Detecting the Chemoselective Ligation of Peptides to Silicon with

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Langmuir 2006, 22, 7059-7065

7059

Detecting the Chemoselective Ligation of Peptides to Silicon with the Use of Cobalt-Carbonyl Labels Christophe Olivier,† Aurore Perzyna,‡ Yannick Coffinier,§ Bruno Grandidier,*,§ Didier Stie´venard,§ Oleg Melnyk,† and Jean-Olivier Durand*,‡ UMR CNRS 8161, Biological Institute of Lille, 1 rue du Professeur Calmette, 59021 Lille, France, UMR CNRS 5637, UniVersite´ Montpellier 2, Place Euge` ne Bataillon, 34095 Montpellier Cedex 05, France, and Institut d’Electronique et de Microe´ lectronique et de Nanotechnologie, IEMN, (CNRS, UMR 8520), De´ partement ISEN, 41 bouleVard Vauban, 59046 Lille Cedex, France ReceiVed February 8, 2006. In Final Form: May 19, 2006 While fluorescent-based methods are generally used to detect the immobilization and the interactions of biomolecules to solid supports, recent studies have shown their limitations in the case of silicon surfaces. As an alternative, we investigated the synthesis of peptides labeled with a metal transition complex and their subsequent immobilization to the silicon surfaces. The feasibility of using such probes has been explored by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). By starting with hydrogen-terminated or oxidized silicon surfaces, we functionalized those surfaces with semicarbazide groups and showed the site-specific linkage of glyoxylyl peptides labeled with a Co2(CO)6 moiety.

Introduction The development of strategies to immobilize biomolecules to various surfaces is of great importance in many different fields of research and has dramatically increased the rate and scope of discoveries in basic and applied science. In particular, the development of an efficient surface chemistry for biomolecule anchoring is often a key step in micro- or nanotechnology projects.1-3 Usually, a high surface coverage by the biomolecules is preferred to reach high detection sensitivities. This is best achieved by combining an efficient method to functionalize the surface and linking the biomolecules to this modified surface. In this context, well-defined silicon surfaces are increasingly used.4 The design of novel methods for their functionalization and the characterization of their reactivity or immobilization efficiency are thus of great interest. In such a goal, a variety of interesting methods have been designed to first modify the silicon surface with reactive groups through the formation of Si-C bonds and then covalently immobilize biomolecules, such as DNA,5-9 oligosaccharides,10 or polypeptides. Site-specific anchoring of biomolecules constitutes an attractive approach for preparing well-defined biomolecular interfaces. In this context, we recently described the site-specific anchoring of peptides on * Corresponding author. E-mail: [email protected] (J.-O.D.); [email protected]. (B.G.). † Biological Institute of Lille. ‡ Universite ´ Montpellier 2. § Institut d’Electronique et de Microe ´ lectronique et de Nanotechnologie. (1) Emili, A. Q.; Cagney, G. Nat. Biotechnol. 2000, 18, 393. (2) Cui, Y.; Wei, Q.; Park, H.; Lieber, C. M. Science 2001, 293, 1289. (3) Cui, D.; Gao, H. Biotechnol. Prog. 2003, 19, 683. (4) Buriak, J. M. Chem. ReV. 2002, 102, 1271. (5) Strother, T.; Cai, W.; Zhao, X.; Hamers, R. J.; Smith, L. M. J. Am. Chem. Soc. 2000, 122, 1205. (6) Strother, T.; Hamers, R. J.; Smith, L. M. Nucleic Acids Res. 2000, 28, 3535. (7) Yin, H. B.; Brown, T.; Wilkinson, J. S.; Eason, R. W.; Melvin, T. Nucleic Acids Res. 2004, 32, e118. (8) Voicu, R.; Boukherroub, R.; Bartzoka, V.; Ward, T.; Wojtyk, J. T. C.; Wayner, D. D. M. Langmuir 2004, 20, 11713. (9) Pike, A. R.; Ryder, L. C.; Horrocks, B. R.; Clegg, B. A. Chem.sEur. J. 2005, 11, 344. (10) 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.; Sudholter, E. J. R. J. Am. Chem. Soc. 2003, 125, 13916.

