Article pubs.acs.org/Langmuir
Redox-Induced Conformational Change in Mercaptoalkanoic Acid Multilayer Films Steven Johnson,*,† Agnieszka Bronowska,‡ Jocelyn Chan,† David Evans,† A. Giles Davies,† and Christoph Wal̈ ti*,† †
School of Electronic and Electrical Engineering, University of Leeds, Leeds LS2 9JT, U.K. Heidelberg Institute for Theoretical Studies gGmbH, D-69118 Heidelberg, Germany
‡
ABSTRACT: We discuss the assembly, structure, and stability of multilayer molecular films formed from multiple mercaptoalkanoic acid monolayers ligated via carboxyl and thiol interactions with divalent copper ions. Using dual-polarization interferometry to study the assembly of multilayer films in real time, we observe a clear linear relationship between the number of layers within a film and the overall average film thickness. Unexpectedly, however, we find a restructuring of the lower monolayer upon association of the Cu2+ ions to form the Cu carboxylate surface. In particular, the thickness of the lower monolayer was found to decrease significantly, accompanied by an increase in the film density. The conformation of the monolayer subsequently recovered to that observed originally following the reduction of the Cu ion to Cu+ upon chemisorption of the adlayer. Comparable restructuring was also observed in molecular dynamics simulations of a bilayer film assembled on a gold surface. Our combined experimental and theoretical study suggests that the observed restructuring is a result of charge−charge interactions between adjacent Cu ions that exist in the +2 oxidation state in the copper carboxylate surface and in the +1 oxidation state following chemisorption of the adlayer.
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INTRODUCTION Assemblies of organic compounds that order spontaneously into 2D crystalline molecular films have become a major focus of modern surface chemistry and nanoscience.1−3 Early studies of these self-assembled monolayers addressed the fundamental questions that underpin their assembly: the kinetics and thermodynamics of monolayer formation, the organization of the molecular adsorbates, and the properties of the molecular film. Even today, the development and understanding of selfassembling molecular systems remains a major research activity, albeit increasingly focused on the application of SAMs as novel materials for nanoscience and nanotechnology. For many proposed applications, it is necessary to engineer the composition and properties of the exposed SAM surface through a chemical modification of the assembled monolayer. For example, the modification of SAMs to present complex ligands and molecules, such as DNA oligomers or proteins, is used widely in the study of biochemical and biological processes.4−6 Similarly, electrochemically active SAMs formed by the addition of electroactive species, such as ferrocene or ruthenium pentaamine, have played a critical role in the development of molecular electronics.7 A range of covalent and noncovalent modification strategies have been developed for engineering the SAM surface chemistry and attaching appropriate ligands,8 and the molecular conformation and organization of the underlying SAM are typically assumed to be conserved following modification. Here, we show that rather than being immutable, molecular films are very dynamic and sensitive to remarkably subtle chemical modifications, and similar behavior is likely for a wide © 2012 American Chemical Society
range of self-assembling materials and chemical functionalization strategies. In particular, we have investigated the assembly of 3D organic films assembled by the stacking of multiple selfassembled monolayers (SAMs) through ligation between adjacent monolayers using the interaction of divalent metal ions (Cu2+) with thiol and carboxylic acid functional moieties (Figure 1).9−13 A monolayer of alkanethiols functionalized with an acid terminal group, [HS(CH2)nCOOH], is assembled onto a gold surface through the spontaneous formation of the gold− thiolate bond. The exposed carboxylic acid surface is subsequently modified by exposure to a solution containing Cu2+ ions to form a Cu carboxylate to which a second layer of HS(CH 2)n COOH molecules can be chemisorbed. The assembly of a bilayer molecular film is shown schematically in Figure 1. By the sequential adsorption of mercaptoalkanoic acid molecules and Cu2+ ions, one can thus form multiple monolayers ligated through carboxyl and thiol interactions with Cu ions. One of the key discussions originally surrounding multilayer SAM assembly concerned the oxidation state of the copper. It is now recognized that the copper remains in the +2 oxidation state after complexing with the carboxylic acid terminal group but reduces to Cu+ upon chemisorption of the upper HS(CH2)nCOOH adlayer.14−16 Lateral interactions between adjacent terminal moieties can be critical in driving the organization of molecules within a monolayer film. For such multilayer films, this terminal group can be either Cu2+, Received: February 3, 2012 Published: March 26, 2012 6632
