Colloids and Surfaces B: Biointerfaces 122 (2014) 552–558

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Membrane protein resistance of oligo(ethylene oxide) self-assembled monolayers Amit Vaish a,b , David J. Vanderah c,d,∗ , Ryan Vierling c,d,1 , Fay Crawshaw c,d,2 , D. Travis Gallagher c,d , Marlon L. Walker e,∗∗ a

National Institute of Standards and Technology (NIST) Center for Neutron Research, Gaithersburg, MD 20899, USA Department of Materials Science and Engineering, University of Maryland, College Park, MD 20742, USA c Biomolecular Measurement Division, Material Measurement Laboratory, NIST, Gaithersburg, MD 20899, USA d Institute for Bioscience and Biotechnology Research (IBBR), Rockville, MD 20850, USA e Materials Measurement Science Division, Material Measurement Laboratory, NIST, Gaithersburg, MD 20899, USA b

a r t i c l e

i n f o

Article history: Received 12 March 2014 Received in revised form 11 July 2014 Accepted 19 July 2014 Available online 31 July 2014 Keywords: Membrane protein resistance Oligo(ethylene oxide) self-assembled monolayers

a b s t r a c t As part of an effort to develop biointerfaces for structure-function studies of integral membrane proteins (IMPs) a series of oligo(ethylene oxide) self-assembled monolayers (OEO-SAMs) were evaluated for their resistance to protein adsorption (RPA) of IMPs on Au and Pt. Spectroscopic ellipsometry (SE) was used to determine SAM thicknesses and compare the RPA of HS(CH2 )3 O(CH2 CH2 O)6 CH3 (1), HS(CH2 )3 O(CH2 CH2 O)6 H (2), [HS(CH2 )3 ]2 CHO(CH2 CH2 O)6 CH3 (3) and [HS(CH2 )3 ]2 CHO(CH2 CH2 O)6 H (4), assembled from water. For both substrates, SAM thicknesses for 1 to 4 were found to be comparable indicating SAMs with similar surface coverages and OEO chain order and packing densities. Fibrinogen (Fb), a soluble plasma protein, and rhodopsin (Rd), an integral membrane G-protein coupled receptor, adsorbed to the SAMs of 1, as expected from previous reports, but not to the hydroxy-terminated SAMs of 2 and 4. The methoxy-terminated SAMs of 3 were resistant to Fb but, surprisingly, not to Rd. The stark difference between the adsorption of Rd to the SAMs of 3 and 4 clearly indicate that a hydroxy-terminus of the OEO chain is essential for high RPA of IMPs. The similar thicknesses and high RPA of the SAMs of 2 and 4 show the conditions of protein resistance (screening the underlying substrate, packing densities, SAM order, and conformational mobility of the OEO chains) defined from previous studies on Au are applicable to Pt. In addition, the SAMs of 4, exhibiting the highest resistance to Fb and Rd, were placed in contact with undiluted fetal bovine serum for 2 h. Low protein adsorption (≈12.4 ng/cm2 ), obtained under these more challenging conditions, denote a high potential of the SAMs of 4 for various applications requiring the suppression of non-specific protein adsorption. Published by Elsevier B.V.

1. Introduction

Abbreviations: RPA, resistance to protein adsorption; OEO, oligo(ethylene oxide); TLC, thin layer chromatography; SAM, self-assembled monolayer; AAO, anodized aluminum oxide; IMP, integral membrane protein; FBS, fetal bovine serum; Fb, fibrinogen; Rd, rhodopsin; CA, contact angle. ∗ Corresponding author at: Biomolecular Measurement Division, Material Measurement Laboratory, NIST, Gaithersburg, MD 20899, USA. Tel.: +1 240 314 6266. ∗∗ Corresponding author. Tel.: +1 301 975 5593. E-mail addresses: [email protected], [email protected] (D.J. Vanderah), [email protected] (M.L. Walker). 1 Current address: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 2 Current address: James Madison University, Department of Chemistry and Biochemistry, Harrisonburg, VA 22807, USA. http://dx.doi.org/10.1016/j.colsurfb.2014.07.031 0927-7765/Published by Elsevier B.V.

