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One-Step Photochemical Attachment of NHS-Terminated Monolayers onto Silicon Surfaces and Subsequent Functionalization Menglong Yang,† Rosalie L. M. Teeuwen,†,‡ Marcel Giesbers,† Jacob Baggerman,† Ahmed Arafat,† Frits A. de Wolf,§ Jan C. M. van Hest,‡ and Han Zuilhof*,† Laboratory of Organic Chemistry, Wageningen UniVersity and Research Center, Dreijenplein 8, 6703 HB Wageningen, The Netherlands, Department of Organic Chemistry, Radboud UniVersity Nijmegen, ToernooiVeld 1, 6525 ED Nijmegen, The Netherlands, and Agrotechnology and Food InnoVations, Wageningen UniVersity and Research Center, Bornsesteeg 59, 6708 PD Wageningen, The Netherlands ReceiVed February 12, 2008. ReVised Manuscript ReceiVed April 17, 2008 N-Hydroxysuccinimide (NHS)-ester-terminated monolayers were covalently attached in one step onto silicon using visible light. This mild photochemical attachment, starting from ω-NHS-functionalized 1-alkenes, yields a clean and flat monolayer-modified silicon surface and allows a mild and rapid functionalization of the surface by substitution of the NHS-ester moieties with amines at room temperature. Using a combination of analytical techniques (infrared reflection absorption spectroscopy (IRRAS), extensive X-ray photoelectron spectroscopy (XPS) in combination with density functional theory calculations of the XPS chemical shifts of the carbon atoms, atomic force microscopy (AFM), and static contact angle measurements), it was shown that the NHS-ester groups were attached fully intact onto the surface. The surface reactivity of the NHS-ester moieties toward amines was qualitatively and quantitatively evaluated via the reaction with para-trifluoromethyl benzylamine and biotin hydrazide.
Introduction The functionalization of inorganic surfaces via molecular selfassembly is of growing importance, in the respect of the development of molecular electronics, biosensors, and biochips.1 A major route to the immobilization of amine-containing biomolecules such as proteins or amine-end-capped DNA oligomers, as important examples of biofunctional molecules, is the use of activated esters derived from N-hydroxysuccinimide (NHS).2 Given the ubiquitous use of silicon in microelectronic devices and given the possibility to easily obtain very flat surfaces that facilitate scanning probe-based studies, it is of significant interest to study the functionalization of silicon. In the most stable manner, this can be accomplished via the use of Si-C bound organic monolayers (without involvement of an interfacial silicon oxide layer), which allows the formation of NHS-activated surfaces. To obtain such silicon-bound NHS-functionalized monolayers, four approaches have been reported: (1) A multistep procedure in which an ω-ester-terminated 1-alkene/1-alkyne is attached onto a hydrogen-terminated silicon surface with subsequent hydrolysis of the ester to a carboxylic acid3 and surface-bound conversion of that carboxylic acid to an NHS-ester.4 While the attachment reaction proceeds without any problems with, for example, methyl ester-derived alkenes,3 the * To whom correspondence should be addressed. Telephone: +31-317482367. Fax: +31-317-484914. E-mail:
[email protected]. † Laboratory of Organic Chemistry, Wageningen University and Research Center. ‡ Radboud University Nijmegen. § Agrotechnology and Food Innovations, Wageningen University and Research Center. (1) (a) Aswal, D. K.; Lenfant, S.; Guerin, D.; Yakhmi, J. V.; Vuillaume, D. Anal. Chim. Acta 2006, 568, 84–108. (b) Sassolas, A.; Leca-Bouvier, B. D.; Blum, L. J. Chem. ReV. 2008, 108, 109–139. (2) Sullivan, T. P.; Huck, W. T. S. Eur. J. Org. Chem. 2003, 17–29. (3) Sieval, A. B.; Demirel, A. L.; Nissink, J. W. M.; Linford, M. R.; Van der Maas, J. H.; De Jeu, W. H.; Zuilhof, H.; Sudho¨lter, E. J. R. Langmuir 1998, 14, 1759–1768. (4) (a) Wei, F.; Sun, B.; Liao, W.; Ouyang, J. H.; Zhao, X. S. Biosens. Bioelectron. 2003, 18, 1149–1155. (b) Liao, W.; Wei, F.; Qian, M. X.; Zhao, X. S. Sens. Actuators, B 2004, 101, 361–367. (c) Liao, W.; Wei, F.; Liu, D.; Qian, M. X.; Yuan, G.; Zhao, X. S. Sens. Actuators, B 2006, 114, 445–450.
