Effect of Surface Wettability on Ion-Specific Protein Adsorption

Sep 19, 2012 - by the Sauerbrey equation(34) (1)where f0 is the fundamental .... (44) In other words, the adsorption of BSA at pH 7.4 is governed by t...
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Effect of Surface Wettability on Ion-Specific Protein Adsorption Xiaowen Wang,† Guangming Liu,*,† and Guangzhao Zhang*,‡ †

Department of Chemical Physics, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, P. R. China 230026 ‡ Faculty of Materials Science and Engineering, South China University of Technology, Guangzhou, P. R. China 510640 S Supporting Information *

ABSTRACT: We have systematically investigated the effect of surface wettability on ion-specific adsorption of bovine serum albumin (BSA) by using quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR). The changes in frequency (Δf) and resonance unit (ΔRU) show a nonmonotonous change of the adsorbed amount of BSA as a function of molar fraction of 1-dodecanethiol (xDDT) of the self-assembled monolayer at pH 3.8, while the amount of adsorbed protein gradually increases with the xDDT at pH 7.4. The small changes of dissipation (ΔD) indicate that BSA molecules form a quite rigid protein layer on the surfaces, which results in only a slight difference in the adsorbed mass between the mass-uptake estimations from the Sauerbrey equation and the Voigt model. The difference in the adsorbed mass between QCM-D and SPR measurements is attributed to the coupled water in the protein layer. On the other hand, specific anion effect is observed in the BSA adsorption at pH 3.8 with the exception of the surface at xDDT of 0%, but no obvious cation specificity can be observed at pH 7.4. The ΔD−Δf plots show that the BSA adsorption at pH 3.8 has two distinct kinetic processes. The first one dominated by the protein−surface interactions is an anionnonspecific process, whereas the second one dominated by the protein structural rearrangements is an anion-specific process. At pH 7.4, the second kinetic process can only be observed at the relatively hydrophobic surfaces, and no cation specificity is observed in the first and second kinetic processes.

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proteins; for example, β-casein is an amphiphilic protein with chain structure, whereas HSA is a well-folded globular protein whose hydrophobic groups are not open on the surface.13,14 After adsorption, HSA may change its conformation to expose its hydrophobic groups to increase the hydrophobic interaction with the surface, thereby leading to a stronger protein adsorption on a more hydrophobic surface.15 Besides, other studies also demonstrate that no obvious trend of protein adsorption can be observed with the surface wettability because the protein adsorption usually involves multipoint electrostatic, hydrophobic, and hydrogen-bonding interactions between protein molecules and surfaces.7 On the other hand, a few studies have been performed on the ion-specific protein adsorption at solid surfaces12,16−22 due to the potential applications in column chromatography23 and marine antifouling systems.24 In general, the ions would follow the so-called Hofmeister series,25 and they are usually categorized as chaotropes or kosmotropes based on their perceived influence on water structure.26,27 The chaotropes have weak interactions with water molecules, whereas the kosmotropes are strongly hydrated by water molecules.28 At a hydrophilic surface, the measurements of protein adsorption

INTRODUCTION Protein adsorption at solid/water interfaces has attracted extensive attention due to its importance in many natural and industrial processes.1−4 However, there are still some contradictory opinions in the understanding of protein adsorption in terms of the structural rearrangements, cooperative adsorption, adsorption kinetics, and protein aggregation on the surfaces.5 No matter in order to inhibit or to enhance the protein adsorption, understanding the mechanism of protein adsorption at solid surfaces under various conditions is a prerequisite to control such adsorptions. Generally, the adsorption of proteins is influenced not only by the surface properties but also by the solution conditions since the driven forces may arise from electrostatic interactions, hydrophobic interactions, and hydrogen-bonding interactions.6 It is reported that surface wettability has a significant effect on the protein adsorption, though no agreement has been reached.7−9 Whitesides et al. reveal that the adsorbed amount of proteins on the hydrophobic surfaces is higher than that on the hydrophilic surfaces,10 which is consistent with the result for the adsorption of human serum albumin (HSA) at the solid surfaces.11 In contrast, the surface excess on the hydrophilic surface is higher than that on the hydrophobic surface when βcasein is adsorbed at the solid/aqueous interface.12 The difference in protein adsorption between HSA and β-casein might be attributed to the different structures of these two © 2012 American Chemical Society

Received: July 25, 2012 Revised: September 13, 2012 Published: September 19, 2012 14642

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fundamental resonant frequency ( f 0) when a RF voltage is applied across the electrodes near the resonant frequency. The addition of a small layer to the electrodes would induce a decrease in resonant frequency (Δf) which is proportional to the mass change (Δm) of the layer. In vacuum or air, for a rigidly adsorbed layer which is evenly distributed and much thinner than the crystal, Δf is related to Δm and the overtone number (n = 1, 3, 5....) by the Sauerbrey equation34

show that the amount of adsorbed protein decreases from kosmotropic to chaotropic ions.22 This result is explained by the concept that water is more structured in the presence of kosmotropes, which acts as a driving force, moving the protein to the surface, whereas the adjacent water of chaotropes has less structuring than pure water and the driving force for protein adsorption is less.22 However, there is no single Hofmeister effect on the protein adsorption that ranges from kosmotropic to chaotropic ions at a hydrophobic surface.18 More specifically, kosmotropic ions reduce the protein adsorption by disfavoring a conformational adaptation and a dehydration of the protein molecules at the surface, while chaotropic ions shield hydrophobic interactions between the protein and the surface by saturating hydrophobic patches on the protein surface.18 Clearly, the surface wettability would significantly influence the ion-specific protein adsorption, though the exact mechanism still remains unclear. Actually, the ion-specific protein adsorption is expected to be determined by the combined effects between the protein− surface, lateral protein−protein, and protein−ion interactions. Because the protein−surface interactions are dependent on the hydrophobic/hydrophilic properties of the solid surfaces,10,29 the ion specificity in the protein adsorption would be related to the surface wettability. However, no systematic study on the effect of surface wettability on the ion-specific protein adsorption has been conducted to date. In the present work, we have systematically investigated the adsorption of bovine serum albumin (BSA) at the solid surfaces with different wettabilities in the presence of different ions by using quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR). We are interested in how the ionspecific protein adsorption at the solid surfaces is influenced by the surface wettability.

