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J. Phys. Chem. C 2008, 112, 7158-7167

Amidation of Monolayers on Silicon in Physiological Buffers: A Quantitative IR Study A. Moraillon, A. C. Gouget-Laemmel,* F. Ozanam, and J.-N. Chazalviel Physique de la Matie` re Condense´ e, Ecole Polytechnique, CNRS, 91128 Palaiseau, France ReceiVed: December 21, 2007; In Final Form: February 13, 2008

Mixed carboxyl-terminated monolayers grafted onto monocrystalline (111) silicon are prepared by photochemical hydrosilylation of undecylenic acid/1-decene mixtures on hydrogenated surfaces. The attachment of a simple primary amine (hexylamine) to the mixed-acid-terminated monolayers is achieved in a physiological buffer by a two-step process using the water-soluble coupling agents N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl terminations. The chemical compositions of the acid-terminated, activated, and amidated monolayers are studied carefully as a function of the morphology of the initial hydrogenated surface (rough or flat) by quantitative FTIR spectroscopy (ATR geometry) coupled with AFM imaging. For the amidation reaction, in situ IR experiments are performed in hexylamine solutions at three different concentrations. A simple model is proposed to explain the sublinear variation of the reaction rate as a function of the amine concentration.

Introduction There is presently strong interest in the investigation of the grafting of organic groups onto solid substrates for their potential use as recognition layers for the development of biological sensors.1-9 In this framework, surfaces bearing a carboxyl termination are especially appealing because they can be modified easily for the controlled covalent attachment of biological probes at the surface via amide bond formation. This is usually carried out by the activation of the carboxylic acid groups under mild conditions using the so-called peptide coupling reagents (carbodiimides, aminium or phosphonium salts of benzotriazole derivatives, uronium salts...),10-13 allowing for amide bond formation with primary amines. In the literature, many examples have been described using glass,14 nanoparticles,15 metallic (gold),16 or semiconductor surfaces (diamond,17 silicon,18 carbon nanotubes,19...) as solid substrates. Previously, we have studied well-defined mixed carboxylterminated alkyl monolayers on monocrystalline silicon surfaces prepared by direct hydrosilylation of ω-alkenoic acids on hydrogenated silicon surfaces.20 We have demonstrated the excellent electronic properties of the interface (notably a low density of surface states), the good compactness of such monolayers with a total molecular surface density of 2.7 1014 cm-2, and the absence of an interfacial silicon oxide layer. All of these characteristics are necessary in order to use the silicon as a transducer for the electrical detection of biological recognition. Here, the controlled attachment of biological molecules (like DNA) is performed through a two-step activation protocol developed by C. Douarche21 using the water-soluble carbodiimide EDC (N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide) in the presence of the coupling agent N-hydroxysuccinimide NHS. The latter improves the EDC-mediated coupling reaction by the formation of a stable NHS-ester intermediate that is amine-reactive.22,23 The purpose of the present article is the * Corresponding author. E-mail: anne-chantal.gouget@ polytechnique.edu. Phone : +33 1 69 33 46 80. Fax: +33 1 69 33 47 99.

quantitative analysis of the activation of mixed acid/alkylterminated monolayers followed by their amidation with simple primary amines. Such a purpose could have been reached using quantitative XPS.24,25 Here we preferred using IR spectroscopy in attenuated total reflection geometry (see Figure 1). Our goal is to reach good control of the molecular surface composition before and after the chemical modifications by careful calibration of the IR intensity and by AFM imaging. To our knowledge, it is the first time that such a quantification of the activated and amidated surface is performed by IR spectroscopy. Our starting substrates are the hydrogenated monocrystalline (111) silicon surfaces obtained by etching the oxidized silicon substrates either in hydrogen fluoride HF or in ammonium fluoride NH4F. Different mixed-acid monolayers were prepared starting from 1-decene (CH2dCH-(CH2)7-CH3)/undecylenic acid (CH2dCH-(CH2)8-COOH) mixtures. The coverage of these monolayers was studied as a function of the state of the starting hydrogenated surface (rough or flat) and the dilution of undecylenic acid in 1-decene. The surface composition after the activation of these different mixed-acid monolayers was investigated. Then, the amidation reaction was explored by ex situ and in situ IR spectroscopy. Experimental Section General Information. The chemicals ammonium sulfite monohydrate (92%), hexylamine (99%), NHS (97%), Nsuccinimidyl palmitate (∼98%), and EDC (98%) were purchased from Sigma-Aldrich, the undecylenic acid (99%) from Acros Organics, the alkene 1-decene (97%) from Fluka, and the 10X PBS buffer (pH 7.4) from Ambion. All cleaning (H2O2, 30%; H2SO4, 96%; acetic acid, 100%) and etching (NH4F, 40%; HF, 50%) reagents were of VLSI grade and supplied by Merck. Ultrapure water (MilliQ, 18.2 MΩ cm) was used for all rinses. The silicon samples (Siltronix, France) were cut either from one-side polished n-type (111) silicon wafers (Cz, 5-10 Ω cm, 525 µm) with a miscut of 0.2° toward the (112h) direction (for AFM imaging) or from double-side polished float zone 800 Ω cm n-type (111) silicon (for IR spectroscopy).

10.1021/jp7119922 CCC: $40.75 © 2008 American Chemical Society Published on Web 04/17/2008

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Figure 1. Scheme of the-acid modified monolayer followed by the amidation via EDC/NHS coupling.

