Effect of Surface Potential on Extracellular Matrix Protein Adsorption

Article pubs.acs.org/Langmuir

Effect of Surface Potential on Extracellular Matrix Protein Adsorption Jiun-Hao Lin,†,‡ Hsun-Yun Chang,§,∥,⊥ Wei-Lun Kao,†,‡,⊥ Kang-Yi Lin,‡ Hua-Yang Liao,‡ Yun-Wen You,‡ Yu-Ting Kuo,†,‡ Ding-Yuan Kuo,‡ Kuo-Jui Chu,†,‡ Yi-Hsuan Chu,†,‡ and Jing-Jong Shyue*,†,‡ †

Department of Materials Science and Engineering, National Taiwan University, Taipei 106, Taiwan Research Center for Applied Sciences, Academia Sinica, Taipei 115, Taiwan § Department of Engineering and System Science, National Tsing Hua University, Hsinchu 300, Taiwan ∥ Nano Science and Technology Program, Taiwan International Graduate Program, Institute of Physics, Academia Sinica, Taipei 115, Taiwan ‡

ABSTRACT: Extracellular matrix (ECM) proteins, such as fibronectin, laminin, and collagen IV, play important roles in many cellular behaviors, including cell adhesion and spreading. Understanding their adsorption behavior on surfaces with different natures is helpful for studying the cellular responses to environments. By tailoring the chemical composition in binary acidic (anionic) and basic (cationic) functionalized self-assembled monolayer (SAM)-modified gold substrates, variable surface potentials can be generated. To examine how surface potential affects the interaction between ECM proteins and substrates, a quartz crystal microbalance with dissipation detection (QCM-D) was used. To study the interaction under physiological conditions, the ionic strength and pH were controlled using phosphate-buffered saline at 37 °C, and the ζ potentials of the SAM-modified Au and protein were determined using an electrokinetic analyzer and phase analysis light scattering, respectively. During adsorption processes, the shifts in resonant frequency (f) and energy dissipation (D) were acquired simultaneously, and the weight change was calculated using the Kelvin−Voigt model. The results reveal that slightly charged protein can be adsorbed on a highly charged SAM, even where both surfaces are negatively charged. This behavior is attributed to the highly charged SAM, which polarizes the protein microscopically, and the Debye interaction, as well as other short-range interactions such as steric force, hydrogen bonding, direct bonding, charged domains within the protein structure, etc., that allow adsorption, although the macroscopic electrostatic interaction discourages adsorption. For surfaces with a moderate potential, proteins are not significantly polarized by the surface, and the interaction can be predicted through simple electrostatic attraction. Furthermore, surfaceinduced self-assembly of protein molecules also affects the adsorbed structures and kinetics. The adsorbed layer properties, such as rigidity and packing behaviors, were further investigated using the D−f plot and phase detection microscopy (PDM) imaging.

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INTRODUCTION The interaction between the cell and material surfaces is one of the key factors in regulating cell behavior, and it has increasingly garnered attention over recent years due to the development of biomaterials for applications such as cell sorting, implantation, biochips, and biosensors. The cellular response to material surfaces is mediated by adsorbed proteins; hence, the cell adhesive extracellular matrix (ECM) proteins, such as fibronectin, laminin, and collagen IV, play important roles in regulating cell adhesion, cell spreading, and matrix assembly.1 Therefore, many studies have focused on adsorption of these proteins over the past decade.2 Various materials have been used to study protein adsorption,3−6 and polymers have received the most interest because they have highly variable surface properties.7,8 However, polymer surface properties are controlled by both molecular structure and functional groups, and it is difficult to differentiate which factor more significantly affects protein adsorption behavior. Furthermore, between two sets of © 2014 American Chemical Society

polymers, while both the mechanical and chemical properties are altered, it is difficult to examine which cue dominates in controlling the interaction. Finally, for a randomly oriented coil and backbone, it is difficult to define the region where the polymer structure interacts with the protein. In a more recent work,9 gradients of β-SiC and diamond were grown on a Si substrate and the effect of the density of surface hydroxyl groups on the protein adsorption was examined. Although the mechanical and chemical properties are decoupled, the choice of chemical properties is limited in this system. Because of their ordered, close-packed structure and welldefined surface chemistry,10 self-assembled monolayers (SAMs) have been widely used to modify surface properties of solid materials and have been useful in studies on interactions between biomolecules and materials in recent years.11−13 In Received: May 26, 2014 Revised: July 18, 2014 Published: August 11, 2014 10328

