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J. Phys. Chem. B 2008, 112, 8241–8249

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Fibronectin Adsorption on Tantalum: The Influence of Nanoroughness Mads Bruun Hovgaard,† Kristian Rechendorff,†,‡ Jacques Chevallier,† Morten Foss,*,† and Flemming Besenbacher*,† Interdisciplinary Nanoscience Center (iNANO) and Department of Physics and Astronomy, UniVersity of Aarhus, DK-8000 Aarhus C, Denmark, and Laboratory of Physics of LiVing Matter, IPMC, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland ReceiVed: February 6, 2008; ReVised Manuscript ReceiVed: April 10, 2008

The complex mechanisms of protein adsorption at the solid-liquid interface is of great importance in many research areas, including protein purification, biocompatibility of medical implants, biosensing, and biofouling. The protein adsorption process depends crucially on both the nanoscale chemistry and topography of the interface. Here, we investigate the adsorption of the cell-binding protein fibronectin on flat and nanometer scale rough tantalum oxide surfaces using ellipsometry and quartz crystal microbalance with dissipation (QCMD). On the flat tantalum oxide surfaces, the interfacial protein spreading causes an increase in the rigidity and a decrease in the thickness of the adsorbed fibronectin layer with decreasing bulk protein concentration. For the tantalum oxide surfaces with well-controlled, stochastic nanometer scale roughness, similar concentration effects are observed for the rigidity of the fibronectin layer and saturated fibronectin uptake. However, we find that the nanorough tantalum oxide surfaces promote additional protein conformational changes, an effect especially apparent from the QCM-D signals, interpreted as an additional stiffening of the formed fibronectin layers. 1. Introduction The adsorption of proteins on solid surfaces plays a critical role in many applications, such as protein purification, pharmaceutical design, biosensing, food and biochemical processing, and development of new medical implants. For instance, a detailed understanding of the protein-surface interactions is essential to design self-assembled nanoconstructs1–3 and for use in sensors and diagnostics.2 Protein adsorption is of utmost relevance to the design and optimization of new and improved biomaterials, since the introduction of a foreign implant surface into the body fluid will cause a layer of biomolecules, mainly proteins, to be formed prior to any cellular response. The subsequent cellular attachment is indeed mediated by interactions with this protein layer.4–6 Protein adsorption depends strongly on both the specific chemical and morphological surface characteristics on the nanometer (nm) length scale, the latter documented from studies on model surfaces with a regular surface morphology, e.g., silica beads of well defined sizes,7–13 nanopyramids,14 or surfaces with regular nanostructured patterns.15 Fewer studies have addressed the question how surfaces with a random stochastic surface roughness on the nanometer length scale affect protein adsorption.16,17 Here, we have investigated by means of the ellipsometry and quartz crystal microbalance with dissipation (QCM-D) techniques the adsorption of fibronectin on tantalum oxide surfaces being either flat or with a well-controlled nanoscale roughness. In particular, we focus on the conformational changes of fibronectin during adsorption and the viscoelastic properties of the protein layer formed on the surfaces. Fibronectin is a flexible, oblate ellipsoid-like, high molecular weight glycoprotein consisting of two nearly identical ∼250 * Corresponding authors. E-email: [email protected] (M.F.); [email protected] (F.B.). † University of Aarhus. ‡ Ecole Polytechnique Fe ´ de´rale de Lausanne (EPFL).

kD subunits, which are mainly comprised of three types of subdomains arranged like beads on a string (types I, II, and III),18 with disulfide bridges cross-linking the dimer structure near the C-terminus. Fibronectin is a key component of the extra-cellular matrix (ECM) of all connective tissues, and it is known to play a key role in many essential cellular functions, including adhesion, growth, migration, and differentiation.18–20 Fibronectin contains among others, binding sites for heparin, fibrin, collagen, and integrins.18 Tantalum has proven to be biocompatible21 and has been used, for e.g., in implant coatings.22,23 The quantitative adsorption of fibronectin on tantalum has to our knowledge only been investigated once before,24 where the focus was mainly on the subsequent cellular attachment. These facts make fibronectin adsorption studies on tantalum surfaces highly relevant to a wide range of biomedical applications and an interesting model system for the study of protein-interface interactions. At pH and ionic strength values resembling physiological conditions, fibronectin is known to adopt a compact structure stabilized dominantly by intermolecular ionic interactions,25 resulting in an oblate ellipsoidal shape26 with the dimensions of 1.41 nm × 13.9 nm (minor and major axis, respectively). However, at increased ionic concentrations or upon the addition of urea, fibronectin undergoes a conformational transition to a more extended configuration with the otherwise hidden cellbinding RGD tripeptide site becoming functionally available.26,27 This unfolding, and subsequent RGD site exposure, can occur as a response to the adhesion of the fibronectin protein on surfaces, often referred to as a surface activation.28,29 On the single-molecule level, the influence of surface chemistry on the adsorbed protein conformation has previously been investigated using both electron microscopy and atomic force microscopy (AFM).30–32 In general, the adopted surface configuration of fibronectin is shown to depend on the particular physicochemical properties of an interface and the protein is found to adopt a

10.1021/jp801103n CCC: $40.75  2008 American Chemical Society Published on Web 06/20/2008

