Electrochemical Investigations into the Binding of Some Nonredox

J. Phys. Chem. C 2007, 111, 4235-4245

4235

Electrochemical Investigations into the Binding of Some Nonredox Active Metal Ions to Surface-Bound Glutamic Acid Conjugates Francis E. Appoh and Heinz-Bernhard Kraatz* Department of Chemistry, UniVersity of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada ReceiVed: August 18, 2006; In Final Form: December 5, 2006

A number of ferrocene glutamic acid cystamine conjugates suitable for the preparation of thin film on gold were prepared and fully characterized electrochemically and spectroscopically. In this study, we evaluate the interactions of nonelectroactive metal ions such as Ca2+ and Tb3+ ions using a combined electrochemical and spectroscopic approach. Cyclic voltammetry studies show a reversible one-electron redox behavior associated with the ferrocene group. These peptide interfaces exhibit a considerable affinity to bind Ca2+ and Tb3+ ions from aqueous solutions with log K ∼ 3. In the presence of metal ion, the charge associated with oxidation of the ferrocene group decreases in line with a blockage of the formation of Fc+/ClO4- ion pair by the formation of an overlayer on top of the film, which essentially shuts down the oxidation of the Fc group. The resulting increase in the film thickness is monitored by X-ray photoelectron spectroscopy (XPS) and by a decrease in the capacitance as monitored by electrochemical impedance spectroscopy. XPS measurements show that the metal ions bind exclusively to the carboxylic acid and that the amide group is not involved in metal binding.

Introduction The interaction of metal ions with peptides has been studied extensively and has been exploited more recently to study the interaction of metal ions with surface-bound peptides. The ability to alter amino acid sequences within oligopeptides offers considerable control over the affinities of ligands for metal ions. Voltammetric methods have been used extensively and often exploit the inherent redox activity of some metal ions. For example, the interaction of Cu2+ with L-cystein linked to Au surfaces gives a measureable electrochemical response down to concentrations of below 5 ppb.1 Films of poly(L-aspartate) conjugates of 3-mercaptopropionic acid exhibit a detection limit of 0.2 ppb for Cu2+.2 For the Cu-binding oligopeptide GlyGly-His, a Cu2+ specific electrochemical response was observed down to the sub-ppt level.3 Ab initio calculations indicate that binding of the metal to the backbone Ns is responsible for the selectivity.4 The detection of electrochemical inactive metal ions at monolayer interfaces is often facilitated by the use of solutionbased redox probes, such as [Fe(CN)6]3-/4- or [Ru(NH3)6]3+/2+ or by having a redox-active group being an integral part of the film itself. For example, the nonredox active metal ions La3+, Eu3+, Lu3+, Ca2+, Mg2+, and K+ are detected using glutathionemodified Au surfaces in the presence of [Fe(CN)6]3-/4- as a solution probe, showing a good selectivity for lanthanides over alkali and alkaline eath metal ions.5-7 Films that contain redox active groups as well as metal binding sites have shown to be particularly useful. There are several examples of metal ion detection at electropolymerized films possessing a metal binding site. Examples include the detection of Cu2+ and Ni2+ using films prepared from thioazophenol8 and the detection of alkaline metals using films of * Corresponding author. E-mail: [email protected].

tetrathiofulvalene (TTF)-dithia-crown ethers.9 Majoral and coworkers demonstrated the detection of Ba2+ using electrodeposited films of a third generation TTF containing dendrimer.10 Another interesting example is the detection of alkaline and alkaline earth metals at ferrocene (Fc)-crown polypyrrole films prepared by electropolymerizing a substituted pyrrol.11,12 We wanted to explore an alternative approach involving selfassembled peptide films on gold in which the peptide is modified by a redox-active group and decided to make use of thin films self-assembled on gold from suitable Fc-peptides conjugates and explore the ability of such films to interact with metal ions. In principle, this should enable us to monitor the binding of redox and nonredox active metal ions avoiding the use of redox probes in solution that results in the ion-gated detection of metal ions. We were particularly interested in investigating Fcconjugates of glutamic acid, an amino acid that is known to interact with Ca2+ in proteins and with lanthanide ions. From a recent solution study of the interaction of Fc-glutamic acid conjugates-peptide conjugates with alkaline earth and lanthanide metal ions, we have learned that these systems give a well-defined electrochemical response. Binding of the metal ion to the glutamic acid residue causes a strong anodic shift of the Fc’s halfwave potential.13 In this paper, we describe the synthesis and characterization of Fc-glutamic acid cystamine conjugates suitable for surface studies and a study of their interactions with metal ions. We are particularly interested in addressing questions related to the nature of the metal-peptide interaction and its effect on the redox properties of the Fc group. Experimental Section UV-vis Spectroscopy. Measurements of absorption spectra were carried out on a CARY 500 Scan-UV/vis near-infrared (NIR) double beam spectrophotometer. The UV-vis absorption

10.1021/jp065363l CCC: $37.00 © 2007 American Chemical Society Published on Web 02/28/2007

4236 J. Phys. Chem. C, Vol. 111, No. 11, 2007

Appoh and Kraatz

SCHEME 1: Schematic Presentation of the Stepwise Synthesis of [Fc-CO-Glu(G1OBz)-CSA]2 (1) and [Fc-CO-Glu(G1OH)-CSA]2 (2)a

a (i) Cystamine-HCl/Et3N, EDC/HOBt in CH2Cl2, 36 h; TFA, FcCOOH, EDC/HOBt in CH2Cl2, 48 h, yield 63%; (ii) NaOH in MeOH, 0 °C, 8 h, yield 65%.