hydrolysis at the surface has only been proven to be complete under basic conditions that also diminish the quality of the organic monolayer.5 A second disadvantage of any indirect method is that, in the process of modifying the intermediary carbodiimideactivated acid group with NHS, NHS will stick rather persistently to the surface. As a result, such monolayers display a significantly increased roughness as can, for example, be seen by AFM.6 (2) A two-step procedure in which direct attachment of an ω-acid-terminated 1-alkene/1-alkyne to a H-terminated silicon surface is performed, followed by a surface-bound conversion of that carboxylic acid to an NHS-ester.7 While the direct attachment of acids (ω-acid-terminated 1-alkenes/1-alkynes) eliminates the hydrolysis step, such monolayers do typically display water contact angles > 50°, whereas fully acid-terminated monolayers on Au display water contact angles < 10°.8 This may reflect a small fraction of upside-down addition and/or effects of low grafting density.7 In addition, contamination of the surface by adsorbed NHS remains nonideal. (3) Direct thermal attachment of an ω-NHS-functionalized 1-alkene onto a H-terminated silicon surface. While this directly yields NHS-ester-terminated monolayers on silicon,9 this does not allow photoinduced patterning of the surface. In addition, ω-NHS-functionalized 1-alkenes may suffer from partial decomposition upon prolonged heating. (5) Liu, Y.-J.; Navasero, N. M.; Yu, H. Z. Langmuir 2004, 20, 4039–4050. (6) Roughness of surface after indirect attachment of NHS due to adsorption thereof. (7) (a) Voicu, R.; Boukherroub, R.; Bartzoka, V.; Ward, T.; Wojtyk, J. T. C.; Wayner, D. D. M. Langmuir 2004, 20, 11713–11720. It has been reported that “no reaction occurs between the carboxyl group and the silicon surface” (detailed study in ref 7b), indicating 50°) obtained under these conditions are then largely attributed to the exposure of the otherwise hidden CH2 groups in the alkyl chains. (b) Faucheux, A.; Gouget-Laemmel, A. C.; de Villeneuve, C. H.; Boukherroub, R.; Ozanam, F.; Allongue, P.; Chazalviel, J.-N. Langmuir 2006, 22, 153–162. (c) Allongue, P. Personal communication. (8) Mendoza, S. M.; Arfaoui, I.; Zanarini, S.; Paolucci, F.; Rudolf, P. Langmuir 2007, 23, 582–588. (9) (a) Bo¨cking, T.; James, M.; Coster, H. G. L.; Chilcott, T. C.; Barrow, K. D. Langmuir 2004, 20, 9227–9235. (b) Wojtyk, J. T. C.; Morin, K. A.; Boukherroub, R.; Wayner, D. D. M. Langmuir 2002, 18, 6081–6087.
10.1021/la800462u CCC: $40.75 2008 American Chemical Society Published on Web 07/12/2008
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Figure 1. Schematic representation of the formation of a mixed monolayer terminated with NHS-ester moieties (a) and of the subsequent substitution of the NHS-ester moiety by para-trifluoromethyl benzylamine (TFBA) (b) or biotin hydrazide (c).
(4) Direct photochemical attachment of an ω-NHS-functionalized 1-alkene using 254 nm light. Such photochemical attachment is, of course, of interest for the possible formation of patterned activated surfaces.10 While this leads to the attachment of NHS-terminated monolayers at room temperature, XPS analysis of the resulting monolayers reveals up to 30% decomposition of the NHS-ester-terminated monolayer and photoinduced oxide formation in the interface between the monolayer and the silicon substrate.11 Given the optical absorption spectrum of NHS-ester moieties, photochemical reactions could play a role under these conditions. Therefore, in the present work, we propose a mild one-step attachment of NHS-ester-terminated monolayers onto silicon surfaces via irradiation with visible light. As we have recently shown,12,13 this provides a mild, room-temperature alternative for the binding of covalently attached, Si-C linked monolayers onto silicon. The resulting NHS-ester-terminated monolayers were further modified by two amines, namely, para-trifluoromethyl benzylamine and biotin hydrazide (see Figure 1). All monolayers were characterized in detail by water contact angles, infrared reflection absorption spectroscopy (IRRAS), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) analyses.