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compact, globular form when adsorbed to hydrophobic substrates, in contrast to a more expanded, often linear configuration on hydrophilic surfaces.30–32 The adsorption of fibronectin on surfaces has additionally been investigated using a variety of different techniques including QCM-D, laser scanning microscopy, optical waveguide light mode spectroscopy (OWLS), and X-ray photoelectron spectroscopy (XPS) on a multitude of substrates, such as POMA (poly(octadecene-alt-maleic anhydride)) and PPMA (poly(propene-alt-maleic anhydride)),33 titanium oxide,34 stainless steel,35 and silica nanoparticles.13 2. Materials and Methods 2.1. Proteins. Human plasma fibronectin (FN, Sigma-Aldrich) and monoclonal mouse antihuman fibronectin (IgG, clone IST-4, Sigma-Aldrich) were used as delivered and dissolved in Tris buffer (10 mM Tris, 0.1 M NaCl, pH 7.4, Sigma-Aldrich). For nonspecific antibody adsorption, rabbit antihuman albumin (IgG fraction only, Sigma-Aldrich) dissolved in the same buffer was used. 2.2. Tantalum Substrates. Both for the QCM-D and the ellipsometry experiments Ta films were coated on standard 5 MHz AT-cut gold-coated quartz crystals (model QSX 301, Q-Sense AB, Gothenburg, Sweden).36 The quartz crystals were coated with 80 nm of Ta (target from Goodfellow; purity 99.9%) by e-gun evaporation at room temperature with a base pressure of about 10-8 bar and a constant deposition rate of 15 Å/s. The distance between the evaporation source and the substrate was 25 cm, and the evaporation was performed at 10° and 90° of oblique incidence, measured as the angle between the direction of evaporation and the substrate surface. As previously demonstrated,37 this process of oblique angle deposition leads to Ta films of varying, well-controlled, stochastic surface roughness on the nanometer scale. The subsequent exposure of the crystals to ambient air results in the formation of a native tantalum oxide film, typically of a 2-5 nm thickness.37 The surface topography of the substrates were determined using a Nanoscope IIIa MultiMode AFM (Veeco Instruments, Santa Barbara, CA) operated in the tapping mode under ambient conditions. The AFM imaging was performed at scan frequencies of 1-2 Hz, with minimal loading forces applied using optimized feedback parameters. Silicon nitride cantilevers (NSG01, NT-MDT, Russia) with a typical resonance frequency of 150 kHz, a spring constant of 5.5 N/m, aspect ratio of 3:1, and a typical tip radius below 10 nm were used. AFM images with a 1 µm linear scan range and a 512 × 512 pixel resolution were obtained from several locations across the films to ensure good statistics. The root-mean-square (rms) value of the surface roughness, denoted w, was subsequently determined using the commercial scanning probe image processor software (SPIP, Image Metrology ApS, version 4.2, Lyngby, Denmark)38 and found to be 1.01 ( 0.10 nm and 4.93 ( 0.14 nm for films deposited at 90° and 10 degrees°, respectively, with corresponding z-ranges (peak-tovalley) of 12 ( 2 nm and 44 ( 2 nm and a roughness factor (normalized increase in surface area) of 1.001 and 1.239, respectively (Figure 1 and Table 1). In the following, the 90° and 10° deposited films are referred to as flat and rough Taoxide films, respectively. All films were UV-ozone treated (Bioforce Nanosciences UVO cleaner) for 40 min immediately before use to remove any hydrocarbon contaminants and to ensure a clean hydrophilic surface. 2.3. Ellipsometry. With the ellipsometry technique, the change in the polarization state of a light beam upon the reflection from a sample of interest is measured. The change in the polarization state is determined by the properties of the solid-liquid interface. The ratio of the reflection constants of

Figure 1. (top) AFM image (1 µm × 1 µm) of a rough tantalum film along with typical linescans (bottom) of both flat (90° dep.) and rough films (10° dep.) for direct comparison.

TABLE 1: Surface Topographical Characteristicsa for Each Choice of Deposition Angle deposition angle (deg)

w (nm)

z-range

R

10 90

4.93 ( 0.14 1.01 ( 0.10

44 ( 2 12 ( 2

1.239 1.001

a Measured mean values ( std error by AFM based on a minimum of three independent measurements across a minimum of five crystals of each deposition.

p- and s-polarized light (Rp and Rs, respectively) is related to the measured ellipsometric angles Ψ and ∆ through the equation Rp/Rs ) tanΨ exp(i∆).39 Shifts in the ellipsometric angles Ψ and ∆ are described using a four-layer model (buffer-protein-Ta oxide-Ta). The refractive indices pertaining to the Ta films are found initially by employing varying angle ellipsometry. The refractive indices used for the protein layer and the buffer were 1.465 and 1.335, respectively.40,41 With these values, the optical thickness of the protein layer can be determined and converted into a surface mass density, ΓEllip, using the relation:42

ΓEllip ) h1

n1 - n2 dn ⁄ dc

(1)

where h1 is the thickness of the protein layer, n1 and n2 are the respective refractive indices for the protein layer and buffer, and dn/dc is the refractive index increment for a concentration change. The latter parameter is nearly constant for most protein solutions, with a value of 0.18 cm3/g.43 2.4. Quartz Crystal Microbalance with Dissipation (QCMD). The QCM-D technique44 has proven to be well suited for in situ dynamic monitoring of both mass and mechanical

