Nanostructured Composite Layers of Mussel Adhesive Protein and

Jul 1, 2013 - These groups can form complexes with metal ions(15) and form hydrogen ... as measured by a Zetasizer Nano ZS device (Malvern, U.K.). ...
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Nanostructured Composite Layers of Mussel Adhesive Protein and Ceria Nanoparticles Olga Krivosheeva,† Majid Sababi,† Andra Dedinaite,*,†,‡ and Per M. Claesson†,‡ †

Division of Surface and Corrosion Science, Department of Chemistry, School of Chemical Sciences, Royal Institute of Technology, SE-100 44 Stockholm, Sweden ‡ Chemistry, Materials and Surfaces, SP Technical Research Institute of Sweden, Box 5607, SE-114 86 Stockholm, Sweden S Supporting Information *

ABSTRACT: Mussel adhesive proteins are known for their high affinity to a range of different surfaces, and they therefore appear as ideal candidates for producing thin inorganic− organic composite films with high robustness. In this work we explore the possibility of making cohesive films utilizing layerby-layer deposition of the highly positively charged mussel adhesive protein, Mef p-1, and negatively charged ceria nanoparticles. This particular material combination was chosen due to recent findings that such films provide good corrosion protection. Quartz crystal microbalance with dissipation monitoring (QCM-D) was used for following the film formation process in situ on silica surfaces. A close to linear growth of the film with number of deposited layers was found for up to 18 deposition steps, the highest number of depositions investigated in this work. The Mefp-1 concentration during film deposition affected the film properties, where a higher protein concentration resulted in a stiffer film. It was also found that the added mass could be amplified by using a Mefp-1 solution containing small aggregates. The surface nanomechanical properties of dried multilayer films were investigated using peak force QNM (quantitative nanomechanical mapping) in air. Homogeneous surface coverage was found under all conditions explored, and the Young’s modulus of the outer region of the coating increased when a higher Mefp-1 concentration was used during film deposition. The nature of the outermost surface layer was found to significantly affect the surface nanomechanical properties. The abrasion resistance of the coating was measured by using controlled-force contact mode AFM. contains 75−85 repeating decapeptide units13 (see Supporting Information). It is highly positively charged at neutral pH due to a high content of lysine (21 mol %). It also contains a large fraction of 3,4-dihydroxyphenylalanine (DOPA) residues (13 mol %)13 and proline residues that counteract formation of large secondary structures. The hydrodynamic size of Mef p-1 has been determined to be 10.5 nm by dynamic light scattering.14 It is generally accepted that the outstanding adhesion properties arise from the high content of DOPA residues. These groups can form complexes with metal ions15 and form hydrogen bonds with polar surfaces.16 Electrostatic forces also contribute to Mef p-1 adsorption to negatively charged surfaces.17 The other component of the nanocomposite film, consisting of ceria nanoparticles, has been shown to improve the corrosion resistance for different kinds of materials,18,19 is known for its oxygen storage capacity,20 and can change the oxidation state from Ce4+ to Ce3+. Mef p-1/ceria nanoparticle composite layers have recently shown promising anticorrosion effects.21 In addition, there are no reported health

1. INTRODUCTION Layer-by-layer (LbL) deposition is widely used for surface modification. It offers a versatile approach to form thin surface films with desired properties on a wide variety of substrates. This method was first visualized by Iler1 for building colloidal particle multilayers, but it did not become widespread until the publication of Decher et al.,2 that demonstrated the possibility of utilizing oppositely charged polyelectrolytes as a versatile route for controlling surface properties. Since that time the LbL method has been used for surface modification of organic, inorganic, polymeric, and biological substrates for further applications in biosensors,3 catalysis,4 drug delivery systems,5 anticorrosion coatings,6 and others. It allows the use of different combinations of species, often of opposite charge, to create thin films with diverse properties, e.g., polyelectrolytes combined with clay sheets,7 polyelectrolytes and metal nanoparticles,8 proteins and nanoparticles,9,10 etc. In the current study, the LbL method was used to construct nanocomposite films consisting of a mussel adhesive protein (Mef p-1) and ceria nanoparticles. Mef p-1 is one of several mussel adhesive proteins that can be found in the byssus by means of which the mussels attach to a variety of surfaces under water.11 The chemical structure of this protein has been identified,12 and it has a molecular weight of 110 kDa and © 2013 American Chemical Society

Received: May 3, 2013 Revised: July 1, 2013 Published: July 1, 2013 9551

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risks associated with ceria nanoparticles,22 making the Mef p-1/ ceria composite layer attractive also from this perspective. A thorough understanding of the basic film formation process and of the mechanical and structural properties of the deposited layer is required for successful use of Mef p-1/ nanoparticle composite films in surface modification applications including corrosion protection.21 To this end we utilized the quartz crystal microbalances with dissipation monitoring (QCM-D) for following the film formation process on an inert silica surface. This choice of substrate allows us to focus on the interactions between Mef p-1 and ceria nanoparticles without any influence of ions that may leach out from more reactive substrates such as carbon steel. Nanomechanical properties of dried nanostructured composite films were determined with atomic force microscopy (AFM) utilizing the peak force tapping method, which is a relatively new imaging mode23,24 that allows analysis of mechanical properties and their variations across a surface layer with nanometer scale lateral resolution. It is thus possible to map spatial variations in material properties across the interface of a structured surface layer, like that formed in this investigation. AFM in contact mode was utilized for testing the robustness of the composite film under the combined action of load and shear.

