Protein Adsorption on Oligo(ethylene glycol)-Terminated

Jan 20, 2005 - Chain Flexibility in Self-Assembled Monolayers Affects Protein Adsorption and Surface Hydration: A Molecular Dynamics Study. Wesley Bec...
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J. Phys. Chem. B 2005, 109, 2934-2941

Protein Adsorption on Oligo(ethylene glycol)-Terminated Alkanethiolate Self-Assembled Monolayers: The Molecular Basis for Nonfouling Behavior Lingyan Li, Shengfu Chen, Jie Zheng, Buddy D. Ratner,* and Shaoyi Jiang* Department of Chemical Engineering, UniVersity of Washington, Seattle, Washington 98105 ReceiVed: June 18, 2004; In Final Form: NoVember 17, 2004

A study of protein resistance of oligo(ethylene glycol) (OEG), HS(CH2)11(OCH2CH2)nOH (n ) 2, 4, and 6), self-assembled monolayers (SAMs) on Au(111) surfaces is presented here. Hydroxyl-terminated OEG-SAMs are chosen to avoid the hydrophobic effect observed with methyl-terminated OEG-SAMs, particularly at high packing densities. The structure of the OEG-SAM surfaces is controlled by adjusting the assembly solvent. These SAMs were characterized by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Protein adsorption on these surfaces was investigated by surface plasmon resonance (SPR). OEGSAMs assembled from mixed ethanol and water solutions show higher packing density on gold than those from pure ethanol solution. For EG2OH- and EG4OH-SAMs, proteins (i.e., fibrinogen and lysozyme) adsorb more on the densely packed SAMs prepared from mixed ethanol and water solutions, while EG6OH-SAMs generally resist protein adsorption regardless of the assembly solvent used.

1. Introduction An important challenge to biomaterials scientists is the prevention of nonspecific protein adsorption on surfaces. Nonspecific protein adsorption degrades the performance of surface-based diagnostic devices and may have an adverse effect on the healing process for implanted biomaterials. Since the early 1980s, surface-grafted poly(ethylene glycol) (PEG)-based materials have been used to inhibit protein adsorption from biological media.1,2 Subsequently, OEG (oligo(ethylene glycol))terminated alkanethiolate self-assembled monolayers (SAMs) were also found to resist protein adsorption.3-5 The fouling resistance of PEG polymers is often explained by a “steric” repulsion model.6,7 In this model, steric repulsion, resulting from the compression of PEG chains as protein approaches the surface, was mainly responsible for prevention of protein adsorption. This model is suitable for those systems with long PEG chains. Experimental and simulation results show that the protein resistance of PEG polymers depends on the density and thickness of the PEG chains.6,8-10 PEG polymers with high surface densities and long chain lengths exhibit optimal protein resistance. Szleifer et al.11,12 improved the model of Jeon et al.6 using the single-chain mean field (SCMF) theory. They found that the surface density of the grafted polymer was the most important parameter in preventing protein adsorption while chain length had a weak effect. In the Jeon and Szleifer models, protein-PEG interactions are assumed to be repulsive; that is, PEG chains resist protein adsorption. They studied the effects of various physical parameters such as the length and density of surface-grafted PEG chains on protein adsorption. While the Jeon and Szleifer models provide some insight regarding the mechanism of surface resistance to protein adsorption, their models do not provide molecular-level information.13 In their work, water was treated either as a continuous medium or as a homogeneous spherical noninteracting molecule, and the detailed conformational changes of the polymer chains were ignored. * To whom correspondence should be addressed. E-mail: (S.J.) [email protected]; (B.D.R.) [email protected].