Etching of the Oxidized Silicon Substrate in HF. The Si(111) sample was cleaned in a 1/3 H2O2/H2SO4 piranha solution at 100 °C for 30 min and then rinsed copiously with water. Subsequently, it was immersed in a HF solution for 30 s and then quickly rinsed in water and blown dry under nitrogen. Photochemical Hydrosilylation Reaction. Described in ref 20. Activation. A freshly prepared acid-terminated alkyl surface was transferred into a degassed Schlenk tube containing 7 mL of cold EDC (10-2M). After 30 min of argon bubbling, 3.5 mL of cold NHS (10-3M) was added and the system was kept under bubbling for 1 h at room temperature. The activated surface was copiously rinsed in water (2 min) and blown dry under nitrogen. Amidation. A fresh solution of hexylamine (10-3M, 20 mL) in 1X PBS buffer was prepared. The pH was adjusted to 7 with a solution of HCl (2N). This solution was transferred into a degassed Schlenk tube, and bubbling was continued for several minutes. The activated surface was immersed into the solution, and after 10 min of bubbling the Schlenk tube was closed overnight. The resulting amidated surface was copiously rinsed in water (2 min) and blown dry under nitrogen. Infrared Spectroscopy. FTIR-ATR spectra were recorded using a Bomem MB100 FTIR spectrometer equipped with a liquid-nitrogen-cooled MCT photovoltaic detector. Spectra were recorded with p and s polarization over the 900-4000 cm-1 spectral range (4 cm-1 resolution). A typical sample is a prism of ∼14 mm width and 46° bevels on the two opposite sides, giving N ≈ 26 reflections. In all of the displayed spectra, the reference spectrum was always recorded on a freshly hydrogenated Si(111) surface. The computer software OPUS 4.2 was used for the spectral fits. The calibration and the in situ measurements were performed in a homemade PTFCE IR cell of ∼5 mL volume. On the top and the bottom of the cell, a PTFE tube (0.8 mm diameter) is connected allowing for the addition of different solutions without breaking the spectrometer purge. On the side, there is an opening of 9 mm diameter against which the prism is pressed via a nitrile O-ring seal. 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 ) 0.2 N m-1) and in a N2 atmosphere. Results and Discussion 1. Preparation of Hydrogenated Silicon Surfaces. The hydrogen-terminated silicon surfaces are prepared by etching the native silicon oxide layers on Si(111) surfaces in aqueous 40% ammonium fluoride26 or in 50% hydrogen fluoride solution. In the first case, the ammonium fluoride treatment yields ideal hydrogenated Si(111) surfaces where all Si-H bonds located on flat terraces are perpendicular to the surface (one sharp peak

observed at 2083 cm-1 by IR-ATR in p polarization). As evidenced by AFM, the topography has a staircase structure with smooth terraces separated by rectilinear parallel monatomic steps of 0.31 nm height.27 The advantage of such an “Si-H” surface is that we can have a perfect monitoring of the surface state after each chemical modification by AFM imaging (presence of physisorbed residues after the activation, loss of the homogeneity of the grafted monolayers ...), which is very important for quantitative IR studies. With the HF treatment, the obtained surface is “rough” on an atomic scale (no structuration of the surface). In IR-ATR spectra, this roughness is revealed by the presence of a broad band containing three main peaks related to the different stretching modes of the monohydrides, dihydrides, and trihydrides.28 We have decided to work on these “Si-Hx” surfaces because they appear less specific than the (111)Si-H: the results obtained on an SiHx surface will plausibly be transposable to other crystallographic orientations and also to amorphous silicon surfaces, which may be of interest for future applications. Moreover, the etching rate of silicon in HF is much slower than that in NH4F, allowing for indefinite reusability of our prisms. For an easier comparison of the coverages obtained on the two types of surfaces (atomically flat and atomically rough), we will take the number of hydrogenated atoms on the flat Si-H surfaces, that is, 7.83 1014 cm-2, as the reference for unit coverage (this is actually an underestimate for the Si-Hx surface, but this is a suitable convention because the coverages are limited by steric hindrance between the grafted groups rather than by the number of anchoring sites available on the surface).29 2. Preparation of Mixed-Acid-Terminated Monolayers. We have prepared different monolayers by UV irradiation of Si-Hx surfaces in the presence of the following alkenes: undecylenic acid, 1-decene and undecylenic acid/1-decene in proportions 50/50 and 25/75. For the acid and acid/alkyl surfaces, we have demonstrated that a final rinse in hot acetic acid leads to the formation of a monolayer perfectly free of physisorbed contaminants (the initial structure of the Si-H surface is preserved as evidenced by AFM imaging).20 The IR study of these acid monolayers in the range 900-4000 cm-1 clearly shows that the grafting occurs via the reaction of the double bond CdC (presence of the CdO stretching mode of hydrogen-bonded carboxylic acids at 1715 cm-1 and of the C-O-H in-plane modes at 1280 and 1410 cm-1) and that the interface is essentially oxide-free.20 In the case of the acid monolayers on Si-Hx (see Figure 2), we notice that the oxidation is negligible for the 100% acidterminated surface but increases with decreasing volume concentration of acid in 1-decene. For the decyl monolayer (0% acid terminated monolayer), we observe a significant peak characteristic of the surface oxidation. We can roughly estimate the oxide thickness from the intensity of the band related to the

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Moraillon et al. TABLE 1: Summary of the IR Absorption Frequencies of the CdO and νsCH2 Stretching Modes and Their Integrated Absorbances for 100% Decyl (0% COOH), Mixed 10-Carboxydecyl/decyl (25% and 50% COOH) and 100% Acid-Terminated Monolayers Grafted on Si-Hx frequency in cm-1; intensity in mAbs in p pol. (area in cm-1)a %COOH

νCO, I

0 25 50 100 a

Figure 2. IR-ATR spectra of the 100%, 50%, 25%, and 0% acidterminated monolayers in s polarization (after baseline correction). The reference spectra are the hydrogenated Si-Hx surfaces.