Fibronectin Adsorption on Tantalum

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properties such as viscoelasticity of absorbed biomolecules.16,45,46 The AT-cut quartz crystal is piezoelectric and deforms mechanically when exposed to an electrical field. By applying an (RF) ac voltage across the crystal, a shear oscillation is induced at the fundamental resonance frequency (first) and at the third, fifth, and seventh etc., overtones; for the crystals used here, the fundamental resonance frequency is ∼4.95 MHz. Information about the adsorption process is obtained by the simultaneous measurement of changes in both the resonance frequency (∆F N) and the dissipation factor (∆DN) at each overtone (N ) 1,3,5,7). The dissipation factor is given by the ratio between the sum of all energy losses in the system per oscillation cycle and the total energy stored in the oscillator, and it is defined in relation to the Q-factor of the oscillation, D ) 1/Q. Changes in dissipation, ∆DN, provide information about the viscoelastic properties of the adsorbed layer. For rigid films displaying little shift in dissipation, the total adsorbed surface mass density (ΓQCM-D) is well estimated by the change in the Nth overtone resonance frequency, ∆FN, via the simple Sauerbrey equation:41,47

∆FN ) -N

2F12 (F0 µ0 )1 ⁄ 2

ΓQCM-D

(2)

where F0 and µ0 are the density and shear modulus of quartz, respectively, F1 is the fundamental resonance frequency, and ΓQCM-D is the surface mass density detected by QCM-D. Throughout this study, ΓQCM-D is found using eq 2 for the seventh overtone (N ) 7) in accordance with previous QCM-D studies.40,41,48 With respect to the viscoelastic properties of the adsorbed layer, it is often useful to consider the normalized dissipation values |∆DN/∆FN| values instead of the relative shifts (∆DN) in dissipation. The quantity |∆DN/∆FN| expresses the dissipation shift per frequency unit, which makes comparison between adsorbed layers more straightforward. Within the context of viscoelastic materials, |∆DN/∆FN| estimates a characteristic relaxation time of the adsorbed layer.49 In contrast to the ellipsometry technique, the QCM-D technique is sensitive to both the mass of the adsorbed biomolecules (termed the dry mass) as well as to any solvent molecules, for example water, rigidly bound or dynamically coupled to the interfacial layer. This total hydrated mass present is often referred to as the wet mass.40,41 The difference in the adsorbed mass detected by the ellipsometry and QCM-D techniques can be utilized to obtain further insight into the resulting protein layer, in particular the hydrated mass density (Flayer) and hydrated thickness (hlayer) of the adsorbed protein layer can be estimated. These parameters (Flayer and hlayer) provide more realistic information about the protein layer properties than their protein-only analogues (i.e., when disregarding any solvents in the layer) and can be estimated by:16,41

1 Flayer

)

Vprotein + Vsolvent ΓEllip 1 ) ( )+ Mprotein + Msolvent Fprotein ΓQCM-D 1 ΓQCM-D - ΓEllip ( ) (3.1) Fsolvent ΓQCM-D hlayer )

ΓQCM-D Flayer

(3.2)

Here Mprotein and Msolvent are the dry and solvent mass present, occupying the respective volumes Vprotein and Vsolvent in the hydrated layer, Fprotein ) 1.33540,41 and Fsolvent ) 1.00 g/cm3.

3. Experimental Details Fibronectin was diluted to concentrations of 10 µg/mL, 25 µg/mL, and 100 µg/mL in Tris-buffer, while the IgG antibodies (antifibronectin and antialbumin) were diluted to a concentration of ∼230 µg/mL. The concentrations 10, 25, and 100 µg/mL are referred to as low, medium, and high concentrations, respectively. Fibronectin adsorption was investigated on the flat Ta-oxide sample for all concentrations using both the ellipsometry and QCM-D techniques, while on rough substrates, it was investigated only at high and low concentrations, with a focus on the most pronounced cases, using QCM-D exclusively. At all concentrations and surface morphologies, the subsequent adsorption of both antifibronectin and antialbumin antibodies was investigated. For the ellipsometry studies, a single wavelength ellipsometer (model ELX-02C from DRE GmbH, Germany, λ ) 632.8 nm) was used. The Ta-oxide sample was mounted in a liquid cell, and a fixed angle of 70° relative to the substrate normal was used due to the construction of the cell. After a stable baseline buffer signal was achieved, both for the fibronectin and for the antibodies, samples with a volume of 1 mL were injected by very briefly pausing the measurement for less than 5 s and replacing the entire cell volume. The QCM-D experiments were carried out using two axial flow chambers (models QAFC 301 and QAFC 302, Q-Sense AB, Gothenburg, Sweden).36 After a stable buffer-signal baseline (drift less than 0.1 Hz/min on the third overtone) was obtained, the samples were thermally equilibrated and subsequently injected into the measurement chamber. Both in case of QCM-D and ellipsometry, the experiments were performed at room temperature under static conditions. The adsorption process was monitored until saturation, i.e., when the signal increment was less than or comparable to the natural drift of the experimental setup (in case of QCM-D < 0.1 Hz/min on the third overtone). Since we wanted to directly compare the results obtained from the ellipsometry and QCM-D techniques, we investigated whether the protein layers were truly saturated using both techniques under similar experimental conditions. The QCM-D studies showed that at low concentration a secondary injection of fibronectin led to a small additional adsorption. The additional adsorption observed was in average approximately 12%. This behavior differs from the ellipsometry results, where a secondary injection had no observable effect. This difference is most likely due to the differences in measurement-cell design between the QCM-D and ellipsometry set-ups, more precisely the higher surface-to-volume ratio in the case of the QCM-D measurement chamber compared to that of the ellipsometry liquid cell. To facilitate a more straightforward comparison of saturated surface mass densities between the two techniques, a secondary fibronectin injection for the QCM-D studies at low concentration after 120 min was performed, ensuring a full, reproducible saturation after 180 min. For high and medium concentrations, the process was monitored for a period of 120 min after a single injection only for both experimental set-ups. The subsequent adsorption of monoclonal IgG isotype fibronectin antibodies and IgG isotype antihuman albumin for investigation of specific and nonspecific recognition was in all cases executed 180 min after the initial fibronectin injection. Saturated values for the antibody uptake were recorded after 60 min of antibody exposure, and neither the QCM-D nor the ellipsometry results indicated any depletion behavior (data not shown). For each adsorption study, at least five experiments were carried out and mean value ( standard error was reported.