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was reached. This was followed by an 80% ethanol/water solution injected over both waveguides for 2 min before reverting to 50% ethanol/water. Water (150 μL) was then injected over both waveguides for 2 min before returning again to the 50% ethanol/ water solution. This procedure enabled the refractive index response of both the sensor chip and the 50% ethanol/water buffer to be calibrated. All experiments were performed with a temperature control of ±0.002 °C and with a set point of 20 °C, as controlled by the AnaLight Bio200 thermal control system. For the immobilization of the first MHDA monolayer onto the silicon oxynitride waveguide surface, the sensor chips were cleaned first by immersion in piranha solution (70% H2SO4, 30% H2O2) for 10 min and rinsed subsequently by sonicating in water, ethanol, and isopropyl alcohol (IPA) for 10 min each. The cleaned sensor chips were then immersed in a 4% solution of (3-mercaptopropyl)trimethoxysilane in IPA for 18 h, followed by thorough rinsing in IPA to provide thiol functionalization. Following calibration, each thiol-modified waveguide sensor was subjected to repeated 150 μL injections of 5 mM copper(II) perchlorate (Cu(ClO4)2) hexahydrate ethanoic solution for 15 min before returning to the 50% ethanol/ water solution to generate a Cu ion-functionalized surface onto which the SAM can be chemisorbed. Note that Cu2+ reduces to Cu+ following chemisorption onto the thiol-functionalized waveguide surface. It was found to be necessary to repeat the Cu2+ ion injection at least twice in order to saturate the surface fully with Cu+ ions. The first MHDA monolayer was assembled on the Cu+ surface by the injection of 150 μL of a 1 mM ethanolic solution of MHDA for 15 min before reverting back to 50% ethanol/water running buffer. Similar investigations in which monolayers of MHDA were assembled onto a thiol-functionlized waveguide surface using a homobifunctional maleimide cross-linker showed identical results to the Cu + immobilization strategy. For the investigation of multilayer films, we adopted the following sequential functionalization procedure: (1) injection of 150 μL of a 5 mM solution of Cu(ClO4)2 in ethanol for 15 min before returning to the 50% ethanol/water running buffer and (2) injection of 150 μL of a 1 mM MHDA solution in ethanol for 15 min before returning to the 50% ethanol/water running buffer. By repeating steps 1 and 2, it was possible to assemble multilayer SAM films. Molecular Dynamics Simulation Protocol. Molecular dynamics (MD) simulations were carried out using AMBER 8 with the parm99SB force field. Initially, a nanometer-scale section of Au(111), consisting of 500 gold atoms, was established using the parameters for gold as published by Bizzarri et al.20 All systems to be modeled (i.e., a single MHDA monolayer [Au−S(CH2)15COOH], a single MHDA monolayer coordinated with Cu2+ ions [Au−S(CH2)15COOH−Cu2+], a single MHDA monolayer coordinated with Cu+ ions [Au− S(CH 2 ) 1 5 COOH−Cu + ], a MHDA bilayer film [Au−S(CH2)15COOH−Cu+−S(CH2)15COOH], and finally an MHDA bilayer film coordinated with Cu2+ ions [Au−S(CH2)15COOH− Cu+−S(CH2)15COOH−Cu2+]) were immersed in a rectangular box of TIP3P water (in all systems, the boundary was 5 Å) and subjected to 1500 cycles of molecular mechanical energy minimization. All simulations were carried out at constant temperature and pressure (293 K, 1 atm) with periodic boundary conditions, a 12 Å cutoff for nonbonded interactions, and a 2 fs time step. To replicate the experimental procedure, counterions required to maintain charge neutrality were not included. Particle mesh Ewald was used and SHAKE constraints were applied to all hydrogen atoms during MD simulations to eliminate the fastest X−H vibrations and allow a longer simulation time step. Translational and rotational center-of-mass motion was removed every 10 ps. Equilibration started with the gradual heating of each system to the target temperature while the atomic positions of the heavy atoms were harmonically restrained. As the temperature increased, the restraints were gradually released, from 25 kcal/mol/Å2 to zero. The equilibration period took 500 ps, and the production phase took 2.5 ns. The coordinates were saved every 1 ps of the production phase. To understand the influence of the Cu2+ and Cu+ ions on the properties of an MHDA monolayer and MHDA bilayer, the MD
Figure 1. Schematic diagram of a multilayer molecular film assembled by the sequential adsorption of n-alkanethiolate monolayers and divalent metal ions, here Cu2+. The Cu2+ ion is reduced to Cu+ following chemisorption of the upper monolayer. We note that the divalency of the Cu2+ ions associated with the acid surface prohibits the formation of a complete copper layer and hence leads to an incomplete assembly of the subsequent monolayer.16
following the formation of the copper carboxylate surface, or Cu+, after the chemisorption of the adlayer. Here we present the results of a combined experimental and molecular dynamics study that reveals for the first time a change in the conformation of each monolayer driven by the charge state of the copper ion. Similar molecular restructuring is expected for a range of SAMs and chemical modifications that are used routinely for surface functionalization, and our findings thus have widespread implications.
