Electron Transfer Across α-Helical Peptide Monolayers: Importance

Article pubs.acs.org/Langmuir

Electron Transfer Across α‑Helical Peptide Monolayers: Importance of Interchain Coupling Jan Pawlowski, Joanna Juhaniewicz, Dagmara Tymecka, and Slawomir Sek* Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland ABSTRACT: Four helical peptides with the general formula (Boc)-Cys-(S-Acm)-(Ala-Leu)n-NH-(CH2)2-SH (n = 4−7) were synthesized and further used for the preparation of self-assembled monolayers (SAMs) on gold substrates. The electron-transfer behavior of these systems was probed using current-sensing atomic force microscopy (CS-AFM). It was found that the electron transmission through SAMs of helical peptides trapped between an AFM conductive tip and a gold substrate occurs very efficiently and that the distance dependence obeys the exponential trend with a decay constant of 4.6 nm−1. This result indicates that the tunneling mechanism is operative in this case. Conductance measurements under mechanical stress show that peptide-mediated electron transmission occurs with the possible contribution of intermolecular electron tunneling between adjacent helices. It was also demonstrated that an external electric field applied between metallic contacts can affect the structure of the peptide SAM by changing its thickness. This explains the asymmetry of the current−voltage response of metal−monolayer−metal junction.

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INTRODUCTION Peptides are known to be crucial components of proteins that provide different functions in biological systems. These include enzymatic catalysis, control of mass transport, adhesion, regulation of biochemical processes, energy storage, and electron transfer.1 Such a broad range of functions results from the diversity of the peptides and their ability to adopt numerous structural motifs. Given the above, peptides seem to be excellent components that can be suitably designed to provide specific properties that are useful in nanoscale electronics and biosensing devices.2,3 In most cases, the successful application of peptides in such nanodevices requires the adsorption of molecules on a conductive substrate in a conformation that enables the efficient mediation of the electron-transfer process. It was demonstrated in numerous papers that peptides assembled into molecular layers immobilized on a metallic surface can act as electron-transfer mediators.4−10 Moreover, the efficiency of this process can be modulated by the changes in the secondary structure and length of the peptide.11−13 Among the variety of peptide structural motifs, helical structures seem to be the most efficient electron-transfer mediators. As reported by Kimura’s group, αhelical peptides organized within a self-assembled monolayer (SAM) enable long-range charge transport over an enormous distance of 10 nm.14,15 The good mediating properties of helices are also confirmed by the relatively weak distance dependence of electron transfer along the peptide bridge. The decay factors reported for helical peptides are in the range of 0.2−5.0 nm−1.8,9,12,14 The large spread in the reported decay constants results from the fact that the overall electron transfer through peptides can be affected by two mechanisms: tunneling and hopping.6 Their contribution varies with the length of the mediating bridge.16 Tunneling dominates for short bridges, © 2012 American Chemical Society

giving rise to a sharp exponential distance dependence. For longer bridges, the hopping contribution prevails, resulting in a much weaker distance dependence. Unfortunately, it is difficult to indicate the sharp transition between these two mechanisms. Usually for helical bridges the increased contribution of hopping, recognized as a weakening of the distance dependence, become apparent for bridges exceeding 3.0 nm in length.9,14,17 Nevertheless, this number cannot be considered to be a stiff limit because the relative changes in the contributions of two mechanisms occur gradually. Another important factor that needs to be considered in a description of electron transfer through the helix is related to the motional freedom of the adsorbed peptide. Molecular dynamics was demonstrated to have a large impact on electron-transfer behavior, and the restriction of some vibrational modes of the molecule results in the suppression of the electron-transfer rate.9,18 Additionally, the whole picture is complicated by the fact that the structure of the peptide can also be affected by an external electric field. For example, the variation of the helical peptide SAM thickness induced by the potential applied to the underlying substrate was reported by Wain et al.19 Moreover, Kimura and coworkers have demonstrated that the peptide molecule placed between a metallic substrate and an STM tip changes its conformation from an α-helix to a 310-helix in response to the applied bias voltage.20 It is known that helical peptides possess large dipole moments along their molecular axes, with a partial positive charge at the N terminus and a partial negative charge at the C terminus. Therefore, the helix can be compressed or stretched depending on whether the external electric field is Received: July 6, 2012 Revised: September 28, 2012 Published: November 26, 2012 17287