Despite extensive research in this area, a molecular-level understanding of the nonfouling mechanism for these more densely packed, shorter OEG-SAMs is lacking. It has been generally accepted that water at the protein-OEG interface plays a very important role in the resistance of OEG to protein adsorption.13-17 The water barrier theory is also used to interpret the nonfouling behavior of a surface; that is, protein cannot adsorb on the OEG surface because there is a layer of tightly bound water molecules around OEG chains. Therefore, the protein resistance of OEG-SAMs is mainly due to the difficulty in the dehydration of both the ethylene glycol chains/segments and the protein. Conformational flexibility may not be required for the OEG surface to be protein resistant because densely packed, shorter OEG-SAMs have less freedom for conformational change upon protein adsorption.18 This hypothesis is based on the assumption that OEG-terminated alkanethiols form densely packed SAMs on gold surfaces, similar to the structure of methyl-terminated alkanethiols. However, it has been shown that OEG-SAMs prepared from pure ethanol solution are not highly ordered and form incomplete monolayers.3,19,20 It was shown in our previous work and the work of others that the size of well-ordered domains and the packing conformation of SAMs largely depend on assembly solution conditions.19,21,22 Both the polarity of the solvent and the assembly solution temperature can affect the structure of SAMs at the nanoscale level.21,22 These nanoscale structures of SAMs could affect protein adsorption behavior.21 Recently, it was reported that higher protein adsorption was observed on methoxy-terminated OEG-SAM surfaces with higher lateral packing density or more ordered structures.19,20,23,24 Harder et al.20 and Herrwerth et al.23 reported that OEG-SAMs generally have a higher packing density on silver than on gold. Methoxy-terminated OEG-SAMs are protein resistant when assembled on gold, but not on silver. However, in contrast to the methoxy-terminated OEG-SAMs, hydroxyl-terminated OEGSAMs show full protein resistance on both gold and silver. Vanderah et al.19 reported that ordered or disordered methylterminated OEG-SAMs can be formed by assembly from

10.1021/jp0473321 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/20/2005

Protein Adsorption on Alkanethiolate SAMs solvents other than ethanol. In their work, lysozyme adsorbed to the SAMs assembled from 95% ethanol/5% water and pure ethanol, while BSA adsorbed to the SAMs assembled from 95% ethanol only. For methoxy-terminated OEG-SAMs, the increase in protein adsorption could be due to the increase in the hydrophobic sites on the surfaces or the combined effects of hydrophobicity and the surface packing density, which induce changes in the chain flexibility and associated water layers. For protein adsorption measurements, both Herrwerth23 and Vanderah19 used ex-situ techniques, ellipsometry, and reflectionabsorption infrared spectroscopy, respectively. A surface plasmon resonance (SPR) sensor was used in our work to monitor the process of protein adsorption on surfaces. SPR allows for real-time and label-free detection of protein adsorption and its kinetics with high sensitivity (about 1 pg/1 mm2). In this work, the protein resistance of OEG-SAMs with different ethylene glycol repeat units was systematically studied. The structures of the OEG-SAM surfaces were altered by adjusting the assembly solvent. These SAMs were characterized by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Protein adsorption on these surfaces was measured by SPR. In addition to the experimental studies, we also carried out molecular simulations of protein interactions with OEG-SAMs in the presence of explicit water molecules and ions to examine the mobility and hydration of OEG-SAMs. Simulation results provide valuable information, which is often difficult to obtain from laboratory experiments.25 On the basis of our experimental and simulation results, we would like to address the following important issues: why and under what conditions are OEG-SAMs nonfouling, and whether there is a correlation between bound water molecules around OEG chains and the nonfouling behavior of OEG-SAMs. 2. Experimental Section Materials. (1-Mercaptoundec-11-yl)di(ethylene glycol) (EG2OH), (1-mercaptoundec-11-yl)tetra(ethylene glycol) (EG4OH), and (1-mercaptoundec-11-yl)hexa(ethylene glycol) (EG6OH) were purchased from Prochimia (Poland) and used as received. 11-Mercaptoundecanol (C11OH), dodecanethiol (C12), and hexadecanethiol (C16) were purchased from Aldrich Chemical Co. Fibrinogen (fraction I from bovine plasma), lysozyme (L6876), bovine serum albumin (BSA, A7638), and phosphate buffered saline (PBS, 138 mM NaCl, 2.7 mM KCl, pH 7.4) were purchased from Sigma Chemical Co. Ethanol (absolute 200 proof) was purchased from AAPER Alcohol and Chemical Co. Au(111) Preparation. For XPS and AFM experiments, gold substrates were prepared by the vapor deposition of gold onto freshly cleaved mica (Asheville-Schoonmaher Mica Co.) in a high-vacuum evaporator (BOC Edwards Auto306) at ∼10-7 Torr. Mica substrates were preheated to 325 °C for 2 h by a radiation heater before deposition. Evaporation rates were 0.10.3 nm/s, and the final thickness of gold films was around 200 nm. Gold-coated substrates were annealed in an H2 flame for 1 min before use. SAM Preparation. SAMs were formed by soaking goldcoated substrates (immediately after vacuum deposition or annealing by an H2 flame) in the solutions of alkanethiols (0.2 mM) overnight at room temperature in one of three different solvents: absolute ethanol, 95% ethanol and 5% distilled water, or distilled water. SAMs were then rinsed extensively with water and ethanol and were dried in a stream of N2. X-ray Photoelectron Spectroscopy (XPS). XPS spectra were obtained on a Surface Science Instruments (SSI) S-Probe XPS.