Surfaces resistant to nonspecific protein adsorption are an essential component of non-crystallographic structure-function studies of integral membrane proteins (IMPs). IMPs comprise a significant portion of the human genome (20–30%) and play vital roles in many functions of the cell [1]. Our objective is the creation of a biosurface containing oriented IMPs embedded in a membrane-like matrix on a high surface area substrate. Such membrane-functionalized constructs enable the application of neutron scattering and solid-state nuclear magnetic resonance (NMR) techniques to study proteins that are not amenable to crystallization as well as protein–antigen, protein–agonist, and protein–protein interactions in biologically-relevant milieus. Construction of the desired biosurface (Fig. 1) involves several steps. Briefly, an anodized aluminum oxide (AAO) surface is initially

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Fig. 1. Schematic of biosurface containing an oriented IMP such as a G-protein coupled receptor (GPCR) in a lipid-like bilayer on Pt-coated AAO membranes.

coated with a metal such as Pt, to which is chemisorbed a mixed self-assembled monolayer (SAM) containing a capture agent, in our case compounds with a nitrilotriacetic acid (NTA) terminus, laterally diluted with molecules that exhibit high resistance to protein adsorption (RPA) [2]. Exposure of the capturing SAM to polyhistidine-tagged (his-tag) IMPs, after activation by nickel to the NTA–Ni+ complex, results in the well-established oriented protein capture via the strong NTA–Ni+ –his-tag interaction. Finally, a bilayer is constructed around the captured IMP to sustain it in its native (active) conformation. It is imperative that nonspecific protein adsorption be minimized in the protein capture step for meaningful interpretation of the scattering or NMR data. Many surfaces modified by polymers or SAMs have been shown to be protein resistant [3–15]. Surfaces modified with chains of the ethylene oxide [EO] motif, i.e., (CH2 CH2 O)n , are the most well studied and understood and, generally, are the surfaces to which other surfaces are ultimately compared. However, the EO motif does not create a wholly sufficient condition for high RPA, i.e., not all oligo(ethylene oxide) [OEO] or poly(ethylene oxide) [PEO]/poly(ethylene glycol [PEG] surfaces are protein resistant [16–19] and because polymer–protein interactions are inherently difficult to define [20], the mechanism of protein resistance has lacked clarity for the larger PEGs. Studies on SAMs of OEO-terminated compounds have led to a better definition of the structural and conformational requirements of the OEO segments such as chain length, packing density, conformational order, and, indirectly, the complex OEO–water interactions necessary for high RPA [17,19,21–24]. While full clarification is still lacking [25], it is now well-established that the condition of high RPA exists when the OEO segments uniformly cover the substrate and are (a) not tightly packed [3,16,17,23], (b) disordered, i.e., composed of neither the 7/2 helical nor the all-trans extended conformation over any significant ensemble of molecules [17], (c) conformationally mobile [24], and (d) hydrated/interactive with water [16,26]. In addition, earlier reports established that RPA was largely independent of the OEO end group for (CH2 CH2 O)n H (hydroxy-terminated) and (CH2 CH2 O)n CH3 (methoxy-terminated) SAMs [27]. Maximum RPA was observed at approximately 55% surface coverage for SAMs of the general formula HS(CH2 )3 O(CH2 CH2 O)x CH3 , {1, x = 6 (Fig. 2); x = 5 (Ref. [24])} on Au. However, these highly resistant SAMs of 1 were difficult to reproduce [short (≤1 s), manuallyperformed immersion times in the SAM-forming solutions] and, more importantly, unstable. Protein adsorption was observed within hours of the initially-prepared SAMs as a result of surface reorganization forming ordered domains of 1 (OEO segment adopting the 7/2 helical conformation [24]) and poorly covered or bare Au patches. SAMs of the subsequent bis-sulfur OEO-compound, N,N(bis 3 -thioacetylpropyl)-3,6,9,12,15,18-hexaoxanonadecanamide (BTHA) [28], in which the optimal OEO packing is encoded in the

Fig. 2. Structures of the OEO compounds.

