Study of Peptide Dendrimers Having a Ferrocene Core Supported on

binding to a functionalized nanostructured surface. Mahmoud Labib , Sanela Martić , Patrick O. Shipman , Heinz-Bernhard Kraatz. Talanta 2011 85, ...
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Langmuir 2006, 22, 10515-10522

10515

Study of Peptide Dendrimers Having a Ferrocene Core Supported on Mercaptoundecanoic Acid† Francis E. Appoh, Yi-Tao Long, and Heinz-Bernhard Kraatz* Department of Chemistry, UniVersity of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan, Canada S7N 5C9 ReceiVed April 25, 2006. In Final Form: August 4, 2006 Hydrogen-bonding interactions between the carboxylic acid groups of mercaptoundecanoic acid (MUA) coated gold substrates and the ester surface of peptide dendrimers allows the formation of glutamic acid dendrimers films. Dendrimer films were prepared for generations 1-6 (G1-G6) and analyzed by spectroscopic and electrochemical techniques. Electrochemical studies using cyclic voltammetry and differential pulse voltammetry show that all films except those of G6 were electrochemically active. Lack of activity of G6 films is rationalized by the total encapsulation of the ferrocene redox probe by the dendritic sheath and lack of ion pairing, which prevents its oxidation.

Introduction Dendrimers have been exploited in an attempt to rationalize some of the properties of naturally occurring proteins. In particular, dendrimers having a redox-active core exhibit some of the properties displayed by some redox proteins. For example, in redox proteins, the redox properties of the redox group are modulated by the peptide matrix,1 whereas in dendrimers possessing a redox-active core, the dendrimer sheath influences the redox properties of the core. For high dendrimer generations, the central core is completely encapsulated and thus isolated from the exterior by the dendrimer sheath.2-7 However, there is a general lack of generality of this phenomenon which is rationalized by different conformational changes that may occur in the dendrimer sheath or by orientation effects between the dendrimer and the electrode surface.8 Some studies of redoxactive dendrimers immobilized on surfaces have appeared in the literature.9,10 In general, the resulting dendrimer films possess a high mechanical stability and can be functionalized without any loss of dendrimer from the surface.11,12 This is perhaps not too surprising since films prepared from redox proteins, such as Cyt c, are robust and can maintain their redox activity in the presence of redox mediators such as bipyridines.13-15 However, †

Part of the Electrochemistry special issue. * To whom all correspondence should be addressed. E-mail: kraatz@ skyway.usask.ca. (1) Andreas, R. P.; Bielefiled, J. D.; Henderson, J. I.; Janes, D. B.; Kolgunta, V. R.; Kubiak, C. P.; Mahoney, W.; Osifchin, R. G. Science 1996, 271, 16901693. (2) Cardona, C. M.; Kaifer, A. E. J. Am. Chem. Soc. 1998, 120, 4023-4024. (3) Cardona, C. M.; Mendoza, S.; Kaifer, A. E. Chem. Soc. ReV. 2000, 29, 37-42. (4) Cardona, C. M.; McCarley, T. D.; Kaifer, A. E. J. Org. Chem. 2000, 65, 1857-1864. (5) Stone, D. L.; Smith, D. K.; McGrail, P. T. J. Am. Chem. Soc. 2002, 124, 856-864. (6) Jayakumar, K. N.; Bharathi, P.; Thayumanavan, S. Org. Lett. 2004, 6, 2547-2550. (7) Jayakumar, K. N.; Thayumanavan, S. Tetrahedron 2005, 61, 603-608. (8) Camaron, C. S.; Gorman, C. B. AdV. Funct. Mater. 2002, 12, 17-20. (9) Chasse, T. L.; Gorman, C. B. Langmuir 2004, 20, 8792-8795. (10) Wang, Y.; Candona, C. M.; Kaifer, A. E. J. Am. Chem. Soc. 1999, 121, 9756-9757. (11) Wells, M.; Crooks, R. M. J. Am. Chem. Soc. 1996, 118, 3988-3989. (12) Zhou, Y.; Bruening, M. L.; Bergbreiter, D. E.; Crooks, R. M.; Wells, M. J. Am. Chem. Soc. 1996, 118, 3773-3774. (13) Armstrong, F. A.; Hill, H. A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21, 407. (14) Chen, X.; Ferrigno, R.; Yang, J.; Whitesides, G. M. Langmuir 2002, 18, 7009-7015.