silicon surfaces based on the chemoselective ligation of semicarbazide-functionalized surfaces with peptides derivatized by an R-oxo aldehyde group.11 Once the successful attachment of biomolecules is achieved, the optimization of the surface functionalization and biomolecule site-specific anchoring requires a simple method for detecting the immobilized biomolecules. Fluorescence detection is often used for this purpose. For silicon surfaces, however, the optical signal emitted by the fluorescently labeled biomolecules can be low because of the proximity of the dye molecule to the silicon surface, which prevents a high excitation of its fluorescence.12,13 In addition, the signal can vary considerably from one biomolecule to another because the emitted fluorescence depends critically on, among other parameters, the distance between the dye and the silicon surface. An alternative for the detection of immobilized biomolecules could be based on the use of transition metal carbonyl complex labels. Fourier transform infrared spectroscopy (FTIR) has been shown to be sensitive enough to the detection of metallic transition moieties when they are bound to a self-assembled layer on a metallic surface.14 As a result, the linkage of such moieties to biomolecules should offer a complementary approach to the existing surface-based methods used for biomolecule detection. In this article, we describe the preparation of peptides modified by a Co2(CO)6 moiety and study the binding of two types of hexacarbonyldicobalt-labeled peptides to silicon surfaces modified with a semicarbazide group. This reactive group is provided through the direct formation of silicon carbon bonds11 to a silicon surface or the use of a trichlorosilane isocyanate derivative to modify hydroxylated Si surfaces.15 For both surfaces, we show that the Co2(CO)6 group is easily detected by FTIR and X-ray photoelectron spectroscopy (XPS). We take advantage of the (11) Coffinier, Y.; Olivier, C.; Perzyna, A.; Grandidier, B.; Wallart, X.; Durand, J.-O.; Melnyk, O.; Stievenard, D. Langmuir 2005, 21, 1489. (12) Lambacher, A.; Fromherz, P. Appl. Phys. A 1996, 63, 207. (13) Volle, J. N.; Chambon, G.; Sayah, A.; Reymond, C.; Fasel, N.; Gijs, M. A. M. Biosens. Bioelectron. 2003, 19, 457. (14) Yam, C. M.; Zheng, L.; Salmain, M.; Pradier, C. M.; Marcus, P.; Jaouen, G. Colloids Surf., B 2001, 21, 317. (15) Ardes-Guisot, N.; Durand, J.-O.; Granier, M.; Perzyna, A.; Coffinier, Y.; Grandidier, B.; Wallart, X.; Stievenard, D. Langmuir 2005, 21, 9406.

10.1021/la060370m CCC: $33.50 © 2006 American Chemical Society Published on Web 06/28/2006