structure, required no exacting preparation conditions, sustained their physical properties when stored in preparatory solutions for weeks, and exhibited high RPA. Essentially all of the previous protein adsorption studies of OEO SAMs utilized plasma soluble proteins, most frequently fibrinogen (Fb) and bovine serum albumin (BSA). Recognizing the need to establish SAMs that will minimize nonspecific protein adsorption for IMPs, this work reports the RPA properties of four OEO SAMs (Fig. 2): 1-mercaptopropyl-OEO 1 and 2 and 4-(1,7dimercaptoheptan-4-oxy)-OEO 3 and 4 on Au and Pt substrates. For direct comparison to the plasma soluble protein literature we used Fb [(340 kD)] and for the IMP we used the readily available G-protein coupled receptor (GPCR) rhodopsin (Rd) [37 kD]. Compounds 1 and 2 provide a direct correlation to our previous work with monothiol OEO SAMs while 3 and 4 correlate to the BTHA SAMs, respectively [28]. We use in situ and ex situ spectroscopic ellipsometry (SE) to evaluate the RPA of the SAMs of 1–4, on Au and Pt surfaces. The evaluation of Pt surfaces was necessitated by our ability to obtain more uniform Pt deposition in the AAO membrane pores using an organometallic vapor deposition procedure relative to Au deposited by plasma sputtering. 2. Experimental [29] Materials and Reagents. Except for silica gel and FBS (fetal bovine serum), all chemicals, including fibrinogen and solvents, were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO) and were used as received. Silica gel (40 ␮m, 7024-02) was purchased from J.T. Baker (Mallinckrodt Baker, Inc., Phillipsburg, NJ). FBS with Roswell Park Memorial Institute media and associated antibiotics were purchased from Life Technologies (Carlsbad, CA). Tetrahydrofuran (THF) was distilled under N2 from CaH2 immediately before use. Compounds 1 and 2 were prepared as outlined in Scheme 1 and compounds 3 and 4 as outlined in Scheme 2. The preparation of 1 [4,7,10,13,16,19,22-heptaoxatricosane-1-thiol (M CH3 )] was described earlier [30]. Details of the preparation of 2 to 4 and spectral data for all compounds are given in the

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constraints of an assumed refractive index of 1.45 for all organic compounds.

Scheme 1. Synthesis of 1 and 2. (a) NaH/THF, ally bromide; (b) thioacetic acid, azobisisobutyronitrile, h; (c) 0.1 M HCl/MeOH.

supplemental information (SI) section. Structural assignments were made from proton nuclear magnetic resonance (1 H NMR) [270 MHz (JOEL, USA, Inc, Peabody, MA) or Bruker Avance 500 MHz (Bruker, Inc., Billerica, MA) spectrometer in CDCl3 containing 0.03% (v/v) tetramethylsilane (TMS); recorded chemical shifts (ı) are relative to TMS] alone or in combination with mass spectrometry data. Sample purity (>98%) was determined from thin-layer chromatography (TLC) analysis (single spot by TLC). All reactions were carried out under nitrogen. 2.1. Synthesis of 1–4 2.1.1. Sample preparation Pt substrates were prepared by depositing Pt onto Si wafers using a FlexAL atomic layer deposition tool (Oxford Instruments America Inc, Concord, MA) [>200 cycles, which corresponds to a Pt thickness ≈9 nm] and used immediately [31]. The ALD method utilizes a surface reaction between the substrate and the precursor molecules that does not require any adhesion layer to provide adhesive strength to the Pt thin film. Our Pt films passed the “scotch tape test”. Au substrates [100 nm Au on 5 nm Ti adhesion layer on Si wafers were purchased (Platypus Technologies, Madison, WI)] and cleaned as described previously [32] prior to use. SAMs were formed on the Au and Pt substrates by immersion into 0.2 mM aqueous solutions of 1–4 for ≥18 h. Previous studies show small, water soluble thiols such as 1 and 2 reach their “final state”, fully-formed SAMs in less than 12 h [30]. SAM completion for compounds 3 and 4 can be expected to be comparable or faster, than 1 and 2, due to increased reactivity with the Au (two thiols per molecule). 2.1.2. Spectroscopic ellipsometry (SE) Multiple wavelength ellipsometric measurements were performed on a J. A. Woollam Co., Inc. (Lincoln, NE) M2000 spectroscopic ellipsometer, as described earlier [17]. Monolayer thicknesses are thus reported as optical thicknesses under the

Scheme 2. Synthesis of 3 and 4. (a) H+ ,3,4-dihydro-2H-pyran, CHCl3 ; (b) methanesufonyl choride, pyridine, CHCl3 ; (c) NaHTHF followed by 1,6-heptadien4-ol/THF; (d) thioacetic acid, azobisisobutyronitrile, h; (e) 0.1 M HCl/MeOH. 8: CH2 CHO(EO)5 CH3

