Oligo(ethylene oxide) Self-Assembled Monolayers with Self-Limiting

Apr 9, 2009 - Zhu , B., Eurell , T., Gunawan , R. and Leckband , D. J. Biomed. Mater. Res. 2001, 56, 406– 416. [Crossref], [PubMed], [CAS]. 10. Chai...
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Oligo(ethylene oxide) Self-Assembled Monolayers with Self-Limiting Packing Densities for the Inhibition of Nonspecific Protein Adsorption David J. Vanderah,*,† Ryan J. Vierling,† and Marlon L. Walker‡ †

Biochemical Science Division and ‡Surface and Microanalysis Science Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, Maryland 20899 Received November 25, 2008. Revised Manuscript Received March 4, 2009 We have created a molecule that forms self-assembled monolayers (SAMs) on Au, possessing the characteristics for inhibition of nonspecific protein adsorption, i.e., uniformly distributed, loosely packed, conformationally mobile, hydrated ethylene oxide (EO) chains of near optimal packing densities. SAMs of the bipodal molecule CH3O (CH2CH2O)5CH2CON(CH2CH2CH2SCOCH3)2 [N,N-(bis-30 -thioacetylpropyl)-3,6,9,12,15,18-hexaoxanonadecanamide (BTHA)] on polycrystalline Au are described. Spectroscopic ellipsometry (SE) and reflection-absorption infrared spectroscopy data indicate that BTHA SAM thickness and EO chain disorder closely match that of partially formed monothio-(EO)5-6CH3 SAMs when they exhibit maximum inhibition of protein adsorption. However, in contrast to the monothio-(EO)5-6CH3 SAMs, the BTHA SAM thickness and EO chain disorder remain constant in the presence of unbound molecules because of the structurally imposed upper limit of one EO chain per two Au occupancy sites. SE data indicate high resistance to protein adsorption for bovine serum albumin, fibrinogen, and a mixture of the two, suggesting uniform EO surface coverage on a length scale at least equal to the smallest dimension of these proteins.

Introduction Surfaces coated with poly(ethylene oxide) (PEO)- or oligo (ethylene oxide) (OEO)-terminated compounds have been broadly studied for use in applications where control of protein adsorption is required.1-3 Protein resistance is not an intrinsic property of the ethylene oxide (-CH2CH2O-, EO) structural motif because PEO- and OEO-coated surfaces can be either protein-adsorbing or protein-resistant.4-9 The condition of resistance to protein adsorption appears to be dependent upon the EO chain length,10 packing density,9 conformational mobility,3 and hydration.11,12 Optimization of these parameters for complete resistance to the nonspecific adsorption of protein for extended periods of time continues to be pursued through theoretical13-17 *To whom correspondence should be addressed. Telephone: (301) 975-6438. Fax: (301) 975-8246. E-mail: [email protected]. (1) Poly(ethylene glycol): Biotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenum Press: New York, 1992. (2) Nogaoka, S.; Mori, Y.; Takiuchi, H.; Yokota, K.; Tanazawa, H.; Nishiumi, S. Interaction between blood components and hydrogels with poly(oxyethylene) chains. In Polymers as Biomaterials; Shalaby, S., Hoffman, A., Ratner, B. D., Horbett, T. A., Eds.; Plenum Press: New York, 1984. (3) Andrade, J. D.; Hlady, V.; Jeon, S. I. Hydrophilic Polym. 1996, 248, 51–59. (4) Pasche, S.; De Paul, S. M.; Voros, J.; Spencer, N. D.; Textor, M. Langmuir 2003, 19, 9216–9225. (5) Unsworth, L. D.; Tun, Z.; Sheardown, H.; Brash, J. L. J. Colloid Interface Sci. 2005, 281, 112–121. (6) Vanderah, D. J.; Valincius, G.; Meuse, C. W. Langmuir 2002, 18, 4674–4680. (7) Vanderah, D. J.; La, H. L.; Naff, J.; Silin, V.; Rubinson, K. A. J. Am. Chem. Soc. 2004, 126, 13639–13641. (8) Huang, N. P.; Michel, R.; Voros, J.; Textor, M.; Hofer, R.; Rossi, A.; Elbert, D. L.; Hubbell, J. A.; Spencer, N. D. Langmuir 2001, 17, 489–498. (9) Kenausis, G. L.; Voros, J.; Elbert, D. L.; Huang, N. P.; Hofer, R.; Ruiz-Taylor, L.; Textor, M.; Hubbell, J. A.; Spencer, N. D. J. Phys. Chem. B 2000, 104, 3298–3309. (10) Zhu, B.; Eurell, T.; Gunawan, R.; Leckband, D. J. Biomed. Mater. Res. 2001, 56, 406–416. (11) Heuberger, M.; Drobek, T.; Voros, J. Langmuir 2004, 20, 9445–9448. (12) Kim, H. I.; Kushmerick, J. G.; Houston, J. E.; Bunker, B. C. Langmuir 2003, 19, 9271–9275. (13) Fang, F.; Szleifer, I. Langmuir 2002, 18, 5497–5510. (14) Halperin, A.; Leckband, D. E. C. R. Acad. Sci., Ser. IV 2000, 1, 1171–1178. (15) Halperin, A.; Fragneto, G.; Schollier, A.; Sferrazza, M. Langmuir 2007, 23, 10603–10617. (16) Morra, M. J. Biomater. Sci., Polym. Ed. 2000, 11, 547–569. (17) Szleifer, I. Biophys. J. 1997, 72, 595–612.

