Article pubs.acs.org/Langmuir
Functionalized Hydrogen-Bonding Self-Assembled Monolayers Grafted onto SiO2 Substrates Michael̈ A. Ramin, Gwénael̈ le Le Bourdon, Karine Heuzé, Marie Degueil, Colette Belin, Thierry Buffeteau, Bernard Bennetau, and Luc Vellutini* Université de Bordeaux 1, ISM, UMR 5255, F-33400 Talence, France CNRS, ISM, UMR 5255, F-33400 Talence, France S Supporting Information *
ABSTRACT: A novel urea coupling agent possessing a vinylterminal group and trimethoxysilyl anchoring group was synthesized and grafted onto SiO2/Au substrates. This ureido coupling agent exhibits a good capacity to directly yield homogeneous SAMs with a surface smoothing. Polarization modulation infrared reflection−absorption spectroscopy (PMIRRAS) was used to monitor these SAMs. Indeed, the different functional groups (alkyl chain, urea, and vinyl) of this coupling agent were clearly observed in the PM-IRRAS spectra. Chemical modifications of the terminal function for the covalent immobilization of biomolecules were monitored by PM-IRRAS for the first time. We have demonstrated the successful reactions of the conversion of the vinyl-terminated SAMs successively into SAM-COOH and SAM-NHS without any degradation of the monolayer. The reactivity of activated esters was successfully investigated in order to immobilize the protein A.
1. INTRODUCTION In the fields of biotechnology and nanotechnology, the control of surface properties represents an important parameter for their development. Self-assembled monolayers (SAMs) on silicon based surfaces provide molecularly defined platforms for chemical derivatization.1,2 SAMs are largely used to immobilize in a covalent way biomolecules, such as DNA or proteins, on flat surfaces in view to be used in optical and electronic biosensors.3,4 More particularly, SAMs grafted onto oxide surfaces via covalent siloxane bonding are generally more stable than thiol-based compounds deposited onto gold surfaces. Indeed, the stability of SAMs based-organosilanes depends on the covalent nature of the grafting process, which induces a covalent anchoring with the silanol groups onto the substrate and on the partial cross-linkage of the molecules through Si− O−Si bonds. These SAMs grafted onto SiO2 surfaces can undergo chemical modifications without deterioration of the organic monolayers.2 Various reports have pointed out the crucial role of the molecular structure of precursors on self-assembly, and it has been shown that the chain length is an important factor for the formation of high quality SAMs. Biological applications are strongly dependent on the surface properties of SAMs, including wetting and adhesion. These properties are essentially determined by the terminal functional groups present at the SAM-air interface, by the composition of the monolayers, and by the packing properties.5−15 © 2012 American Chemical Society
From a synthetic point of view, the control of the chain lengths usually involves the coupling of halogenated derivatives via organometallic intermediates. However, it should be noticed that the routes leading to long-chain alkyl groups are timeconsuming and give relatively poor yields with poorly soluble silylated precursors.16 Concerning the formation of a monolayer, the molecular self-assembly is mainly controlled by the intermolecular van der Waals interactions of the long alkyl chains. Nevertheless, the assembly of SAMs can be also obtained by electrostatic interactions or hydrogen bonding. Organosilane based SAMs containing internal amide17−22 or urea23−25 groups have received much attention since they allow hydrogen bonds between adjacent molecules. The selfassembled monolayers containing hydrogen bonds lead to the formation of a network of lateral cross-links within the film which could improve the stability of the SAMs.18 More particularly, the urea groups self-assemble strongly via hydrogen bonds into a one-dimensional α-network between neighboring urea groups of adjacent molecules.26,27 These intermolecular interactions are generally stronger than those obtained with amide groups. Recently, we have described the synthesis of ester-terminated organosilane coupling agents containing urea moiety in the linear alkyl chains to prepare functionalized hydrogen-bonding SAMs on a silica surface.28 The IR studies Received: September 21, 2012 Revised: November 28, 2012 Published: November 28, 2012 17672
