Colloids and Surfaces B: Biointerfaces 66 (2008) 53–59

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Colloids and Surfaces B: Biointerfaces

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Bovine serum albumin adsorption on nano-rough platinum

surfaces studied by QCM-D

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A. Dolatshahi-Pirouz, K. Rechendorff, M.B. HovgaarInterdisciplinary Nanoscience Center (iNANO) and Department of Physics and Astronom

a r t i c l e i n f o

Article history:Received 9 April 2008Received in revised form 16 May 2008Accepted 16 May 2008Available online 24 May 2008

Keywords:BSANano-topographyQCM-DPlatinumProtein adsorption

a b s t r a c t

The adsorption of bovine sranging from 1.49 nm to(QCM-D). Two different BSwere complemented by mprocess was significantly imass density of the adsorba close examination of theit was additionally observadsorption phases revealmorphology during the adat the low (50 �g/ml) BSAa significant influence on

1. Introduction

Adsorption of proteins on surfaces constitutes an important and

very active research field not least due to its impact on areas such ase.g. protein purification, design of food processing equipment, bio-compatibility, and biosensors [1]. For example, the biocompatibilityof an artificial material is closely related to the cellular responsewhen the material is put into contact with a biological system.The cellular response is in turn influenced by the proteins thatadsorb at the implant interface from the biofluid [2]. Both the type,amount, orientation and the conformational state of the proteinsare important in the cell-protein mediated response [3,4]. Also,from a more fundamental research perspective the protein adsorp-tion process is of interest due to the complex nature of the system,and ideally one would like to understand in detail how the proteinconcentration, buffer, pH, ionic strength, etc., influence the pro-tein adsorption. Therefore, numerous studies have been conductedin the past decades in order to examine the effect of such exper-imental conditions on the protein adsorption [5–9]. Even thoughthe effect resulting from a change in one or more of the aboveparameters depends to a large extent on the actual protein in ques-tion, some general features of the adsorption process have beenelucidated [1]. Furthermore, the chemical and morphological prop-

∗ Corresponding author. Tel.: +45 8942 3698, fax: +45 8942 3690.E-mail address: foss@inano.dk (M. Foss).

0927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.colsurfb.2008.05.010

. Foss ∗, J. Chevallier, F. Besenbacheriversity of Aarhus, DK-8000 Aarhus C, Denmark

albumin (BSA) on platinum surfaces with a root-mean-square roughnessm was investigated using quartz crystal microbalance with dissipationcentrations, 50 �g/ml and 1 mg/ml, were used, and the adsorption studiesring the antibody interaction with the adsorbed BSA layer. The adsorptionnced by the surface nano-roughness, and it was observed that the surfaceSA layer is enhanced in a non-trivial way with the surface roughness. Fromgy dissipation vs. frequency shift plot obtained by the QCM-D technique,

at the BSA adsorption on the roughest surface is subject to several distincte presence of structural changes facilitated by the nano-rough surface

ion process. These changes were in particular noticeable for the adsorptionntration. The results confirm that the nano-rough surface morphology has

the BSA mass uptake and the functionality of the resulting protein layer.© 2008 Elsevier B.V. All rights reserved.

erty of the substrate on which protein adsorption occurs is also ofcrucial importance. The effect of the surface chemistry has beenaddressed extensively in the past, e.g. by studying adsorption on dif-ferent metal (oxide) surfaces or chemically functionalized surfacesand is rather well understood [10–12]. The effect of substrate mor-phology on the protein adsorption process has also been studied

extensively in the recent years [13–19], but so far no general consen-sus has been reached yet on how the detailed surface morphologyand nanostructure influence the interfacial protein adsorption pro-cess.

