monolayers (SAMs). The characterization of binary SAMs obtained with HS-(CH2)11-EG6-N3 and HS-(CH2)11-EG4-OH (EG, ethylene glycol) was performed using a quartz crystal microbalance with dissipation monitoring (QCM-D), cyclic voltammetry, and contact angle measurements. The functionalization of SAMs with β-CD was made via the “click” reaction between the β-CD monoalkyne derivative and azide groups exhibited by SAMs. The formation of the host-guest complex between SAM-β-CD and Fc-derivatized polymers (chitosan (CHI) and poly(allylamine hydrochloride) (PAH)) was studied by QCM-D. The viscoelastic model of Voinova was used to fit QCM-D curves recorded during the adsorption and electrochemically controlled desorption of CHI-Fc and PAH-Fc on SAM-β-CD. Using QCM-D coupled to cyclic voltammetry, we demonstrated that CHI-Fc and PAH-Fc can be successfully deposited on a SAM-β-CD-coated gold surface forming a stable multivalent inclusion complex between Fc moieties of polymer and β-CD cavities of SAM. We also showed that all specifically attached polymer chains can be detached from the SAM-β-CD-coated gold surface by applying an electric field.
Introduction The design of kinetically stable nanostructures on solid surfaces while keeping binding control is of high current interest for potential applications in molecular electronics, biosensors, and biomedical science.1-4 Supramolecular chemistry is particularly attractive for such applications because it possesses controllable molecular recognition properties with structural modification abilities on a specific area of a nanoassembly.5 By taking advantage of multivalent host-guest interactions, the group of Huskens and Reinhoudt introduced an electrochemically controlled attachment/detachment of nanostructures onto “molecular printboards” consisting of self-assembled monolayers (SAMs) of heptathioether-functionalized β-cyclodextrin (β-CD) on gold surfaces.6 The term molecular printboard was used to designate these SAMs according to their ability to bind molecules with ultimate control over binding thermodynamics and kinetics and positioning with molecular accuracy. It was demonstrated that preliminarily adsorbed Fc-functionalized poly(propylene imine) (PPI(Fc)n, n = 4-64) dendrimers can be successfully removed from (1) Tang, C. S.; Schmutz, P.; Petronis, S.; Textor, M.; Keller, B.; V€or€os, J. Biotechnol. Bioeng. 2005, 91, 285. (2) Guillaume-Gentil, O.; Akiyama, Y.; Schuler, M.; Tang, C.; Textor, M.; Yamato, M.; Okano, T.; V€or€os, J. Adv. Mater. 2008, 20, 560–565. (3) Richert, L.; Lavalle, Ph.; Vautier, D.; Senger, B.; Stoltz, J.-F.; Schaaf, P.; Voegel, J.-C.; Picart, C. Biomacromolecules 2002, 3, 1170–1178. (4) Boulmedais, F.; Tang, C. S.; Keller, B.; V€or€os, J. Adv. Funct. Mater. 2006, 16, 63–70. (5) (a) Schneider, H. J.; Yatsimirsky, A. K. Principles and Methods in Supramolecular Chemistry; John Wiley & Sons: Chichester, England, 2000. (b) Reinhoudt, D. N.; Crego-Calama, M. Science 2002, 295, 2403–2407. (6) (a) Nijhuis, Ch. A.; Huskens, J.; Reinhoudt, D. N. J. Am. Chem. Soc. 2004, 126, 12266–12267. (b) Nijhuis, Ch. A.; Yu, F.; Knoll, W.; Huskens, J.; Reinhoudt, D. N. Langmuir 2005, 21, 7866–7876. (c) Nijhuis, Ch. A.; Boukamp, B. A.; Ravoo, B. J.; Huskens, J.; Reinhoudt, D. N. J. Phys. Chem. C 2007, 111, 9799–9810. (d) Ling, X. Y.; Reinhoudt, D. N.; Huskens, J. Chem. Mater. 2008, 20, 3574–3578.
13976 DOI: 10.1021/la102026h
β-CD SAMs by electrochemical oxidation of the redox-active Fc end groups. The authors then extended this concept to the reversible assembly of β-CD-functionalized nanoparticles on preadsorbed PPI-(Fc)16 dendrimers at β-CD SAM. This study shows that guest dendrimers can be advantageously used as multivalent “reversible supramolecular glue” to control the attachment/ detachment process of nanostructures at interfaces.6 Fc-functionalized nanotubes were also found to be convenient electroactive building blocks for designing tunable host-guest assemblies by complexation with β-CD SAM.7 Regarding host- or guest-functionalized linear polymers, we and other authors reported the formation of stable multivalent complexes with guest- or host-modified solid surfaces.8,9 However, the possibility of the electrochemical detachment of functionalized linear polymers based on switching the redox state of electroactive guest groups has not been reported yet. One can point out that the processes underlying the adsorption and electrochemical detachment of Fc-functionalized dendrimers/ nanoparticles/nanotubes interacting through complementary groups with β-CD functionalized surfaces are quite different from the ones involving linear polymers. In the case of dendrimers/ nanoparticles/nanotubes, because of the geometrical constraints, only limited numbers of host/guest inclusion complexes can be formed with a functionalized surface. It was shown that for PPI-(Fc)n dendrimers the number of interactions with the β-CD host surface varies from 2 to 7 (or 8) when the number of Fc end groups in the dendrimers varies from 4 to 64.6 For polymers, (7) Chen, Y.-f.; Banerjee, I. A.; Yu, L.; Djalali, R.; Matsui, H. Langmuir 2004, 20, 8409–8413. (8) Crespo-Biel, O.; Peter, M.; Bruinink, Ch. M.; Ravoo, B. J.; Reinhoudt, D. N.; Huskens, J. Chem.;Eur. J. 2005, 11, 2426–2432. (9) Van der Heyden, A.; Wilczewski, M.; Labbe, P.; Auzely, R. Chem. Commun. 2006, 3220–3222.