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and experimental efforts,18,19 as well as from kinetic and thermodynamic perspectives.20,21 Surfaces modified with OEO segments have shown protein resistance comparable to that of PEO.22-26 Because selfassembled monolayers (SAMs) afford greater reproducibility and control of surfaces, the specific aim of many of these studies was to probe further the molecular details of the protein-surface interactions and establish optimal packing densities. SAMs of HS (CH2CH2O)6CH3, hereafter designated EO6, were the first to show that protein resistance is lost at high packing densities,6 confirming earlier predictions from theoretical models,27 and clearly indicate that protein resistance requires loosely packed, disordered OEO chains. Subsequently, SAMs of HS(CH2)3O (EO)5CH3, hereafter designated as C3EO5, allowed studies of protein resistance as a function of the surface coverage from 0 to 100% with the added convenience of assembly from water. The highest protein resistance was obtained at the surprisingly low coverage of 55 ( 10%,7 where 100% coverage equals approximately 4.5  1014 molecules/cm2, the packing density of alkanethiols on Au(111).28 The 55 ( 10% coverage corresponds to approximately one C3EO5 molecule bound per two Au occupancy sites, and the high protein resistance is consistent with uniformly (18) Unsworth, L. D.; Sheardown, H.; Brash, J. L. Langmuir 2008, 24, 1924–1929. (19) Feng, W.; Nieh, M. P.; Zhu, S.; Harroun, T. A.; Katsaras, J.; Brash, J. L. Biointerphases 2007, 2, 34–43. (20) Latour, R. A. J. Biomed. Mater. Res., Part A 2006, 78A, 843–854. (21) Fang, F.; Satulovsky, J.; Szleifer, I. Biophys. J. 2005, 89, 1516–1533. (22) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426–436. (23) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003, 125, 9359–9366. (24) Valiokas, R.; Svedhem, S.; Svensson, S. C. T.; Liedberg, B. Langmuir 1999, 15, 3390–3394. (25) Vanderah, D. J.; Walker, M. L.; Rocco, M. A.; Rubinson, K. A. Langmuir 2008, 24, 826–829. (26) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12–20. (27) Jeon, S. I.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142, 159–166. (28) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1103–1169.

Published on Web 4/9/2009

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Article Scheme 1. Synthesis of BTHA

Figure 1. Structure of BTHA.

distributed C3EO5 molecules that adequately screen the underlying substrate by eliminating openings on a length scale of the smallest dimension of the proteins. While these previous SAMs established a strong correlation between packing density and protein resistance, they required narrowly controlled preparation conditions and exhibited changes with time to a more non-uniform coverage. In this report, we describe SAMs of N,N-(bis-30 -thioacetylpropyl)-3,6,9,12,15,18-hexaoxanonadecanamide [IUPAC: S-20(3-acetylthio)propyl-19-oxo-2,5,8,11,14,17-hexaoxa-20-azatricosan-23-yl ethanethiolate] (Figure 1), hereafter referred to as BTHA, and their resistance to protein adsorption. As suggested from recent reports,29 packing densities on Au can be controlled with molecules possessing more than one sulfur atom. In the case of BTHA, one penta(ethylene oxide) chain per two sulfur atoms can be expected to result in uniformly distributed EO chains that should not exceed 50% coverage, near the midpoint of the optimal packing density range. In addition, protein-rejecting BTHA SAMs should not require carefully controlled formation conditions nor change significantly with time. Our data provide evidence that BTHA SAMs exhibit these desirable characteristics and high resistance to protein adsorption.