Experimental Section Materials. Petroleum ether 40/60, dichloromethane, and ethyl acetate were distilled before use. 1,2,4-Trichlorobenzene was distilled twice and stored on CaCl2. 1-Decene (Fluka, 97%) and 1-undecylenic acid (UA) (Acros, 99%) were distilled twice at reduced pressure; acetone (Acros, 99+%), 2-propanol (Fisher, p.a.), NH4F (Sigma, 98+%), N-hydroxysuccinimide (NHS) (Sigma), N,N′-dicyclohexylcarbodiimide (DCC) (Sigma, 99%), EZ-Link biotin hydrazide (Pierce), and para-trifluoromethyl benzylamine (TFBA) (Aldrich, 97%) were used as obtained. Single polished n-type Si(111) substrates (10) Yin, H. B.; Brown, T.; Wilkinson, J. S.; Eason, R. W.; Melvin, T. Nucleic Acids Res. 2004, 32, e118. (11) Decomposition percentage of the NHS-ester moieties based on detailed XPS analysis of the C narrow spectrum and C/O ratio in the Supporting Information of ref 9a. (12) Sun, Q.-Y.; De Smet, L. C. P. M.; Van Lagen, B.; Giesbers, M.; Thu¨ne, P. C.; Van Engelenburg, J.; De Wolf, F. A.; Zuilhof, H.; Sudho¨lter, E. J. R. J. Am. Chem. Soc. 2005, 127, 2514–2523. (13) (a) De Smet, L. C. P. M.; Stork, G. A.; Hurenkamp, G. H. F.; Sun, Q.-Y.; Topal, H.; Vronen, P. J. E.; Sieval, A. B.; Wright, A.; Visser, G. M.; Zuilhof, H.; Sudho¨lter, E. J. R. J. Am. Chem. Soc. 2003, 125, 13916–13917. (b) Sun, Q.-Y.; de Smet, L. C. P. M.; van Lagen, B.; Wright, A.; Zuilhof, H.; Sudho¨lter, E. J. R. Angew. Chem., Int. Ed. 2004, 43, 1352–1355. (c) Eves, B. J.; Sun, Q. Y.; Lopinski, G. P.; Zuilhof, H. J. Am. Chem. Soc. 2004, 126, 14318–14319. (d) De Smet, L. C. P. M.; Pukin, A. V.; Sun, Q.-Y.; Eves, B. J.; Lopinski, G. P.; Visser, G. M.; Zuilhof, H.; Sudho¨lter, E. J. R. Appl. Surf. Sci. 2005, 252, 24–30. (e) Scheres, L.; Arafat, A.; Zuilhof, H. Langmuir 2007, 23, 8343–8346.