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EXPERIMENTAL SECTION
Materials. Ethanol (99%) and isopropanol (99%) from Fisher (Loughborough, U.K.); acetone (98%), (3-mercaptopropyl)trimethoxysilane (95%), mercaptohexadecanoic acid [MHDA] (99%), copper(II) perchlorate hexahydrate (98%), sulfuric acid, and hydrogen peroxide from Sigma (Gillingham, U.K.); and n-doped silicon(100) capped with a 300-nm-thick thermal oxide from Compart Technology (Peterborough, U.K.) were used as received. All water used in this study was Milli-Q water with a resistance of at least 18.2 MΩ cm. Dual-Polarization Interferometry. The assembly of multilayer mercaptoalkanoic acid SAMs was interrogated in real time using an AnaLight Bio200 optical evanescent dual-polarization interferometer (DPI)17 (Farfield Sensors Ltd., Crewe, U.K.). Briefly, DPI is based on the analysis of interference patterns resulting from coherent laser light propagating along two vertically stacked, independent optical waveguides. The interaction of the evanescent wave with a molecular layer attached to the upper, sensing waveguide results in a change in the relative phase of light propagating along the two waveguides, leading to a shift in the interference pattern observed in the far field. By measuring this shift for two different optical polarizations (the transverse electric and transverse magnetic polarizations), it is possible to determine both the thickness and refractive index of the adsorbed molecular film and provide a quantitative analytical measurement related directly to the structure of materials immobilized on the sensor surface. Each DPI sensor chip comprises two channels (i.e., two stacked waveguides and hence two exposed waveguides) connected to a microfluidics cell consisting of a Rheodyne HPLC injector valve and an external pump (Harvard Apparatus, PHD2000). Prior to each experiment, the sensor chips were calibrated as follows. A running buffer a comprising 50% ethanol/water solution was passed over both sensor waveguides at a flow rate of 50 μL min−1 until a steady baseline 6633
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change significantly following the addition of Cu2+ ions because the interaction between the alkane SAM and the electromagnetic field dominates. Unexpectedly, however, a significant change in the refractive index of the layer and a change in the structure of the MHDA monolayer were observed upon exposure to Cu2+ ions. In particular, the measured thickness of a single MHDA monolayer following the formation of the Cu carboxylate surface was found to reduce to 1.7 nm. This reduction in layer thickness was accompanied by an increase in the measured monolayer density, which more than doubles from 0.16 to 0.36 g/cm3 as shown in Figure 2. The assembly of the second MHDA monolayer onto the exposed copper carboxylate surface reverses this change (i.e., the density of the bilayer MHDA film returns to that observed for a single monolayer), and the total film thickness corresponds to two MHDA monolayers with the expected thickness. We note that the calculations of thickness and density assume a film with an isotropic refractive index and do not account for the birefringence of the MHDA monolayers. The magnitude of the thickness and density changes as calculated are thus expected to be overestimated.18 This oscillation in thickness and density following the formation of the Cu carboxylate surface and the subsequent assembly of an additional adlayer were seen to repeat with the formation of each new MHDA monolayer. Figure 3 shows the thickness and density of a four-layer MHDA SAM. Measurements were made after the assembly of each monolayer and following the transformation of the exposed carboxylic acid surface to copper carboxylate. As with previous studies of multilayer assembly, we found that the total film thickness
trajectories were postprocessed and analyzed using the ptraj module of AMBER. Solvent molecules were removed, and gold atoms in the first monolayer were superimposed onto the corresponding reference structure. rms deviations, atomic fluctuations, torsional angle fluctuations, and the average thickness of each monolayer were calculated directly.