SCHEME 2: Synthetic Scheme for [Fc-CO-Glu(G2OMe)2-CSA]2 (3) and [Fc-CO-Glu(G2OH)2-CSA]2 (4)a

a (i) N-Glu(OMe)2, Et3N, EDC/HOBt, CH2Cl2, 48 h, yield 78%, (ii) Pd/C in wet MeOH, H2 60 PSI, 10 h, yield 83%; (iii) cystamine-HCl/Et3N, EDC/HOBt, CH2Cl2, 36 h, yield 85%; (iv) TFA/Et3N; Fc-COOH, EDC/HOBt, CH2Cl2, 48 h, yield 64%; (v) NaOH, wet MeOH, 6 h, yield 64%.

spectra were recorded in the wavelength range of 300-600 nm in the absorbance mode using quartz cuvettes of 1 mm path length. All measurements were done in MeOH in concentration ranges of 1.0-6.0 mM of compounds with background subtraction of MeOH. IR Spectroscopy. IR experiments were carried out on Biorad FTS-40 Fourier transform spectrometer as KBr disc. Preparation of Electrodes for Surface Studies. Gold electrodes (Bioanalytical Systems, geometric area 0.025 cm2) working electrodes were used for all surface work. These were polished with a slurry of 1.0 and 0.05 µm Al2O3, washed ultrasonically in water, and then were reduced electrochemically in 0.5 M KOH by linear sweep between the potential range of 0 to -1.4 V versus Ag/AgCl at a scan rate 100 mV s-1. The gold electrodes were subjected to cyclic voltammetry (CV) scans between 0.1 and 1.5 V vs Ag/AgCl in 1.0 M H2SO4 solution until a stable CV was obtained. Surface Roughness Measurements by Under Potential Deposition (UPD). 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 method using chronoamperometry was performed in 1.0 mM of Cu(ClO4)2‚6H2O in 0.1 M HClO4 from a potential of 500 to 0 mV for 30 s to deposit a monolayer of copper atoms on the surface of the gold electrode. After, a linear sweep voltammetry (LSV) was used to strip off the copper from 150 mV to 500 mV at a scan rate of 100 mV/s. The amount of charge for the deposition of copper by UPD on a bare gold electrode was obtained by integration of the cathodic peak from

LSV. The ratio between experimental and geometrical surface areas provided electrode surface roughness. The surface roughness of the electrodes, determined by Cu UPD, were between 1.3 and 1.5. Characterization of Fc-CO-Dendrimer Cystamine Films. Gold electrodes were modified with Fc-peptide dendrimer cystamine were placed in 5 mL 2.0 M NaClO4 (aq) or 2.0 M Na2SO4 (aq) solution at pH 6.8-7.0 at 23 °C ( 2 °C and characterized by CV. Blocking Studies. Gold electrodes were modified with Fcpeptide dendrimer cystamine and blocking studies were done by running CV experiments in a solution of K4[Fe(CN)6]‚3H2O and K3 [Fe(CN)6]‚3H2O (1 mM each) in 2.0 M aqueous NaClO4 as a supporting electrolyte. The CV curve was first recorded in 2.0 M NaClO4 (aq) solution, and then in 1.0 mM K4[Fe(CN)6]‚ 3H2O and K3[Fe(CN)6]‚3H2O/2.0 M NaClO4. Metal Titration at Fc-CO-Dendrimer Cystamine Films. Metal ion interactions at Fc-peptide acid cystamine films were placed in 5 mL 2.0 M NaClO4 (aq) solution and titrated by incremental addition of 5 µL aliquots of 125.0 mM of metal salts aqueous solutions at pH 6.8-7.0 at 23 °C ( 2 °C. The solutions were stirred for 30 s and allowed to stand for 2 min before each measurement. CV/differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) spectra were taken before and after each addition of metal ions. Surface Regeneration. Regeneration of the Fc-CO-peptide film was performed by soaking film in 0.125 M metal ion solution for 30 min followed by stripping the adsorbed metal ion by soaking in a solution of 1.0 mM ethylenediaminetetracetic

Binding of Metal Ions to Glutamic Acid Conjugates

J. Phys. Chem. C, Vol. 111, No. 11, 2007 4237

Figure 1. Left: typical CVs of a film of compound 2 on a BAS gold electrode (geometric area 0.025 cm2) at scan rates from 100 to 500 mV/s; right: plot of anodic and cathodic peak currents vs scan rate. 2.0 M NaClO4 as supporting electrolyte at pH 7.0, Ag/AgCl reference electrode, and Pt wire counter electrode.

acid (EDTA) solution for 30 min. The films were washed with Millipore water and scaned by DPV to monitor the nature of Fc-CO-peptide film, the Fc-CO-peptide-Mn+ film and the regenerated films. Films are about 85% regenerated after 30 min. Preparation of Fc-CO-Peptide Dendrimer-Au Films on Substrates. Gold substrates were cleaned with Piranha solution (HNO3/ 30% H2O2 3:1),14,15 followed by washing with copious amount of water. Fc-CO-peptide films on gold were formed by soaking the gold substrate in 1.0 mM ethanolic solutions of the compound for 5 days, then were washed with copious amounts of ethanol and the water. X-ray Photoelectron Spectroscopy (XPS). 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 constant analyzer pass energy of 160 eV and highresolution spectra of Au4f, C1s, N1s, O1s, S2p, Fe2p, Tb3d, and Ca2p were acquired with a pass 20 eV and a down time of 200 ms. For films containing Tb3+, survey scans were extended to 1350 eV. 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. The 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 thickness, XPS spectra were measured by rotating sample holder. The spectra were measured at takeoff angles (θ) of 0o, 30o, 45o, 60o, and 90o. Electrochemical Measurements. Electrochemical experiments were carried out at room temperature (23 ( 1 °C) on a CHI 600 electrochemical analyzer using a conversional 3-electrode cell system of working electrode, Pt wire as counter electrode, and Ag/AgCl (3.0 M NaCl, BAS) as a reference electrode using 2.0 M NaClO4 (aq) adjusted to pH 7.0 as supporting electrolyte and solvent. CV were scanned generally at a scan rate of 100 mV s-1 and at a scan rate of 100-500 mV for variable scanning experiments. DPV were used and scanned at a scan rate of 20 mV/s and pulse amplitude of 25 mV. Electrochemical Impedance Measurements. The impedance spectra are acquired at 20 frequencies ranging from 0.1 Hz to 100 kHz at an applied potential equivalent to the Eo of the individual Fc-peptide cystamine dendrimer. Modeling was achieved using ZsimpWin program.