(475-550 µm thick, resistivity 1.0-5.0 Ωcm) were obtained from Addison Engineering, San Jose, CA. Synthesis of N-Succinimidyl Undecyl-1-enate (NHS-UA). The preparation procedure was as described before.14 In brief, UA (3.64 g, 19.8 mmol) and NHS (2.53 mg, 22.0 mmol) were dissolved in 100 mL of ethyl acetate. DCC (4.54 g, 22.0 mmol) was added to the solution on ice, and the mixture was stored at 4 °C overnight. The reaction mixture was filtered to remove depositions and evaporated in a low-pressure rotavapor to remove solvents. The resulting waxy solid was recrystallized from 20 mL of 2-propanol, collected by vacuum filtration with a Büchner funnel, and rinsed with a small amount of water. After drying in vacuum overnight, NHS-UA was obtained (3.37 g, 12.0 mmol, 61%) with purity > 99% (GC). 1H NMR (300 MHz, CDCl3): δ 5.80 (m, 1H), 4.97 (m, 2H), 2.85 (s, 4H), 2.62 (t, 2H), 2.05 (q, 2H), 1.76 (m, 2H), 1.33-1.42 (m, 10H) (See Figure S1 in the Supporting Information for the full 1H NMR spectrum, which is in agreement with literature.9). Monolayer Preparation. The approach is indicated schematically in Figure 1. The detailed procedure was as described before.12 In brief, oxygen plasma-cleaned Si(111) chips were etched to a H-terminated surface by immersion in an argon-saturated 40% aqueous NH4F solution for 15 min under an argon atmosphere. Monolayers were prepared by immersing the freshly prepared H-terminated Si(111) chips into a deoxygenated solution of NHSUA and/or 1-decene in 1,2,4-trichlorobenzene, followed by irradiation with 447 nm light for 16 h. The total concentration of alkenes was 0.5 M, while the alkene composition was varied with different NHSUA ratios of 0, 50, and 100%. Attachment of para-Trifluoromethyl Benzylamine. NHS-esterterminated monolayer samples were placed in a solution of ∼20 mM para-trifluoromethyl benzylamine (TFBA) in dichloromethane. After incubation overnight, the samples were cleaned by sonication in dichloromethane, rinsed copiously with dichloromethane and acetone, and dried by nitrogen flushing. Attachment of Biotin Hydrazide. NHS-ester-terminated monolayer samples were placed in a solution of ∼5 mM biotin hydrazide in 100 mM sodium acetate buffer pH 5.5. After incubation overnight, the samples were cleaned by sonication in sodium acetate buffer, rinsed copiously with H2O and CH2Cl2, and dried by nitrogen flushing. Analysis of the Monolayers. Contact angle measurements were ¨ SS, Germany). carried out with a DSA 100 instrument (KRU Infrared reflection absorption spectroscopy (IRRAS) measurements were performed with a Bruker Tensor 27 instrument with a liquid-nitrogen-cooled mercury cadmium telluride (MCT) detector (Bruker, Germany), using a commercial variable-angle reflection unit (Auto Seagull, Harrick Scientific). A Harrick grid polarizer was installed in front of the reflection unit and was used for measuring spectra with p-polarized (parallel) radiation at an (14) Macossay, J.; Shamsi, S. A.; Warner, I. M. Tetrahedron Lett. 1999, 40, 577–580.
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incident angle of 68° with respect to the normal of the sample surface. Single channel transmittance spectra were collected using a spectral resolution of 4 cm-1 and 1024 scans in each measurement and using a oxygen plasma-cleaned Si(111) chip as reference. The XPS analysis of the surfaces was performed using a JPS9200 Photoelectron spectrometer (JEOL, Japan). The highresolution spectra were obtained at takeoff angle of 80° under ultrahigh vacuum (UHV) conditions using monochromatic Al KR X-ray radiation at 12 kV and 25 mA, using an analyzer pass energy of 20 eV. Atomic force microscopy (AFM) images were obtained with a JSPM-5400 microscope (Jeol, Japan) with silicon cantilevers operating in the alternating current (AC) mode in air. The scan size was 1 × 1 µm2 at a scan rate of 1.0 Hz. Electronic Core Level Calculations. All calculations were done with the GAUSSIAN03 program.15 The geometries of the different systems were optimized at the B3LYP/6-311G(d,p) level of theory. Natural bond orbital (NBO) analysis16 was employed to obtain the core orbital energies.