J. Phys. Chem. B, Vol. 109, No. 7, 2005 2935 An aluminum KR 1, 2 monochromatized X-ray source is used to stimulate photoemission. The energy of the emitted electrons is measured with a hemispherical energy analyzer at pass energies ranging from 50 to 150 eV. Lower pass energies provide high spectral resolution but slow data acquisition and less accurate quantitative analysis. The binding energy (BE) scale is referenced by setting the peak maximum in the C1s spectrum to 285.0 eV. Spectra are collected with the analyzer at 55° with respect to the surface normal of the sample. Typical pressures in the analysis chamber during spectral acquisition are 10-9 Torr. Data analysis software from Service Physics, Inc. is used to calculate elemental compositions from the peak areas. Surface Plasmon Resonance (SPR) Sensor. A home-built SPR sensor was used for measurements of protein adsorption.26,27 For the SPR, a parallel polychromatic light beam passes through an optical prism coated with a thin gold layer and excites surface plasmons at the interface between the gold layer and the analyte. Excitation of the surface plasmon is accompanied by the transfer of optical energy into the surface plasmon and the dissipation of optical energy in the metal layer, resulting in a narrow dip in the spectrum of reflected light. The wavelength at which resonant excitation occurs depends on the refractive index of the analyte in the proximity of the SPR surface. Thus, the analytes at the interface can be quantified by measuring the refractive index change-induced shift in resonant wavelength. SPR chips were prepared by coating glass substrates with an adhesion-promoting chromium layer (thickness ∼2 nm) and a surface plasmon active gold layer (thickness ∼48 nm) using electron beam evaporation in a vacuum followed by further surface functionalization. A chip was attached to the base of the prism, and optical contact was established using refractive index matching fluid (Cargille). A dual-channel Teflon flow cell with two independent parallel flow channels (one is the sensing channel, while the other is the reference channel) was used to contain liquid sample during experiments. A peristaltic pump (Ismatec) was utilized to deliver liquid sample to the two channels of the flow cell. Protein solutions of ∼1.5 mg/mL in PBS at pH 7.4 were flowed over SAM surfaces at a flow rate of 0.05 mL/min. 3. Results and Discussion Characterization of SAMs. SAMs of HS(CH2)11(CH2CH2O)nOH (where n ) 2, 4, and 6) were prepared using three separate solvents: 100% ethanol, 95% ethanol, and distilled water. Table 1 shows the (C+O)/Au ratio of various OEGSAMs calculated from the XPS compositional data. Previously, the C/Au ratio has been used to monitor the increase of carbon on the surface and the attenuation of the underlying gold signal for alkanethiol SAMs. For methyl-terminated alkanethiol selfassembled on gold surfaces, Graham found that the C/Au ratio changes linearly with the carbon chain length.28 For OEGSAMs, we will use the (C+O)/Au ratio instead to monitor their relative thickness. From Table 1, it can be seen that the (C+O)/ Au ratio increases as a function of the number of ethylene glycol units, which suggests a consistent increase in the thickness of OEG-SAMs. Furthermore, the (C+O)/Au ratio is higher for OEG-SAMs prepared from 95% ethanol than those from pure TABLE 1: (C+O)/Au Ratio from XPS (C+O)/Au

EG2OH

EG4OH

EG6OH

ethanol 95% ethanol H2O

3.1 ( 0.2 3.4 ( 0.3 2.7 ( 0.2

3.6 ( 0.2 4.8 ( 0.3 4.2 ( 0.3

5.8 ( 0.4 7.2 ( 0.4 7.4 ( 0.4

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Li et al. TABLE 2: C-O/C-C Ratio for OEG-SAMs from High-Resolution C1s XPS Scans C-O/C-C

EG2OH

EG4OH

EG6OH

ethanol 0.62 ( 0.03 1.12 ( 0.05 1.36 ( 0.07 experiment 95% ethanol 0.63 ( 0.03 1.15 ( 0.05 1.8 ( 0.1 theoretical value 0.5 0.9 1.3

Figure 1. High-resolution spectra of the C1s region from XPS for OEGSAMs prepared in ethanol solution.

