Influence of the Molecular Design on the Antifouling Performance of

Sep 18, 2012 - Cp(EG)nOMe layers exhibit a lower surface concentration than .... for 15 min (leading to an atomically flat hydrogenated surface, terme...
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Influence of the Molecular Design on the Antifouling Performance of Poly(ethylene glycol) Monolayers Grafted on (111) Si Emmanuel Perez, Khalid Lahlil, Cyrille Rougeau, Anne Moraillon, Jean-Noel̈ Chazalviel, François Ozanam,* and Anne Chantal Gouget-Laemmel* Physique de la Matière Condensée, École Polytechnique, CNRS, 91128 Palaiseau, France S Supporting Information *

ABSTRACT: Various poly(ethylene glycol) monomethyl ether moieties were grafted onto hydrogenated silicon surfaces in order to investigate the influence of the molecular design on the antifouling performance of such coatings. The grafted chains were either oligo(ethylene oxide) chains (EG)nOMe bound to silicon via Si−O−C covalent bonds, or hybrid alkyl/oligo(ethylene oxide) chains Cp(EG)nOMe bound via Si−C covalent bonds (from home-synthesized precursors). Quantitative IR spectroscopy gave the molecular coverage of the grafted layers, and AFM imaging demonstrated that a proper surfactinated rinse yields Cp(EG)nOMe layers free of unwanted residues. The protein-repellent character of these grafted layers (here, toward BSA) was studied by IR and AFM imaging. Cp(EG)nOMe layers exhibit a lower surface concentration than (EG)nOMe layers, because of the presence of a solvent in the grafting solution; they however demonstrate high resistance against BSA adsorption for high values of the n/p ratio and a higher stability than (EG)nOMe. This behavior is consistently explained by the poor ordering capability of the alkyl part of the layer, contrary to what is observed for similar layers on Au, and the key role of an entangled arrangement of the ethylene oxide chains which forms when these chains are long enough.

1. INTRODUCTION Coatings based on poly(ethylene glycol) (PEG) are nowadays used in biomedical and biosensor applications because of their ability to prevent protein adsorption or bacterial and cell adhesion.1−3 The “antifouling” behavior of these coatings is generally ascribed to the hydrophilic and highly hydrated nature of the PEG arising from their structure and conformational flexibility which are thought to favor the formation of a water interfacial layer preventing direct contact between the surface and the proteins.4−10 They have been used on many substrates, such as glasses,11,12 noble metals,13,14 metal oxides,15,16 polymers,17,18 semiconductors,19−21 and carbon nanotubes.22,23 As it is the case for most functional layers, the performances of these coatings depend on the layer organization and its packing density. In order to give a useful insight into a generic system, and not only to assess the performances of a coating obtained in specific conditions, it is needed to apply methods giving quantitative access to the layer structure and to check the absence of any unwanted residues. With respect to this approach, well-defined silicon surfaces have proved their interest since they allow for easier quantification of molecule surface concentration and constitute a demanding benchmark for AFM imaging. In order to obtain a functional layer, molecules to be grafted are generally composed of a functional termination, a backbone favoring the packing and organization of the layer, and a surface-reactive headgroup. Here we will use hydrogenterminated (111) silicon surfaces as substrates, since this allows for the attachment of organic molecules in a controllable way © 2012 American Chemical Society

through hydrosilylation of 1-alkene precursors and formation of robust Si−C covalent bonds.24−28 Therefore, we will use oligo(ethylene oxide) monomethyl ether moieties as functional groups, alkyl chains as backbones, and a vinyl group (abbreviated as Vi) as headgroup. We investigated various films made by grafting hybrid molecules abbreviated as ViCp−2(EG)nOMe, where Cp−2 stands for p − 2 methylene units and (EG)n for n ethylene oxide (−O−CH2−CH2−) units, with p varying from 3 to 11 and n from 3 to 16. We also look for the role of the backbone by investigating pure poly(ethylene glycol) monomethyl ethers abbreviated as H(EG)nOMe. As shown in Figure 1, the former molecules yield a molecular layer denoted as Cp(EG)nOMe with p methylene units and n ethylene oxide units attached to the surface via a Si−C bond, and the latter ones yield a molecular layer denoted as (EG)nOMe with only n ethylene oxide units attached to the surface via a Si−O−C bridge. We will use quantitative IR spectroscopy in the attenuated total reflection (ATR) geometry and atomic force microscopy (AFM) imaging to carefully analyze these layers, determine their molecular coverage as a function of the chain composition, and correlate these characteristics to a quantitative evaluation of the proteinrepellent properties of the obtained films. In order to assess the protein-repellent character of the molecular layers grafted on silicon, we have chosen as model protein the serum albumin (in Received: July 30, 2012 Revised: September 13, 2012 Published: September 18, 2012 14654

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ultrapure water, the hydrogenated silicon surface was transferred into the Schlenk tube. It was then kept at room temperature under continuous argon bubbling for 15 min, and then the vessel was hermetically closed and irradiated for 3 h in a UV reactor (6 mW cm−2, 312 nm). After the UV treatment, the sample was rinsed either with toluene (60 °C, 15 min), EtOH (65 °C, 2 × 15 min) and finally CH2Cl2 (5 min, room temperature) for ViCp−2(EG)nOMe or with EtOH (65 °C, 2 × 15 min) and CH2Cl2 (5 min, room temperature) for H(EG)nOMe. Complementary rinses were actually found to be necessary for ViCp−2(EG)nOMe (described in Results and Discussion section 2). The sample was finally blown dry under nitrogen. 2.4. BSA Adsorption Assay. The modified samples were immersed for 1 h in a BSA solution (1 mg/mL) in 1× PBS buffer. The solution was removed and the samples were rinsed for 1 min in 1× PBS buffer, followed by a quick rinse in Milli-Q-water. 2.5. Water Contact Angle Measurements. The static contact angles were measured in a homemade goniometer after depositing a 10 μL water drop on the surface. For each sample, at least three spots were taken on the surface. 2.6. Infrared Spectroscopy. ATR-FTIR spectra were recorded using a Bomem MB100 FTIR spectrometer equipped with a liquidnitrogen-cooled MCT photovoltaic detector. All spectra were recorded with s- and p-polarization over the 900−4000 cm−1 spectral range (100 scans, 4 cm−1 resolution). For the IR measurements, silicon prisms were prepared as follows: double-side polished float zone 30− 40 Ω cm n-type (111) silicon (Siltronix, France) wafers were shaped as 15 × 15 × 0.5 mm3 platelets, then two opposite sides were bevelled. The bevel angle was carefully measured after polishing in order to compute the actual number of internal reflections (∼25). This angle was 46° for the results presented here and 46.5° for our calibration experiments. 2.7. Contact Mode AFM Imaging. AFM images were obtained using a Pico SPM microscope (Molecular Imaging, Phoenix, AZ) in contact mode, with silicon nitride cantilevers (Nanoprobe, spring constant = 0.12 N m−1) under a N2 atmosphere. The silicon sample was cut from one-side polished n-type (111) silicon wafers (Cz, 5−10 Ω cm, 525 μm) with a miscut of 0.2° toward the (112̅) direction for obtaining a staircase structure.