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QCM-D was used to examine laminin, fibronectin, and collagen IV adsorption. To examine this interaction under physiological conditions, the adsorption was conducted at 37 °C in phosphate-buffered saline (PBS). To understand the electrostatic interactions, the ζ potentials of the binary-SAM-modified Au and proteins were measured using an electrokinetic analyzer and phase analysis light scattering, respectively, under physiological conditions. The structure of the adsorbed protein film was then imaged using phase detection microscopy (PDM).

addition, SAM molecules can spontaneously bond substrates via chemisorption at room temperature through a simple procedure, and the functional groups can be transformed in situ using simple reactions to produce surfaces with different chemical properties; hence, it is a flexible platform for various applications.14,15 Using SAM-modified surfaces, most previous studies on surface−molecule interactions have concentrated on the effect from hydrophobicity/hydrophilicity.8,11,16 In addition, SAMs were typically deposited using one specific molecule; hence, fine-tuning the surface properties was not possible unless partial surface functionalization is employed.17 Although the surface properties could be tuned with incomplete SAM coverage18 or incomplete in situ functional group replacement,19 the resulting properties were related to the extent of reaction progression that may be difficult to control due to the nonequilibrium state. With a controlled immersion time for SAM deposition followed by filling the uncoated sites with another molecule, surfaces with gradient properties were also reported.20 A more straightforward protocol is to codeposit multiple surfactants onto the surface, and the surface properties can be tailored with the mixing ratio of the functional groups. Focusing on the wettability, CH3 and (C2H2O)n mixed SAM was developed.21 Similarly, focusing on the surface charge, weak acid (COOH and SH) and weak base (NH2) were also codeposited to produce surfaces with tunable potential, isoelectric point, and effective surface dipole on gold and silicon substrates.22−26 In addition to the ease of preparation, the surface composition is thermodynamically controlled and is less sensitive to the experimental variables. Furthermore, the mixed COOH and NH2 SAMs have similar wettability.27 These tunable surfaces allow the examination of how the surface potential alone affects protein adsorption. To examine protein adsorption kinetics, optical methods are often used, such as surface plasmon resonance (SPR),7,11−13 ellipsometry6 and dual polarization interferometry.28 However, SPR requires Au for its surface plasmon, and ellipsometry requires a reflective surface; hence, the substrates are limited. For their high mass sensitivity29 and broad range of available substrates, a quartz crystal microbalance with dissipation detection (QCM-D) has garnered significant attention in the study of biomolecule adsorption.5,6,8,30,31 Because the mass change is directly correlated to the resonant frequency shift, QCM was originally used as a film thickness gauge for growing rigid film. Unfortunately, for soft materials that are elastic and easily deformed on the oscillating crystal, especially in a viscous environment, the Sauerbrey equation fails to generate reliable results because the elastic film does not oscillate with the crystal, and the oscillation energy quickly dissipates due to deformation of the elastic films. Hence, the effective mass change calculated from the frequency shift differs from the real mass change.32−34 Through detecting the energy dissipation and applying the established viscoelastic model, it is currently feasible to convert the frequency shift and level of energy dissipated into the actual mass change. Furthermore, the dissipation signal provides additional information on the viscoelastic properties of the adsorbed layer.34−36 In this work, Au surfaces were modified with homogeneously mixed amine and carboxylic acid functional groups with the same carbon chain lengths in the SAM. The surface chemical composition was verified using X-ray photoelectron spectrometry (XPS). To study the way that the surface potential affects ECM formation, which, in turn, affects cellular interactions,