Molecular Modeling. Geometry optimization of the threedimensional (3D) conformational structure of the dendrimer was performed in a vacuum by a molecular mechanics method (Merck Molecular Force Field) in Spartan’05 (Wavefunction, Inc., Irvine, CA). [Spartan Program; Wavefunction, Inc.: Irvine, CA, 1999]. The input data was derived from the builder routine in the package. In these calculations, the individual cyclopentadienyl rings and the Fe of the core were forced into the sandwich geometry using the dihedral angle constrain option to prevent deformations. The S-Au linkage was also constrained at an angle of 30o relative to the -Au-Au- bond. The 3D shape of all compounds has been simulated by energy minimization of the molecules. Solvent effects were excluded in the calculations. Results and Discussion Synthesis and Characterization. Schemes 1 and 2 summarize the synthetic procedures leading to the Fc glutamic acid cystamine conjugates [Fc-CO-Glu(G1OBz)-CSA]2 (1), [FcCO-Glu(G1OH)-CSA]2 (2), [Fc-Glu(G2OMe)2-CSA]2 (3), and [Fc-Glu(G2OH)2-CSA]2 (4). Coupling of Boc-Glu(OBz)-OH with cystamine followed by N-coupling to ferrocene-carboxylic acid results in the formation of the desired [Fc-CO-Glu(G1OBz)-CSA]2 (1) from which the free acid [Fc-CO-Glu(G1OH-CSA]2 (2) is obtained cleanly by base hydrolysis. Similarly, N-terminal attachment of an Fc group to a γ-linked glutamic acid dipeptide cystamine conjugate results in the formation of [Fc-CO-Glu(G2OMe)2-CSA]2 (3), which upon base hydrolysis gives the desired free acid of [FcCO-Glu(G2OH)2)-CSA]2 (4) (see Supporting Information for spectroscopic characterization). Surface Immobilized Films. Films of the Fc-CO-peptide cystamines 1-4 were prepared by exposing gold surfaces to 1 mM ethanolic solutions of the conjugates for 5 days, followed by thorough washing of the films to remove any physisorbed material. A representative set of CV of a film of compound 2 recorded in a 2.0 M aqueous NaClO4 solution at various scans rates and a plot of the oxidation and reduction peak currents versus scan rate are shown in Figure 1. The CVs show a reversible one-electron oxidation-reduction redox couple associated with the oxidation of ferrocene. A plot of ip for both the cathodic and anodic versus scan rate are linear at the sweep rate investigated (Figure 1) and that the ratios are unity shows that the dendrimers are surface-confined.16 Ideally, CV of adsorbed redox species should exhibit a sharp, symmetric anodic and cathodic peak with a peak separation ∆E ) 0 mV.17a


Appoh and Kraatz

TABLE 1: Summary of the Electrochemical Properties of Films of Fc-CO-glutamic Acid Cystamine Conjugates 1-4a

films 1 2 3 4

E0

∆E

467 ( 20 73 ( 5 425 ( 18 47 ( 5 455 ( 15 109 ( 12 424 ( 15 42 ( 6

∆Efwhm

ΓFc

163 ( 18 125 ( 25 184 ( 20 130 ( 28

3.12 ( 0.40 3.75 ( 0.09 3.35 ( 0.20 3.03 ( 0.40

calc specific specific area area 530 ( 60 440 ( 70 500 ( 50 550 ( 40

390 370 480 440

a Supporting electrolyte 2.0 M NaClO4 at pH 7.0, Ag/AgCl reference electrode, film modified BAS Au working electrode, Pt wire counter electrode. Surface concentrations ΓFc in 10-11 mol cm-2, specific area and calculated specific areas in Å2. Eo, ∆E, and ∆Efwhm in mV.

This is however rarely observed in most CV experiments. Reversibility is usually observed in cases in which peak potentials are constant with increasing scan rates. The points at which potential changes with increasing scan rates represents the onset of quasi-revesible behavior. From Figure 1, one observes a near constant ∆E with increasing scan rate from 100 to 500 mV s-1 which suggests a reversible behavior. The electrochemical reversibility of Fc-monolayers has been attributed to the rapid lateral transport of the electrons between adjacent Fc groups, following the electron shuttling from the gold surface to the Fc.17b A nonzero peak separation ∆E was observed for all films of these compounds and may be the result of a variety of factors such as solvation, ion pair effects, acidbase equilibria, and lateral interactions.17c-f,19 The electron-transfer rate associated with surface confined redox active centers is known to be related to the ∆E value.18 The peak separation ∆E for the acids 2 and 4 is noticeably lower than that for the corresponding esters 1 and 3, suggesting faster electron-transfer kinetics for the acid films compared with that of the ester analogues. The formal potentials Eo and the peak separation ∆E obtained at a scan rate of 100 mV s-1 for films of compounds 1-4 are listed in Table 1. All films exhibit a reversible redox behavior with formal potentials Eo ranging from 467-424 mV versus a Ag/AgCl reference electrode. It is noticeable that the Eo for the films of the acids 2 and 4 are cathodically shifted by about 30-40 mV compared to the corresponding esters 1 and 3. The ∆Efwhm is used to evaluate the organizational behavior of monolayers. The parameter provides a quantitative measure of the relative interactions taking place between the electroactive groups. In systems having minimal intermolecular interactions, the ideal value for ∆Efwhm is 90.3/n, where n is the number of electrons.18 Generally, a ∆Efwhm in the range of 90-100 mV for ferrocenecontaining films indicates a homogeneous environment around the Fc centers.19 For films of compounds 1-4, values of ∆Efwhm in the range of 125-184 mV were observed, suggesting some inhomogeneity of the Fc environments, which is commonly observed in Fc-peptide films.20 The surface concentrations ΓFc can be obtained from the charge associated with Fc/Fc+ redox couple determined by integrating the area under the anodic peak of the CVs 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 by integration of the faradaic peak currents. We observed values of ΓFc between 3.0 and 3.7 × 10-11 mol cm-2, which correspond to specific areas in the range of 440-820 Å2 per molecule. These values are close to footprints derived from

Figure 2. CV of a film of 2 before and after metal ion titration. 2.0 M NaClO4 supporting electrolyte at pH 7.0, vs Ag/AgCl scan rate 100 mV/s.