film formation is generally aided by providing a surface suitable for hydrogen bonding. Mercaptoundecanoic acid (MUA) provides such a surface. Cyt c adsorbs easily onto MUA, which allows H-bonding interactions between the amide and carboxylate groups of the protein and the carboxylate surface.16 MUA modified surfaces also provide a suitable substrate for film formation with some dendrimers. Intermolecular H-bonding interactions between the terminal amino groups of poly(iminopropane) dendrimers with the carboxylate surface of MUA films resulted in facile film formation.17 Merz et al. have extended this procedure to study the adsorption of porphyrin dendritic protein mimics.18 H-bonding interactions between carboxylic acid and amide groups in peptides have been exploited extensively to generate robust architectures.19,20 We were interested in using this approach for the study of films of peptide dendrimers supported on MUA and investigate if peptide dendrimers having a redox core remain redox active once supported on such a surface. We have reported the synthesis and characterization of the glutamic acid-based peptide dendrimers G1-G6 (Scheme 1) which possess a ferrocene (Fc) at the center of the dendrimer and showed that backfolding and intramolecular H-bonding increases with increasing generations,21 which in essence is responsible for the formation of a globular structure of these peptide dendrimers in the higher generations22 and the encapsulation of the redox core.21 In contrast, lower generations possess an open architecture which makes the Fc group readily accessible to the environment. Although the redox properties are modulated by the peptide sheath, dendrimers up to G6 remain redox active. In this paper, we take advantage of MUA surfaces to immobilize Fc-peptide dendrimers exploiting the H-bonding interactions between the carboxylate groups of MUA and the amide NH and CdO of the peptide dendrimers. This raises questions regarding the nature and strength of the interaction between the surface (15) Eddowes, M. J.; Hill, H. A. O. Chem. Commun. 1977, 3154. (16) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 1847-1849. (17) Tokuhisa, H.; Crooks, R. M. Langmuir 1997, 13, 5608-5612. (18) Merz, L.; Hitz, J.; Hubler, U.; Weyermann, P.; Diederich, F.; Murer, P.; Seebach, D.; Widmer, I.; Sto¨hr, M.; Gu¨ntherodt, H.-J.; Hermann, B. A. Single Mol. 2002, 3, 295-299. (19) Ren, Z.; Ma, D.; Yang, X. Polymer 2003, 44, 6419-6425. (20) Vishweshwar, P.; Nangia, A.; Lynch, V. M. Cryst. Growth Des. 2003, 3, 783-790. (21) Appoh, F. E.; Thomas, D. S.; Kraatz, H.-B. Macromolecules 2005, 38, 7562-7570. (22) Cavillo, L.; Fraternali, F. Chem. Eur. J. 1998, 4, 927-934.

10.1021/la061114c CCC: $33.50 © 2006 American Chemical Society Published on Web 09/12/2006

10516 Langmuir, Vol. 22, No. 25, 2006 Scheme 1. Glutamic Acid Dendrimers G1-G6 Having a Ferrocene Corea

a

The exterior surface of the dendrimer is ethylester terminated.

and the dendrimers, the stability of the resulting films, and the redox activity of the Fc-peptide dendrimers. We are addressing these questions using a combination of spectroscopic and electrochemical techniques, beginning with a study of the interaction of Fc-peptide dendrimers and MUA in solution, followed by an investigation of the immobilized Fc-peptide dendrimers on MUA. Experimental Section General. Fc-peptide dendrimers G1-G6 (Scheme 1) were prepared as described previously.21 NMR spectra were recorded on a Bruker Avance 500 spectrometer operating at 500 MHz (1H). Peak positions are reported in ppm (δ) relative to TMS. 1H NMR spectra of Fc-peptides are referenced to the CH2Cl2 resonance (δ 5.32) of an external standard (CDCl3/CH2Cl2). 1H NMR Determination of H-Bonding by Dendrimer-MUA Mixtures. A 1:1 composite mixture of the dendrimers and 11-mercaptoundecanoic acid (MUA) HS(CH2)10COOH in CDCl3 was stirred for 30 min. The mixture was investigated for H-bonding interactions between the amide NH and COOH in the regions between 6.0∼12.00 ppm. Preparation of Surfaces and Films. Gold electrodes were purchased from Bioanalytical Systems (geometric area 0.025 cm2). Prior to film deposition, the electrodes were polished with a slurry of 1.0 and 0.05 µm Al2O3, washed with Millipore water and then cleaned electrochemically in a 0.5 M KOH by applying a linear sweep in the potential range of 0 to -1.4 V vs Ag/AgCl at a scan rate of 0.1 V s-1. The gold electrodes were subjected to cyclic voltammetry (CV) sweeps between 0.1 and 1.5 V vs Ag/AgCl in 1.0 M H2SO4 solution until a stable CV was obtained. Au coated silicon wafers (Platypus) were cleaned with piranha solution (H2SO4: 30% H2O2 3:1)25,26 followed by washing with copious amount of water. Under potential deposition (UPD) of copper was used to determine the surface roughness of the gold electrode after cleaning. The surfaces of gold electrodes were cleaned as described above. A potential step (23) Schulze, J. W.; Dicertmann, D. Surf. Sci. 1976, 54, 489-492. (24) Bediako-Amoa, I.; Sutherland, T. C.; Li, C.-Z.; Silerova, R.; Kraatz, H.-B. J. Phys. Chem. B, 2004 108, 704-714. (25) Liu, D.; Szuiczewski, G. J.; Kispert, L. D.; Primak, A.; Moore, T. A.; Moore A. L.; Gust, D. J. Phys. Chem. B 2002, 106, 2933-2936. (26) Degenhart, G. H.; Dordi, B.; Scho¨nherr, H.; Vansco, G. J. Langmiur 2004, 20, 6216-6224.