TO vibration mode at 1035 cm-1 in s polarization, knowing that an absorbance of 10-3 per reflection corresponds to ∼1.5 Å of SiO2.30 We found an equivalent oxide thickness of ∼2 Å, 1 Å, and less than 0.4 Å for the 0%, 25%, 50% acid-terminated monolayers, respectively. The oxidation peak is weaker when the 1-decene is first purified before its grafting on Si-Hx (as shown on Si-H surfaces).20 A. QualitatiVe Analysis. In the region of the alkyl CH stretching modes (between 2850 and 2980 cm-1), the packing of the alkyl chains on silicon can be qualitatively estimated from the position of the asymmetric stretching mode νaCH2.31 A monolayer is considered to be compact when the frequency is near that of the corresponding crystalline material at 2917 cm-1. In the monolayers grafted on Si-H, the νaCH2 position is located between 2921 cm-1 for the 100% decyl monolayer and 2923 cm-1 for the 100% carboxydecyl monolayer, whereas it is shifted to 2925 and 2927 cm-1, respectively, in the case of mixed-acid monolayers grafted on Si-Hx. At first glance, the monolayers on Si-Hx appear to be less packed and ordered than those on Si-H. As expected, we observe an increase in the intensity of the νCO stretching mode and a decrease in the νaCH3 at 2961 cm-1 as a function of the increasing volume concentration of acid in the grafting solution. However, the intensities of the νaCH2 bands of the decyl monolayer are significantly lower than those of the mixed-acid monolayers. This suggests a lower compactness of the alkyl monolayers, a behavior at variance with that observed on Si-H.20 B. QuantitatiVe Analysis. We have quantitatively calculated the molecular coverage of the layers from the analysis of the area of the carbonyl and the methylene absorption peaks (for more details, see ref 20 and the Appendix). The intensity of the stretching mode of the carbonyl at ca. 1715 cm-1 is used to determine the absolute areal density of the acid chains immobilized on the surface. The νCO mode is fitted by using the superposition of a Gaussian and a Lorentzian profile. The intensity of the symmetric νsCH2 mode at ca. 2855 cm-1 is used to determine the total areal density of the methylene groups on the surface (from the decyl chains and from the acid-terminated decyl chains). In this case, the overall νCH stretching vibrations are fitted by using a model consisting of a linear baseline and the superposition of five pseudo-Voigt functions representing the four CH2 and CH3 modes and the main Fermi-resonance

1714.5; 0.28 (0.0131; 0.0116) 1715.4; 0.39 (0.0155; 0.0133) 1715.8; 0.68 (0.0272; 0.0251)

νsCH2, I 2854.1; 0.24 (0.00639; 0.00567) 2854.3; 0.32 (0.00876; 0.00843) 2854.0; 0.37 (0.00917; 0.00844) 2855.9; 0.34 (0.00916; 0.00832)

Area in p polarization; s polarization.

enhanced overtone at ∼2900 cm-1. The frequencies and the integrated intensities of the νCH and νCO modes are listed in Table 1, and the fitting parameters are given in Tables A-C in the Supporting Information. In a second step, we have performed two calibrations in liquids, namely in dodecane and in 1% dodecanoic acid in dodecane, for which we know the concentration of the CH2 and CO groups. We measure the area of the CH2 and CO IR peaks by fitting them with the same profiles as described above. Then, by comparing these areas with those of the CH2 and CO of the grafted layer, we can calculate the number of methylene groups per unit area originating from the alkyl and acid chains, NCH2 and the number of acid chains, NCO. Both can be decomposed into two contributions, N// and N⊥, corresponding to the components parallel and perpendicular to the surface. In the first case

NCH2 ) N// + N⊥

(1)

with N// ) 1.80 1017 × As and N⊥ ) 1.75 1017 × (1.96Ap 1.78As) where As,p stands here for the integrated absorbance per reflection of the νsCH2 mode (expressed in cm-1) measured in s or p polarization, and in the second case

NCO ) N// + N⊥

(2)

with N// ) 6.57 1015 × As and N⊥ ) 6.32 1015 × (1.96Ap 1.78As) where As,p stands for the integrated absorbance per reflection of the νCO mode (expressed in cm-1) measured in s or p polarization (all of these numbers stand for our samples with 46° incidence angles). From these results, we derived the surface concentration of alkyl chains accounting for the fact that there are 10 methylene groups per acid chain and 9 per alkyl chain. We deduce the final coverage θ, knowing that the maximum attainable surface coverage is ∼50% on (111)Si, corresponding to 3.9 1014 Si-C per cm2.29 The surface densities of acid-terminated chains, decyl chains, and the total density of molecular chains are plotted in Figure 3 as a function of the undecylenic acid volume concentration in the grafting solution. Several points have to be emphasized: first, we verify that the number of acid chains increases whereas the number of decyl chains decreases with increasing acid content. Second, the overall coverage is weakly sensitive to the dilution of acid in 1-decene. We obtain a coverage between 25 and 27% for the mixed-acid-terminated monolayers, which is somewhat lower than the coverage obtained at flat Si-H surfaces. Third, as already observed for the flat surfaces, we obtain a nonlinear relation between the