Several studies both theoretical and experimental have beenconducted on more simple polymers adsorbing on silica beads ofdifferent sizes to gain a fundamental understanding of the inter-action between polymers and nanostructured substrates [20,21].These studies have proven that spherical structures can lead toa curvature-induced enhancement of the amount of polymersadsorbing on top of the spherical structures when appropriatebead sizes were chosen [22]. Since proteins in contrast to mostother simple polymers have a well-defined, 3D configuration ofminimal energy with a highly complex structure, only a crudeunderstanding of how proteins might behave on nano-structuredsurfaces can be established from such model studies. Neverthe-less, curvature dependent adsorption characteristics have in factalso been observed for proteins such as fibrinogen on hydrophilicand hydrophobic silica beads in free solution [13], and for lysosymeand human carbonic anhydrase II (HCAII) when adsorbed on nano-particles in free solution, where the particle curvature was reported

d Surf

54 A. Dolatshahi-Pirouz et al. / Colloids an

to induce an altered protein conformation and to reduce the enzy-matic activity of the proteins [14,15]. However, a main obstacleconcerning protein studies on nano-particles is the electrostaticinteraction between proteins and nano-particles in free solution,which varies significantly depending on the particle size [20]. Thishas proven to make a direct understanding of how nano-curvatureinfluences the protein adsorption from measurements conductedon nano-particles rather uncertain. Another problem associatedwith nano-sized beads as a model system arises when a direct com-parison is attempted towards solid substrates that are utilised inbiomaterial applications.

Protein adsorption studies on solid surfaces are highly depen-dent on the system chosen for the experiments. For instance inRef. [16] from an interplay of QCM-D studies and Monte Carlosimulations it is concluded that the geometrical orientation ofadsorbed fibrinogen is influenced significantly by the nano-roughsurface morphologies of tantalum surfaces due to the anistropi-cal shape of fibrinogen. On the other hand in Refs. [17,18], wherethe adsorption of bovine serum albumin (BSA) has been inves-tigated on rough titanium surfaces and surfaces fixed with silicaparticles using a bicinchoninic acid assay and QCM-D, respectively,the authors find that the surface roughness does not affect theadsorption process. However, the full complexity of the surfaceroughness induced effects on BSA adsorption is still not unravelled[16,19]. For example, the authors in Ref. [16] conclude that the BSAadsorption increases with surface roughness, apparently due to anincreased surface area, while Ref. [19] reports that the character-istic nano-rough surface features provides nucleation sites, whichresults in an increase in the BSA adsorption beyond the surface areaincrease.

Here, we have fabricated quartz crystal surfaces with well-defined, nano-rough surface morphologies which were usedas sensor surfaces in QCM-D measurements combined withantigen–antibody assays to investigate how nano-rough platinumsurfaces influence the adsorption of a model protein, BSA, at twodifferent concentrations. The dissipation of the protein film is usedto obtain further information on the interaction of proteins withnano-rough surfaces. Platinum (Pt) was chosen as the substratematerial since it is biocompatible and frequently used as the con-ducting material in implant electrodes for nerve stimulations [23].

2. Materials and methods

2.1. Proteins and antibodies

Bovine serum albumin (BSA) with a purity of 98% and rab-bit IgG polyclonal anti-BSA were purchased from Sigma–Aldrich(Denmark). To test for non-specific binding, mouse IgG monoclonalanti-fibrinogen, clone FG-21, was also obtained from Sigma–Aldrich(Denmark). BSA and the antibodies were stored at −50 ◦C beforeuse. The BSA was dissolved in a 10 mM Tris buffer with 1 mM CaCl2and 100 mM NaCl adjusted with HCl and NaOH to pH 7.4 at 37 ◦C.BSA concentrations of 50 �g/ml and 1 mg/ml were used (subse-quently referred to as low and high concentration, respectively),and the polyclonal antibodies were diluted 1:280 in a Tris buffer.The final protein and antibody solutions were stored at 4 ◦C, and nosolutions were used after more than 10 days after dilution. Duringthat period of time no sign of protein degradation was observed inthe QCM-D measurements.

The dimensions of an IgG antibody is 5.9 nm ×13.1 nm × 14.3 nm, and it is commonly accepted that BSA attainsthe shape of an oblate ellipsoid in liquid with the dimensions of14 nm × 4 nm [24]. The molecular weight of BSA and IgG antibodiesis 65 kD and 150 kD, respectively [24,25].

aces B: Biointerfaces 66 (2008) 53–59

2.2. Preparation and characterisation of surfaces

Platinum films were grown by e-gun evaporation with anoblique angle of incidence between the evaporation flux and thesubstrate in order to generate well-controlled nano-rough plat-inum substrates. The deposition angle was determined with aprecision of ∼1◦. As substrates, gold-coated, AT-cut quartz crys-tals with a diameter of 14 mm (Q-Sense AB, Gothenburg, Sweden,model QSX 301) and a root-mean-square (RMS) roughness around1–2 nm was used.