Results and Discussion Formation and Characterization of Mixed Monolayers. Mixed monolayers were obtained from the photochemical reaction (λ ) 447 nm) of a hydrogen-terminated silicon Si(111) surface with a mixture of N-succinimidyl undecylenate (NHS-esteralkene) and 1-decene in different ratios. The mole fraction of the terminal NHS-ester groups on the resulting mixed monolayers was expected to approximate to the mole fraction of NHS-esteralkene in the reaction mixture.5 As expected, depending on the composition of reaction solutions, the water contact angle of the resulting mixed monolayers varied from 110° (0% NHS-esteralkene) to 52° (100% NHS-ester-alkene). In comparison with the pure 1-decene-derived monolayer (i.e., 0% NHS), infrared reflection absorption spectroscopy (IRRAS) clearly revealed the NHS-ester functionalities in the pure NHS-UA monolayer (i.e., 100% NHS) by the appearance of the characteristic CdO stretching vibrations at 1817, 1788, and 1745 cm-1 (Figure 2) belonging to the ester carbonyl stretch and the symmetric and asymmetric carbonyl stretches in the succinimidyl end groups, respectively.17 This technique also revealed that the NHS-esterterminated monolayers were not well-ordered, as the antisymmetric and symmetric CH2 stretching vibrations showed up at ∼2926 and ∼2854 cm-1, respectively.9,18 AFM analysis showed that the resulting surfaces were clean and flat with, for example, easily recognizable Si(111) edge steps and with an average line roughness of 0.06 nm on the terrace surfaces and an average area roughness of 0.09 nm over the whole measured surface of a 100% NHS-ester-terminated monolayer. (15) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian03, revision D.01; Gaussian, Inc.: Wallingford, CT, 2004. (16) Glendening, E. D.; Reed, A. E.; Carpenter, J. E.; Weinhold, F. NBO, version 3.1. (17) (a) Guo, D.-J.; Xiao, S.-J.; Xia, B.; Wei, S.; Pei, J.; Pan, Y.; You, X.-Z.; Gu, Z.-Z.; Lu, Z. J. Phys. Chem. B 2005, 109, 20620–20628. (b) Duhachek, S. D.; Kenseth, J. R.; Casale, G. P.; Small, G. J.; Porter, M. D.; Jankowiak, R. Anal. Chem. 2000, 72, 3709–3716. (18) (a) Snyder, R. G.; Strauss, H. L.; Elliger, C. A. J. Phys. Chem. 1982, 86, 5145–5150. (b) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559–3568.
Figure 2. IRRA spectra of the pure NHS-UA monolayer (100% NHS, with the characteristic triple-peak carbonyl pattern of NHS-esters) and the pure 1-decene monolayer (i.e., 0% NHS) attached on Si(111) (top). AC mode AFM topographic image of 100% NHS-ester-terminated surface (1 × 1 µm2; color scale from 0 to 0.60 nm) (bottom). Average area roughness (A Ra) and average line roughness (L Ra) (along the arrow) are marked in the image.
A schematic depiction of the types of carbon atoms that are distinguishable by XPS is shown in Figure 3.19 Figure 4 shows the XPS narrow scans of C1s, N1s, F1s, and O1s of a 100% NHSester-terminated monolayer on Si(111) before and after binding of TFBA. The C1s signal of the NHS-ester-terminated monolayer (a) can be deconvoluted into three peaks (Figure 4 (NHS C1s)), from low to high binding energy (BE), as (i) a peak at 285.1 eV (with a full width at half-maximum (fwhm) of 1.4 eV) for carbons in the aliphatic hydrocarbon chain; (ii) a peak at 286.4 eV for R-carbons adjacent to the carbonyl carbon atoms (These three R-carbons shift much more than an ordinary R-CH2 (general shift of 0.4∼0.7 eV)20 in an alkyl chain because of the strong electron-withdrawing effect from the imide group in the NHS moiety, in line with the calculated ∆BEs of 1.1∼1.5 eV (see (19) As observable with our XPS set-up. See for highly detailed other XPS analyses of organic monolayers on silicon: (a) Wallart, X.; de Villeneuve, C. H.; Allongue, P. J. Am. Chem. Soc. 2005, 127, 7871–7878. (b) Webb, L. J.; Nemanick, E. J.; Biteen, J. S.; Knapp, D. W.; Michalak, D. J.; Traub, M. C.; Chan, A. S. Y.; Brunschwig, B. S.; Lewis, N. S. J. Phys. Chem. B 2005, 109, 3930–3937. (c) Webb, L. J.; Michalak, D. J.; Biteen, J. S.; Brunschwig, B. S.; Chan, A. S. Y.; Knapp, D. W.; Meyer, H. M., III; Nemanick, E. J.; Traub, M. C.; Lewis, N. S. J. Phys. Chem. B 2006, 110, 23450–23459.