Materials and Methods30 Materials. Penta(ethylene oxide) monomethyl ether was purchased from TCI America (Portland, OR). Bovine serum albumin (BSA) and fibrinogen (Fb) were purchased from Sigma-Aldrich (St. Louis, MO). Tetrahydrofuran (THF) (Mallinckrodt AR) was purchased from North Strong Scientific (Phillipsburg, NJ) and distilled from CaH2 immediately before use. All other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used as received. Synthetic compounds were purified by chromatography [silica gel, J.T. Baker (Phillipsburg, NJ), 40 μm; column = 33  3 cm] and characterized by proton nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy, or mass spectroscopy (MS), as necessary. NMR spectra were obtained on a JEOL 270 MHz spectrometer (JEOL, Ltd., Tokyo, Japan) in CDCl3 solutions containing 0.03% tetramethylsilane (TMS). Chemical shifts (δ) are in parts per million (ppm) relative to TMS, and coupling constants (J) are in Hertz (Hz). Mass spectra were obtained from the Department of Chemistry and Biochemistry, University of Maryland, College Park, MD. Stock solutions of BSA and Fb were 10 mg/mL in 50 mM phosphate buffer solution (PBS) at pH 7.4 prepared with a phosphate buffer mix (Sigma) and ultra-pure 18.2 MΩ water. Protein adsorption experiments were performed in PBS. Sample Preparation. For spectroscopic ellipsometry on dried SAMs (ex situ SE), silicon (100) wafers (Silicon, Inc., Boise, ID) were initially coated with chromium (∼2 nm) and then with gold (∼200 nm) by magnetron sputtering (Edwards Auto 306, U.K.), as described previously.31 The freshly coated (29) Park, J. S.; Vo, A. N.; Barriet, D.; Shon, Y. S.; Lee, T. R. Langmuir 2005, 21, 2902–2911. (30) Certain commercial materials, instruments, and equipment are identified in this paper to specify the experimental procedure as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology (NIST) nor does it imply that the material, instruments, or equipment identified is necessarily the best available for the purpose. (31) Vanderah, D. J.; Meuse, C. W.; Silin, V.; Plant, A. L. Langmuir 1998, 14, 6916–6923.