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2. EXPERIMENTAL SECTION
have demonstrated that the carbonyls of the urea groups were preferentially oriented parallel to the surface, allowing strong hydrogen bonding between the molecules. In addition, the presence of the urea group improves the solubility of coupling agents in organic solvents and facilitates the synthesis of silylated precursors by using isocyanate chemistry. A wide range of chemical groups (amines, alcohols, carboxylic acids, ...) can be introduced as a terminal function of the monolayer films. Nevertheless, the terminal functional group of the silylated coupling agent must be chemically compatible with the very electrophilic trichlorosilane group. This restriction involves protecting the terminal functional group in a non-nucleophilic form before grafting or the required terminal functional group must be generated after the SAM formation. According to the latter, the self-assembled vinylterminated monolayers have received much attention in the past decade to generate polar terminal functional groups such as alcohol by hydroboration and oxidation,29−31 aldehydes by ozonolysis,32 and carboxylic acids by oxidation.33−36 ω-Alkenealkyltrichlorosilanes are mainly used to prepare vinyl-terminated SAMs.30,34,37−39 The incorporation of the anchoring group occurs generally at the last step of the synthesis to avoid the possible hydrolyzation of the silylated coupling agent. Alkoxysilanes can also be used to incorporate the anchoring group since they are more stable with respect to the hydrolysis than chlorosilanes.40 The chemical modification of the terminal function is necessary in order to attach biomolecules by covalent links. The different steps of this chemical modification (oxidation of the vinyl groups, activation of carbonic acids, and immobilization of the biomolecules) should be carefully monitored to ensure the quality of the biosensor. A large number of techniques can be used to investigate and characterize selfassembled films, such as FTIR spectroscopy, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), Brewster angle microscopy (BAM), ellipsometry, and contact angle measurements. Among these techniques, polarization modulation infrared reflection−absorption spectroscopy (PMIRRAS) is a very simple and nondestructive way of acquiring molecular information on these bidimensional systems, such as the formation of chemical bonds with the substrate, the hydrogen-bonded structures, the conformation of the alkyl chains and the orientation of the functional groups. Due to its high sensitivity in surface absorption detection, PM-IRRAS has been used successfully to obtain vibrational spectra of Langmuir−Blodgett (LB) monolayers, lipid bilayers and SAMs deposited onto metallic substrates,41−49 as well as onto oxide surfaces (SiO2/Au or TiO2/Au).50−52 The very high sensitivity of PM-IRRAS allows us to monitor easily the chemical modification of the terminal function of SAMs grafted onto oxide surfaces, as demonstrated for the first time in this study. In the present paper, we report the design of a novel urea coupling agent possessing a vinyl-terminal group and a trimethoxysilyl anchoring group. We have investigated the preparation of self-assembled vinyl-terminated monolayers onto silica surfaces and their conversion into carboxylic acid groups in order to attach biomolecules by covalent links. The identification of the vinyl-terminated monolayers onto the surface as well as its subsequent chemical modifications have been performed using PM-IRRAS.