Consistent with previous studies [26,27] we find that theRMS-roughness of the grown platinum films increases when thedeposition angle is decreased. By choosing the depositing anglesof 35◦ and 10◦, substrates with two different surface roughnessvalues were synthesised, in the following denoted surface F andsurface R (for flat and rough), respectively. The morphology of thefilms was analysed with atomic force microscopy (AFM) using acommercial Nanoscope IIIA Multimode SPM (Veeco instruments,Santa Barbara, CA). AFM images were acquired in tapping mode atscan frequencies 1–2 Hz under ambient conditions applying a sil-icon cantilever (NSG01, NT-MDT, Russia) with a typical resonancefrequency around 150 kHz, a spring constant of 5.5 N/m and a tipradius below 10 nm. The AFM images, all 512 × 512 pixels, werequadratic with linear dimensions of 1 �m and 5.5 �m. To deter-mine the RMS-roughness of the Pt films and the roughness factorR, defined as the ratio between the surface area of the rough sub-strates and the surface area of a completely flat surface, the AFMimages were analysed using a home-written plug-in to the imageanalysis software package, Scanning Probe Image Processor (SPIP)[28]. Further details about the film growth and roughness analysishave been published previously [26,27].

2.3. Quartz crystal microbalance with dissipation (QCM-D)

The QCM-D (quartz crystal microbalance with dissipation) tech-nique relies on a sensor consisting of a piezoelectric quartz crystaloscillating in a shear mode [29]. An increase in adsorbed mass onthe QCM sensor surface will in most cases induce a decrease inresonance frequency and visa versa [30]. In the case of a thin non-dissipative adsorbed layer with no-slip condition, the adsorbedmass, �m is directly proportional to the frequency shift, �f,through the simple Sauerbrey equation [31]:

�m = −C�f

n(1)

where the mass sensitivity constant is denoted C (17.7 ng/cm2 Hz−1

for a 5 MHz crystal), and n (1, 3, 5. . .) is the overtone number. The 3rdovertone (15 MHz) was chosen whenever the Sauerbrey equationwas employed in the data analysis. In addition to the frequencyshift, the dissipation factor, D, was also monitored, which is definedas

D = Elost

2�Estored, (2)

where Elost is the energy dissipated during each oscillation cycle,and Estored is the total energy of the system. For Eq. (1) to be validthe dissipation value �D is required to be low, which is the casein this study. The advantage of the simple Sauerbrey equation Eq.(1) is that the adsorbed mass is obtained directly from the mea-sured frequency shift without involving any complex modelling.All crystals applied in the QCM-D studies were ozone-cleaned for30 min before use, to remove any hydrocarbon contamination layerand each experiment was performed at 37 ◦C under static (no flow)conditions, and repeated 3–5 times to assure good statistics.

d Surfaces B: Biointerfaces 66 (2008) 53–59 55

atinum surfaces (F and R) used in the QCM-D experiments. (A) 35◦ deposition, surface F.

A. Dolatshahi-Pirouz et al. / Colloids an

Fig. 1. Representative atomic force microscopy (AFM) images of the nano-rough pl(B) 10◦ deposition, surface R.