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MATERIALS AND METHODS

Specimen Preparation. A gold-coated AT-cut quartz crystal with 5 MHz fundamental resonance frequency (QSX 301, Q-sense, Sweden) was used in this study. The crystals were exposed to an UV−ozone cleaner (193 nm) for 10 min, chemically cleaned with a solution composed of NH3:H2O2:H2O = 1:1:5 at 75 °C for 5 min, and again exposed to UV−ozone for 10 min to eliminate organic contamination on the surfaces.37 The as-cleaned crystal was immediately immersed overnight in an ethanol solution composed of 6-amino-1-hexanethiol hydrochloride (HS(CH2)6NH2·HCl, SigmaAldrich, St. Louis, MO) and 6-mercaptohexanoic acid (HS(CH2)5COOH, Sigma-Aldrich) for SAM deposition at room temperature. The total thiol concentration was fixed at 1 mM and the ratio of thiol surfactants varied. After SAM formation, the crystal was removed from the deposition solution, thoroughly rinsed with absolute ethanol to remove noncovalent bonded molecules, and then dried in a stream of nitrogen. XPS Measurements. The elemental composition of the SAMmodified gold surface was recorded immediately after its preparation using a PHI 5000 VersaProbe (ULVAC-PHI, Chigasaki, Japan) system to avoid photooxidation of the amine functional groups.38 The microfocused Al Kα X-ray (25 W, 100 μm) and analyzer were rastered on the same 300 × 300 μm area using a dynamic emittance matching (DEM) control. The takeoff angle of the photoelectron was fixed at 45°. A dual beam charge neutralizer (7 V Ar+ ions and 1 V flooding electron beam) was used to compensate for the charge-up effect. N(1s), S(2p), Au(4f), and C(1s) spectra were collected on each specimen using an analyzer pass energy of 58.7 eV. The peak intensity ratio was converted to an atomic ratio using the built-in sensitivity factors in the associated Multipak software. Electrokinetic Measurements. The ζ potential of the modified gold surface was determined based on the streaming current using a clamping cell in an electrokinetic analyzer (SurPass, Anton Paar GmbH, Graz, Austria). The sample was a polycarbonate substrate with a gold layer deposited onto the surface in a high vacuum e-beam evaporator. The measurements were performed by pumping phosphate-buffered saline (PBS, Biological Industries, Israel, pH 7.4) through the microchannel formed with the SAM-modified Au surface using syringe pumps. A pressure ramp from 0 to 300 mbar was employed to force the electrolyte solution through the channel. The measured streaming currents in 4−10 individual pressure ramps were converted to ζ potentials using the Helmholtz−Smoluchowski equation.39 Phase Analysis Light-Scattering Measurements. Laminin (Sigma-Aldrich), fibronectin (Sigma-Aldrich), and collagen IV (Sigma-Aldrich) were dispersed in PBS (pH 7.4) and the ζ potential of each protein was determined through phase analysis light scattering (90-Plus ZetaPALS, Brookhaven Instruments Corp., Holtsville, NY) with 5−10 individual runs. The ζ potential was calculated from the electrophoretic mobility using the Smoluchowski equation. QCM-D Measurements. The frequency shift and energy dissipation of the SAM-modified Au-coated quartz crystal were monitored using a QCM-D instrument (Q-sense E4, Q-sense, Sweden) in odd overtones from the 1st to the 13th. The blank solution (PBS, pH 7.4) and protein solution (2 μg/mL in PBS, pH 7.4) were pumped into a flow cell (QFM 401) containing the quartz crystal using a peristaltic pump at the flow rate 0.1 mL/min. The flow 10329

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cell was maintained at 37 °C by the thermoelectric circuit, while the stock solutions were temperature-controlled at slightly above 37 °C using a dry bath incubator to prevent liberation of the dissolved air due to the increased temperature within the measurement cell. Before exposing the SAM-modified crystal to the protein solution, the surfaces were equilibrated with the background environment by flushing with the blank PBS solutions for 1 h. After steady-state was obtained and a flat baseline indicating equilibrium was established for at least 20 min, the protein-containing solution was pumped into the system to begin the protein adsorption process. After the adsorption was complete, the acquired frequency and dissipation shifts were fit to the mass change as a function of time using Kelvin−Voigt model34 in the Q-tools software. Identical experiments were carried out at least three times on each specimen to obtain statistical information and representative data curves at median were reported herein. PDM Imaging. After the QCM measurements, the proteinadsorbed Au-coated crystal was extracted and rinsed with DI water to prevent salt crystallization then air-dried for imaging by diInnova (Veeco, Plainview, NY). A cantilever with the force constant ∼3 N/m and resonance frequency ∼75 kHz was used in the phase detection microscopy (PDM) mode.