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Figure 2. Typical adsorption curves on a flat Ta surface obtained from QCM-D and ellipsometry for a 25 µg/mL experiment including the subsequent adsorption of antibodies.

Figure 3. (a) Experimental results of the saturated fibronectin adsorption on flat tantalum oxide obtained using QCM-D and ellipsometry, and (b) the dissipation versus frequency ratios as a function of protein concentration. Each point is the average of at least five measurements (mean ( std error).

4. Results and Discussion 4.1. Adsorption on Flat Ta-Oxide Surfaces. 4.1.1. Fibronectin Adsorption. A typical adsorption curve of fibronectin on a flat Ta-oxide surface with a subsequent injection of antibodies as monitored by the ellipsometry and QCM-D techniques is displayed in Figure 2. A concentration of 25 µg/ mL was used, and the recorded response converted into surface mass densities using eqs 1 and 2. Experimental results for fibronectin adsorption at saturation for all concentrations are summarized in Figure 3. For low-dissipative systems, the Sauerbrey frequency-to-mass conversion is considered an good estimate of the adsorbed surface mass density, and the conversion has previously been applied using the higher frequency response harmonics.40,41,48 Ideally, eq 2 will be overtone independent, but for certain viscoelastic materials displaying a nonzero dissipation shift, ∆DN, some overtone-dependency might be experienced, in turn making the Sauerbrey mass estimate inaccurate. For the present work, the overtone variation within a single experiment was always less than 6% but, more importantly, the variation (less than 2%) was consistent across

Hovgaard et al. the experimental parameters of concentrations and morphologies. In short, the data displayed a very limited frequency dependency, and analysis using the third, fifth, or seventh overtone yielded the same relative increases in surface mass densities (within 2%) and would therefore not significantly impact the analysis or conclusions reached. From an experimental point of view, is was further desirable to follow the adsorption via the seventh overtone, since the higher harmonics were less susceptible to noise and overall provided a better data quality throughout the studies. In general, as displayed in Figures 2 and 3a, the QCM-D technique measures a consistently higher surface mass density as compared to the ellipsometry technique, since the QCM-D technique is sensitive also to coupled liquid. Furthermore, for both experimental techniques, a clear concentration dependency of the saturated surface mass densities is observed, with the fibronectin uptake being higher for increased bulk concentrations. For the ellipsometry results, a total increase in surface mass density of 52% is observed, ranging from 208 ( 4 ng/ cm2 at low concentration to 316 ( 9 ng/cm2 at high concentration. These values are in good agreement with previous results for fibronectin adsorption on different substrates.50,51 The change in saturated fibronectin uptake with varying bulk concentration can be associated to protein-protein interactions and postadsorption configurational changes of the adsorbed fibronectin layer. To further elucidate this aspect of the adsorption process, the fibronectin layer is for simplicity estimated using the model of random sequential adsorption (RSA).52 The QCM-D and ellipsometry data, respectively, showed that a secondary injection of fibronectin lead either to a small or no additional mass uptake (only for low concentration and afterward completely saturated). Although theoretically possible, we find that the build-up of fibronectin multilayers or alternatively inhomogeniously distributed surface aggregates on tantalum are not well-supported by data, in contrast to the claim by Sousa et al.53 for fibronectin adsorption on TiO2. In relation to the RSA model, the fibronectin layer is therefore modeled as a closed-packed single layer of proteins existing in two different forms: either in an oblate ellipsoidal form resembling the configuration in suspension26 with a side-down orientation (with a circular footprint area of 606 nm2) or, conversely, with the molecule in a highly denatured state, as a rod of dimensions 120 nm × 2 nm in a side-down configuration (footprint area of 240 nm2), more resembling the single molecule, low-concentration states previously observed by AFM and electron microscopy.30–32 If one takes the aspect ratio of the model configuration into consideration, a theoretical upper limit of the saturated surface mass densities can be determined in both cases.54 In the first sidedown oblate configuration, a RSA packed monolayer corresponds to an approximate surface mass density of 67 ng/cm2 (54% packing), while for fibronectin in the highly relaxed state, a complete layer corresponds to a surface mass density of approximately 106 ng/cm2 (∼34% packing). The experimentally determined values of the surface mass densities show that in average the fibronectin layer at saturation consists of proteins which occupy less surface area per molecule when compared to both the highly denatured side-down state and the more compact side-down bulk oblate configuration. The concentration dependency of the surface mass densities can be understood in terms of an increased protein footprint at low concentration, with the underlying mechanism being the competition for available binding sites, in turn causing lateral protein-protein interactions. While this mechanism restricts postadsorption