2.1. Synthesis of Ureido Silylated Coupling Agent 2. All solvents were dried and distilled under inert atmosphere, according to literature procedures, immediately before use. 1H and 13C NMR spectra of compounds were recorded on Bruker AC 250 or DPX 300 spectrometers at room temperature in deuterated chloroform and using TMS as an internal reference for the chemical shifts. Chemical shifts, δ, are represented in part per million (ppm) and coupling constants, J, in Hertz (Hz). The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet. High resolution mass spectra were obtained on an hybrid Fisons-Instruments VG Micromass AutoSpecEQ spectrometer using LSIMS+ technique in 3-nitrobenzyl alcohol (NBA) as the matrix. Microanalyses of molecules were performed at the Service Central d’Analyse du CNRS, BP22, 69390 Vernaison. Synthesis of 10-Isocyanatodecene, 1. 10-Undecenoyl chloride (2.69 g, 13.3 mmol) was added slowly at 0 °C to a suspension of sodium azide (1.73 g, 26.6 mmol) in anhydrous toluene (20 mL). The mixture was stirred at room temperature for 4 h and then was transferred under inert atmosphere in a three-necked flask containing anhydrous toluene (200 mL) at 75 °C. The mixture was stirred at this temperature for 20 h. Then, it was filtered through Celite and was concentrated under reduced pressure. The isocyanate was purified by distillation under reduced pressure (105 °C, 0.05 mmHg) as a colorless oil (1.47 g, 61% yield). 1H NMR (300 MHz, CDCl3): δ = 1.30−1.50 (m, 10 H, 5 CH2), 1.63 (q, 2H, CH2CH2CO), 2.05 (q, 2 H, J = 7.0 Hz, CH-CH2), 3.27 (t, 2 H, J = 6.8 Hz, CH2NCO), 4.94− 5.05 (m, 2 H, CH2), 5.79−5.89 (m, 1 H, CH). 13C NMR (75.4 MHz, CDCl3): δ = 26.4−31.2 (7 CH2), 33.7 (CH−CH2), 42.9 (CH2NCO), 114.1 (CH2), 138.9 (CH). FTIR (cm−1): 2268 (νNCO), 2856 (νs CH2), 2928 (νas CH2). Anal. Calcd for C11H19NO (%): C 72.88, H 10.56, N 7.73, O 8.83. Found (%): C 72.69, H 10.63, N 7.73, O 8.84. Synthesis of 1-(Dec-9-enyl)-3-[trimethoxysilylpropyl]urea, 2. To a solution of 10-isocyanatodecene 1 (0.50 g, 2.76 mmol) in dried CH2Cl2 (30 mL) was added slowly at 0 °C (3-aminopropyl)trimethoxysilane (0.52 mL ; 3.04 mmol). The mixture was stirred for 16 h at room temperature. The solvent and the excess of (3aminopropyl)-trimethoxysilane were then removed by Kugelrohr distillation (80 °C, 0.01 mmHg) to afford the compound 2 as a very viscous oil (0.75 g, 80% yield). 1H NMR (300 MHz, CDCl3): δ = 0.63−0.69 (m, 2 H, CH2Si), 1.28−1.66 (m, 14 H, 7 CH2), 2.04 (q, 2 H, J = 6.8 Hz, CH2-CH), 3.11−3.20 (m, 4 H, 2x CH2N), 3.56 (s, 9 H, Si(OCH3)3), 4.32−4.45 (m, 2 H, 2x NH), 4.92−5.02 (m, 2 H, CH2), 5.77−5.86 (m, 1 H, CH). 13C NMR (75.4 MHz, CDCl3): δ = 6.3 (CH2Si), 33.7−23.4 (8 CH2), 33.7 (CH-CH2), 42.8−40.6 (CH2N), 50.5 (Si(OCH3)3), 114.1 (CH2), 139.1 (CH), 158.3 (COurea). 29Si NMR (59 MHz, CDCl3): δ = −42.04. FTIR (cm−1): 1089 (νSi−O), 1576 (δNH, νCN), 1633 (νCO), 2855 (νS CH2), 2928 (νas CH2). Anal. Calcd for C17H36N2O4Si (%): C 56.63, H 10.06, N 7.77, Si 7.79. Found (%): C 56.50, H 10.20, N 7.55, Si 7.75. ESI-MS m/z: 383.3 (M+Na)+. 2.2. Chemical Surface Modification. Materials and Substrates. The SiO2/Au substrates were supplied by Optics Balzers AG. They correspond to Goldflex mirror with SiO2 protection layer (Goldflex PRO, reference 200785). Their absolute reflectance was higher than 98% in the 1.2−12 μm spectral range. The thickness of the SiO2 layer, measured by ellipsometry, was 215 ± 7 Å, using a refractive index of 1.46 (I-elli2000 NFT ellipsometer, λ = 532 nm). A homogeneous surface was observed by atomic force microscopy (AFM) with a rootmean-square (rms) roughness of 9 Å (Thermomicroscope Autoprobe CP Research, Park Scientific). Formation of Self-Assembled Monolayers. The substrates were cleaned and activated just before the grafting. They were treated successively with milli-Q water (18 MΩ.cm) and hot chloroform (10 min at least). Then, the substrate were exposed to UV-ozone (homemade apparatus, λ = 185−254 nm) for 30 min and introduced into the silanization flask immediately. Trichloroacetic acid (TCA, 0.4 mg, 10 mol %) as catalyst was added to a solution of compound 2 17673