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Au wafers were immediately immersed in 0.5 mmol/L BTHA solutions in water for a designated period of time. Prior to measurements, the SAM-coated substrates were removed from the BTHA solutions, rinsed thoroughly with 18.2 MΩ water, and dried in a stream of dry nitrogen. For reflection absorption infrared spectroscopy (RAIRS) measurements and protein adsorption experiments, SAMs were prepared on 100 nm Au substrates (Platypus Technologies, Madison, WS) with a 5 nm Ti adhesion layer, as described previously.25 The Au substrates were initially cleaned with a UV-light/ozone treatment for approximately 15 min, soaked in 18.2 MΩ water for 20 min to reduce any Au oxide formed, placed in the BTHA solution for a designated period of time (1-60 days), rinsed thoroughly with 18.2 MΩ water, and finally dried in a stream of dry nitrogen. Synthesis. BTHA was synthesized as outlined in Scheme 1. All synthetic steps were carried out under nitrogen. Benzyl Azidoacetate (3). To a solution of 3.99 g (0.0207 mol) of 2 and 2.10 g (0.021 mol) of Et3N in 25 mL of acetonitrile was added 4.103 g (0.208 mol) of p-TsN3 dropwise and stirred at room temperature for 1.5 h. A solution of 2.5 g of KOH in 2.0 mL of water was added. After stirring for 2.5 h, the mixture was poured into 50 mL of methyl t-butyl ether (MTBE). The aqueous layer was removed; the MTBE layer washed twice with 2 mL of aqueous KOH (0.3 g/mL), dried over anhydrous sodium sulfate (Na2SO4), and concentrated. Chromatography (20% ethyl acetate/80% hexanes, v/v) afforded 2.18 g (69%) of pure 3 as a yellow liquid. IR: 2110 cm-1 (νazide). 1H NMR δ: 7.36 (br m, 5H, C6H5), 5.20 (s, 2H, CH2C6H5), 4.80 (br s, 1H, N2CHCO). Benzyl-3,6,9,12,15,18-hexaoxanonadecanoate (4). To a solution of 2.442 g (0.00968 mol) of penta(ethylene oxide) monomethyl ether in 7 mL of dry dichloromethane (CH2Cl2) was added 3 drops of boron trifluoroetherate followed by 1.5219 g (0.010 mol) of 3 in 5 mL of CH2Cl2 at room temperature. During the addition, the reaction mixture warmed, the color disappeared, and gas was evolved. After stirring for 4 h, 2 drops of methanol were added and the solution stirred for an additional 1 h. The CH2Cl2 was washed with 10 mL of water, dried (Na2SO4), and concentrated under reduced pressure to give 3.07 g of crude 4. Chromatography (4% methanol/ethyl acetate) afforded 2.5 g (65%) of pure 4. 1H NMR δ: 7.36 (br m, 5H, C6H5), 5.19 (s, 2H, CH2C6H5), 4.20 (s, 2H, OCH2CO2Bn), 3.8-3.5 [br m, 20H, CH3O(CH2CH2O)5], 3.38 [s, 3H, CH3O(EO)5].

N,N-(Bis-2-propenyl)-3,6,9,12,15,18-hexaoxanonadecanamide (5). Hydrogen gas was passed over a solution of 1.186 g (0.00296 mol) of 4 in the presence of 100 mg of 10% Pd/C for 8 h. Filtration and concentration afforded the carboxylic acid. 1H NMR δ: 4.17 (s, 2H, OCH2CO2H), 3.8-3.5 (br m, 20H, CH3O (CH2CH2O)5, 3.38 (s, 3H, CH3O(EO)5). NMR spectroscopic data indicated a small amount of an ethyl ester. Without further purification, the carboxylic acid was refluxed in 10 mL of thionyl chloride for 1 h to afford the crude acid chloride, which was dissolved in 5 mL of THF and added to a solution of 3.1 g (0.032 mol) of diallyl amine in 5 mL of THF at 0 °C, warmed to room temperature, and finally refluxed for 30 min. Removal of solvents and chromatography (30% methanol/ethyl acetate) DOI: 10.1021/la803896a

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afforded 0.807 g of purified 5 (70% from 4). 1H NMR δ: 5.855.68 [m, 2H, 2 (CH2CHdCH2)], 5.3-5.1 [m, 4H, 2 (CH2CHdCH2)], 4.22 (s, 2H, OCH2CON), 4.0-3.85 [4H, 2 doublets, J = 6 Hz, 2(CH2CHdCH2)], 3.8-3.5 (br m, 20H, CH3O(CH2CH2O)5, 3.38 [s, 3H, CH3O(EO)5]. Hindered rotation around the amide linkage places the allyl groups in different magnetic environments, within the time scale of the NMR, resulting in slightly different chemical shifts for the protons of the two allyl groups. This effect is largest (Δδ = 0.10 ppm) for the methylenes bonded to the nitrogen atom. Low-resolution electrospray mass spectrometry (MS) yielded m/z values of 390 (M + H), 407 (M + H2O), and 412 (M + Na).

N,N-(Bis-30 -thioacetylpropyl)-3,6,9,12,15,18-hexaoxanonadecanamide (1, BTHA). A solution of 0.5317 g (0.00136