Appoh et al. method using chronoamperometry was performed in 1.0 mM of Cu(ClO4)2‚6H2O in 0.1 M HClO4 from a potential of 500-0 mV for 30 s to deposit a monolayer of copper atoms on the surface of the gold electrode. Linear sweep voltammetry (LSV) was used to strip off the copper from 150 to 500 mV at a scan rate of 100 mV s-1. The charge Q for Cu UPD was obtained by integration of the cathodic peak in the LSV. The ratio between experimental and geometrical surface areas provided electrode surface roughness of 1.3∼1.5 for all electrodes examined.23,24 Clean Au electrodes and Au-coated silicon wafers were soaked in ethanolic solutions of 5.0 mM MUA for 48 h and washed with ethanol and then water. To ensure a fully protonated MUA carboxylate surface, the modified gold surfaces (electrodes and wafers) were immersed into a 0.3 mM HClO4 solution for 30 s. The adsorption of the Fc-dendrimers G1-G6 were carried out by immersion of the MUA-modified Au surfaces in a 1.0 mM methanol solution of G1G6 for 3 h, after which the electrodes were washed with ethanol to remove any physisorbed material. Electrochemistry of Fc-Peptide Dendrimer Films. Electrochemical measurements were carried out at 22 ( 1 °C on a CHI 660B potentiostat. Gold electrodes (BAS, geometric area 0.025 cm2) modified with Fc-peptide dendrimer films were used as working electrodes. Pt wire was used as a counter electrode. All measurements were carried out in a 2.0 M aqueous solution of NaClO4 and potentials were measured against a Ag/AgCl/3M KCl reference electrode (BAS). All data were analyzed by OriginLab 7.0 software. CV and DPV curves were recorded for the MUA/Au surfaces before and after formation of Fc-peptide dendrimer films. CV and DPV curves are background subtracted. A scan rate of 100 mV-1 was used for all CV experiments with the exception of variable scan rate experiments in which CV curves were recorded at scan rates of 100-500 mV/s. The scan rate in all DPV experiments was 20 mV/s with a pulse amplitude of 25 mV. RAIRS. RAIRS experiments were performed at room temperature using a BioRad FTS-60A FTIR spectrometer. p- Polarized infrared radiation was reflected from the substrates with immobilized MUA film and dendrimers/MUA films at an angle of incidence of 80° with respect to the surface normal. Data were recorded as the co-addition of 5000 scans in a single-beam absorbance mode at a resolution of 4 cm-1 and the final spectrum obtained by baseline correction. X-ray Photoelectron Spectroscopy. XPS measurements were performed using an Axis-165 X-ray photoelectron spectrometer (Kratos Analytical) with a monochromatic Al KR X-ray source (1486.7 eV). Survey spectra (0-1100 eV) were taken at a constant analyzer pass energy of 160 eV, and high-resolution spectra of Au4f, C1s, N1s, O1s, S2p, and Fe2p were acquired with a pass energy of 20 eV and a down time of 200 ms. The takeoff angle measured as the angle between the film surface and the photoelectron energy analyzer was 90°. The typical operating pressure in the analyzing chamber was ∼5 × 10-10 Torr. Number of scans for high resolution required obtaining high signal-noise ratio varied from 4 for Au to 40 for Fe and 60 for S. The binding energies were referenced to Au4f7/2 at 84.0 eV. For measurement of the film thickness, XPS spectra were measured by rotating the sample holder, and spectra were measured at takeoff angles (θ) of 0°, 30°, 45°, 60°, and 90°.

Results and Discussion The interaction of peptide dendrimers G1-G6 with MUA was investigated in solution by 1H NMR spectroscopy prior to depositing the dendrimers onto MUA-modified gold surfaces. The peptide dendrimers as well as MUA have H-bond donor and acceptor groups. In particular, it was thought that the peptide amide NH and the carboxylic acid OH of MUA should be strongly affected by H-bonding interactions. Figure 1 shows partial 1H NMR spectra of a solution of the nonglobular G2, of MUA, and of a solution containing equimolar amounts of G2 and MUA. In the presence of MUA, all three amide signals of G2 experience a downfield shift in the 1H NMR spectrum, whereas the COOH

Study of Peptide Dendrimers

Langmuir, Vol. 22, No. 25, 2006 10517 Scheme 2. Formation of Fc-Peptide Ester Dendrimers on MUA Modified Au Surfacesa

Figure 1. Partial 1H NMR spectra showing the amide NH and carboxylic acid regions of the NMR spectrum of a solution of MUA, an equimolar solution of G2 + MUA, and a solution of G2 in CDCl3. a (1) Immersion of a gold surface in a solution of MUA results in the formation of a MUA film on gold. after washing. (2) The MUA-modified surface is immersed in a solution of the dendrimer. H-bonding interactions between the dendrimer and the acidterminated MUA surface allow the formation of a stable dendrimer film. Three possible forms of H-bonding for the formation of MUApeptide layer (a) amide NH donor-carboxyl acceptor interactions, (b) acid OH donor-carboxyl of amide acceptor interaction, and (c) acid OH donor-ester carboxyl acceptor interaction.