Figure 1. Grafting of poly(ethylene glycol) moieties H(EG)nOMe and ViCp−2(EG)nOMe (with p = 3, 5, 11 and n = 3, n ≈ 7, 12, 16) on a hydrogenated Si(111) surface through Si−O−C and Si−C bonds. The obtained grafted groups are labeled under compact form as (EG)nOMe and Cp(EG)nOMe.

this case from cows, bovine serum albumin, BSA), which is the most abundant protein in blood. BSA is a globular protein with a molecular weight of 66 kDa and is widely used because of its high ability to adsorb easily on different types of surfaces.29,30 The behavior of the different PEG films toward BSA adsorption was studied by IR-ATR and AFM and compared with that of a hydrophobic alkyl monolayer (p = 10) grafted on silicon.

2. EXPERIMENTAL SECTION 2.1. General Information. For the synthesis of ViCp−2(EG)nOMe precursors and the protein adsorption tests, all solvents, chemicals, and the following reagents triethylene glycol monomethyl ether (n = 3, 97%), poly(ethylene glycol) methyl ether PEG 350 (⟨n⟩ ≈ 7), PEG 550 (⟨n⟩ ≈ 12), and PEG 750 (⟨n⟩ ≈ 16) were used without further purification and purchased from Sigma-Aldrich. Thin-layer chromatography (TLC) was performed on precoated plates 60F254 (60 A-15 μm) from SDS and flash column chromatography was performed using silica gel (60 A CC 35−70 μm) from SDS. For the grafting on silicon, the solvents were of HPLC grade and anhydrous toluene was distilled over sodium/benzophenone before use. All cleaning (H2O2, 30%; H2SO4, 96%; EtOH absolute anhydrous) and etching (NH4F, 40%; HF, 50%) reagents were of RSE grade and supplied by Carlo Erba. Ultrapure water (Milli-Q, 18.2 MΩ cm) was used for the preparation of the buffers and for all rinses. 2.2. General Procedure for the Synthesis of ViCp−2(EG)nOMe. In a two-necked round bottomed flask equipped with a reflux condenser, 1 equiv of H(EG)nOMe was gently added to 2 equiv of 50% NaOH in water. The reaction mixture was heated at reflux for 1 h. After cooling down to 60 °C, 2 equiv of bromoalkene was added dropwise under magnetic stirring and the mixture was heated at reflux for ∼20−72 h. The bromoalkene was removed under vacuum (and at 130 °C for 11-bromo-1-undecene) for several hours. Then, the mixture was extracted with CH2Cl2 (3 times). The organic phase was washed with water, dried over MgSO4 and the solvent evaporated. The oily residue was purified by flash column chromatography on silica gel using a 9:1 ethyl acetate/ethanol mixture as the eluent. Different chain lengths of ViCp−2(EG)nOMe were isolated (final yield ∼ 60%). 2.3. Photochemical Hydrosilylation. The Si(111) sample was cleaned in a 1/3 H2O2/H2SO4 piranha solution (at 100 °C for 30 min) and then immersed either in an oxygen-free NH4F solution for 15 min (leading to an atomically flat hydrogenated surface, termed SiH)31 or in HF solution for 10 s (leading to an atomically rough hydrogenated surface, termed SiHx).32 During this time, a 0.1 M solution of ViCp−2(EG)nOMe in freshly distilled toluene (respectively pure H(EG)nOMe) was outgassed under argon in a Schlenk tube for 5 min at 60 °C (respectively at room temperature). After rinsing with

3. RESULTS AND DISCUSSION 3.1. Grafting of Commercial H(EG)nOMe. We first investigated the grafting of simple molecules made of a surface reactive headgroup and an oligo(ethylene oxide) functional termination, without backbone. In order to avoid specific synthesis work, we used an alcohol headgroup, since alcohols can be directly grafted onto hydrogenated silicon surfaces by a thermal33−35 or a photochemical36,37 process. In either case, monolayers are bonded to the silicon substrate via Si−O−C bonds. To our knowledge, in the literature, there are only few examples of the grafting of poly(ethylene glycol) moieties on silicon.38 The thermal grafting of H(EG)nOMe (n ≈ 7) was performed onto hydrogen-terminated silicon surfaces at different temperatures, and the obtained monolayers exhibit excellent protein repellency, as soon as the grafting density is equal to 1.7 × 1014 chains/cm2 or higher.39 Hamers and coworkers reported on the photochemical grafting of an alkene precursor terminated by triethyleneglycol. The grafting occurred via both end groups even though the reaction kinetics was three times as fast via the vinyl as via the hydroxyl termination.40 In contrast, Bernasek and Zhong recently proved that a direct photochemical functionalization of Si(111) with bifunctional 10-undecen-1-ol can be achieved with selective attachment via the vinyl end.41 In this study, commercial poly(ethylene glycol) monomethyl ethers H(EG)nOMe with n = 3 and n ≈ 7, 12, and 16 are grafted in one step onto hydrogenated silicon surfaces. Apart 14655