Published on Web 08/04/2010
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Scheme 1. Schematic Representation of SAM Formation, SAM-β-CD Formation via the Click Reaction, the Adsorption/Desorption of Fc-Modified Polymers (1), and the Chemical Structures of HS-(CH2)11-EG6-N3 (2), HS-(CH2)11-EG4-OH (3), PAH-Fc (4), CHI-Fc (5), β-CD-Alkyne (6), Ethynylferrocene (7), and Triazole Formation (8)
because of their flexibility and longer linear dimension, the number of binding sites with the surface should be much higher. It was demonstrated for poly(isobutene-alt-maleic acid) derivatives containing different numbers of hydrophobic guest groups (from 35 to 164 per polymer chain) that polymer adsorption on β-CD SAM apparently uses most of the hydrophobic groups.8 This may hinder the further detachment of Fc-functionalized linear polymers from a host surface. In the present work, we show the first example of the electrochemically controlled adsorption of Fc-functionalized linear polymers on β-CD-modified SAMs (Scheme 1), with the possibility to detach all specifically adsorbed polymer chains being demonstrated. Because linear (bio)polymer thin films constitute ideal substrates for the specific and nonspecific adhesion of cells,2,3,10,11 the ability to adsorb monolayered and multilayered polymer assemblies reversibly on solid surfaces may pave the way for new applications in the fields of cell growth10,11 and cell sheet engineering.2 The controlled electrodissolution of polymer films also constitutes a platform for surface-initiated drug delivery.4 We selected chitosan (CHI) and poly(allylamine hydrochloride) (PAH) to construct such reversible platforms because these linear polymers have been shown to be promising building blocks for the design of multilayer films as planar substrates for cells adhesion and growth.12 These two polymers possess amine groups that allow for the selective grafting of Fc groups under mild conditions and confer polyelectrolyte character to the chains at acidic pH. Moreover, CHI and PAH present different behavior when they are assembled layer by layer with anionic polyelectrolytes. Although CHI, a natural semirigid polysaccharide derived from chitin, has been shown to form hydrogel-like films because of its exponential growth with the number of deposited layers, films based on PAH have shown linear growth. This suggests the (10) Moby, V.; Boura, C.; Kerdjoudj, H.; Voegel, J.-C.; Marchal, L.; Dumas, D.; Schaaf, P.; Stoltz, J.-F.; Menu, P. Biomacromolecules 2007, 8, 2156–2160. (11) Jessel, N.; Atalar, F.; Lavalle, Ph.; Mutterer, J.; Decher, G.; Schaaf, P.; Voegel, J.-C.; Ogier, J. Adv. Mater. 2003, 15, 692–695. (12) Boudou, Th.; Crouzier, Th.; Ren, K.; Blin, G.; Picart, C. Adv. Mater. 2010, 22, 441–467.
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importance of comparing their adsorption/desorption behavior. Indeed, such a study should provide clues to the elaboration of original self-assemblies stabilized with specific interactions. On the basis of previous results indicating that the growth of assemblies prepared by the alternate deposition of CHI derivatives bearing AD or β-CD moieties depends on the density of AD groups offered by the initial surface,9 we decided to develop conditions for SAM-β-CD formation to allow control of the density of β-CD molecules on solid surfaces. According to the literature, the most common method of functionalizing the solid surface with β-CD is the chemisorption of various monothiolated13,14 and multithiolated14-18 β-CD derivatives on the gold surface, the latter being with14,15 or without16-19 a spacer between thiol/sulfide groups and the β-CD cavity. An alternative method of β-CD immobilization on a gold surface was introduced recently on the basis of the covalent derivatization of a preformed SAM (11-amino-undecanethiol) by di-(N-succinimidyl) carbonate followed by the formation of ester bonds with the primary hydroxyl (13) (a) Henke, C.; Steinem, C.; Janshoff, A.; Steffan, G.; Luftmann, H.; Sieber, M.; Galla, H.-J. Anal. Chem. 1996, 68, 3158–3165. (b) Michalke, A.; Janshoff, A.; Steinem, C.; Henke, Ch.; Sieber, M.; Galla, H.-J. Anal. Chem. 1999, 71, 2528–2533. (c) Endo, H.; Nakaji-Hirabayashi, T.; Morokoshi, S.; Gemmei-Ide, M.; Kitano, H. Langmuir 2005, 21, 1314–1321. (d) Damos, F. S.; Luz, R. S.C.; Tanaka, A. A.; Kubota, L. T. J. Elecroanal. Chem. 2010, 639, 36–42. (14) (a) Nelles, G.; Weisser, M.; Back, R.; Wohlfart, P.; Wenz, G.; MittlerNeher, S. J. Am. Chem. Soc. 1996, 118, 5039–5046. (b) Weisser, M.; Nelles, G.; Wohlfart, P.; Wenz, G.; Mittler-Neher, S. Phys. Chem. 1996, 100, 17893–17900. (c) Weisser, M.; Nelles, G.; Wenz, G.; Mittler-Neher, S. Sens. Actuators, B 1997, 38-39, 58–67. (15) (a) Beulen, M. W. J.; B€ugler, J.; Lammerink, B.; Geurts, F. A. J.; Biemonf, E. M. E. F.; Leerdam, K. G. C.; Van Veggel, F. C. J. M.; Engbersen, J. F. J.; Reinhoudt, D. N. Langmuir 1998, 14, 6424–6429. (b) Beulen, M. W. J.; B€ugler, J.; de Jong, M. R.; Lammerink, B.; Huskens, J.; Sch€onherr, H.; Vansco, G. J.; Boukamp, B. A.; Wieder, H.; Offenh€auser, A.; Knoll, W.; Van Veggel, F. C. J. M.; Reinhoudt, D. N. Chem.;Eur. J. 2000, 6, 1176–1183. (16) D’Annibale, A.; Regioli, R.; Sangiorgio, P.; Ferri, T. Electroanalysis 1999, 11, 505–510. (17) Rojas, M. T.; K€oniger, R.; Stoddart, J. F.; Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 336–343. (18) Choi, S.-W.; Jang, J.-H.; Kang, Y.-G.; Lee, Ch.-J.; Kim, J.-H. Colloids Surf., A 2005, 257-258, 31–36. (19) Domi, Y.; Yoshinaga, Y.; Shimazu, K.; Porter, M. D. Langmuir 2009, 25, 8094–8100.