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sensitivity of this detection method to demonstrate the site-specific anchoring of hexacarbonyldicobalt-labeled glyoxylyl peptides to the semicarbazide-functionalized silicon surface. Materials and Methods Peptide Synthesis. A. General. N-succinimidyl-5-hexynoate (1) and (N-succinimidyl-5-hexynoate)hexacarbonyldicobalt (2) were synthesized as described elsewhere.16 Solid-phase peptide syntheses were performed on an automated peptide synthesizer (model Pioneer, Applied Biosystems, Foster City, CA). Peptides were purified on a C18 12.5 × 300 mm Nucleosil column, with a water/acetonitrile linear gradient containing 0.05% trifluoroacetic acid (TFA) (method 1) or on a 15 × 250 mm C3-Zorbax column, with an A/B linear gradient in which A is pH 7, 10 mM triethylamine acetate, and B is acetonitrile/pH 7, 10 mM triethylamine acetate (95/5 by vol) (method 2). Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectra were recorded on a Applied Biosystems (Foster City, CA) Voyager-DE STR spectrometer. Electrospray mass spectrometry (ES-MS) were recorded on a Micromass Quatro II electrospray mass spectrometer. IR spectra were recorded on a PerkinElmer Spectrum 1000 FT-IR spectrometer (KBr pellets). B. Tartaramide Peptidyl Resin 3. Synthesis was performed using the Fmoc/tert-butyl strategy on 0.909 g (0.220 mmol/g, 0.200 mmol) of Novasyn TG resin (Novabiochem, Switzerland) modified by an isopropylidene tartrate-based linker. Following peptide elongation, the peptidyl resin was deprotected using TFA/water/triisopropylsilane (9.5 mL/250 µL/250 µL; 2h). The resin was washed with CH2Cl2 (4 × 2 min), ether (2 × 2 min), dried under vacuum, and separated into two parts (0.1 mmol each). C. Synthesis of Peptide 4. Tartaramide peptidyl resin 3 (0.1 mmol) was washed with CH2Cl2 (3 × 5 min) and N,N-dimethylformamide (DMF) (2 × 5 min). A solution of 1 (100 mg, 0.478 mmol) and 4-methylmorpholine (73 µL, 0.64 mmol) in DMF (3 mL) was added (three times). The coupling was monitored using the TNBS test.17 The resin was then washed with DMF (3 × 5 min), CH2Cl2 (3 × 5 min), dried under vacuum, and conditioned in 5 mL of AcOH/ water (5/95 by vol). Cleavage of the diol moiety was performed using 123 mg of NaIO4 (0.573 mmol) in 3.5 mL of an AcOH/water (5/95 by vol) mixture. After 2.5 min, the reaction was quenched with 138 µL (2.30 mmol) of ethanolamine. The resin was filtered and washed twice with 4 mL of AcOH/water (5/95 by vol). The combined filtrates were diluted to 50 mL with water and purified by reversed-phase high-performance liquid chromatography (RPHPLC) (method 1) to give 29.3 mg (33% yield) of peptide 4. MALDITOF: [M+H]+ monoisotopic calcd, 828.43; found, 828.44. D. Synthesis of Glyoxylyl Peptide 5. Tartaramide peptidyl resin 3 (0.1 mmol) was washed with AcOH/water (5/95 by vol, 2 × 2 min). A periodate oxidation was performed in 3 mL of AcOH/water (5/95 by vol) containing 135 mg of NaIO4 (0.631 mmol). After 2.5 min, the reaction was quenched with 153 µL (2.53 mmol) of ethanolamine. The resin was filtered and washed twice with 3 mL of AcOH/water (5/95 by vol). The combined filtrates were diluted to 135 mL with water and purified by RP-HPLC (method 1) to give 51.7 mg (51% yield) of peptide 5. MALDI-TOF: [M+H]+ monoisotopic calcd, 734.40; found, 734.42. E. Synthesis of Peptide 6. Synthesis starting from peptide 4. To a stirred solution of peptide 4 (29.3 mg, 31.1 µmol) in anhydrous DMF (1.2 mL) at 4 °C and protected from light, 189 mg (0.552 mmol) of Co2(CO)8 was added in 10 portions for 12 h. Ten milliliters of CH3CN and 40 mL of triethylamineacetate buffer (10 mM, pH 7) were then added. The mixture was centrifuged (3500 rpm, 15 min, 4 °C). The supernatant was filtrated (0.22 µM) to remove the precipitate and purified by RP-HPLC (method 2) to afford, after lyophilization, 6.0 mg (18% yield) of peptide 6. Synthesis from peptide 5. To a stirred solution of 5 (42.4 mg, 44.1 µmol) in 4.25 mL of borate buffer (0.1 M, pH 8.5) at 4 °C and protected from light, a solution of 2 (13.8 mg, 66.0 µmol) in (16) Salmain, M.; Vessiere`re, A.; Butler, I. S.; Jaouen, G. Bioconjugate Chem. 1991, 2, 13. (17) Hancock, W. S.; Battersby, J. E. Anal. Biochem. 1976, 71, 260.