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Figure 2. Grafting of H(EG)12OMe on a hydrogenated Si(111) surface. (A) 4 μm × 4 μm AFM image and (B) IR-ATR spectrum in p-polarization, with the reference spectrum being in this case a SiHx surface. The inset represents the IR-ATR spectra of (EG)nOMe in the νCH region as a function of n (with n = 3, 7, and 16).

for n = 3, all molecules are polydisperse. Notice that, at one end, the alcohol termination is protected by a methoxy group to avoid any competition during the grafting. The grafting reaction is performed either photochemically or thermally (at 150 °C). After the grafting, the surfaces are rinsed in ethanol and dichloromethane. Figure 2 represents a typical AFM image (a) and an IR-ATR spectrum (b) after the photochemical grafting of H(EG)nOMe (here for n ∼ 12). We notice that the obtained surface is clean on the molecular scale. We reproducibly observe the staircase structure of the (111) silicon surface with atomically smooth terraces separated by monatomic steps of a height of 3.14 Å. A line profile of a 500 nm ×500 nm image is shown in Figure A in the Supporting Information. This structure is identical to that of the Hterminated substrate, confirming the formation of a uniform organic layer without adventitious contamination or physisorbed molecules. The water contact angle is about 37° whatever the chain length (n > 3) and the grafting method are. As shown in Figure 2, the IR spectra of (EG)nOMe layers in ppolarization exhibit the characteristic peaks related to ethylene oxide moieties. At ca. 2876 cm−1, we observe a large band corresponding to the symmetric and antisymmetric stretching modes of the CH2 and CH3. The band will be described in detail later. The band at 1461 cm−1 is assigned to the OCH2 scissor mode of the ethylene oxide group. The ether CH2wagging modes (trans at 1325 cm−1 and gauche at 1354 cm−1) and -twisting modes (at 1246 and 1297 cm−1) modes indicate an amorphous structure of the grafted PEGs with gauche and trans conformations.14 The broad band extending from 980 to 1200 cm−1 is related to the stretching modes of the C−O−C (1104 and 1143 cm−1) and Si−O−C groups (1042 cm−1). The surfaces grafted with (EG)nOMe do not exhibit measurable oxidation since the characteristic LO vibration mode of SiOSi at 1250 cm−1 is not discernible in p-polarization (see Figure B in the Supporting Information where p- and s-polarized spectra of grafted (EG)12OME layer are shown). By increasing n from 3 to 12, the IR band intensities characteristic of grafted (EG)nOMe chains also increase (cf. inset in Figure 2B). When the grafting is now performed thermally, the IR intensities are higher than those for the photochemical grafting but oxidation is also present. This increased tendency to

oxidation when thermal activation is used instead of photochemical activation is likely to be paralleled with known differences in the reaction mechanism.27 So, for the following of this work, we have used the photochemical grafting. 3.1.A. Determination of the Surface Coverage. We have quantitatively determined the molecular coverage of the monolayers from the analysis of the area of the ethylene oxide absorption bands. The large band in the 2650−3050 cm−1 region is the most appropriate to be fitted with accuracy and has been used to determine the absolute areal density of the (EG)nOMe chains immobilized on the surface. The following modes are supposed to contribute to this band: the two main symmetric and antisymmetric stretching modes of the CH2 at 2876 cm−1 and 2935−2940 cm−1 respectively; the symmetric and antisymmetric stretching modes of the CH3 at ∼2820 and 2980 cm−1 respectively. Whereas these modes are clearly observed for H(EG)nOMe in solution with small values of n, they tend to broaden with increasing values of n. Their positions are not clearly discernible because they are superimposed. The νasCH3 mode is even not seen. They are also superimposed to other contributions enhanced by Fermi resonance.42 In order to fit correctly the experimental data to a simple model, at least five Voigt functions and a linear baseline are necessary. We actually used the integrated absorbances As,p corresponding to the value of total band area in s- and p-polarization after baseline subtraction. Even though the fit is somehow poorly conditioned, due to the various overlapping contributions, the As,p values appear robust and therefore can be reliably used. We note that, as expected, the integrated intensities increase when n increases. In order to extract the chain density from the integrated absorbances As,p, we made calibrations by measuring the integrated absorbances of the same band A0 in liquids where the concentration of H(EG)nOMe is known (in our case, 0.1 M solutions of PEG 350, PEG 550, and PEG 750 in deuterated chloroform). The spectra can be fitted with the same profiles as described above (five Voigt functions and a linear baseline), but again, we rather considered the integrated area of the whole band after baseline subtraction. By comparing this integrated area with that for the grafted layer, we can calculate the number N of grafted (EG)nOMe chains per unit area (see Supporting 14656

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Table 1. Summary of the Overall IR Integrated Absorbance As,p in the ν-CH Spectral Range (from 2700 to 3000 cm−1) in s- and p-Polarization for Grafted (EG)nOMe and Cp(EG)nOMe layers on SiHxa grafted (EG)nOMe

A0s (H(EG)nOMe) (cm−1)

As,p (cm−1)

0.220 0.306 0.423

0.0342, 0.0522 0.0575, 0.0718 0.0701, 0.0951 As,p (cm−1)

⟨n⟩ ∼ 7 ⟨n⟩ ∼ 12 ⟨n⟩ ∼ 16 grafted Cp(EG)nOMe

A0s (ViCp(EG)nOMe) (cm−1)

C3(EG)3OMe C3(EG)7OMe C3(EG)12OMe C3(EG)16OMe C5(EG)3OMe C5(EG)7OMe C5(EG)12OMe C5(EG)16OMe C11(EG)3OMe C11(EG)7OMe C11(EG)12OMe C11(EG)16OMe

0.122 0.226 0.355 0.459 0.140 0.244 0.374 0.477 0.194 0.298 0.428 0.532

0.0069, 0.0146, 0.0231, 0.0308, 0.0132, 0.0287, 0.0392, 0.0461, 0.0208, 0.0342, 0.0453, 0.0531,

0.0083 0.0183 0.0280 0.0406 0.0160 0.0353 0.0503 0.0601 0.0247 0.0425 0.0584 0.0705

N (×1013cm−2)

θ (%)

21.2 18.7 19.3 N (×1013cm−2)

27.1 23.9 24.6 θ (%)

5.4 5.4 6.7 7.4 9.2 11.7 11.1 10.5 10.1 11.6 11.3 11.1

6.9 6.9 8.6 9.4 11.7 14.9 14.1 13.3 12.9 14.8 14.4 14.1

a The integrated absorbance A0s of H(EG)nOMe molecules are experimental data from a 0.1 M solution in CDCl3, whereas those of ViCp(EG)nOMe molecules are calculated for a similar 0.1 M concentration from eq 5. The number of grafted chains N and the corresponding coverage θ are indicated in the last two columns. The absolute uncertainly of the coverage determination is estimated to be approximately ±1013 cm−2.