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Figure 3. Types of C atoms that can be distinguished by XPS in the NHS-ester-terminated monolayers (a), and after the functionalization with TFBA (b) and biotin hydrazide (c).
Table 2).); and (iii) a peak at 289.7 eV for the carbonyl carbon atoms. The ratio between these three peaks for a 100% NHSester-terminated monolayer was 9.4: 2.7: 2.4, which was within the experimental error equal to the theoretical ratio of 9:3:3 (see Table 1). As presented in Figure 4 (narrow scan N1s region), the nitrogen of the NHS-ester-terminated monolayer (a) has a binding energy of 402.3 eV. Analogous XPS analyses were carried out on a 50% NHS-ester-terminated monolayer (see Figure S2 in the Supporting Information). Approximately the same BEs and chemical shifts were observed in N1s, C1s, F1s, and O1s narrow XPS spectra for the 50% NHS-ester-terminated monolayer, albeit, of course, in different atomic ratios (see Table 1, entries 2 and 4). The assignment of the C1s XPS spectra is supported by density functional theory (DFT) calculations of the core orbital energy levels. Following Koopmans’ theorem,21 the core orbital energy levels are assumed to be directly related to the binding energies of the core electrons (“initial state approximation”). The absolute values of calculated binding energies cannot be compared directly with the experimental data because of the difference in reference energies in theory and experiment.22 However, energy differences (∆BE) can be used for the carbon atoms in comparison to the C1s core orbital energy of a self-chosen point of reference, in this case, the CH2 moiety in the center of the aliphatic hydrocarbon chain. The calculated ∆BEs were used to simulate the XPS spectra assuming an equal contribution from each carbon atom. For every carbon atom, a gaussian centered at the corresponding binding energy was used with a fwhm of 1.2 eV. The sum of all gaussians gave the simulated XPS spectra. The detailed results for all three compounds are included in the Supporting Information. The final simulated C1s XPS spectrum and calculated differences in binding energies for the carbon atoms of a model compound representing the 100% NHS-UA monolayer are shown in Figure 5 and Table 2, respectively. The calculated data are in good agreement with the experimental C1s XPS spectrum and show that the fitted peaks i-iii (Figure 4, top left) actually consist of several subsets of peaks. Peak i is the sum of the carbon (1) bonded to silicon, the CH2 moieties (2-7) in the alkyl chain, and carbon atoms (8 and 9). The other two peaks consist of two different carbon types: peak iii for the carbonyl carbons (11 and 12) and peak ii for R-CH2 (10 and 13) adjacent (20) Brigss, D.; Grant, J. T., Eds.; Surface Analysis by Auger and X-ray Photoelectron Spectroscopy; IM Publications: Chichester, U.K., 2003. (21) Koopmans, T. Physica 1933, 1, 104. (22) Tielens, F.; Costa, D.; Humblot, V.; Pradier, C. M. J. Phys. Chem. C 2008, 112, 182–190.