OliVier et al. acetonitrile (2.8 mL) was added. After 3 h of stirring, the reaction mixture was diluted with 3 mL of water, filtered (0.22 µM), and purified as described above by RP-HPLC (method 2) to afford, after lyophilization, 23.3 mg (51% yield) of peptide 6. IR (KBr): 2092, 2050, 2019; MS (ESI): [M+H2O+H]+ monoisotopic calcd, 1132.3; found, 1132.0. F. Synthesis of Peptide 7. Synthesis was performed on 1.110 g (0.180 mmol/g, 0.200 mmol) of [5-(4-Fmoc-aminomethyl-3,5dimethoxyphenoxy)valeric acid]-poly(ethylene glycol)-polystyrene resin (Fmoc-PAL-PEG-PS resin, Applied Biosystems, Foster City, CA). Following peptide elongation, the peptide was deprotected and cleaved using TFA/water/triisopropylsilane (9.5 mL/250 µL/250 µL) for 2 h. The peptide was precipitated in cold diethyl ether/n-heptane (1/1 by vol) and purified by RP-HPLC (method 1) to afford, after lyophilization, 73.5 mg (43% yield) of peptide 7. MALDI-TOF: [M+H]+ monoisotopic calcd, 621.3; found, 621.4. G. Synthesis of Peptide 8. To a stirred solution of 7 (50.0 mg, 59.0 µmol) in 5 mL of borate buffer (0.1 M, pH 8.5) at 4 °C and protected from light, a solution of 2 (17.3 mg, 82.7 µmol) in acetonitrile (3 mL) was added. After 3 h of stirring, the reaction mixture was diluted with 8 mL of water, filtered (0.22 µM), and purified by RP-HPLC (method 2) to afford, after lyophilization, 32.3 mg (62% yield) of peptide 8. IR (KBr): 2092, 2050, 2019; MS (ESI): [M+H]+ monoisotopic calcd, 1001.2; found, 1001.2. Preparation of Semicarbazide-Terminated Silicon Surfaces. The Boc-protected semicarbazide silicon wafers Sa-Boc and SbBoc were prepared following our recently described methods.11,15 The Boc removal was then performed by dipping the protected semicarbazide-terminated surfaces, Sa-Boc or Sb-Boc, in a TFA/ dichloromethane (4/6 by vol) solution for 4 h. The sample was rinsed in an ultrasonic bath with dichloromethane (5 min), dipped in absolute ethanol (5 min), and then dried under a nitrogen flow. Peptide Immobilization on Semicarbazide-Terminated Silicon Surfaces. The semicarbazide-terminated silicon wafer (Sa or Sb) was immersed for 1 h at 37 °C in a 0.1 mM solution of peptide dissolved in 0.1 M pH 5.5 sodium acetate buffer. The wafer was washed with water (30 min), and then successively washed with 5% aqueous K2HPO4 (1 h), then water (15 min), and then dried under a nitrogen flow. Ellipsometric Measurements. The measurements were made on a Plasmos SD 2300 ellipsometer with a rotating analyzer as the modulating element. The laser source was a helium/neon laser with a wavelength of 632.8 nm. The angle of incidence was 70°, and the spot size was about 2 mm. The substrate was silica that had grown on silicon. The refractive index was 1.46 for silica and 3.83 for silicon. A refractive index of 1.45 was used for the silane layers. In Situ FTIR Measurements. Attenuated total reflectance (ATR) infrared spectra were recorded on an FTIR Perkin-Elmer 2000 spectrometer equipped with a narrow-band liquid nitrogen-cooled mercury-cadmium-telluride detector and a thermoregulated ATR flow cell. The sample compartment was purged with dry air. The flow cell containing the internal reflection element, a trapezoidal Si crystal (72 × 10 × 1 mm3) with the narrow edges polished at a 45° angle, was placed and aligned in the dry air-purged sample compartment of the spectrophotometer and thermoregulated at the convenient temperature of the reaction. A peristaltic pump was used to pump the solutions through the cell. First, the cell was filled with pure solvent to collect background spectra, and then solutions of the reactive materials were pumped and the spectra were recorded. All the spectra were recorded at a resolution of 1 cm-1, and 128 scans were accumulated. To attach the semicarbazide layer through the direct formation of silicon carbon bonds, the crystal was a Si(111) crystal, whereas a Si(100) crystal with an oxide thickness at the crystal surface of 1.8-2.0 nm was used to form a semicarbazide layer through the use of a trichlorosilane isocyanate derivative. Atomic Force Microscopy (AFM). The experiments were performed on a commercial optical deflection microscope (standalone configuration for a large sample, Dimension 3100 with a Nanoscope IIIa, Veeco) operated in ambient conditions. Measurements were performed in the tapping mode, and commercial silicon cantilever probes with a nominal radius of 5-10 nm and a spring

ChemoselectiVe Ligation of Peptides to Silicon

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Scheme 1. Synthesis of Glyoxylyl Peptide 6

Scheme 2. Synthesis of Peptide 8

constant in the range of 20-70 N/m were used. Calibration of the gray scale was obtained from the step height between the monatomic terraces observed in the hydrogen-terminated Si(111) surface. Characterization of the Modified Surfaces by XPS. For XPS measurements, we used a monochromatic Al KR X-ray source and an analyzer pass energy of 12 eV. Under these conditions, the overall resolution as measured from the full width at half-maximum of the Ag 3d5/2 line was 0.55 eV. The binding energy scale was calibrated using the Au 4f7/2 line at 84.0 eV. The acceptance angle of the analyzer was set to 14°, and the angle between the incident X-rays and the analyzer was 90°. The detection angle of the photoelectrons was 25°, as referenced to the sample surface. The intensity of the various XPS core levels (CLs) were measured as the peak area after standard background subtraction, according to the Shirley procedure. For the CL decomposition, we used Voigt functions and a leastsquares minimization procedure. The different components were modeled with the same parameters, that is, the Gaussian and Lorentzian broadenings were kept fixed for each component of a given CL. Ratios between different chemical species are calculated by taking into account the relative intensities for Si, N, O, C, and Co, which are 0.27, 0.48, 0.71, 0.30, and 3.25, respectively.18

Results and Discussion Synthesis of the Peptides Labeled with Co2(CO)6. Peptides 6 and 8 (Schemes 1 and 2) were designed to document the site(18) Moulder, J. F.; Stickle, W. F.; Sobol, P. E. Bomben, K. D. Handbook of X-ray Photoelectron Spectroscopy; Perkin-Elmer Corporation, Physical Electronics Division: Eden Prairie, MN, 1992.