The oscillation of nonrigid (viscoelastic) layers that do not fully couple to the crystal oscillation results in more pronounced oscillation dampening, and therefore the change in energy dissipation (ΔD) needs to be taken into account.28,29 In the QCM-D device ΔD is determined from the oscillation decay rate when the driving voltage is turned off. One theoretical model describing the QCM-D response for viscoelastic layers was developed by Voinova et al.30 We used that model and evaluated the experimental data by describing the surface layer as a Voigt solid (a spring connected in parallel to a dashpot), for which the viscoelastic properties of the adsorbed film are represented by a complex shear modulus: G = G′ + iG″ = μ + i2πfη

where G′ and G″ are the film storage and loss modulus, respectively, μ is the film shear elasticity, and η is the film shear viscosity. Changes in solvent density (ρ) and viscosity (η) give rise to changes in frequency and dissipation that are not related to the adsorbed layer31 as illustrated in Figure 3 (region II). According to Kanazawa and Gordon,32 the frequency shift can be calculated as:

Δfn =

2.1. Materials. The mussel adhesive protein Mef p-1 (molecular weight 110 kDa and isoelectric point 10.3)13 was kindly provided by Biopolymer AB (Gothenburg, Sweden) in 1 wt % citric acid buffer (pH around 2). Two different stock solutions with concentrations 1.2 mg mL−1 (purity 92%) and 10 mg mL−1 (purity 97%) were used in this investigation. The former stock solution contains nonaggregated25 Mefp-1, and the latter contains small aggregates with hydrodynamic radius of 20 nm (see Supporting Information). The stock solutions were kept at +5 °C in darkness and used as received. Before use, aliquots of the stock solution were added into degassed 50 mM citric acid buffers (pH ≈ 5.5) to achieve the desired Mefp-1 concentration. Ceria nanoparticles (NANOBYK-3810) were purchased from BYK Additives and Instruments (Wesel, Germany). The stock solution was kept in darkness at room temperature (≈22 °C). Prior to each experiment dilute nanoparticle solutions with concentration 0.5 mg mL−1 were prepared by mixing the desired amount of stock solution with Milli-Q water followed by 5 min ultrasound treatment to destroy possible agglomerates. The pH of the final solutions was in the range 5.5 to 6 as revealed by a pH sensitive stripe. The particles’ hydrodynamic diameter is about 10 nm as determined by dynamic light scattering measurements, and the zeta-potential is −10 mV in the solution used for LbL deposition (pH 5.5, 0.5 mg mL−1 nanoparticles) as measured by a Zetasizer Nano ZS device (Malvern, U.K.). 2.2. Methods. 2.2.1. Quartz Crystal Microbalance with Dissipation (QCM-D). A QCM-D from Biolin Scientific (Västra Frölunda, Sweden) was used for following the buildup of protein/ nanoparticle composite layers by measuring changes in resonant frequency (Δf) and energy dissipation (ΔD). The principles of the technique have been described by Rodahl et al.26 Briefly, the change in resonance frequency is related to the mass added to the crystal, and thus adsorption or desorption of material induces a frequency shift. For thin, rigid, and evenly distributed layers on the surface the frequency shift (Δf) is directly proportional to the change in sensed mass (Δm) via the Sauerbrey27 equation: Δf n

n f0

⎛ ρ η ⎞1/2 ⎜ ⎟ − ⎝ πμq ρq ⎠

3/2 ⎜ s0 s0 ⎟

n f0

⎛ ρη ⎞1/2 s s ⎟ ⎜ πμ ρ ⎟ ⎝ q q⎠

3/2 ⎜

(3)

where n is the harmonic number, f 0 is the fundamental frequency in air, μq is the shear modulus of quartz, and subscripts q, s0, and s denote quartz, pure solvent, and solution, respectively. The changes in dissipation occurring due to changes in liquid bulk properties have been considered by Rodahl and Kasemo,33 and they suggest the following relation:

2. MATERIALS AND METHODS

Δm = − C

(2)

ΔDn =

1/2 1 ⎛⎜ ηsρs ⎞⎟ − n ρq tq ⎜⎝ 2πfn ⎟⎠

1/2 1 ⎛⎜ ηs0ρs0 ⎟⎞ n ρq tq ⎜⎝ 2πfn ⎟⎠

(4)

where tq is the thickness of the quartz crystal. 2.2.2. Surface Preparations. For all QCM experiments the SiO2coated crystals were cleaned in 2% Deconex for 20 min followed by extensive rinsing with Milli-Q water. The surfaces were then left under water overnight. They were finally rinsed with ethanol and dried with nitrogen gas just prior to use. Silica wafers used in some AFM experiments were cleaned in the same way. 2.2.3. Atomic Force Microscopy (AFM). Peak force tapping mode allows imaging with a controlled feedback force,34 and simultaneously provides access to quantitative mapping of surface material properties.35−38 Briefly, the surface position is modulated by a sine wave with amplitude of approximately 150 nm and a frequency of 1−2 kHz. During each period of oscillation the surface is moved into contact with the AFM tip and the feedback electronics adjust the surface position such that the maximum cantilever deflection (the peak force) equals a predetermine set point value. The force is continuously measured during each oscillation. By calibration of the optical lever sensitivity39 and the cantilever spring constant40,41 the information about cantilever deflection and piezo position can be converted to force vs distance curves describing the tip−sample interaction during approach and separation. From such a force curve one can determine surface deformation, maximum adhesion force between tip and sample, and the amount of energy that is dissipated during the interaction. In addition, the elastic modulus of the sample can be obtained by fitting the Derjaguin−Muller−Toporov (DMT) model42,43 to the part of the force curve where the sample and tip are in contact:

(1)

F=

where C is a constant that depends on the density and thickness of the quartz crystal and equals 0.177 mg m−2 Hz1− for the crystals used in this work, and n is the overtone number. In our experiments the third overtone was used for sensed mass calculations according to eq 1.

4 E* Rd3 + Fadh 3

(5)

where F is the force, R the tip radius, d the deformation value at a given force, Fadh the maximum adhesion force, and E* the effective elastic modulus: 9552

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Figure 1. Frequency (A) and dissipation (B) change versus time for 0.01 (dotted line), 0.05 (dashed line), and 0.1 (solid line) mg mL−1 solutions of nonaggregated Mefp-1 in 1 wt % citric acid buffer, pH 5.5, and ceria nanoparticles (dash dotted line). The adsorption was performed at a flow rate of 100 μL min−1.

1 − vs 2 1 − vt 2 1 = + E* Es Et

these adsorption events are shown as a function of time in Figure 1. As expected, no detectable adsorption of the negatively charged nanoparticles to the negatively charged silica surface was observed, as demonstrated by the lack of frequency and dissipation change during 20 min exposure to the nanoparticle solution, see Figure 1. In contrast, the positively charged Mef p1 adsorbs readily to silica, and more rapidly so the higher the Mef p-1 concentration. Adsorption leads to decrease in frequency and increase in dissipation, and the changes are somewhat larger with increasing protein concentration. The evolution of layer properties during an adsorption process can be followed by optical techniques45 or with QCM-D. With the QCM-D device it can be assessed by plotting the change in dissipation as a function of the changes in frequency, which is a similar type of plot as a refractive index vs thickness plot that is used for analysis of, e.g., ellipsometry data.46 Such plots are shown in Figure 2.

(6)

where νs and Es are the Poisson’s ratio and Young’s modulus of the sample and νt and Et are the Poisson’s ratio and Young’s modulus of the tip. Nanometer scale lateral resolution images of surface topography and surface material properties were obtained using an atomic force microscope, multimode, Nanoscope V, Bruker, operating in peak force tapping mode. Silicon tips, NSC15, Mikromasch, were used for all experiments. The cantilevers were backside coated with a reflective aluminum layer and have an n-type silicon etched (phosphorus doped) tip with a nominal radius of 10 nm and total tip height on the order of 21−25 μm. The nominal spring constant and nominal resonance frequency were 46 N m−1 and 325 kHz, respectively. The actual cantilever spring constant, resonance frequency, and tip radius, as well as the deflection sensitivity, were determined for each cantilever prior to each experiment as explained elsewhere.44 Images with 512 × 512 pixel resolution were recorded using a scan rate of 1.0 Hz. AFM images of composite films were captured in air under ambient conditions with relative humidity of 42%. All AFM data analysis and data processing were made with the NanoScope software version 1.40. An AFM-based robustness test was performed utilizing the same AFM instrument operated in contact mode, using a range of applied forces. An uncoated n-type silicon AFM probe, NSC18 Mikromasch, with measured tip radius of 15 nm and nominal total tip height on the order of 12−18 μm was used for testing the robustness of the composite film. The deflection sensitivity and spring constant of the cantilever were determined by measuring a force curve against a surface with high elastic modulus, sapphire glass, and thermal tuning in air, respectively. The deflection sensitivity was 69.8 nm V−1 and the spring constant was 3.2 N m−1 for the cantilever used in this measurement series. The applied force was calibrated according to the set-point voltage. Different regions of the surface were probed, and each region was scanned with a different applied normal force. The 1 μm × 1 μm area was scanned with 256 parallel lines. Before and after this scratching-type experiment, an area of 10 μm × 10 μm, which included all previously scratched 1 μm × 1 μm areas, was imaged with 512 × 512 pixel resolution with peak force tapping QNM for further analysis.

Figure 2. Change in dissipation as a function of frequency change during adsorption from citric acid buffer, pH 5.5, with Mefp-1 concentrations of 0.01 (◇), 0.05 (●), 0.1 (□) mg mL−1 for the nonaggregated sample, and 0.1 (■) mg mL−1 for the aggregated sample. The dashed line is a linear fit to the middle region of the curve.