Figure 2. High-resolution spectra of the C1s region from XPS for EG6OH-SAMs prepared from 95% ethanol solution (the solid line) and from 100% ethanol solution (the dashed line). The ether signal (OCH2) is stronger for EG6OH-SAMs prepared from 95% ethanol solution than those from 100% ethanol solution.

ethanol, which indicates that the thicknesses of the OEG-SAMs prepared from 95% ethanol solution are higher than those prepared from pure ethanol solution. It also can be seen that the influence of water content in the assembly solution on the (C+O)/Au ratio (or the thickness of the films) increases as the number of ethylene glycol units increases. Besides the (C+O)/Au ratio obtained from the surface compositional data, the C-O/C-C ratio obtained from the peak fitting of the high-resolution C1s spectrum also indicates that the packing density of OEG-SAMs prepared from 95% ethanol is higher than those prepared from pure ethanol. Figure 1 compares the high-resolution C1s spectra for OEG-SAMs with different EG units prepared in pure ethanol. The C1s spectra show two distinct peaks: one is around 285 eV, a characteristic peak of the internal units of the polymethylene chain (CH2CH2CH2) while the other appears around 286.5 eV, corresponding to the ether carbon atoms (OCH2). The relative intensity of the ether peak increases with the number of ethylene glycol units, as expected. Figure 2 shows the high-resolution spectra of the C1s region for EG6OH-SAMs prepared from two different solvents: pure ethanol and 95% ethanol. The highresolution C1s XPS spectra show that the ether signal (OCH2) is stronger for OEG-SAMs prepared from 95% ethanol solution than those from 100% ethanol solution. The C-O/C-C ratios as determined from the peak fitting of the high-resolution spectra of C1s are summarized in Table 2. For the same OEG-SAMs,

the higher C-O/C-C ratio indicates more EG units at the outermost surface of the SAM and/or an OEG-SAM structure with higher total EG unit density. By monitoring the S2p binding energy, the molecular environment of the bond between the sulfur atom and the gold surface can be characterized. Thus, we studied the high-resolution S2p spectrum for all of the OEG-SAMs prepared from different solvents. Figure 3 gives one example for EG2OH-SAMs prepared from 100% and 95% ethanol solutions. The highresolution sulfur data show up to 90% sulfur species at 162 eV, indicating that sulfur is bound to the gold surface.29 The results suggest that the increase in the (C+O)/Au ratio is due to a higher surface packing density rather than unbound alkanethiol molecules on the surface. The XPS results discussed above suggest that the OEG-SAMs assembled from 95% ethanol generally have a higher packing density than those prepared from 100% ethanol. The assembly solvent influences the structure of a monolayer, particularly for those headgroups capable of forming hydrogen bonds. In the case of OEG-SAMs, a thin water film within the ethylene glycol segments could further stabilize a helical or random conformation of the OEG-SAMs, resulting in a compact SAM structure. Moreover, as the number of ethylene glycol units increases, the influence of water content in the assembly solvent on the structure of OEG-SAMs becomes greater. EG6OH-SAMs prepared from pure water have an even higher (C+O)/Au ratio (or packing density) than those prepared from 100% or 95% ethanol solutions. Techniques such as sum frequency generation (SFG) or quartz crystal microbalance (QCM) could be used to probe water molecules trapped around the OEG segments of the SAMs and are currently being applied to this problem. Adsorption of Proteins to SAMs. SPR allows for real-time and label-free detection of protein adsorption and its kinetics with high sensitivity. In SPR experiments, the surfaces were first washed with PBS for 3 min, then a solution of protein was passed through the SPR flow cell for 10 min, and finally the adsorbed layer of protein was washed with buffer for 10 min. The proteins used in this work, that is, fibrinogen, lysozyme, and BSA, were chosen because of their structures, sizes, and pI values. Fibrinogen is a large protein (MW ) 340 kDa, pI ) 5.5) that adsorbs readily onto hydrophobic and charged surfaces. Horbett and co-workers found that the surfaces that are resistant to fibrinogen adsorption also prevent the adhesion of mammalian cells.30 Lysozyme is a small and “hard” protein (MW ) 14.7 kDa, pI ) 11.1) and is positively charged in neutral buffer solutions. It is often used as a model in studies of protein adsorption. BSA (MW ) 67 kDa, pI ) 4.7) is the most abundant protein in blood and has been used widely as a preadsorbed protein in medical applications to inhibit thrombus formation. The structural stability of BSA is lower than that of lysozyme.31 Figure 4 gives representative SPR wavelength shift plots as a function of time (sensorgrams) of fibrinogen (Figure 4a and c) and lysozyme (Figure 4b and d) adsorption onto EG4OH(Figure 4a and b) and EG6OH-SAMs (Figure 4c and d). In this work, the surface coverage of each protein on C16-SAMs (Figure 5a) is used to normalize the amount of adsorbed proteins on each of the OEG-SAMs. The normalized coverage of fibrinogen and lysozyme on EG4OH-SAMs assembled from the