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then in DMF/IPA. Such treatment led to the removal of the Fmoc protecting group, and free amine groups were further coupled with activated Fmoc-L-Ala-OH using the same routine as described above. Monitoring of the completion of the each Fmoc cleavage and of each coupling reaction was performed using the qualitative ninhydrine test (Kaiser test). The entire procedure was repeated until the desired sequence of the peptide was obtained (i.e., including four to seven AlaLeu pairs). Finally, the peptide was coupled with (Boc)-L-Cys-(Acm), sequentially washed with DMF, dichloromethane, methanol, and diethyl ether, and removed from the resin using a 1.1% solution of trifluoroacetic acid in dichloromethane/triisopropylsilane (95:5 v/v). We have synthesized four peptides characterized by the general formula (Boc)-Cys-(S-Acm)-(Ala-Leu)n-NH-(CH2)2-SH, where n = 4−7. For simplicity, the peptides will be abbreviated as AL4, AL5, AL6, and AL7. The identity of the final products was confirmed by mass spectrometry. ESI MS: (AL4), (Boc)-Cys-(S-Acm)-(Ala-Leu)4-NH(CH2)2-SH, [M + Na]+ m/z = 1110.7; (AL5), (Boc)-Cys-(S-Acm)(Ala-Leu)5-NH-(CH2)2-SH, [M + Na]+ m/z = 1295.3; (AL6), (Boc)Cys-(S-Acm)-(Ala-Leu)6-NH-(CH2)2-SH, [M + Na]+ m/z = 1480.0; (AL7), (Boc)-Cys-(S-Acm)-(Ala-Leu)7-NH-(CH2)2-SH, [M + Na]+ m/z = 1665.2. The helicity of the peptides was evaluated on the basis of circular dichroism (CD) spectroscopy experiments. The spectra were collected using a J-815 CD spectrometer (Jasco Inc., Easton, MD) in a 1 cm quartz cell at room temperature. A mixture of TFE/H2O (6:4 v/v) was used as a solvent. The concentration of the peptides in solution was 50 μM. The ellipticity was expressed as the mean residue molar ellipticity (θmre in deg cm2 dmol−1): θmre = θobs /(10ClN), where θobs is the ellipticity in degrees, C is peptide concentration in mol/cm3, l is the optical path in cm, and N is the number of peptide bonds.33 For each peptide studied here, the spectra displaying a double-minimum pattern were observed. Negative peaks were observed at 208 and 222 nm, indicating that the peptides adopt an α-helical structure. The α-helix content of each peptide was calculated on the basis of the mean residue molar ellipticity at 222 nm, f h = θ222 + 2340/(−30 300).34 The values found this way were 22, 50, 52, and 54% for AL4, AL5, AL6, and AL7, respectively. To check whether the helical conformation is sustained after adsorption on a metallic surface, we have carried out additional CD measurements for gold nanoparticles coated with peptides. First, the peptides were immobilized on gold nanoparticles (i.e., a 100 μM solution of a given peptide in a mixture of TFE/H2O (6:4 v/v) was slowly added to a water suspension of ∼0.13 μM gold nanoparticles with an average diameter of 5 nm (Sigma-Aldrich) and stirred for 16 h in darkness. Then the solution was removed, and the peptide-covered nanoparticles were purified by five cycles of washing with water, centrifugation, and removal of the supernatant. The CD spectra for peptide-modified nanoparticles displayed a doubleminimum pattern characteristic of helical conformation. Although the results were obtained on nanoparticles (3D assembly), we assume that the helical structure is also preserved on the planar gold surface (2D assembly). As demonstrated by Mandal and Kraatz, the helicity of the peptide adsorbed on gold can be affected by surface curvature.35 These authors have reported a structural investigation of a Leu-rich peptide organized in both 2D and 3D SAMs. The formation of 3D SAMs involved the immobilization of the peptide on gold nanoparticles with increasing core diameters: 5, 10, and 20 nm. In this way, the curvature between crystallographic faces was gradually increased. What was found is that the degree of surface curvature has a considerable effect on the secondary structure of the peptide. It was demonstrated that the decrease in surface curvature leads to a stabilization of the helical conformation and an increase in the helical content of the peptide. In our case, CD spectra of the 3D SAM displayed a double-minimum pattern similar to that observed for free peptides in solution, indicating that the helical conformation is retained after adsorption on gold nanoparticles. Because we observed the preservation of the helical structure of peptides even on 5 nm gold nanoparticles with high surface curvature, we expect that the helical structure is retained and even improved on the planar gold surface. Recently, Lopez-Perez et al. reported their results of a molecular

oriented parallel or antiparallel to the direction of the dipole moment. The above-mentioned charge separation is responsible for another interesting featurethe directional dependence of helix-mediated electron transfer (i.e., electron transfer parallel to the dipole moment is accelerated compared to that in the antiparallel direction21−23). Generally, peptide-mediated electron transfer is a complex process, and the description of its detailed mechanism is quite complicated. In this article, we report the electron transfer behavior of helical peptides assembled into single molecular layers immobilized on gold surfaces. The aim of this work was to explore the mechanisms controlling the electron transmission through helical peptide monolayers and evaluate the effects induced by an external electric field. In particular, we looked at the effects related to possible interchain electron transport between adjacent helices. The latter has not been considered extensively so far. As we demonstrated very recently, intermolecular interactions may play an important role in determining the overall efficiency of charge transfer.24 This may contribute to the establishment of alternative electron-transfer pathways. We have used methodology based on the molecular junction approach (i.e., the monolayers were sandwiched between the underlying substrate and the conductive goldcoated probe of the atomic force microscope; see scheme 1).25 Scheme 1. Schematic Illustration of a Molecular Junction Formed Using the Current-Sensing AFM Setup

In such case, electron transmission occurs in response to the bias voltage applied between the substrate and the metallized tip. The magnitude of the measured current is proportional to the area of the junction and the efficiency of electron transfer through the monolayer separating the metallic contacts. Such an approach was successfully used for numerous systems including self-assembled monolayers of alkanethiols,26 alkanedithiols,27 oligophenylene derivatives,28 tetrathiafulvalene derivatives,29 peptides,30 proteins,31 and more.