Figure 2. Partial 1H NMR spectra showing the amide NH and carboxylic acid regions of the NMR spectrum of a solution of MUA, an equimolar solution of G5 + MUA, and a solution of G5 in CDCl3.

of the MUA is shifted upfield, indicating H-bonding interactions between G2 and MUA.27 Addition of MUA to a solution of G2 affects the amide NH proximal to the Fc group more than the other two peripheral amide groups. In the absence of a suitable external H-bond acceptor, the amide NH proximate to the Fc moiety is not engaged in H-bonding but addition of MUA allows H-bonding resulting in a 0.4 ppm downfield shift. For the peripheral amide groups, smaller shifts of ∆δ ∼ 0.2 ppm are observed. These downfield shifts indicate that intermolecular H-bonding is formed between the NH of the peptide and the CdO moiety of the MUA.28 The signal due to the carboxyl group of MUA at δ 11.6 experiences an upfield shift, suggesting the disruption of the intermolecular H-bonding interactions of the MUA dimers (COOH‚‚HOOC interactions). In sharp contrast to the lower generation dendrimers having an open architecture, in admixtures of MUA with higher generations G4-G6, which possess a globular and compact architecture, the O-H peak of the MUA is most affected and some changes in the amide region of the dendrimers are observed in the δ 7.30-7.70 region. Figure 2 shows the admixture of MUA with G5 in CDCl3 solution. The MUA signal is broadened, which suggests its involvement in H-bonding, presumably with the ester groups which are on the exterior surface of the peptide dendrimer. In contrast, the 1H NMR signals for the amide NHs in G5 exhibit only small changes, which suggests that the amides NH remain largely involved in intramolecular H-bonding within the dendrimer. Accessible amide NH on the dendrimer surface (27) Westwood, J.; Coles S. J.; Collinson S. R.; Gasser, G.; Green S. J.; Hursthouse, M. B.; Light, M. E.; Tucker, J. H. R. Organometallics 2004, 23, 946-951. (28) Santo, M.; Fox, M. A. J. Phys. Org. Chem. 1999, 12, 293-307.

Figure 3. CV of 1.0 mM [Fe(CN)6]3-/4- in 2.0 M NaClO4 at (a) bare gold electrode (solid) and (b) MUA modified gold electrode (dotted). Scan rate 100 mV/s; Ag/AgCl reference and Pt counter electrodes.

may establish intermolecular H-bonds with MUA. These observations show that MUA is effective in the formation of H-bonds with Fc-peptide dendrimers and is useful for the surface immobilization of these dendrimers on gold. Surface Immobilization of Fc-Peptide Ester Dendrimers. Deposition of MUA onto gold surfaces is expected to provide an acid-terminated substrate to which the peptide dendrimers can H-bond. For this purpose, cleaned gold electrodes with a surface roughness ranging from 1.3 to 1.5 were immersed in a 5.0 M ethanolic solution of MUA for 48 h (see Scheme 2). Typical cyclic CVs of [Fe(CN)6]3-/4- at a bare gold electrode and a MUA-modified gold electrode are shown in Figure 3. At the bare gold, the reduction and oxidation peaks of [Fe(CN)6]3-/4- were observed with a formal potential of 210 mV versus Ag/AgCl with a peak separation of ∆E ) 105 mV and peak current ratio ipa/ipc of unity, indicating a quasireversible redox behavior. For MUA modified surfaces, no redox signal was observed, indicating an effective blocking of the MUA film

10518 Langmuir, Vol. 22, No. 25, 2006

Appoh et al. Table 1. Electrochemical Data of Fc-Petide Ester Dendimers at MUA Monolayer Interface at pH 7.0a films

E0

G1 G2 G3 G4 G5

456 (8) 455 (9) 455 (7) 452 (8) 450(10)

∆E

∆Efwhm

ΓFc

sp. a.

71 (8) 120 (15) 2.95 (10) 560 (70) 59 (6) 130 (20) 1.84 (2) 900 (25) 65 (8) 120 (25) 1.60 (4) 1037 (60) 15 (5) 80 (20) 2.90 (10) 570 (80) 23 (6) 90 (10) 2.70 (8) 615 (60)

cal. sp. a. 60 160 230 350 575

a 0 E ) (Eox + Ered)/2, ∆E ) (Eox - Ered), ∆Efwhm in mV, ΓFc 10-11 mol‚cm-2, calculated specific area (cal. sp. a.) derived from spartan modeling and experimental specific area (sp. a.) are in Å2 (standard deviations in brackets).

Figure 4. Stack plot of CV scans of G5 at scan rates of 100-500 mV s-1 and a plot of ip vs scan rate ν. 2.0 M NaClO4, Ag/AgCl reference and Pt counter electrodes.

Figure 5. CV (i) and DPV (ii) scans of (A) G1, (B) G5, and (C) G6 on MUA-modified Au electrodes (BAS). G6 exhibits no redox activity due to complete encapsulation. For CV: scan rate 100 mV/ s; for DPV: scan rate 20 mV/s, pulse amplitude 25 mV; supporting electrolyte: 2.0 M NaClO4, Ag/AgCl reference and Pt counter electrodes

to electron transfer from the [Fe(CN)6]3-/4- redox probe and a lack of significant pinholes in the film.29,30 Films of peptide dendrimers were prepared by immersion of the MUA-modified gold surfaces into 1.0 mM methanolic solutions of Fc-peptide dendrimers G1-G6 for 3 h, as shown in Scheme 2. We envision several possible H-bonding interactions between the carboxylic acid group of the MUA films and the (29) Cecchet, F.; Rudolf, P.; Rapino, S.; Margotti, M.; Paolucci, F.; Baggerman, J.; Brouwer, A. M.; Kay, E. R.; Wong, J. K. Y.; Leigh, D. A. J. Phy. Chem. B 2004, 108, 15192-15199. (30) Mendes, R. K.; Freire, R. S.; Fonseca, C. P.; Neves, S.; Kubota, L. T. J. Braz. Chem. Soc. 2004, 849-855.