Figure 3. Graph of the apparent surface charge QFc/µC of films of 1(b), 2 (9), and 4 (O), versus molar concentration of metal ion added ([Mn+]/µM). Graph shows decrease in QFc with [Mn+] in 2.0 M NaClO4 at pH 7.0 vs Ag/AgCl (line ) guide).

molecular modeling of the individual Fc-glutamic acid cystamine conjugates using Spartan. Electrochemical Studies of Metal Interactions at Monolayer Interface. The electrochemical response of films of compounds 1, 2, and 4 to Ca2+ and Tb3+ was examined by CV. Figure 2 shows a representative CV of a film of compound 2 before and after addition of 2.0 µM of Tb3+. For all systems investigated, the formal potentials of the Fc/Fc+ couple are slightly anodically shifted. More importantly, there is a drastic decrease in the peak current ip and, hence, the apparent surface charge QFc with each addition of metal ions which eventually reaches a steady-state value (Figure 4). Decreases in the apparent surface charge upon metal binding to surface-confined redox species has been observed before and was rationalized in terms of ion-pairing effects.21-33 In the present study, the reduction of the peak currents ip is due to a reduction in the number of electroactive sites on the electrode caused by the trapping of the Fc group by the metal coordination at the carboxylate terminals. Thus the observed voltammetric response at a stage in the titration arises from unblocked Fc. The Fc groups blocked by the complexation of metal ions are nonelectroactive because they are shielded from the incorporation of the chargecompensating anion from the electrolyte solution during the oxidation of the Fc moiety.33a Essentially, counterions in the supporting electrolyte necessary to support the Fc redox process are blocked with the addition of Mn+ due to the formation of layers of metal ion-counterion Mn+[ClO4-]n on top of the thin


J. Phys. Chem. C, Vol. 111, No. 11, 2007 4239 (SAMs) on Au formed dense overlayers at the interface between the electrolyte and the functionalized monolayer.36 The investigations revealed that strong chemical interactions between the carboxylate groups, the metals, and the counterion induced dense counterion overlayers.36 Figure 3 shows plots of QFc versus the concentrations of metal ion added for films of 1, 2, and 4, showing a decrease in QFc with increasing [Mn+] addition.24,26,27 For films of 1, there is an initial sudden decrease that may be due to reorganization of the film followed by a more gradual decrease. The rates of change for films of 2 and 4 were more gradual throughout the titration. The changes in QFc versus [Mn+] were fitted to a Langmuir adsorption isotherm from which a binding constant for the interaction is obtained. The Langmuir model is based on the assumptions monolayer adsorption occurs at equivalent but independent sites. The complexation equilibrium is given by

Figure 4. Langmuir plots of [Mn+]/QFc of films of 1 (b), 2 (9), and 4 (O) versus [Tb3+] /µM.

Au-(Fc-peptide) + Mn+ (aq) a Au-(Fc-peptide-Mn+)

TABLE 2: Data for Interaction of Tb3+ and Ca2+ with Films of Fc-Glutamic Acid Cystamine Conjugatesa

The equilibrium constant K can be determined using the Langmuir adsorption isotherm equation

1/Tb3+ E0

483 (15) ∆E0 21 (5) K/M 60 (10) log K 1.78 (0.08) a

2/Tb3+

4/Tb3+

2/Ca2+

4/Ca2+

433 (8) 7 (4) 2700 (21) 3.43 (0.02)

440 (10) 15 (6) 2100 (15) 3.32 (0.04)

437 (8) 11 (6) 1600 (68) 3.20 (0.04)

451 (12) 18 (8) 700 (74) 2.85 (0.05)

θ ) K[Mn+]/(1 + K[Mn+])

(2)

where θ is the ratio of the surface charge QFc of metal ion at a concentration [Mn+] to its maximum surface charge Qmax. Equation 2 thus becomes

E0 was recorded after the addition of 2 eq of Mn+.

ClO4-

film. This effectively blocks the penetration of the film by counter anions and oxidation of the Fc, resulting in the decrease in QFc.22,27,34b,35 Ulman and co-workers have shown that binding of Cd2+, Ca2+, Pd2+, and Ba2+ to ω-COOH self-assembled monolayers

[Mn+]/Q ) (1/KQmax) + ([Mn+]/Qmax)

(3)

Figure 4 shows plots of [Tb3+]/QFc versus [Tb3+] for the Fc oxidation from which we were able to determine equilibrium constants for Tb3+ binding. A similar series of plots was obtained for titration experiments with Ca2+. Formal potentials

Figure 5. (A) Impedance plot in the capacitance plane 2 and 2 + Ca2+ (continuous line is fitting to the model shown in (C). (B) Effect of Ca2+ on Ctot of films of conjugates 2 and 4. (C) Equivalent circuit for fitting. (D) Possible interaction of Ca2+ with a film of 2.