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Δm = −

ρq lq Δf f0 n

= −C

Δf n

(1)

where f 0 is the fundamental frequency and ρq and lq are the specific density and thickness of the quartz crystal, respectively. The dissipation factor is defined by30 D=

Ed 2πEs

(2)

where Ed is the energy dissipated during one oscillation and Es is the energy stored in the oscillating system. The measurement of ΔD is based on the fact that the voltage over the crystal decays exponentially as a damped sinusoidal when the driving power of a piezoelectric oscillator is switched off.30 By switching the driving voltage on and off periodically, we can simultaneously obtain a series of changes of the resonant frequency and the dissipation factor. Δf and ΔD values from the fundamental were discarded because they were usually noise due to insufficient energy trapping.35 In the present study, all the changes of Δf and ΔD were obtained from the measurements at the third overtone (n = 3), and all the experiments were conducted at ∼25 °C. The typical protein adsorption isotherm measured by QCM-D can be found in the Supporting Information (Figure S1). The Sauerbrey mass (ΔmS) of the adsorbed BSA was calculated based on the value of Δf after the resonator surface was saturated by the protein molecules and was rinsed with PB solution. It is known that the Sauerbrey equation might not be valid for the viscoelastic protein layer.36 Assuming that the adsorbed protein layer is homogeneous and is surrounded by a semiinfinite Newtonian fluid under a no-slip condition, the hydrodynamic thickness (dh) and the corresponding hydrodynamic mass (ΔmV) can be obtained by fitting the changes of Δf and ΔD at different overtones based on the Voigt model using a Q-tools software from Q-sense AB.31 Surface Plasmon Resonance (SPR). In the present study, SPR measurements were carried out on a Biacore X (Biacore AB).37−39 The gold-coated sensor chip is attached to a glass prism with a silicone opto-interface between the sensor chip and the prism to ensure good matching of their refractive indices. Light from a near-infrared lightemitting diode (λ = 760 nm, p-polarized) is focused through the prism onto the sensor chip surface in a wedge-shaped beam to give a fixed range of incident light angles. Light reflected from the sensor chip is monitored by a linear array of light-sensitive diodes covering the range of incident light angles. The SPR angle (θSPR) is defined as the angle of minimum intensity of the reflected light beam. The response of θSPR is measured in resonance units (RU), where 1000 RU corresponds to an angle change of ∼0.1°. The typical protein adsorption isotherm measured by SPR can be found in the Supporting Information (Figure S2). If the gold-coated chip surface is coated with a protein layer whose thickness is less than the effective penetration depth, the change of RU can be converted to the adsorbed mass.37,40 At an alkanethiol self-assembled monolayer covered chip surface, the change of 1000 RU corresponds to the adsorption of ∼0.65 ng/mm2 of BSA.40 Thus, the SPR mass (ΔmSPR) for the adsorbed protein could be calculated from the value of ΔRU after the chip surface was saturated by the protein molecules and was rinsed with PB solution. The signal drift of SPR during the protein adsorption is typically within ∼40 RU. Here, all the experiments were performed at ∼25 °C. Preparation and Characterization of Self-Assembled Monolayers. Before preparing the self-assembled monolayers (SAMs) on the surfaces, the QCM-D and SPR sensor chips were cleaned in a piranha solution at ∼55 °C for ∼10 min and then ultrasonically cleaned in water and dried with nitrogen. The SAMs were prepared by immersing the sensor chips in the mixtures of HUT and DDT with

EXPERIMENTAL SECTION

Materials. BSA (Mw ∼ 68 kDa, pI ∼ 4.8) was purchased from Hualvyuan Biotechnology Co. and used as received. The phosphate buffer (PB) solutions (40 mM) with different pH values were prepared using phosphoric acid (H3PO4), potassium phosphate (KH2PO4), and sodium phosphate dibasic (Na2HPO4·12H2O). Sodium chloride (NaCl), sodium bromide (NaBr), sodium nitrate (NaNO3), potassium chloride (KCl), and cesium chloride (CsCl) were all analytical reagent (AR) grade (Sinopharm Chemical Reagent Co.) and used as received. Tetrahydrofuran (THF) was refluxed in the presence of sodium wire and distilled prior to use. 11-Hydroxyundecane-1-thiol (HUT, SigmaAldrich) and 1-dodecanethiol (DDT, Sinopharm) were used as received without further purification. The water used was purified by filtration through the Millipore gradient system after distillation, giving a resistivity of 18.2 MΩ·cm. When studying anion-specific protein adsorption, we employed sodium salts so that the influence of cations was constant; similarly, chloride salts were used when investigating cation-specific protein adsorption. In the present work, the concentration of BSA used in the protein adsorption was fixed at 1.0 mg/mL and the salt concentration was fixed at 0.2 M. Quartz Crystal Microbalance with Dissipation (QCM-D). QCM-D measurements were conducted on a Q-sense E1.30 The quartz crystal (AT-cut) with a fundamental resonant frequency of 5 MHz and a diameter of ∼14 mm was mounted in a fluid cell with one side exposed to the solution.30,31 The resonator had a mass sensitivity constant (C) of ∼17.7 ng cm−2 Hz−1.32 The uncertainty for the QCMD experiments mainly comes from the instrumental drifts which are typically ∼2 Hz and ∼1 × 10−7 for the frequency and dissipation, respectively, during the protein adsorption. The effects of surface roughness were neglected because the resonator surfaces were highly polished with a root-mean-square (rms) roughness less than 3 nm.33 A quartz crystal is excited to oscillate in the thickness shear mode at its 14643