specific ligation of glyoxylyl peptides with the prepared semicarbazide surfaces. The glyoxylyl or amide groups were placed at the C-terminus of peptides 6 or 8, respectively. The side-chain of the N-terminal Lys residue of both peptides was derivatized with an activated ester incorporating an acetylenic moiety. The acetylenic moiety served to install a cobalt hexacarbonyl complex through the reaction with Co2(CO)8. Compounds 1 and 2 were synthesized using published procedures.16 Since the Co2(CO)6 complex is not stable in the conditions used for the solid-phase peptide synthesis, the Co2(CO)6 label was introduced in solution on a fully deprotected peptide. The starting material for the synthesis of peptide 6 is peptidyl resin 3, which was prepared using published solid-phase methods. Rapidly, the peptide chain was assembled on a Novasyn TG resin using standard Fmoc/tert-butyl chemistry19 and an isopropylidene tartrate linker (Scheme 1).20-22 The peptide chain and the diol moiety of the tartaramide linker were deprotected using TFA to give peptidyl resin 3. The synthesis of peptide 6 was envisaged using two different paths (Scheme 1). In the first approach, the  amino group of the (19) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161. (20) Fruchart, J.-S.; Gras-Masse, H.; Melnyk, O. Tetrahedron Lett. 1999, 40, 6225. (21) Melnyk, O.; Fruchart, J.-S.; Grandjean, C.; Gras-Masse, H. J. Org. Chem. 2001, 66, 4153-4160. (22) Urbe`s, F.; Fruchart, J.-S.; Gras-Masse, H.; Melnyk, O. Lett. Pept. Sci. 2002, 8, 253.

7062 Langmuir, Vol. 22, No. 16, 2006 Scheme 3. Preparation of Semicarbazide-Functionalized Si(111) Wafers

N-terminal Lys residue was derivatized on the solid-phase with compound 1 to give peptide 4 after oxidative cleavage of the tartaramide diol with periodate (overall yield: 32.6%). Complexation of the acetylenic group by Co2(CO)8 was attempted in DMF solution, and indeed peptide 6 could be isolated with an 18.5% overall yield starting from peptide 4 and following RPHPLC purification.23 However, this strategy led to a low yield. Indeed, the decomposition of Co2(CO)8 during the reaction required the use of a large excess of reagent, making difficult the separation of the target peptide 6 from the Co2(CO)8 byproducts. As a result, path 2 was investigated. This alternative strategy was based on the use of activated ester 2 already incorporating the Co2(CO)6 complex.14,16 Peptidyl resin 3 was subjected to oxidative cleavage in the presence of periodate to give glyoxylyl peptide 5 (51% overall yield). Reaction of the  amino group of the N-terminal Lys residue with 2 led to the formation of peptide 6 (51% yield following RP-HPLC purification). Thus, this second strategy based on the coupling in solution of a preformed Co2(CO)6 complex led to better yields. This procedure was thus also used for the synthesis of control peptide 8 using peptide 7 as the starting material (Scheme 2, 62% yield following RP-HPLC purification). Peptides 6 and 8 were obtained as brown powders following lyophilization and were characterized by IR (strong absorption bands at 2092, 2050, and 2019 cm-1, typical for the Co2(CO)6 complex), ES-MS, RP-HPLC, and capillary zone electrophoresis. Site-Specific Immobilization Studies. Sa-Boc and Sb-Boc surfaces were prepared following our recently described methods.11,15 Briefly, the Sa-Boc semicarbazide-modified silicon surface is obtained by the reaction of Si(111)-H surfaces with olefin-terminated semicarbazide molecules under UV light (Scheme 3), whereas the Sb-Boc surface is achieved by the silanization of an oxidized Si(100) surface with a trichlorosilane isocyanate derivative and its subsequent reaction with a protected hydrazine compound (Scheme 4). In both cases, since the semicarbazide group might react to the surfaces, it was protected to avoid any side reactions. The deprotection by an acid treatment was successfully monitored by XPS and FTIR experiments.11,15 As a further means of investigation, we measured the variation of the layer thickness before and after the removal of the Boc group for the Sb surface by ellipsometry. The thickness was found to decrease by 3 Å (Table 1), in agreement with the deprotection of the semicarbazide group.24 As a result of the deprotection, the Sa and Sb surfaces are terminated by semicarbazide groups. These semicarbazide groups are known to react chemoselectively with glyoxylyl peptides (Scheme 5).25,26 Since the chemical modification of a Si(111) (23) Le Borgne, F.; Beaucourt J. P. Tetrahedron Lett. 1998, 29, 5649. (24) Bo¨cking, T.; James, M.; Coster, H. G. L.; Chilcott, T. C.; Barrow, K. D.; Langmuir 2004, 20, 9227. (25) Melnyk, O.; Duburcq, X.; Olivier, C.; Urbe`s, F.; Auriault, C.; GrasMasse, H. Bioconjugate Chem. 2002, 13, 713.