Fibronectin Adsorption on Tantalum dynamics (spreading) for high protein concentrations, the competition for available binding sites is expected to diminish with bulk protein concentration due to the decreasing deposition flux, allowing the postadsorption spreading effects to become more pronounced. From the dampening of the oscillating QCM-D crystal, the shifts in dissipation for all the protein concentrations can further be studied. In general, the dissipative losses of an oscillating quartz crystal during protein adsorption can be directly related to the viscoelastic properties of the resulting interfacial molecular layer. Several dissipative channels are present with the major contributions being the viscoelasticity of the adsorbed proteins along with additional dampening caused by water coupled within the layer.40,55 Overall, less dissipative layers are considered more rigid and potentially more compact than systems displaying higher dissipative losses. The dissipation shifts of the seventh overtone increase with concentration, from (2.49 ( 0.19) × 10-6 at 10 µg/mL to (3.11 ( 0.07) × 10-6 at 25 µg/mL to (3.97 ( 0.05) × 10-6 at 100 µg/mL, in fairly good agreement with previous results of fibronectin adsorption onto hydrophilic polymer substrates.33 The concentration dependency of the resulting protein layer is confirmed by the normalized dissipation values |∆D7/∆F7| as displayed in Figure 3, ranging from 6.6 ( 0.2 ns at 10 µg/mL to 7.49 ( 0.08 ns at 100 µg/mL. These findings show a more rigid conformation of the fibronectin layer at lower concentrations and support the hypothesis of an increased protein surface spreading when adsorbed from decreasing bulk concentrations. 4.1.2. Density and Thickness Adsorbed Protein Layers. The observed difference in surface mass densities measured by the ellipsometry and QCM-D techniques can be attributed to the difference in detection principles between the two techniques and their sensitivity to solvent molecules effectively contained in the adsorbed layer. The ratio of the observed surface mass FN ⁄ ΓFN densities ΓQCM-D Ellip expresses the relative amount of coupled solvent molecules in the protein layers, and as displayed in Figure 4a, the experiments show an almost constant coupling factor of solvent across the concentration range with ratios ranging from 4.6 ( 0.2 to 4.29 ( 0.14 from low to high protein concentration, respectively. At a more quantitative level, the mass density (Flayer) and average thickness (hlayer) of the hydrated protein layer can be estimated using eqs 3.1 and 3.2, respectively, and in correspondence with the surface mass density ratios, the fibronectin adsorption saturates at constant mass densities of approximately 1.06 g/cm3 for all concentrations FN (Table 2). The ratio ΓQCM-D ⁄ ΓFN Ellip is highly sensitive to changes in the coupled solvent content of the adsorbed protein FN ⁄ ΓFN layers. Accordingly, the maximum and minimum ΓQCM-D Ellip ratios observed for fibronectin adsorption (4.7 and 4.29, respectively) are equivalent to a narrow mass density interval of only 0.005 g/cm3 (from 1.056 g/cm3 to 1.061 g/cm3). The FN variations observed in ΓQCM-D ⁄ ΓFN Ellip across the concentration range thus signify only small changes in the amount of coupled solvent (water) to the fibronectin layers formed at all bulk concentrations on the Ta-oxide surfaces. Interestingly, as displayed in Figure 4b, the saturated adsorption shows a hydrated thickness depending on bulk fibronectin concentration, with the effects of an increased spreading of the adsorbed proteins directly evident from the observed decrease in layer thickness by a total of ∼29% from 12.8 ( 0.2 to 9.10 ( 0.34 nm from high to low fibronectin bulk concentrations, respectively. Comparing the layer thickness to the size of the fibronectin molecule in suspension, it is evident that the fibronectin proteins in average spread upon adsorption, as the

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Figure 4. (a) Surface mass density ratios and (b) hydrated thickness of the interfacial protein layer at saturation as estimated using eqs 3.1 and 3.2.

TABLE 2: Protein Mass Densitiesa for for the Fibronectin (FN) Layer with and without Antibodies Flayer (g/cm3)

a

conc (µg/mL)

FN

FN with antibodies

10 25 100

1.057 ( 0.010 1.056 ( 0.012 1.062 ( 0.008

1.050 ( 0.010 1.048 ( 0.008 1.055 ( 0.011

Mass densities estimated using eqs 3.1 and 3.2.

TABLE 3: Surface Mass Densities for the Saturated Uptake anti of Antibodies (anti) as Determined Using Ellipsometry (ΓEllip ) anti and/or QCM-D (ΓQCM-D ) along with Estimated Number of Antibodies Binding per Fibronectin Protein anti ⁄ Fn anti ⁄ Fn (ΓEllip or NQCM-D ) Determined by the Two Techniques, Respectively conc (µg/mL) on flat Ta 10 25 100 on rough Ta 10 100

anti ΓQCM-D (ng/cm2)

anti⁄FN NQCM-D

anti ΓEllip (ng/cm2)

anti⁄FN NEllip

494 ( 45 533 ( 12 480 ( 2

1.69 ( 0.08 1.30 ( 0.02 1.07 ( 0.02

71 ( 6 66 ( 6 69 ( 15

1.01 ( 0.09 0.69 ( 0.18 0.53 ( 0.18

466 ( 9 505 ( 22

1.41 ( 0.03 0.98 ( 0.03

layer appears significantly thinner than the major axis of the oblate fibronectin configuration in suspension (length 27.8 nm), but still does not bind in a side down or completely relaxed orientation which would have a thickness of 2.42 nm or less. 4.1.3. Antibody Adsorption. Results from the subsequent exposure of the saturated fibronectin layers to IgG-isotype antibodies are summarized in Table 3. The QCM-D technique consistently measures higher surface mass densities for each concentration due to the coupled solvents. In addition, as the antibodies are expected to recognize specific epitopes on each