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Figure 1. Characterization of the solid surfaces covered with the self-assembled monolayers. (a) XPS O1s spectra of the mixed monolayers on the gold surfaces as a function of the molar fraction of 11-hydroxyundecane-1-thiol (xHUT) in the THF solution. Inset: relationship between the xHUT in the THF solution and the xHUT on the surface calculated from the XPS spectra. (b) Relationship between the water contact angle on the surface and the molar fraction of 1-dodecanethiol (xDDT) in the THF solution. different percentages in THF at a concentration of 1.0 mM for ∼18 h.41 After the preparation of SAMs, the surfaces were in turn washed with THF and water and then blown dry with a stream of nitrogen. The X-ray photoelectron spectroscopy (XPS) measurements were conducted on an ESCALAB-250 spectrometer using a monochromatic Al Kα (1486.6 eV) as radiation source. The composition of SAMs was calculated using the O1s peak intensity of each mixed monolayer normalized to the O1s peak intensity of the monolayer prepared from the pure HUT solution.11 The contact angles formed by water droplet (∼4 μL) on the monolayer surfaces were measured using a KSV (Helsinki, Finland) CAM 200 contact angle goniometer to characterize the surface wettability.

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RESULTS AND DISCUSSION Surface Wettability of SAMs. It is reported that a gradual variation of surface wettability from hydrophilic to hydrophobic

Figure 3. Changes in frequency (−Δf), dissipation (ΔD), and Sauerbrey mass (ΔmS) as a function of xDDT for the adsorption of BSA at pH 3.8 in the presence of different anions.

character can be achieved by modifying the gold surface using the mixtures of methyl- and hydroxyl-terminated alkanethiols in different relative concentrations in THF.41 In Figure 1a, the intensity of O1s peak of the mixed SAMs increases with the molar fraction of HUT (xHUT) in THF solution, indicating that the xHUT of the mixed monolayer on the surface increases with the xHUT in the solution. The inset in Figure 1a shows that the xHUT on the surface linearly depends on the xHUT in the solution; that is, the surface composition of the mixed SAMs can be well controlled via tuning the ratio of HUT to DDT in the THF solution. Figure 1b shows that the contact angle of water droplet on the surface linearly increases from ∼26° to ∼109° as the molar fraction of DDT (xDDT) in the solution increases from 0% to 100%, implying that the surface wettability from hydrophilic to hydrophobic character can be

Figure 2. Changes in resonance unit (ΔRU) and SPR mass (ΔmSPR) as a function of xDDT for the adsorption of BSA at pH 3.8 and 7.4 in the presence of different ions: (a) for the anions at pH 3.8; (b) for the cations at pH 7.4. 14644

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performed under pH 3.8 or 7.4 in the present work; thus, the hydroxyl group at the end of HUT will keep an uncharged state during the protein adsorption because its pKa is ∼13.7 Nevertheless, the electrostatic dipole−charge interactions between the hydroxyl groups of HUT and the protein molecules may also occur when HUT is introduced to the monolayer. Besides, previous streaming potential measurements showed that the inert hydrophobic surfaces may carry a negative charge.42 Hence, the hydrophobic surfaces prepared in the present study may have a negative charge and electrostatically interact with the protein molecules. However, the electrostatic interactions mentioned above may not significantly influence the BSA adsorption because the Debye length is reduced to less than 0.7 nm at the electrolyte concentration of 0.2 M and the electrostatic interactions would be largely screened. SPR Studies on BSA Adsorption. It is well-known that the shift of RU is sensitive to the change in the refractive index of the protein layer on the surface; thus, the “dry” mass of the adsorbed BSA can be determined using SPR.43 That is, the coupled water molecules are not included in the mass determination by this technique. Figure 2 shows the changes in ΔRU and ΔmSPR as a function of xDDT for the adsorption of BSA at pH 3.8 and 7.4 in the presence of different ions. Note that the error bars plotted in this figure and in the following figures are based on the experimental uncertainty of SPR or QCM-D measurements. At pH 3.8, BSA is a positively charged protein due to the protonation of amino groups, and the protonation of carboxylate groups would strengthen their capability to form hydrogen bonds.44 Furthermore, BSA is in its F-form with a partially unfolded conformation at pH 3.8.45,46 This may lead BSA to expose more hydrophobic residues, thereby strengthening the hydrophobic interactions between BSA and the surface.47 ΔRU and ΔmSPR for the anions increase as xDDT increases from 0% to 50%, and then they decrease with the further increase of xDDT from 50% to 100% (Figure 2a). Namely, the strongest protein adsorption occurs at the surface with the xDDT of 50%. Here, the protein adsorption is driven by the hydrophobic and hydrogen-bonding interactions. At the pure HUT surface, it is expected that the adsorption of BSA is mainly driven by the hydrogen-bonding interactions between the hydroxyl groups at the end of HUT and the carboxylic acid groups on the protein surface. When xDDT increases to 25%, both the hydrophobic and the hydrogen-bonding interactions would contribute to the adsorption of BSA, giving rise to larger values of ΔRU and ΔmSPR compared with that for the pure HUT surface. This result indicates that the combined effect between the hydrophobic and the hydrogen-bonding interactions would enhance the BSA adsorption, and such a combined effect leads to the maximum adsorption of BSA at the xDDT of 50%. The combined effect is gradually weakened with the further increase of xDDT, as indicated by the fact that ΔRU and ΔmSPR gradually decrease with the increasing xDDT from 50% to 100%. On the other hand, no obvious anion specificity can be observed at the hydrophilic HUT surface; for example, Cl−, Br−, and NO3− have similar values of ΔRU and ΔmSPR on this surface. However, the specific anion effect is observed on other surfaces when DDT is introduced to the SAMs. Specifically, ΔRU and ΔmSPR decrease following the order NO3− > Br− > Cl− for the same xDDT in the range of xDDT between 25% and 100%. Previous study demonstrated that specific ion effect would follow the direct or inverse Hofmeister series in protein