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RESULTS AND DISCUSSION Surface Composition Characterization. According to previous work,25 self-assembled monolayers (SAMs) formed Table 1. Relationship between the Deposition Solution Mixed Ratios and the SAM Functional Group Ratios Prepared Using Such Deposition Solutionsa,b ratio of COOH in solution (%) ratio of COOH in SAM (%) ζ potential (mV) a b

0

20

0

18 ± 4 −28 ±3

6.8 ± 2.8

40

60

80

34 ± 7 62 ± 2 83 ± 2 −42 ± 10

−79 ± 10

−121 ±9

100

Figure 1. Laminin (ζ potential = +6.2 mV) adsorption on substrates with different surface potentials obtained using different functional group ratios. (a) Adsorbed mass vs exposure time and (b) D−f plot at the 7th overtone.

100 −187 ±7

The ζ potential of each surface measured in PBS is also presented. COOH% + NH2% = unity.

Table 2. ζ Potentials of Protein Measured in PBS protein

laminin

fibronectin

collagen IV

ζ potential (mV)

6.2 ± 0.9

−5.7 ± 0.5

5.1 ± 0.5

using a mixed HS(CH 2 ) 8 NH 2 and HS(CH 2 ) 15 COOH surfactant can be used to generate gold substrates with a series of ζ potentials. However, the chain length difference between the two types of thiols may induce side effects, such as exposure of the carbon chain and altitude disparity at the atomic level. Therefore, equal length thiol molecules with NH2 and COOH functional groups were used to prepare the binary SAMs in this study and the surface morphology and roughness of all surfaces remain identical to the bare Au-coated crystal at ∼1 nm (RMS) that indicated the formation of uniform monolayer. As revealed in previous work,25 the deposition rate of the two types of thiols can differ; hence, SAM surface chemical compositions can deviate from the deposition solution. Therefore, specimens from deposition solutions with a series of thiol ratios were prepared for surface composition characterization through XPS to determine the actual functional group ratios on the modified surface. Through the XPS analysis, the deposition kinetics for the two types of thiols were measured, and suitable deposition conditions for the desired surface chemical composition were selected. The quantity of COOH and NH2 deposited on the Au substrate can be calculated based on the atomic ratio of sulfur (S) and nitrogen

Figure 2. Adsorbed weight of laminin, fibronectin, and collagen IV as a function of surface potential. The error bars indicated the standard deviation. For each protein, p < 0.005 in one-way ANOVA. *p < 0.05 in t-test.

(N),25 and macroscopic phase segregation was not observed. Table 1 summarizes the quantification results. Unlike the pervious report,25 significant concentration difference between the SAMs and deposition solutions was not observed. The similarity in deposition kinetics between the NH2-SAMs and COOH-SAMs in the system presented herein is attributed to their equal chain lengths and similar molecular weights while molecules with longer chain lengths are deposited faster than molecules with shorter chain lengths.25 10330

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Figure 3. Representative PDM images of the adsorbed laminin layer on substrates with different surface potentials obtained using different functional group ratios. Scale bar: 250 nm.

ζ Potentials of Binary-SAM-Modified Au Surfaces. Table 1 also showed the ζ potentials of substrates prepared from deposition solutions with different functional group ratios in physiological phosphate-buffered saline (PBS). The ζ potentials gradually shifted from 6.8 mV (pure NH2) to a more negative −187 mV (pure COOH). Therefore, it was possible to generate gold substrates with weak positively charged surfaces to strong negatively charged surfaces using this mixed binary-SAM system. ζ Potentials of ECM Proteins. Protein suspensions in physiological PBS were used to measure the electrophoretic mobilities of protein particles and then convert the mobilities to ζ potentials. Table 2 shows the measured ζ potentials of proteins used in this study. Because the sum of charges on individual amino acid residues contributes to the total charge of a protein, the charge from different parts of the protein can cancel each other out; hence, the ζ potential is found to be a minute value with a positive or negative sign. Effect of Surface Potential on Laminin Adsorption. Figure 1a shows the change in adsorbed mass as a function of time during laminin adsorption on substrates with different functional group ratios and surface potentials. The final adsorption weight with the statistical variation is summarized in Figure 2. The analysis of variance (one-way ANOVA) revealed that the surface potential has a significant effect on the laminin adsorption at the 0.005 level. The initial adsorption rate and final adsorbed quantity increased with COOH loading in the SAMs, except that the final quantity adsorbed on the 80% COOH substrate was slightly higher than that on the pure COOH substrate. This result is consistent with the prediction from electrostatic interactions between SAMs and laminin, where the positively charged protein experienced a greater attractive force when the substrate surface charge was more negative; hence, laminin deposited at higher rates and formed thicker films with an increasing COOH percentage on the surface.