2.1.3. Contact angle measurements Contact angles were determined either with a Ramé-Hart model 110-00-115 (Ramé-Hart Instrument Co., Succasunna, NJ) or a FTA100 (First Ten Angstroms, Inc., Portsmouth, VA) goniometer at room temperature and ambient relative humidity (40 ± 5) % using water as the probing liquid. Contact angles () were measured by lowering a 2–3 ␮L drop onto the surface from a blunt-ended needle attached to a 2 mL syringe. The contact angle was recorded immediately after the drop detached from the needle tip. The value of  was the average of at least four measurements (SD = ±2◦ ). 2.1.4. Protein adsorption The Fb and Rd adsorption studies were performed ex situ using either 1 mg/mL Fb in 0.2 M phosphate buffer (PBS, pH = 7.2; Sigma Aldrich, St. Louis, MO) or 0.39 mg/mL bovine Rd in solution as a protein-detergent complex [50 mmol/L [4-(2-hydroxyethyl)1-piperazine-ethanesulfonic acid] (HEPES), pH 7.0, 3% (w/v) n-octylglucoside]. The SAMs were incubated with the protein solutions for 1 h followed by rinsing with water and drying under a stream of nitrogen. Error bars represent the standard error of the mean of three measurements per sample. All Rd experiments were carried out in the dark to prevent denaturation. Protein adsorption to bare Au and Pt substrates is given in Table S1 in the SI section. The FBS absorption experiments were carried out in situ in a custom built cell and monitored ellipsometrically in real-time [28,33]. Either bare Au substrates (initially cleaned in a UV-ozone cleaner, exposed to distilled water to reduce any Au oxide present after ozone treatment) or functionalized Au substrates were mounted in the in situ cell. The cell was filled with PBS and the substrate allowed to equilibrate. After equilibration, the PBS was removed via a sterile syringe and the cell filled with 100% FBS, for approximately 130 min. The extent of protein adsorption was determined after the cell contents were converted back to protein-free PBS (removal of protein solution and multiple re-fills with fresh PBS) and temperature re-stabilization. 3. Results and discussion The SAMs of 1 to 4 were characterized by spectroscopic ellipsometry (SE) and contact angle (CA) measurements (Fig. 3A and B, respectively). The ellipsometric or optical thickness, is hereinafter simply referred to as “thickness”. Fig. 3A shows that the SAMs of all four compounds are similar on Au and Pt with the SAMs of 1 thicker than those of 2–4. SAM thickness differences on Au and Pt for 1–4 are small, similar to that observed for n-alkanethiol SAMs [34], and support the use of Pt for further studies involving IMPs (Fig. 1). Previous work on OEO SAMs of the general structures HS(EO)x CH3 and HS(CH2 )y O(EO)x CH3 , x = 3–9 and y = 3 or 11, correlate SAM thickness with structure, i.e., surface packing density and OEO chain order and hydration [16,24,28,35,36]. Because each compound exhibits essentially identical thicknesses (Fig. 3A) on both substrates, the structure of each SAM is comparable on Au and Pt, which have identical crystal structure [face-center cubic] with nearly identical atomic radii (174 pm and 177 pm, respectively) [37]. Studies on Au (and Ag) have shown that ordered OEO SAMs exhibit higher thicknesses than disordered OEO SAMs, regardless of whether the OEO segment adopts the 7/2 helix or the all-trans extend conformation [16,17,19,24]. The thicker SAMs of 1 indicate more densely packed SAMs relative to those of 2–4 [24]. The comparable thicknesses for 2–4 indicate that these SAMs possess similar order and surface coverage despite the fact that 2 is a monothiol and 3 and 4 are bisthiols.

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A

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Ellipsometric Thickness (nm)

2.5 2 1.5 1 0.5 0

1

2

3

4

SAM

B Sa m p le

Compound 1

Wafer A Wafer B Wafer C

Au 43.8 (0.5) 46.4 (0.9) 44.4 (0.5)

2 Pt ND

Au 28.9 (0.8) 25.8 (2.5) 28.8 (1.4)

3 Pt 21.8 (3.4)

Au 41.8 (0.7) 42.2 (1.3) 41.5 (1.0)

4 Pt 43.8 (1.6)

Au 27.8 (0.7) 29.1 (1.0) 23.0 (2.4)

Pt 35.2 (4.4)

Fig. 3. (A) SE thicknesses of the SAMs of 1–4, assembled from water onto Au (black columns) and Pt (gray columns). SAM formation in 0.2 mM thiol or dithiol solutions for 18–24 h. (B) Contact angle data for SAMs of 1–4 on Au and Pt.