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Figure 1. Synthesis of ureido silylated coupling agent. (i) NaN3, toluene, 0 °C to RT, 4 h then, toluene, 75 °C, 20 h. (ii) (3-aminopropyl)trimethoxysilane, CH2Cl2, RT, 16 h. (2.5 × 10−4 mol.L−1) in anhydrous toluene (100 mL). This solution was introduced into the silanization flask at 18 °C under inert atmosphere. The substrates were immersed for 12 h. They were sonicated in toluene (5 min), chloroform (5 min) and milli-Q water (10 min) and then dried under vacuum for 10 min. Oxidation of the Vinyl Group to Form a Carboxylic Acid Group. The substrates were placed in an oxidizing solution containing 2 mL of 5 mM KMnO4, 2 mL of 195 mM NaIO4, 2 mL of 18 mM K2CO3, and 14 mL of milli-Q water. After 24 h, the substrates were washed several times with 0.3 M NaHSO3 to remove the excess of potassium permanganate. After, they were sonicated in milli-Q water (5 min) and dried with a stream of argon. Activation of Carboxylic Acids. The acid-terminated surfaces were immersed in a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 50 mg/mL) and N-hydroxysuccinimide (NHS, 25 mg/mL) in 0.3 M MES buffer (2-(N-morpholino)ethanesulfonic acid) for 2 h. Then, the resulting surfaces were rinsed thoroughly with milli-Q water and sonicated in chloroform (5 min) prior to characterization. Reaction with Decylamine. The NHS-terminated surfaces were immersed in 4 mL of DMF containing 60 μL of decylamine and 5 μL of triethylamine. After 2 h at room temperature, the substrates were rinced with milli-Q water, sonicated in chloroform (5 min), and then dried under a stream of argon. Immobilization of Protein A. The protein A was purchased from Sigma-Aldrich. This protein was diluted with 10 mL of phosphatebuffered saline (PBS: 0.14 M NaCI, 0.01 M PO4, 0.003 M KCI), adjusted to pH 8 with 0.5 M KOH. The NHS-terminated surfaces were then immersed in this mixture at 37 °C for 2 h. The samples were washed abundantly with milli-Q water to remove the salts, and then they were sonicated in milli-Q water (5 min) to remove the unbound protein. 2.3. PM-IRRAS Experiments. PM-IRRAS spectra were recorded on a ThermoNicolet Nexus 670 FTIR spectrometer at a resolution of 4 cm−1, by coadding several blocks of 1500 scans (30 min acquisition time). All spectra were collected in a dry-air atmosphere after 30 min of incubation in the chamber. Experiments were performed at an incidence angle of 75° using an external homemade goniometer reflection attachment, adding a ZnSe photoelastic modulator (PEM, Hinds Instruments, type III) after the polarizer.41 The parallel beam was directed out of the spectrometer with an optional flipper mirror and made slightly convergent with a first ZnSe lens (191 mm focal length). A BaF2 wire grid polarizer (Specac) was used to select the ppolarized radiation. After reflection on the sample, the infrared beam is focused with a second ZnSe lens (38.1 mm focal length) onto a photovoltaic MCT detector (Kolmar Technologies, model KV104) cooled at 77 K. The PEM oscillates at ωm = 31 kHz and changes the polarization from parallel to perpendicular at 62 kHz. The polarization modulated signal IAC was separated from the low frequency signal IDC (ωi between 500 and 5000 Hz) with a 40 kHz high pass filter and then demodulated with a lock-in amplifier (Stanford Model SR 830). The output time constant was set to 100 μs to pass all the frequencies ωi. The two interferograms are high-pass and low-pas filtered (Stanford Model SR 650) and simultaneously sampled in the dual channel electronics of the spectrometer. In all experiments, the PEM was adjusted for a maximum efficiency at 2500 cm−1 to cover the mid-IR range in only one spectrum. For calibration measurements, a second linear polarizer (oriented parallel or perpendicular to the first preceding the PEM) was inserted between the sample and the second