Amidation of Monolayers on Silicon in Buffers

Figure 3. Composition of monolayers grafted from undecylenic acid/ 1-decene mixtures: Number of grafted acid (b), decyl (9) chains and total number of grafted chains (() as a function of the undecylenic acid fraction in the grafting solution. The final coverage (2) is given by the right-hand-side vertical scale.

concentration of acid chains on the surface and in the grafting solution, indicating that the mixed monolayers are richer in acid chains than the grafting solution. This is plausibly due to a stronger physisorption of the acid as compared to 1-decene.20 Finally, the stronger oxidation of the surface during the alkyl grafting may explain why the pure alkyl monolayers on SiHx are less compact (θ∼20%) than the mixed-acid monolayers. 3. Activation of Mixed-Acid-Terminated Monolayers. As described in Figure 1, the acid-terminated surfaces can now be activated by the carbodiimide EDC in a two-step protocol using NHS. First, the acid functions react with EDC to produce a reactive O-acylisourea, which can be viewed as a carboxylic ester with an activated leaving group. Then, NHS is added to form the more stable NHS-ester with release of urea. The presence of this coupling agent avoids side reactions of the O-urea derivative (such as fast hydrolysis and/or rearrangement in an N-acylurea).32 A. QualitatiVe Analysis. Figure 4 shows the AFM image and the IR-ATR spectrum in p polarization of a 50% NHS-esterterminated monolayer with respect to the flat hydrogenated surface. In our experimental conditions, the atomically flat structure is preserved, indicating the absence of unwanted materials. The IR-ATR spectrum exhibits three new peaks at 1746, 1788, and 1819 cm-1, corresponding to the vibrations of the NHS-ester bonds. The exact attribution of these peaks is controversial.33,34 Most often, the 1746 and 1788 cm-1 modes are assigned, respectively, to the asymmetric and symmetric Cd O stretches of the cycle, by analogy with succinimide and succinimide compounds.35,36 The third high-frequency mode (1819 cm-1) is attributed to the “ester” CdO stretch. Alternatively, a plausible interpretation seems to assign the 1819 cm-1 mode to the symmetric CdO stretch of the NHS cycle. Indeed, this mode is expected to exhibit a low dynamic dipole (weak IR activity) and a significantly higher frequency than the CdO ester stretch. The other two modes may result from the hybridization of the asymmetric CdO stretch of the NHS and the CdO ester stretch: the 1746 cm-1 mode arises from the in-phase coupling of the two modes, resulting in a large dynamic dipole and, conversely, the 1788 cm-1 mode can be ascribed to the out-of-phase coupling of the two modes. Whatever the attribution is, the set of these three characteristic peaks of NHSester can be used simultaneously for the quantitative measurement of the activation efficiency as discussed below. In the

J. Phys. Chem. C, Vol. 112, No. 18, 2008 7161 region of 1050-1400 cm-1, we observe the appearance of different peaks, which can be attributed to C-N-C stretching bands of the succinimidyl end group and the ester C-O-N stretching band.33 We also observe the band at 1715 cm-1 arising from the unreacted acid groups. Decreasing the amount of acid in 1-decene (from 100% to 25% NHS-ester) does not lead to a conspicuous improvement of the activation yield. The use of the shorter 1-octene instead of 1-decene does not appreciably favor the EDC attack on the carboxyl end groups. Once again, the activation is not complete. Therefore, the activation yield seems to be essentially limited by the weak concentrations of EDC and NHS and not by the steric hindrance between the bulky acid groups and EDC. The IR-ATR spectra of the mixed NHS-ester-activated surfaces grafted on Si-Hx exhibit some differences as compared to those on Si-H (cf. Figure 5): (i) the bands are broader, which makes the remaining acid appear as a shoulder on the main peak at 1746 cm-1 and not as a distinct peak, (ii) the activation reaction tends to oxidize the surfaces somewhat more strongly, especially the acid surfaces already oxidized after the grafting, (iii) in the case of highly hydrophilic surfaces (such as the oxidized mixed-acid monolayers on Si-Hx and the 100% acid monolayers on Si-H), we observe the appearance of two weak extra bands located at 1645 and 1550 cm-1. They are indicative of urea traces on the surface (CdO stretch at 1645 cm-1 and CNH mode at 1550 cm-1). The first band may also be attributed to the absorption of liquid water. B. QuantitatiVe Analysis. The p and s spectra of the NHSester monolayers have been analyzed quantitatively by fitting the NHS-ester bands and the stretching CH bands as pseudoVoigt functions and determining the area of each relevant band. The absorption intensities of the νCH bands are calibrated as described for the acid-terminated monolayers. The absorption intensities of the NHS-ester bands are calibrated from a solution of N-succinimidyl palmitate in THF, measured in the same ATR geometry. Details of the calibration procedure and the fitting parameters are given in the Appendix and in Tables D-G (cf. Supporting Information). To summarize, the range of the NHSester IR modes is fitted as a linear baseline and a superposition of three pseudo-Voigt functions representing the three NHSester vibrations and two Gaussian profiles representing the remaining acid and the liquid water absorptions. In some cases, the addition of a Gaussian profile corresponding to the vibration band located at ∼1550 cm-1 is necessary. The intensities of the two NHS-ester bands at 1788 and 1819 cm-1 are less sensitive to the other fitting parameters than that of the first NHS-ester mode at 1746 cm-1, which presents the inconvenience of a significant overlap with the remaining acid band. They can be used for measuring the surface concentration of NHS-ester chains in the grafted layers. Table 2 shows the integrated intensities in p and s polarization of the νsCH2 and the two νCO-NHS modes for the full and mixed NHS-ester terminated monolayers grafted on Si-Hx and Si-H. The activation yield can be deduced from the area values of the two NHS-ester bands. The latter ones are converted into absolute numbers corresponding to the parallel and perpendicular NHSester groups from equation A7 (cf. Appendix). Knowing the number of starting acid chains (before the activation), we deduce the ratio of activated sites to nonactivated sites. The activation yield is ∼50% for the 100% and 50% NHS-ester monolayers grafted on Si-Hx or Si-H. It appears just slightly higher (59%) for the 25% NHS-ester monolayer. This improvement, attributable to a higher accessibility of the acid sites for the chemical modification, is actually much weaker than expected. In order