Figure 4. Changes in frequency (−Δf), dissipation (ΔD), and Sauerbrey mass (ΔmS) as a function of xDDT for the adsorption of BSA at pH 7.4 in the presence of different cations.

Figure 5. Change in the adsorbed mass of BSA (ΔmV) estimated based on the Voigt model as a function of xDDT at pH 3.8 and 7.4 in the presence of different ions: (a) for the anions at pH 3.8; (b) for the cations at pH 7.4.

well controlled by the composition of mixed alkanethiols in the THF solution. Note that all the protein adsorptions were 14645

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Figure 6. Comparison between the changes in the Sauerbrey mass (ΔmS), the hydrodynamic mass (ΔmV), and the SPR mass (ΔmSPR) for the adsorption of BSA at pH 3.8 and 7.4 as a function of xDDT in the presence of different ions: (a) for the anions at pH 3.8; (b) for the cations at pH 7.4.

and the surfaces. Consequently, ΔRU and ΔmSPR increase with the xDDT because a more hydrophobic surface would exhibit a stronger hydrophobic interaction. Here, the fact that the pure HUT and DDT surfaces have the weakest and strongest protein adsorption, respectively, further indicates that the electrostatically attractive dipole-negative charge interaction and the electrostatically repulsive hydrophobic surface-negative charge interaction may not significantly influence the protein adsorption. Furthermore, no obvious cation specificity can be observed in the changes of ΔRU and ΔmSPR, which is consistent with the general knowledge that cations usually have weaker Hofmeister effect than anions.53 QCM-D Studies on BSA Adsorption. Figure 3 shows the changes in −Δf, ΔD, and ΔmS as a function of xDDT for the adsorption of BSA at pH 3.8 in the presence of different anions. Obviously, there is no monotonous trend in the changes of −Δf and ΔmS with the xDDT. For Cl−, Br−, and NO3−, −Δf and ΔmS respectively locate around 25 Hz and 1.5 ng/mm2 at the pure HUT surface, and then they reach the maximum value (i.e., −Δf ∼ 75 Hz and ΔmS ∼ 4.5 ng/mm2) at the surface with the xDDT of 25%, followed by the gradual decreases of −Δf and ΔmS with the further increase of xDDT from 25% to 100%. Here, the strongest protein adsorption occurs at the surface with the xDDT of 25%, which is different from that observed in the SPR measurements. However, such a discrepancy between QCM-D and SPR measurements is understandable because the massuptake estimation by the former technique includes both the adsorbed protein and the coupled water molecules, and the latter technique only senses the mass change of the adsorbed protein. Therefore, the appearance of the strongest adsorption of BSA at different xDDT between QCM-D and SPR measurements is probably due to the difference in the relative water content (RWC) of the adsorbed protein layer at different surfaces. For example, the RWC of the adsorbed BSA layer at

systems depending on the solution pH.48 When solution pH is below the pI of protein, an inverse Hofmeister effect would be observed and chaotropic anions are more effective to salt out proteins than kosmotropic anions.49 It was previously reported that the positively charged amino group is weakly hydrated by water molecules.50 According to Collins’ concept of matching water affinities, the strength of interactions between amino group and anions increases following the series Cl− < Br− < NO3− because the extent of hydration of anions decreases along this series.28 That is, the effectiveness of anions to screen the positive charges on the BSA surface and to induce the saltingout of protein increases following the order Cl− < Br− < NO3−, such that the hydrophobic interactions between BSA and the monolayer surface become stronger from Cl− to NO3−. Meanwhile, the more effective screening of lateral intermolecular electrostatic repulsions between the proteins on the surface would also lead to a lager amount of adsorbed protein by an increase in the packing density.51,52 Consequently, the amount of adsorbed BSA increases following the order Cl− < Br− < NO3− for the same xDDT at the surfaces where the hydrophobic interactions are introduced. When solution pH is increased to 7.4, BSA becomes a negatively charged protein due to the deprotonation of carboxylic acid groups. Figure 2b shows that ΔRU and ΔmSPR gradually increase as the xDDT increases from 0% to 100%, suggesting that the protein adsorption becomes stronger with the gradual change from a hydrophilic to a hydrophobic surface. For the pure HUT surface, ΔRU and ΔmSPR for the cations are respectively equal to ∼85 and ∼0.06 ng/mm2. This fact implies the weak hydrogen-bonding interactions between the protein and the surface at pH 7.4 due to the reduced fraction of carboxylic acid group on the protein surface.44 In other words, the adsorption of BSA at pH 7.4 is governed by the hydrophobic interactions between the protein molecules 14646