ZnSe lens. This procedure was used to calibrate and convert the PMIRRAS signal in terms of the IRRAS signal (i.e., 1 − Rp(d)/Rp(0), where Rp(d) and Rp(0) stand for the p-polarized reflectance of the film/substrate and bare substrate systems, respectively).28,53
3. RESULTS AND DISCUSSION 3.1. Synthesis of Ureido Silylated Coupling Agent (2). ω-Alkene-alkylsilanes are mainly used to prepare vinylterminated SAMs but the synthetic route involved multiple steps leading to a moderate overall yield.37,39,54 In this work we describe a simple way to prepare a vinyl alkoxyorganosilane via the isocyanate chemistry according to the literature in the case of hybrid materials55 or monolayers.23,28 This synthesis is performed in only two steps from commercially available starting compounds. The unsaturated isocyanate 1 is synthesized in a good yield by the classical Curtius reaction using the starting acid chlorides via the acyl azides intermediate (Figure 1). The ureido silylated coupling agent 2 was obtained by the condensation of (3-aminopropyl)-trimethoxysilane with the unsaturated isocyanate 1 in high yield (80%). The ability of ureido silylated coupling agent 2 to autoassociate through hydrogen-bonds was shown from FTIR experiment (Supporting Information, Figure S-1). At low concentration (5 × 10−3 M in CDCl3), the amide I and amide II bands are observed at 1663 and 1536 cm−1, respectively, which is expected for the amide vibrations of free urea groups. The shoulder observed at 1640 cm−1 is associated with the νCC vibration of the vinyl groups. On the other hand, in the solid state, the amide I band shifts toward shorter wavenumbers (1630 cm−1), whereas the amide II band shift toward higher wavenumbers (1574 cm −1 ) in agreement with strong association of the urea groups.55 3.2. Characterization of Vinyl-Terminated Monolayers. The silylated coupling agents are most often grafted onto glass substrates, but the molecular information obtained by transmission FTIR experiment is limited only to the stretching vibrations of the alkyl chains.14 Recently, we have reported that SAMs grafted onto SiO2/Au substrates can be characterized by PM-IRRAS which allows the detection of different functional groups over the mid-IR spectral range.28 The PM-IRRAS spectrum of a vinyl-terminated monolayer is presented in Figure 2 in the 3100−2700 cm−1 region characteristic to the methylene vibrations (Figure 2A) and in the 1700−1500 cm−1 region characteristic to the amide I and amide II bands (Figure 2B). Specific information about the conformation of the alkyl chains can be obtained from the methylene vibrations. The frequency, width, and intensity of the antisymmetric (νa CH2) and symmetric (νs CH2) methylene stretching bands are sensitive to the gauche/trans conformer ratio and to the packing density of alkyl chains, respectively. Shifts to lower wavenumbers of these modes (i.e., 2920 and 2850 cm−1 for the νa(CH2) and νs(CH2) vibrations, respectively) are indicative of highly ordered conformations 17674
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Figure 2. PM-IRRAS spectra of 2 grafted onto SiO2/Au substrate, in the (A) 3100−2700 and (B) 1700−1500 cm−1 spectral ranges.
isotropic compact monolayer are reported in Supporting Information (Figures S-2 and S-3). Using the theoretical approach previously described,28 we find that the calculated tilt angle between the carbonyl of the urea groups and the normal of the substrate surface makes an angle of 76°. Therefore, the CO and N−H chemical groups are preferentially oriented parallel to the surface, favoring intermolecular hydrogen bonding. From this result and considering the disorder of the alkyl chains, we can propose the schematic representation of the vinyl-terminated SAMs on silica surface reported in Figure 3.