Figure 3. Amount of adsorbed BSA on various Si(111) surfaces grafted with H(EG)nOMe with n = 3 and n ≈ 7, 12, and 16, measured by IR-ATR and normalized to the BSA adsorbed on a hydrogenated SiHx surface (n = 0) where the retention is maximal. (A) IR spectra for n = 0 and n = 12. (B) Percentage of BSA adsorption versus n. The inset represents a 4 μm × 4 μm AFM image of the BSA adsorption for n ≈ 12.

on Si(111), corresponding to 3.91 × 1014 Si−C/cm2.43 Table 1 summarizes the overall integrated absorbance, the molecular density, and the corresponding coverage of the various grafted (EG)nOMe chains. We obtain a density of about 2 × 1014 cm−2 corresponding to a coverage of about 25% for all of the grafted (EG)nOMe chains.44 The obtained coverage is in good agreement with coverage values of clean monolayers obtained by thermal grafting of poly(ethylene glycol) methyl ether, estimated by X-ray reflectometry.39 These monolayers also reach coverage values as high as those obtained on monolayers photochemically grafted via Si−C bonds on similar silicon surfaces.45 In summary, grafting of H(EG)nOMe molecules on Si surfaces appear to yield very smooth and regular molecular layers, with a fairly high surface concentration in grafted chains. 3.1.B. BSA Adsorption on Grafted (EG)nOMe. The water wettability of the different Si(111) surfaces modified with H(EG)nOMe (n = 3 and n ≈ 7, 12, and 16) was first measured after immersion in a BSA solution for 1 h. The contact angles allow the changes in the surface chemical state to be

Information part C for more details). This number can be decomposed into two contributions, N∥ and N⊥, corresponding to the equivalent number of vibrators associated with the values of the components of dynamic dipole parallel and perpendicular to the surface. In the case of the grafting of (EG)nOMe chains, As n

(1)

⎛ Ap A ⎞ N⊥ = 2.18 × 1016⎜2.11 − 1.91 s ⎟ n n⎠ ⎝

(2)

N = 2.33 × 1016

and

where n stands for the number of ethylene oxide units and As,p for the overall integrated absorbance per reflection of the νCH2 and νCH3 mode (expressed in cm−1) measured in s- or ppolarization. From these results, we deduce the final coverage θ, knowing that the maximum attainable surface coverage is ∼50% 14657

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between commercial H(EG)nOMe (n = 3 and n ≈ 7, 12, and 16) and a bromo-alkene precursor (p = 3, 5, and 11).2 After purification by column chromatography, they present a narrow molecular weight distribution. For example for ViC3EG7OMe, we can successfully separate molecules with n = 7 ± 1 from others with n > 8 and n < 6, as determined by 1H NMR (see Supporting Information, Figure D). The grafting of such Cp(EG)nOMe chains was achieved photochemically in toluene (0.1 M). The solvent easily dissolves these amphiphilic molecules and is also inert toward the hydrogenated surfaces. Although it is well-suited for the grafting, a toluene rinse is not sufficient to clean the grafted surfaces. Adding the standard rinse in EtOH and CH2Cl2, which was efficient for the grafted (EG)nOMe layers, is not successful to remove all residues from the surface on Cp(EG)nOMe layers. These residues are thought to be ViCp−2(EG)nOMe molecules which probably form micelles and are well-physisorbed on Cp(EG)nOMe layers. Protic solvents (like water, PEG derivates, decanol...) largely improve the cleanliness of the surface but lead to nonreproducible results. Only a rinse in a saline buffer in the presence of an amphiphilic surfactant like SDS allows for obtaining perfectly clean surfaces. The AFM images of surfaces grafted with C5EG16OMe (Figure 4A) and C11EG16OMe (Figure 4B) chains

determined and the antifouling properties of the surfaces to be eventually correlated with their hydrophilicity. The static water contact angle stays constant at 37° when n > 7 and increases to 42° for lower values of n. We also measured the water contact angle of a decyl-terminated surface which is supposed to be prone to protein adsorption. This angle actually drops from 110° to 46° after BSA immersion. By contrast, this result suggests that BSA does not adsorb on the (EG)nOMe monolayers. In view of obtaining a more quantitative assessment of BSA adsorption (contact angle measurements hardly allow to discriminate between clean PEG surfaces and surfaces with adsorbed BSA, because the measured contact angles differ by less than 10°), the amount of adsorbed BSA on the grafted (EG)nOMe layers was determined by IR-ATR as shown in Figure 3. The intensities of the amide I and II bands of the protein were quantified relative to those measured for BSA adsorbed on a decyl monolayer grafted on the Si(111) surface (n = 0), for which retention is maximal. We also tested the hydrogenated SiHx surface as a reference, and found that the amount of adsorbed BSA was the same as for the decylterminated surface. As expected, increasing n from 3 to 12 lowers the protein adsorption. For n ∼ 12, only 4% of BSA are physisorbed. The corresponding AFM image (Figure 3B) shows that some white spots with heights comprised between 3 and 9 nm, plausibly corresponding to the aggregation of BSA, are deposited onto the surface which remains relatively clean. For n ∼ 16, the surface is perfectly repellent to BSA. Although these surfaces exhibit antifouling properties, they are not chemically stable. Namely, after several hours of immersion in saline solution, the surfaces are degraded (loss of (EG)nOMe chains together with oxidation) leading to a loss in efficiency toward the non specific adsorption. Surfaces covered with acidterminated monolayers of similar density obtained by hydrosilylation are found to be stable as long as pH does not exceed 10.46 Therefore, the degradation observed here is primarily ascribed to the hydrolysis of Si−O−C bonds at the surface. In conclusion, in spite of their fairly high surface concentration, (EG)nOMe layers do not retain their antifouling properties for the long-term. This weakness is ascribed to the chemical vulnerability of the bond between surface and molecules. It is also an indirect consequence of the absence of a backbone promoting molecular organization, as shown by the stability of Si−OC bonds when they are protected by a properly organized alkyl chain.33,36 3.2. Grafting of ViCp−2(EG)nOMe. In this context, we grafted various hybrid molecules consisting of a methoxypoly(ethylene glycol) fragment attached to a backbone alkyl chain bearing a terminal vinyl group, ViCp−2(EG)nOMe. The latter provides a stable chemical link with the silicon surface via the formation of Si−C bonds yielding the grafting of Cp(EG)nOMe chains on the surface. In the literature, several groups reported on the grafting of PEG coatings composed of long alkyl chains (9−11 carbons) necessary to passivate the substrate and of several ethylene oxide units (from 3 to 9) terminated by various end groups (OMe, NH2, OH, COOH, ...).47−49,40 It has been reported that at least six units of EG are necessary for negligible protein adsorption.50,51 Until now, there is no study on the influence of the length of the alkyl versus PEG chains toward the nonspecific adsorption on silicon substrate. We therefore varied the length of the hydrophobic aliphatic alkyl chain from 3 to 11 and that of the ethylene glycol moieties from 3 to 16. These amphiphilic molecules are synthesized in one step via the Williamson ether reaction