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the anions at the hydrophilic HUT surface are attributed to the low adsorbed amount of BSA at this surface. At the surface with the xDDT of 25%, the values of ΔD are slightly lower than that for the pure HUT surface, though −Δf has higher values at this surface. This unexpected result is probably because a more rigid and dense protein layer is produced on the surface due to the stronger interactions between the protein molecules and the surface induced by the combined effect between the hydrophobic and the hydrogen-bonding interactions. As xDDT increases from 25% to 75%, ΔD increases with the xDDT, which indicates that the BSA molecules form more swollen protein layer with the increasing xDDT. However, ΔD decreases again as the xDDT increases from 75% to 100%, which is attributed to the reduced amount of adsorbed BSA on the pure DDT surface. In addition, no obvious specific anion effect is observed in the change of ΔD. Figure 4 shows the changes in −Δf, ΔD, and ΔmS as a function of xDDT for the adsorption of BSA at pH 7.4 in the presence of different cations. Obviously, the increases of −Δf and ΔmS for the cations with the increasing xDDT from 0% to 100% are due to the gradual strengthening of the hydrophobic interactions from a hydrophilic to a hydrophobic surface. Moreover, no cation specificity is observed in the changes of −Δf and ΔmS at this pH. Clearly, the results obtained in the QCM-D measurements are similar to that in the SPR measurements. At pH 7.4, ΔD increases with the xDDT for all the cations, implying that more proteins are adsorbed on the surface and dissipate more energy of the oscillation with the increase of xDDT. All the values of ΔD are less than 0.9 × 10−6, which also indicates the formation of a quite rigid protein layer on the surfaces. Interestingly, ΔD decreases along the series Cs+ > K+ ≈ Na+ for the same xDDT. This fact indicates that BSA has a similar conformation in the presence of K+ and Na+, and K+ and Na+ may not influence the protein structure significantly. The larger ΔD in the presence of Cs+ indicates a relatively swollen structure of BSA on the surfaces. It is reported that carboxylate group on the protein surface is highly hydrated.50 Therefore, according to the law of matching water affinities, the electrostatic interactions between the carboxylate group and Cs+ are expected to be weaker than K+ and Na+ because Cs+ has a relatively low extent of hydration than K+ and Na+. Therefore, the relatively swollen structure of BSA in the presence of Cs+ might be due to the weaker charge screening effect on the intramolecular electrostatic repulsions by Cs+. The specific influence of Cs+ on the interaction of BSA with the interface might also lead to a swollen structure of the protein by changing the protein conformation. No doubt, understanding the exact mechanism would need further investigations to provide more direct experimental evidence. Additionally, we have investigated the adsorption of BSA at the surfaces in the presence of different anions at pH 7.4, and no obvious specific anion effect is observed in the changes of −Δf and ΔD (data not shown). Generally, the estimation of the mass of a viscoelastic protein layer by the Sauerbrey equation would generate a deviation from the “real” mass due to two possible reasons.40 The first one is related to the coupled water molecules in the adsorbed protein layer because QCM is sensitive not only to the mass of the adsorbed protein molecules but also to the mass of the coupled water molecules which are trapped in the hydration shell of the protein or in the cavities of the protein layer.56−58 The second one is correlated with the propagation of the shear

Figure 7. Change in the relative water content (RWC) of the adsorbed BSA layer as a function of xDDT at pH 3.8 and 7.4 in the presence of different ions: (a) for the anions at pH 3.8; (b) for the cations at pH 7.4.

the xDDT of 25% is higher than that at the xDDT of 50%, leading to the strongest adsorption occurs at the xDDT of 25% in the QCM-D measurements but at the xDDT of 50% in the SPR measurements (see below). Again, no anion specificity is observed at the pure HUT surface; for example, Cl− and NO3− have similar values of −Δf and ΔmS, but Br− exhibits relatively low values of −Δf and ΔmS compared to Cl− and NO3−. In Figure 2, the SPR result shows similar amounts of adsorbed BSA in the presence of Cl−, Br−, and NO3− at the HUT surface, which is different from that observed here. The apparent result that BSA adsorption is suppressed by Br− at the HUT surface in the QCM measurement might be due to the relatively low RWC of the adsorbed BSA layer in the presence of Br− (see below for details). In the range of xDDT between 25% and 100%, −Δf and ΔmS decrease along the series NO3− > Br− > Cl− for the same xDDT, which is the same as the observation in the SPR measurements and could also be attributed to the difference in the effectiveness of charge screening by the anions. It is known that the shift in ΔD is related to the structure of protein layer on the surface.54,55 A rigid and dense protein layer would have a small dissipation factor, whereas a soft and swollen protein layer would give rise to a large one.54 Besides, for the protein molecules with a similar structure on the surface, the larger the amount of adsorbed protein is, the higher the value of ΔD would be resulted.40 In Figure 3, all the values of ΔD are less than 0.8 × 10−6, indicating the formation of a quite rigid protein layer on the surfaces. The small values of ΔD for 14647

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Figure 8. ΔD−Δf plots for the BSA adsorption at pH 3.8. (a) xDDT: 0%; (b) xDDT: 25%; (c) xDDT: 50%; (d) xDDT: 75%; (e) xDDT: 100%. (f) Changes of the slopes of the ΔD−Δf plots for the first (k1) and second (k2) kinetic processes as a function of xDDT for the anions. The solid lines with arrows are provided to guide the eye.