mol) of 5, 2.13 g (0.028 mol) of thioacetic acid, and 25 mg of 2,20 azobisisobutyronitrile in 10 mL of THF was irradiated (λ = 280-750 nm with 40-43% in the visible range) using a watercooled jacketed, medium pressure quartz Hg-vapor lamp (Ace Glass 7825-34, Vineland, NJ) in a pyrex absorption sleeve) for 7 h under nitrogen. Filtration followed by purification (20% methanol/ethyl acetate) afforded 0.48 g (65%) of pure BTHA. 1 H NMR δ: 4.20 (s, 2H, OCH2CON), 3.8-3.5 [br m, 20H, CH3O (CH2CH2O)5], 3.38 [s, 3H, CH3O(EO)5], 3.40-3.26 [m, 4H, 2 (NCH2CH2CH2S)], 2.85 [t, 4H, 2 (NCH2CH2CH2S)], 2.372.32 (2 s, 6H, 2 SCOCH3), 1.91-1.76 [2 pentets, 4H, 2 (NCH2CH2CH2S)]. Hindered rotation around the amide linkage in 1 produced the same effect in the NMR for the 30 acetylpropyl protons as that described above for allyl protons in 5. The difference (Δδ) was 0.03 ppm for the thioacetyl methyl groups. HR fast atom bombardment MS [M + H] Calcd. for C23H44NO9S, 542.24575; found, 542.24491. Ex Situ SE. Multiple wavelength ex situ ellipsometric measurements were performed on the SAMs using a J. A. Woollam Co., Inc. (Lincoln, NE) M-44 spectroscopic ellipsometer, as described earlier.6 Thicknesses were calculated relative to the thickness of a (C18D37S)2 SAM on Au. The optical constants of the gold were determined using a two layer model, for which we assumed a thickness of 2.3 nm and a refractive index of 1.45 for the (C18D37S)2 SAM. The optical constants for the Au were then used to determine the thicknesses of the BTHA SAMs, also assuming a refractive index of 1.45. In Situ SE Protein Adsorption. Multiple wavelength in situ ellipsometric measurements of protein adsorption on the BTHA SAMs were performed in an in situ cell, under static conditions, using a Woollam M2000D spectroscopic ellipsometer, as described earlier.25 Briefly, preformed BTHA SAM substrates were positioned in a custom-built Teflon cell, placed on the instrument platform, and filled with PBS. The system was allowed to stabilize before adding protein. To minimize perturbation of the in situ cell temperature, protein was added by quickly mixing an aliquot of the stock solution (see above) with a significant volume of the PBS buffer solution withdrawn from the cell, reintroducing this volume into the cell, and fully mixing. All adsorbed protein layer thickness values are reported after rinsing by draining and refilling the cell several times with protein-free PBS. Protein layer thicknesses were calculated using a three-phase substrate-dielectric-buffer model with a Cauchy function of the form ε(λ) = (no + b/λ2)2, employing software default coefficients n0 = 1.45 and b = 0.01 nm2 for an organic-based film and n0 = 1.344 and b = 0.0031 nm2 experimentally determined for the PBS solution. Because of assumptions made concerning the value of the indices of refraction and uncertainties regarding the densities of coverage, the calculated protein thicknesses should be taken as an approximation but should be proportional to the actual physical thicknesses. The mass of the adsorbed protein per cm2, Γ, is calculated by eq 1 Γ ¼

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ðnA -nc ÞdA dn=dc

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ð1Þ

Table 1. Ex Situ SE BTHA SAM Thickness (dSE) from Water as a Function of Time thickness, dSE ((0.1 nm)a

time (days) 1 3 6 10 42 a Uncertainty = one standard deviation.

0.8 0.9 1.0 1.0 1.1

where nA is the assumed index of refraction of the adsorbate film, nc is the index of refraction of the solution, dA is the thickness of the adsorbed layer (nm), and dn/dc is the refractive index increment of the adsorbate, taken to be 0.187 cm3/g.32 RAIRS. The RAIRS data were obtained using a Nicolet Magna-IR model 860 spectrometer (Thermo Nicolet, Madison, WI) with a 80° grazing angle external reflection accessory and mercury cadmium telluride (MCT/A) detector. A wire grid polarizer ensured that only p-polarized light reached the detector. Spectra were acquired at 4 cm-1 resolution between 4000 and 700 cm-1 as a summation of 512 scans using HappGenzel apodization and no zero filling. Background spectra (Ro) were taken using a freshly UV-ozone-cleaned Au substrate, soaked in 18.2 MΩ water for 20 min, and then blown dry with nitrogen. The sample spectra (R) were acquired under identical equipment conditions and compared to background spectra to obtain spectra of -log(R/Ro) versus wavenumber (cm-1).