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RESULTS AND DISCUSSION Dual-Polarization Interferometry. DPI allows quantitative information regarding the density and thickness of a molecular layer to be extracted simultaneously and is thus a very powerful technique for probing both the kinetics and conformation of assembled molecular systems. Real-time DPI measurements showing the thickness and density changes occurring during the assembly of a bilayer MHDA film are shown in Figure 2. The thicknesses of the first and second monolayers were measured to be 2.4 and 2.2 nm, respectively, as shown in Figure 2. These values agree well with previous measurements of MHDA monolayers assembled on Au and suggest the formation of a well-packed monolayer.2 The refractive index of the MHDA monolayer is not expected to
Figure 2. Real-time DPI measurement showing the (top) thickness and (bottom) density of an MHDA bilayer formed on a Cu2+functionalized waveguide sensor. The start and end of each injection are indicated by the broken arrows. The large discontinuities that occur at the start and end of each sample injection reflect mostly the change in refractive index of the bulk solution rather than the formation of the new layer on the waveguide surface. Steady-state, calibrated measurements of each layer are obtained from the plateau regions between injections.
Figure 3. DPI experimental data showing the change in thickness and density of a multilayer film assembled from four MHDA monolayers. The dashed line in the upper graph shows the straight line fitted to the total film thickness measured following the assembly of each new monolayer (regions i, iii, v, and vii). The assembly mechanism is shown schematically in the upper panel where open (closed) circles represent copper ions in the +2 (+1) oxidation state, respectively. 6634
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increased linearly with the number of layers within the film (dashed line in Figure 3), with each new monolayer contributing an additional 2.2 nm to the total film thickness of the multilayer film, equivalent to the thickness of an MHDA monolayer.2,9 Figure 3 also clearly demonstrates the reorganization of the multilayer film that occurs following the addition of copper ions and upon the assembly of each new layer. In particular, the film is seen to oscillate between films of low density and high thickness, corresponding to a monolayer terminated with carboxylic acid (regions i, iii, v, and vii in Figure 3), and films of high density and reduced thickness following the formation of the copper carboxylate complex on the uppermost monolayer surface (regions ii, iv, and vi in Figure 3). The transformation between films of high and low density persists over the growth of four monolayers, although the amplitude of the oscillation in density decreases with an increasing number of layers. Molecular Dynamics Simulation. A change in the thickness of a SAM accompanied by a corresponding inverse shift in density is indicative of a conformational change in the immobilized layer. For example, a reorientation of the polymethylene chains that increases the tilt angle relative to the surface normal is consistent with an observation of decreased SAM thickness coincident with an increase in the monolayer density. For a molecular conformation in which the polymethylene chains are arranged more parallel to the surface normal, one would expect to see the reverse response. To clarify the conformational changes that occur during multilayer film assembly and to confirm the driving force leading to reorganization, detailed investigations of the structure of the monolayer and bilayer MHDA films assembled on a gold surface were carried out using molecular dynamics simulations.19,20 Time-averaged measurements of the monolayer thickness and tilt angle for each system simulated are summarized in Table 1, and representative snapshots of MD simulations of
Figure 4. Representative snapshots of an MD simulation showing a single MHDA monolayer assembled on gold (a) before and (b) after association with Cu2+ ions (blue spheres). Note the 2:1 ratio of MHDA molecules to Cu2+ ions.
