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Article Cite This: Langmuir 2018, 34, 1953−1966

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Dominant Albumin−Surface Interactions under Independent Control of Surface Charge and Wettability Shanshan Guo,† Dicky Pranantyo,‡ En-Tang Kang,‡ Xian Jun Loh,∥,⊥,# Xiaoying Zhu,*,§ Dominik Jańczewski,*,¶ and Koon Gee Neoh*,†,‡ †

NUS Graduate School for Integrative Science and Engineering, National University of Singapore, Kent Ridge, 117576, Singapore Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 119260, Singapore § Department of Environmental Science, Zhejiang University, Hangzhou 310058, China ∥ Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 2 Fusionopolis Way, 138634, Singapore ⊥ Department of Materials Science and Engineering, National University of Singapore, 9 Engineering Drive 1, 117576, Singapore # Singapore Eye Research Institute, 11 Third Hospital Avenue, 168751, Singapore ¶ Laboratory of Technological Processes, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland ‡

S Supporting Information *

ABSTRACT: Understanding protein adsorption behaviors on solid surfaces constitutes an important step toward development of efficacious and biocompatible medical devices. Both surface charge and wettability have been shown to influence protein adsorption attributes, including kinetics, quantities, deformation, and reversibility. However, determining the dominant interaction in these surface-induced phenomena is challenging because of the complexity of inter-related mechanisms at the liquid/solid interface. Herein, we reveal the dominant interfacial forces in these essential protein adsorption attributes under the influence of a combination of surface charge and wettability, using quartz crystal microbalance with dissipation monitoring and atomic force microscopy-based force spectroscopy on a series of model surfaces. These surfaces were fabricated via layer-by-layer assembly, which allowed two-dimensional control of surface charge and wettability with minimal cross-parameter dependency. We focused on a soft globular protein, bovine serum albumin (BSA), which is prone to conformational changes during adsorption. The information obtained from the two techniques shows that both surface charge and hydrophobicity can increase the protein−surface interaction forces and the adsorbed amount. However, surface hydrophobicity triggered a greater extent of deformation in the adsorbed BSA molecules, leading to more dehydration, spreading, and resistance to elution by ionic strength changes regardless of the surface charge. The role played by the surface charge in the adsorbed protein conformation and extent of desorption induced by changes in the ionic strength is secondary to that of surface hydrophobicity. These findings advance the understanding of how surface chemistry and properties can be tailored for directing protein−substrate interactions. involving the surface, proteins, and solvent.2,9 Thus, proteins can attach to a surface in diverse quantities and conformations depending on the local conditions. Both the adsorbed quantities and conformations are reported as important factors in implant biocompatibility.10 Surface wettability (or hydrophobicity), among the various surface properties, has been the subject of many studies. A number of proteins, including lysozyme3,11 and bovine serum

1. INTRODUCTION Protein adsorption at aqueous−solid interfaces plays an important role in various areas of contemporary technology such as biosensors,1 medical implants,2−4 or protein purification.5 For example, preadsorbed proteins on implant surfaces mediate subsequent cell adhesion and tissue integration and thus affect healing. Protein adsorption is a complex process involving hydrophobic, electrostatic, and van der Waals interactions as well as hydrogen bonding.6 In addition, the initial attachment is commonly followed by conformational rearrangement (e.g., unfolding and spreading) of proteins7,8 because of changes in the thermodynamic state of the system © 2018 American Chemical Society

Received: December 1, 2017 Revised: January 4, 2018 Published: January 10, 2018 1953

DOI: 10.1021/acs.langmuir.7b04117 Langmuir 2018, 34, 1953−1966

Article

Langmuir

Figure 1. Schematic illustration of model surfaces: (a) polyanion and polycation used in the LbL assembly; (b) four surfaces employed in this work, characterized by both pI and θ.

albumin (BSA),11 adsorbed in higher amounts and were stronger bound on hydrophobic polystyrene11 or methyl-coated silica surfaces3 compared to hydrophilic silica surfaces as a reference. Typically, proteins, for example lysozyme and αlactalbumin, also deformed faster on hydrophobic silane-coated surfaces than on hydrophilic silica surfaces.7 However, these studies used a bare silica surface, which was negatively charged at physiological pH. Thus, it is difficult to distinguish the hydrophobic interaction of proteins with the surface from that caused by electrostatic interaction. Other studies of protein adsorption as a function of surface wettability showed that protein adsorption (e.g., BSA) did not increase monotonically with surface hydrophobicity,8 likely because the model surfaces used were not well-defined in terms of other possible relevant surface parameters such as surface charge and roughness.12 Self-assembled monolayers (SAMs) have been used as welldefined model systems to study protein adsorption on surfaces. The wettability of such surfaces is controlled by the terminal functional group of the SAM, for example, polar functional group OH and nonpolar group CH3.8−10,13 A higher amount of protein (e.g., collagen13 and BSA9) was adsorbed on hydrophobic CH3 than on hydrophilic OH surfaces. These studies also showed that the degree of denaturation increased with surface hydrophobicity for proteins such as BSA,8,9,14 fibrinogen,9,14 and collagen.13 Studies covering more extensive terminal functional groups, including both ionic and nonionic chemical groups15,16 or a mixture of two terminal groups,14 demonstrated a direct correlation between the amount of protein adsorbed and surface hydrophobicity.15,16 However, when SAMs with different terminal groups are used to achieve variation of a single surface parameter, for example hydrophobicity, there is limited control over other properties. For example, substitution of alkyl for the hydroxyl group affects not only the surface hydrophobicity but also the surface zeta potential and the ability to participate in hydrogen bonding. The layer-by-layer (LbL) technique, consisting of selfassembled, sequential adsorption of oppositely charged polymers, offers opportunities to form coatings with tunable