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EXPERIMENTAL SECTION

The chemicals used in this work were purchased from Aldrich, Merck, Fluka, ChemPur, and POCh Gliwice. We have synthesized four peptides based on the Ala-Leu sequence because these two amino acids are known from their high α-helix-forming propensities.32 The synthesis of the peptides was based on the active esters method and was performed by solid-phase synthesis techniques using cysteamine 4methoxytrityl resin. In the first stage, the carboxylic group of Fmoc-LLeu-OH was activated in the presence of 1-hydroxybenzotriazole (HOBt), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU), and diisopropylethylamine (DIPEA). This step was performed in N,N-dimethylformamide (DMF). The resulting solution was mixed with cysteamine 4-methoxytrityl resin and left for 24 h to complete its coupling. Then the resin was sequentially washed with DMF, a 20% solution of piperidine in DMF, again in DMF, and 17288

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dynamics simulation that also confirms that the α-helical conformation is stabilized in densely packed 2D systems.24 The adsorption of the peptides on gold was investigated using the quartz crystal microbalance. The measurements were carried out using an Autolab PG STAT 302N equipped with an EQCM-oscillator (Metrom Autolab). The working crystals were commercially available 6 MHz gold-coated AT-cut quartz crystals with an area of 0.36 cm2. The surface of the crystal was cleaned by electrochemical treatment in 1 M H2SO4 and 1 M NaOH and washed thoroughly with Milli-Q water. The measurements were performed in a mixture of trifluoroethanol/water (6:4 v/v), and the viscosity and density of the solution were kept constant during the experiments. Before each experiment, the frequency response of the crystal was stabilized in a pure solvent for 2 to 3 h. The frequency change corresponding to the adsorption of the peptide on the gold surface was recorded after the addition of an aliquot of peptide solution to the cell. For an assumed ideal system, the mass change was calculated according to the frequency−mass relationship described by the Sauerbrey equation.36 The area occupied by a single molecule determined from QCM experiments is shown in Table 1.

Figure 1. Exemplary STM images of (A) bare gold and (B) an AL6 self-assembled monolayer on gold. Images were recorded in air using electrochemically etched tungsten tips. Tunneling conditions: (A) it = 0.6 nA, Ebias = 0.3 V and (B) it= 0.02 nA, Ebias= 1.0 V. sensors) silicon nitride probes for contact AFM and nanoshaving. The force constants for the cantilevers used in this work were in the range of 0.1−0.5 N/m. To verify whether the Acm deprotection procedure was successful, force−distance curves were recorded using gold-coated probes, and the adhesion forces were determined. It was found that the average adhesion forces are 6 and 12 nN for Acm-protected and deprotected samples, respectively. The higher adhesion force observed after the removal of Acm groups reflects the stronger interaction between the tip and the monolayer, which is reasonable because the chemisorption of free thiol groups on gold-coated probes is expected.

Table 1. Thickness of the Peptide-Based SAMs and Estimated Tilt Angles of the Molecules peptide forming the SAM (no. of amino acid residues) AL4 AL5 AL6 AL7

(9) (11) (13) (15)

length of the peptide molecule/ nma 1.90 2.20 2.50 2.80

molecular area estimated from QCM experiment/ nm2 per molecule 1.23 1.23 1.23 1.17

± ± ± ±

0.06 0.05 0.06 0.08

SAM thickness under 1 nN applied load/ nm

tilt angle at 1 nN load/ deg

± ± ± ±

41 42 43 45

1.43 1.64 1.81 1.98

0.01 0.02 0.01 0.01

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RESULTS AND DISCUSSION To determine the thicknesses of the peptide monolayers, we have performed measurements utilizing an AFM-based nanometer-scale lithography technique (i.e., nanoshaving37). In detail, a given area of the film was scanned using a silicon nitride probe under a relatively high applied load. Usually, forces in the range of 50−60 nN were required. Because of the high forces applied, the molecules were removed from the substrate by the scanning probe. In this way, the so-called shaved region was obtained. Then, the applied load was decreased down to 1 nN and the scanned area was increased to get the topography of the shaved region with the surrounding nonperturbed monolayer. Such images were subjected to crosssectional analysis, which allows us to measure the height differences between the bare and monolayer-coated substrate. Said differences correspond to the monolayer thickness measured under a 1 nN load applied to the AFM tip.38 The results of the thickness analysis are shown in Table 1. As expected, the film thickness changes proportionally to the length of the peptide molecules forming the SAM (i.e., the addition of two amino acid residues results in approximately a 0.2 nm increase in film thickness). Using these data, we were able to estimate the average tilt angle of the peptide molecules with respect to the surface normal. As we found, it ranges from 41 to 45°, indicating a significant deviation from the upright orientation. The ability of the peptide-based SAMs to mediate electron transfer was investigated using CS-AFM. We have measured the current−voltage (I−V) characteristics for the monolayers entrapped between two metallic contacts (i.e., a gold substrate and a gold-coated conductive probe as the bottom and top contacts, respectively (Scheme 1). The electrical measurements were performed at a 1 nN loading force. We have used five tips, and the measurement were performed using a so-called blind approach procedure (i.e., without imaging/scanning of the surface). Such an approach allows us to minimize tip wear, which would result in a considerable variation of the junction