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to the carbonyls. The binding energies of the two R-carbons (13) in the NHS moiety are shifted more than the R-carbon (10) in the alkyl chain, as evidenced by the calculated ∆BE of 1.5 eV for each R-carbon (13) and 1.1 eV for R-carbon (10) (Table 2). The average of the calculated ∆BEs for all three R-carbons is ∼1.4 eV, in good agreement with the experimental results of peak ii in the C1s spectra of the NHS-ester-terminated monolayer (Figure 4, top left). These results show that DFT calculations are a simple and effective tool in the assignment of XPS peaks. Modification with para-Trifluoromethyl Benzylamine (TFBA). Confirmation that the NHS-ester moieties were attached fully intact comes from a qualitative and semiquantitative XPS analysis of surfaces in which the NHS-ester-containing monolayer was allowed to react with TFBA. The narrow C1s spectrum of the TFBA-modified 100% NHS-ester-terminated monolayer (b) is shown in Figure 4 (TFBA C1s). TFBA quantitatively replaces the NHS moiety, and yields for both the 100% and 50% NHS monolayers ratios between the expected carbon atoms that are in excellent agreement with theoretical predictions (see Table 1, entries 3 and 5). After bonding of TFBA (i.e., monolayer b), the appearance of a new carbon peak (iv) at a high BE of 293.4 eV indicates the presence of the CF3 group. The other three carbon peaks were at 288.6 (iii), 286.6 (ii) and 285.4 (i) eV. The BE of carbonyl carbon (iii) decreased by about 1 eV (from 289.7 to 288.6 eV) in going from the NHS-ester to a TFBA-substituted monolayer, since the release of the electronegative oxygen of the NHS-ester decreases the electron-withdrawing effect on the carbonyl. Comparison of the experimental XPS data with the simulated C1s XPS spectrum (see Figure S6 in the Supporting Information) shows that the peak (ii) at 286.6 eV was attributable to the benzylic carbon bound to the electronegative nitrogen atom and the aromatic carbon bound to the CF3 group, as illustrated in Figure 3b. The chemical shift of ∼1.6 eV is in agreement with the calculated energy differences (∆BE; average of 1.5 eV) for these two carbons. The carbon peak (i) at 285.4 eV was assigned to comprise all other carbons with calculated ∆BEs of less than 1 eV in order to compare it with peak (i) of the initial NHS-ester-terminated monolayer. Peak (i) of the TFBA sample displays an additional shift of 0.3 eV and a broadened fwhm (∼1.6 eV) after the bonding of TFBA. This is caused by the attachment of aromatic carbons (not directly bound to CF3) and the R-carbon atom (bound to the amide carbonyl) with a calculated average ∆BE of 0.7 eV (part i,b in Figure 3b), adjacent to the aliphatic carbons (parts i,a and i,c in Figure 3b). The atomic ratio between these four C1s peaks (Ci:Cii:Ciii:Civ in Table 1) was 15.0:2.0:1.0:1.1, very close to the theoretical ratio of 15:2:1:1. This carbon assignment is confirmed by a more detailed quantitative analysis of the carbon atomic ratios. Deconvolving peak (i) into three subpeaks of (i,a), (i,b), and (i,c) gives results that are in agreement with theoretically expected atomic ratios and simulated XPS spectra, as reported in Figure S6 in the Supporting Information. The presence of a CF3 group in monolayer (b) was further confirmed by the appearance of a strong fluorine peak at 688.8 eV in Figure 4 (F1s), while a small but detectable trace at a low BE of 686.3 eV (about 3% of the total F signal), also present in the initial NHS-ester-terminated monolayers, could be assigned to the contamination of fluoride ions from the etching procedure in the preparation of the hydrogen-termianted silicon surface. Furthermore, the release of the NHS-ester moiety was seen from the O1s spectra. The oxygen signal of the NHS-ester-terminated monolayer (a) in Figure 4 (O1s) was composed of two peaks at 533.0 and 535.5 eV, with a ratio of 4:1 (larger than the theoretical ratio of 3:1). The high BE peak at 535.5 eV was characteristic
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Figure 4. XPS narrow scans of C1s of a 100% NHS-ester-terminated monolayer (top left) and a TFBA-modified monolayer (top, right). (bottom) N1s, O1s, and F1s narrow range scans of a 100% NHS-ester-terminated monolayer (upper curves; red) and a TFBA-modified monolayer (lower curves; green). Table 1. Atomic Ratios of 100% and 50% NHS-Ester-Terminated Monolayer by XPS Analyses F/C 100% NHS 100% NHS + TFBA 50% NHS 50% NHS + TFBA
Ci:Cii:Ciii:Civ
theor
exp
theor
exp
0:15a 3:19 0:25a 3:29
0.2:14.8 3.3:18.6 0.1:24.8 3.2: 28.8
9:3:3:0b 15:2:1:1 19:3:3:0b 25:2:1:1
9.4:2.7:2.4:0b 15.0:2.0:1.0:1.1 19.3:3.3:2.4:0b 25.0:2.0:1.1:1.2
a 15 and 25 are the theoretical numbers of carbon atoms in NHS-UA and NHS-UA/decene (1:1), respectively. b 0 means no Civ in monolayers in theory or detected in experiments.