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Figure 4. AFM image (on the left) of a 50% NHS-ester-terminated monolayer grafted on Si-H and IR-ATR spectra (on the right) in p polarization of (a) a 50% acid-terminated monolayer and (b) a 50% NHS-ester-terminated monolayer grafted on a Si-H surface. The reference spectra are the hydrogenated surface Si-H.

Figure 5. IR-ATR spectra of the 100%, 50%, and 25% activated monolayers in p polarization. The reference spectra are the hydrogenated Si-Hx surfaces.

TABLE 2: Summary of the IR Integrated Absorbance of the Two NHS-Ester Stretching Modes for the 100%, 50%, and 25% NHS-Ester-Terminated Monolayers Grafted on Si-Hx and for the 100% and 50% NHS-Ester-Terminated Monolayers Grafted on Si-H integrated absorbance in p polarization; s polarization (in cm-1) %COOH Si-Hx Si-H

25 50 100 50 100

νCO(1788)

νCO(1819)

νsCH2

0.00190; 0.00156 0.00169; 0.00124 0.00279; 0.00236 0.00288; 0.00266 0.00442; 0.00355

0.00243; 0.00201 0.00233; 0.00211 0.00475; 0.00423 0.00307; 0.00285 0.00460; 0.00358

0.0117; 0.0103 0.0093; 0.0090 0.0082; 0.0074 0.0124; 0.0120 0.0131; 0.0121

to check the reliability of our calculations, we can take into account the calibration of the CH bands allowing for the determination of the overall areal density of methylene groups on the surface. The calibration shows that there is no loss or addition of chains, in accordance with AFM images. As a last remark, we observe that there is no correlation between the

activation yield and the state of the hydrogenated surfaces, whereas the grafting yield is really higher on Si-H than on Si-Hx. This confirms once again that the activation efficiency is not limited by steric hindrance problems but more plausibly here by the low concentrations used for the reagents EDC and NHS. Some experiments have been performed with higher EDC and NHS concentrations, and larger yields have indeed been obtained. However, this progress in the activation yield was made at the expense of a good reproducibility in the surface cleanliness, that is, surface contamination was sometimes detected by AFM. For this reason, the present study was limited to the above-given low EDC and NHS concentrations. An optimization of the procedure, yielding a high activation yield together with a low level of surface contamination, is presently in progress. 4. Amidation of the NHS-Ester-Terminated Monolayers. We have chosen hexylamine as a model precursor for the amidation of the NHS-ester-terminated monolayers. We have noticed that amine coupling takes place only when the pH of the amine solution is lower than the pKa of the corresponding amine (10.56). In our case, the amidation reactions have been performed at pH ∼ 7 in water or in the well-known phosphate buffer 1X PBS. In these conditions, the AFM images of the amidated monolayers reveal once again that the atomically flat structure is preserved and the surfaces are reasonably clean for an amine concentration lower than ∼10-3 M. At concentrations higher than 5 × 10-3 M, the hexylamine is less soluble in water and AFM imaging shows the presence of physisorbed materials even after rinses in different organic solvents. Figure 6 represents the AFM image and the IR-ATR spectra in p polarization of a 50% acid monolayer grafted on Si-H after amidation. Spectrum a corresponds to the activated surface and spectrum b to the amidation in 10-3 M hexylamine in a phosphate buffer. The reference spectra are the hydrogenated surface. The three bands related to the NHS-ester vibrations fully disappear for the benefit of two characteristic vibrations of amide I (at 1648 cm-1) and amide II (at 1547 cm-1). The amidation reaction is complete. We still observe the broad peak assigned to the remaining acid at 1715 cm-1. Fitting this peak as a Gaussian profile yields an integrated intensity similar to that obtained after the activation. This suggests that there is no hydrolysis of the ester.

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Figure 6. AFM image of a 50% amidated monolayer grafted on Si-H surface (on the left) and IR-ATR spectra (on the right) in p polarization of a 50% NHS-ester-terminated monolayer (a) and a 50% amidated monolayer grafted on Si-H surface (b). The reference spectra are the hydrogenated surface Si-H.

Figure 7. In situ IR following of the amidation of a 50% activated monolayer in 10-3M nhexNH2 in PBS at pH ∼ 7: (a) t ) 0 min; (b) t ) 15 min; (c) t ) 30 min; (d) t ) 45 min; (e) t ) 60 min; (f) t ) 90 min; (g) t ) 120 min; (h) t ) 140 min. The reference spectra are the activated surface.