ester and amide groups that are present on the surface of the dendrimers, as shown in Scheme 2. To probe the successful formation of dendrimer films, variable scan rate experiments were carried out. A typical stack plot of CVs at various scan rates for G5 is shown in Figure 4. The relationship between the peak currents ip and scan rate ν is linear for all systems investigated, demonstrating successful film formation. G6 did not exhibit any redox activity on MUAmodified gold surfaces (vide infra). Figure 5 shows the CV and DPV of dendrimers G1, G5, and G6 supported on MUA-modified gold electrodes recorded in a region of 0.2-0.8 V (versus Ag/AgCl). Under the experimental conditions, the MUA is electrochemically inactive and the redox activity is due to the adsorption of the Fc-peptide ester dendrimers, indicating the ET from the Fc-core of the dendrimer to the surface of the gold electrode. With the exception of G6, all dendrimers exhibit a redox signal attributed to the Fc/Fc+ redox couple of the dendrimers (Table 1). The formal potential E° of G1-G5 is observed at ca. 455 mV (versus Ag/AgCl), which is typical for Fc-peptides. It is interesting to note that the peak half-widths (∆Efwhm) vary, with those of G1-G3 being broader than those of G4 and G5. ∆Efwhm can be used to evaluate the organizational behavior of films. In well-ordered systems with minimal interactions between the redox centers, ∆Efwhm ) 90.3/n, when n is the number of electrons.34 Disordered systems have higher ∆Efwhm values. The observed ∆Efwhm values for films of G4 and G5 are close to the ideal value of 90 mV, suggesting formation of uniform and homogeneous films, while values for films of lower generations G1-G3 are larger indicating disordered and potentially heterogeneous interactions between Fc centers. The ∆E of films of lower generations G1-G3 are higher compared to those of G4 and G5, supporting the formation of more homogeneous films for the higher generation dendrimers. These observations can be rationalized by considering that lower generations possess a more open architecture and are more likely to interact with the MUA surface via their amide NH and in particular their Fc-proximal NH, which possibly causes the Fc group to be drawn into the MUA layer. Generally, Fc groups embedded in alkyl monolayers give rise to a broad CV signal.31,32 Formation of multilayers and interactions between Fc centers may also account for peak broadening. However, we have no evidence to suggest the formation of Fc-peptide dendrimer multilayers. In contrast, the higher generation Fc-peptide esters G4-G6 assume a globular structure due to backfolding of the peptide dendrons and have increased intramolecular H-bonding. This protects the Fc core from the interaction with the MUA surface. (31) Creager, S. E.; Rowe, G. K. Anal. Chim. Acta 1991, 246, 233. (32) Rowe, G. K.; Creager, S. E. J. Phys. Chem. B 1994, 98, 5500. (33) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M. J. Am. Chem. Soc. 1990, 112, 4301. (34) Sabapathy, R. C.; Bhattacharyya, S.; Leavy, M. C.; Cleland, W. E.; Hussey, C. L. Langmuir 1998, 14, 124.

Study of Peptide Dendrimers

Langmuir, Vol. 22, No. 25, 2006 10519

Figure 6. Partial RAIRS of a MUA film showing a sharp signal at 3577 cm-1 and a signal for the acid CdO 1747 cm-1 with a small fraction face-to-face dimer at 1710 cm-1.

Thus, for higher generations, it is plausible to assume interactions between the ester CdO on the dendrimer surface with the acid terminated MUA surface. As expected for such an interaction, the resulting film is more compact.33 The Faradic charge associated with Fc/Fc+ electron-transfer processes was determined by integrating the anodic peak area of the CVs from which the surface concentration ΓFc was obtained, according to eq 1

ΓFc ) Q/nFA

(1)

where ΓFc is the surface concentration associated with the redox activity, Q is the surface charge, n is the number of electrons (n ) 1 for Fc), F is Faraday’s constant, and A is the area of the electrode. The values of calculated and experimentally determined specific areas of coverage of adsorbed Fc-peptide dendrimers are listed in Table 1. Comparing the experimental and theoretical footprints reveals that films of G4 and G5 have approximate monolayer coverage, whereas the films of lower generations G1-G3 exhibit submultilayer coverage. The results of our electrochemical study raise the question why films of G6 on MUA-modified surfaces are electrochemically inactive whereas G6 is reversibly oxidized in solution.21 To rationalize our observations, various factors should be considered: (i) the influence of the donor-acceptor distance on redox activity between the Fc core and electrode, (ii) the migration of counterions in and out of the film during oxidation and reduction of the Fc group, and (iii) potential differences in the rigidity of the dendrimers supported on the surface. The Fc is encapsulated by the peptide sheath and prevents access of the Fc group by the electrolyte. The encapsulation of the Fc group decreases the propensity of the formation of ionpairs between the Fc+ and the ClO4- counterions,35,36 thereby preventing oxidation of the Fc core. It is expected that, in the absence of ion-pairing, no redox signal will be observed. The critical role of ion-pairing between Fc and the electrolyte anion in Fc films was demonstrated before.36,37 In solution, the dendrimers diffuse to the surface of the electrode, but since G6 is globular with a diameter of 34 Å,21 electrons will have to tunnel through the sheath to the electrode, but when immobilized on the MUA surface, the additional distance created by the alkanethiol barrier significantly reduces redox activity with the electrode. In addition, the dendrimer may not be able to explore the conformation necessary for efficient electron transfer from the asymmetrically placed Fc core to the surface. This rigidity may be at least partially responsible for the loss of the redox activity on for G6. (35) Guo, L.-H.; Facci, J. S.; McLendon, G. J. Phys. Chem. 1995, 99, 41064112. (36) Rowe, G. K.; Creager, S. E. Langmuir 1991, 7, 2307-2312. (37) Uosaki, K.; Sato, Y.; Kita, H. Langmuir 1991, 7, 1510-1514.