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Figure 3. High-resolution spectra of the S2p region from XPS for EG2OH-SAMs prepared from (a) 100% ethanol and (b) 95% ethanol solutions. The peaks were fit using two S2p doublets with 2:1 area ratio and splitting of 1.2 eV.29 The high-resolution sulfur data show up to 90% sulfur species at 162 eV, indicating that the majority of sulfur is bound to the gold surface.

95% ethanol solution is about 8.5% and 6.4%, respectively. Table 3 shows the coverage of various proteins adsorbed on OEG-SAMs prepared from different solvents. The results in Table 3 and Figure 4 show that fibrinogen and lysozyme adsorb to the EG2OH and EG4OH-SAM surfaces assembled from 95% ethanol but not on the OEG-SAMs assembled from either 100% ethanol or distilled water. However, BSA does not adsorb to any of the OEG-SAMs assembled from the three different solvents. Prime and Whitesides5 observed that mixed SAMs of EG6OH and HS(CH2)10CH3 begin to adsorb protein as the mole fraction of EG6OH decreases to 0.5, when the density of OEGSAMs is too low to cover the surface. Previously, OEG-SAMs were often prepared in ethanol solution. It has been shown that

OEG-SAMs prepared from pure ethanol solution are not highly ordered and form incomplete monolayers.3,19,20 The results in Tables 1-3 and Figure 4 show that the EG2OH- and EG4OHSAMs assembled from a mixed ethanol and water solution have higher surface packing density than those formed in ethanol solution and adsorb ∼6-9% of a monolayer of proteins, such as fibrinogen and lysozyme as compared to less than ∼1% in the case of OEG-SAMs formed in pure ethanol solution. For the HS(OCH2CH2)6CH3 system, Vanderah and co-workers19 also found that bovine serum albumin adsorbed to the most ordered, helical HS(OCH2CH2)6CH3 SAMs, but did not adsorb to the disordered HS(OCH2CH2)6CH3 SAMs. The results show that when the surface density of OEG-SAMs is too high, OEGSAMs would adsorb a certain amount of protein. Figure 6 shows

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Figure 4. SPR sensorgrams of fibrinogen (a and c) and lysozyme (b and d) adsorbed onto EG4OH- (a and b) and EG6OH-SAMs (c and d) prepared from 95% ethanol solution (solid lines) and 100% ethanol solution (dashed lines).

the amount of adsorbed fibrinogen versus the normalized surface packing density of EG4OH. The surface packing density of EG4OH is normalized to that of EG4OH prepared from 95% ethanol solution. EG4OH-SAMs with lower packing densities were prepared by adding C12 to the EG4OH, while EG4OH-SAMs with higher surface packing densities were controlled by varying assembly solvents, as discussed previously. Results show that these SAMs resist protein adsorption within a certain range of the normalized surface OEG densities (∼0.6-0.8), yet adsorb proteins when the OEG surface density is too high or too low. In parallel, molecular simulations were performed to study the interactions between protein (e.g., lysozyme) and CH3-, OH-, or (OCH2CH2)4OH-terminated SAMs in the presence of explicit water molecules and ions.25 The surface density of (OCH2CH2)4OH groups was controlled by adjusting the surface composition of mixed SAMs of SH(CH2)4(OCH2CH2)4OH and SH(CH2)4OH. In the MD study, the mobility of chains was monitored by the root-mean-square derivations (RMSD) values. The RMSD value was calculated by translating and rotating the coordinates of the instantaneous structure to superimpose the reference structure with maximum overlap. A geometric criterion was used to identify hydrogen bonds. A hydrogen bond exists if the donor-acceptor distance is less than 0.35 nm and the hydrogen donor-acceptor angle is smaller than 60°. Our simulation results show that the total number of hydrogen bonds per unit area between water molecules and OEG chains is higher around xOEG ) 0.5 and 0.8, but decreases for densely packed pure OEG-