The amidation reaction in hexylamine solution was studied by monitoring the in situ IR absorption at three concentrations 10-3 M, 10-4 M, and 10-5M. In Figure 7, the IR spectra of the amidation of a 50% NHS-ester monolayer grafted on Si-Hx in 10-3 M hexylamine solution are represented at different times. The reference spectra are the activated monolayers. The disappearance of the NHS-ester peaks, concomitant with the appearance of the CH2 stretching and scissor modes and the amide II mode as a function of time, confirms the amide formation. The amide I IR mode is unfortunately partly masked because of the absorption of liquid water. After 2 h and 30 min, there is no more evolution. For all amidation reactions, the kinetics is determined by measuring the decrease of the absorbance of the NHS IR band as a function of time as shown in Figure 8a. The three obtained curves can be fitted as exponentials, indicating a first-order kinetics. We deduce time constants equal to 50, 130, and 345

min at 10-3 M, 10-4 M, and 10-5 M, respectively. The rate constant, k, taken as the inverse of these times, has been represented as a function of the amine concentration c on a log log plot (cf. Figure 9). Discussion. k appears to behave as a power law of c, with an exponent on the order of one-half. This behavior is somewhat surprising. The most naive guess would be k ∝ c. Alternately, one may think that the grafting reaction is a two-step reaction, consisting of a fast physisorption step, followed by a slow, ratelimiting surface reaction. In such a scheme, the reaction rate would be determined by the surface concentration of physisorbed amine molecules, weakly dependent on amine concentration in solution. The fact that the observed behavior is intermediate between these two extreme cases suggests that an investigation of the physisorption/reaction scheme must be done in more detail. Let us focus on the equilibrium between the physisorbed phase and the solution. The physisorbed phase can be characterized by a coverage θp, where we have assumed for convenience that the surface is divided in a fixed number NS of physisorption sites. Neglecting interactions between physisorbed molecules, θp is governed by a Langmuir isotherm

θp c c ) or θp ) 1 - θp c0 c0 + c

(3)

where c0 ) exp(∆G0/kBT) is a constant in mol L-1 determined by the standard free enthalpy of physisorption ∆G0. Now the grafting kinetics is ruled by a differential equation

dθg ) kgθp(1 - θg) dt

(4)

where θg is the coverage of grafted molecules (taken as unity at maximum coverage) and kg a kinetic constant. This integrates to

θg ) 1 - exp(-kgθp t)

(5)

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Figure 8. Kinetics of the amidation of a 50% activated monolayer in nhexNH2 at 10-3 M (b), 10-4 M (9), 10-5 M (() in PBS at pH ∼7. (a) Absorbance of the NHS ester IR peak at 1746 cm-1 as a function of time. (b) Determination of the rate constant from the slope of a log linear plot (n0 represents the maximal absorbance of amide).

CH, where CH is the Helmholtz capacitance, then the above formulas can be modified by incorporating the interaction term e 2NSθp/CH into ∆G (Frumkin isotherm), whence

(

)

θp e 2NSθp c ) exp 1 - θp c0 kBTCH

(7)

where kB is Boltzmann’s constant and T is the absolute temperature. Note that a similar treatment accounts for proton adsorption at an acid-terminated silicon surface.37 The modified relation between k and c can be written in closed form as

(

e 2NS k k exp c ) c0 kg - k kBTCH kg

Figure 9. Plot of the rate constant as a function of amine concentration on a log log plot. The dashed straight lines correspond to the two extreme cases (k ∝ c and k ) cst). Points (b) are experimental data, the dotted curve corresponds to the Langmuir isotherm (adsorption without interaction), and the solid curve to the Frumkin isotherm (adsorption with electrostatic interactions).

The kinetics are exponential, as observed indeed, and the rate constant is given by

k ) kgθp )

kg c c0 + c

(6)

For low amine concentrations, k is proportional to c. At higher concentrations, θp reaches unit coverage and k saturates to a constant value. This corresponds indeed to a behavior intermediate between the two extreme cases mentioned above. However, the change in behavior from linear to constant appears much too sharp to account for the experimental data. The agreement with experiment can be improved if one realizes that in the operating conditions the amine molecules are actually in cationic form, and the interaction between charged physisorbed species cannot be neglected. If the electrostatic potential in the adsorbed layer is taken as eNSθp/

)

(8)

This dependence again exhibits two limiting regimes, a linear regime at low c, given by k ≈ kgc/c0, and a saturation regime k ≈ kg at high c, but now the transition between the two regimes appears much more progressive. Reasonable values of the parameters [c0 ) 1.4 × 10-4 mol/L, kg ) 0.045 min-1, e 2NS/ (kBTCH) ) 5] can give a fair fit to the experimental data (Figure 9). The presence of unsubstituted acid groups at the surface may somewhat complicate the picture because at the operating pH part of them will be in the negatively charged state COO-. These effects can be incorporated into the above treatment by writing now

(

) (

e 2(NSθp - NAθi) θp c ) exp 1 - θp c0 kBTCH and

)

[H + ]θi e 2(NSθp - NAθi) ) KA exp (9) 1 - θi kBTCH

where [H+] represents the proton concentration in solution, NA is the surface concentration of acid sites, θi is their ionized fraction, and KA is the acido-basic equilibrium constant (∼10-6). The modified system of eq 9 has been solved in parametric form, by using θi as a parameter. The results for k(c) were found to be intermediate between the two above cases. Namely, the linear-to-constant transition in k(c) becomes sharper as NA is taken larger. However, up to NA ∼ NS the transition remains