properties such as surface wettability17 and charge2,18−22 to manipulate protein adsorption. The effect of LbL surface charge on the adsorption behavior of different types of proteins, including BSA, fibrinogen, and lysozyme has been studied. The results show that surfaces of charge opposite to that of the protein were more effective at promoting protein adsorption for different LbL systems.2,18−22 The effect of the surface charge on the secondary structure of the adsorbed protein has also been investigated. For LbL films assembled from poly(allylamine hydrochloride) and poly(styrenesulfonate), the structural changes of BSA were found to be larger when the charges of the LbL terminal layer and the protein were opposite.20 Surface wettability effects have been studied by altering the composition of the LbL films. For example, surface hydrophobicity was increased by increasing the poly(styrene sulfonate) content in LbL films prepared from poly(styrene sulfonate) and poly(acrylic acid), and the adsorption of immunoglobulin G increased with surface hydrophobicity.17 Hydrophilic surfaces fabricated from LbL films of a diblock copolymer comprising a hydrophilic poly(ethylene oxide) block have been shown to minimize protein (e.g., BSA and fibrinogen) adsorption.18 Less is known about the effect of LbL surface hydrophobicity on protein conformational aspects upon adsorption. Moreover, many studies focusing on engineering the surface wettability largely overlooked the influences on other surface parameters. For example, changes to surface wettability of LbL films assembled from poly(styrene sulfonate)/poly(acrylic acid)17 or alkylated polyethylenimine/ poly(acrylic acid)23 were accompanied by a significant change in the surface roughness. Past studies using different engineered surfaces mainly focused on altering one parameter, and much less is known about the relative and synergistic influence of surface charge and wettability on protein adsorption. Because of inter-related mechanisms involved in a multiparameter study, a twodimensional independent control over both charge and wettability is needed for minimizing cross-parameter influence. In our recent study, we developed a surface fabrication strategy 1954

DOI: 10.1021/acs.langmuir.7b04117 Langmuir 2018, 34, 1953−1966

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PBS for 3 h. BSA was chemically immobilized on the probe surface by formation of imine linkages between the primary amine groups of BSA and the aldehyde groups of glutaraldehyde. After rinsing with PBS, the BSA-coated probes were used immediately for AFM force spectroscopy measurements. Force maps of 64 force versus distance ( f−d) curves were recorded on areas of 64 μm2, with a constant vertical scan rate of 1 Hz and an approach and retraction speed of 1 μm s−1. Adhesion force histograms were obtained using ∼250 f−d curves from three or four samples. The samples were tested on three separate occasions using a freshly prepared AFM probe on each occasion. Before the force measurement, all systems were allowed to equilibrate for at least 1 h. The spring constant of the colloidal probe was measured using the thermal noise method, and the measured values were in the range of 0.08−0.12 N m−1. To obtain the f−d curves, the JPK SPM Data Processing software (v. 4.3.25) was used to process the raw AFM data, acquired in terms of a photodetector signal in volts, versus the relative piezo position (details on data processing are given in the Supporting Information). The effects of ionic strength and pH on BSA adhesion forces on the LbL surfaces were investigated at three salt concentrations (10, 150, and 510 mM) and two pH values (3.6 and 7.4). The composition of the buffer solutions used is summarized in Table 1.

that allowed us to independently adjust both surface charge and wettability using an LbL protocol24 with a similar smoothness and chemical composition. In the current work, we used these model surfaces to study protein adsorption via quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM)-based force spectroscopy. Application of these techniques allowed us to study the essential protein adsorption attributes (e.g., kinetics, quantities, deformation, and reversibility). By investigating the two relevant parameters collectively, the relative contribution of forces and the dominant interactions acting between the proteins and surfaces can be altered to provide a more complete picture of how the parameters affect protein adsorption. This will enhance our ability to predict or control protein adsorption attributes in a rational way. Our work is focused on a “soft” and globular protein, BSA, which is one of the most abundant blood proteins that potentially affects cell adhesion on surfaces.25 The effect of ionic strength and pH on the BSA−surface interaction was also investigated.

2. MATERIALS AND METHODS

Table 1. Composition of the Buffer Solutions Used in AFM Measurements

2.1. Materials. Polyethylenimine (PEI, Mw 25 kDa, branched), poly(isobutylene-alt-maleic anhydride) (PIAMAn, Mw 60 kDa), 6aminocaproic acid (Mw 131.17 Da), (3-aminopropyl)-trimethoxysilane (APTMS, 97%), and BSA (Mw 66 kDa, lyophilized powder, ≥96%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aminepoly(ethylene glycol) (PEG)-carboxylic acid (NH2-PEG-CM, Mw 2000) was purchased from Laysan Bio Inc. Ethanol and toluene were purchased from Tedia. Silicon wafers were purchased from Latech Scientific Supply Pte. Ltd. 2.2. LbL Film Preparation and Characterization. The polyanions PIAMA-C5 and PIAMA-PEG (chemical structures shown in Figure 1a) were synthesized as described previously.24 These polyanions were paired with branched PEI in the LbL assembly. Before LbL assembly, the silicon substrates were ultrasonicated in water and ethanol (10 min each) separately. After drying with nitrogen, they were subjected to oxygen plasma treatment for 3 min at 160 W. The plasma-treated samples were coated with a precursor layer of positively charged amine by immersing in toluene containing 10 mM APTMS for 3 h. The polyions were then deposited by immersing the substrates in polycation and polyanion solutions (1.0 mg mL−1) for 5 min in a cyclic manner, up to 6.5 or 7 bilayers. The deposition of 6.5 bilayers resulted in films with a polyanionic outermost layer, whereas 7 bilayers resulted in films with a polycationic outermost layer. Ultrapure water rinsing was performed between each immersion for 1 min. The pH of the polyelectrolyte solutions was adjusted by adding HCl or NaOH aqueous solution (0.1 M) and was selected from a larger library of conditions used for fabricating LbL surfaces previously.24 The detailed fabrication conditions are presented in Table S1. Surface zeta (ζ) potential was measured by an electrokinetic analyzer (SurPASS, Anton Paar). The electrolyte solution used was 0.001 M KCl. The pH of the KCl solution was controlled by auto pH titration with HCl (0.01 M). The average ζ potential value at a given pH was calculated from four repeats. Contact angles were measured using a goniometer (250-F1 from Ramé-Hart Instrument Co.) via the static sessile drop method. The average contact angle (θ) was calculated from four measurements on different locations. 2.3. AFM. Adhesion force measurements were performed using a JPK NanoWizard 3 NanoOptics AFM system in a liquid cell. Silicon dioxide colloidal probes (diameter 2.5 μm, Novascan Technologies, Inc.) were functionalized with BSA molecules according to a procedure reported previously.26 Briefly, the probes were treated with oxygen plasma at 120 W for 120 s, followed by vapor deposition of APTMS at 50 °C and curing at 90 °C to impart amine groups to the surfaces. The amine-functionalized probes were then immersed in glutaraldehyde solution (2.5%) for 2 h, rinsed with phosphate-buffered saline (PBS, pH 7.4), and immersed in BSA solutions (1 mg mL−1) in