OliVier et al. Scheme 4. Preparation of Semicarbazide-Functionalized Si(100) Wafersa

a (i) Cl-CHdCCl , 5 °C; (ii) Boc-NH-NH , Cl-CHdCCl ; 2 2 2 (iii) TFA, CH2Cl2.

Table 1. Ellipsometric Measurements surface

thickness (Å)

Sb-Boc Sb Sb-peptide 6

18 15 26

Scheme 5. Reaction of Peptide 6 with Sa and Sb Surfaces

surface by a semicarbazide alkyl groups does not alter the initial morphology of the surface, the immobilization of peptide 6 was first investigated by AFM (Figure 1). An AFM image of the Sa surface (Figure 1a) shows a succession of flat terraces separated by monatomic steps as well as a few triangular pits, consistent with the morphology of hydrogen-terminated Si(111) surfaces etched in basic fluoride solutions.27 After the reaction of the Sa surface with peptide 6 and the subsequent washing of the surface, its roughness changes significantly (Figure 1b). The step edges are not visible any more, and the surface exhibits a grain-like structure, typical of a surface fully covered by peptides.11 Scanning the Sa-peptide 6 surface on a larger scale shows the triangular stacking pits generally observed for the chemically prepared Si(111)-H surfaces (Figure 1c), thus indicating that the peptide layer is quite thin and that a physisorbed overlayer of peptides is unlikely. Such observations suggest that the semicarbazide groups of the Sa surface have reacted with the glyoxylyl groups of the peptides, thus allowing the covalent immobilization of peptide 6 onto the Sa surface. Characterizing the Sb surface after its incubation with peptide 6 by ellipsometric measurements yields an increase in the organic layer thickness of 11 Å (Table 1). Like surface Sa, such a small thickness suggests the formation of a sub- or monolayer of peptide 6 linked to the Sb surface, which precludes the adsorption of further peptides on top of this monolayer. (26) Duburcq, X.; Olivier, C.; Desmet, R.; Halasa, M.; Carion, O.; Grandidier, B.; Heim, T.; Stie´venard, D.; Auriault, C.; Melnyk, O. Bioconjugate Chem. 2004, 15, 317. (27) Flidr, J.; Huang, Y. C.; Hines, M. A. J. Phys. Chem. 1999, 108, 5542.

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Figure 2. ATR-FTIR spectra obtained for (a) the Sa surface terminated with either Boc, peptide 6, or peptide 8 and (b) the Sb surface terminated with either Boc, peptide 6, or peptide 8. Table 2. Integrated Peak Areas of the Cobaltcarbonyl Bands Measured by FTIR for peptide 6 and Peptide 8 Adsorbed to Both Sa and Sb Surfaces peptide 6

Figure 1. Tapping-mode AFM image showing (a) the semicarbazidemodified Si(111) Sa surface and (b,c) the same surface after the immobilization of peptide 6. Grey scale ranges are (a) 7.8 nm, (b) 18.3 nm, and (c) 33 nm.

To demonstrate the covalent ligation of peptide 6 to the semicarbazide-modified surfaces, the Sa,b surfaces were also characterized by FTIR ATR analysis after their incubation with peptide 6. Three vibration bands at 2020, 2050, and 2090 cm-1, which were observed for neither the Sa-Boc and Sb-Boc surfaces nor the Sa and Sb surfaces, are clearly resolved in Figure 2a. These bands are characteristic of the Co2CO6 groups28 and consistent with the bands of vibration observed for peptide 6 (see Materials and Methods). Abundant washings with water or K2HPO4 did not lead to a significant decrease in the band intensity, thus indicating a strong binding of peptide 6 to the surfaces. While the vibration bands in the region of the NH and CO groups are not visible, since the experiments were carried out in water, it is interesting to note the intensity of the peaks for the Co2(CO)6 (28) Yam, C. M.; Pradier, C. M.; Salmain, M.; Fisher-Durand, N.; Jaouen G. J. Colloid Interface Sci. 2002, 245, 204.

surface

in solution (A‚cm-1)

after washing (A‚cm-1)