Figure 4. Grafted C5(EG)16OMe (small p) and C11(EG)16OMe (large p) monolayers. (A, B) 2 μm × 2 μm AFM images after a surfactinated rinse. (C) IR-ATR spectra in p-polarization, the reference spectra being SiHx surfaces.

show that the staircase structure of the silicon is preserved and that the surfaces are free from unwanted residues. These results are confirmed by IR-ATR spectroscopy where the global intensities of the bands characteristic of ethylene oxide moieties decrease after the surfactinated rinse due to the removal of the physisorbed contaminants or micelles (results not shown). Thus, this specific rinse is of prime importance if one wants to determine the molecular density of the grafted chains from the area of the ethylene oxide absorption peaks. In practice, the optimized rinse is the following: a first rinse in 1× PBS/0.1% SDS for 5 min, a second one in 0.2× PBS for 1 min then in 0.1× PBS for 1 min, and finally a quick rinse in water. Figure 4C shows the IR-ATR spectra of two Si(111) surfaces modified with a methoxy-poly(ethylene glycol) (n ≈ 16) terminated by a pentyl chain (p = 5) or an undecyl chain (p = 11). Here the νCH band now contains contributions from the alkyl chain backbone and from the ethylene oxide units. Figure 5 shows the 14658

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of ViC3(EG)8OMe in the 2700−3000 cm−1 range, recorded in s-polarization, and the results of the decomposition of a possible fit (five Voigt functions as previously for the various νC−H modes of the ethylene oxide units, plus two Voigt functions for those of the methylene groups of the alkyl chain, and a linear baseline). As a matter of fact, nine O−CH2−C units are expected to contribute to the ethylene oxide-type vibrational modes and only two C−CH2−C units of the alkyl chains are expected to contribute to the methylene-type modes. Notice that the C−H bonds of the vinyl group have a very low infrared absorption that is outside the spectral range considered here, and therefore do not contribute to the band shown in Figure 6A. If one makes the reasonable assumption that the intensity of the ethylene oxide-type modes is proportional to the number of contributing O−CH2−C units (and respectively the intensity of the methylene-type modes is proportional to the number of contributing C−CH2−C units), one could hope to extract the infrared cross section of a single O−CH2−C unit and that of a single C−CH2−C unit from the decomposition shown in Figure 6A. It is clear that such a procedure is questionable in view of the ill-conditioned mathematical problem associated with the above fit. Therefore, in the following, only the overall integrated absorbance As0 is considered (in s-polarization and after baseline subtraction). Generally speaking, for a ViCp−2(EG)nOMe molecule, there are p − 3 C−CH2−C units, 2n + 1 O−CH2−C units, and one O− CH3 unit contributing to the νCH absorption in the 2700− 3000 cm−1 range. Making the further assumption that the contributions of O−CH2−C and O−CH3 units are identical, it comes:

Figure 5. IR-ATR spectra in the νCH range recorded in p-polarization of various grafted Cp(EG)nOMe with p = 3, 5, 11 and n = 3 and n ≈ 7, 12, 16.

spectra plotted in the νCH range for various grafted Cp(EG)nOMe. When p increases, the intensities of the CH2 deformation and stretching modes of the alkyl chains also increase. The position of the antisymmetric stretching mode at 2926 cm−1 suggests that the alkyl chains have gauche defects and are not closely packed. In addition to the band associated with C−O−C stretching vibrations from ethylene oxide units (Figure 4C), a vibration related to the oxide appears as a shoulder at ∼1049 cm−1 for p = 3 suggesting that, for this low value of p, surfaces grafted with Cp(EG)nOMe are more prone to oxidation than those grafted with H(EG)nOMe. However, in this case, the LO mode detected in p-polarization appears at a frequency (∼1155 cm−1) significantly lower than that of a continuous oxide layer. Such a low TO-LO splitting demonstrates that only minute oxidation has taken place at the surface.52 3.2.A. Determination of the Surface Coverage. As for (EG)nOMe layers, the IR CH-stretching region of the alkyl and ethylene oxide chains is used to determine the grafted molecules concentration. Similarly, calibration of infrared cross sections of related molecules in solution is needed to determine the surface concentrations. For that purpose, we recorded infrared spectra of some of the ViCp−2(EG)nOMe molecules used as precursors for the grafting (0.1 M solution in CDCl3). As an example, Figure 6A shows the IR-ATR spectrum

A s0 = A(2n + 2) + B(p − 3)

(3)

where n stands for the number of ethylene oxide units and p − 2 for the number of methylene units in the ViCp−2(EG)nOMe molecule. A and B are coefficients accounting for the IR cross section of the vibrational modes of interest for O−CH2−C and C−CH2−C units, respectively. For all n and p values, we can obtain the values of A and B from