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groups of β-CD.20 Whereas the formation of compact and homogeneously distributed β-CDs was demonstrated, the redox responses of electroactive guest probes at the SAM-β-CD sensors were shown to appear at very high potentials. The authors suggested that this problem can be due to the high degree of aggregation of the β-CDs that could be overcome by improving the design of the device.20 In the present work, we thus developed another methodology for the formation of the SAM-β-CD on a gold surface based on the selective grafting of a monofunctionalized β-CD derivative on a preformed SAM via the “click” reaction, allowing perfect control of the amount and accessibility of the immobilized β-CD cavities. Our methodology thus relied on a Huisgen 1,3dipolar cycloaddition reaction between β-CD monosubstituted by an alkyne group on the primary face and azide-containing binary SAMs (Scheme 1). This click reaction is efficiently catalyzed by copper(I), resulting exclusively in the formation of 1,4bisubstituted 1,2,3-triazole rings (Scheme 1). Because triazole formation is irreversible and usually quantitative, this method can be advantageously used for surface modification.21 The click reaction was successfully applied to functionalize azide-terminated binary SAMs with Fc21-23 and cell adhesion peptide,24 allowing for control over the density of azide and, in turn, the density of Fc or peptide on the substrates. In this work, we first produced and characterized binary SAMs made of HS-(CH2)11-EG4-OH and HS-(CH2)11-EG6-N3 (EG, ethylene glycol) by the use of a quartz crystal microbalance with dissipation monitoring (QCM-D), cyclic voltammetry (CV), and contact angle measurements. Oligoethylene glycol (OEG)-terminated alkanethiol was chosen as a diluting component of SAMs to minimize nonspecific adsorption phenomena, which is important for control over the specific interactions between host-modified SAMs and guest-containing polymers. Indeed, it was shown that using OEG-terminated SAMs prevents the nonspecific adsorption of proteins on a modified surface.25 It was also demonstrated that terminal OEG chains of alkanethiol SAMs prevent the deposition of strong polyelectrolytes,26 whereas the deposition of weak polyelectrolytes (polyamines) is influenced by the degree of ionization depending on the pH.27 The click functionalization of binary SAMs with β-CD and the electrochemical characterization of the obtained SAM-β-CD are described in the second section of the article. In the third section, we report the study of the adsorption/desorption behavior of CHI and PAH functionalized with Fc moieties, CHI-Fc, and PAH-Fc on a SAM-β-CD platform (Scheme 1). The attachment/detachment process was monitored in situ by QCM-D coupled to electrochemistry.
Experimental Section Materials. Poly(allylamine hydrochloride) (PAH, Mw ≈ 70 000 g/mol), ferrocene (Fc), ferrocene methanol, ferrocenecarboxaldehyde, ethynylferrocene, diisopropylcarbodiimide (DIC), propargylamine, sodium cyanoborohydride (NaCNBH3), hydroxybenzotriazole (HOBt), (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), Na4[Fe(CN)6] 3 3H2O, sodium ascorbate, copper sulfate (CuSO4 3 5H2O), and all other chemicals were purchased from Sigma-Aldrich Fluka. Chitosan (CHI) protasane (20) Campi~na, J. M.; Matrins, A.; Silva, F. Electrochim. Acta 2009, 55, 90–103. (21) Collman, J. P.; Devaraj, N. K.; Chidsey, Ch. E. D. Langmuir 2004, 20, 1051–1053. (22) Collman, J. P.; Devaraj, N. K.; Eberspacher, T. P. A.; Chidsey, Ch. E. D. Langmuir 2006, 22, 2457–2464. (23) Chelmowski, R.; K€afer, D.; K€oster, S. D.; Klasen, T.; Winkler, T.; Terfort, A.; Metzler-Nolte, N.; W€oll, Ch. Langmuir 2009, 25, 11480–11485. (24) Hudalla, G. A.; Murphy, W. L. Langmuir 2009, 25, 5737–5746. (25) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714–10721. (26) Clark, S. L.; Hammond, P. T. Adv. Mater. 1998, 10, 1515–1519. (27) Clark, S. L.; Hammond, P. T. Langmuir 2000, 16, 10206–10214.
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with a degree of N-acetylation (DA) of 0.09 was purchased from FMC BioPolymer AS, Novamatrix (Norway). Its viscosity-average molecular weight was determined to be Mw ≈ 180 000 g/mol from the intrinsic viscosity measured in 0.3 M AcOH/0.1 M AcONa at 25 °C (assuming K = 0.074 and a = 0.76).28 HS(CH2)11-EG4-OH and HS-(CH2)11-EG6-N3 were purchased from Prochimia (Poland). For simplicity, the terms “thiol-PEG-OH” and “thiol-PEG-N3” are used to refer to HS-(CH2)11-EG4-OH and HS-(CH2)11-EG6-N3, respectively, in the sequel. β-Cyclodextrin (β-CD) was kindly supplied by Roquette Freres (Lestrem, France). β-CD monoalkyne was synthesized from a β-CD monocarboxylic acid derivative described previously.29 Synthesis of β-CD Monoalkyne. To a solution of β-CD monocarboxylic acid (0.2 g, 0.165 mmol) in dry DMF (16 mL) were added HOBt (0.046 g, 0.33 mmol), DIC (0.084 g, 0.66 mmol), and propargylamine (0.011 g, 0.215 mmol). The resulting mixture was stirred under nitrogen at room temperature overnight. After evaporation of most of the solvent, the residual syrup was poured into acetone (200 mL). The white precipitate was collected by filtration, washed three times with acetone, and dried to give pure β-CD monoalkyne (0.172 g, 79%). 1 H NMR (400 MHz, D2O): δ 7.65 (d, 1H, J 7 Hz, -CHd), 4.95-4.92 (m, 7H, anomeric protons of β-CD), 4.43-4.25 (m, 2H, CH2-CO), 4.00-3.39 (m, 50 H, other protons of β-CD, CH2-CtH), 2.7 (s, 1H). MS-MALDI-TOF: [M þ Na]þ calculated for C47H74N2O36Na, 1265.39; found, 1265.56. Synthesis of PAH-Fc. PAH (0.1 g, 1.069 mmol repeating units) was dissolved in H2O (7 mL). After 4 h of stirring, ethanol (EtOH) (4 mL) was slowly added to the polymer solution. Then, ferrocenecarboxaldehyde (0.0183 g, 0.0855 mmol) dissolved in EtOH (1 mL) was added dropwise. The pH of the mixture was adjusted to 5.1 using a 0.5 M NaOH aqueous solution, and NaCNBH3 (0.1 g, 1.6 mmol) was added. After 24 h of stirring at room temperature, the pH was adjusted to 7.0 using 0.1 N NaOH. Then, the reaction mixture was ultrafiltered through an Amicon YM10 ultramembrane in an 8200 Amicon cell equipped with an Amicon RS4 tank filled with an EtOH/H2O (2/3 v/v) mixture and then with pure water. The ultrafiltration was stopped when the filtrate conductivity was lower than 10 μS, and PAH-Fc was recovered by freeze drying as a yellow powder (0.095 g, yield 82%). The degree of substitution of PAH-Fc was found to be to 0.08 ( 0.01 by 1H NMR. 1 H NMR (400 MHz, D2O): δ 4.38-4.17 (m, protons of ferrocene), 2.85 (m, CH2 of PAH), 1.90-0.97 (m, CH and CH2 of PAH). Synthesis of CHI-Fc. CHI protasan (0.1 g, 0.6 mmol of repeating units) was first solubilized in aqueous 0.2 M CH3CO2H (7 mL). After stirring overnight at room temperature to allow the perfect solubilization of chitosan, absolute ethanol (4 mL) was slowly added, followed by the addition of ferrocenecarboxaldehyde (0.0104 g, 0.0485 mmol) solubilized in 1 mL of EtOH. The pH was then adjusted to 5.1 by the dropwise addition of 0.5 M NaOH, and then NaCNBH3 (0.0565 g solubilized in 1 mL of water) was added. The reaction mixture was stirred at room temperature for 24 h. The reaction was quenched by the precipitation of the CHI derivative with 0.5 M NaOH, and the yellow product was collected by centrifugation (6000 rpm for 12 min) and washed successively with 3/2, 7/3, and 9/1 (v/v) ethanol/water mixtures using the same centrifugation procedure (6000 rpm for 12 min) for each washing step. The precipitate was dissolved in 30 mL of ethanol and filtered. After drying for 24 h at room temperature, CHI-Fc was recovered as a red powder (0.094 g) in 89% yield. Owing to the overlapping of the NMR signals of HOD and Fc at 80 °C and errors in integration values at lower (28) Brugnerotto, J.; Desbrieres, J.; Roberts, G.; Rinaudo, M. Polymer 2001, 42, 9921–9927. (29) Charlot, A.; Heyraud, A.; Guenot, P.; Rinaudo, M.; Auzely-Velty, R. Biomacromolecules 2006, 7, 907–913.