with preferential all-trans conformation. In contrast, the frequency and width of νa(CH2) and νs(CH2) vibrations increase with the chain disorder. The methylene stretching bands are observed at 2928 cm−1 (νa(CH2)) and at 2856 cm−1 (νs(CH2)) in Figure 2A. These wavenumbers suggest that the alkyl chains are quite disordered in the grafted molecules and contain significant gauche defects. The van der Waals interactions between the alkyl chains are weak due to the presence of urea groups within the monolayer which induce a steric hindering effect and a more complex interchain interaction. Indeed, the distance between urea groups (typically 4.5−5 Å) is larger than the distance between two silicon atoms of siloxane bridge (∼3.2 Å), which may explain the disorder observed for the alkyl chains. Thus, the distance between the alkyl chains is too large to establish van der Waals interactions and, consequently, does not favor the all-trans conformation of the chains. Contact angle measurements confirm this result. Indeed, this technique can be sensitive to the degree of alkyl chains order in the outer part of the layers. We measured a value of 78° (with water) for the vinyl-terminated SAMs, which is consistent with disorder alkyl chains since the value is much lower than the one (i.e., 103°) previously reported in the literature for a well-ordered vinyl-terminated monolayer on glass.56 The observed bands at 1630 and 1577 cm−1 (Figure 2B) are attributed to the amide I (νCO) and amide II (δNH) modes of the urea group, respectively. The width of these bands is relatively large, revealing a distribution of the hydrogen bonds in the SAMs. The Δν (νamide I − νamide II) value of 55 cm−1 is indicative of strong association of the urea groups by hydrogen bonds in the vinyl-terminated SAMs. This value appeared similar to the one (56 cm−1) measured for the precursor in the solid state, significantly lower that the Δν = 127 cm−1 measured for the free urea groups in diluted chloroform solution. The νCC stretching vibration of the vinyl group appears at 1640 cm−1 with a weak intensity, as a shoulder of the amide I band. The presence of these bands confirms that the molecules are grafted on the surface. We have shown in a previous study28 that the orientation of the urea groups can be determined from the intensities of the amide I and amide II bands measured in the experimental PMIRRAS spectrum and in the IRRAS spectrum calculated from the isotropic optical constants of the studied molecules. The isotropic optical constants of the ureido silylated coupling agent determined from ATR experiments as well as the IRRAS spectrum calculated from these optical constants for an
Figure 3. Schematic representation of the vinyl-terminated SAM on silica surfaces showing the intermolecular hydrogen bonds between the adjacent urea moieties within the monolayer assembly.
The vinyl-terminated monolayer was also characterized by atomic force microscopy (AFM) in the tapping mode. The AFM height image (Figure 4, right) reveals that the monolayer is homogeneous without particular flaws. The root-mean-square (rms) roughness of this monolayer is significantly lower (0.45 nm) than the value for a bare substrate (0.90 nm), suggesting a surface smoothing. 17675
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Figure 4. AFM height image (2 × 2 μm) in the tapping mode of SiO2/Au substrate (left) and vinyl-terminated monolayer (right).
Figure 5. Reaction scheme for the immobilization of biomolecules on the functional groups of the exposed SAM surface.