3. Results and discussion

3.1. AFM analysis of the surface morphologies

In Fig. 1, representative AFM images of the platinum-coatedcrystal sensor surfaces (F and R) are depicted. For both surfacesmorphologies nano-rough surface features of varying size in thenanoscale regime are observed. The respective surface areas and theroot-mean-square value, the RMS-value, are presented in Table 1.From Table 1 it is seen that the surface roughness and sur-face area increase as the depositing angle decreases, as expected[26,27]. The surface roughness for a deposition angle of 10◦ was4.62 ± 0.03 nm, decreasing to 1.49 ± 0.02 nm when the depositionangle was increased to 35◦ and the corresponding increase in sur-face area when comparing the F and the B surfaces corresponds to13%. Since an RMS-value at 1.49 ± 0.02 nm is a very low value forthe surface roughness and close to the roughness of the uncoatedsensor surface, surface F was used as a flat reference. Moreover

the AFM line scans, see Fig. 2, show that the characteristic size ofthe nano-rough surface features, like the peak-to-valley distanceand the size of the individual surface features on surface F is muchsmaller as compared with the size of a BSA molecule, while the sur-face features on surface R have a size that is comparable with thedimensions of a BSA molecule.

3.2. The QCM-D results for BSA adsorption

QCM-D is a well-suited technique for monitoring the time-resolved adsorption kinetics of proteins onto a surface, and it haspreviously with success been used to measure protein adsorptionon rough surfaces, avoiding some of the difficulties that tradi-tional optical methods such as ellipsometry and surface plasmonresonance (SPR) present [16,18]. However, one major challenge con-cerning the QCM-D technique is that the shift in frequency is dueto all mass coupled to the surface, including hydrated proteins andwater trapped in the pores of the protein film [32,33]. In Fig. 3, therecorded change in the resonance frequency, �f, and in the dis-sipation, �D, is shown when BSA at a concentration of 1 mg/ml is

Table 1The root-mean-square roughness value (RMS-value) and the surface area relative toa completely flat surface (roughness factor R = Asurface/Aflat surface)

Substrate R RMS-value (nm)

F (flat) 1.03 ± 0.004 1.49 ± 0.02R (rough) 1.16 ± 0.03 4.62 ± 0.03

Fig. 2. Line scans of the different substrates with the BSA molecule drawn as anoblate ellipsoid with dimensions of 14 nm × 4 nm in accordance with Ref. [24]. Theheight curves were obtained by AFM measurements and presented herein as anexcision of a height curve where the x and y scales are identical.

adsorbed onto the “flat” platinum surface (Surface F) followed by aninjection of polyclonal antibodies. The initial baseline correspondsto the situation with only pure buffer in the adsorption chamber,prior to the inlet of protein solution. An immediate decrease infrequency along with an increase in dissipation is observed bothwhen BSA is injected, and likewise when a subsequent polyclonal

Fig. 3. The shift in dissipation, �D, and frequency, �f, when 1 mg/ml BSA adsorbs ona flat surface (surface F) from a Tris solution, and a subsequent polyclonal anti-BSAinjection is made.

56 A. Dolatshahi-Pirouz et al. / Colloids and Surfaces B: Biointerfaces 66 (2008) 53–59

increase in the frequency shift on the rough surface compared to the

Fig. 4. (A) The scaling of the frequency shift, �f, for the overtones n = 3, 5, 7 ofsurfaces F and R at a high concentration. (B) The scaling of the frequency shift, �ffor the overtones n = 3, 5, 7 of surfaces F and R at a low concentration.

anti-BSA injection is made. A test for non-specific polyclonal anti-BSA binding showed a non-specific adsorption ranging from 9% to35% of the surface mass uptake of anti-BSA. No clear trend wasobserved between non-specific binding and surface type/proteinconcentration (data not shown). Typical frequency shifts for thescaled overtones n = 3, 5, and 7 are shown in Fig. 4 for surfaces F andR, and the two concentrations (50 �g/ml and 1 mg/ml). It is immedi-

ately observed that the scaled overtones are almost superimposedfor both surfaces and at both concentrations. This superimpositionis also reflected in the low dissipation shifts and dissipation shiftper frequency shift (Fig. 5), indicating that the Sauerbrey equa-tion, Eq. (1), is an appropriate approximation to employ for theconversion of frequency shifts to adsorbed surface mass densities.The converted frequency shifts using the 3rd overtone are shownin Table 2 for both surfaces, F and R, at high and low concentra-tion. A clear increase in mass uptake is evident when the surfaceroughness increases (F → R). The mass uptake increase is 51 ± 15%at high concentration (from 453 ng/cm2 to 685 ng/cm2) similar tothe 46 ± 13% (from 340 ng/cm2 to 496 ng/cm2) found at low con-centration. To be able to differentiate clearly between the influencefrom the surface morphology and the increase in surface area onthe protein adsorption, the average saturation surface mass den-sity on the different surfaces is normalized by the roughness factorR (see Table 2). It is observed that the increased adsorption doesnot only reflect an increase in surface area, since the normalizedsurface mass densities increase (F → R) by 34 ± 18% and 29 ± 14% athigh and low concentration, respectively. Moreover, the enhance-