Table 1. Molecular Dynamics Simulation Data Summarizing the Height and Tilt Angle of an MHDA Monolayer and MHDA Bilayer Film before and after the Adsorption of Cu2+ and Cu+ system (a) (b) (c) (d) (e) (f)
MHDA MHDA MHDA MHDA MHDA MHDA
monolayer monolayer + Cu2+ monolayer + Cu+ bilayer (bottom layer) bilayer (top layer) bilayer + Cu2+ (top layer)
layer height (Å)
tilt angle (deg)
18.3 15.2 19.2 18.9 17.6 15.8
24 27 23 23 25 27
Figure 5. Representative snapshots of the MD simulation of a bilayer MHDA film assembled on gold (a) before and (b) after association with Cu2+ ions (blue spheres). Internal Cu+ ions ligating the two MHDA monolayers are represented by the orange spheres located at the interface between the two monolayers.
monolayer and bilayer MHDA films before and after the adsorption of Cu2+ are shown in Figures 4 and 5, respectively. MD simulations of an MHDA monolayer, shown in Figure 4a, were found to be stable but also very dynamic, similar to that observed for lipid monolayers/bilayers. From regular sampling of the MD trajectories, the average monolayer thickness and tilt angle were found to be 18.3 Å and 24°, respectively. Here, the thickness and angles are calculated from the sulfur moiety to the C atom of the carboxyl group. These values are in good agreement with previous studies of MHDA monolayers and with the MHDA monolayer thickness measured here using DPI. Having established a stable MHDA
monolayer, the Cu2+ ions were added to the single-layer system, as shown in Figure 4b. Here a total of 48 Cu2+ ions were introduced into the system, equal to the number of MHDA molecules within the monolayer; however, only 26 Cu2+ ions on average associate with the exposed carboxylic acid surface. This is equivalent to a surface coverage of around 50%, 6635
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reflecting the predicted reaction stoichiometry of RCOO−/ Cu2+ = 2:1 and in agreement with our AFM studies of island growth in bilayer MHDA films.16 Cu ion surface coverages approaching 70% were obtained when the carboxylic acid surface is exposed to monovalent Cu ions. In addition to demonstrating 50% surface coverage, MD simulations also establish a reorganization of the underlying MHDA monolayer upon association with Cu2+ ions. In particular, the thickness of the Au−S(CH2)15COOH−Cu2+ monolayer was found to be around 20% less than that of the equivalent monolayer prior to Cu2+ association. This is in qualitative agreement with that observed experimentally, as shown in Figure 3. We again stress that the calculation of film thickness and density from the DPI experimental data assumes that the refractive index of the MHDA monolayer is isotropic. The MHDA layer is, however, birefringent, and the magnitudes of the thickness and density changes as calculated are thus expected to be overestimated.18 Restructuring is suppressed when the ratio of MHDA molecules to Cu2+ ions is reduced below the maximum 2:1. That the reorganization of the monolayer occurs only at a high Cu2+ surface coverage suggests that interactions between adjacent Cu2+ ions are critical in driving the observed structural changes. MD simulations of bilayer MHDA films, shown in Figure 5, were generated by adding Cu+−S(CH2)15COOH molecules to the underlying MHDA monolayer. As with the experiment, the thickness of the bilayer (36.5 Å) is found to be exactly twice that of the monolayer (18.3 Å), and the conformation of the upper and lower MHDA monolayers in a bilayer film are almost identical to that of a single MHDA monolayer (tilt angles of 25 and 23°, respectively, compared to 24° for a single monolayer). Importantly, our MD simulations also predict a restructuring of the MHDA monolayer upon association with Cu2+ ions. In particular, the SAM is found to tilt further away from the surface normal by approximately 3° (rows b and f in Table 1). This reorientation of the molecular layer and the corresponding decrease in monolayer thickness are in precise qualitative agreement with our experimental findings. It is worth noting that the MHDA monolayer was assembled onto the DPI sensor surface via a (3-mercaptopropyl)trimethoxysilane SAM. Furthermore, the density of the MHDA monolayer assembled onto the DPI sensor surface was calculated to be 8 × 1013 molecules/ cm 2 . Although this is typical of a SAM assembled experimentally, the packing density of the molecular model is around 4 × 1014 molecules/cm2. It is thus likely that the experimental MHDA monolayer is less constrained geometrically and thus possess greater dynamic freedom than an equivalent SAM assembled directly onto an Au surface, as with simulation. In addition to time-averaged structural details, MD simulations can also provide critical insights into the dynamic behavior of multilayer molecular films. The rms deviation of the heavy atoms for a single MHDA monolayer, for an MHDA monolayer following Cu2+ adsorption, and for the two MHDA layers in a bilayer film following exposure to Cu2+ is shown in Figure 6. The single MHDA monolayer was found to be very stable, with an average rms deviation of