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fibronectin molecule, it is important to consider the number of antibodies adsorbed per fibronectin molecule:

Nanti ⁄ FN )

(

)(

∆mantibody mfibronectin ∆mfibronectin mantibody

)

(4)

Here, mfibronectin and mantibody are the masses of the fibronectin (450 kD) and the monoclonal antibody (150 kD), respectively, and ∆mantibody and ∆mprotein are the experimentally measured adsorbed antibody and protein masses. In the case of the ellipsometry technique, the ratio ∆mantibody/∆mfibronectin equals anti FN the ratio of adsorbed surface mass densities ΓEllip /ΓEllip . As is evident from top section of Table 3, the number of antibodies bound per protein molecule bears a marked dependency on the preadsorbed fibronectin layer, with a decreasing antibody recognition for increasing protein uptake. Specifically, the ellipsometry Nanti⁄FN ratios range from 1.01 ( 0.09 antibodies Ellip per protein at 10 µg/mL to 0.53 ( 0.18 antibodies per protein at 100 µg/mL, corresponding to a 48% decrease. As the monoclonal antibodies specifically recognize the fifth type III repeat unit on each fibronectin monomer, a maximum of two binding sites per fibronectin molecules exists, which is in agreement with the observed antibody per fibronectin ratios ranging between 0.53 and 1.01. Furthermore, as the antibodyprotein interaction is very specific, the antibody adsorption is expected to be sensitive to the availability of binding sites, either influenced by changes in the geometrical configuration of the adsorbed proteins or by steric hindrance from protein crowding. The present findings of increasing antibody recognition for decreasing bulk fibronectin concentrations are consistent with the conformational analysis presented so far, that is, the observed decrease in number of antibodies binding to high concentration layers is most likely caused by steric hindrance, where the more tightly packed fibronectin molecules at high concentration limits the availability of exposed binding sites. At lower fibronectin concentrations, the more spread-out state of the adsorbed fibronectin molecules allows for an easy access for the antibodies, as previously observed from fibronectin adsorption on hydrophilic PMMA.33 This trend is again confirmed with the anti ⁄ FN QCM-D results, although with consistently higher NQCM-D ratios observed for all concentrations. The number of antibodies per fibronectin molecule now ranges from 1.69 ( 0.08 to 1.07 ( 0.02 for low to high concentrations, respectively. In general, the application of eq 4 for the analysis of the QCM-D results is not straightforward, as the subsequent antibody adsorption may induce a change in the resulting protein layer hydration, which effectively changes the mass density of the hydrated fibronectin layer. This additional hydration of the adsorbed fibronectin and antibody layer is directly confirmed by the significant increase FN+anti ⁄ ΓFN+anti upon adsorpin surface mass density ratios ΓQCM-D Ellip tion of antibodies depicted in Figure 4a, again with an approximately constant ratio for all concentrations. Furthermore, by application of eqs 3.1 and 3.2, the effective mass density and the hydrated thickness of the resulting layer can be estimated, and from the results summarized in Table 2 (FN with antibodies section), it is found that the mass density after adsorption of the antibodies has a constant value of approximately 1.05 g/cm3 across the whole concentration range. The larger antibody/protein ratios as determined by the QCM-D technique can thus largely be associated with an increased hydration, while the similarity in trends in the antibody/protein ratios as obtained from the QCM-D and ellipsometry results pertains to the antibody-induced hydration being approximately independent of fibronectin concentration. As evident from Figure 4b, the antibody adsorption is further accompanied by an