Figure 6. (A) IR-ATR spectrum of ViC3(EG)8OMe in the νCH range recorded in s-polarization and results of the decomposition of the fit (contribution of 7 pseudo-Voigt peaks superimposed on a linear baseline). (B) Ratio of the total area of the νCH band, A0s , over 2n + x as a function of (p − 3)/(2n + x), with p number of CH2 units and n number of OCH2CH2 units in the ethylene oxide chain for various H(EG)nOMe (p = 0, x = 1) and ViC−2(EG)nOMe (p ≥ 3, x = 2) molecules. Points (■) are experimental data, point (red ◆) corresponds to the average value of H(EG)nOMe and the solid line corresponds to eq 5. 14659

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A s0 p−3 =A+B 2n + 2 2n + 2

chains is very low (∼7 × 1013 cm−2) whatever the value of n is. This weak grafting yield can plausibly be ascribed to the absence of local ordering when the alkyl chain is too short: the absence of sizable interaction between neighboring grafted chains favors a configuration flat on the surface and limits the density of grafted molecules. Alternately, one might also consider that for p = 3, the single carbon inserted between the vinyl group and the first oxygen atom of the ethylene oxide units does not completely prevent the weakening of the CC reactivity induced by the electronegativity of the oxygen atom. When p increases, the density of grafted chains increases to 1.1 × 1014 cm−2 corresponding to a coverage of about 14%. These values are lower than those of (EG)nOMe chains obtained after grafting of H(EG)nOMe molecules. This is probably due to the difference in concentration of the solutions used for the grafting, since the commercial H(EG)nOMe are used pure whereas the synthesized ViCp−2(EG)nOMe are used diluted. When the grafting of PEG550 is done at 0.1 M in toluene, the coverage is found to be 13%, confirming the impact of the molecule concentration during the grafting on the density of the grafted layers. Conversely, one may infer that using a solvent-free grafting technique inspired from that described by Yam et al,51,53 it would be possible to obtain Cp(EG)nOMe of similar concentration than those reached for (EG)nOMe layers (∼2 × 1014 cm−2, see above). This figure is still lower than that reported by others.51 We think that this discrepancy mostly comes from uncertainties associated with the XPS determination of surface coverages.14,24,34,54 However, in spite of their limited surface concentration, the monolayers grafted via Si−C are very stable as expected. After 15 days of storage (in air or under nitrogen atmosphere), the corresponding IR spectra revealed no appearance of oxidation. The grafted Cp(EG)nOMe layers are also stable in water for more than 8 days without any sizable oxidation. 3.2.B. BSA Adsorption on Grafted Cp(EG)nOMe. We tested the resistance of the grafted Cp(EG)nOMe monolayers toward BSA adsorption. Test experiments performed on grafted C5(EG)12OMe and C5(EG)16OMe revealed that the contact angle (θ = 38°, close to the values recorded on (EG)nOMe layers) does not change upon immersion in BSA solution. In order to go beyond these first indications, the amount of adsorbed BSA on grafted Cp(EG)nOMe was quantified by IRATR spectroscopy which is more sensitive and quantitative than the contact angle method. Once again, the adsorption amounts are normalized to that of BSA adsorbed on a hydrogenated SiHx surface. Figure 7 shows the results for (EG)16OMe and Cp(EG)16OMe with p = 5 and 11 after the standard rinse (in 1× PBS) and the surfactinated rinse (i.e., phosphate buffer containing 0.1% SDS as previously used). The latter rinsing conditions are more efficient to remove physisorbed proteins and are currently used in protein chips applications. Whereas there is almost no adsorption for grafted (EG)nOMe, the amount of BSA significantly increases from 25% for p = 5 to 50% for p = 11 after a normal rinse. For p = 3 (results not shown), the BSA adsorption is even higher than for p = 5. This behavior can be traced back to a low concentration of grafted C3(EG)nOMe chains and a higher surface oxidation, as revealed by infrared measurements and already discussed. For a given value of p, less adsorption is observed when n increases. After a surfactinated rinse, the protein repellent character is largely improved: for p = 5, there is no physisorption and for p = 11, the amount of BSA drops to 10%. In contrast, there is no change in amide band intensity on

(4)

In Figure 6B, + 2) is plotted against (p − 3/2n + 2) for various ViCp‑2(EG)nOMe molecules: (ViC(EG)8OMe and ViC(EG)10OMe for (p − 3/2n + 2) = 0, ViC3(EG)16OMe for (p − 3/2n + 2) = 0.059, ViC3(EG)8OMe for (p − 3/2n + 2) = 0.11, ViC3(EG)6OMe for (p − 3/2n + 2) = 0.14, ViC9(EG)8OMe for (p − 3/2n + 2) = 0.44). The alignment of all the experimental points of the plot along a straight line supports the previous assumptions. A linear fit of this plot allows for the determination of the A and B coefficients (in cm−1): (A0s /2n

A s0 p−3 ≈ 0.0130 + 0.0090 2n + 2 2n + 2

(5)

Consistently, the contribution of one O−CH2−C unit, as determined under the same assumptions from the infrared spectra of solutions of H(EG)nOMe molecules (as reported in section 3.1.A) is in agreement with the extrapolation of the plot of Figure 6B to a vanishing number of contributing C−CH2−C units (see the circle at the origin of the plot). This relation allows for determining the Aos value of a 0.1 M solution of ViCp−2(EG)nOMe molecule in the same geometry as in the above calibration experiments, without making an actual experiment. After reaction of ViCp−2(EG)nOMe molecules with the hydrogenated silicon surface, Cp(EG)nOMe chains are grafted on the surface. Therefore, p − 1 C−CH2−C units, 2n + 1 O− CH2−C units, and one O−CH3 unit contribute to the νC−H absorption of these chains in the 2700−3000 cm−1 range. This contribution is proportional to that of a ViCp(EG)nOMe molecule, and in order to determine the surface concentration of the grafted Cp(EG)nOMe chains, the IR absorbance of the Cp(EG) nOMe chains has to be calibrated against the absorbance of a solution of ViCp(EG)nOMe molecules at a known concentration. Such an absorbance, As0, can be computed for a 0.1 M solution from eq 5. The absorbance As,p in s- and p-polarization, integrated from 2700 to 3000 cm−1, is determined from the IR-ATR spectra of the grafted chains Cp(EG)nOMe shown in Figure 5. Then, as described in the Supporting Information, the combination of these various quantities yields the numbers of vibrators parallel and perpendicular to the surface, i.e., for values of incidence angles corresponding to our experimental conditions (46° for the measurement after grafting, 46.5° for the calibration experiment): N = 6.06 × 1014