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Dubacheva et al. temperatures, the degree of substitution of CHI-Fc could not be determined by 1H NMR. It was thus derived from elemental analysis. From the hydration rate of CHI-Fc determined by thermogravimetry (9.9% w/w H2O) and the elemental analysis data, the DS value was estimated to be 0.06 as indicated below. Elemental analysis calculated for C6.77H12.05O4.08NFe0.06 3 H2O: C, 41.99; H, 6.73; N, 7.24; Fe, 1.73; found: C, 41.06; H, 6.81; N, 6.96; Fe, 1.75. Substrate Preparation. The electrochemically active (geometrical) area of gold-coated quartz crystals (Q-Sense, Sweden) was determined by cyclic voltammetry at 25 °C in potassium phosphate buffer (I = 0.1 M, pH 8.0) from the slope of the linear plot of the anodic peak current versus (scan rate)1/2 for the reversible electrooxidation of ferrocene methanol (0.2 mM). The diffusion coefficient measured for ferrocene methanol was found to be 6.7 10-6 cm2 s-1. Using this value, we determined A = 1.01 ( 0.02 cm2 (ø = 1.13 ( 0.01 cm). The roughness of the gold surface was studied by atomic force microscopy (AFM). AFM images (SI, Figures S1 and S2) were analyzed using Gwyddion 2.15 software, with the roughness being determined as the ratio of a real surface area to a geometrical one. The maximal roughness obtained for new gold crystals was 1.01, with the maximal rms being 0.79 nm. Gold crystals exposed several times to a regeneration procedure exhibited a maximal roughness of 1.04, with the maximal rms being 2.01 nm. Because roughness factors were found to be less than 5%, the gold surface was assumed to be flat. SAM Formation and Click Reaction. SAM-coated gold electrodes were prepared by dipping a preliminary cleaned gold surface (new or regenerated not more than five times) overnight in a solution of the desired thiol or a mixture of thiols (1 mM total thiol concentration in absolute ethanol). Two thiols were used for SAM preparation: HS-(CH2)11-EG4-OH and HS-(CH2)11-EG6N3. The cleaning procedure included UV-ozone treatment for 5 min followed by the immersion of sensors in ethanol for 20 min with stirring. The regeneration of gold sensors was performed by keeping them overnight in a 2% SDS solution followed by ultrasonication in ethanol for 5 min, placing them in 2% Hellmanex for 5 min, UV-ozone treatment for 10 min, and finally immersion in ethanol for 20 min with stirring. To obtain SAM-β-CD (or SAM-Fc) on a gold surface, after overnight SAM formation, gold sensors were rinsed with ethanol, dried under nitrogen, and immersed at room temperature in a water/t-butanol (1:2) solution containing 1 mM β-CD monoalkyne (or 1 mM ethynylferrocene), 1 mM CuSO4, 1 mM TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, which is a commonly used ligand for stabilizing the Cu(I) catalyst), and 7 mM sodium ascorbate (which is necessary for generation of the Cu(I) catalyst) for 5 h. Exposure to light was kept to a minimum in order to prevent photooxidation of the SAM. No attempt was made to exclude the oxygen. After reaction, monolayers were rinsed with ethanol, water, dichloromethane, and ethanol to ensure that all physisorbed molecules were washed off. NMR Spectroscopy. 1H NMR experiments were performed using a Bruker DRX400 spectrometer operating at 400 MHz. Chemical shifts (δ) were given relative to external tetramethylsilane (TMS = 0 ppm), and calibration was performed using the signal of the residual protons of the solvent as a secondary reference. Deuterium oxide was obtained from SDS (Vitry, France). Mass Spectrometry. MALDI-TOF measurements were performed on a Bruker Daltonics Autoflex apparatus using 2,5dihydroxybenzoic acid as a matrix for the analysis of modified β-CD. Thermogravimetry. The water content of CHI-Fc was determined by thermogravimetry (Setaram, France). Elemental Analysis. Elemental analysis of CHI-Fc was performed by the Service Central d’Analyze du CNRS (Solaize, France). Langmuir 2010, 26(17), 13976–13986
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Contact Angle Measurements. The contact angle of water was measured using a FTA188 video tensiometer (First Ten A˚ngstoms, Inc.). Contact angle measurements were performed directly on the gold sensors with a 2 μL drop of ultrapure water. The contact angle value (θ) was calculated from the average of five measurements, with drops being positioned on different places on the gold surface. Electrochemistry. Electrochemical experiments were performed with a conventional three-electrode potentiostatic system. The equipment was a CHI 440 potentiostat (CH-Instruments, Inc.). Electrode potentials were measured with reference to Ag/ AgCl/KCl (3 M). The counter electrode was platinum, and the working electrode was the gold sensor activated and functionalized as described previously. The electrochemical cell was homemade, with the working electrode being at the base of the cell covered with the electrolyte solution in which the counter and reference electrodes were immersed. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). QCM-D measurements were performed at 24 °C using gold-coated quartz crystals (Q-Sense, Sweden) with a 5 MHz resonance frequency as the substrate. Before the experiment was started, the resonance frequency and dissipation found for each overtone were set equal to zero. All QCM-D experiments were repeated at least two times. Monitoring of SAM formation was performed in absolute ethanol using QCM-D D300 (Q-Sense, Sweden). The measurements were made in batch mode using pure ethanol as a solvent. QCM-D signals were recorded for the fundamental resonance frequency as well as for the third, fifth, and seventh overtones (n = 3, 5 and 7). Data were fitted according to the Sauerbrey equation, which can be used to obtain adsorbed masses (ΔΓ) in the case of homogeneous, quasi-rigid films with low thickness ΔΓ ¼ - CΔFn=n -2
ð1Þ 1-
with a mass sensitivity of C = 17.7 ng cm Hz for F1 = 5 MHz. All experiments in aqueous solutions were performed using the QCM-D E4 system equipped with four flow chambers (Q-Sense, Sweden). Overtones n = 3, 5, 7, 9, 11, and 13 were recorded in addition to the fundamental resonance frequency to improve stability and sensitivity. QCM-D measurements were made in flow mode (50 μL/min) using 10 mM HCl with 0.15 M NaCl (pH 2) as the medium. All solutions were also thermostatted at 24 °C using a thermomixer (Ependorf, France). All polymers were dissolved overnight in 10 mM HCl with 0.15 M NaCl (pH 2) at room temperature. SAM-β-CD-functionalized gold sensors were mounted in the QCM-D chamber. Experimental data were fitted according to the viscoelastic model of Voinova30 using Q-tools modeling software. χ2 was defined by the Q-tools software as χ2 ¼
X
½ðYtheory, i - Ymeas, i Þ=σi 2
ð2Þ
i
where the sum is taken over all measured points (index i), σi is the measurement error, or standard deviation of the ith data point, and Ytheory,i and Ymeas,i are the calculated and measured Y values, respectively. A combination of electrochemical and QCM-D measurements (E-QCM-D) was performed using the electrochemical QCM-D module (Q-Sense, Sweden) connected to a CHI 440 potentiostat (CH-Instruments, Inc.) as described above. Electrode potentials were measured with reference to Ag/AgCl/KCl (3 M) (Q-Sense, Sweden). The counter electrode was platinum, and the working electrode was the gold sensor (Q-Sense, Sweden). The working electrode mounted in an electrochemical QCM-D module was covered with the electrolyte solution in which the counter and the reference electrodes were immersed. (30) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scr. 1999, 59, 391–396.