SAMs or dilute mixed OEG-SAMs at xOEG ) 0.2 (Figure 7). Furthermore, our simulation results also show that those highly hydrated chains in mixed OEG-SAMs exhibit larger mobility at xOEG ) 0.5 and 0.8 (Figure 7). By comparing these experimental and simulation results, it appears that there is a correlation between the resistance of OEG-SAMs to protein adsorption and the amount of hydrogen bonds (or the mobility of OEG chains); that is, the highly mobile and highly hydrated OEG-SAMs have better nonfouling properties. The recent simulation work by Grunze et al.15 also pointed out the importance of the surface density of hydrogen bonds to the surface resistance to protein adsorption. Their results showed that more water molecules penetrated into helical (on Au) than all-trans (on Ag) OEG-SAMs to form hydrogen bonds with OEG chains, leading to the prevention of protein adsorption on the OEG-covered gold surface. However, it was pointed out later that the conformation of EG chains in aqueous solutions is either a random coil or a helix,23,32 not a zigzag all-trans conformation as reported previously.15 Here, we will use fibrinogen as an example to present our nonfouling mechanism of OEG-SAMs. Figure 8 shows the amount of adsorbed fibrinogen as a function of the number of ethylene glycol units of OEG-SAMs prepared from two assembly conditions. An OH-terminated SAM is also included in Figure 8 as a reference for comparison with the OEG-SAMs. Protein adsorption on the OH-terminated SAMs is much smaller than on CH3-, NH2-, or COOH-SAMs. Therefore, OH-

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Figure 6. The amount of adsorbed fibrinogen versus the surface packing density of EG4OH. The surface packing density of EG4OH is defined as the ratio of the area under the O1s peak from the SAM surface to that of the pure EG4OH-SAM surface prepared from 95% ethanol solution, the surface packing density of which is taken to be 100%.

Figure 5. SPR sensorgrams of fibrinogen (solid lines) and lysozyme (dashed lines) adsorption onto C16- (a) and C11OH-SAM surfaces (b).

TABLE 3: Percentage of the Surface Coverage of Proteins on OEG-SAMs Measured by SPR and Normalized to the Adsorbed Amount on C16/Au EG2OH

EG4OH

EG6OH

Fg LYZ BSA Fg LYZ BSA Fg LYZ BSA ethanol 0 95% ethanol 6.6 H2O 0

0 5.0 0

0 0 0

0 8.5 0

0 6.4 0

0 0 0

0 0 0

0 0 0

0 0 0

terminated SAMs are often used to mimic a “low-fouling” surface. As can be seen, when n g 2, OEG-SAM surfaces significantly reduce protein adsorption, which is consistent with previous findings.3,5,23 Our SPR results show that proteins (i.e., fibrinogen and lysozyme) adsorb on OEG-SAMs (n ) 2 and 4) prepared from 95% ethanol but not on those prepared from 100% ethanol solutions. Although the adsorbed amount of protein is small (∼18 ng/cm2 or ∼8% surface coverage) on the OEG-SAMs prepared from the 95% ethanol solution, it was shown previously that as little as 10 ng/cm2 adsorbed fibrinogen could still induce monocyte adhesion in vitro.30 However, longer-chain OEG-SAMs (e.g., n ) 6) resist protein adsorption even for those prepared from 95% ethanol and pure water solutions. The different adsorption behaviors of proteins on C11OH and OEG-SAMs prepared from different solvents suggest that the mobility and hydration of the ethylene glycol chains are important for a surface to resist protein adsorption. This is consistent with our recent molecular simulation studies.25

Figure 7. The number of hydrogen bonds with associated water molecules (white bars) and the mobility of OEG chains (black bars) at different OEG compositions calculated from molecular simulations.

Figure 8. The amount of adsorbed fibrinogen versus the number of ethylene glycol units for OEG-SAMs prepared from 95% ethanol (the dashed line) and 100% ethanol solution (the solid line).