This was estimated assuming an α-helical conformation with dihedral angles of φ = −58°, ψ = −47°, and ω = 180°, resulting in a 0.15 nm increase per residue. The errors indicated in the table represent standard deviations of the measured values.

a

For AFM studies, the monolayers of peptides were immobilized on gold substrates purchased from Arrandee (Werther, Germany). Each substrate was carefully flame annealed before the deposition of the peptide. The self-assembled monolayers (SAMs) were prepared from 0.1 mM solutions of peptides in TFE/H2O (6:4 v/v) for 24 h. It was demonstrated in our previously published papers that the presence of the Acm group protecting sulfur at the cysteine residue hinders the adsorption of the peptides through the N terminus.12 As a result, peptides bind to gold using the free thiol group at the cysteamine linker, and the monolayer is formed by the molecules with uniform orientation. However, the presence of free thiol groups in the external plane of the monolayer is crucial to the formation of the metal− monolayer−metal junctions. Therefore, the monolayer-modified samples were thoroughly rinsed with water and subjected to an Acm deprotection procedure. The monolayer-covered substrates were placed in deoxygenated water, and the pH was adjusted to 4.0 using small amounts of ammonia and acetic acid. Then, mercury(II) acetate was added, and the resulting solution together with the substrates was bubbled with argon for 45 min. After that, an aliquot of βmercaptoethanol was added, and the solution was further bubbled with argon for the next 10 min. Finally, the substrates were removed from the solution, washed with water, and dried in a stream of argon. The quality of the monolayers that were formed was checked by STM imaging in air. Exemplary images are shown in Figure 1. Dry samples were ready to use for AFM experiments and the formation of the metal−monolayer−metal junctions. Atomic force microscopy (AFM) experiments were carried out with a 5500 AFM (Agilent Technologies, Santa Clara, CA, USA). The AFM data was recorded in air using PPP-CONTAu (Nanosensors) goldcoated conductive probes for current-sensing AFM, SCM-PIC (Pt/Ir)coated tips (Bruker AFM Probes), and PPP-CONTAuD (Nano17289

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area. The data was collected on five different spots on each sample, and on average, 10 I−V curves were recorded at 1 location. To minimize the effects connected to the tip-to-tip variation in the junction area, the data obtained with each tip was analyzed separately. Figure 2 presents example I−V curves

that the conductance of the junction decays exponentially with increasing SAM thickness and the decay factor varies from 4.0 to 5.1 nm−1 with an average of 4.6 ± 0.3 nm−1. This value is quite low but close to that reported by our group earlier for the helical peptides entrapped within STM-based molecular junctions (i.e., 5.0 nm−1).12 An even lower value of the decay constant (3.2 nm−1) was reported by Kimura and co-workers for SAMs of Ala-Aib helical peptides.9 As suggested by these authors, such a weak distance dependence may result from the considerable contribution of the hopping mechanism, which is opposite to the tunneling and is characterized by a weak distance dependence. The difference comes from the fact that hopping occurs through sequential electron transfer between adjacent amide bonds that act as hopping sites. In other words, this mechanism assumes the localization of electrons or holes at specific sites along the peptide chain, and the reversible oxidation and reduction of amide bonds is involved. The latter becomes possible because the α-helical structure possesses a large dipole moment that causes a decrease in the redox potential of the amide groups to a level comparable to heterocyclic and sulfur-containing side chains.39,40 Unfortunately, it is quite difficult to evaluate whether such a mechanism is operative in the case of the junction system presented in this work. The conductance was determined from the measurements performed in air and at a relatively low bias voltage. Such conditions may not be sufficient to meet the requirements for charge hopping. Therefore, we conclude that the dominating mechanism for ET through AL4−AL7 peptide SAMs is simply tunneling. However, Gray and Winkler suggested that the upper limit of the tunneling distance is 2 nm,41 and the length of the peptides used here spans the range from 1.90 to 2.80 nm (Table 1). Thus, in the case of simple through-bond electron transmission, tunneling should be negligible for AL5, AL6, and AL7. This prompted us to consider the shortest possible ET pathway, which involves intermolecular electron jumps between neighboring molecules. In such case, the tunneling distance is defined by the SAM thickness and does not exceed the limit of 2 nm (Table 1). Slowinski and co-workers proposed a twopathway model including through-bond and through-space coupling.42 The first pathway involves electron tunneling along the molecular backbone, and thus its length is constant for a given molecule. The second pathway involves chain-to-chain coupling, and it depends on the tilt angle of the molecules. To verify whether the contribution from chain-to-chain coupling exists, we have performed conductance measurements for a SAM of AL6 under increasing applied load. It can be expected that compression leads to tilting and/or structural changes in the molecules forming the film. In the latter case, the helical structure is destroyed and the random coil conformation becomes dominant. However, it should be noted that such a structural transition can alter the stiffness of the film under compression. As demonstrated by Oelker and co-workers, the ratio of the elastic moduli for helical and random coil hydrogel network is ∼2.2, which means that the helical conformation contributes to a higher stiffness compared to that of a random coil.43 On the basis of this observation, we conclude that the transition between helical and random coil conformations should be reflected by changes in the mechanical properties of the SAM. Indeed, the plot presented in Figure 4 demonstrates the sudden change in the elastic properties of the AL6 SAM at load exceeding 4 nN. Considering the above, we ascribed the elastic deformation of the film under low forces mainly to the variation of the molecular tilt angle. In this way, by increasing