Sa Sa-Boc Sb

1.51 1.72 1.36

0.96 1.00 1.12

peptide 8 ratio (%)

in solution (A‚cm-1)

after washing (A‚cm-1)

ratio (%)

64 58 82

0.53 1.87 0.35

0.06 0.12 0.02

11 6 5

groups in comparison with the peaks observed in the C-H stretching region. Such a result reveals the high sensitivity of the FTIR technique to the Co2(CO)6 moiety when a concentration on the order of one monolayer of peptides is bound to the Sa,b surfaces. Taking advantage of the sensitivity of the FTIR experiments to the Co2(CO)6 moiety, we investigated further the ability of the Sa,b surfaces to immobilize peptides derivatized with two different groups. As shown in Table 2, by comparing the integrated peak areas before and after washing the surfaces incubated with peptide 6, we find ratios of 64% for the Sa surface and 82% for the Sb surface. In contrast to the immobilization of peptide 6, when the semicarbazide-terminated Sa or Sb surfaces were reacted with the control peptide 8, it was difficult to resolve the three vibration bands of the cobalt carbonyl complex (Figure 2). The comparison of the integrated peak areas before and after washing

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Figure 4. XPS spectra of (a) Si(2p), (b) O(1s), (c) C(1s), and (d) N(1s) CLs from the Sa surface. Table 3. Normalized Intensities of the C1s, N1s, O1s, Si2p, and Co2p Peaks

Figure 3. XPS survey of the (a) Sa, (b) Sa-peptide 6 and (c) Sa-peptide 8 surfaces.

the surfaces incubated with peptide 8 yields ratios of 11% and 5% for the Sa and Sb surfaces, respectively (Table 2). Since the chemical structures of peptide 6 and 8 differ only by their reactive group, we can assume that the concentrations of peptides 6 and 8 involved in the absorption of the infrared signal within the same sampling volume are similar at the end of the incubation process for both the Sa and Sb surfaces. As a result, the ratios obtained for the immobilization of peptide 6 and peptide 8 can be compared. For both the Sa and Sb surfaces, we find that the concentration of adsorbed peptides is higher for peptide 6 than for peptide 8. Since peptide 8 is not functionalized with the

surfaces

C1s

N1s

O1s

Si2p

Co2p

Sa Sa-peptide 8 Sa-peptide 6

4793 13150 24753

1258 3017 6406

11000 9839 8748

28752 21681 12848

0 150 761

glyoxylyl moiety, this result demonstrates the site-specific ligation of peptide 6 to the semicarbazide surface. Table 2 also shows that the Sa-Boc surface was able to immobilize peptides 6 and 8. But the concentration of adsorbed peptides is lower than the ones obtained for the fully deprotected surfaces. This result suggests that a covalent linkage between the aldehyde group of peptide 6 and the protected semicarbazide groups can be formed, in agreement with previous studies, where aminal derivatives could be formed in solution by the reaction of aldehydes with N,N′-diacylhydrazines.29,30 In addition to the FTIR experiments, we performed an XPS analysis of the surfaces. Figure 3 showed the XPS surveys of Sa surfaces incubated with peptides 6 and 8. In Figure 3b, the immobilization of peptide 6 on the Sa surface is clearly demonstrated with the observation of strong peaks with binding energies in the range of 770-810 eV, related to Co species present on the surface.31,32 In contrast, the incubation of the Sa surface with peptide 8 yields a survey, where much smaller peaks are visible in the Co(2p3/2) and the Co(2p1/2) regions (Figure 3c). The Co(2p) intensity after incubation of the Sa surface with peptide 6 is 5 times those obtained for peptide 8 (Table 3). Such a result confirms the FTIR experiments, where no covalent immobilization of peptide 8 is expected because of the lack of the glyoxylyl moiety on peptide 8. More quantitative information can be obtained from analysis of the peak areas and intensities in the XPS spectra. Figure 4 shows the XPS spectra of the Sa surface, corresponding to the regions of the silicon (2p), oxygen (1s), carbon (1s), and nitrogen (29) Wuensch, B.; Nerdinger, S.; Hoefner, G. Liebigs Ann. Org. Biorg. Chem. 1995, 7, 1303. (30) Gadzhiev, G. Y.; Budagov, V. A.; Dzhalilov, E. Y. J. Org. Chem. USSR 1977, 13, 1445. (31) Klebanoff, L. E.; Van Campen, D. G.; Pouliot R. J. Phys. ReV. B 1994, 49, 2047. (32) Wu, N.; Fu, L.; Su, M.; Aslam, M.; Chun Wong, K.; Dravid, V. P. Nano Lett. 2004, 4, 383.