(λD: debay length)

salt concentration buffer type ionic strength variation

pH variation

phosphate buffer (10 mM) phosphate buffer (10 mM) phosphate buffer (10 mM) acetic acid buffer (10 mM) phosphate buffer (10 mM)

NaCl (mM)

total (mM)

pH

λD (nm)

500

510

7.4

∼0.4

140

150

7.4

∼0.7

0

10

7.4

∼2

140

150

3.6

∼0.7

140

150

7.4

∼0.7

The integrity of the BSA coating on the AFM probe was assessed by comparing field emission scanning electron microscopy (FESEM) images of the probes before and after the AFM experiments (Figure S1). From a comparison of the FESEM images of an AFM colloidal probe fully coated with BSA before f−d measurements and a similar probe after ∼1000 f−d measurements (Figure S1a,b, respectively), it can be concluded that the BSA protein remained on the probe after ∼1000 f−d measurements. On the other hand, partial desorption of BSA molecules after ∼2000 f−d measurements was observed (Figure S1c). Thus, the AFM measurements were kept within ∼1000 f−d curves. In addition, the solutions and surfaces were exchanged in random order to check for any irreversible effect on the adsorbed protein layer, and the measured forces showed random variation of adhesion forces without a systematic time-dependent change. This indicates that proteins did not desorb or undergo irreversible conformational changes during our AFM measurements. 2.4. QCM-D. QCM-D was performed using a Q-Sense E4 multichannel instrument. Quartz crystal disks coated with silicon dioxide (fundamental frequency 4.95 MHz) were used as sensor chips. The sensor surface was coated with LbL films before use, according to the procedure described in section 2.2. Protein adsorption experiment was conducted at pH 7.4 in a buffer solution consisting of 10 mM phosphate buffer and 140 mM NaCl. Before the protein adsorption measurement, the pure buffer solution was flowed into the QCM cell until a stable frequency (Δf) and dissipation (ΔD) baseline reading was reached. Afterward, the protein solution (100 μg mL−1) was passed through the measuring chamber. The adsorption process was carried out until surface saturation was achieved on each surface, and the required time differed among the 1955

DOI: 10.1021/acs.langmuir.7b04117 Langmuir 2018, 34, 1953−1966

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Langmuir

Figure 2. Average values and histograms of the adhesion forces measured between BSA-coated AFM colloidal probes and LbL films in a buffer solution (pH 7.4) comprising 10 mM phosphate buffer and 140 mM NaCl. For LbL C and D, the arrows refer to the highest number of events in the respective histogram (values exceed the y-axis scale). surfaces. After surface saturation, the pure buffer solution was flowed into the cell to remove the loosely attached molecules. Changes in Δf and ΔD of the sensor chip were recorded in real time. The QCM experiments were performed on two or three independent samples. After protein adsorption, the adsorbed proteins on the chips were subjected to two cycles of buffer solution rinse at pH 7.4 using buffer solutions consisting of 10 mM phosphate buffer with different concentrations of NaCl. The proteins were first stabilized in buffer solutions with 10 mM phosphate buffer and 140 mM NaCl (the condition under which the proteins were adsorbed on the chips). The first cycling was between a buffer solution with high ionic strength (10 mM phosphate buffer and 500 mM NaCl, pH 7.4) and the adsorption buffer solution. The second cycling was between a buffer solution with low ionic strength (10 mM phosphate buffer without NaCl, pH 7.4) and the adsorption buffer solution. Each injection was carried out for 30 min to 1 h to reach stable frequency readings. The flow rate was controlled at 200 μL/min, which was high enough to allow a quasi-constant bulk concentration in the vicinity of the surface.27 The temperature was kept at 20.0 ± 0.1 °C. To calculate the hydrated mass of adsorbed proteins, the Sauerbrey equation was used for rigid adsorbed layers (ΔD/Δf ≪ 4 × 10−7