Amidation of Monolayers on Silicon in Buffers progressive, so we think that these improvements in the model can be dropped in a first approach. However, note that for given values of the other parameters an increase in NA leads to an increase in k. For example, at vanishingly small amine concentrations (c and θp , 1), one has θp ≈ (c/c0) exp(e 2NAθi/kBTCH). Under typical experimental conditions, the exponential factor may reach values on the order of 3, which means that the grafting rate is plausibly favored by the presence of residual acid groups left after the activation step. Conclusions We have prepared different mixed-acid-terminated monolayers grafted onto (111) silicon by direct photochemical hydrosilylation of undecylenic acid/1-decene mixtures on hydrogenated silicon surfaces. The composition of the grafted layers was determined from the quantitative analysis of the COOH- and CH-related IR bands. We have observed that (i) the compactness of the final layers is lower when the grafting is performed onto hydrogenated surfaces obtained in HF rather than in NH4F and (ii) the monolayers are richer in acid chains than the starting grafting mixture. The activation reaction of these mixed-acidterminated monolayers with NHS/EDC was investigated by a careful analysis of the NHS-ester IR bands. We have proposed a method of calibration to determine quantitatively the amount of NHS-ester molecules grafted onto silicon. In our experimental conditions (dilute concentrations of EDC and NHS in water), the activation yield is found to be ∼50%. This figure can probably be improved by increasing the NHS and EDC concentrations, provided that surface cleanliness can be preserved in such conditions. Finally, we have studied the amine coupling between the hexylamine and the terminal NHS-ester groups. In situ IR experiments reveal that the kinetic constant of the amidation reaction exhibits a sublinear behavior as a function of amine concentration. A kinetic model was proposed by considering that the amidation mechanism is initiated by the physisorption of the protonated amine and by taking into account the electrostatic interaction between physisorbed species. Acknowledgment. We thank P. Allongue and C. Henry de Villeneuve for fruitful discussions and for providing assistance for AFM imaging, and C. Douarche for giving experimental details on the amidation procedure prior to publication. Appendix. Calibration of the IR Absorption Intensity of Adsorbates In ATR geometry, the density of molecules grafted onto a surface can be calculated from the integrated absorbance of the signal associated with a vibrational mode of the molecules. The infrared cross section of this mode has to be known first, which can be determined if the absorption of the considered mode can be measured in a liquid-phase experiment in the same ATR geometry (calibration). In our study, we are interested in determining the number of acid and decyl chains grafted onto Si-Hx and the number of activated NHS-ester chains. Several calibrations are then performed for each of the IR modes that we want to study (CO, CH, or CO-NHS). 1. Acid-Terminated Monolayers. The details of the calibration procedures for the decyl and acid chains have already been described for the mixed-acid monolayers grafted on Si-H.20 Here we recall how the integrated absorbance of the spectra corresponding to the monolayers grafted on Si-Hx is computed because the fitting procedure had to be adjusted with respect to those on Si-H due to the lower signal-to-noise ratio and the broadening of the CO and CH bands.

J. Phys. Chem. C, Vol. 112, No. 18, 2008 7165 Fitting Parameters. Integrated absorbances have been extracted from the data by fitting the experimental vibrational signals to simple line shapes. To obtain a reliable determination, we simultaneously fit the spectral baseline and the vibrational lines in the region of interest. (a) CO Band. The line shape is fitted in the whole 15501820 cm-1 range. We use the superposition of a Lorentzian profile centered at ca. 1715 cm-1 and a Gaussian profile at ca. 1710 cm-1 (width e60 cm-1). The sum of the area of these two profiles gives the integrated absorbance per reflection of the νCO mode (expressed in cm-1) in p and s polarization (Ap and As). Table A summarizes the fitting data for a 50% acidterminated monolayer grafted on Si-Hx (see the Supporting Information). (b) CH Bands. The whole line shape is fitted in the 27903010 cm-1 range as a superposition of five pseudo-Voigt profiles, four of them accounting for the fundamental modes and the fifth one accounting for a Fermi-resonance enhanced combination mode at ca. 2900 cm-1. As shown in Table B (in the Supporting Information) some of the parameters, such as the width, position, or nature of the profile, need to be enforced in order to obtain a reliable fit. As an example, Table C gives the result of the fitting data of the νCH modes for a 50% acidterminated monolayer grafted on Si-Hx (see the Supporting Information). The IR νsCH2 mode is considered to be the most reliable experimental quantity, so the surface concentration of the methylene groups in the grafted layers is determined from its integrated absorbance per reflection in p and s polarization (Ap and As). 2. NHS-Ester-Terminated Monolayers. Calibration. The density of activated grafted chains is determined from the area of the three characteristic NHS-ester absorption peaks located at 1746, 1788, and 1819 cm-1. In this case, the sum of the integrated absorbances of the three peaks is used for the calibration. We have performed a calibration in a solution of 92 mM N-succinimidyl palmitate (CH3-(CH2)14-CO-NHS) in THF where the concentration of NHS-ester is 5.5 × 1019 cm-3. The infrared absorption of the liquid (in s polarization) is measured in an attenuated total reflection configuration at the interface between the silicon (n1 ) 3.42) and the THF solution (n2 ) 1.4075). For the accuracy of the calibration, the exact value of the ATR prism angle (which determines the incidence angle φ and the number of reflections) is measured and is equal to φ ) 46°. The liquid absorbance per reflection (Absp0 and Abss0) depends on the penetration depth (δ) of the infrared electric field, given by