decreases following the order NO3− > Br− > Cl− for the same xDDT; namely, the specific anion effect is observed. At pH 7.4, ΔmV for the cations gradually increases with the increase of xDDT from 0% to 100%, and no obvious cation specificity is observed in the change of ΔmV. Clearly, all the results observed in Figure 5 are similar to that in Figures 3 and 4. Comparison between ΔmS, ΔmV, and ΔmSPR. The detailed comparison between ΔmS and ΔmV at pH 3.8 and 7.4 is shown in Figure 6. It is evident that ΔmV is similar to or slightly larger than ΔmS in all the cases, indicating that the estimation of the adsorbed mass of BSA using the Sauerbrey equation does not induce a large deviation from that evaluated by the Voigt model. This is understandable because the protein molecules form a quite rigid layer on the surfaces at both pH 3.8 and 7.4, as reflected by the small values of ΔD (Figures 3 and 4). This can be further indicated by the overtoneindependent responses of frequency and dissipation during the BSA adsorption (Figure S3 in Supporting Information). In addition, it is known that BSA is heart-shaped globular protein

wave in the viscoelastic protein film; namely, the adsorbed mass depends on how the shear wave propagates through the film as the wave energy would be damped by the viscoelastic layer.59−61 To look into how the amount of adsorbed protein estimated from the Sauerbrey equation is influenced by the viscoelastic properties of the protein layer, the so-called Voigt model is used to analyze the protein adsorption by fitting the changes of Δf and ΔD at different overtones. Figure 5 shows the amount of adsorbed BSA (ΔmV) estimated based on the Voigt model as a function of xDDT at pH 3.8 and 7.4 in the presence of different ions, where the density of the BSA layer is evaluated to be ∼1000 kg/m3.62 At pH 3.8, ΔmV for the anions increases as xDDT increases from 0% to 25%, followed by a gradual decrease of ΔmV with the further increase of xDDT from 25% to 100%. That is, the strongest protein adsorption occurs at the surface with the xDDT of 25%. At the pure HUT surface, the value of ΔmV for Cl− is similar to that for NO3− but is larger than that for Br−. In the range of xDDT between 25% and 100%, ΔmV 14648

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Figure 9. ΔD−Δf plots for the BSA adsorption at pH 7.4. (a) xDDT: 0%; (b) xDDT: 25%; (c) xDDT: 50%; (d) xDDT: 75%; (e) xDDT: 100%. (f) Changes of the slopes of the ΔD−Δf plots for the first (k1) and second (k2) kinetic processes as a function of xDDT for the cations. The solid lines with arrows are provided to guide the eye.

dehydration of BSA should be, and consequently more coupled water molecules in the protein layer would be released.65 This is why RWC has a minimum value at the xDDT of 50% for the anions. Additionally, the higher RWC at the xDDT of 25% compared with that at the xDDT of 50% may result in the appearance of the strongest adsorption of BSA at the xDDT of 25% in the QCM-D measurements (Figures 3 and 5). At the HUT surface, the lower RWC for Br− compared with Cl− and NO3− explains why the BSA adsorption is apparently suppressed by Br− at this surface in the QCM measurement (Figure 3). Globally, for the same xDDT, Cl− has a relatively high value of RWC than Br−, whereas NO3− has a relatively low value of RWC than Br−. This fact indicates that the extent of hydration of the adsorbed protein layer decreases following the order Cl− > Br− > NO3−. According to the concept of matching water affinities, the strength of anions to interact with the positively charged amino groups on the BSA surface increases along the series Cl− < Br− < NO3−.28 The stronger interactions would lead to more effective screening of the charge-dipole

that can be approximated as an equilateral triangle with sides of ∼8.0 nm and a depth of ∼3.0 nm.63,64 At both pH 3.8 and 7.4, the maximum hydrodynamic thickness (dh) of the adsorbed protein layer estimated using the Voigt model is ∼4.5 nm which roughly matches the size of BSA molecules, indicating that BSA molecules adsorb as a monolayer on the surfaces. The detailed comparison between ΔmV and ΔmSPR is also shown in Figure 6. It can be seen that ΔmSPR is much smaller than ΔmV in all the cases, indicating that the adsorbed protein layer contains large amounts of water. The RWC of the protein layer can be calculated by RWC = [(ΔmV − ΔmSPR)/ΔmV] × 100% (Figure 7). At pH 3.8, RWC for the anions decreases from ∼90% to ∼80% with the increasing xDDT from 0% to 50%, and then it increases from ∼80% to ∼90% with the further increase of xDDT from 50% to 100%. The values of RWC of the adsorbed BSA layer observed here are similar to that for other protein films.40 In Figure 2, the change of ΔmSPR shows that BSA has the strongest adsorption at the surface with the xDDT of 50%. The stronger adsorption BSA has, the larger extent of 14649

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can be seen from Figure 8f, for all the anions, k2 decreases with the increase of xDDT from 0% to 25%, and then it gradually increases with the increasing xDDT from 25% to 100%. At the pure HUT surface, BSA has the weakest adsorption compared with that at other surfaces (Figure 6), so that there is an enough space to accommodate the structural rearrangements of the protein molecules, giving rise to a high k2. At the xDDT of 25%, the protein adsorption has the largest hydrodynamic mass (i.e., protein plus coupled water), as shown in Figure 5. Therefore, only very limited space could be provided for the structural rearrangements, thereby leading to the lowest value of k2. As xDDT increases from 25% to 100%, the hydrodynamic mass of the adsorbed protein layer gradually decreases; thus, more space on the surface is available to the structural rearrangements with the xDDT. This is why k2 gradually increases with the increase of xDDT from 25% to 100%. On the other hand, for the same xDDT, k2 decreases following the order Cl− > Br− > NO3−; namely, the specific anion effect can be observed even for the hydrophilic HUT surface. This fact indicates that the adsorbed protein layer is more difficult to deform when the anion changes from Cl− to NO3−. As we know, the anions would follow an inverse Hofmeister series in the BSA system at pH 3.8, so that the ability of anions to stabilize the protein structure increases along the series Cl− < Br− < NO3−.49 Consequently, the deformation or structural rearrangement of the protein layer becomes more difficult from Cl− to NO3−, and k2 decreases following the order Cl− > Br− > NO3− for the same xDDT. Figure 9 shows the relationship between ΔD and −Δf for the adsorption of BSA at pH 7.4 on the surfaces in the presence of different cations. Previous study suggested that BSA would keep a more stable conformation at pH 7.4 than that at pH 3.8.44,68 Hence, the adsorbed BSA layer may be difficult to carry out the structural rearrangements on some surfaces. As can be seen from Figure 9, only the first kinetic process is observed at the surfaces with the xDDT of 0% and 25%, while both the first and the second kinetic processes can be observed at other surfaces with higher values of xDDT. This result implies that no obvious structural rearrangements of the protein layer occur at the surfaces when xDDT is less than 25%. At pH 7.4, the hydrophobic interactions between the protein and the surface are the main driven force for the protein adsorption. Thus, at the surfaces with the xDDT of 0% and 25%, the weak driven force might not be enough to induce the structural rearrangements during the protein adsorption. In the range of xDDT from 50% to 100%, the strong hydrophobic interactions between the BSA molecules and the surfaces would lead to a strong protein adsorption and the subsequent structural rearrangements. In Figure 9f, k1 for the cations gradually decreases with the increasing xDDT from 0% to 100%. This is because the strength of initial protein adsorption increases with the xDDT due to the increasing hydrophobic interactions, thereby forming a relatively dense and rigid protein layer with the xDDT in the first kinetic process. From Figures 2, 4, and 5, the amount of adsorbed BSA gradually increases with the increasing xDDT from 50% to 100%; thus, the free space available to the structural rearrangements gradually decreases with the xDDT. Therefore, k2 gradually decreases as xDDT increases from 50% to 100%. In addition, no obvious cation specificity is observed in the change of either k1 or k2 at pH 7.4.