Results and Discussion BTHA SAMs attain a SE thickness, dSE, of 1.0 ( 0.1 nm over several days and remain constant thereafter in the presence of unbound BTHA molecules (Table 1). The final state BTHA thickness closely matches those of the self-limiting [S(EO)6]2 SAMs and the 55 ( 10% coverage C3EO5 SAMs (dSE = 1.2 ( 0.1 nm)7,25 and is well-below that for 24 atoms [from S to C (methyl group) ≈ 8 EO monomers] in an all-trans conformation of ∼2.8 nm (0.36 nm  8, where 0.36 nm = Δd/EOtrans22) or an all-helical conformation of ∼2.2 nm (0.278 nm  8, where 0.278 nm = Δd/EOhelical33), suggesting that the OEO chains are loosely packed and disordered. Using the formula of Cuypers and co-workers34 and the SE thickness, we calculate an OEO chain surface coverage of ∼1.6  1014/cm2  1.6 EO chains/nm2. RAIRS data for the BTHA SAMs from 1800 to 800 cm-1 (midrange region) and from 3200 to 2600 cm-1 (C-H stretch region) are shown in Figures 2 and 3, respectively. Previous studies have used these regions to assess the conformational order of OEO SAMs, identifying the EO chains in ordered (7/2 helical and all-trans) conformations and disordered (amorphous) conformations.22,35-37 In the midrange region (Figure 2), the BTHA SAM spectrum (solid black line) exhibits a broadband from 1140 to 1122 cm-1, assigned as the νC-O and νC-C stretching bands of the EO chain. The breadth of this band (top and bottom) is characteristic of a disordered (amorphous) EO chain adopting many (32) Defeijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759–1772. (33) Takahashi, Y.; Tadokoro, H. Macromolecules 1973, 6, 672–675. (34) Cuypers, P. A.; Corsel, J. W.; Janssen, M. P.; Kop, J. M. M.; Hermens, W. T.; Hemker, H. C. J. Biol. Chem. 1983, 258, 2426–2431. (35) Zolk, M.; Eisert, F.; Pipper, J.; Herrwerth, S.; Eck, W.; Buck, M.; Grunze, M. Langmuir 2000, 16, 5849–5852. (36) Vanderah, D. J.; Arsenault, J.; La, H.; Gates, R. S.; Silin, V.; Meuse, C. W.; Valincius, G. Langmuir 2003, 19, 3752–3756. (37) Vanderah, D. J.; Parr, T.; Silin, V.; Meuse, C. W.; Gates, R. S.; La, H. Y.; Valincius, G. Langmuir 2004, 20, 1311–1316.

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Figure 2. RAIRS spectra of a BTHA SAM (black solid line) from water (5 days) and a partially formed EO6 (p-f EO6) SAM (gray dashed line) from 95% ethanol (1 h) from 1800 to 800 cm-1.

Figure 3. RAIRS spectra of a BTHA SAM (black solid line) from water (5 days) and a partially formed EO6 (p-f EO6) SAM (gray dashed line) from 95% ethanol (1 h) from 3200 to 2600 cm-1.

conformational states.38 A comparison of the νC-O and νC-C stretching band of the BTHA SAMs to that in the p-f EO6 spectrum at 1129 cm-1 indicates that, although both SAMs are disordered, they are different.6 The prominence of the 1129 cm-1 band in the p-f EO6 spectrum indicates more order as might be expected of a slightly thicker SAM (see above) that has been shown, under longer assembly conditions (g3 h), to attain a final state dSE = 2.0 nm (100% coverage).36 The spectra of both SAMs show the presence of gauche conformations by the bands at 1460 and 1352 cm-1, assigned as EO CH2 scissor and wag bending modes, respectively.22 The shape of the νC-O and νC-C stretching bands and the presence of gauche conformations are strong evidence that the EO chains in the BTHA SAMs are highly disordered. The bands at 1660, 1540, and 1424 cm-1 in the BTHA SAM spectrum are assigned as the νCdO amide I, νCdO amide II, and νC-N stretching bands, respectively. The 1660 cm-1 band is similar to that found in the spectra of HSCH2CONHR SAMs, R = n-alkyl,39 and indicates polarization of the CdO along the (38) Dissanayake, M. A. K. L.; Frech, R. Macromolecules 1995, 28, 5312–5319. (39) Tamchang, S. W.; Biebuyck, H. A.; Whitesides, G. M.; Jeon, N.; Nuzzo, R. G. Langmuir 1995, 11, 4371–4382.