Figure 2. Current−voltage characteristics of the peptide monolayers (AL4−AL7). Vertical dotted lines border the low-bias region with the linear part of the I−V curves. The inset shows an example gold-coated AFM probe used for electrical measurements.

obtained with a single tip on gold substrates modified with SAMs AL4−AL7 in which each curve is an average of 10 individual current−voltage traces. It is apparent that the current flow through the junction decreases with increasing SAM thickness. This simply shows that the electron transmission is affected by the width of the barrier established by the peptide film (i.e., a thicker film means a broader barrier for electron transfer). To evaluate the efficiency of electron transmission through AL4−AL7 films, we have analyzed the changes in conductance as a function of the monolayer thickness. The values of the conductance were obtained from the slopes of the linear parts of I−V curves (within the ±0.25 V range). Figure 3 presents the data obtained with five independent tips. It is clear

Figure 3. Changes in the junction conductance as a function of the SAM thickness. The decay constants determined from the slopes vary between 4.0 and 5.1 nm−1. 17290

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Figure 5. Current density as a function of tip-compression-induced changes in the AL6 SAM thickness. The current density was determined at a bias of 0.1 V. The thickness of the AL6 SAM at different loads was obtained using AFM by imaging previously nanoshaved regions of the SAM. Imaging was carried out at 0.5, 1, 2, and 4 nN, and then the thickness was determined from the crosssectional profiles.

Figure 4. Thickness of the AL6 self-assembled monolayer as a function of the load applied between the tip and the sample. The monolayer is elastically deformed as the load is increased because the thickness changes proportionally to F2/3. The inflection point corresponds to a load of 4.4 nN, and it reflects a sudden change in the mechanical properties of the SAM.

the force applied between the tip and the sample from 0.5 to 4 nN, the tilt angle of the molecules with respect to the surface normal will be increased. If interchain jumps contribute to the overall electron transmission, then this should result in the growth of the measured conductance because the tunneling pathway is shortened as the load is increased. However, it is well known that the changes in junction conductance are also related to the variation in the junction area. The latter increases proportionally to the applied load. According to the Johnson− Kendall−Roberts (JKR) model, the contact radius (a) between the probe and the sample can be calculated as44 R⎡ ⎤ a3 = ⎣L + 2La + 2 LaL + La 2 ⎦ (1) K where R is the mean radius of tip curvature (in our case, it was not greater than 35 nm), L and La represent the external load and adhesive force, respectively, and K is an effective modulus defined as 2 (1 − νs 2) ⎤ 4 ⎡ (1 − νt ) ⎥ K= ⎢ + 3⎣ Et Es ⎦

the exponential growth of the current density when the applied load was increased from 0.5 to 4 nN. The slope yields a decay factor of 3.6 ± 1.1 nm−1. This result indicates that the tunneling mechanism prevails and that the increase in the tilt angle affects the junction conductance by shortening the electron-transfer pathway. The latter is a consequence of interchain coupling between neighboring helices because through-bond electron transfer makes a constant contribution and does not affect the conductance when the tilt angle is changing. It is noticeable that the value of the decay constant does not differ statistically from that measured for AL4−AL7 systems, indicating that the height of the tunneling barrier is similar. This leads us to the conclusion that the structural integrity of the monolayer is largely preserved during compression and the observed increase in conductance is attributed mainly to the changes in the width of the barrier (i.e., the tunneling distance). The tilted orientation of the molecules and interchain coupling would also explain the very poor current rectification observed for AL4−AL7 monolayers. As already noted, the molecules of α-helical peptides possess a large dipole moment directed along the molecular axis from the C-terminus to the N-terminus. Therefore, when considering electron transmission through a helical peptide we can expect some influences from the electric field induced by the dipole. In other words, the electron transport in a direction aligned with the dipole should be faster than in the opposite direction. This would result in asymmetric current−voltage characteristics. Such rectification behavior was reported for helical peptides immobilized on conductive surfaces.21−23 Nevertheless, the analysis of the current−voltage traces obtained for AL4−AL7 monolayers (Figure 2) indicates that the asymmetry is noticeable but quite small. The currents measured for negative bias are at most 2 times higher than those obtained for positive bias. This indicates that the influence of the dipole is suppressed, which may result from the fact that the electron-transfer pathway is not fully aligned with the direction of the molecular axis of the peptide molecule. Such an interpretation seems to support an interchain mechanism.