3. RESULTS AND DISCUSSION 3.1. Mef p-1 Adsorption at Different Concentrations. The first step in the multilayer formation process, adsorption of Mef p-1, was investigated using three different protein concentrations, 0.01, 0.05, and 0.1 mg mL −1, of the nonaggregated Mef p-1 sample. In separate experiments attempts were made to adsorb ceria nanoparticles to the bare silica surface. The changes in frequency and dissipation during

The ΔD−Δf plots for the three different concentrations of the nonaggregated sample and also for the aggregated Mef p-1 sample overlap, suggesting that the mass adsorbed, but not the adsorption kinetics or the presence of small aggregates in bulk solution, determines the layer structure. At the initial stage (between 0 and −5 Hz) the derivative of the ΔD vs Δf curve is small. The small change in energy dissipation per added mass 9553

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(frequency change) suggests that the Mef p-1 molecules at low surface coverage adsorb in a flat conformation, which previously has been discussed for adsorption of other proteins47 and polymers.48 A further decrease in frequency, to about −25 Hz, results in an almost linear increase in dissipation with frequency with a derivative larger than in the first region, suggesting a continuous buildup with no dramatic structural changes in the layer.47,49 At the two highest Mef p-1 concentrations a third region, where Δf is less than −25 Hz, is observed. In this region the dissipation changes significantly with a small change in frequency, suggesting that some adsorbed proteins adopt a conformation that extends further from the surface. Such conformational changes have been reported for other proteins using optical techniques.45 It is only in this final stage that the results for the nonaggregated and aggregated Mef p-1 sample differ, with the aggregated sample leading to somewhat larger frequency and dissipation changes. Further analysis of the data was performed by means of the viscoelastic Voigt model with a fixed layer density of 1200 kg m−3, with which the best fitting of the data was obtained. The evaluated sensed mass of Mef p-1, including hydrodynamically coupled water, and the layer thickness after rinsing with water were 3.0 mg m−2 and 2.5 nm when the layer was formed from 0.01 mg mL−1 solution, increasing to 4.8 mg m−2 and 4 nm when the layer was formed from 0.1 mg mL−1 Mef p-1 solution. Previously, Fant et al. using Mef p-1 concentration 0.025 mg mL−1 in 0.1 M acetate buffer with pH 5.5 obtained a sensed mass and a hydrodynamic thickness equal to 3.5 mg m−2 and 3.3 nm, respectively, assuming a layer density of around 1300 kg m−3.50 The optical thickness of adsorbed Mef p-1 layers at pH 5.5 has also been determined with dual polarization interferometry and found to be 2.0−2.5 nm for the nonaggregated samples.17 The optical thickness is slightly smaller than the hydrodynamic thickness due to the larger sensitivity to the dilute outer region in the QCM measurement. 3.2. Layer-by-Layer (LbL) Formation: Changes in Frequency and Dissipation. The sequential deposition of Mef p-1 and ceria nanoparticles on SiO2 surfaces was followed using the QCM-D technique. These experiments were conducted utilizing two different Mef p-1 concentrations, 0.01 or 0.1 mg mL−1 in different experiments, and a constant ceria nanoparticle concentration of 0.5 mg mL−1. A representative plot of changes in frequency and dissipation during formation of the first double layer is shown in Figure 3. First the baseline for pure water (region I) was established, and then 1 wt % citric acid buffer (region II) was introduced into the cell and a decrease in frequency (≈ −10 Hz) and an increase in dissipation (≈ 5 × 10−6) were observed. This could be either due to adsorption of citric acid or due to changes in bulk viscosity and density. The effect due to changes in bulk properties was estimated using eqs 3 and 4. In these calculations we used the density and viscosity of 1 wt % citric acid at room temperature (around 20 °C), 1002 kg m−3 and 0.00103 Pa s, respectively,51 and the corresponding values for pure water, 998 kg m−3 and 0.001002 Pa s, respectively.52 The calculated change in frequency equals −19 Hz, and that in dissipation equals 5 × 10−6. Comparison of these values with those observed experimentally (see Figure 3, region II) reveals that a somewhat lower than expected frequency change was observed, whereas the measured change in dissipation agrees very well with the predicted one. We conclude that the QCMD response observed when changing between water and citric acid is a bulk effect and not an indication of adsorption. The

Figure 3. Changes in frequency (solid curve) and dissipation (dashed curve) during sequential adsorption of nonaggregated Mef p-1 and ceria nanoparticles. Regions I and II show the water and 1 wt % citric acid baselines, respectively, region III shows the protein adsorption step (0.01 mg mL−1), regions IV and VI are rinsing steps with water, and region V shows ceria nanoparticle adsorption using a particle concentration of 0.5 mg mL−1.