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Figure 9. Tapping mode AFM images obtained in water for EG2OH- (a), EG4OH- (b), and EG6OH-SAMs (c) on Au(111) prepared from 95% ethanol solution, with a z bar of 5 nm. The assembly solvent does not affect the surface morphology of these OEG-SAMs.

Protein adsorption on the OEG-SAM surface is disfavored in two aspects. If a protein molecule adsorbs on the OEG surface, water molecules associated with the OEG chains will be released into the bulk and the OEG chains will be somewhat compressed. An increase in enthalpy due to chain dehydration and a decrease in entropy due to chain compression (even though the latter term could be small) are both thermodynamically unfavorable, although an increase in entropy due to the release of water molecules associated with the OEG chains to the bulk favors protein adsorption. In the case of the OEG-SAMs, the factors unfavorable to protein adsorption prevail. Thus, an OEG-SAM surface resists protein adsorption. As compared to OEG-SAMs, the OH-terminated SAM is a more rigid surface and retains few water molecules. Therefore, it adsorbs a greater amount of protein. When the assembly solvent is changed to form OEGSAMs with a more ordered structure on gold surfaces, the packed chains leave little room for water penetration and also lose some of their mobility, leading to an increase in protein adsorption. XPS results show that OEG-SAMs prepared from 95% ethanol are thicker (or higher packing density or of a more

ordered structure) as compared to those prepared from 100% ethanol. Figure 8 also shows that the extent of surface resistance to protein adsorption depends not only on the packing density but also on the number of the repeat ethylene glycol units. EG6OH-SAMs generally resist protein adsorption regardless of the assembly solvent used. EG6OH-SAMs may have lower packing densities than EG2OH- and EG4OH-SAMs even for those prepared from 95% ethanol and pure water due to the longer repeat unit of the ethylene glycol group. These less densely packed OEG-SAMs exhibit higher mobility and trap more water molecules, leading to a reduction in protein adsorption. Therefore, very high or low OEG surface densities will lead to protein adsorption. Appropriate OEG surface densities are required for its nonfouling behavior as illustrated in Figure 6. Figure 9 shows AFM images obtained in water for EG2OH(Figure 9a), EG4OH- (Figure 9b), and EG6OH-SAMs (Figure 9c) on Au(111). The AFM images show that, regardless of the assembly solvent, the surface is featureless for EG2OH- and EG4OH-SAMs on gold except for defects and domains from the gold substrates. However, for EG6OH-SAMs on gold, the

Protein Adsorption on Alkanethiolate SAMs AFM image shows that they form small domains or terraces on the surface about 9-15 nm in width. EG6OH-SAMs prepared from 95% ethanol show the same terrace structure, but with a larger width of 13-25 nm. We acquired AFM images of these SAMs in both liquid and air and in both tapping- and contactmode AFM. Results show that these features as described above do not depend on scanning conditions (i.e., scanning medium or operating mode). As the chain length of the ethylene glycol terminal group increases, the longer chain length will lead to lower OEG-SAMs densities. Thus, it is expected that for EGnOH-SAMs, where n g 6, they will maintain their ability to resist protein adsorption due to their lower packing density regardless of the assembly solvent used. Our SPR results in Table 3 show that BSA is not sensitive to the small structure changes in OEG-SAMs. Vert and Domurado pointed out that albumin and PEG polymers were compatible in PBS at room temperature whereas fibrinogen and PEG phase-separated and were incompatible.33 They argue that the stealth effect of PEG is primarily due to the compatibility between PEG and albumin, thus transitioning the PEG-coated surfaces to appear like native albumin. This may explain why BSA behaviors differently from fibrinogen and lysozyme, that is, OEG-SAMs prepared from 95% ethanol resist BSA adsorption, but adsorb fibrinogen and lysozyme in the range 6-9% of the saturation coverage. 4. Conclusions In this work, we studied the adsorption of three proteins, that is, fibrinogen, lysozyme, and BSA, onto EGnOH-SAM (where n ) 2, 4, and 6) surfaces. The surface coverage of OEG-SAMs is controlled by changing the assembly solvent. OEG-SAMs assembled from 95% ethanol solution show a higher packing density on gold than from 100% ethanol solution, based on XPS measurements. AFM images show that the EG6OH-SAM has a surface morphology different from those of the EG2OH- and EG4OH-SAMs. Exposure of various OEG-SAMs to proteins establishes a correlation between OEG-SAM packing structure on gold and protein resistance. Our SPR results show that proteins (i.e., fibrinogen and lysozyme) adsorb more on the densely packed SAMs of EG2OH and EG4OH. The extent of the surface resistance to protein adsorption depends not only on the packing density but also on the number of the repeat ethylene glycol units. EG6OH-SAMs generally resist protein adsorption regardless of the assembly solvent used due to lower packing density. For OEG-SAMs, both the hydration and the conformational flexibility of the ethylene glycol chains are responsible for their resistance to protein adsorption, which is consistent with our recent molecular simulation results. Acknowledgment. We gratefully acknowledge the National Science Foundation for financial support under CTS-0308598, CTS-0092699, and EEC-9529161 (UWEB). We thank Professor