increase in the hydrated layer thickness with a dependency on the preadsorbed fibronectin uptake. To confirm the specificity of the antibody adsorption, rabbit antihuman albumin (IgGisotype fraction only) was adsorbed on the fibronectin layers formed at all concentrations. The results confirm the specificity by the significantly decreased adsorption of rabbit antihuman albumin when compared to the specific binding of the antihuman fibronectin with a maximal nonspecific contribution of 26% at low concentration. 4.2. Adsorption of Fibronectin on the Nanorough TaOxide Substrates. The introduction of surface roughness on the nanometer scale can influence protein adsorption via a multitude of complex mechanisms. The morphology of the surface can appear in many forms, from ultraflat single-crystal surfaces to nearly random rough structures resembling those often found in nature. In most cases, previous work concerning protein adsorption on structured surfaces have been limited to surfaces with a regular structure, such as groove-,15 pyramid-,14 or colloidal7–9,11–13 nanotopographies, whereas only a few studies have be focused on random microstructures.16,17,56 Generally, protein adsorption is found to be highly sensitive to the exact geometry of the surface topography on the nanometer scale. While the increased surface area of a nanostructured surface is often expected to enhance the protein uptake,16,17 several additional effects have also been taken into account, including protein alignment,15 reduced adsorption,13,15 enhanced surface packing,12,14,16 and various curvature dependent conformational changes of the adsorbed protein.7–13 In a recent study,14 Mu¨ller et al. investigated protein adsorption on solids with varying densities of nanopyramids present, and the authors observed a 2-3 fold increase in the protein adsorption for an only 7% increase in the surface area. The dramatic increase in adsorption was suggested to be caused by a super-packing introduced by the kink sites on the pyramids, which additionally had the effect of decreasing the biofunctionality of the adsorbed proteins. This finding emphasizes another important point, that the functionality of the proteins is closely linked to their structure when attached to a surface and therefore susceptible to conformational changes by the introduction of nanoroughness or nanostructures in general. In a QCM-D study by Lord et al.13 of fibrinogen, albumin, and fibronectin adsorption on colloidally structured silica surfaces (of radius 7, 14, and 21 nm), no significant differences were found in the amount of protein adsorbed onto surfaces structured on 7 and 14 nm large silica colloids but a decrease in fibrinogen uptake was noted on 21 nm colloids. A further investigation of the biological functionality of a fibronectin layer, however, revealed that the RGD sites on fibronectin adsorbed onto the nanocolloids were sterically concealed due to protein-protein interactions or being hidden by adsorptioninduced conformational changes on the single protein level. Additionally, the interplay between local curvature and protein conformation has been investigated in a number of studies concerning protein adsorption onto nanocolloids.7–12 In many cases, small globular proteins tend to preserve their native shape, whenadsorbedonhighcurvature(smalldiameter)nanoparticles.10–12 Roach et al. 12 studied the adsorption of fibrinogen and BSA adsorption on a series of colloids with diameters of 15-165 nm in both a hydrophilic and hydrophobic versions and showed that BSA retained most of its native structure on the smallest, high curvature, nanoparticles with an increasing tendency to spread on the larger colloids. These findings are in contrast to the case of fibrinogen, where the smallest 15 nm nanoparticles guided a side-down, relaxed orientation of the protein with a

Fibronectin Adsorption on Tantalum

J. Phys. Chem. B, Vol. 112, No. 28, 2008 8247

Figure 5. Direct comparison of protein adsorption on the flat and nanoscale rough substrates (a-c). The introduction of nanoroughness is seen to induce an increase in protein adsorption (a) and a stiffening of the formed fibronectin layer at saturation (b). The presence of different surface protein conformations on the rough substrates is supported by the differences in antibody adsorption on the nanorough versus flat substrates (c).

transition to a densely packed, dominantly side-up protein orientation for adsorption onto larger colloids. These results10–17 clearly demonstrate that other mechanisms besides the increase in surface area are pivotal for the understanding of protein adsorption on nanostructured surfaces. Several coexisting effects can influence the protein adsorption depending on the surface and protein under consideration. For instance, an enhanced protein adsorption caused by an increase in surface area may easily be accompanied by a reorientation of the proteins or even curvature-dependent super-packing of the proteins. The detailed nanotopography may thus have a nontrivial influence on the adsorbed protein structure, saturation coverage, and in particular biological functionality. 4.2.1. Fibronectin Adsorption. The adsorption of fibronectin onto nanorough Ta-oxide films with an rms roughness of 4.93 ( 0.14 nm (cf. Table 1) was studied by means of the QCM-D technique at concentrations of 10 and 100 µg/mL. The main results are displayed in Figure 5. Even though we have previously quantified the adsorption of fibrinogen on Ta films with a similar rms roughness using the ellipsometry technique,16 we were not able to unequivocally model the adsorption of the fibronectin protein in the present case. Often, the Ψ-∆ diagrams were very noisy, and in the best case, modeling of the data would constitute a very approximate and highly speculative estimate of the adsorbed surface mass density. If attention is turned to the results obtained by QCM-D, the fibronectin uptake on the nanorough tantalum oxide surfaces is found to increase with increasing bulk fibronectin concentration, similar to the case of fibronectin adsorption on the flat Ta crystals. The average surface mass density rises from 973 ( 36 ng/cm2 at low concentration to 1550 ( 57 ng/cm2 at high concentration, corresponding to a 59% increase. Clearly, the concentrationdependent spreading of the protein layers and postadsorption dynamics also occur at the nanorough substrates. Concerning the viscoelastic properties of the fibronectin layers, the saturated shifts in dissipation factor (∆D7) are observed to rise from (2.14 ( 0.11) × 10-6 at low concentration to (3.9 ( 0.4) × 10-6 at high concentration. The normalized dissipation shifts |∆D7/∆F7| increase from 5.7 ( 0.3 ns to 6.5 ( 0.2 ns from low to high concentrations, respectively. As for the adsorption on the flat Ta surfaces, more rigid fibronectin layers form at low concentrations. 4.2.2. Antibody Adsorption on the Nanorough Ta-Oxide Surfaces. Subsequent adsorption of antihuman fibronectin (IgG, clone IST-4) was performed for fibronectin layers formed at both high and low concentrations. As observed from the results summarized in the lower section of Table 3, an almost identical antibody adsorption is observed for both fibronectin concentrations. However, as fibronectin adsorption at high bulk concentration is accompanied by a larger surface uptake, it is important