Conversion of Vinyl-Terminated SAM to Acid Monolayers. The conversion of the double bond into the carboxylic acid groups was achieved using similar procedures developed by Whitesides by reacting the vinyl-end groups with oxidant solution of KMnO4/NaIO4.34 After this treatment the contact angles decreased from 78° to 61°. These values showed that the surface became more hydrophilic due to the presence of acid carboxylic groups. This result is close to those observed in the literature for oxidation of alkene-terminated SAMs prepared with n-alkenyltrichlorosilane (56°)39 and for the hydrolysis reaction of ester-terminated monolayers (58°).57 The PM-IRRAS spectrum of the conversion of SAM-vinyl to SAM-COOH is shown in Figure 6. The successful reaction was confirmed in Figure 6A by the almost total disappearance of the shoulder at 1640 cm−1 associated with the CC vibration of the vinyl group and by the appearance of a new broad band around 1710 cm−1 (CO stretching vibration) characteristic of acid groups. The width of the latter band is indicative of different environment for acid groups involving non-hydrogenbonded and hydrogen-bonded carbonyls.58
3.3. Chemical modification of Vinyl-Terminated Monolayers. The previous section shows that PM-IRRAS experiments can be used to characterize SAMs grafted onto SiO2/Au substrates. Indeed, PM-IRRAS spectra are recorded with a very good signal-to-noise ratio, allowing the identification of the different functional groups of the investigated molecules (alkyl chains, urea, and terminated functional groups). Additional information on the molecular orientation can be obtained by comparison of the experimental PM-IRRAS spectrum with the one calculated for an isotropic orientation of the molecules. PM-IRRAS experiments can be also used to follow chemical modifications of the terminal group of the SAM in view to immobilize biomolecules, as reported in Figure 5. In the first step, the vinyl-terminated SAM is treated with KMnO4 as a reducing agent to generated carboxylic acid groups. The second step consists of the activation of acid groups with the carbodiimide reagent (EDC) and N-hydroxysuccinimide (NHS) to form the NHS ester. Finally, these SAM-NHS surfaces react with amine-containing protein A to form amide linkages. 17676
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Figure 6. PM-IRRAS spectra of SAM-CC and SAM-COOH (A) in the 1800−1500 cm−1 and (B) in the 3050−2750 cm−1 spectral ranges.
Figure 7. PM-IRRAS spectra of acid- and NHS-terminated monolayers, in the (A) 1850−1500 and (B) 3050−2750 cm−1 spectral ranges.
literature.60,61 The PM-IRRAS spectrum of the SAM-NHS is shown in Figure 7. We observe in Figure 7A a complete disappearance of the band associated with the acid groups (around 1710 cm−1) and the appearance of three new bands (at 1816, 1785, and 1737 cm−1) assigned to the stretching modes of the carbonyl of succinimidyl ester moiety. The region of methylene stretching modes (see Figure 7B) does not reveal any degradation of the monolayer during the chemical activation of the acid function. Covalent Immobilization of Decylamine. The accessibility of activated esters has been investigated for covalent attachment by using decylamine with the formation of an amide bond. The coupling is clearly evidenced in Figure 8, considering the following spectral modifications. First, we observe an almost complete disappearance of the three carbonyl bands of the succinimidyl ester group. Second, the intensity of the bands attributed to stretching vibrations of CH2 groups increased and we observe the stretching vibrations of the methyl group of decylamine. Third, the successful reaction is confirmed by the increases of intensity of the amide 1 and amide 2 bands due to the new amide bond formation. These results demonstrate the good accessibility and reactivity of these NHS-SAMs. The reactivity of activated ester surface could be explained by the presence of the buried urea group in the molecule which induces disorder of the alkyl chains. Indeed, this disorder decreases the steric hindrance of the terminal head groups, which is responsible for the hight reactivity of the surfaces. The steric hindrance of the terminal groups can be avoided by the
The conversion of vinyl-terminated SAM to acid monolayer does not modify the position of the amide I and amide II bands of the urea groups, revealing that the self-assembly by the intermolecular hydrogen bonds is preserved after the oxidation reaction. In addition, the wavenumbers and the relative intensity of methylene stretching bands (Figure 6B) observed at 2928 cm−1 (νa(CH2)) and 2856 cm−1 (νs(CH2)) for vinylterminated SAM and SAM-COOH are similar, indicating that the organization of the grafted molecules onto SiO2/Au substrate are not modified. This feature is confirmed by AFM since the rms measured for the two SAMs are quite similar. Activation of Acid Monolayers. The SAM-COOH can be used to immobilize in a covalent way chemical and biological species on the silica surfaces. However, due to its weak reactivity, the carboxylic acid groups need to be activated on the surface. Carbodiimides chemistry is the most popular method to perform this activation since it is compatible with biological applications.59 The acid-terminated SAM was chemically activated with an aqueous solution of EDC/NHS for 2 h at room temperature. The EDC reagents generate a reactive O-acylurea intermediate which can react with amine groups to form an amide bond. Nevertheless, the conversion rate of this reaction can be limited in aqueous media with the hydrolysis of the O-acylurea intermediate. The NHS reagent displaces the O-acylurea intermediate to give a succinimide ester which is less prone to hydrolysis and sufficiently reactive with amino groups. The water contact angle was 58° ± 1° for the NHSterminated SAM which is consistent with those reported in the 17677
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Figure 8. PM-IRRAS spectra of NHS-terminated monolayers before (black) and after attachment of decylamine (red) in the (A) 1850−1500 and (B) 3050−2750 cm−1 spectral ranges.