Ellipsometric Thickness Increase (nm)

SAMs [38]. Importantly, the thickness for the SAMs of 2 is within the optimal range for high RPA expected for compounds of this OEO chain length (1.3–1.6 nm for 6 EO units) [17,24,28]. The selflimiting nature of the SAMs of 2 coupled with the decrease in hydrophobicity and expected greater hydration of the terminus are the main factors for the high RPA of these SAMs, although adsorption of Fb slightly above the detection limits of our measurements is observed. The SAMs of 4 exhibit even higher RPA than the SAMs of 2, with SE thickness changes at or below the limits of the ellipsometer

2.5 2

A

1.5 1 0.5 0

1

2

3

4

3

4

SAM Ellipsometric Thickness Increase (nm)

Detailed characterization of disordered OEO SAMs lacking order over any significant segment of the EO chain is difficult. Reflection–adsorption infrared spectroscopy of disordered OEO SAMs in the C H stretch (2700–3100 cm−1 ) and midrange regions (900–1400 cm−1 ) exhibit poorly resolved, broad, overlapping bands [24,35] and CA values remain relatively constant over surface coverages from 55 to 85% [24]. However, on the basis of our protein adsorption data and discussions (vide infra) we characterized the SAMs of 1–4 by CA measurements (Fig. 3B). CA values for OEO SAMs vary widely from 70◦ ± 2◦ for highly ordered, near crystalline methyl-terminated SAMs [17] to 50%. Increased hydrophobicity will result in stronger interactions between proteins and the methyl-presenting surface [42,43]. This will be especially true for IMPs, which have a large hydrophobic surface area. The grand average hydrophilicity index [44] for Rd is −0.81 (negative values typically indicate membrane proteins) and +0.47 for Fb (see Table S3 for a comparison with other proteins). The positive value for Fb indicates no significant

hydrophobic surface area and, in turn, adsorption mediated by factors other than a hydrophobic effect. Fig. 6 illustrates two possible scenarios for Rd adsorption to the SAMs of 3: an induced orientation of the surface-bound 3 molecules or a simple insertion into the SAM. In the case of the induced orientation scenario, as Rd approaches the SAM the terminal methyl groups of 3 orient toward the approaching protein, especially toward the hydrophobic amino acids of the hydrophobic surface. A more uniform orientation (ordering) of methyl groups of methoxy-terminated SAMs along the substrate normal increases the hydrophobicity of the SAM as indicated by the higher CA values for the SAMs of HS(CH2 )3 O(EO)5 CH3 at the higher packing densities [>85% coverage (Fig. 5, black squares). Also, CA for 100% coverage HS(EO)6 CH3 SAMs = 70◦ ± 2◦ (Ref. [17]). Analogous increases in CA values with packing densities and SAM order that orient methyl groups of simpler alkanethiols on Au are well known [45]]. Increases in hydrophobicity by local ordering of the surfacebound 3 in the vicinity of Rd could facilitate interactions with the hydrophobic amino acids resulting in multiple sites of attachment, i.e., irreversible adsorption. Our data indirectly supports this hypothesis. As can be seen in Fig. 4 A, significantly more Rd adsorbed to the SAMs of 1 and 3 on Pt than on Au. Previous work indicates that SAMs on Pt are oriented more along the normal to the substrate on Pt than on Au [34], which may facilitate the induced ordering hypothesis and result in increased protein adsorption. Alternatively, Rd may simply insert into the SAM with hydrophobic amino acid-3 interactions along the EO chain as well as the chain terminus. Although the EO segments are not as hydrophobic as the terminal methyl group (CA ≥ 45◦ at lower packing densities of HS(CH2 )3 O(EO)5 CH3 ; Fig. 5, black squares), direct contact between Rd and the outer segments of several molecules of 3 could be sufficient for increased protein–SAM interaction leading to irreversible adsorption, as Rd inserts further into the SAM. In addition, since the SAMs of 3 are only ≈1.5 nm thick, insertion into the SAM by the much larger protein could result in displacement of some of the surface-bound molecules resulting in direct protein–Au interactions. An increase in SAM hydrophobicity with increasing packing density/orientation (order) of the OEO terminus is not observed for hydroxy-terminated SAMs as the CA values for 2 remain the same, or slightly decrease (Fig. 5, red circles). Indeed, the RPA of the SAMs of 2 and 4 are high for Rd as was the case for the SAMs of 2 and the GPCR deca(histidine-tagged)-cannabinoid receptor type II [2]. In addition, hydroxy-terminated SAMs can be expected to be more hydrated at the terminus, as has been suggested from molecular simulation studies [46], which would provide a barrier toward protein insertion into the SAM. Our data indicate the SAMs of 4 to be our best candidate for further studies toward bioscience/biotechnology applications. However, the full measure of the RPA for the SAMs of 4 is not revealed in the single protein adsorption experiments (Fig. 4) because thickness increases were at or below the limits of detection of our instrument (±0.03 nm). To further probe the low adsorption properties of this SAM, we exposed it to undiluted FBS – a complex biofluid containing many proteins and other components, representative of in vivo conditions – in our in situ SE cell for ≈130 min. Fig. 7 shows, after rinsing with protein-free PBS, adsorption of 0.20 nm, which corresponds to approximately 12.4 ng/cm2 (Ref. [47]). This low level of adsorption is ≈103 fold less than that recently reported from serum to a PEG-functionalized surface [48] and defines more clearly the magnitude of RPA for the SAMs of 4. Larger “apparent” adsorption to bare Au and the SAMs of 4, prior to the rinse (Fig. 7), is due to the combination of reversible and irreversible protein adsorption as well as any refractive index difference between the FBS solution and the protein-free PBS buffer. Noteworthy, for the SAMs of 4, is that the apparent adsorption