Figure 4. Fibronectin (ζ potential = −5.7 mV) adsorption on substrates with different surface potentials obtained using different functional group ratios. (a) Adsorbed mass vs exposure time and (b) D−f plot at the 7th overtone.

As the actual ratios of the two functional groups were uniformly distributed in the range 0−100%, these specimens were selected for subsequent studies. 10331

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Figure 5. Representative PDM images of the adsorbed fibronectin layer on substrates with different surface potentials obtained using different functional group ratios. Scale bar: 250 nm.

The adsorbed laminin layer is highly hydrated due to laminin’s significant carbohydrate component;40 hence, the “wet mass” measured using QCM-D must also consider the contribution from hydrated water. In Figure 1b, the energy dissipation shift (ΔD) during laminin adsorption is plotted with respect to the corresponding frequency shift (Δf), and the socalled D−f plot is presented. The D−f plot provides information on the evolution of the layer properties during adsorption, and its relative slope is the most important information. Because energy dissipation is related to the adsorbed layer elasticity, and the frequency shift is correlated to the mass change, the slope of D−f plot (energy dissipation per unit frequency shift) indicates the layer rigidity. A smaller slope is expected for a more compact and rigid layer, and a higher slope value indicates a looser and more elastic layer. Except for 80% COOH, the slope increased with the COOH functional group concentration. This trend could be attributed to the difference in water content, which indicates that a highly charged substrate will enhance the level of hydration and lead to a looser packed adsorbed laminin layer. As a result, on the repulsive NH2 surface, most rigid film was observed due to the less water content. For the 80% COOH substrate, considering that the surface is microscopically composed of the positively and negatively charged functional groups of NH2 and COOH, the charge distribution within the protein could allow laminin to arrange its orientation to match the surface charge distribution; hence, higher adsorption and a denser layer were observed. Figure 3 presents the morphology of laminin adsorbed onto substrates with different surface potentials. The protein domains are smaller on NH2 and 20% COOH than the other sample. Such a small cluster feature is consistent with the slope difference in the D−f plot, where a more rigid and less hydrated structure is observed with low COOH loading. The higher water content structure formed on the higher COOH loading surfaces, which renders the adsorbed laminin softer and easy to aggregate to a larger domain, while the domain remains small

Figure 6. Collagen IV (ζ potential = +5.1 mV) adsorption on substrates with different surface potentials obtained using different functional group ratios. (a) Adsorbed mass vs exposure time and (b) D−f plot at the 7th overtone.

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Figure 7. Representative PDM images of the adsorbed collagen IV layer on substrates with different surface potentials obtained using different functional group ratios. Scale bar: 250 nm.

significantly, and the microscopic attraction between SAMs and domains within the protein17 allows a more significant adsorption of fibronectin. In addition, similar to the former case (laminin), the 80% COOH substrate also yields higher adsorption levels compared with the pure COOH substrate and may be attributed to the protein structure arrangement. Figure 4b shows that the slopes are almost identical among these substrates, and the value is low (∼4 × 10−8 Hz−1), which indicates that the adsorbed fibronectin layer is dense, and its packing structure will not change significantly with the substrate electrostatic potential. Figure 5 also shows a similar morphology for adsorbed fibronectin on substrates with different potentials, which indicates that the surface potential does not significantly affect the structure of adsorbed fibronectin. Effect of Surface Potential on Collagen IV Adsorption. While collagen IV is macroscopically positively charged (Table 2), Figure 6a shows that adsorption decreases with an increasing negative charge on the surface. The final adsorption weight with the statistical variation is summarized in Figure 2. The analysis of variance (one-way ANOVA) revealed that the surface potential has a significant effect on the collagen IV adsorption at the 0.005 level. Using t-test, it is noted that below −42 mV (> 40% COOH), there is no significant difference on the adsorption amount. Although the ζ potential of collagen IV is similar to that of laminin discussed previously, the effect of surface potential on their adsorption shows the opposite trend. With a strong tendency to self-assemble, collagen IV molecules tend to organize into large fibril-like structures on an NH2 substrate, while sponge-like structures are observed on COOH substrates. Furthermore, NH2-induced self-assembly yields higher adsorption compared with the COOH substrate.42 The similarity among the 40−100% COOH adsorption curves is attributed to the decrease in collagen assembly, which is compensated by an increase in the Debye and short-range interactions with an increasing COOH concentration.