As A s0

(6)

⎡ 2.11A p − 1.91A s ⎤ ⎥ N⊥ = 5.68 × 1014⎢ A s0 ⎣ ⎦

(7)

and

The total number of grafted Cp(EG)nOMe chains per unit area can finally be deduced as the sum of N∥ and N⊥. Table 1 gives the calculated values of A0s for 0.1 M solutions of ViCp(EG)nOMe molecules, according to eq 5, the experimental values As,p obtained after photochemical hydrosilylation of ViCp−2(EG)nOMe molecules, and the corresponding surface concentrations of grafted Cp(EG)nOMe chains deduced from eqs 6 and 7. For p = 3, the density of grafted 14660

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Such phenomena could be avoided by imaging in tapping mode. The ability of the grafted Cp(EG)nOMe layer with high n/p value to retain their protein repellent properties has also been verified. After 15 days of storage (in air or under nitrogen atmosphere), the corresponding IR spectra revealed no amide peaks after immersion in BSA followed by a surfactinated rinse. This stability is to be paralleled with the chemical stability of the layer itself. In contrast, the grafted (EG)nOMe layers exhibit limited chemical stability, resulting in a poor retention of their protein repellent properties. 3.2.C. Discussion. For grafting oligo(ethylene oxide) molecules on silicon, the presence of a minimum backbone aimed at favoring packing and organization of the molecular layer seems mandatory. However, it appears that the role of this backbone is mostly to provide a vinyl headgroup allowing for robust chemical coupling to the surface via Si−C bonds. Very short backbones (p = 3) appear to be impracticable, yielding poorly dense layers, with detectable substrate oxidation and limited resistance to protein adsorption. This deficiency can be ascribed either to a lack of molecule organization during the grafting, or to a decrease in the CC reactivity due to the presence of a nearby oxygen atom of the first ethylene oxide unit. The use of long enough backbones could plausibly favor the inner organization of the molecular layers. This does not appear to be the case, since similar surface concentrations of grafted molecules are obtained for p = 5 and p = 11. This could be ascribed to the bulky presence of the disordered oligo(ethylene oxide) units. This is however questionable since very compact C11EG6OH layers are obtained on Au(111) and even denser layers on Ag(111).14 On metals, such an organization is promoted by favorable van der Waals interactions between the chains and a high packing densely formed favored by the dense arrangement of surface metal atoms. On silicon, the lower surface atom concentration and the stronger covalent character of the molecule-surface bonding are less favorable to the establishment of high molecular packing density and strong van der Waals interactions, as it is well-known for alkyl chains. We therefore suggest that the presence of oligo(ethylene oxide) units makes this balance even less favorable and prevents the van der Waals interactions among alkyl chains to promote a significant ordering of the layer, limiting surface concentration to values reached in the absence of backbone (∼2 × 1014 cm−2). With respect to the capacity of resisting protein adsorption, high n/p ratios seem to be the best solution. This trend may appear of common sense in view of the known role of the ethylene oxide units in the repellency of proteins. Nevertheless, the present specific results are at variance with previous reports in which protein resistance was acquired in most cases when n reaches a value of ∼6.50,51,55 These trends can be consistently explained in view of the limited surface concentration of grafted molecules in our layers. Short oligo(ethylene oxide) chains are expected to adopt a brushlike conformation at the surface. If their structure is too open, it offers some access for protein to the inner part of the layer (or even the substrate), favoring thereby their adsorption. Longer chains are expected to adopt a more entangled conformation.14 This will be plausibly form a more efficient shield and account for the good resistance of the layer against protein adsorption. Intuitively, besides the importance of having stable Si−C bonds at the surface, the chemical stability of the surface is also thought to be associated with the presence of the water-repellent alkyl layer. But it has been demonstrated

Figure 7. Amount of adsorbed BSA on various Si(111) surfaces grafted with (EG)16OMe and with Cp(EG)16OMe with p = 5 and 11, normalized to the BSA adsorbed on a hydrogenated SiHx surface, for which the retention is maximal: after standard (yellow) and surfactinated (cyan) rinse. For grafted (EG)16OMe and after the surfactinated rinse of C5(EG)16OMe, there is no detectable adsorption.

a decyl terminated surface, indicating that on decyl surfaces, proteins are strongly adsorbed. These results clearly demonstrate that the antifouling performance of the surface depends on the density of the grafted Cp(EG)nOMe chains which should be higher than 1.1 × 1014 cm−2, a threshold slightly lower than that given in reference 39, and on the n/p ratio: the higher this ratio, the more efficient the monolayer is to prevent the nonspecific adsorption of proteins. Whereas Cai and coworkers51 demonstrated that a dense C11(EG)6OMe layer reduced the adsorption of protein to ∼3% monolayer, we show here that no protein adsorption takes place on a much less dense C5(EG)16OMe film (no detectable proteins by IR, i.e., an amount lower than 1%). The AFM images of Si(111) modified with C5(EG)16OMe after BSA adsorption are shown in Figure 8

Figure 8. 4 μm × 4 μm AFM images of the BSA adsorption on Si(111) surfaces modified with C5(EG)16OMe: after (A) standard and (B) surfactinated rinse.

after the standard rinse (A) and after the surfactinated rinse (B). In both cases, we can easily distinguish the staircase structure of the grafted silicon. However, the amount of protein is clearly more important in (A) than in (B). Although the IR amide bands of the BSA were almost undetectable after the surfactinated rinse, AFM indicated that some traces of proteins are still present. One explanation is that the images were obtained in contact mode. So even if there are small protein clusters, they can be wiped by the tip scanning, resulting in some meandering white lines running across the surface image. 14661

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that this layer brings long-term stability for dense enough layers only,56 which is not the case here. Therefore, the entangled conformation of the ethylene oxide chains on top of the layer plausibly plays an active role in the chemical stability of the surface, also providing an efficient shield against water penetration. In this picture, water access to the interface is not impeded by the close packing of the alkyl chains, like in dense alkyl layers, but by the upper entangled ethylene oxide chains. In conclusion, it appears that a good resistance to protein adsorption can be achieved with a modest surface concentration of molecules ViCp−2(EG)nOMe containing ethylene oxide moieties. In this case, the presence of Si−C bonds appears to play a major role in the resistance to protein adsorption, plausibly through the ability of these chains to adopt a disordered entangled conformation at the surface. The alkyl backbone seems to act as a (required) linker rather than an entity promoting organization in the inner part of the layer.