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Fig. 6. Cartoon illustration of the hydrophobic effect proposed for Rd adsorption to SAMs of 3 (methoxy-terminated) via local orientation of surface-bound OEO SAM molecules or by partial insertion into the SAM.

adsorption propensity for the SAMs of 4 of ≈12 ng/cm2 , as defined by exposure to undiluted FBS, indicate its potential usefulness in the development of biocompatible, durable, nonfouling surfaces. Finally, this work establishes use of Pt in surface assemblies designed to contain oriented IMPs for structure-function studies, high-throughput screening of pharmaceutical targets, and future applications. Acknowledgments

Fig. 7. In situ cell, real-time trace of the SE thickness increase of a bare Au surface (red) and a SAM of 4 (blue) on Au in contact with undiluted FBS and, after rinsing, (conversion back to protein-free buffer). Arrows indicate time of addition of FBS solution to the cell and initiation of the rinse. The higher apparent thickness in both traces before rinsing is due to reversibly- and irreversibly-bound protein as well as to differences in the refractive index of the FBS and protein-free buffer solutions. The minimal thickness difference before and after rinsing for the bare Au surface indicates almost all of the protein is chemisorbed (irreversibly-bound). Traces are offset from t = 0 for clarity and to show baseline stability. After rinsing, adsorption to bare Au = 301.1 ng/cm2 (Ref. [47]). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

(≈0.60 nm) is established in less than 1 min after FBS exposure and remains essentially constant thereafter (texposure ≈ 130 min). The absence of any change after ≈1 min suggests that the irreversible adsorption component may occur quickly and, once complete, essentially passivates the surface toward further adsorption of any of the serum components. 4. Conclusions We have investigated the protein adsorption characteristics for SAMs of the general formulas HS(CH2 )3 O(EO)x M and [HS(CH2 )3 ]2 CHO(EO)x M, where M H or CH3 , for both plasma and IMP proteins on Au and Pt. We establish that the terminus of the OEO segment must be a major consideration in the design of future protein resistant surfaces. We show that methoxy-terminated OEO surfaces adsorb IMPs even though formed with bismercapto-OEO molecules that structurally encode optimal OEO surface coverage for high RPA. The high RPA for the bismercapto compounds 3 and 4 to Fb indicates the substrate is essentially fully-screened from direct protein-substrate contact at length scales equal to the smallest axis of the protein structure. Taken together, our SE data on compound 4 define a set of molecular requirements needed toward the goals of structural studies of IMPs. The low

A. V. acknowledges the National Institute of Standards and Technology-American Recovery and Reinvestment Act (NISTARRA) fellowship. R. J. V. and F. C. were NIST summer undergraduate research fellows (SURF) [R. J. V. (2006 and 2007) and F. C. 2011]. We thank Dr. Klaus Garwisch, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD for the generous gift of purified rhodopsin and Alessandro Tona and Dr. John Elliott for the gift of fetal bovine serum. We thank Dr. Lee Richter for helpful suggestions and comments. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.07.031.

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