−1

(2)

where ν and E are Poisson's ratio and Young's modulus, with indexes t and s corresponding to the tip and the sample, respectively. For the system studied here we have used following values: Poisson’s ratio was νt = 0.42 for gold and νs = 0.33 for helical peptide SAM,45 and Young's modulus was Et = 69 GPa for a gold-coated probe46 and Es = 1.2 GPa for a helical peptide.47 The effective modulus was calculated to be 1.8 × 109 N/m2. The resulting value of the contact radius (a) was used to estimate the area of circular contact between the tip and the sample. Furthermore, the measured current was normalized by dividing the determined value by the contact area. We have noted that the contact area changed on average by 10% when increasing the loading force from 0.5 to 4 nN. Figure 5 illustrates the changes in the current density as a function of the SAM thickness determined at the given force. The SAM thickness was determined using the same experimental procedure as described earlier. We have observed 17291

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the monolayer thickness has to be decreased. Then, by applying a positive bias, the situation is reversed (i.e., the top part of the molecule is repelled from the positively charged substrate). Thus, the molecules respond either by stretching the helix or decreasing the tilt angle. As a result, the monolayer is thicker. Such a variation in monolayer thickness would involve an ∼0.3 nm change in the length of the molecule or an ∼6° change in the tilt angle. Of course, by using AFM data we are not able to distinguish which scenario is correct; however, our results confirm that the molecular motion is an important factor in determining the efficiency of peptide-mediated electron transfer. It should also be noted that the changes in monolayer thickness can explain the asymmetry of the I−V curves recorded in our experiments. At negative bias, electron transmission occurs through a thinner film; therefore, the distance for electron transfer is shorter compared to the positive bias when the monolayer becomes thicker. As a result of the shorter pathway, the current is higher at negative voltage. Moreover, our results would also explain why the rectification ratio for peptide monolayers is negligible at low and moderate voltages and becomes significant at higher biases.21−23 At low voltages, we can expect rather small changes in the SAM thickness because of the relatively weak electric field, but said changes become more pronounced when the strength of the electric field is increased.

In the molecular junction, the monolayer is entrapped between two metallic contacts, and a certain bias voltage is applied between them. This means that the molecules are exposed to the electric field present between the two electrodes. Considering that helical peptides possess a large dipole moment, such an external electric field may influence both their structure and orientation. We have verified this assumption by performing AFM measurements of the AL6 monolayer thickness while applying bias voltages with opposite signs. First, the given area of the monolayer was subjected to a nanoshaving procedure under a high load (i.e., 50 nN). Then the scanning area was increased, and after the system was stabilized for drift, the shaved region together with the surrounding intact monolayer was imaged using an SCM-PIC conductive tip at −1.5 and +1.5 V bias voltages. The imaging was performed under a 1 nN applied load on exactly the same spot on the sample. Furthermore, we have performed crosssectional analysis of the images and compared the profiles extracted from the scan lines corresponding to the same position on images recorded at −1.5 and +1.5 V (Figure 6). We

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CONCLUSIONS We have demonstrated that electron transmission through helical peptide monolayers entrapped between two metallic contacts occurs very efficiently. The dominant mechanism of electron transport is tunneling, as indicated by the exponential distance dependence. The small value of the decay constant (i.e., 4.6 nm−1) indicates that the tunneling barrier is relatively low. Our results show that interchain electron jumps should also be considered to be a possible electron-transfer pathway for peptide SAMs. Such a mechanism is promoted by the tilted orientation of the molecules forming the assembly. It was also demonstrated that the external electric field can affect the structure of the peptide monolayer, resulting in the asymmetry of the current−voltage response.

Figure 6. Changes in the AL6 monolayer thickness after switching the bias voltage from +1.5 to −1.5 V. Height profiles were taken along the white lines indicated in the AFM images. The red image was recorded at +1.5 V, and the blue one was recorded at −1.5 V. The crosssectional profiles utilize the same color code.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

have found that the AL6 monolayer becomes 0.21 ± 0.07 nm thicker on average when switching the bias from negative to positive. This result seems to confirm the data reported by Kraatz18,19 and Kimura,20 who observed the structural transition of the helical peptides subjected to an external electric field. The changes in the AL6 monolayer thickness can be explained either by the structural transformation or the variation of the tilt angle of the peptide molecules in response to the external electric field. Helical peptide AL6 and other peptides studied in this work are oriented on gold with the C terminus located close to the electrode surface whereas the N terminus is near the external plane of the monolayer. This leads to the orientation of the dipole moment with the negative partial charge close to the substrate and the positive partial charge in the top part of the assembly. By applying a negative bias (i.e., a negatively charged substrate), the positively charged top part of the peptide is attracted to the gold surface. This results either in a slight compression of the helix or an increase in its tilt angle with respect to the surface normal. In both cases,

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Higher Education (grant no. N N204 138137). This project was also operated within the Foundation for Polish Science MPD Program cofinanced by the EU European Regional Development Fund. Research was carried out with the use of CePT infrastructure financed by the European Unionthe European Regional Development Fund within the Operational Program “Innovative Economy” for 2007−2013.