structure-controlled and well-blended films with tailored physicochemical properties via modulation of the assembling conditions.30−34 Custom-synthesized polyanions bearing PEGor alkyl-carboxylic side chains (Figure 1a) were used to pair with the polycation, PEI, to modulate the water contact angle (θ) of the surfaces in the range of ∼35° to ∼70°. Surface isoelectric point (pI) was fine-tuned in the pH range from ∼5 to ∼9 by controlling the amount of polyelectrolytes deposited through the adjustment of the dipping solution pH (Table S1). To fabricate positively charged surfaces with pI ≈ 9 (LbL A and B), we deposited polyanion and PEI at a high pH of 8−10 compared to their pKa values and terminated the film with PEI at pH 10 to reach seven bilayers. Depositing the polyelectrolytes at a low pH led to the formation of negatively charged LbL C and D. Representative ζ potential curves are included in the Supporting Information (Figure S2). The fabricated model surfaces LbL A−D are characterized by a well-defined contact angle and surface ζ potential (Figure 1b). The surfaces are also characterized by low surface roughness (Table S2), exhibiting essential characteristics for a comparative study on protein adsorption using independent variation of the surface charge and wettability as surface parameters with a minimal cross-parameter dependency. As all subsequent QCMD and AFM measurements were conducted in aqueous buffer solutions, film swelling was also monitored using QCM-D. The swelling effect may lead to protein entrapment within the expanded film and complicate the comparison among the model surfaces.35 The four surfaces exhibit a similar degree of dissipation increase (softness) upon water uptake in the swelling process, regardless of surface hydrophilicity, which further confirms the suitability of the chosen model surfaces (Figure S3). The components of surface tension including dispersive and polar interactions, as estimated from the contact angle measurements with water and methylene iodide in our earlier work,24 are given in Table S2. The results show that surface wettability correlates well with the changes in the polar

Δf

Hz28): Δm = − C nn , where C is the sensor-specific proportionality constant (17.7 ng cm−2 Hz−1) and n is the overtone number. The Voigt model was used for soft adlayers with larger ΔD/Δf values. The 3rd, 5th, 7th, 9th, and 11th overtones were used in the Voigt model using QTools software (Q-Sense). The Voigt model was also used to estimate the thickness of the adsorbed layers using QTools software. The thickness fitting range was set between 1 × 10−10 and 1 × 10−6 m.29 The adsorbed layer density, solution density, and solution viscosity were assumed as 1200 kg m−3,29 1000 kg m−3, and 0.001 kg m s−1, respectively.

3. RESULTS AND DISCUSSION 3.1. Surface Fabrication and Characterization. To study the independent surface parameters, namely surface charge and wettability, as factors influencing protein adsorption, we utilized the LbL protocol we developed earlier for fabricating well-defined polyelectrolyte film architecture (Figure 1).24 The LbL technique offers the benefit of fabricating 1956

DOI: 10.1021/acs.langmuir.7b04117 Langmuir 2018, 34, 1953−1966

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Langmuir

Figure 3. Three representative f−d curves acquired with BSA-coated colloidal probes on different LbL films in a buffer solution of 10 mM phosphate buffer and 140 mM NaCl, pH 7.4. Dotted and solid lines represent the approaching and retracting traces, respectively. The insets provide a pictorial depiction of the interactions between the BSA-coated probe and the different surfaces.

Table 2. Summary of AFM Force Measurements and QCM-D Adsorbed Mass and Dissipation Changesa AFM

QCM-D b

−2

sample

average adhesion forces (nN)

pull-out distance (nm)

LbL A

3.40 ± 0.46

89.8 ± 35.9

12.00 ± 1.95

LbL B LbL C

5.81 ± 1.76 0.16 ± 0.15

167.7 ± 63.29 70.5 ± 25.23

7.07 ± 0.33 5.93 ± 0.18

LbL D

0.40 ± 0.22

171.4 ± 65.8

adsorbed wet mass (ng mm )

a

1.05 ± 0.22

|∂D/∂f | (10

−6

−1 c

Hz )

|∂D/∂f |I = 0.025 |∂D/∂f |II = 0.179 |∂D/∂f | = 0.019 |∂D/∂f |I = 0.180 |∂D/∂f |II = 0.760 |∂D/∂f | = 0.050

final |ΔD/Δf | (10−6 Hz−1)d 0.091 0.035 0.425 0.178

b

Buffer solution: 10 mM phosphate buffer and 140 mM NaCl, pH 7.4. Pull-out distance is defined as the separation distance at which the force returns to the zero force line in the retracting traces of f−d curves. c∂D/∂f values are obtained by fitting a line with r2 values > 0.96 for all cases except for LbL D with r2 ≈ 0.6 (drawn in Figure 5, at the third overtone). A good fit was difficult to acquire for small signal changes such as those on LbL D.44 Subscripts I and II refer to the two regimes exhibited by the ΔD/Δf curves in Figure 5. dFinal |ΔD/Δf | values were calculated for the third overtone.

As shown in Figure 2, strong adhesion forces were recorded on the positively charged LbL A and B with average values of 3.40 ± 0.46 and 5.81 ± 1.76 nN, respectively. On the negatively charged LbL C and D, the adhesion forces were weak with average values 100 nm, Table 2) during retraction. By contrast, the f−d curves on the hydrophilic surfaces, LbL A and C, returned to zero force at shorter distances. Such steplike features and large pull-out distances on LbL B and D suggest that the protein molecules were stretched and probably deformed during retraction.41,42 This is consistent with previous studies on protein unfolding triggered by surface hydrophobicity,9,14 which increases the surface area of protein molecules available for interacting with the surface.43 Thus, the BSA structure can be easily deformed on the hydrophobic surfaces, LbL B and D, leading to a variety of binding sites. This also accounts for the wider variability in the adhesion forces for the hydrophobic surfaces in comparison to the similarly charged hydrophilic surfaces, as shown in Figure 2. Considering the surface charge effect on the negatively charged surfaces, LbL C and D, electrostatic repulsion led to a small area of contact and a weak adhesion. For the positively charged surfaces, LbL A and B, their net charge is opposite to that of BSA. The resulting electrostatic attraction led to an increase in the area of contact and a concomitant increase in the adhesion forces. For LbL A, the profile of the f−d curves tends to be sharp, with few sequential and multiple rupture events and a strong electrostatic attraction. This indicates that the dominant electrostatic attraction has a limited influence on protein unfolding in AFM measurements. On the other hand, surface hydrophobicity changes the interaction landscape substantially, leading to accelerated unfolding of BSA and larger adhesion forces. This can be seen (Figure 3) when a comparison is made between the two pairs, LbL A versus B and LbL C versus D, where increasing the surface hydrophobicity induced a larger area of contact and deformation because of the entropy gain from the dehydration of hydrophobic surfaces and reduction in the unfolding free energy barrier.9 Elevated attraction forces observed on LbL B, which is more hydrophobic than LbL A and has a net charge opposing that 1958