when water content is lower on low COOH-loaded surfaces. Moreover, a relatively small domain and ordered packing on the 80% COOH substrate and then 40%, 60%, and pure COOH were observed, which is consistent with the viscoelastic properties observed in the QCM-D experiments where stronger laminin−surface interaction is revealed. Effect of Surface Potential on Fibronectin Adsorption. Both the initial adsorption rate and final adsorbed quantity of fibronectin increased with an increasing negative charge on the surface, as shown in Figure 4a. The final adsorption weight with the statistical variation is summarized in Figure 2. The analysis of variance (one-way ANOVA) revealed that the surface potential has a significant effect on the fibronectin adsorption at the 0.005 level. While fibronectin was negatively charged in this environment (Table 2), the observed trend contradicts the prediction from the electrostatic interactions; hence, it cannot explain the experimental result. Furthermore, the final weight of the adsorbed fibronectin layer is significantly lower than for laminin. This behavior is attributed to the highly charged SAM, which microscopically polarized the protein, and the Debye interaction allows adsorption; however, the macroscopic electrostatic repulsion discourages adsorption. Nevertheless, once the macroscopic repulsion is overcome by the Debye interaction, the formation of hydrogen bonds or direct chemical bonding between SAM and protein could further enhance its adhesion. In addition, other short-range forces such as steric, solvation, etc., could also aid the adsorption.41 Furthermore, as macromolecules, proteins usually have charged domains microscopically17 that could yield short-range attraction although the net-charge yields macroscopic repulsion. For SAM-modified surfaces with low potentials, proteins are not strongly polarized by the surface, and the macroscopic electrostatic interaction dominated; hence, less adsorption was observed. Therefore, 20% COOH and 40% COOH adsorbed slightly less but comparable fibronectin to that of the pure NH2 surface. With increasing COOH loaded on the surface, the stronger surface charge polarizes the protein more 10333