Appoh and Kraatz

TABLE 3: Data for Impedance Titration of Tb3+ and Ca2+ at Monolayer Interfaces of 2 and 4a Rs CPE int N CPEmono N Ri Rm a

2

2 + Tb3+

2 + Ca2+

4

4 + Tb3+

4 + Ca2+

10.9(0.5) 43.5 (0.2) 0.85 9.5 (0.2) 0.90 0.6 (0.3) 0.2 (0.1)

11.3 (0.3) 21.8 (0.4) 0.90 8.3 (0.1) 0.95 0.6 (0.2) 0.3 (0.1)

11.9 (0.4) 20.8 (0.1) 0.80 7.4 (0.1) 0.90 0.5 (0.2) 0.3 (0.2)

5.1 (0.2) 14.2 (0.4) 0.80 11.4 (0.2) 0.90 1.5 (0.4) 1.7 (0.3)

5.1 (0.2) 11.9 (0.2) 0.95 11.9 (0.2) 0.85 1.5 (0.3) 2.0 (0.1)

5.1 (0.5) 10.9 (0.2) 0.90 10.9 (0.1) 0.80 1.3 (0.2) 1.9 (0.3)

CPEint (µF cm-1), CPEmono (µF cm-1), Rdl (µΩ cm2), Rct (102Ω cm2), Rs (Ω cm2) from EIS Studies Taken at E0 of Fc Redox Probe.

and binding constants are summarized in Table 2. As expected a film of the ester 1 showed only weak binding to the metal ions. Binding constants for films of the acids 2 and 4 are significantly higher. In addition, films of 2 give higher binding constants for a given metal compared to films of 4. The log K values for Ca2+ and Tb3+ interaction with 2 and 4 are comparable to the literature values for metal-carboxylate interactions for some mono- and dicarboxylate ligands, including that of glutamic acid and aspartic acid.37-41 The decrease in the observed charge QFc upon increasing metal ion concentration is related to a lack of oxidation of the Fc center. This can be rationalized by the inability of the ClO4counterion to pass through the film in the presence of the metal ions. Presumably, the formation of a tight overlayer of Mn+[ClO4-]n and the potential compactness of the Glu-metal ion complex formed at the film interface prevent efficient diffusion of the counterion into the film. The ion pair theory was invoked by Dong and co-workers to explain the behavior of adsorption of various surfactants at coassembled Fc-teminated alkanethiol-alkylthiolphene thiols SAMs on gold. It was proposed that the adsorption of surfactants at the mixed monolayer created a barrier that inhibited the diffusion of counterions present in solution from diffusing into the adsorbed layer as compensation for the oxidation of the Fc to Fc+ ions.29 The creation of such a barrier layer at an interface can be tested by EIS in which an increase in the film thickness will result in the reduction of the total capacitance. The addition of EDTA to such a system should in principle chelate the metal ions and restore the properties of the film. Because films of the esters do not interact strongly with the metal ions, we focused our attention to EIS studies of films of the acids 2 and 4 and monitored the capacitive changes that occur upon addition of Ca2+ and Tb3+. Binding of metal ions to the immobilized Fc-Glu-conjugates 2 and 4 is expected to affect the interfacial properties of the film through ionic interactions. These changes are monitored by changes in the capacitance of the film.42,43 The total capacitance Ctot as described by eq 4 is composed of a two of capacitances. Cmomolayer is the capacitance of the monolayer and Cinterface is the capacitance due to the interfacial layer formed by the protonation/deprotonation of the COOH group at pH 7.20,44-46 For a capacitance in series

1/Ctot ) 1/Cmonolayer + 1/Cinterface

(4)

Ctot ) o/d

(5)

where is the dielectric constant of the monolayer, o is the dielectric constant of the solution, and d is the film thickness. Cmonolayer and Cinterface is defined by the circuit diagram as shown in Figure 5. The circuit diagram contains constant phase elements (CPE) instead of capacitance. This means that the capacitance is inversely proportional to the thickness and one would expect a decrease in capacitance with increasing layer formation.

Figure 6. Normalized DPV scans showing typical results of regeneration experiments of films of 2. (A) Film of 2, (B) 2 + Tb3+. After soaking in a 1.0 mM EDTA solution for 30 min, the signal recovers (EDTA). DPV scan rate of 20 mV/s and pulse amplitude of 25 mV at film-modified gold electrodes (BAS with geometric area 0.025 cm2), Ag/AgCl reference, and Pt wire counter electrode, aqueous 2.0 M NaClO4 solution pH 7.0.

The impedance spectra are acquired at twenty frequencies ranging from 0.1 Hz to 100 kHz at an applied potential equivalent to the Eo of the individual Fc-peptide cystamine dendrimer. Figure 5A is a typical example of the impedance plot in the capacitance plane for films of 2 in the absence and the presence of Ca2+ ions. Fitting of the experimental impedance spectra to an equivalent circuit makes it possible to evaluate the individual components of the equivalent circuit. The CPE is used here is to account for inhomogeneity of the film due to surface roughness effects. Therefore the CPEinterface or CPEmono are pseudocapacitances which require an additional exponent n. Values of n from fittings are in the range 0.85-0.95 (Table 3), which is sufficiently close to unity for the CPE to be interpreted as capacitance. The titration with metals does not change the equivalent circuits, indicating that conformational integrity on the surface is maintained.42,43,47 As was observed in metal titrations of other Fc-films,2,3 the capacitance of the systems decreased significantly with the addition of the Ca2+ and Tb3+ (Figure 5B). The most reasonable explanation for the observed drop in capacitance is the more effective neutralization of the surface charge by Ca2+ and Tb3+ compared to Na+ the supporting electrolyte35,47,48 and presumably formation of a surface confined metal ion complex. This poses the question as to the nature of this complex. In addition, we observe differences in the solution resistance Rs for systems of 2 and 4. However, that is not too surprising given that for films of shorter molecules the resistive parameters are often indistinguishable, as is the case for films of (CH2)13-COOH and HS(CH2)5-COOH reported by Nahir and Bowden.48



Figure 7. (A) Deconvoluted high-resolution C1s spectra of films of 2 and in the presence of Ca2+ and Tb3+; (B) corresponding O1s spectra analysis.