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Figure 4. Additional RAIRS spectra of a BTHA SAMs from water (5 days) from 3100 to 2700 cm-1 (νC-H region) and from 1400 to 900 cm-1.

Figure 5. In situ SE adsorption of Fb (1.0 mg/mL) to a BTHA SAM (solid line) and to bare Au (dashed line). (Inset) Plot of Fb adsorption to the BTHA SAM at smaller thickness (y axis) (for clarity) and showing times of Fb addition and rinse (see Figures 1S and 2S in the Supporting Information for corresponding psi and del changes at 633.2 nm).

substrate normal. Previous OEO SAMs containing an amide linkage did not display an amide I band and were attributed to alignment of the CdO bond parallel to the surface.40 In the C-H stretch region (Figure 3), the νCH2 stretching band of the BTHA SAM spectrum (dark black line) is broad, from 3000 to 2800 cm-1, and nearly featureless. The νCH2 maximum appears below 2880 cm-1 in distinct contrast to that for SAMs with the EO segment in the 7/2 helix conformation, which exhibit a prominent νCH2 maximum at 2893 ( 2 cm-1.36 The similarity of the νCH2 stretching band envelope from 2970 to 2830 cm-1 in the BTHA spectrum to that of p-f EO6 (gray dashed spectrum) indicates comparable coverage for both SAMs. In contrast, the two SAMs display different signatures for the νCH3 stretching bands. For the p-f EO6 SAM, the νCH3 asymmetric and symmetric stretching bands at 2984 and 2818 cm-1, respectively, indicate polarization along the substrate normal of an upright phase, with some of the molecules in the helical conformation. An EO chain in (40) Valiokas, R.; Svedhem, S.; Svensson, S. C. T.; Liedberg, B. Langmuir 1999, 15, 3390–3394.

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Vanderah et al. Table 2. In Situ SE Protein Adsorption of Fb and BSA to BTHA SAMs and Bare Au

immersion time (days)

protein

exposure time (min)

protein concentration (mg/mL)

thickness adsorbed protein (nm)a,b

mass adsorbed protein (ng/cm2)b

1-2 Fb 30 0.5 0.02 ( 0.01 1.2 ( 0.6 1.0 0.04 ( 0.02 2.5 ( 1.2 5-6 Fb 60-90c 1.0 0.05 ( 0.04 3.1 ( 2.5 >8 Fb 60-90c 1.0 0.05 ( 0.03 3.1 ( 1.9 5-6 BSA 30-60c c 1.0 and 1.0 0.08 ( 0.01 5.0 ( 0.6 5-6 BSA and Fb 30-60 60 Fb 50 0.5 0.06 3.7d Au Fb 30 0.5 7.8 ( 0.1 483.9 ( 6.2 Au BSA 30 1.0 2.1 ( 0.1 130.3 ( 6.2 a b c d After washing with PBS (several cell volumes). Uncertainties = one standard deviation. Range over several experiments. One experiment.