J. Phys. Chem. B, Vol. 109, No. 7, 2005 2941 Allan Hoffman and Tom Horbett for helpful discussions. The XPS experiments were performed at the National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/ BIO), which is supported by NIBIB grant # EB02027. References and Notes (1) Harris, J. M. Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; Plenum: New York, 1992. (2) Bailey, F. E., Jr.; Koleske, J. Y. Poly(ethylene Oxide); Academic: New York, 1984. (3) Palegrosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12. (4) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (5) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (6) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; De Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (7) Jeon, S. I.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142, 159. (8) Shalaby, S. W. Polymers as Biomaterials; Plenum: New York, 1984. (9) Gombotz, W. R.; Guanghui, W.; Hoffman, A. S. J. Appl. Polym. Sci. 1989, 37, 91. (10) Leckband, D.; Sheth, S.; Halperin, A. J. Biomater. Sci., Polym. Ed. 1999, 10, 1125. (11) Szleifer, I. Biophys. J. 1997, 72, 595. (12) McPherson, T.; Kidane, A.; Szleifer, I.; Park, K. Langmuir 1998, 14, 176. (13) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605. (14) Feldman, K.; Hahner, G.; Spencer, N. D.; Harder, P.; Grunze, M. J. Am. Chem. Soc. 1999, 121, 10134. (15) Pertsin, A. J.; Grunze, M. Langmuir 2000, 16, 8829. (16) Pertsin, A. J.; Hayashi, T.; Grunze, M. J. Phys. Chem. B 2002, 106, 12274. (17) Luk, Y. Y.; Kato, M.; Mrksich, M. Langmuir 2000, 16, 9604. (18) Kane, R. S.; Deschatelets, P.; Whitesides, G. M. Langmuir 2003, 19, 2388. (19) Vanderah, D. J.; Valincius, G.; Meuse, C. W. Langmuir 2002, 18, 4674. (20) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426. (21) Li, L. Y.; Chen, S. F.; Jiang, S. Y. Langmuir 2003, 19, 2974. (22) (a) Yamada, R.; Wano, H.; Uosaki, K. Langmuir 2000, 16, 5523. (b) Yamada, R.; Sakai, H.; Uosaki, K. Chem. Lett. 1999, 667. (23) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9359. (24) After the acceptance of this paper, we noticed that the effect of OEG density on protein adsorption was also discussed in a related paper: Vanderah, D. J.; La, H.; Naff, J.; Silin, V.; Rubinson, K. A. J. Am. Chem. Soc. 2004, 126, 13639. (25) Zheng, J.; Li, L. Y.; Chen, S. F.; Jiang, S. Y. Langmuir 2004, 20, 8931. (26) Homola, J. Sens. Actuators, B 1997, 41, 207. (27) Homola, J.; Dostalek, J.; Chen, S. F.; Rasooly, A.; Jiang, S. Y.; Yee, S. S. Int. J. Food Microbiol. 2002, 75, 61. (28) Graham, D. University of Washington, personal communications. (29) Castner, D. G.; Hinds, K.; Grainger, D. W. Langmuir 1996, 12, 5083. (30) Shen, M. C.; Pan, Y. V.; Wagner, M. S.; Hauch, K. D.; Castner, D. G.; Ratner, B. D.; Horbett, T. A. J. Biomater. Sci., Polym. Ed. 2001, 12, 961. (31) Norde, W.; Anusiem, A. C. I. Colloids Surf. 1992, 66, 73. (32) Tasaki, K. J. Am. Chem. Soc. 1996, 118, 8459. (33) Vert, M.; Domurado, D. J. Biomater. Sci., Polym. Ed. 2000, 11, 1307.