Figure 9. PM-IRRAS spectra of NHS-terminated monolayers before and after immobilization of protein A, in the (A) 1850−1500 and (B) 3050− 2750 cm−1 spectral ranges.
formation of mixed monolayers.62 However, this method can generate surfaces with nanodomains of chemical functionality.63 Covalent Immobilization of Protein A. We employed the NHS-terminated SAMs to immobilize the protein A as a model. Figure 9 shows PM-IRRAS spectra of the SAM-NHS and SAM after the immobilization of protein A (SAM-PA). The intensity of the amide bands at 1659 cm−1 (amide 1) and 1548 cm−1 (amide 2) for SAM-PA strongly increases in Figure 9A primarily due to the amide bonds of the protein A and also to the contribution of amide linkage formed between the protein and the activated acid SAM. The presence of a shoulder at 1742 cm−1 characteristic of NHS ester indicates that all the NHS moieties have not been converted into amides. This residual band of unreacted NHS can be explained by the steric hindrance of protein A immobilized onto the surfaces which limit the accessibility of NHS ester to other biomolecules. Moreover, two new bands appear in Figure 9B at 2964 and 2877 cm−1 corresponding to the asymmetric and symmetric stretching vibrations bands of CH3 groups, respectively, which is consistent with the presence of amino acids such as leucine, isoleucine, methionine, and valine of the primary structure of protein A.64
anchoring group has been described. This ureido coupling agent exhibits a good capacity to directly yield homogeneous SAMs with a surface smoothing, as demonstrated by AFM experiments. PM-IRRAS was used to characterize SAMs grafted onto SiO2/Au substrates. PM-IRRAS were recorded with very good signal-to-noise ratio, allowing the identification of the different functional groups of the investigated molecules. Chemical modifications of the terminal functions for the covalent immobilization of biomolecules were followed by PMIRRAS for the first time. The conversion of the vinylterminated SAMs to acid monolayers was confirmed by the appearance of the new band at 1710 cm−1, associated with the CO vibration of acid groups. The activation of acid monolayers to obtain SAM-NHS was demonstrated by the appearance of the three bands at 1816, 1785, and 1737 cm−1, assigned to the stretching mode of carbonyls of succimidyl ester moiety. No degradation of the monolayers was observed during the chemical modifications of surface since the wavenumbers and the intensity of methylene stretching bands remained unchanged. The reactivity of activated esters was successfully investigated through reaction with decylamine in order to immobilize the protein A. Functionalized hydrogen-bonding self-assembled monolayers leads to a new platform with potential for the development of smooth silica-based surfaces in view of the covalent grafting of biomolecules for biotechnology applications.
4. CONCLUSIONS In this article, a novel and simple synthesis of urea coupling agent possessing a vinyl-terminal group and a trimethoxysilyl 17678
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ASSOCIATED CONTENT
S Supporting Information *
IR spectrum of 2 in CDCl3 at low concentration. S-polarized ATR spectrum of 2 in the solid state. Isotropic optical constants of 2. IRRAS spectrum calculated for an isotropic monolayer of 2. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial supports of the “Ministère de la Recherche”, the CNRS, and ANR-P2N2010.
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REFERENCES
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