fact that this effect is significant means that the sensed mass of the Mef p-1 layer adsorbed from 1 wt % citric acid solution must be evaluated relative to the baseline obtained in the protein-free 1 wt % citric acid solution, whereas the sensed mass of Mef p-1 after rinsing with pure water and all data for ceria nanoparticles (adsorbed from water and rinsed with water) must be evaluated against the baseline obtained in pure water. Introduction of Mef p-1 containing 1 wt % citric acid (pH 5.5) into the measurement chamber (region III) results in a further decrease in frequency (≈−27 Hz) and a moderate increase in dissipation (≈1.7 × 10−6) (no significant changes in bulk viscosity and density occur in this case since both the Mef p-1 and the preceding buffer solution contain 1 wt % citric acid). Once surface saturation was achieved, the excess of the protein as well as citric acid were removed by rinsing with pure water (region IV). Part of the large shifts in frequency and dissipation observed in this region is due to changes in bulk viscosity and density due to removal of citric acid from the bulk, but also some desorption of Mef p-1 occurs. At the next step, negatively charged CeO2 nanoparticles were adsorbed onto the protein layer (region V), which resulted in a rapid decrease in frequency, and a small decrease in dissipation. Thus more mass is added to the layer but nevertheless the dissipation decreases, showing that the layer rigidity increases. The energy dissipation decreases from 0.89 ± 0.14 × 10−6 (region IV) to 0.62 ± 0.01 × 10−6 (region VI). More pronounced changes in dissipation were observed for subsequent layers (Figure 4A−C).The subsequent rinsing with pure water (region VI) had almost no effect on the frequency and dissipation values, demonstrating the firm attachment of the Mef p-1/ceria composite layer. 3.3. Effect of Mef p-1 Concentration and Mef p-1 Aggregation on LbL Deposition. The adsorption cycle presented in Figure 3 was repeated several more times, and the total change in frequency and dissipation after each step are illustrated in Figure 4, which compares the results obtained for two different Mef p-1 concentrations, and for nonaggregated and aggregated Mefp-1 at the same bulk concentration. These values were calculated from the plateau regions during adsorption and rinsing steps relative to the relevant buffer solutions. The error bars on the graphs were obtained on the basis of at least two independent experiments. The total dissipation change (see Figure 4A,B) remains low (about 2 × 10−6) throughout, demonstrating the rigid nature of 9554

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Figure 4. Total energy dissipation (A, B, C) and frequency (D, E, F) change as a function of layer number. Filled symbols correspond to data obtained after the adsorption of Mef p-1 and CeO2 nanoparticles, and open symbols to data obtained after the following rinsing step. The Mefp-1 concentration (nonaggregated sample) during adsorption was 0.01 mg mL−1 (A, D) and 0.1 mg mL−1 (B, E). The Mefp-1 concentration from the aggregated sample (C, F) was 0.1 mg mL−1.

Figure 5. Changes in sensed mass as a function of layer number, calculated by using Voigt (filled symbols) and Sauerbrey (open symbols) models. The Mefp-1 concentration in the nonaggregated sample was 0.01 mg mL−1 (A) or 0.1 mg mL−1 (B). The concentration in the aggregated sample was 0.1 mg mL−1 (C).

the nanocomposite layer formed by nonaggregated Mef p-1 and ceria nanoparticles. Adsorption of aggregated Mef p-1 leads to higher ΔD values, close to 3 × 10−6, indicating formation of a more viscoelastic layer. The dissipation increases with each Mef p-1 adsorption step and decreases with each ceria nanoparticle deposition. This shows that the overall rigidity increases when nanoparticles are added and decreases when Mef p-1 is adsorbed as the last constituent, and suggests that adsorption of ceria nanoparticles onto Mef p-1 results in a less extended conformation of the protein that becomes trapped between two interfaces, i.e., between the substrate and the nanoparticle or between two nanoparticles. The dissipation increase due to Mef p-1 addition is more pronounced for the higher protein concentration and even more so when an aggregated Mef p-1 sample is used. In the sequential adsorption experiment the dissipation with Mef p-1 as last added constituent is remarkably similar to that of the Mef p-1 layer adsorbed to silica independent of how many previous adsorption steps that has been carried out. This suggests that the underlying composite formed by the previous Mef p-1 and ceria nanoparticle depositions is rigid and does not contribute significantly to the energy dissipation. Rinsing of the first Mef p-1 layer results in more pronounced changes in dissipation and frequency compared to rinsing of subsequent Mef p-1 layers (Figure 4). This suggests more significant desorption from silica than from the Mef p-1/ceria

composite layer, and stronger interactions between Mef p-1 and ceria than between Mef p-1 and silica. The sensed mass presented in Figure 5 was calculated by means of either the Sauerbrey or the Voigt model, and the data obtained with these two models are very similar due to the high rigidity of the nanocomposite layer. The values reported were obtained for the layers after rinsing with water and represent the average value from at least two independent measurements. There is a close to linear increase in sensed mass with number of adsorption steps for all cases, and the ten times difference in the Mef p-1 concentration in the two sets of experiments did give rise to a moderate difference in the total sensed mass, as seen in Figure 5. For instance, after six adsorption steps the sensed mass equals 27 and 32 mg m−2 when a Mefp-1 concentration of 0.01 and 0.1 mg mL−1, respectively, was used. The sensed mass obtained when the aggregated Mef p-1 sample was used equals 35 mg m−2, which is larger than for the case when nonaggregated Mef p-1 was utilized. An estimate of the thickness of the adsorbed layers can be obtained from the Voigt model. However, unlike the sensed mass, the thickness value will strongly depend on the assumed layer density. Our QCM-D data for the initial Mefp-1 layer is best reproduced with a layer density of 1200 kg m−3, and the density of the Mef p-1 ceria nanocomposite layer should be higher than this. Nevertheless, we calculated the upper bound of the layer thickness using a density of 1200 kg m−3, and this 9555

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Figure 6. (A) Sensed mass as a function of layer number, calculated using the Voigt (filled symbols) and the Sauerbrey (open symbols) models. The sensed mass obtained with these two models overlaps, and therefore only one symbol is seen in most cases. (B) Total energy dissipation change vs layer number.