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(6) Hook, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. A comparative study of protein adsorption on titanium oxide surfaces using in situ ellipsometry, optical waveguide lightmode spectroscopy, and quartz crystal microbalance/dissipation. Colloids Surf., B 2002, 24 (2), 155−170. (7) Green, R. J.; Davies, J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Surface plasmon resonance for real time in situ analysis of protein adsorption to polymer surfaces. Biomaterials 1997, 18 (5), 405−413. (8) Osaki, T.; Renner, L.; Herklotz, M.; Werner, C. Hydrophobic and electrostatic interactions in the adsorption of fibronectin at maleic acid copolymer films. J. Phys. Chem. B 2006, 110 (24), 12119−12124. (9) Wang, T.; Handschuh-Wang, S.; Yang, Y.; Zhuang, H.; Schlemper, C.; Wesner, D.; Schonherr, H.; Zhang, W. J.; Jiang, X. Controlled surface chemistry of diamond/β-SiC composite films for preferential protein adsorption. Langmuir 2014, 30 (4), 1089−1099. (10) Brzoska, J. B.; Benazouz, I.; Rondelez, F. Silanization of solid substrates. A step toward reproducibility. Langmuir 1994, 10 (11), 4367−4373. (11) Arima, Y.; Iwata, H. Effect of wettability and surface functional groups on protein adsorption and cell adhesion using well-defined mixed self-assembled monolayers. Biomaterials 2007, 28 (20), 3074− 3082. (12) Ostuni, E.; Grzybowski, B. A.; Mrksich, M.; Roberts, C. S.; Whitesides, G. M. Adsorption of proteins to hydrophobic sites on mixed self-assembled monolayers. Langmuir 2003, 19 (5), 1861−1872. (13) 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 (1), 94−103. (14) Balachander, N.; Sukenik, C. N. Monolayer Transformation by nucleophilic-substitution. Applications to the creation of new monolayer assemblies. Langmuir 1990, 6 (11), 1621−1627. (15) Shyue, J. J.; De Guire, M. R. Acid−base properties and zeta potentials of self-assembled monolayers obtained via in situ transformations. Langmuir 2004, 20 (20), 8693−8698. (16) 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 (14), 3464−3473. (17) Meder, F.; Kaur, S.; Treccani, L.; Rezwan, K. Controlling mixedprotein adsorption layers on colloidal alumina particles by tailoring carboxyl and hydroxyl surface group densities. Langmuir 2013, 29 (40), 12502−12510. (18) Gozlan, N.; Haick, H. Coverage effect of self-assembled polar molecules on the surface energetics of silicon. J. Phys. Chem. C 2008, 112 (33), 12599−12601. (19) Puniredd, S. R.; Platzman, I.; Tung, R. T.; Haick, H. Bidirectional control of silicon’s surface potential by means of molecular coverage. J. Phys. Chem. C 2010, 114 (43), 18674−18678. (20) Beurer, E.; Venkataraman, N. V.; Sommer, M.; Spencer, N. D. Protein and nanoparticle adsorption on orthogonal, charge-densityversus-net-charge surface-chemical gradients. Langmuir 2012, 28 (6), 3159−3166. (21) Capadona, J. R.; Collard, D. M.; Garcia, A. J. Fibronectin adsorption and cell adhesion to mixed monolayers of tri(ethylene glycol)- and methyl-terminated alkanethiols. Langmuir 2003, 19 (5), 1847−1852. (22) Kuo, C. H.; Chang, H. Y.; Liu, C. P.; Lee, S. H.; You, Y. W.; Shyue, J. J. Effect of surface chemical composition on the surface potential and iso-electric point of silicon substrates modified with selfassembled monolayers. Phys. Chem. Chem. Phys. 2011, 13 (9), 3649− 3653. (23) Kuo, C. H.; Liu, C. P.; Lee, S. H.; Chang, H. Y.; Lin, W. C.; You, Y. W.; Liao, H. Y.; Shyue, J. J. Effect of surface chemical composition on the work function of silicon substrates modified by binary selfassembled monolayers. Phys. Chem. Chem. Phys. 2011, 13 (33), 15122−15126. (24) Lee, S. H.; Lin, W. C.; Chang, C. J.; Huang, C. C.; Liu, C. P.; Kuo, C. H.; Chang, H. Y.; You, Y. W.; Kao, W. L.; Yen, G. J.; Kuo, D.

From Figure 6b, the slope of the pure COOH substrate differs from the other slopes, and the higher slope value indicates a less dense structure. This result is consistent with the literature, which has demonstrated that COOH is expected to produce randomly oriented collagen IV.42 Figure 7 shows structures of adsorbed collagen IV and indicates that the protein domain on the pure COOH substrate is smaller than that of the other substrate, which might abrogate the selfassembly effect and, therefore, discourage aggregation.

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CONCLUSIONS In this study, the adsorption behaviors of ECM proteins were examined, including laminin, fibronectin, and collagen IV, on Au substrates modified with binary NH2 and COOH selfassembled monolayers. The surface potential was controlled by the chemical composition; hence, the effect of surface potential on ECM adsorption was studied. The substrates presented herein include surface potentials that range from slightly positively charged to strongly negatively charged in physiological phosphate-buffered saline. The adsorption kinetics were examined using QCM-D, and the resulting film morphology was determined using PDM. The viscoelastic properties of adsorbed protein were further discussed based on the slope in D−f plot. Electrostatic attraction could play an important role and dominate the adsorption kinetics. However, the strongly charged substrate could encourage adsorption of proteins with the same macroscopic charge sign due to the Debye interaction with highly polarizable proteins, and other short-range interactions such as steric force, hydrogen bonds, charged domain within protein, etc., could enhance its adhesion. Furthermore, surface-induced self-assembly of protein molecules could also overcome electrostatic repulsion and promote protein adsorption on a surface with the same sign of macroscopic charge.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +886(2)2787-3137. E-mail: [email protected]. edu.tw. Author Contributions ⊥

These authors contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge support from the Academia Sinica and Taiwan Ministry of Science and Technology through grant number 100-2113-M-001-030-MY3.

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REFERENCES

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dx.doi.org/10.1021/la5020362 | Langmuir 2014, 30, 10328−10335