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Figure 1. QCM-D response: frequency (a) and dissipation (b) shifts (fifth overtone) obtained for the adsorption of thiol-PEG-OH (thick lines) and thiol-PEG-N3 (thin lines) on a gold surface in ethanol at room temperature; V, thiol injection; v, rinsing. Variations in dissipation over the time range of 250-500 min are attributed to temperature changes occurring due to the long experiment time.
Results and Discussion Characterization of SAMs. The formation of SAMs on a gold surface was characterized by QCM-D, cyclic voltammetry (CV), and contact angle measurements (CAM). Figure 1 presents typical QCM-D profiles obtained during the adsorption of thiolPEG-OH and thiol-PEG-N3 on gold sensors using ethanol as a solvent. For clarity, only the data recorded at one overtone (n = 5) are shown, and the two other overtones (n = 3, 7) exhibited the same behavior. It can be seen that after a fast negative shift corresponding to the fixation of alkanethiols on the surface, normalized frequency F5/5 decreases slowly during the following hours. We interpret this as the rearrangement of the monolayers into highly ordered structures. Indeed, two distinct types of adsorption kinetics have been observed during alkanethiol adsorption onto Au(111): a very fast step, which takes a few minutes, by the end of which the thickness can reach 80% of its maximum, and a slow step, which takes several hours, at the end of which the thickness reaches its final value.31 Rinsing the measurement chamber with ethanol after the overnight adsorption of alkanethiols did not induce a significant change in frequency. Therefore, we demonstrated that SAMs obtained with thiol-PEG-OH or thiolPEG-N3 exhibit high stability during both monolayer formation and rinsing. Regarding energy dissipation, different behaviors were observed for thiol-PEG-OH and thiol-PEG-N3. In the case of thiol-PEG-OH, the dissipation decreases from 0.5 10-6 to 0.1 10-6 after ethanol rinsing (Figure 1b), which fully agrees with the formation of a very rigid thiol-PEG-OH SAM. In the case of thiol-PEG-N3, a higher dissipation of 0.8 10-6 was recorded even after rinsing with ethanol (Figure 1b), which could be a consequence of longer EG moieties (6 EG as compared to 4 EG in thiol-PEG-OH) as well as different interfacial interactions of the SAM with bulk ethanol as a result of the presence of azide end functions. However, it was observed that despite this relatively high dissipation, normalized frequency shifts were identical for different harmonic numbers (SI, Figure S3), which is in good agreement with rigid behavior. Therefore, we used the Sauerbrey equation to calculate a surface acoustic mass corresponding to SAM formation. (31) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321–335.
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Using the averaged data from two different QCM-D experiments and taking into account normalized frequency shifts at three overtones (n = 3, 5, and 7), we obtained a surface acoustic mass of 230 ( 13 ng/cm2 for the thiol-PEG-OH SAM and 360 ( 30 ng/cm2 for thiol-PEG-N3 SAM formation. Assuming in a first approximation that the contribution of the solvent acoustically coupled to the monolayer is negligibly small, we obtain (6.0 ( 0.3) 10-10 mol/cm2 for the thiol-PEG-OH SAM and (7.3 ( 0.6) 10-10 mol/cm2 for the thiol-PEG-N3 SAM. When a monolayer √ √of well-packed alkanethiol is entirely composed of the 3 3 R30° structure of Au(111) and so consists of 4.67 1014 molecules/cm2, 7.76 10-10 mol/cm2 is obtained.32 Therefore, the surface concentration of thiol-PEG-N3 appears to be in better agreement with the theoretical estimate than that of thiol-PEG-OH. One explanation is that thiol-PEG-N3 is able to form more densely packed SAMs on gold surfaces than thiol-PEG-OH: the average molecular surface areas calculated from the surface concentration are 22.8 A˚2 for thiol-PEG-N3 and 27.7 A˚2 for thiol-PEG-OH. Using X-ray photoelectron spectroscopy, Grunze and coworkers33 characterized the packing density of PEG-terminated SAMs obtained with HS-(CH2)11-EG2-OH, HS-(CH2)11-EG3OH, and HS-(CH2)11-EG6-OH: molecular surface areas were found to be respectively 25.4, 26.4, and 28.9 A˚2. In view of the decreasing packing density of EGn-OH- and EGn-OCH3-terminated SAMs with increasing length of the PEG tail,33 the molecular surface area determined by QCM-D for SAM-EG4-OH (27.7 A˚2) is in good agreement with the literature, which reinforces our hypothesis that the contribution of ethanol acoustically coupled to thiol-PEG-OH SAM is negligibly small. To investigate the structure of the SAMs and their molecular organization, CV experiments were performed in aqueous electrolytic solutions using bare gold electrodes and gold electrodes modified with the thiol-PEG-OH SAM, the thiol-PEG-N3 SAM, and a mixed SAM prepared with a thiol-PEG-N3/thiol-PEG-OH ratio in solution of 0.50. First, we examined the interfacial capacitance of bare and SAM-covered gold electrodes by recording their CV responses in 0.1 M potassium phosphate buffer (pH 7.0) at different scan rates. As expected, the capacitive current intensities were proportional to the potential scan rate (SI, Figure S4), from which the interfacial capacitances (C) were estimated: CAu = 51.2 ( 0.2 μF cm-2, CSAM-PEG-N3 = 5.4 ( 0.1 μF cm-2, CSAM-PEG-OH = 6.9 ( 0.1 μF cm-2, and Cmixed SAM = 5.6 ( 0.1 μF cm-2. The fact that we obtained a lower C value for thiol-PEG-N3 than for thiol-PEG-OH can be explained by their different lengths (d) because the behavior of alkanethiol SAMs appears to be similar to that predicted by the Helmholtz theory (C µ d-1).34 Another reason can be the difference in packing density that was demonstrated by QCM-D experiments (Figure 1). The fact that the interfacial capacitance obtained for SAM-covered gold electrodes is 7-10 times less when compared to that of bare gold proves a homogeneously distributed and rather close monolayer structure of the SAMs. Thus, the interfacial capacitance of gold electrodes covered with (3-mercaptopropyl)sulfonate (MPS) SAM in phosphate buffer is divided only by a factor of 2 as compared to that of bare gold.35 A QCM-D and electrochemical study showed that this result is due to the open structure of MPS (32) Strong, L.; Whitesides, G. M. Langmuir 1988, 4, 546–558. (33) Herrwerth, S.; Eck, W.; Rainhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9359–9366. (34) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, Ch. E. D. J. Am. Chem. Soc. 1987, 109, 3559–3568. (35) Mokrani, C.; Fatisson, J.; Guerente, L.; Labbe, P. Langmuir 2005, 21, 4400–4409.