interactions between the amino groups and water molecules, resulting in a weaker hydration of the protein.66 Consequently, the RWC of the protein layer for the same xDDT decreases following the order Cl− > Br− > NO3−. At pH 7.4, the values of RWC of the adsorbed protein layer for the cations are also located between ∼80% and ∼90%. As the xDDT increases from 0% to 100%, RWC gradually decreases with the exception of the cases of K+ and Cs+ at the surfaces with the xDDT of 25% and 100%. As can be seen from Figures 2, 4, and 5, the amount of adsorbed protein for the cations gradually increases with the xDDT at pH 7.4; thus, the extent of adsorption induced dehydration of the protein is expected to increase with the xDDT. Consequently, more coupled water molecules would be released from the adsorbed BSA layer at the higher xDDT, resulting in the gradual decrease of RWC with the xDDT. In addition, no obvious specific cation effect is observed in the change of RWC at pH 7.4. Kinetic Processes of BSA Adsorption. Protein adsorption usually has two distinct processes, that is, the fast binding of the protein molecules onto the surface and the subsequent structural rearrangements or conformational changes within the adsorbed layer.5 It is known that the different kinetic processes during the protein adsorption can be viewed by the ΔD−Δf plot in the QCM-D measurements.54 Figure 8 shows the relationship between ΔD and −Δf for the adsorption of BSA at pH 3.8 on the surfaces in the presence of different anions. Obviously, two distinct kinetic processes can be observed during the protein adsorption, as reflected by the two different slopes in the ΔD−Δf plots. In Figure 8f, the first kinetic process with a lower slope (k1) corresponds to the initially fast adsorption of protein molecules onto the surface and the second kinetic process with a higher slope (k2) relates to the structural rearrangements or conformational changes. The relatively large value of k2 compared to k1 indicates that the structural rearrangements upon the protein adsorption would lead to a less rigid and dense protein layer than that in the first kinetic process. This result may be due to the fact that the deformation of BSA is likely to accelerate extension of polypeptide chains with the loss of α-helical content accompanied by the formation of a more random and softer protein structure.67 The value of k1 reflects the rigidity of the protein layer formed in the first kinetic process.54 It can be seen from Figure 8f that k1 for the anions decreases as xDDT increases from 0% to 25%, and then it exhibits a slight increase with the further increase of xDDT from 25% to 100%. The relatively high values of k1 at the pure HUT surface imply the formation of a less dense and rigid protein layer due to the relatively weak interactions between the protein molecules and the surface. At the xDDT of 25%, the lower values of k1 indicate that the protein molecules would form a denser layer on the surface in the first kinetic process due to the increasing interactions generated by the combined effect between hydrogen-bonding and hydrophobic interactions. The slight increase of k1 with the increase of xDDT from 25% to 100% implies the formation of a less dense protein layer with the xDDT, which might be due to the slight decrease of the interactions between the protein molecules and the surface with the increasing xDDT in the first kinetic process. Additionally, no obvious anion specificity can be observed in the change of k1. This is because k1 is mainly determined by the surface−protein interactions instead of the anion−protein interactions. The value of k2 is an indicative of the degree of deformation or structural rearrangements of the adsorbed protein layer. As 14650