Table 2Results from the protein adsorption at high (1 mg/ml) and low concentration (50 �g/mmorphologies

Concentration 1 mg/ml

Substrate R Protein (ng/cm2) Protein (ng/cm2)/roughness factor R

Antibod

Surface F 1.03 ± 0.004 453 ± 28 440 ± 27 0.97 ±Surface R 1.16 ± 0.03 685 ± 21 591 ± 35 0.39 ±

Fig. 5. (A) The dissipation shift �D for surface F (flat) and R (rough) at high (1 mg/ml)and low (50 �g/ml) concentrations. (B) The dissipation shift per frequency shift�D/�f for surfaces F and R at low and high concentrations.

ment in protein uptake on the rough surface R relative to the “flat”surface F is similar for the two chosen concentrations. Several fac-tors, when excluding the change in surface area, may explain the

flat surface: (i) The resolution of the AFM technique is limited dueto the finite radius of the cantilever tip, and this might underesti-mate the measured surface area. However, although it is importantto recognize this effect, it is highly unlikely that it can account for adifference in 34% in the normalized surface mass uptakes. (ii) TheQCM-D technique measures both the amount of adsorbed proteinsand the water coupled to the protein film. The enhanced proteinadsorption (increased frequency shift) on the rough surface R couldtherefore be ascribed to additional water coupled to the protein filmon surface R. (iii) The differences in detected protein surface massuptake between surfaces F and R could be due to a more denselypacked protein layer induced by the nano-rough surface morphol-ogy of surface R. This scenario is rather plausible since BSA is a softprotein, which is known to undergo conformational changes afterit adsorbs on a surface [34–36] with each adsorption state havinga different contact area (footprint) with the surface [37]. (iv) Bycombining scenario (ii) and (iii) the non-trivial adsorption on sur-face R could also be explained, since conformational changes in theprotein structure arising from the surface roughness upon surfacebinding might lower the hydration level in the protein film.

l) including antibody-protein binding on the two different nano-rough surface

50 �g/ml

y/protein Protein (ng/cm2) Protein (ng/cm2)/roughness factor R

Antibody/protein

0.17 340 ± 14 330 ± 10 1.21 ± 0.050.03 496 ± 23 427 ± 34 0.82 ± 0.07

d Surf

A. Dolatshahi-Pirouz et al. / Colloids an

To obtain further insight into the adsorption of BSA on therespective substrates, the dissipation shift per frequency shift�D/�f-values are explored in detail. It has previously been shownthat the dissipation �D and the endpoint �D/�f value increases foran increasing water content in the protein film and decreases as theprotein film becomes stiffer [10,38–40]. From the results depictedin Fig. 5A and B we observe that the endpoint �D/�f values forboth protein concentrations are significantly lower for the rough

surface R as compared to the flat surface F. This could imply thatthe proteins on surface R constitute a stiffer protein layer, possi-bly with a lower water content which would disfavour scenario (ii)discussed above. The results displayed in Fig. 5 on the other handis in favour for scenario (iv), since the endpoint �D/�f value arelowest for surface R in accordance with structural changes in theBSA molecule upon surface binding leading to a more dense pro-tein layer on surface R and perhaps even a decrease in the watercontent in the protein film, facilitated by the structural changes inthe BSA molecule. Furthermore, it has earlier been demonstratedwith the QCM-D technique, by controlling the packing density offerritin molecules with the salt concentration of the buffer that amore densely packed protein layer leads to a lower endpoint �D/�fvalue [8].