Next, we attempted the regeneration of the Fc-CO-Glu films using EDTA, which is a strong chelating agent for metal ions such as lanthanides and alkaline earth metals (binding constant EDTA-Ln3+ about log K ) 18.0).49 Figure 6 shows DPV scans for films of 2 before and after Tb3+ was added (A, film of 2; B, after addition of Tb3+). After addition of Tb3+, the resulting film was then soaked in 1.0 mM EDTA solution for 30 min. Addition of EDTA results in a partial recovery of the signal, lending further support to the importance of ion-pairing effects. We want to add that a complete recovery of the redox signal to its original intensity was never observed. Potential causes may involve the formation of EDTA-peptide metal complexes or potential H-bonded adducts between EDTA and the peptides, thus trapping some metal ions in the process. The use of strong acids coupled with mechanical shaking has been reported for

stripping lanthanides bound to glycine-functionalized sorbent materials.50 The use of strong acids, however, leads to the decomposition of the Fc moiety and cannot be applicable in this experiment. Our experiments so far have shown that Ca2+ and Tb3+ cause significant changes in the electrochemistry of the films of the Fc-Glu conjugates 2 and 4. In solution studies, we have shown that metal ions coordinate to the carboxylate groups of the FcGlu conjugate but not to the amide groups. To get insight into the coordination behavior at the interface, we carried out XPS of films before and after the addition of Ca2+ and Tb3+. The immobilized dendrimers films of 2, 4, and their metal ion complex were further examined by XPS. At the beginning of each surface characterization experiment, core level survey spectra were collected using a clean gold surface as a reference.


Appoh and Kraatz

TABLE 4: XPS Analysis of Films of 2, 4 and Their Interactions with Ca2+ and Tb3+ (All Binding Energies Are in eV) C1s

O1s

N1s

films

CH-

C-O/C-N

CdO

C-O-H

CdO

N

2 2-Ca2+ 2-Tb3+ 4 4-Ca2+ 4-Tb3+

284.68 288.70 284.68 284.78 284.81 284.78

285.95 286.15 286.10 285.49 286.20 286.35

288.03 288.38 288.43 288.09 288.43 288.49

532.61 532.96 533.43 532.65 533.04 533.04

531.23 531.66 531.69 531.28 531.76 531.82

399.74 399.79 399.78 400.01 399.96 399.98

To characterize species present on the surfaces, high-resolution spectra were recorded for the main core level peaks of C, O, N, S, Fe, and the metal ions Tb, Ca that formed complexes with the films. All spectra were calibrated with reference to the Au4f7/2 peak at 84.00 eV. For the purpose of this study, the spectra analysis will emphasize the carboxylate (CdOOH) and the amide region (C(d)-NH-) as they are the ligand functional groups suitable for metal ion coordination. Studies by Winograd and co-workers describing the interaction of Al3+ with mercaptohexadecanoic acid (MPA) films showed that C1s and O1s core-level peaks shifted in the presences of Al3+.51 Figure 7 shows the C1s core peaks for films of 2 in the absence and presence of Ca2+ and Tb3+. The C1s core level of films of 2 was deconvoluted into three peaks, 284.68, 285.95, and 288.03 eV, corresponding to CH-, C-O/ C-N, and CdO moieties respectively.52,53 Table 4 lists the deconvoluted peaks associated with the C1s, O1s and N1s core elements. The C1s peaks for the C-O and CdO experience shifts toward higher binding energies upon addition of Ca2+ and Tb3+ (Figure 7). This is consistent with changes associated with metal ion coordination as observed for Al3+ on MPA surfaces.51 Shifts of 0.35-0.40 eV were observed for CdO components that compares with the 0.45 eV for Al3+MPA and the 0.5 eV for Cu2+-N-(amino-1-carboxypentyl)iminodiacetic acid (ANTA) films.54 Shifts associated with the C-O/C-N peak for the Ca2+ and Tb3+ of 0.71-0.89 eV with films of 4 were significantly higher than the smaller shifts of about 0.15-0.20 eV for films of 2. Interactions with NH of the peptides are unexpected but a differentiation between the C-N and C-O groups is not possible in this study. The O1s spectrum, shown in Figure 7, is deconvoluted into two peaks: the double bonds CdO at 531.23 and 531.28 eV and the single bonded C-O-H centered at 532.61 and 533.65 eV for films of 2 and 4 respectively.55 It has been suggested that the changes observed in these peaks after coordination to metal ions are indications of the involvement of the oxygen in the metal-binding process.55 From Table 4, we observe shifts of O peaks shifts toward higher binding energies, thereby increasing the peak separations between them. Increases in peak separation observed for Al3+MPA and Cr2+-11-mercaptoundecanoic acid (MUA) films have been associated with bidentate coordination51,56 while decreases in peak separation observed for Cu2+-MUA complexes have been linked with monodendate coordination.56 The bidentate complexation mode of Ca2+ and Tb3+ with the acid terminals of Fc-CO-peptide acids from the IR and NMR studies have been alluded to in previous solution studies.31 On the strength of metal in titration studies in solution, the IR and NMR studies of metal complex and the XPS surface studies of these metal interactions, a bidentate mode of coordination of Ca2+ and Tb3+ with Fc-peptide acid cystamine films is reasonable and is consistent with bidendate metal coordination exhibited by Fc-CO-GluOH.

Figure 8. N1s core-level spectra of films of 2 and in the presence of Ca2+ and Tb3+.