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(6) Roach, P.; Farrar, D.; Perry, C. C. Interpretation of Protein Adsorption: Surface-Induced Conformational Changes. J. Am. Chem. Soc. 2005, 127, 8168−8173. (7) Silin, V.; Weetall, H.; Vanderah, D. J. SPR Studies of the Nonspecific Adsorption Kinetics of Human IgG and BSA on Gold Surfaces Modified by Self-Assembled Monolayers (SAMs). J. Colloid Interface Sci. 1997, 185, 94−103. (8) Ostuni, E.; Yan, L.; Whitesides, G. M. The Interaction of Proteins and Cells with Self-Assembled Monolayers of Alkanethiolates on Gold and Silver. Colloids Surf., B 1999, 15, 3−30. (9) Scotchford, C. A.; Gilmore, C. P.; Cooper, E.; Leggett, G. J.; Downes, S. Protein Adsorption and Human Osteoblast-Like Cell Attachment and Growth on Alkylthiol on Gold Self-Assembled Monolayers. J. Biomed. Mater. Res. 2002, 59, 84−99. (10) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. Effect of Surface Wettability on the Adsorption of Proteins and Detergents. J. Am. Chem. Soc. 1998, 120, 3464−3473. (11) Martins, M. C. L.; Ratner, B. D.; Barbosa, M. A. Protein Adsorption on Mixtures of Hydroxyl- and Methylterminated Alkanethiols Self-Assembled Monolavers. J. Biomed Mater. Res., Part A 2003, 67A, 158−171. (12) Kull, T.; Nylander, T.; Tiberg, F.; Wahlgren, N. M. Effect of Surface Properties and Added Electrolyte on the Structure of BetaCasein Layers Adsorbed at the Solid/Aqueous Interface. Langmuir 1997, 13, 5141−5147. (13) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M. Neutron Reflectivity of Adsorbed Beta-Casein and Beta-Lactoglobulin at the Air/Water Interface. J. Chem. Soc., Faraday Trans. 1995, 91, 2847−2854. (14) He, X. M.; Carter, D. C. Atomic-Structure and Chemistry of Human Serum-Albumin. Nature 1992, 358, 209−215. (15) Baszkin, A.; Boissonnade, M. M.; Kamyshny, A.; Magdassi, S. Native and Hydrophobically Modified Human Immunoglobulin G at the Air/Water Interface - Sequential and Competitive Adsorption. J. Colloid Interface Sci. 2001, 239, 1−9. (16) Moreira, L. A.; Böstrom, M.; Ninham, B. W.; Biscaia, E. C.; Tavares, F. W. Effect of the Ion-Protein Dispersion Interactions on the Protein-Surface and Protein-Protein Interactions. J. Brazil. Chem. Soc. 2007, 18, 223−230. (17) Nylander, T.; Tiberg, F.; Su, T. J.; Lu, J. R.; Thomas, R. K. BetaCasein Adsorption at the Hydrophobized Silicon Oxide-Aqueous Solution Interface and the Effect of Added Electrolyte. Biomacromolecules 2001, 2, 278−287. (18) Evers, F.; Steitz, R.; Tolan, M.; Czeslik, C. Analysis of Hofmeister Effects on the Density Profile of Protein Adsorbates: A Neutron Reflectivity Study. J. Phys. Chem. B 2009, 113, 8462−8465. (19) Salis, A.; Bhattacharyya, M. S.; Monduzzi, M. Specific Ion Effects on Adsorption of Lysozyme on Functionalized SBA-15 Mesoporous Silica. J. Phys. Chem. B 2010, 114, 7996−8001. (20) Heath, M. D.; Henderson, B.; Perkin, S. Ion-Specific Effects on the Interaction between Fibronectin and Negatively Charged Mica Surfaces. Langmuir 2010, 26, 5304−5308. (21) Poleunis, C.; Rubio, C.; Compère, C.; Bertrand, P. Role of Salts on the BSA Adsorption on Stainless Steel in Aqueous Solutions. II. ToF-SIMS Spectral and Chemical Mapping Study. Surf. Interface Anal. 2002, 34, 55−58. (22) Wendorf, J. R.; Radke, C. J.; Blanch, H. W. The Role of Electrolytes on Protein Adsorption at a Hydrophilic Solid-Water Interface. Colloids Surf., B 2010, 75, 100−106. (23) Tsumoto, K.; Ejima, D.; Senczuk, A. M.; Kita, Y.; Arakawa, T. Effects of Salts on Protein-Surface Interactions: Applications for Column Chromatography. J. Pharm. Sci. 2007, 96, 1677−1690. (24) Poleunis, C.; Rubio, C.; Compère, C.; Bertrand, P. ToF-SIMS Chemical Mapping Study of Protein Adsorption onto Stainless Steel Surfaces Immersed in Saline Aqueous Solutions. Appl. Surf. Sci. 2003, 203, 693−697. (25) Hofmeister, F. Zur Lehre von der Wirkung der Salze Zweite Mitteilung. Arch. Exp. Pathol. Pharmakol. 1888, 24, 247−261.

CONCLUSION We have systematically studied the effect of surface wettability on the ion-specific BSA adsorption. At pH 3.8, the combined effect between the hydrophobic and hydrogen-bonding interactions leads to a nonmonotonous change in the adsorbed mass of protein with the gradual change of surface wettability. At pH 7.4, the protein adsorption is dominated by the hydrophobic interactions, and the adsorbed mass gradually increases as the surface changes from hydrophilic to hydrophobic character. Moreover, the specific anion effect is observed in the BSA adsorption at pH 3.8 when the hydrophobic interactions are introduced between the protein molecules and the surfaces; i.e., the amount of adsorbed BSA is larger in the presence of a more chaotropic anion, but no cation specificity can be observed at pH 7.4. Besides, two distinct kinetic processes are observed at pH 3.8 during the BSA adsorption. The first one dominated by the protein−surface interactions is an anion-nonspecific process, whereas the second one dominated by the protein structural rearrangements is an anion-specific process and the adsorbed protein layer is more difficult to deform on the surfaces in the presence of a more chaotropic anion. At pH 7.4, the second kinetic process can only be observed at the relatively hydrophobic surfaces, and no cation specificity is observed in the first and second kinetic processes.

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ASSOCIATED CONTENT

S Supporting Information *

Typical BSA adsorption isotherms in the QCM-D and SPR measurements and the responses of frequency and dissipation at different overtones during the BSA adsorption. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (G.M.); [email protected] (G.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of National Program on Key Basic Research Project (2012CB933800), the National Natural Science Foundation of China (21004058, 91127042, and 21234003), Scientific Research Startup Foundation of the Chinese Academy of Sciences, and the Fundamental Research Funds for the Central Universities (WK2060030008) is acknowledged.

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