Figure 7. Peak force QNM AFM images of bare QCM crystal: (A-I) height, (A-II) adhesion, and (A-III) deformation. The peak force set point was 750 nN. Images of bare silica wafer (B-I) height, (B-II) adhesion, and (B-III) deformation. The peak force set point was 830 nN.

Table 1. Summary of Surface Properties of Bare Silica Surfaces and of Mef p-1/Ceria Nanocomposite Layers Determined by Means of AFM Peak Force QNM Tapping in Aira roughness (nm) Ra

Rq

0.5 ± 0.1 0.1 ± 0.03

0.4 ± 0.1 0.1 ± 0.03

substrate QCM crystal silica wafer MC10-6Q on QCM MC100-6Q on QCM MC100-17Q on QCM MC100-17W on silica MC100-18Q on QCM MC100-18W on silica a

2.0 2.1 2.2 3.2 2.7 2.4

± ± ± ± ± ±

0.1 0.1 0.2 0.5 0.4 0.2

1.6 1.6 2.8 2.3 2.2 2.3

± ± ± ± ± ±

0.1 0.1 0.3 0.2 0.3 0.3

elastic modulus (GPa) 78 ± 8 113 ± 7 4.5 6.5 4.0 3.7 4.4 4.7

± ± ± ± ± ±

0.6 0.3 0.7 0.9 0.9 0.8

adhesion (nN)

deformation (nm)

peak force set point (nN)

17 ± 2 11.6 ± 0.6

1.3 ± 0.2 1.0 ± 0.1

750 830

± ± ± ± ± ±

33 30 35 34 34 35

2.8 2.6 7.2 5.7 1.9 2.3

± ± ± ± ± ±

0.4 0.3 1.6 0.9 0.7 0.7

1.3 1.3 1.2 1.8 1.5 1.6

0.1 0.1 0.21 0.26 0.21 0.22

The thickness of the 6-layer coating was estimated to 17 nm and that of the 18-layer coating to 55 nm as described in the Supporting Information.

thickness of 17 nm (see Supporting Information for details). This should be compared with the average hydrodynamic diameter of the ceria nanoparticles in the aqueous dispersion,

suggests that the layer formed after 6 adsorption steps is less than 25 nm. Indeed, by removing an area of the film and measuring the thickness with AFM in air we find a film 9556

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Table 2. Information on the Mef p-1 and Ceria Nanocomposite Layers Analyzed with Peak Force QNM

about 10 nm, and the thickness of the Mef p-1 layer that after rinsing is 2−2.5 nm for nonaggregated Mef p-1 as obtained in this work and in measurements using dual polarization interferometry.17 Thus, if complete surface coverage is achieved at each adsorption step one would expect the total film thickness after six steps to be in the range 36−39 nm. This value is significantly larger than the upper bound of the layer thickness calculated from the Voigt model, and demonstrates that less than perfect coverage is obtained at each adsorption step. Thus, the nanocomposite film does not consist of a perfectly layered structure, but should rather be viewed as an entangled network of Mef p-1 and ceria nanoparticles. Since the aggregated Mefp-1 sample provided the largest added mass, we explored this case further and increased the number of sequential adsorption steps to 18, and the results obtained are shown in Figure 6. A close to linear growth of the film was observed with increasing layer number as illustrated in Figure 6A. The dissipation value increases after the protein addition and decreases with addition of ceria nanoparticles (Figure 6B). However, after layer number 6 there is also an overall increasing dissipation with increasing number of deposited double layers. It is tempting to suggest that this is due to growth of the film beyond complete monolayer coverage after layer number 6. 3.4. Nanomechanical Mapping. Peak force tapping AFM was used to gain information on physical and mechanical properties of the Mef p-1/ceria nanocomposite layers. First, the surfaces of bare QCM crystals and silica wafers were probed as references, and maps of the height, adhesion, and deformation are shown in Figure 7. In order to have reliable results for the elastic modulus, the deformation value was kept in the range of 1−2 nm for all measurements. It can be seen in Table 1 that the required peak force set point for deforming the bare QCM crystal and silica wafer was much higher than needed for achieving similar deformation of the nanocomposite layers, reflecting that the composite layer is significantly softer than the bare silica crystals. Moreover, Table 1 includes average values for surface arithmetic roughness, Ra, and root-mean-square roughness, Rq. The QCM crystal and silica wafer have Rq values of 0.4 ± 0.1 nm and 0.1 ± 0.03 nm, respectively. Thus, the silica wafer is flatter than the QCM silica crystal. The average elastic modulus was found to be lower for the QCM silica crystal than for the silica wafer. The difference between the elastic modulus of these two silica substrates is due to having silicon oxide with different properties. The silicon dioxide QCM crystal surface is made of physical vapor deposition (PVD), and here we obtain an elastic modulus of 78 ± 8 GPa. The uncertainty represents the standard deviation over at least three different measurements. This value is in good agreement with data obtained with other methods.53−55 In contrast, the silica wafer consists of silicon oxide (with 30 nm thickness) thermally grown on a single silicon crystal with FCC structure with elastic modulus of 130 GPa56 in the direction normal to the top (100) surface. 3.4.1. Effect of Mef p-1 Concentration. Nanomechanical properties of the Mef p-1/ceria composite films were also evaluated. These films were prepared using the same adsorption procedure as in the QCM measurements, but using a dipping robot to allow deposition also on flat silica wafers. In the following, we will refer to the six samples that we discuss with names based on their composition (see Table 2). The samples are abbreviated as MCX-YZ, where MC stands for Mef p-1/ceria composite, X is the Mef p-1 concentration used during

sample MC106Q MC1006Q MC10017Q MC10018Q MC10017W MC10018W

Mef p-1 concn (mg mL−1)