DOI: 10.1021/acs.langmuir.7b04117 Langmuir 2018, 34, 1953−1966

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Langmuir

Figure 5. Viscoelastic changes ΔD as a function of Δf (third overtone). For the two hydrophilic surfaces (LbL A and C), the ΔD/Δf curves are divided into two regimes labeled as I and II, with the time period indicated. Time increases from left to right as shown by the arrows. The fitted lines have r2 values > 0.96 for all cases, except for LbL D with r2 ≈ 0.6. Buffer solution: 10 mM phosphate buffer and 140 mM NaCl, pH 7.4.

conformational changes on hydrophobic surfaces and that its hydration is reduced upon surface contact.10,29 The mass adsorption kinetics on the hydrophilic and hydrophobic surfaces shows different patterns (Figure 4). On the hydrophobic surfaces, LbL B and D, adsorption attained saturation rapidly within several minutes, whereas on the hydrophilic surfaces, LbL A and C, the initial rapid adsorption was followed by a slower increase in the mass before finally levelling off after typically several hours. This behavior is independent of protein adsorption concentrations (Figure S4), indicating that it is insensitive to molecular packing over a range of concentrations. To further analyze the differences in the BSA adsorption kinetics, ΔD versus Δf curves (D−f plots) were plotted to present the dissipation change per unit of adsorbed mass on each surface (Figure 5).15,55 The slopes of the D−f plots (∂D/ ∂f) reflect the dynamic changes in the protein layers’ conformation.55 The two hydrophobic surfaces, LbL B and D, exhibit a low ∂D/∂f value (Table 2), denoting the formation of a rigid protein layer, with protein adopting a more spread out and deformed conformation.55,56 This is also consistent with previous studies on methyl-terminated SAMs.9,29 By contrast, for the two hydrophilic surfaces, LbL A and C, the D−f plots can be divided into two regimes: an initial fast regime with relatively low ∂D/∂f and a second slow regime with linear and high ∂D/∂f. The high ∂D/∂f value in the second regime indicates a significant increase in the softness of the adsorbed protein, denoting a conformation transition in accordance with the adsorption of a second protein layer, which is more readily penetrated by water, more loosely bound, and more nativelike.55 The differences observed in the D−f plots may be due to the different adsorption kinetics on the hydrophobic and hydrophilic surfaces. As BSA adsorption progressed rapidly toward a deformed state on the hydrophobic surfaces, further structural rearrangements to accommodate more protein adsorption were inhibited.10 By contrast, on the hydrophilic surfaces, the slower and weaker adsorption allowed a small

of protein layers. The BSA solution was passed over the QCM sensor, until equilibrium adsorption was reached. For all four surfaces, BSA injection led to a Δf decrease and a ΔD increase, indicating BSA adsorption on all LbL-coated QCM sensors.10 The saturated mass adsorbed on each surface can be calculated from the Sauerbrey equation or Voigt model using QTools software (Q-Sense).28 A greater adsorbed mass was observed on the positively charged surfaces (12.00 ± 1.95 ng mm−2 for LbL A and 7.07 ± 0.33 ng mm−2 for LbL B) than on the negatively charged surfaces (5.93 ± 0.18 ng mm−2 for LbL C and 1.05 ± 0.22 ng mm−2 for LbL D). The QCM-D adsorption results complemented the AFM protein adhesion results and the results of our earlier work in which adsorption of fluorescein isothiocyanate (FITC)-labeled proteins was studied (Table S3).24 Both the direct adhesion forces and the adsorbed mass increased with the positive surface charge, confirming electrostatic interactions as important components of BSA−surface interactions. However, the QCM-D and FITC-labeled protein adsorption results are different when surface wettability is considered as a parameter. The FITC-labeled BSA assay indicates that a larger amount of BSA was adsorbed on the hydrophobic surfaces, whereas the QCM result shows a slightly higher mass on the hydrophilic surfaces. This difference in the results can be explained by the hydration effect of BSA molecules.35,52 The FITC-labeled BSA assay measures the “dry” protein mass adsorbed, whereas QCM-D measures the total hydrated mass, which includes both the protein and the associated water. The lower mass on the hydrophobic surfaces compared to that on the hydrophilic surfaces as measured by QCM-D suggests that surface hydrophobicity triggered BSA denaturation and reduced hydration of BSA adsorbed on the surface. This is also reflected by a minimal increase in ΔD (softness) upon BSA adsorption and overlapping Δf and ΔD curves under different overtones53,54 for the hydrophobic surfaces, LbL B and D (Figure 4). This is consistent with earlier reports that BSA undergoes 1959