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REFERENCES

(1) Whitford, D. Proteins: Structure and Function; John Wiley & Sons: Chichester, U.K., 2005. (2) Pepe, B. J.; Fairman, R. Peptides as materials. Curr. Opin. Struct. Biol. 2009, 19, 483−494. 17292

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(3) Cui, Y.; Kim, S. N.; Naik, R. R.; McAlpine, M. C. Biomimetic peptides nanosensor. Acc. Chem. Res. 2012, 45, 696−704. (4) Morita, T.; Kimura, S. Long-range electron transfer over 4 nm governed by an inelastic hopping mechanism in self-assembled monolayers of helical peptides. J. Am. Chem. Soc. 2003, 125, 8732− 8733. (5) Galka, M. M.; Kraatz, H. B. Electron transfer studies on selfassembled monolayers of helical ferrocenoyl-oligoproline-cystamine bound to gold. ChemPhysChem 2002, 3, 356−359. (6) Long, Y.-T.; Abu-Rhayem, E.; Kraatz, H. B. Peptide electron transfer: more questions than answers. Chem.Eur. J. 2005, 11, 5186−5194. (7) Dey, S. K.; Long, Y.-T.; Chowdhury, S.; Sutherland, T. C.; Mandal, H. S.; Kraatz, H. B. Study of electron transfer in ferrocenelabeled collagen-like peptides. Langmuir 2007, 23, 6475−6477. (8) Kai, M.; Takeda, K.; Morita, T.; Kimura, S. Distance dependence of long-range electron transfer through helical peptides. J. Pept. Sci. 2008, 14, 192−202. (9) Takeda, K.; Morita, T.; Kimura, S. Effects of monolayer structures on long-range electron transfer in helical peptide monolayer. J. Phys. Chem. B 2008, 112, 12840−12850. (10) Okamoto, S.; Morita, T.; Kimura, S. Electron transfer through a self-assembled monolayer of a double-helix peptide with linking the terminals by ferrocene. Langmuir 2009, 25, 3297−3304. (11) Sek, S.; Sepiol, A.; Tolak, A.; Misicka, A.; Bilewicz, R. Distance dependence of the electron transfer rate through oligoglycine spacers introduced into self-assembled monolayers. J. Phys. Chem. B 2004, 108, 8102−8105. (12) Sek, S.; Misicka, A.; Swiatek, K.; Maicka, E. Conductance of αhelical peptides trapped within molecular junctions. J. Phys. Chem. B 2006, 110, 19671−19677. (13) Juhaniewicz, J.; Sek, S. Peptide molecular junctions: distance dependence electron transmission through oligoprolines. Bioelectrochemistry 2011, 87, 21−27. (14) Arikuma, Y.; Nakayama, H.; Morita, T.; Kimura, S. Electron hopping over 100 Å along an α helix. Angew. Chem., Int. Ed. 2010, 49, 1800−1804. (15) Arikuma, Y.; Nakayama, H.; Morita, T.; Kimura, S. Ultra-longrange electron transfer through a self-assembled monolayers on gold composed of 120-Å-long α-helices. Langmuir 2011, 27, 1530−1535. (16) Petrov, E. G.; Shevchenko, Y. V.; Teslenko, V. I.; May, V. Nonadiabatic donor-acceptor electron transfer mediated by a molecular bridge: a unified theoretical description of the superexchange and hopping mechanism. J. Chem. Phys. 2001, 115, 7107− 7122. (17) Paul, A.; Watson, R. M.; Wierzbinski, E.; Davis, K. L.; Sha, A.; Achim, C.; Waldeck, D. H. Distance dependence of the charge transfer rate for peptide nucleic acid monolayers. J. Phys. Chem. B 2010, 114, 14140−14148. (18) Mandal, H. S.; Kraatz, H. B. Electron transfer across α-helical peptides: potential influence of molecular dynamics. Chem. Phys. 2006, 326, 246−251. (19) Wain, A. J.; Do, H. N. L.; Mandal, H. S.; Kraatz, H. B.; Zhou, F. Influence of molecular dipole moment on the redox-induced reorganization of α-helical peptide self-assembled monolayers: an electrochemical SPR investigation. J. Phys. Chem. C 2008, 112, 14513− 14519. (20) Kitagawa, K.; Morita, T.; Kimura, S. A helical molecule that exhibits two lengths in response to an applied potential. Angew. Chem., Int. Ed. 2005, 44, 6330−6333. (21) Kitagawa, K.; Morita, T.; Kimura, S. Molecular rectification of a helical peptide with a redox group in the metal-molecule-metal junction. J. Phys. Chem. B 2005, 109, 13906−13911. (22) Sek, S.; Swiatek, K.; Misicka, A. Electrochemical behavior of molecular junctions incorporating α-helical peptide. J. Phys. Chem. B 2005, 109, 23121−23124. (23) Wu, J.-C.; Chen, C.-C.; Chen, K.-H.; Chang, Y.-C. Controlled growth of aligned α-helical-polypeptide brushes for tunable electrical conductivity. Appl. Phys. Lett. 2011, 98, 133304.