Ceria concn (mg mL−1)

no. of layers

0.01

0.5

6

Ceria

QCM

0.1

0.5

6

Ceria

QCM

0.1

0.5

17

Mef p-1

QCM

0.1

0.5

18

Ceria

QCM

0.1

0.5

17

Mef p-1

wafer

0.1

0.5

18

Ceria

wafer

the last layer

substrate

deposition in mg mL−1, and Y is the total number of layers deposited. Z refers to type of underlying substrate where Q means QCM crystal and W silica wafer. The effect of Mef p-1 concentration during LbL deposition on the nanomechanical properties of the composite layer is illustrated in Figure 8, where images displaying height, elastic modulus, adhesion, and deformation are summarized. The layers were prepared on the QCM crystals and characterized in terms of sensed mass, see Figure 5. In the modulus (Figures 8A-II and 8B-II), adhesion (Figures 8A-III and 8B-III), and deformation (Figures 8A-IV and 8B-IV) images a domain like pattern is observed, which reflects that of the bare QCM crystal (Figure 7A). This supports the suggestion from the QCM-D data that close to monolayer coverage is found at this stage. The surface properties like modulus and roughness are very different with and without the nanocomposite layer (see Table 1). The presence of this layer leads to increase in surface roughness Rq = 1.6 ± 0.1 nm, compared to Rq = 0.4 ± 0.1 nm for bare QCM crystal. Moreover, the average of the elastic modulus for the composite films is much lower than for the bare QCM crystal, and the tip−sample adhesion is reduced in the presence of the nanocomposite film (Table 1 and Figure 8). Figure 8 includes images of the nanocomposite film formed with different Mef p-1 concentrations, MC100-6 and MC10-6, respectively. The average tip−layer adhesion is unaffected by the Mef p-1 concentration in this concentration interval. However, the elastic modulus for the composite layer formed using the higher protein concentration, 6.5 ± 0.3 GPa, which is higher than the elastic modulus of the nanocomposite built with lower Mef p-1 concentration, 4.5 ± 0.6 GPa (Table 1). We note that also the dissipation values determined by QCM-D are lower for the higher protein concentration (Figure 4A,B). This can be rationalized by increased number of ceria−Mef p-1 crosslinks at the higher protein concentration, and these bonds contribute to increased stiffness of the composite layer. 3.4.2. Effect of Number of Depositions and Nature of Last Added Constituent. Figure 9 shows the elastic modulus for the nanocomposite films formed by seventeen and eighteen consecutive adsorptions of Mef p-1 and ceria nanoparticles on QCM silica crystals and silica wafers. These data illustrate the effect of the underlying substrate and nature of the last added layer. First, increasing the number of consecutive adsorption steps leads to moderately rougher surfaces with Rq = 2.8 ± 0.3 nm (MC100-17Q) and Rq = 2.2 ± 0.3 nm (MC100-18Q) in comparison to layers formed with the same concentration of 9557

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Figure 8. Peak force QNM AFM images of MC10-6: (A-I) height, (A-II) modulus, (A-III) adhesion, (A-IV) deformation, and peak force set point = 33 nN. MC100-6: (B-I) height, (B-II) modulus, (B-III) adhesion, (B-IV) deformation, peak force set point = 30 nN.

Figure 9. Peak force QNM AFM images of DMT modulus, (A-I) bare QCM crystal, (A-II) 17 Mefp-1/ceria layers on QCM crystal, (A-III) 18 Mef p1/ceria layers on QCM crystal, (B-I) bare silica wafer, (B-II) 17 Mef p-1/ceria layers on silica wafer, and (B-III) 18 Mefp-1/ceria layers on silica wafer.

Mef p-1 but lower number of layers (Rq = 1.6 ± 0.1 nm for MC100-6Q). Further, it is noteworthy that the adhesion value is a factor of 2−3 higher compared to when ceria nanoparticles are the last layer (see Table 1). This suggests that Mef p-1 contributes more to the sample−tip adhesion than ceria nanoparticles. The elastic modulus of the layer decreases from 6.5 ± 0.3 GPa to 4.4 ± 0.9 GPa with increasing number of layers, which is attributed to the increased thickness. A more homogeneous surface with less clear domain structures is obtained when Mef p-1 is added last, compared to when ceria nanoparticles are added last, see Figure 9. It is interesting that the initial surface features can affect the layer even after 18 deposition steps, with smaller domains being seen on the QCM crystal substrate than on the more flat silica wafer. The elastic modulus image is

obtained by fitting the DMT model to the deformation curve obtained as the tip is retracted after the initial deformation. Thus, these images are related to the mechanical properties of the material in the part of the sample that is influenced by the deformation. 3.5. Robustness of the Nanocomposite Coating. A sample with eighteen layers of Mefp-1/ceria on a QCM crystal (MC100-18Q) was selected for determination of the scratch resistance of the nanocomposite coating in air, where contact mode AFM was used to abrade the layer. Figure 10A,B shows how the topography of the surface area changes due to scratching with different applied normal forces. The applied normal forces increase from right to left and from top to bottom in the sequence of 1 μm × 1 μm squares. It is clear 9558