DOI: 10.1021/acs.langmuir.7b04117 Langmuir 2018, 34, 1953−1966

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Langmuir extent of structural rearrangements to accommodate the additional protein, giving rise to a second adsorption regime. Moreover, the transition takes place when the adsorbed mass reaches a critical f (surface coverage), indicating that the interactions between the adsorbed protein molecules play a role in this transition. The differences in the conformation rearrangements of the protein layers led to different final ΔD/Δf ratios (Table 2), which indicate the ultimate viscoelastic nature of the adsorbed protein layers. The final ΔD/Δf ratios were also lower for the more hydrophobic surfaces than for the hydrophilic ones with similar charges, indicating the formation of a less hydrated and stiffer protein layer on the hydrophobic surfaces.57 Thus, the QCM-D results show that surface hydrophobicity increased the adsorbed protein rigidity because of surface-induced deformation, regardless of the surface charge. On the hydrophobic surfaces, entropy gains from exclusion of water allow a greater proportion of the protein to interact with the surface,9 which may help to explain the greater conformational change of BSA on the hydrophobic surfaces LbL B and D. Upon binding to these hydrophobic surfaces, as a result of protein conformational changes, the exposure of inner regions of the protein allows for additional hydrophobic and electrostatic interactions. On the other hand, the surface charge opposing that of the protein increased the adsorbed protein mass, but its role in adsorbed protein conformation is secondary to surface hydrophobicity. Unlike the QCM-D adsorption experiment, AFM force spectroscopy measures the interaction strength between the protein and surface, which would not be affected by long-term conformational changes,37 lateral interactions between the adsorbed proteins,7 or multilayer formation processes58 that may be present in the former. Thus, it simulates the very early stage of protein adsorption.39 In general, the AFM force measurements agree with the macroscopic capacity of the different LbL surfaces to adsorb the BSA molecules, as measured by QCM-D and FITC-labeled BSA assay. Both the positive surface charge and hydrophobicity increased the BSA− surface interaction forces and the adsorbed amount. However, as suggested by the AFM and QCM-D experiments, BSA molecules were more deformed on the hydrophobic surfaces, irrespective of the surface charge, compared with those on the hydrophilic surfaces. The proposed mechanism also agrees with the protein layer thickness estimated using the Voigt model. On the hydrophobic LbL B and D, the estimated thickness values are lower, reaching ∼5 and ∼2 nm, respectively. On the hydrophilic LbL A and C, the estimated thickness is ∼11 and ∼9 nm, respectively. All of the observed values fall within the range of published data.29,59 The low thickness values on the hydrophobic surfaces also suggest that BSA molecules adopted a more spread out and deformed conformation (Figure 6). BSA is known as a “soft” protein prone to conformational changes during adsorption depending on the surface properties.29 The results above are consistent with previous studies showing that protein deformation/spreading on hydrophobic surfaces occurs to a greater extent20,55 and at a higher rate than on hydrophilic surfaces.7,14 3.3. BSA Desorption. Removing preadsorbed proteins from a surface involves contributions from irreversible protein conformational changes and protein−protein interactions. To understand the role of the interfacial forces, protein unfolding and protein−protein interactions in protein desorption, the

Figure 6. Schematic of BSA molecular rearrangement on surfaces as a function of surface pI and θ.

effect of ion concentration on desorption of the preadsorbed BSA molecules on the surfaces was investigated using QCM-D (Figure 7). As indicated in Figure 4, rinsing the BSA-loaded surfaces with a BSA-free physiological buffer solution generated negligible desorption, which is typical of adsorbed BSA on a variety of surfaces.60 To study BSA desorption at varying ionic strengths, the BSA-loaded LbL surfaces were first equilibrated in buffer solutions with 10 mM phosphate buffer and 140 mM NaCl (the buffer in which BSA was adsorbed, λD ≈ 0.7 nm, pH 7.4) to obtain the baseline. The BSA-loaded surfaces were then treated with buffer solutions characterized by a high ion concentration (10 mM phosphate buffer and 500 mM NaCl, λD ≈ 0.4 nm, pH 7.4) and a low ion concentration (10 mM phosphate buffer without NaCl, λD ≈ 2 nm, pH 7.4). As shown in Figure 7, for the two hydrophobic surfaces, LbL B and D, the adsorbed mass (Δf) shows reversible changes upon exposure to high and low ion concentration solutions, indicating no appreciable protein desorption throughout the ionic strength cycle.61,62 This implies that the BSA molecules adsorbed on the hydrophobic surfaces were resistant to elution by ionic strength changes, regardless of the surface charge. This is consistent with a lower ∂D/∂f value, that is, a more spread out and deformed conformation of the adsorbed BSA molecules, which maximized their footprint on the hydrophobic surfaces, as observed in previous sections. The reversible steep drops or rises observed in the adsorbed mass (Δf) during cycling (Figure 7) is due to the effects of salt mass and film swelling when the added mobile ions disrupt some of the polycation−polyanion linkages in the film.63,64 Similar reversible mass changes (Δf) were observed on the native LbL films when exposed to the same ionic strength changes (Figure S5), indicating that the native LbL films maintained their structural integrity during the ionic strength cycling. By contrast, for the two hydrophilic surfaces, LbL A and C, the changes in the adsorbed mass were irreversible, indicating mass losses from the adsorbed proteins triggered by changes in the ion concentration. This is consistent with the more hydrated and nativelike conformation of BSA molecules adsorbed in the second layer. The extent of mass losses was ∼12% and ∼25% for LbL A and C, respectively. BSA molecules desorbed from LbL C with an increase in the ion concentration. This desorption could be attributed to competitive adsorption of Cl− that displaced BSA from the surface or screened localized electrostatic attraction.65 By contrast, on LbL A, the BSA molecules partially desorbed at a lower ion concentration. Lowering the ion concentration would increase the local electrostatic attraction between the surface 1960

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Figure 7. Adsorbed BSA molecules on the LbL surfaces exposed to buffer solutions of different ionic strengths. To obtain the baseline, BSA-loaded surfaces were stabilized in buffer solutions with 10 mM phosphate buffer and 140 mM NaCl (the condition under which the proteins were adsorbed). This is defined as the “rinse” step in the figure. High ion concentration (10 mM phosphate buffer and 500 mM NaCl) is denoted as “500” in the first step. Low ion concentration (10 mM phosphate buffer without NaCl) is denoted as “0” in the second step. Buffer solutions of pH 7.4 were used in these experiments.