Figure 7. Partial RAIRS for (A) G1 and (B) G5 deposited unto MUA modified Au substrates showing the (i) amide A and C-H and (ii) ester, amide I and II regions. Table 2. Selected Data from RAIRS for MUA Films and Fc-Peptide Dendrimers G1-G6 Deposited onto MUA Filmsa film

amide A

CdO(a)

MUA G1 G2 G3 G4 G5 G6

3312 3327 3335 3368 3210 3262

1747, 1710 1745* 1747* 1743* 1745 1745 1745

CdO(b)

amide I

amide II

1720 1712 1700

1660 1664 1683, 1632 1683, 1627 1668, 1628 1661, 1624

1586, 1556 1550 1595, 1556 1557 1568, 1557 1556, 1535

ν in cm-1. C)O(a) is assigned to MUA; CdO(b) is assigned to the ester. *CdO for the acid and the ester can not be differentiated. a

The stability of the dendrimer films of MUA was evaluated by repeated cycling in the potential range of 200-800 mV. In this potential range, our dendrimer films experience less than a 5% loss of coverage as judged by integration of the faradaic current. The interactions of the Fc-peptide dendrimers with the MUAfilm were studied by reflection-absorption IR spectroscopy (RAIRS). In addition, X-ray photon spectroscopy (XPS) was also used to gain information about the elemental composition and film thickness of the dendrimer films. Scheme 2 shows some plausible H-bonding interactions between MUA/MUA, MUAamide, and MUA-ester.38-40 RAIRS were recorded for MUAmodified gold surfaces and Fc-peptide dendrimers deposited onto MUA-modified gold surfaces. Figure 6 shows the partial RAIRS for the MUA film on Au, which is characterized by an intense absorbance at 1747 cm-1 with a shoulder at 1710 cm-1. The peaks of MUA at 1740 cm-1 is consistent with the CdO stretch of non-H-bonded acid groups. The shoulder peak at 1710 cm-1 indicates the presence of a small fraction of face-to-face H-bonded dimers.38-40 Also present is an intense sharp absorbance at 3577 cm-1 and a set of peaks in the 2950-2800 cm-1 region. Although, we are not making any effort in providing a detailed assignment (38) Nakayama, D.; Nishio, Y.; Watanabe, M. Langmuir 2003, 19, 85428549. (39) Me´thivier, C.; Beccard, B.; Pradier, C. M. Langmuir 2003, 19, 88078812. (40) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558-559.

10520 Langmuir, Vol. 22, No. 25, 2006

Appoh et al.

Figure 8. Typical core level XPS spectra of S2p, O1s, N1s, and Fe2p of G5 deposited onto MUA-modified Au surfaces. Table 3. Assignments of XPS Spectra (Binding Energy in eV) for MUA and Fc-Dendrimers G1-G6 Deposited on MUA-Au Modified Surfaces (* ) Low Signal-to-Noise) film

CHs

C-O/C-N

CdO

MUA G1 G2 G3 G4 G5 G6

284.7 284.8 284.7 284.7 284.6 284.9 284.9

285.9 286.3 285.9 285.9 285.5 286.4 285.8

288.9 288.9 288.8 289.3 288.5 289.0 288.8

O1s 533.0 532.9 532.9 533.2 533.4 533.9 533.3

of the signals, it is likely that the band at higher wavenumbers is due to the carboxylate OH (acidic υ(O-H)), whereas the absorptions at lower wavernumbers are most lilely the result of symmetric and asymmetric methylene stretching vibrations. Aroca and co-workers showed that IR surface immobilized acids showed a peak around 3500 cm-1 for the υ(O-H) of the acid and that the intensity of this peak and the related peak 1767 cm-1 for υ(CdO) decreases with increasing pH.41 Figure 7 shows typical partial RAIRS for films of dendrimers G1 and G5 on MUA/Au. After deposition of the dendrimers G1-G6, significant changes in the RAIRS spectra were observed. For all dendrimers, the peak at 3577 cm-1 is absent. The appearance of broad bands in the 3300-3200 cm-1 range is assigned to H-bonded NH and/or H-bonded OH groups, in accordance with the literature.42 The carbonyl region shows some distinct peaks due to the presence of dendrimer esters as well as to the amide groups. For the lower generations G1-G3, a broad peak around 1745 cm-1 is observed and is assigned to carbonyls of the ester linkages, as well as the MUA sublayer. For films of G5 and G6, the peak at 1710 cm-1 is more prominent. This may be due to H-bonding to the carbonyls of the ester. The amide I band appears in the region between 1660 and 1670 cm-1, whereas the amide II band occurs in the 1535-1590 cm-1 region of the spectrum.38 Table 2 summarizes the RAIRS signals for all films. The dendrimer films were further examined by XPS. Initially, core level survey spectra were collected for all dendrimer films G1-G6 using an unmodified gold surface as reference. To characterize the species present on the surfaces, high-resolution spectra were recorded for the main core level peaks of C, O, N,