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Figure 2. CVs recorded for a bare gold electrode (a) and for gold electrodes covered with the thiol-PEG-OH SAM (b), thiol-PEG-N3 SAM, (c) and mixed SAM obtained using the mixture of 50% thiol-PEG-OH and 50% thiol-PEG-N3 (d) in 0.1 M potassium phosphate buffer (pH 7.0) containing 5.0 10-4 M Fe(CN)64-. The scan rate is 100 mV/s. The given scale (20 μA) is the same in a-d for clarity.
SAMs (surface coverage of 60%) that can be penetrated by the ionic species.35 To gain further information about the molecular organization of SAMs on gold surfaces, we investigated the CV responses of modified gold electrodes using Fe(CN)64- as an electrochemically reversible redox probe diffusing in solution. Figure 2 shows the comparison between the typical voltammetric profiles obtained on gold electrodes covered with the thiol-PEG-OH SAM, thiolPEG-N3 SAM, and mixed SAM (with a thiol-PEG-N3/thiolPEG-OH ratio of 0.50 in solution) and that on a bare gold electrode. There is a dramatic difference between the currentpotential (I-E) responses for the electroactive substance at SAMcovered electrodes and a bare gold surface. The fact that the electrochemistry of Fe(CN)64- is strongly depressed on modified electrodes confirms the rather homogeneous distribution of SAMs on a gold surface. The SAM surface coverage percentage can be quantified by the parameter θ whereas (1 - θ) represents the corresponding percentage of pinholes and defects. Assuming that all of the current is passing through the bare spots on the electrode, it has been shown36 that at a high surface coverage (θ ≈ 1) the decrease in the apparent heterogeneous electron-transfer rate constant for a redox probe diffusing in a solution is related to θ and is given by the following expression k°SAM ¼ ð1 - θÞk°Au
ð3Þ
Figure 3. Relation between the thiol-PEG-N3 mole fraction in the solution used to prepare binary SAMs and the thiol-PEG-N3 mole fraction on a gold surface after SAM formation obtained by CAM. The solid line represents the nonlinear fitting (sigmoidal). The number of samples measured is 3 for fthiol-PEG-N3 = 0, 30, 50, and 100%, 15 for fthiol-PEG-N3 = 20%, and 1 for other fthiol-PEG-N3 species in solution.
surface by SAMs with a very small number of pinholes or defects (40%, higher values are detected for the surface than for the solution. It is known that depending on the nature of the thiols their ratio in solution differs from that in the resulting SAM.22,24 For instance, using the same thiol-PEG-N3 and a shorter thiol-PEG-OH (HS-(CH2)11-EG3OH), Hudalla and Murphy24 demonstrated that the mole fraction of thiol-PEG-N3 in the resulting SAM is higher than in the solution along the whole range of thiol azide concentrations. They also showed that the adsorption of thiol-PEG-N3 is faster than that of thiol-PEG-OH, estimating a ratio of 1.69 for the adsorption rate constants. Similar curve shapes as presented in Figure 3 were obtained by Collman and co-workers22 for different pairs of thiols such as HS-(CH2)9-CH3 and HS-(CH2)11-N3, HS-(CH2)11-OH and HS-(CH2)11-N3, and HS-(CH2)10-COOH and HS-(CH2)11-N3. However, for HS-(CH2)7-CH3 and HS-(CH2)11-N3 the curve profile was found to be similar to that reported by Hudalla and Murphy.22,24 Therefore, the extent of preferential alkanethiol adsorption during binary SAM formation cannot be generalized because it is alkanethiol-dependent. However, we showed in this work that the number of azide functional groups present in pegylated SAM can be easily controlled by varying the ratio of thiol-PEG-N3 to thiol-PEG-OH in the solution used to form SAMs. Functionalization of SAMs with β-CD. In this section, we electrochemically characterized two processes: (1) the click reaction used to functionalize azide-terminated pegylated SAMs with β-CD and (2) the inclusion of guest molecules by β-CDs attached to the surface after the click reaction. In both cases, Fc was used as an electroactive probe, also acting in the second case as a guest molecule to be bound by β-CD. It was demonstrated that the efficiency of click modification of azide-terminated SAMs can be controlled electrochemically using alkyne derivatives of redox probes such as Fc.22 We thus studied the efficiency of the click modification of binary pegylated SAMs obtained in our work by performing the click reaction between SAMs having various numbers of azide groups and ethynylferrocene. The number of Fc molecules attached to the surface was determined electrochemically by calculating the anodic charge values associated with the conversion of Fc to Fc 3 þ. It is known that the electroactive properties of Fc-terminated SAMs are very sensitive to the ionic composition of the solution. Such poorly hydrated anions as PF6- and ClO4- are the most preferable ones 13982 DOI: 10.1021/la102026h
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Figure 4. Relation between the thiol-PEG-N3 mole fraction in the solution used to prepare binary SAMs and the surface concentration of Fc after SAM formation and click functionalization calculated from the voltammetric response in 0.1 M KPF6. The scan rate is 100 mV/s. The solid line represents the nonlinear fitting (sigmoidal). The number of samples measured is 3 for fthiol-PEG-N3 = 0, 30, 50, and 100%, 15 for fthiol-PEG-N3 = 20%, and 1 for other fthiol-PEG-N3 species in solution.