the helical conformation does not orient the methyl group along the substrate normal, allowing observation of the symmetric and asymmetric bands. In the BTHA SAM spectrum, the νCH3 asymmetric and symmetric stretching bands are both attenuated to e10% of those of the p-f EO6 monolayer. This is likely due to the combination of two factors. Because BTHA has two anchors, there are half as many methyl groups per coverage. In addition, the two anchors impose spatial separation of the EO segments that should allow for a wider distribution of orientation of the methyl transition moments. While the substrate surface and entropic considerations make isotropic orientation of the methyl groups unlikely, a “net” orientation of ∼54.7° approximates an isotropic orientation and significantly reduces all of the band intensities.41 Figure 4 shows additional BTHA RAIRS spectra, indicating variability from substrate to substrate. Variability for disordered OEO SAMs characterized by broad peaks, maxima and attendant shoulder peaks that shift over a range of wavenumbers, and inconsistent baselines because of the random nature of the surface has been observed previously.22,35-37 A comparison of the data in Figure 4 to the BTHA spectra in Figures 2 and 3 indicate similar spectral features in both regions with the greatest variability seen at 1100 ( 50 cm-1, where different conformations of OEO segments are expected to exhibit different νC-C and νC-O band frequencies. Regardless of these differences, the general properties of the BTHA surfaces, as discussed below, are maintained. The BTHA SAMs are highly resistive to protein adsorption (Figure 5 and Table 2). Figure 5 compares the adsorption of Fb onto a BTHA SAM to that for bare Au. Table 2 summarizes our protein adsorption data onto BTHA SAMs for BSA, Fb, and a mixture of the two. The protein adsorption values for the BTHA SAMs are comparable to that observed for BSA on the self-limiting [S(EO)6]2 SAMs (in situ SE data25), BSA and lysozyme on the p-f EO6 (RAIRS data6), and BSA and Fb on the 55 ( 10% C3EO5 (surface plasmon resonance data7). However, unlike the EO6 and C3EO5 SAMs that progress to higher OEO packing densities, attain dSE ≈ 2.0 nm, and become protein-adsorbing under longer assembly conditions (t g 3 h), the BTHA SAMs exhibit constant, lower OEO packing densities and SE thickness (Table 1) in contact with solutions of unbound BTHA molecules over many days and maintain their resistance to protein adsorption. In the idealized case of Au(111) with a BTHA molecule occupying adjacent Au occupancy sites via its thioacetates, a complete BTHA SAM limits the EO chain surface coverage to ≈2.25  1014 EO chains/cm2 ≈ 2.25 EO chains/nm2 ≈ 11 EO

units/nm2. This upper limit surface coverage is in good agreement for predicted protein resistance by PEO chains from single-chain mean field approximations17 (12-20 EO units/nm2) and found for PEO polymers on oxidic surfaces (15-20 units/nm2).42-44 However, the BTHA SAMs are protein-resistant at ≈1.6 EO chains/nm2, which is ≈30% below the upper limit and corresponds to ≈8 EO units/nm2. This suggests that the lower limit of EO surface coverage, effective in resisting protein adsorption, remains to be determined and presents an opportunity for further exploration experimentally and theoretically. Two possibilities may account for BTHA EO chain packing densities of ≈1.6 chains/nm2. First, our Au is polycrystalline and can be expected to have other Au faces at the surface, as well as step edges and defect sites. It is well-established that packing densities on many of these faces as well as at or near step edges and defect sites are lower than on atomically flat Au(111) terraces.28 Second, the BTHA SAMs may be self-limiting beyond that imposed structurally. This possibility is more speculative because the underlying reason(s) for SAM self-limitation, such as that observed for [S(EO)6]2 SAMs,25 are unknown. Self-limitation may be simply due to SAM disorder that screens the Au to full coverage of BTHA molecules (self-rejection), and our data indicate conformationally mobile EO chains, similar to the p-f EO6 and the [S(EO)6]2 SAMs.

(41) Parikh, A. N.; Allara, D. L. J. Chem. Phys. 1992, 96, 927–945. (42) Pasche, S.; De Paul, S. M.; Voros, J.; Spencer, N. D.; Textor, M. Langmuir 2003, 19, 9216–9225.

(43) Malmsten, M.; Emoto, K.; Van Alstine, J. M. J. Colloid Interface Sci. 1998, 202, 507–517. (44) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998, 31, 5059–5070.

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DOI: 10.1021/la803896a

Conclusions RAIRS and SE data show that [CH3COS(CH2)3]2NCOCH2(EO)5CH3 (BTHA) SAMs are stable films that inhibit nonspecific protein adsorption for BSA, Fb, and mixtures of the two. The high resistance to protein adsorption is strong evidence for uniform coverage by loosely packed, disordered, conformationally mobile, hydrated OEO chains. The possible long-term stability of these films makes them attractive for a variety of applications, where elimination of protein adsorption and subsequent fouling is desired. Acknowledgment. R.J.V. was supported by the NIST summer undergraduate research fellow (SURF) program (2006 and 2007). We thank Dr. Lee Richter for helpful discussions and use of instrumentation. Supporting Information Available: Corresponding plots of psi and del for the protein adsorption data in Figure 5. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2009, 25(9), 5026–5030