In a study of lanthanide selective sorbents, Fryxell and coworkers showed that self-assembly of glycinate-based (similar to films of 2 with only one carboxylate group) monolayers on mesoporous materials bound Eu3+ in an 8-coordinated fashion in which the close proximity of the ligands allowed four bidentate ligands to chelate the lanthanide cation.50 De´rue et al. also have shown from scanning force microscopy that elaidic acid monolayers prepared on a terbium-containing subphase indicate the formation of a complex between one terbium and three elaidic acid molecules in a bidentate mode.57 The films exhibit a single peak for the N1s peak between 399.74 and 400.01 eV due to the various amide functional groups (Figure 8). Individual amide groups are not distinguishable. The peak position is consistent with the literature as the amide groups give a peak at 400.0-400.5 eV.58 The peak position and shape did not change in the presence of Ca2+ and Tb3+, which suggests that the amide groups are not involved in metal binding. This however is significantly different from the films of amino acids/peptide, such as Cys, Gly-Cys, and Val-Cys films in which terminal NH3+ provides an additional binding site. The N1s peaks for these films are observed at 402.2-401.4 eV and are shifted upon coordination to Cu2+ to 399.4, 399.5, and 399.8 eV.3,59 It is however consistent with the observed nonshifting behavior of the 13C NMR spectra of the amide CdO of Fc-CO-GluOH in the presence Ca2+ and Tb3+.13 The Tb3d and Ca2p XPS spectrum for films of 2 and 4 in the presence of Tb3+ and Ca2+ is shown in Supporting Information, Figure S1. The peaks related to the Tb3d occur as a doublet at 1243.62 and 1277.43 eV in 2-Tb film and 1242.41 and 1277.13 eV in 4-Tb film displaying the spin-orbit splitting of 33.81 and 34.72 eV, respectively, compared to literature value of 34.6 eV which is characteristic of binding energies for with trivalent metal ions.60,61 The signals related to the 2p of Ca2+ are centered at 343.32-347.13 eV62,63 and demonstrate their low intensity masked by the presence of the strong interference from the Au4d peaks, which occur in the same region. From the XPS section, the attenuation of the CdO and O1s strongly



Figure 9. XPS stackplots of Au4f region for 2, 2-Tb3+, and 2-Ca2+ films on gold at various angles. Graphs of relation between 1/sinθ and logarithm of the integrated peak intensities of the Au4f in the XPS of 2, 2-Tb3+, and 3-Ca2+ films on gold at various angles.

suggest that both oxygen atoms of the acids functional group reacts with the metal ion to form the metal-carboxylate complexes. Calculations of compositional ratios of Ca2+ and Tb3+/S show a 1:1 ratio for films of 2 and 2:1 ratio for films of 4. Angle-resolved XPS (ARXPS) measurements were used to measure the thickness of the films of 2 and 4 in the presence and absence of the metal ions from the attenuation of the Au signals. 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 664,65

ln I ) -d/(λ sin θ) + ln Io

(6)

where Io 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) according to eq 8. Figure 9 shows the Au4f XP spectra for films of 2, 2-Tb3+, and 2-Ca2+. Au measured at various θ angles and the plots ln I versus 1/sinθ. The two peaks due to Au4f5/2 and Au4f7/2 are at 87.55 as well as 83.83 eV, respectively. The intensities of these peaks decreased with decreasing θ and plots of ln I versus 1/sinθ are linear. The slope of -d/λ from the ln I versus 1/sinθ plot for the Au4f7/2 at 83.83 eV shows that the slope for films containing metal ions increases considerably. Using a λ value of 42 Å,66,67 the thicknesses are calculated and compared with those from Spartan

TABLE 5: Calculated Thickness from XPS and Spartan Modeling (Errors (2 Å) XPS

Spartan

film

slope

λ

dÅ

dÅ

2 2-Tb3+ 2-Ca2+ 4 4-Tb3+ 4-Ca2+

1.63 3.38 2.42 1.86 3.85 2.51

42 42 42 42 42 42

7 14 10 8 16 11

9 9

modeling (Table 5). Films of conjugate 2 and 4 exhibit a film thickness of approximately 7 Å, which compares well with calculated molecular dimensions. In the presence of metal ions, the film thickness increases to about 10 Å for Ca2+ and 14 Å for Tb3+. These increases in thickness of the Fc-dendrimer films in the presence of metal ions confirm the observation from the titration monitored by electrochemical impedance. Differences in the thickness may well be related to the differences in coordination behavior between the two metal ions. Using ARXPS studies, we hoped to find out more details of where the metal ions are located within the film. Figure 10 shows the angular dependence of the Tb3d5/2 core signal for films prepared from the two acids 2 and 4. The spectra show an increasing contribution from the Tb3d5/2 core signal with increasing detecting angle and thus with decreasing probing depth of the spectrometer (see Supporting Information, Figure S4). This behavior clearly suggests that the Tb layer is located


Appoh and Kraatz studies were not aimed at probing the selectivity of the metalpeptide interaction, the system clearly displays a selectivity for Tb over Ca, which is not too surprising giving the high affinity of Tb for oxo-based ligands. Under present conditions, the films exhibit a detection limit for Tb3+ of approximately 10 µM. Presumably this detection limit is set by the formation of the overlayer. Thus, changes in the supporting electrolyte should significantly alter the sensitivity of the system to metal ions. At present we are investigating this further. Acknowledgment. The authors thank NSERC for financial support. H.-B.K. is the Canadian Research Chair in Biomaterials. We thank Dr. Dimitre Karpuzov, University of Alberta, for the XPS measurement.

Figure 10. Graph of normalized Tb3d5/2 core signal intensities as a function of photoelectron detection angle. Films of 2 with Tb3+ (O), and films of 4 with Tb3+ (0).