ChemoselectiVe Ligation of Peptides to Silicon

Figure 5. XPS spectra of C(1s) and Co(2p) CLs measured after the immobilization of (a,b) peptide 6 and (c,d) peptide 8 on the Sa surface.

(1s) species. These spectra are consistent with the ones discussed in ref 11. In the Si(2p) spectrum (Figure 4a), the appearance of a broad peak at the binding energy of 103.4 eV indicates that the acid treatment induced a partial oxidation of the silicon surface with respect to the protected semicarbazide Sa-Boc surface, with the total area in the 102-104 eV range being approximately 0.16 times the total Si(2p) area. Analysis of the C(1s) region for the Sa surface (Figure 4c) yields four peaks for the C(1s) spectrum at energies of 284.3, 285.5, 286.3, and 289.1 eV. These peaks are assigned to the Si-C*, C-C*, C*-N, and N-C*(dO)-N carbon atoms, respectively.11 After the incubation of the surface Sa with peptide 6, the C(1s) peak intensity increased (Figure 5a). Ligation of peptide 6 to the Sa surface increases the number of carbon atoms from 11 to 55 in the chain linked to the Si surface (Scheme 5). Comparing the C(1s) peak intensities for the Sa-peptide 6 and the Sa surfaces gives a factor of 4.1, in agreement with an increase by a factor of 44/11 for the total number of carbon atoms per immobilized peptides. Taking into account the ratio of the different moieties consisting of carbon atoms in peptides 6 and neglecting the contribution of moieties with a small occurrence in the peptide, the C(1s) spectrum can then be fitted with four peaks. The comparison of the N(1s) peak intensities shows a similar relative increase by a factor of 4 between the Sa-peptide 6 and the Sa surfaces. This value is again close to the theoretical factor of 3.7, since, after the linkage of peptide 6, the number of N atoms is 11 instead of 3 per chain linked to Si surface. As a result, the increased concentration of C and N species is directly related to an increased concentration of peptides on the Sa surface. To get a more accurate estimation of the peptide concentration, we then

Langmuir, Vol. 22, No. 16, 2006 7065

have to consider the peak intensity ratio between the Co and C species. A value of 0.038 is found, which is a little smaller than the expected value of 0.045, presumably due to hydrocarbon contaminants, which are physically adsorbed on the Sa surface during the incubation process. Similarly to what has been done for peptide 6, by measuring the C(1s) and N(1s) peak intensities in the case of peptide 8, we find the intensity ratios to be 1.7 and 1.4 between the Sapeptide 8 and the Sa surfaces for the C(1s) and N(1s) components, respectively. These values are smaller than the theoretical values of 3.5 and 3.3, respectively, which are expected for a full coverage of the surface by the peptide. To determine the quantity of carbon species directly related to peptide 8, we can then compare the peak intensity ratio between the Co and C species. A ratio of 0.018 instead of 0.05 for two Co atoms per peptide 8 was obtained, indicating that a large amount of hydrocarbon contaminants are physically adsorbed on the Sa surface. On the basis of these ratios, we can crudely estimate that the amount of peptide 6 immobilized on the Sa surface is 5 times higher than the amount of peptides 8, a value consistent with the value of 5.8 obtained from the FTIR experiments. These results clearly show a higher coverage of peptides for the Sa surface incubated with peptide 6 in comparison with the Sa surface incubated with peptide 8, demonstrating again the selective reaction of the semicarbazide group with the glyoxylyl moiety of peptide 6.

Conclusion Two peptides labeled with a single Co2(CO)6 moiety were synthesized and used to study the site-specific ligation of a glyoxylyl peptide to semicarbazide-functionalized silicon surfaces. This study shows that both the FTIR and XPS techniques are sensitive to the Co2(CO)6 group for submonolayer coverages of peptides adsorbed on a silicon surface and that small coverages are already distinguishable when the measurements are performed in situ with the FTIR technique. Such sensitivity permits the comparison of the amount of immobilized glyoxylyl and control peptides on functionalized silicon surfaces, highlighting the sitespecific immobilization of glyoxylyl peptides through the R-oxo semicarbazone ligation. As fluorescent detection methods do not allow such a direct comparison, because silicon quenches the fluorescence signal emitted by the dye molecules, the use of metal transition probes seems to be well suited to investigate the site-specific anchoring of biomolecules to silicon. Our results suggest that this type of label could offer a good alternative to address the issue of the reactivity of solid supports. Acknowledgment. This work was partly supported by the French Ministry of Research and grants from the EEC and the Region Nord Pas-de-Calais. We also used the microarray platform facilities of the Institute of Biology (Lille, France). LA060370M