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Figure 10. Surface height (A) before scratching and (B) after scratching with different applied contact pressures, which are marked (in MPa) next to the different areas. The contact pressure increases from right to left and from top to bottom. (B-I) Average scratch depth vs contact pressure. (B-II) Roughness in the scratched area vs contact pressure. The error bars are based on three separate measurements; the surface height and surface roughness for the undisturbed surface are 0.0 ± 0.5 and 2.5 ± 0.2 nm, respectively.

Thus the tip continues to abrade and plastically deform the coating. Also, the amount of debris particles around the abraded area is increased. We conclude that the layers are plastically deformed at low pressures, below 16 MPa, but do not abrade until the contact pressure exceeds about 80 MPa.

from Figure 10B that applying normal forces causes plastic deformation of the layers. In order to discuss the amount of plastic deformation we first convert the applied force to contact pressure. We have a hard spherical tip (ESi(100) = 130 GPa)56 in contact with the Mef p-1/ ceria coating (EMC100‑18Q = 4.4 GPa, see Table 1), and considering that surface forces are negligible in comparison to the applied normal forces, it is possible to estimate the average contact pressure using uniaxial normal stress, dividing the normal force by the tip surface area (2πr2), where r is the measured tip radius. The average scratch depth is plotted vs contact pressure in Figure 10B-I, and Figure 10B-II shows the rms surface roughness inside the scratched area as a function of contact pressure. The surface height and surface roughness for the undisturbed surface are 0.0 ± 0.5 and 2.5 ± 0.2 nm respectively. At the lowest contact pressure, 16 MPa, the scratch depth is about 2 nm and the surface roughness is somewhat lower than on the original surface. Thus, some plastic deformation has occurred but the surface roughness is only moderately affected. With increasing contact pressure the surface roughness decreases significantly whereas the scratch depth increases only slightly up to a pressure of 79 MPa, which shows that the applied pressure primarily causes surface flattening due to plastic deformation of the protruding surface features. Increasing the contact pressure to more than 79 MPa causes significant increase in the scratch depth and a sudden increase in surface roughness, which indicate that the composite coating starts to be abraded by the tip. It is noteworthy that at this pressure some debris particles appear at the edge of the scratched region. Increasing the contact pressure to more than around 143 MPa caused a further decrease in scratch depth and recovery of a flat scratched area.

4. CONCLUSIONS In this work the layer-by-layer approach was used to build a composite film consisting of Mefp-1 and ceria nanoparticles. Ceria nanoparticles do not adsorb to silica whereas Mef p-1 does. Thus, the first step in the LbL process is Mef p-1 adsorption. The effect of protein concentration and aggregation of Mef p-1 on the added mass was investigated, and it was found that an increase in Mef p-1 concentration, in the range 0.01 to 0.1 mg mL−1, had a moderate effect on the sensed mass. The sensed mass also increases if an aggregated Mef p-1 sample is used. Sequential adsorption of Mefp-1 and ceria nanoparticles occurs readily, and a close to linear growth was observed for up to 18 layers (the largest numbers of layers deposited in this study). The formed composite layers were rigid as demonstrated by the low dissipation value, and the rigidity increases at each ceria nanoparticle deposition step, and decreases each time Mef p-1 is added. The protein concentration used during deposition affects the properties of the Mef p-1/ceria nanocomposite layer. In particular, the elastic modulus increases with increasing protein concentration, which is attributed to increased number of Mef p-1 cross-links binding the ceria nanoparticles together. Peak force QNM imaging demonstrates that Mef p-1 as the last layer results in a less rigid surface compared to ceria nanoparticles as the last layer, which is consistent with the 9559

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decrease in dissipation value found in the QCM experiments each time ceria nanoparticles were added. The texture of the surface layer is also affected by the last added component, where more clear domain sizes are observed when the ceria nanoparticles are added last. The robustness of the nanocomposite layer was evaluated by AFM. It was found that the layers deform plastically under pressures as low as 16 MPa (the lowest pressure investigated). In particular, the protruding features are flattened, which lowers the surface roughness. Abrasion of the layers was found to start at a pressure of about 80 MPa, demonstrating high internal cohesion. Thus, our data suggest that Mef p-1, which is known for its high affinity to many surfaces, is an excellent component in a polymer−particle composite film when high strength is required. When a large adsorbed amount is needed, it is an advantage to work with a Mef p-1 sample containing small aggregates, and this does not seem to compromise either the homogeneity or the cohesive strength of the film.

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ASSOCIATED CONTENT

S Supporting Information *

Dynamic light scattering data from the Mef p-1 solutions. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +46 8 790 9905. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the program “Microstructure, Corrosion and Friction Control” supported by the Swedish Foundation for Strategic Research, SSF. A.D. acknowledges VINNOVA for a VINNMER grant. Eva Blomberg is acknowledged for helpful advice regarding QCM.

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REFERENCES

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