Figure 8. Average adhesion forces measured between BSA-coated AFM colloidal probes and LbL films under different ion concentrations. Buffer solution: 10 mM phosphate buffer with additional 0, 140, or 500 mM NaCl, pH 7.4.

in the dissipation loss/gain, the reversible changes in the viscoelasticity (ΔD) of the adsorbed layers indicate that these processes were reversible upon ionic strength changes. For LbL A and C, this also indicates that the partial protein desorption caused minimal changes to the total viscoelasticity of the adsorbed layers. The QCM desorption results show that whether the adsorbed BSA molecules on the surfaces could be removed by changes in the ion concentration was primarily determined by the degree of protein deformation, dictated by surface hydrophobicity, regardless of the surface charge or multilayer

and BSA molecules, which is expected to increase their binding and resistance to desorption. However, because of the large amount of BSA molecules adsorbed on LbL A, the electrostatic repulsion among adsorbed BSA molecules could increase with a decrease in the ion concentration, which might lead to partial desorption of the outer protein molecules.66 The viscoelasticity changes of the adsorbed layers (ΔD) were reversible upon ionic strength changes for all surfaces (Figure 7). As changing ion concentration could cause rearrangements of the protein layers66 and LbL films63,64 and solvent/ counterion expulsion/entrapment,62 which would be reflected 1961

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Figure 9. Average adhesion forces and force histograms measured between BSA-coated AFM colloidal probes and different LbL films (LbL A and B) at pH 7.4 and 3.6 with the same ion concentration. Buffer solution for pH 7.4: 10 mM phosphate buffer and 140 mM NaCl; buffer solution for pH 3.6: 10 mM acetic acid buffer and 140 mM NaCl.

protein residues, leading to a more compact protein conformation, which impeded adhesive bond formation with the surface.71 The specific effect of NaCl as a weak salting-out (protein-stabilizing) agent also contributes to the formation of a more compact conformation at a high ion concentration.72 Thus, the decrease in the interaction forces with increasing NaCl concentration is a combination of ions screening the interactions between BSA and LbL A and a more compact protein conformation. This result indicates that the adhesion forces were mainly derived from electrostatic interactions between BSA and LbL A. A rather different picture is observed for LbL B, where the BSA adhesion forces on the positively charged and hydrophobic LbL B were increased by the elevated ion concentration. A likely explanation for this effect is that at low concentration, ions bind strongly to the charged protein residues because of limited shielding.68 As a result, there is a higher energy penalty associated with the release of ions that accompanies protein adsorption, thus lowering the adhesion forces.73 However, at a high ion concentration, the electrostatic attraction between the ions and charged protein residues is reduced because of charge shielding. Water-ion pairs are formed, which reduce the number of water molecules around the protein and disrupt the hydration layer of proteins.74 This facilitates the interaction between the protein hydrophobic residues and the surface, thus enhancing protein affinity for hydrophobic surfaces.74 Moreover, across the ion concentration range tested, steplike behaviors during retraction and large pull-out distances were observed in the f−d curves (Figures 3, S6, and S7), consistent with a greater deformation driven by surface hydrophobicity. On the other hand, on the negatively charged surfaces, LbL C and D, the average adhesion forces remained weak over the ion concentration range investigated. Because of the like charges of the protein and the surfaces, electrostatic repulsion dominated the interactions between the protein and the surfaces. The repulsive forces were reduced with increasing ion concentration, accounting for the slight increase in

formation. We can conclude that fairly stable protein layers composed of denatured BSA molecules were formed on the hydrophobic surfaces, regardless of their surface ζ potential. This is also in agreement with the earlier research suggesting that the effects of surface hydrophobicity could even dominate over protein−protein interactions on protein unfolding,67 thus extending this surface-induced effect to the proteins adsorbed in multilayers. 3.4. Effect of Ionic Strength and pH on BSA−Surface Interaction. The effects of ionic strength and pH on the protein−surface interaction were examined by the determination of “pull-off” forces between the protein and surfaces after immediate contact, using AFM force spectroscopy. Information on the strength and nature of the interaction and the changes in the dynamics of the interacting protein can be obtained. The effects of ionic strength on protein−surface interactions include contributions from ionic screening, ion binding to the charged protein and surface, and protein conformational changes.68 Thus, AFM force measurements performed over an extended ionic strength range helps in understanding the competitive contributions from various interfacial forces and the importance of protein conformation for protein binding.69 We examined the changes in the adhesion forces at different ion concentrations (Figure 8) at pH 7.4. The ion concentration was adjusted by adding NaCl to 10 mM phosphate buffer up to 0, 140, and 500 mM NaCl, which corresponds to λD of ∼2, 0.7, and 0.4 nm, respectively. Phosphate buffer at pH 7.4 was used to maintain structural stability of BSA during the measurements.70 As shown in Figure 8, for the positively charged and hydrophilic LbL A, the adhesion forces decreased with increasing ion concentration. At pH 7.4, BSA is negatively charged. Therefore, there is an attractive electrostatic interaction between BSA and LbL A. Increasing ion concentration screened the attractive interactions between them, leading to lower adhesion forces. Moreover, high ion concentration screened the electrostatic repulsion between 1962