to normalize the results in order to gain further insight. If we tentatively use eq 4 and assume an unchanged hydration for the full concentration range as found in the case of fibronectin adsorption on a flat Ta-oxide surface, one finds that the number of antibodies per fibronectin molecule decreases by 30%, from 1.41 ( 0.03 to 0.98 ( 0.03, on going from a low to high bulk concentration. These results for the variation in antibody recognition support the fact that configurational and/or steric hindrance differences exist between the fibronectin layers formed at high and low concentrations. To verify the specificity of the antibody recognition, the adsorption of rabbit antihuman albumin (IgG fraction only) onto preadsorbed fibronectin layers was studied at all concentrations using the QCM-D technique. The specificity of the antibody recognition was confirmed by the significant decrease in adsorption of the nonspecific rabbit antihuman albumin when compared to the specific binding of the antihuman fibronectin, with a maximum nonspecific contribution of 31% at low concentration. 4.3. Fibronectin Adsorption on Nanorough vs Flat TaOxide Surfaces. To further elucidate the mechanisms underlying the adsorption of fibronectin on the nanorough substrates, we have carried out a direct comparison between nanorough and flat topographies for similar concentrations. 4.3.1. Protein Adsorption. If we compare the normalized shifts in dissipation |∆D7/∆F7| for the flat and nanorough surfaces, as displayed in Figure 5b, we find that the nanorough surface is found to significantly influence the protein layer formed and induce the formation of a more rigid fibronectin layer at both concentrations. These observations are in good accordance with previous investigations by Lord et al.13 of fibronectin adsorption on nanometer colloidal structures, where the authors found a similar stiffening of the adsorbed protein layer on the nanostructured surfaces. If we additionally compare, as seen in figure Figure 5a, the saturated surface mass densities between topographies directly, a 14 ( 5% increase is observed for the high concentration adsorption on the nanoscale rough Ta surfaces. While the observed increase in mass can be ascribed to the geometrical effect of the larger surface area present (Table 1), the enhancement is still lower than expected, as the 24% extra surface area measured by AFM, as depicted from Table 1, is a lower limit due to the finite size of the AFM tip apex. At low protein concentration, this discrepancy is even more pronounced, since the introduction of nanoroughness in this case seems to have no significant effect on the saturated fibronectin uptake (Figure 5a). This apparent discrepancy could be due to a change in fibronectin surface conformation possibly followed by a change in protein layer hydration. However, if we rely on the findings from the fibronectin adsorption results on flat Ta and tentatively assume that the additional mass detected by the QCM-D, due to coupled solvents, is largely independent of the

8248 J. Phys. Chem. B, Vol. 112, No. 28, 2008 fibronectin surface conformation, the observed effects on surface mass densities ΓQCM-D and the stiffening of the protein layers are consistent with a roughness promoted enhanced protein relaxation rate. Here, the proteins effectively occupy more surface area per protein on the rough substrates. Such a mechanism would suppress the effect of an increased surface area and may account for the adsorption of fibronectin to be lower than expected when compared to the adsorption on flat Ta-oxide surface. At low protein concentration, where the adsorption process is more affected by the postadsorption relaxation effects and less restricted by the flux of incoming proteins, such an additional spreading is expected to be even more pronounced, in accordance with the trends observed. 4.3.2. Antibody Adsorption. At both low and high fibronectin concentrations, the normalized antibody adsorption (antibodies per protein) is observed to decrease on the rough substrates when compared to the flat surfaces (Figure 5c) by 8 ( 4% at high concentration and by 17 ( 4% at low concentration. This decrease in antibody recognition between the flat and rough topographies further supports the presence of a conformational change in protein configuration on the nanoscale rough substrates and furthermore indicates a comparatively large influence of the nanoroughness on the protein adsorption at low concentration. 5. Conclusions The adsorption of fibronectin on flat and nanoscale rough Ta surfaces has been investigated by QCM-D and ellipsometry. The nanoscale roughness is found to have a significant influence on the adsorption of fibronectin and (i) induce an increased stiffening of the saturated fibronectin layers, both for high and low concentrations, and to (ii) significantly change the saturated fibronectin uptake, with the increase in surface mass densities being lower than the increase in available surface area, especially at low concentration, where the nanoroughness had very little observable effect on the saturated protein uptake. The observations are consistent with the presence of a competing mechanism in terms of a roughness promoted, enhanced, protein relaxation rate suppressing the enhanced surface area effect and leading to a more spread out, rigid protein conformation. Finally, the changes in fibronectin layer conformation on the flat versus the nanorough Ta-oxide surfaces were further supported by the diffrences in antibody adsorption. The present results provide an improved fundamental insight into the mechanisms of protein adsorption on stochastic nanorough surfaces, with specific relevance to understanding and guiding the complex mechanism of fibronectin surface activation. In connection with cellular attachment and biomaterials studies, our findings demonstrate that nanoscale topographical features can have a significant effect on protein adsorption and potentially on the subsequent material biofunctionality. Acknowledgment. We acknowledge financial support from the Danish Research Council to the Interdisciplinary Nanoscience Center (iNANO), the ”Large Interdisciplinary Research Groups” program (2052-01-0006), the Carlsberg Foundation, and the European Commission (FP6 STREP project: NANOCUES). F. Lyckegaard is acknowledged for growing the thin Ta films. References and Notes (1) Lin, C.; Liu, Y.; Rinker, S.; Yan, H. ChemPhysChem 2006, 7, 1641– 1647. (2) Asuri, P.; Bale, S. S.; Karajanagi, S. S.; Kane, R. S. Curr. Opin. Biotechnol. 2006, 17, 562–568.

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