on top of the film rather than underneath the SAM. The fact that the films of 4 with Tb3+ shows a much steeper slope is a further evidence of a much thicker layer being formed compared to films of 2. Conclusions In this paper, surface characterization of disulfide-linked Fcpeptide conjugates [Fc-CO-Glu(G1OBz)-CSA]2 (1), [FcCO-Glu(G1OH)-CSA]2 (2), [Fc-CO-Glu(G2(OMe)2)-CSA]2 (3) and [Fc-CO-Glu(G2OH)2-CSA]2 (4) was presented. These systems present low generation glutamic acid based dendrimers of generation 1 and 2 and have a disulfide group that allows their immobilization on Au. All films exhibited a reversible oneelectron redox wave due to the Fc/Fc+ redox couple. All systems showed significant blocking to [Fe(CN)6]3-/4- as is evident by a lack of a signal due to the [Fe(CN)6]3-/4- redox couple. Ion pairing has a strong influence on the redox chemistry of the dendrimer films. This is reflected in the dependence of the formal potentials on the nature and the concentration of the counterion in solution. Under the experimental conditions, films of the esters experience stronger ion-pairing effects than the acids. The redox properties of the films of the acids [Fc-CO-Glu(G1OH)-CSA]2 (2) [Fc-CO-Glu(G2OH)2-CSA]2 (4) were monitored by CV and show an attenuation in the presence of Ca2+ and Tb3+, which is caused by changes in the ion pairing between Fc+ and ClO4-. These changes are reversible. The addition of EDTA will virtually regenerate the original film. Binding constants are slightly higher for Tb3+ than for Ca2+ and are comparable with those lanthanide and alkaline earth metal-dicarboxylic acid-metal interactions. Expectedly, the interactions of the films with the metal ions lead to an increase in the film thickness as reflected in a decrease in the total capacitance Ctot from EIS studies and from XPS depth-profiling experiments. XPS analysis of the films in the presence of Ca2+ and Tb3+ indicates that binding takes place at the carboxylate site and that the amide group is not involved in binding. Further compositional analysis of films of [Fc-CO-Glu(G1OH)CSA]2 and [Fc-CO-Glu(G2OH)2-CSA]2 in the presence of Tb3+ indicate stoichiometric interactions between the Tb3+ and the carboxylic acid groups. Two Tb3+ are bound per molecule in films of [Fc-CO-Glu(G2OH)2-CSA]2, possessing two carboxylic acid groups, while only one Tb3+ per molecule was bound for films of [Fc-CO-Glu(G1OH)-CSA]2. Our work on Fc-peptide films clearly shows their utility for the study of metal-peptide interactions. Although present

Supporting Information Available: Detailed information about the synthesis and characterization of all Fc-glutamic acid conjugates, 1H NMR spectra of 1 and 3 in CDCl3, IR, and UV spectroscopy results, ion-pair effect for 3 and 4, impedance plots of 4 and 4 + Tb3+, high-resolution XPS of Tb3d and Ca2p of Au surfaces modified with 2-Tb3+, 4-Tb3+, 2-Ca2+and 2-Ca2+ films, XPS of S2p, normalized Tb3d5/2 core signal intensities as a function of photoelectron detection angle. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Liu, A.-C.; Chen, D.-C.; Lin, C.-C.; Chou, H.-H.; C.-H., C. Anal. Chem. 1999, 71, 1549-1552. (2) Yang, W.; Gooding, J. J.; Hibbert, D. B. Analyst 2001, 126, 15731577. (3) Yang, W.; Jaramillo, D.; Gooding, J. J.; Hibbert, D. B.; Zhang, R.; Willett, G. D.; Fisher, K. J. Chem. Commun. 2001, 1982-1983. (4) Kim, H. T. Bull. Korean Chem. Soc. 2005, 26, 679-681. (5) Takehara, K.; Ide, Y.; Aihara, M. Bioelectrochem. Bioenerg. 1992, 29, 113-120. (6) Takehara, K.; Ide, Y.; Aihara, M.; Obunchi, E. Bioelectrochem. Bioenerg. 1992, 29, 103-111. (7) Takehara, K.; Aihara, M.; Ueda, N. Electroanalysis 1994, 6, 10831086. (8) Wang, Z.; Cook, M. J.; Nygård, A.-M.; Russell, D. A. Langmuir 2003, 19, 3779-3784. (9) Liu, S.-G.; Liu, H.; Bandyopadhyay, K.; Gao, Z.; Echegoyen, L. J. Org. Chem. 2000, 65, 3292-3298. (10) Le Derf, F.; Levillian, E.; Trippe´, G.; Gorgues, A.; Salle´, M.; Sebestian, R.-M.; Caminade, A.-M.; Majoral, J.-P. Angew. Chem. Int. Ed. 2001, 40, 224-227. (11) Ion, A.; Ion, I.; Moutet, J.-C.; Pailleret, A.; Popescu, A.; SaintAman, E.; Ungureanu, E. M.; Siebert, E.; Ziessel, R. Sens. Actuators, B 1999, 59, 118-122. (12) Ion, A. C.; Moutet, J.-C.; Pailleret, A.; Popescu, A.; Saint-Aman, E.; Siebert, E.; Ungureanu, E. M. J. Electroanal. Chem. 1999, 464, 2430. (13) Appoh, F. E.; Sutherland, T. C.; Kraatz, H. J. Organomet. Chem. 2004, 690, 1209-1217. (14) Degenhart, G. H.; Dordi, B.; Scho¨nherr, H.; Vansco, G. J. Langmuir 2004, 20, 6216-6224. (15) Liu, D.; Szuiczewski, G. J.; Kispert, L. D.; Primak, A.; Moore, T. A.; A. L., M.; Gust, D. J. Phys. Chem. B 2002, 106, 2933-2936. (16) Gomez, M. E.; Kaifer, A. E. J. Chem. Educ. 1992, 69, 502-505. (17) (a) Ravenscoft, M. S.; Finklea, H. O. J. Phys. Chem. 1994, 98, 3843-3850. (b) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski T. M.; Mujsce A. M. J. Am. Chem. Soc. 1990, 112, 4301. (c) Laviron, E. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1982; Vol. 12, pp 53-157. (d) Murray R. W. In Electroanalytical Chemistry, Bard, A. J., Ed.; Marcel Dekker: New York, 1982; Vol. 13, pp 191-368. (e) Honeychurch, M. J.; Rechnitz, G. A. Electroanalysis 1998, 10, 285-293. (f) Honeychurch, M. J.; Rechnitz, G. A. Electroanalysis 1998, 10, 453457. (18) Sabapathy, R. C.; Bhattacharyya, S.; Leavy, M. C.; Cleland, W. E.; Hussey, C. L. Langmuir 1998, 14, 124-136. (19) Finklea, H. O.; Liu, L.; Pavenscroft, M. S.; Punturi, S. J. Phy. Chem. 1996, 100, 18852-18858. (20) Finklea, H. O. In Electroananlytical Chemistry; Bard, A. J., Rubinstein, I., Eds.; Marcel Dekker: New York, 1996; Vol. 19, pp 110337.

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