To examine further how well scenario (iv) correlates with ourQCM-D data we shift to the �D vs. �f plot at high and low BSAconcentration presented in Fig. 6A and B, respectively. In particu-lar we examine if any changes in the slope in the �D vs. �f plotoccur during the adsorption process, since such changes typicallyindicate that the proteins binding to the surface undergo structuralrearrangements during the adsorption process [40,41]. From Fig. 6A

Fig. 6. (A) �D/�f plot for the adsorption at high concentration onto surfaces F (flat)and R (rough). (B) �D/�f plot for the adsorption at low concentration onto surfacesF and R. The adsorption starts at 0 Hz, and the arrows indicate the direction of theadsorption and the different adsorption phases.

aces B: Biointerfaces 66 (2008) 53–59 57

and B it is seen that surface F displays a simple behaviour witha linear �D − �f relation both for high and low BSA concentra-tion. For surface R a more complex behaviour is seen, where theslope (�D/�f) changes as a function of frequency shift during theadsorption Especially in Fig. 6B, which represents the �D vs. �fplot at low BSA concentration, a markedly different behaviour onsurface R is observed consisting of two parts: an initial part exhibit-ing very low dissipation as compared to surface F, and a secondpart with an increasing �D/�f value. This indicates that the aver-age structural conformation of the protein layer is altered duringthe adsorption process leading to, in average, a less compact andsofter adsorbed protein film. It is furthermore interesting to note,that the slope of the last part of the curve in Fig. 6B for the sub-strate R is very similar to substrate F, which may indicate, that thenano-roughness influences the initial adsorption significantly, butis less important in the later stages of the adsorption process at lowconcentrations.

For high concentration the �D vs. �f plot for the R substrate isalmost linear—with a tendency to level off at high coverage. Similarobservations have previously been observed [5,10,40,41] and havebeen attributed to structural rearrangements in the protein filmresulting in stiffer protein films.

The (�D/�f) plots indicate that the route which leads to thefinal protein arrangement on substrate R is strongly influenced bythe protein concentration, since apparently different adsorptionphases dominate the overall adsorption at high and low concen-tration on surface R. Moreover, the results presented in Fig. 6A andB also support the interpretation that that the enhanced normal-ized surface mass uptake observed on the rough surface R is in factdue to a non-trivial adsorption scenario and does not result froman underestimate of the measured value of the surface area withAFM as one might initially propose.

We can thus conclude that the adsorption kinetics on the roughsurface R exhibit a more complex behaviour in comparison to the“flat” surface F and the nano-rough surface morphology on surfaceR favours changes in the structural properties of the protein layerduring the adsorption process.

3.3. Probing the protein film structure with polyclonal antibodies

To examine the resulting BSA layer in more detail, a subsequentinteraction with polyclonal BSA-antibodies was studied on all pro-tein layers. An estimate of the number of antibodies that bind to

each BSA molecule is obtained from the recorded �f values, underthe assumption that the water content in the protein layer doesnot change when antibodies are added. The number of antibodiesbound to every BSA molecule is then given as

Nantibody

NBSA= �fantibody

�fBSA

mBSA

mantibody

where mBSA = 65 kD, mantibody = 150 kD, and Nantibody and Nprotein isthe number of antibodies and proteins, respectively. From Table 2it is observed that the number of antibodies per protein moleculestrongly depends on the surface roughness both for high and lowconcentration, similar to the results reported in Ref. [19]. It has alsobeen shown that the ability of antibodies to recognize a proteinlayer correlates well with the orientation/conformation of the pro-teins in that layer [11]. It might be argued that the large differenceobserved in the number of antibodies binding per protein betweensurfaces F and R were due to conformational differences as thosereported in Ref. [11].

At low concentration the number of antibodies per protein isstill larger on surface F as compared to surface R. Furthermore, weobserve an increase in the number of antibodies per protein for

d Surf

58 A. Dolatshahi-Pirouz et al. / Colloids an

both surfaces, when going from high to low concentrations. Whenthe concentration is low, the competition between the proteins inthe search for available surface area in the initial phases of theadsorption process is reduced, leaving the proteins free to estab-lish more contact points with the surface and thus enhancing theprotein contact area with the surface [1,42]. A higher degree of pro-tein denaturation may lead to the formation of a more open proteinfilm with less steric hindrance between the antibodies and proteinson the surface, which means that there are more epitopes availablefor the antibodies to reach. This could explain the enhancementin the number of antibodies binding to the protein films that areformed on surface F and R, after going from high to low concentra-tion.