(24) Lopez-Perez, D. E.; Revilla-Lopez, G.; Jacquemin, D.; Zanuy, D.; Palys, B.; Sek, S.; Aleman, C. Intermolecular interactions in electron transfer through stretched helical peptides. Phys. Chem. Chem. Phys. 2012, 14, 10332−10344. (25) Wold, D. J.; Frisbie, C. D. Formation of metal-molecule-metal tunnel junctions: microcontacts to alkanethiol monolayers with a conducting AFM tip. J. Am. Chem. Soc. 2000, 122, 2970−2971. (26) Wold, D. J.; Frisbie, C. D. Fabrication and characterization of metal-molecule-metal junctions by conducting probe atomic force microscopy. J. Am. Chem. Soc. 2001, 123, 5549−5556. (27) Engelkes, V. B.; Beebe, J. M.; Frisbie, C. D. Length-dependent transport in molecular junctions based on SAMs of alkanethiols and alkenedithiols: effect of metal work function and applied bias on tunneling efficiency and contact resistance. J. Am. Chem. Soc. 2004, 126, 14287−14296. (28) Luo, L.; Choi, S. H.; Frisbie, C. D. Probing hopping conduction in conjugated molecular wires connected to metal electrodes. Chem. Mater. 2011, 23, 631−645. (29) Ditzler, L. R.; Karunatilaka, C.; Donuru, V. R.; Liu, H. Y.; Tivanski, A. V. Electromechanical properties of self-assembled monolayers of tetrathiafulvalene derivatives studied by conducting atomic force microscopy. J. Phys. Chem. C 2010, 114, 4429−4435. (30) Shlizerman, C.; Atanassov, A.; Berkovich, I.; Ashkenasy, G.; Ashkenasy, N. De novo designed coiled-coil proteins with variable conformations as components of molecular electronic devices. J. Am. Chem. Soc. 2010, 132, 5070−5076. (31) Zhao, J.; Davis, J. J.; Sansom, M. S. P.; Hung, A. Exploring the electronic and mechanical properties of protein using conducting atomic force microscopy. J. Am. Chem. Soc. 2004, 126, 5601−5609. (32) Pace, C. N.; Scholtz, J. M. A helix propensity scale based on experimental studies of peptides and proteins. Biophys. J. 1998, 75, 422−427. (33) Kelly, S. M.; Jess, T. J.; Price, N. C. How to study proteins by circular dichroism. Biochim. Biophys. Acta 2005, 1759, 119−139. (34) Chen, Y.-H.; Yang, J. T.; Martinez, H. M. Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry 1972, 11, 4120−4131. (35) Mandal, H. S.; Kraatz, H. B. Effect on the surface curvature on the secondary structure of peptides adsorbed on nanoparticles. J. Am. Chem. Soc. 2007, 129, 6356−6357. (36) Sauerbrey, G. Verwendung von schwingquartzen zur wagung dunner schichten und zur mikrowagung. Z. Phys 1959, 55, 206−222. (37) Liu, G. Y.; Xu, S.; Qian, Y. Nanofabrication of self-assembled monolayers using scanning probe lithography. Acc. Chem. Res. 2000, 33, 457−466. (38) However, if we include the adhesion of the tip to the Acmdeprotected peptide monolayer, then the total force is in fact 13 nN. (39) X. Chen, X.; Zhang, L.; Zhang, L.; Sun, W.; Zhang, Z.; Bu, X.; Cukier, R. I. α-Helix C-terminus acting as a relay to mediate longrange hole migration in proteins. J. Phys. Chem. Lett. 2010, 1, 1637− 1641. (40) Lauz, M.; Eckhardt, S.; Fromm, K. M.; Giese, B. The influence of dipole moment on the mechanism of electron transfer through helical peptides. Phys. Chem. Chem. Phys. 2012, 14, 13785−13788. (41) Gray, H. B.; Winkler, J. R. Electron tunneling through peptides. Q. Rev. Biophys. 2003, 36, 341−372. (42) Slowinski, K.; Chamberlain, R. V.; Miller, C. J.; Majda, M. Through-bond and chain-to-chain coupling. Two pathways in electron tunneling through liquid alkanethiol monolayers on mercury electrodes. J. Am. Chem. Soc. 1997, 119, 11910−11919. (43) Oelker, A. M.; Morey, S. M.; Griffith, L. G.; Hammond, P. T. Helix versus coil polypeptide macromers: gel networks with decoupled stiffness and permeability. Soft Matter 2012, 8, 10887−10895. (44) Johnson, K. L.; Kendall, K.; Roberts, A. D. Surface energy and the contact of elastic solids. Proc. R. Soc. London, Ser. A 1971, 324, 301−313. (45) Kharakoz, D. P. Protein compressibility, dynamics and pressure. Biophys. J. 2000, 79, 511−525. 17293

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(46) Salvadori, M. C.; Brown, I. G.; Vaz, A. R.; Melo, L. L.; Cattani, M. Measurement of the elastic modulus of nanostructured gold and platinum thin films. Phys. Rev. B 2003, 67, 153404. (47) Ptak, A.; Takeda, S.; Nakamura, C.; Miyake, J.; Kageshima, M.; Jarvis, S. P.; Tokumoto, H. Modified atomic force microscope applied to the measurement of elastic modulus for a single peptide molecule. J. Appl. Phys. 2001, 90, 3095−3099.

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dx.doi.org/10.1021/la302716n | Langmuir 2012, 28, 17287−17294