531.8 531.9 531.8 532.1 531.9 532.3 532.2

N1s

Fe 2p

S2p

400.2 400.2 400.1 400.1 400.3 400.3

713.2, 723.6 711.3, 723.6 * * 710.2, 723.2 *

161.9, 163.0 161.9, 163.1 161.9, 163.0 162.0, 163.1 161.9, 163.1 161.9, 163.3 161.9, 163.3

S, and Fe. The XPS data is summarized in Table 3. The sulfur signal is of interest because it forms the basis of the covalent formation of the thiolate bond between the S of the MUA and Au substrate, whereas the presence of C, N, and Fe indicate the presence of the deposited Fc-peptide dendrimers on the MUAmodified Au surfaces. High-resolution XPS spectra for the G5 film are shown in Figure 8. Peaks in the region of 161.5∼162.5 eV indicate goldthiolate interactions due to the MUA on the gold surface.29,42,43 We observed that the peak due to the thiolate occurred as a doublet at 161.9 and 163.3 eV for the S2p3/2 and S2p1/2 with a ratio of 2:1, which supported the formation of a Au-S thiolate bond.44 Some unbound thiolate is also observed at 168.3 eV (see unfitted raw data in the Supporting Information Figure S1). The O1s spectrum was deconvoluted into two peaks, at 533.9 eV due to the double-bonded (sCdO), and at 532.3 eV due to the singlebonded oxygen (OH/CsO). Other evidence of immobilization of the various films was also provided by the N1s peaks derived from the dendrimers’ amide Ns. A single peak is observed for the N1s at 400.1∼400.3 eV, consistent with literature observations for N1s spectra of PAMAM dendrimers 399.9 eV, amide NH at 400.1 eV for benzyl amide macrocylics on MUA-Au films,43 amide NH at 399.2∼399.8 eV for amino-spirobifluorene molecules on (41) Alvarez-Pueble, R. A.; Garrido, J. J.; Aroca R. F. Anal. Chem. 2004, 76, 7118-7125. (42) Cho, Y.; Ivanisevic, A. J. Phy. Chem. B 2005, 109, 6225-6232. (43) Cecchet, F.; Pilling, M.; Hevesi, L.; Schergna, S.; Wong, J. K. Y.; Clarkson, G. J.; Leigh, D. A.; Rudolf, P. J. Phy. Chem. B 2003, 107, 10863-10872. (44) Liu, D.; Szulczewski, G. J.; Kispert, L. D.; Primak, A.; Moore, T. A.; Moore, A. L.; Gust, D. J. Phy. Chem. B 2002, 106, 2933-2936.

Study of Peptide Dendrimers

Langmuir, Vol. 22, No. 25, 2006 10521

Figure 9. Core level XPS spectra of C1s for (a) MUA and for peptide dendrimers on MUA films (b) G2, (c) G6, and (d) histogram plot of the areas of the deconvoluted C1s showing increases in the areas of the components.

The XPS C1s exhibits a broad peak centered around 285 eV which was deconvoluted into three signals assigned to various functional groups of aliphatic C-H, amide C-N/C-O, and carbonyl CdO. Figure 9a-c shows the XPS spectra C’s of a MUA film for G2 and G6 films supported on MUA. The dendrimer films showed three signals assigned to the aliphatic CH groups (below 285 eV), the amide C-O/C-N groups (above 285 eV), and the CdO groups (above 288 eV). A histogram plot of the areas of the deconvoluted C1s signals is shown in Figure 9d and demonstrates an incremental increase in the signals intensity with increasing dendritic generation for all three types of carbon.49,50 XPS angle-resolved (ARXPS) measurements were used to determine the thickness of the dendrimer film from the attenuation of the Au4f signals of the gold substrate. The photoelectron intensity from the thin film-covered substrate varies with the takeoff angle, θ (taken as the angle between the surface plane and entrance to the analyzer), and is given by eq 248,51

ln(I) ) -d/(λ sin θ) + ln(I0)

Figure 10. ARXPS showing the Au4f for the dendrimer films on (a) MUA-gold, (b) G1, (c) G6 measured at various θ’s. (d) Graph of 1/sin θ and ln of the peak intensity of Au4f7/2 of 9, MUA; O, G1; 2, G2; 4, G3; [, G4; 0, G5; ], G6.

MUA-Au films,29 and amide NH at 399.8 eV for TAT peptides supported on MHA-Au films.29 The Fe core levels signals provide further evidence of the adsorption of the dendrimers on the MUA surface. In the Fe2p XPS, two peaks were observed for Fe2p3/2 and Fe2p1/2 peaks at 710.2 and 723.2 eV, respectively (Figure 8),45-48 clearly confirming the presence of the Fc dendrimer on the surface. (45) Qi, H.; Sharma, S.; Li, Z.; Snider, G. L.; Orlov, A. O.; Lent, C. S.; Fehlner, T. P. J. Am. Chem. Soc. 2003, 125, 15250-15259. (46) Lenhard, J. R.; Murry, R. W. J. Am. Chem. Soc. 1978, 100, 7870-7875.