for studying the electroactivity of SAM-Fc due to the formation of surface ion pairs between the ferricenium cation (Fc 3 þ) and hydrophobic anions that directly influence the stability of SAMFc as well as the positions of Epa and Epc and the symmetry of CV curves.38 Therefore, we used 0.1 M KPF6 (pH 5.2) as a medium to determine the surface concentration of Fc. All CVs obtained for SAM-Fc in 0.1 M KPF6 exhibited stable behavior during repetitive cycling. The relation between the Fc surface concentration and the thiol-PEG-N3 molar fraction in solution used to prepare SAMs is presented in Figure 4. One can notice that the evolution of the Fc surface concentration follows the same trend as that obtained for the azide surface concentration (Figure 3) (i.e., the Fc surface concentration increases proportionally to the azide molar fraction in a binary SAM). This observation demonstrates the efficiency and selectivity of the interfacial click reaction between binary azide-terminated SAMs and ethynylferrocene. The maximal surface coverage of Fc determined from three identical electrochemical experiments using gold sensors coated with SAM based on 100% thiol-PEG-N3 is (6.1 ( 0.3) 10-10 mol/cm2 (SI, Figure S8). By treating the Fc group as a sphere with a 0.66 nm limiting diameter, a coverage of 4.5 10-10 mol/cm2 is obtained.39 Thus, the surface density of Fc in SAMs is greater in our case than that of the limiting model, which is in agreement with the literature and can be attributed to a twisting about the long-chain terminus to accommodate a higher packing density of the Fc moieties.40 By taking into account the surface concentration of thiol-PEG-N3 determined by QCM-D ((7.3 ( 0.6) 10-10 mol/cm2), 84% of the thiol-PEG-N3 monolayer functionalized by redox-active Fc molecules is obtained. The first explanation of this limit is that the surface concentration of thiol-PEG-N3 determined by QCM-D is a little overestimated because we did not take into account the presence of solvent molecules that can be acoustically coupled with SAM. A second explanation is that the steric bulk of Fc prevents the further conversion of azides to triazoles. Thus, Collman and co-workers determined that only 55% of SAMs formed by HS-(CH2)11-N3 can be functionalized with Fc because of this limitation.22 (38) Valincius, G.; Niaura, G.; Kazakevicien_e, B.; Talaikyt_e, Z.; Kazem_ekait_e, M.; Butkus, E.; Razumas, V. Langmuir 2004, 20, 6631–6638. (39) Seiler, P.; Duntitz, J. D. Acta Crystallogr. 1979, B35, 1068–1074. (40) Walczak, M. M.; Popenoe, D. D.; Deinhammer, R. S.; Lamp, B. D.; Chung, Ch.; Porter, M. D. Langmuir 1991, 7, 2687–2693.
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Figure 5. CVs recorded for gold sensors coated with SAM-β-CD in 0.2 M Na2SO4 (---) and in 0.2 M Na2SO4 containing 5 μM Fc (-) at v = 500 mV/s (a). Anodic peak current vs scan rate obtained for the SAM-β-CD-coated sensor in 0.2 M Na2SO4 containing 5 (9), 20 (b), and 60 (2) μM Fc (b). The solid lines represent the linear fittings.
From these results, it can be concluded that the curve in Figure 3 can be used as a calibration plot to control the molar fraction of azide groups present in mixed SAMs and, in turn, the surface concentration of Fc groups attached to the surface after the click reaction. Because a direct transposition of the Fc-alkyne reactivity results to the β-CD-alkyne molecules reactivity cannot be made, we performed additional CAM experiments with SAM-β-CD samples obtained using SAMs with different numbers of azide end groups (SI, Figure S7). We observed an exponential decrease in the contact angles measured on a SAM-β-CD-coated gold surface upon increasing the thiol-PEG-N3 surface concentration. Contact angles measured after the click reaction between azide-containing SAMs and β-CD-alkyne decrease from 32 ( 1° (0% thiol-PEGN3) to 15 ( 1° (23% thiol-PEG-N3). The observed decrease in the contact angles corresponds to the stepwise increase in the number of surface-attached β-CDs, which exhibit hydrophilic exterior properties. We did not detect a further decrease in contact angles at higher thiol-PEG-N3 surface concentrations, which can be attributed to the formation of a β-CD monolayer preventing further conversion of the remaining azide end functions to β-CD ones. However, the obtained curve (SI, Figure S7) shows that the surface concentration of β-CD molecules can be controlled by varying the surface concentration of thiol-PEG-N3 in the range from 0 to 25%, which corresponds to the molar fraction of thiolPEG-N3 in solution ranging from 0 to 30% (Figure 3). Moreover, the amount of surface-attached Fc also increases exponentially in this concentration range (Figure 4). On the basis of these results, we assumed that in a first approximation the initial part of the curve presented in Figure 3 can be used as a calibration plot to control the molar fraction of azide groups and, in turn, surfaceattached β-CD groups. To obtain both a high surface density of β-CD molecules and their spatial separation, we used a theoretical estimate of 8.1 10-11 mol/cm2 for the maximum number of hexagonally close packed β-CD molecules with a flat orientation.19 This value corresponds to 11 ( 1% of the maximal amount of thiol-PEGN3 deposited on a gold surface ((7.3 ( 0.6) 10-10 mol/cm2 as determined by QCM-D, Figure 1). According to the calibration plot (Figure 3), this surface concentration corresponds to 20% of thiol-PEG-N3 in the solution used to prepare binary SAMs. Therefore to produce SAM-β-CD, we performed the click reaction between β-CD-alkyne and an azide-terminated mixed SAM using a thiol-PEG-N3/thiol-PEG-OH molar ratio in the solution equal to 0.20. Because of the existence of some variations in the final azide surface concentrations (previous section), each mixed SAM prepared using thiol-PEG-N3/thiol-PEG-OH = 0.20 in solution was first analyzed by CAM and only the samples exhibiting azide surface concentration in the range from 10 to 15% (as determined from CAM) were used. Langmuir 2010, 26(17), 13976–13986