AUTHOR INFORMATION

Corresponding Author

*(A.C.G.-L.) E-mail: [email protected]. Phone: +33 1 69 33 46 80. Fax: +33 1 69 33 47 99. (F.O.) E-mail: [email protected]. Phone: +33 1 69 33 47 04. Fax: +33 1 69 33 47 99. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS E.P. thanks the Direction Générale de l’Armement for Ph.D. financial support. The authors thank the OSEO (ARTAMIS no. A0605054Q grant) and the “Conseil général de l’Essonne” (“Sibioslide” ASTRE grant).

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REFERENCES

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4. CONCLUSION In this work, we investigated the influence of the molecular composition of grafted poly(ethylene glycol) moieties on (111) Si on their final antifouling performances toward BSA. Two types of monolayers were studied. The first one is obtained by photochemical grafting of neat dispersions of commercial H(EG)nOMe. The surface coverage of the resulting (EG)nOMe monolayers, quantitatively determined by IR-ATR spectroscopy, is about 2 × 1014 cm−2 irrespective of the chain length. The monolayers exhibit excellent repellent properties toward BSA for n > 7. Unfortunately, the grafted layers are not stable as a function of time because of the hydrolysis of the Si−O−C bonds. The second kind of layers was obtained by photochemical hydrosilylation of synthesized ViCp−2(EG)nOMe leading to robust monolayers attached via Si−C bonds. The surface state was checked by AFM imaging and we proved the importance of a surfactinated rinse to remove all physisorbed molecules after grafting. The surface concentration of the grafted layers was lower, from 7 × 1013 (for p = 3) to 1.1 × 1014 cm−2 (for p ≥ 5). However, their antifouling performance was largely improved: for high values of n/p (such as for C5(EG)16OMe), the nonspecific adsorption of BSA is prevented in spite of the limited value of the molecular surface concentration. At variance with common belief, these results show that a high concentration of grafted ethylene oxide chains is not required for achieving a good resistance against protein adsorption. We suggest that, for molecular layers on silicon, which are always much less dense than their analogues on Au, the alkyl chains in the inner part of the layer do not have the capability of promoting a specific organization of the upper ethylene oxide part, but when the ethylene oxide chains are long enough, they plausibly adopt an entangled, amorphous configuration which accounts for their antifouling behavior. It can be inferred that, for Cp(EG)nOMe layers denser than ours (obtained, e.g., by a solvent-free grafting process), this amorphous entangled conformation would also be formed for somewhat shorter ethylene oxide chains.

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ASSOCIATED CONTENT

* Supporting Information S

Additional information as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. 14662

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UV-Induced Formation of Oligoethylene Oxide Mono layers. ACS Appl. Mater. Interfaces 2011, 3 (3), 697−704. (50) Clare, T. L.; Clare, B. H.; Nichols, B. M.; Abbott, N. L.; Hamers, R. J. Functional monolayers for improved resistance to protein adsorption: Oligo(ethylene glycol)-modified silicon and diamond surfaces. Langmuir 2005, 21 (14), 6344−6355. (51) Yam, C. M.; Lopez-Romero, J. M.; Gu, J. H.; Cai, C. Z. Proteinresistant monolayers prepared by hydrosilylation of alpha-oligo(ethylene glycol)-omega-alkenes on hydrogen-terminated silicon (111) surfaces. Chem. Commun. 2004, 21, 2510−2511. (52) Ozanam, F.; Chazalviel, J.-N. Insitu Infrared vibrational study of the early stages of silicon oxidation at the interface with a non-aqueous electrolyte. J. Electroanal. Chem. 1989, 269 (2), 251−266. (53) Yam, C. M.; Gu, J. H.; Li, S.; Cai, C. Z. Comparison of resistance to protein adsorption and stability of thin films derived from alpha-hepta-(ethylene glycol) methyl omega-undecenyl ether on HSi(111) and H-Si(100) surfaces. J. Colloid Interface Sci. 2005, 285 (2), 711−718. (54) Note: The XPS determination of the molecular coverage according to the procedure described in ref 34 and used in ref 50 requires the knowledge of various parameters including the attenuation length for photoelectrons in the molecular layer, atomic sensitivity factors which are known to be instrument dependent, and a reliable determination of the molecular layer thickness. Alternate methods described in ref 24 get rid of thickness determination and atomic sensitivity factor but require calibration against a standard and in most cases a comparison to molecular conformation on gold, a not trivial task for oligo(ethylene oxide) layers for which the conformation is known to sensitively depend on the molecule architecture (see ref 14). (55) Kilian, K. A.; Bocking, T.; Gaus, K.; Gal, M.; Gooding, J. J. Si-C linked oligo(ethylene glycol) layers in silicon-based photonic crystals: Optimization for implantable optical materials. Biomaterials 2007, 28 (20), 3055−3062. (56) Gorostiza, P.; Henry de Villeneuve, C.; Sun, Q. Y.; Sanz, F.; Wallart, X.; Boukherroub, R.; Allongue, P. Water exclusion at the nanometer scale provides long-term passivation of silicon(111) grafted with alkyl monolayers. J. Phys. Chem. B 2006, 110 (11), 5576−5585.

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dx.doi.org/10.1021/la303022a | Langmuir 2012, 28, 14654−14664