for oxygen in C-O-N bonds of the NHS-ester, while the low BE peak at 533.0 eV was attributed mainly to the oxygens in carbonyls. The excess amount observed at 533.0 eV is attributed to airborne contaminations. As expected, the oxygen peak at 535.5 eV disappeared after bonding of TFBA, and a carbonylic oxygen peak of monolayer (b) showed up at lower binding energies (532.4 eV) caused by the resonance effects of the amide functionality. The complete substitution of the NHS-ester moieties by TFBA was finally confirmed by the chemical shift of the N1s peak in the N1s narrow spectra (Figure 4, N1s). After removal of the NHS-ester moiety (in which the N atom is bound to an electronegative O atom) and the subsequent formation of an amide bond in monolayer (b), nitrogen signals were only found at a lower BE of 400.5 eV. Analogous XPS analyses were also carried out on a TFBAmodified 50% NHS-ester-containing monolayer and pure 1-decene-derived monolayer (i.e., 0% NHS) (see Figure S4 in the Supporting Information). Approximately the same BEs and chemical shifts were observed in N1s, C1s, F1s, and O1s narrow XPS spectra for the TFBA-modified 50% NHS-ester-containing monolayer but in different atomic ratios (see Table 1, entries 3 and 5). The fact that the measured atomic ratios are in excellent agreement with theoretical predictions suggests that the TFBA
molecules could quantitatively replace the NHS-ester moieties in both the 100% and 50% NHS-ester-containing monolayers. It also indicates that the TFBA molecule does not exhibit any detectable effect of steric hindrance on the substitution of the NHS-ester moiety. Two possible reasons are (1) TFBA is a primary amine and not much larger than the NHS-ester group in volume and (2) NHS-ester-containing monolayers are not as dense as long-chain alkyl monolayers because of the relative large volume occupancy of terminal NHS-ester groups. However, they are still dense enough to hamper oxidation of the underlying silicon by traces of dissolved water and oxygen in the TFBA solution, as confirmed by the narrow Si2p spectra. The XPS narrow scan of Si2p (see Figure S3 in the Supporting Information) showed only peaks at a BE of ∼100 eV, without detectable peaks corresponding to silicon oxide between 102 and 104 eV, either before or after bonding TFBA. Finally, attachment of TFBA was confirmed by static water contact angle measurements, which yielded a change from 52° for 100% NHS-ester-terminated monolayers to 88° ((1°) after the bonding of TFBA. Modification with Biotin Hydrazide. As a second example (see Figure 1), the functionalization of NHS-ester-terminated monolayers on Si(111) for bioapplications was demonstrated by modifying such monolayers with biotin hydrazide to obtain a biotinylated semiconductor surface. After incubation overnight in a ∼5 mM solution of biotin hydrazide in 100 mM sodium acetate buffer pH 5.5, the static water contact angle of the monolayers was measured. In comparison to 100% NHS-esterterminated monolayers, the water contact angle decreases by 10 ( 3° after modification with biotin hydrazide, as the biotin moiety is more hydrophilic than the NHS-ester. However, no trend in this decrease was discernible in relation to the fraction of NHSester moieties in the monolayers, which can be rationalized by two opposing effects: with increasing NHS-ester content, of course, more polar biotin hydrazide moieties will be attached,
7936 Langmuir, Vol. 24, No. 15, 2008
Yang et al.
Figure 5. (A) Structure of model compound used for the calculation of binding energies of the 100% NHS-UA monolayer. (B) Experimental (gray squares) and simulated (black line) core level C1s XPS spectra for the NHS-UA monolayer, simulation based on DFT calculations of the C1s core level energies for the NHS-UA-ester capped with a trimethylsilyl group. Table 2. Analysis of the Simulated C1s Spectra and Comparison with the Experimental Data (Relative to 285 eV) for NHS-UA Monolayers
but on the other hand at higher NHS-ester content the monolayer itself is already more polar (water contact angle 52-60°), which results in a smaller additional wetting effect of the transformation of NHS-ester into biotin moieties. In this regard, it needs to be stated that the stability of properly made alkyl monolayers on Si (contact angles after preparation, >110°) decreases by