Because Fc is able to form an inclusion complex with β-CD in aqueous media, the binding properties of SAM-β-CD can be determined electrochemically using Fc as a redox probe.17-19 Several electrochemical methods are usually used to perform this characterization, all of which are based on immersing SAM-βCD-coated sensors in a 0.2 M Na2SO4 solution containing Fc in low concentrations.17-19 The majority of these techniques is devoted to the determination of the electroactive surface coverage of Fc from the anodic charge associated with Fc conversion, followed by plotting it as a function of Fc concentration and the calculation of maximum Fc surface coverage.17,18 More complicated methods are also reported in which the Fc surface density is not determined directly from the anodic charge value but by using a sweep rate dependence of the anodic peak current or chronocoulometry that allowed the avoidance of the contributions from Fc in solution.19 To study the inclusion properties of SAM-β-CD, we subjected β-CD-modified gold electrodes to voltammetric experiments in 0.2 M Na2SO4 solutions containing Fc in concentrations low enough that the diffusion-controlled oxidation of dissolved Fc molecules does not give rise to detectable currents in voltammetric experiments.17,18 Figure 5a shows the voltammetric responses of gold electrodes covered with SAM-β-CD in the absence and the presence of 5 μM Fc. The voltammetric response of electrodes covered with SAM-β-CD clearly shows the Fc/Fc 3 þ electroactive couple that is sustained upon repetitive cycling and is characterized by Epa = 0.340 V and Epc = 0.145 V. It can be noted that the same peak-potential values were obtained for other Fc concentrations in the range of 5-60 μM. Integration of the anodic wave affords an electroactive Fc coverage of (2.0 ( 0.3) 10-11 mol/cm2 (calculated from three electrochemical experiments done under the same conditions). This value is 2 times greater than that obtained under the same conditions (0.2 M Na2SO4 with 5 μM Fc, 500 mV/s scan rate) for β-CD SAM produced by the direct adsorption of per-6-thio-βCD on a gold surface.17 Figure 5b shows linear plots obtained for the anodic peak current intensity as a function of the potential scan rate for Fc concentrations ranging from 5 to 60 μM. On the basis of this result and the electrochemical study of SAM permeability properties (see previous section), we assumed that Fc molecules responsible for the observed faradic response are confined to the electrode surface (i.e., in our case, the response does not result from the oxidation of dissolved Fc molecules).18 In addition, the release of the included guest molecules upon oxidation, which is evidenced by the much lower cathodic charge compared to the anodic one (Figure 5a), proves the cyclic adsorption/desorption of Fc/Fc 3 þ on the SAM-β-CD based on monovalent host-guest interactions. The same asymmetric voltammogram shape was reported for Fc molecules attached to a DOI: 10.1021/la102026h
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Figure 6. Measured Fc surface concentration on a SAM-β-CD-coated electrode as a function of Fc concentration in the solution (a) and the corresponding Langmuir plot (b). Medium, 0.2 M Na2SO4; scan rate, 500 mV/s. The solid lines represent the nonlinear (hyperbole) (a) and linear (b) fittings.
Figure 7. Frequency Fn/n (-) and dissipation Dn (---) shifts recorded at six overtones n = 3, 5, 7, 9, 11, and 13 (frequency and dissipation shifts increase with overtone number) during the adsorption/desorption of PAH-Fc (a) and CHI-Fc (b) on a SAM-β-CD-coated gold surface: V, injection of polymer; v, rinsing; T, applying þ0.55 V; triangles represent moments when CVs are recorded (solid ones correspond to CVs presented in Figure 8); 24 °C; 50 μL/min; 10 mM HCl with 0.15 M NaCl; and pH 2.
β-CD SAM produced by the direct chemisorption of per-6-thio-βCD on a gold surface.19 Thus, surface concentrations of Fc were determined directly from the anodic charge associated with Fc oxidation (Figure 6). The graph obtained for the Fc surface concentration as a function of Fc concentration in solution exhibits a shape similar to that expected for a Langmuir adsorption isotherm (Figure 6a). Because of the low solubility of Fc in aqueous media, it is not possible to determine the binding saturation region in which the Fc surface coverage would remain constant at increasing Fc concentrations in solution. However, these data can be successfully treated according to the equation C=Γ ¼ 1=ðKΓmax Þ þ C=Γmax
ð5Þ
17-19
where C is the Fc bulk derived from the Langmuir isotherm, concentration, Γ is the Fc surface coverage determined from the experimental data, Γmax is the maximum Fc surface coverage, and K is the equilibrium constant for the adsorption process. Linear regression analysis of the Langmuir plot presented in Figure 6b yields an equilibrium constant from the intercept of K = (5.9 ( 0.5) 104 M-1, which is 1 order of magnitude greater than that between Fc and β-CD in solution (4 103 M-1).41 The same discrepancy, explained by the negative change in entropy after β-CD immobilization, is reported for β-CD SAMs obtained with per-6-thio-β-CD: K = 3.9 104 M-1,17 K = (7.6 ( 1.3) 104 M-1.19 The maximum amount of surface-attached Fc calculated from the linear slope (eq 5, Figure 6b) is (8.6 ( 0.5) 10-11 mol/cm2. The linear regression of the Langmuir isotherm reported in the literature yielded the following maximum amounts of Fc attached (41) Osella, D.; Carretta, A.; Nervi, C.; Ravera, M.; Gobetto, R. Organometallics 2000, 19, 2791–2797.
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to β-CD SAMs produced by the chemisorption of per-6-thio-βCD on a gold surface: (2.5 ( 1.0) 10-11 mol/cm2,17 4.7 10-11 mol/cm2,18 and (6.8 ( 0.3) 10-11 mol/cm2.19 According to CAM, the SAM used in this experiment contained ∼15% thiolPEG-N3. Because the maximal surface concentration of thiolPEG-N3 was determined to be 7.3 10-10 mol/cm2 (Figure 1), ∼15% of the immobilized thiol-PEG-N3 corresponds to the maximal thiol-PEG-N3 amount of 1.16 10-10 mol/cm2. Therefore, at least 80% of β-CD molecules that hypothetically attached to the surface after the click reaction are able to form an inclusion complex with Fc. The fact that almost all β-CDs are accessible for binding Fc, along with common (not overstated) values obtained for the Fc oxidizing potential (Figure 5a), provides evidence of the homogeneous spatial distribution of β-CDs that could not be achieved by using a one-component SAM followed by chemical derivatization.20 Indeed, the obtained amount of surface-attached Fc ((8.6 ( 0.5) 10-11 mol/ cm2) is very close to the theoretically estimated surface concentration of hexagonally close-packed β-CDs with a flat orientation (8.1 10-11 mol/cm2). This SAM-β-CD platform was further used to study the adsorption behavior of Fcmodified linear polymers and its control by applying an electric field. Polymer Attachment/Detachment on SAM-β-CD. Using QCM-D coupled to electrochemistry (E-QCM-D), we studied the in situ adsorption and electrochemical detachment of CHI-Fc (DS = 0.06) and PAH-Fc (DS = 0.08) on SAM-β-CD. Because CHI and CHI-Fc are soluble only at pH