(2)

where I0 and I are the intensities of the photoelectron from the clean substrate and from the covered substrates respectively, d is the film thickness, and λ is the inelastic mean free paths of photoelectrons. Accordingly, ln(I) should be linearly related to 1/sin θ with a slope of -d/λ.Thus, the ratio -d/λ allows for an evaluation of the film thickness. Figure 10 shows the ARXPS for Au4f core levels for the MUAAu film, and dendrimers supported on MUA-Au films, measured at various θ angles and the related plot of ln(I) versus 1/sin θ (47) Voicu, R.; Ellis, T. H.; Ju, H.; Leech, D. Langmuir 1999, 15, 8170-8177. (48) Yanagida, M.; Kanai, T.; Zhang X.-Q.; Kondo, T.; Uosaki, K. Bull. Chem. Soc. Jpn. 1998, 71, 2555-2559. (49) Yau, J.; Tender, L. M.; Hampton P. D.; Lo´pez, G. P. J. Phys. Chem. B 2001, 105, 8905-8910. (50) Wen, X.; Linton, R. W.; Formaggio, F.; Toniolo C.; Samulski, E. T. J. Phys. Chem. B 2004, 108, 9673-9681. (51) Laibinis, P. L.; Bain, C. D.; Whitesides, G. M. J. Phys. Chem. 1991, 95, 7017-7021.

10522 Langmuir, Vol. 22, No. 25, 2006

Appoh et al.

Table 4. Comparison between Experimental and Theoretical Film Thickness from ARXPS Measurements MUA and Fc-Peptide Dendrimers Adsorbed onto MUA-Au Films (Standard Deviations in Brackets) slope exp.(Å) theor. (Å)

MUA

G1

G2

G3

G4

G5

G6

1.1 (0.2) 37 (7) 16

0.8 (0.1) 27 (4) 25

0.7 (0.2) 24 (7) 30

0.8 (0.1) 27 (6) 33

0.9 (0.1) 32 (4) 37

1.0 (0.2) 34 (7) 43

1.2 (0.1) 41 (4) 50

graph. The two peaks due to Au4f5/2 and Au4f7/2 are observed at 87.55 and 83.83 eV, respectively. The intensities of these peaks decreased with decrease in θ and plots of ln(I) verses 1/sin θ were linear for films of MUA, G1-G3, but deviated slightly for G4-G6 at higher sampling angles. The slope of -d/λ, derived from the linear section of the ln(I) vs 1/sin θ plot for the Au4f7/2 at 83.83 eV and an experimental film thickness d assuming a λ value of 34 Å,48 are both listed in Table 4 (calculated diameters for Fc-peptide dendrimers Å from Spartan modeling). The differences between the observed 37 Å and the theoretical 16 Å thickness for MUA is due to the formation of dimers, and this is confirmed by the IR stretching vibrations at 1710 cm-1 and the presence of unbound thiol XPS S2p (supplementary figure S1). The thickness for the adsorbed dendrimer films indicates near-monolayer values. The variations between the theoretical and the experimentally determined may originate from the λ value used in this calculation since the λ value variations between 30 and 56 Å have been reported.52,53 The difference could also be due to deformation and/or tilting of the layers, especially for the higher generation G4-G6. Experimentally determined values for MUA are reported as 10-13 Å.29,54

dendrimers depends on the generation and thus on the structure of the dendrimer. Solution studies provide some important insight into the subtle differences in the interactions. For low generation dendrimers G1-G3, the acid group of MUA is able to interact via H-bonding with the proximal Fc-amide NH, whereas in higher generations G4-G6, the H-bonding interactions involve the ester groups on the surface of the dendrimers and the acid group of MUA. Although films of G4 and G5 were tightly packed giving close to monolayer coverages, films of lower generations G1G3 are less tightly packed having only submultilayer coverages. As expected, the redox activity of the Fc core is influence by the size of the dendrimer. G1-G5 films exhibited electron transfer from the Fc core to the electrode. G6 did not exhibit any redox activity when adsorbed on MUA-modified surfaces due to the encapsulation of the Fc-core by the peptide sheath. The peptide sheath prevents access of solvent and supporting electrolyte to the dendrimer core thus limiting the formation of Fc+ClO4- ion pairs. Rigidity effects and distance effects may also contribute significantly to the loss of the redox signal. Interestingly, the formal potential E0 of surface-immobilized Fc-peptide dendrimers did not change with the size of the dendrimers, in contrast to the observations made in solution.

Conclusions

Acknowledgment. The authors thank NSERC for financial support. H.-B. K. is the Canadian Research Chair in Biomaterials. We also thank Dr Dimitre Karpuzov, University of Alberta, for the XPS measurement.

In this paper, we reported the preparation and characterization of peptide dendrimer films on carboxylic acid group terminated gold surfaces. The interaction between the MUA surface and the (52) Rubin, S.; Bar, G.; Taylor, T. N.; Cutts, R. W.; Zawodzinski, T. A. J. J. Vac. Sci. Technol. A 1998, 14, 1870-1877. (53) Bain, C.; Whitesides, G. M. J. Phys. Chem. B 1989, 93, 1670-1673. (54) Mark, S. S.; Sandhyarani, N.; Zhu, C.; Campagnolo, C.; Batt, C. A. Langmuir 2004, 20, 6808-6817.

Supporting Information Available: Typical XPS showing the S2p of a MUA-modified Au surface; a scheme showing the Glu repeat of the dendrimers. This material is available free of charge via the Internet at http://pubs.acs.org. LA061114C