δi )

λi 2πxn12 sin2 φ - n22

(A1)

where λi is the wavelength of a NHS-ester band. In our geometry, we find δ ) 0.49 µm for the band at 1746 cm-1, δ ) 0.44 µm at 1788 cm-1, and δ ) 0.43 µm at 1819 cm-1. The intensity of the squared electric field at the surface in the (x,y,z) directions is given by the following equations:38

Ix ) 0

4n12 cos2 φ(n12 sin2 φ - n22) n24 cos2 φ + n14 sin2 φ - n12n22 Iy0 )

4n12 cos2 φ n12 - n22

(A2)

(A3)

7166 J. Phys. Chem. C, Vol. 112, No. 18, 2008

Iz0 )

4n14 cos2 φ sin2 φ n24 cos2 φ + n14 sin2 φ - n12n22

Moraillon et al.

(A4)

In our geometry, we find Ix0 ) 1.84, Iy0 ) 2.30, and Iz0 ) 2.73. The infrared absorption of the grafted layer (Absp and Abss) is measured at the interface between the silicon (n1 ) 3.42) and the nonabsorbing medium, that is, vacuum (n2 ) 1). In this case, Ix) 1.90, Iy ) 2.09, and Iz ) 2.27. The actual surface concentration of NHS-ester molecules/cm2 or vibrators is given by N ) N// + N⊥ where N// is the number of vibrators corresponding to the projection of the dynamic dipole of the vibrational mode in the interface plane and N⊥ is that corresponding to the projection of the dynamic dipole along the z direction. Using the sum of the absorbances of the characteristic NHS-ester modes for determining the grafted chain surface concentration, the expressions of N// and N⊥ are the following:

N// )

and N⊥ ) 0 n24 C Iy × × 6 Iy Iz

Iy0 C × × Iy 3

∑

∑ Abss (i) ∑

Abss0(i)

(A5)

δ(i)

repeated once (see Table E, Third Fit), which is enough to check that wl has converged to the same values as for the calibration spectrum. Table F (cf. Supporting Information) gives the final result of the fitting data in p and s polarization for the 50% NHS-ester-terminated monolayer on Si-Hx. (b) CH bands. The fitting procedure for the νCH bands in the 2790-3010 cm-1 range is identical to that for the νCH bands of the acid-terminated monolayers. Table G gives the results of the fitting data for a 50% NHS-ester-terminated monolayer on Si-Hx (see Supporting Information). 3. Determination of the NHS-Ester Surface Concentration. Because of the overlapping of the acid band with the first NHSester band at 1746 cm-1, we regard the sum of the integrated absorbances (Absp and Abss) of the two other NHS-ester modes at 1788 and 1819 cm-1 as the most reliable experimental quantity for measuring the surface concentration of NHS-ester chains in the grafted layers. We end up with the following equations for N// and N⊥ from equations A5 and A6:

N// ) 1.16 × 1016(Abss(1788) + Abss(1819)) and N⊥ ) 9.99 × 1015[2.09(Absp(1788) + Absp(1819)) 1.90(Abss(1788) + Abss(1819))] (A7)

[Iy × Absp(i) - Ix × Abss (i)]

∑

Abss0 (i)

(A6)

δ(i)

Fitting Parameters. (a) NHS Bands. We have first fitted the three NHS-ester vibrational modes of the calibration experiment in s polarization in the 1500-1870 cm-1 range using three pseudo-Voigt functions. The data shown in Table D correspond to the values of the integrated values Abss0, the pseudo-Voigt widths, and the Lorentzian contribution to the pseudo-Voigt profile (see the Supporting Information). The pseudo-Voigt fitting software yields a width w and a “Lorentzian fraction” l, which can be converted to a “Lorentzian width” wl and a “Gaussian width” w(1 - l). When fitting actual surface spectra, it appeared that the integrated area is very sensitive to the value of the Lorentzian width, and we found that more reliable results are obtained by constraining its value. A reasonable assumption was then to assume that it is independent of the vibrator environment. In other words, the change in bandwidth from the liquid (calibration spectrum) to the surface (grafted-layer spectrum) is only due to a change in the Gaussian contribution (inhomogeneous broadening). For the fitting of the actiVated monolayers, the infrared spectra are more complicated because there are the additional bands of the remaining acid at 1715 cm-1 and liquid water at 1645 cm-1. In some cases, we observe an extra band at 1550 cm-1. With the OPUS fitting software, the requirement of a fixed Lorentzian contribution to the bandwidths makes an iterative fitting procedure necessary. The spectra of the grafted layers are first fitted as a linear baseline and a superposition of five (or six) Gaussian profiles. The fitting data of a 50% NHSester monolayer grafted on Si-Hx in p polarization are presented in Table E in the Supporting Information (see First Fit). Let us consider, as an example, the band at 1746 cm-1. In the first fit, this band comes out with a width of 25.34 cm-1, a value larger than that in the liquid (10.17 cm-1). In order to enforce that the Lorentzian width be the same in the two spectra, we take its value for the liquid (wl ) 10.17 × 0.84 ) 8.5 cm-1), calculate the ratio 8.5/25.34 ) 0.34, and impose l ) 0.34 in the second fit (see Table E, second fit). The procedure is

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