The antigen–antibody results are in accordance with the BSAsurface mass uptake results listed in Table 2 wherefrom it wasnoticed that the increase in surface area could not explain theobserved increase in the adsorbed surface mass density when com-paring the results for the flat surface F with those for the roughsurface R, which indicate that other effects are responsible forthe protein adsorption on surface R. This type of complex proteinadsorption behaviour is unlike what was observed for the adsorp-tion of BSA on both spherical surface features and substrates witha more random surface morphology [12,16–18]. The results on theother hand are similar to those published in Ref. [19] on germa-nium nano-pyramid surfaces, in which case an increased amountof BSA was adsorbed on the nano-pyramid surface, with a nearly2–3-fold increased protein adsorption for an only 7% increase in sur-face area. Furthermore, the authors of [19] noticed a large decreasein the ability of antibodies to recognize proteins on the surfaces asthe roughness was increased. These dramatic effects were hypoth-esized to be due to a more efficiently utilisation of the surfacearea on top of pyramid structures, possibly due to the curvatureof the nano-structures. These different conclusions [12,16–18] onhow BSA interacts with nano-structured substrates are most likelydue to the complicated interaction scheme between proteins andnano-rough surface morphologies, which in turn depends on thesurface chemistry and how the dimensions of the actual proteinsin question correlate with the characteristic size of the surface fea-tures of the morphological landscape. Studies on polymers haveshown that a combination of conformational changes of each poly-mer and steric hindrance between the polymers can lead to a moreefficient use of the surface area on top of spherical silica beadstructures due to the curvature of the beads when the bead sizewas in the same order as the dimensions of the polymers [19,20].

Furthermore, protein studies on free nano-particles in solutionhave also shown induced conformational changes and curvatureeffects during protein adsorption [12–14]. We therefore suggesta scenario where the kink sites present on the nano-rough sur-face features residing on surface R induces a close-packed layer ofBSA molecules caused by changes in the orientation/conformationof the molecules. Both the saturated BSA surface mass density(Table 2), the dissipation results from QCM-D (Figs. 5B and 6)and the ability of the polyclonal antibodies to recognize epitopes(Table 2) in the protein layers on the respective surfaces supportsuch an explanation.

4. Conclusion

We have investigated the influence of surface nano-roughnesson the adsorption of bovine serum albumin (BSA) at two differentconcentrations. When comparing the BSA adsorption on a flat and anano-rough surface we found an increase in the surface mass den-sity beyond that corresponding to a simple increase in surface area.After the protein uptake was normalized to the surface area theincrease in surface mass density was 34 ± 18% at the high protein

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aces B: Biointerfaces 66 (2008) 53–59

concentration and 29 ± 14% at the low concentration. The surfacemass densities obtained by the QCM-D technique indicate thatnon-trivial effects influence the protein adsorption on platinumsubstrates with nano-rough surface morphologies. In particular wewere able to successfully exploit the dissipation values monitoredwith the QCM-D apparatus to gain a more thorough understand-ing of how the BSA molecules interacted with the surface, findingthat the surface roughness lead to different prominent adsorptionphases during the adsorption process. The protein and anti-bodyadsorption and the dissipation value �D monitored by the QCM-Dtechnique all favour a scenario where the BSA molecules arrangethemselves in a more densely packed protein layer on the roughsurface R in comparison with the flat surface F.

These results are important both from a fundamental as wellas an applied perspective as we have shown that the surfacenano-roughness has a significant influence on the amount andfunctionality of BSA molecules adsorbed on the respective nano-rough surfaces.

Acknowledgement

We gratefully acknowledge the financial support from the Dan-ish Research Councils to the Centre for NeuroEngineering (CNE)and Interdisciplinary Nanoscience Center (iNANO) and from theEuropean Commission to the FP6 STREP project: NANOCUES. Theauthors would also like to thank Folmer Lyckegaard for the produc-tion of the thin films used in this work.

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