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4. CONCLUSIONS In the present study, we utilized QCM-D and AFM force measurements to investigate BSA adsorption/desorption characteristics driven by a combination of surface charge and wettability using model surface platforms prepared via LbL technique that could simultaneously modulate the two surface variables. The AFM force spectroscopy results reveal the direct contribution of surface effects on protein−surface interaction, which complement the information provided by QCM-D on the adsorbed layer rigidity and packing. Through this combination of techniques, the influence of surface charge and wettability on the process of protein adsorption on the liquid/solid interface was delineated. Electrostatic attraction as the dominant force was found to have limited influence on protein deformation and extent of desorption by changes in the ionic strength. On the other hand, surface hydrophobicity triggered rapid protein denaturation, leading to the formation of a rigid adsorbed layer resistant to elution, regardless of the surface charge. The rigidity of the adsorbed protein layers as well as their desorption behavior on surfaces have strong impacts on their functionality and interactions with cells.25,77 The results obtained in this work may be important for understanding the varying functionality of the protein when adsorbed on different surfaces. For example, research has shown that the rigidity of adsorbed proteins on surfaces78 and the ease in displacing cell-repelling proteins such as BSA25 are important factors determining cell adhesion and tissue integration. We expect the results to contribute to the development of tailor-made surfaces to trigger more desired protein−substrate interactions for biomedical applications.

adhesion forces with increasing ion concentration. Despite the strong electrostatic repulsion, hydrophobicity effects on BSA deformation for the hydrophobic surface LbL D were evident, as shown by the longer pull-out distances in the retracting curves for LbL D than those for the hydrophilic surfaces, LbL A and C (Figures 3, S6, and S7). We further studied the BSA adhesion forces on LbL A and B by recording force curves at pH 3.6 and 7.4 while keeping the solution ion concentration constant at 150 mM. The charge of both the protein and the surface depends on the solution pH. BSA molecules carry a net positive charge at pH 3.6 and a negative charge at pH 7.4. We have chosen LbL A and B (with pI ≈ 9) as model surfaces because they can maintain their positive surface charge at both pH values, with limited variation (Figure S2). Thus, essentially only the charge of the protein was altered, allowing us to isolate the effect of protein properties from that of surface properties. The solution pH affects not only the charge of the protein but also its conformational state.75,76 The average values and histograms of the adhesion forces between the BSA-coated probe and the LbL films in media of different pH values are shown in Figure 9. For both LbL films, the adhesion forces of BSA at pH 7.4 were greater than those at pH 3.6. The electrostatic forces between BSA and the LbL surfaces changed from attractive to repulsive as pH decreased from 7.4 to 3.6. Thus, the electrostatic repulsive force weakened the adhesion forces of BSA with the LbL surfaces at pH 3.6. The hydrophobic LbL B exhibited an elevated adhesion force with BSA when compared with LbL A, for both pH values. The increased surface hydrophobicity promoted BSA adhesion forces, despite pH changes. It is also possible that the BSA conformation exerted an effect on its adhesion forces, particularly at low pH. At pH 3.6, BSA is at its expanded state75,76 and may expose more hydrophobic residues, thereby enhancing the interactions between BSA and hydrophobic surfaces.10 Moreover, for both pH conditions, multiple rupture events and steplike behaviors during retraction and large pullout distances were observed in the f−d curves on the hydrophobic surfaces (Figures 3 and S8), consistent with a greater deformation driven by surface hydrophobicity. Overall, the solution pH and ionic strength affect the electrostatic interactions between the protein and surfaces as well as the conformation of protein. The interplay of these effects is particularly obvious on LbL B, which involves both electrostatic and hydrophobic effects. On this surface, the protein conformation significantly affects the combined influence of these two effects: under an attractive electrostatic effect (pH 7.4), a more easily deformed conformation (at higher ion concentration) led to much larger total adhesion forces because of additional hydrophobic and electrostatic interactions generated by unfolding. However, when electrostatic attraction rather than hydrophobic effects is the dominant force (LbL A), the profile of the force curves is sharp, with few sequential and multiple rupture events at all of the solution pH values and ionic strengths investigated. This indicates that the effect of surface charge mainly alters the strength of protein− surface interaction, with less effect on protein deformation. On the other hand, it was consistently observed that the protein deformation increased with surface hydrophobicity, which suggests that the role of surface charge is secondary to that of surface hydrophobicity in protein deformation on surfaces.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b04117. AFM data processing and colloidal probe; LbL film fabrication; surface free energy and roughness; representative surface ζ potential as a function of pH; film swelling measured by QCM-D; adsorbed mass measured by QCM-D and protein adsorption assay; BSA adsorption at different concentrations; stability of polyelectrolyte layers in salt solutions; and AFM force measurements in buffer solutions with 10 mM phosphate buffer and 500 mM NaCl, in buffer solutions with 10 mM phosphate buffer without NaCl, and in buffer solutions with 10 mM acetic acid buffer and 140 mM NaCl (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86 571 88982651 (X.Z.). *E-mail: [email protected]. Phone: +48 22 234 5583. Fax: +48 22 234 5504 (D.J.). *E-mail: [email protected]. Phone: +65 6516 2176. Fax: +65 6779 1936 (K.G.N.). ORCID

En-Tang Kang: 0000-0003-0599-7834 Xian Jun Loh: 0000-0001-8118-6502 Dominik Jańczewski: 0000-0002-5466-6444 Koon Gee Neoh: 0000-0002-2700-1914 1963

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Langmuir Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Agency for Science, Technology, and Research (A*STAR) for providing financial support and the A*STAR Graduate Academy for the PhD scholarship of S.G.

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DOI: 10.1021/acs.langmuir.7b04117 Langmuir 2018, 34, 1953−1966

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DOI: 10.1021/acs.langmuir.7b04117 Langmuir 2018, 34, 1953−1966