Direct Characterization of Native Chemical Ligation of Peptides on

Article pubs.acs.org/Langmuir

Direct Characterization of Native Chemical Ligation of Peptides on Silicon Nanowires Nabil Dendane,† Oleg Melnyk,† Tao Xu,‡,§ Bruno Grandidier,‡ Rabah Boukherroub,∥ Didier Stiévenard,‡ and Yannick Coffinier*,∥ †

IBL, UMR CNRS 8161, 1 rue du professeur Calmette, Lille, France IEMN, UMR CNRS 8520, Université Lille 1, Cité Scientifique, Avenue Poincaré − BP 60069, 59652 Villeneuve d’Ascq, France § Key Laboratory of Advanced Display and System Application, Shanghai University, 149 Yanchang Road, Shanghai 200072, PR China ∥ Institut de Recherche Interdisciplinaire (IRI), USR CNRS 3078, Université Lille 1, Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq, France ‡

S Supporting Information *

ABSTRACT: We describe the site-specific and chemoselective immobilization of peptides on hydrogen-terminated silicon nanowires (SiNWs) using native chemical ligation (NCL) (i.e., the reaction of a thioester group with a cysteine moiety to give a stable amide bond). The SiNWs investigated in this work were grown via a vapor−liquid−solid mechanism and functionalized with a thioester moiety. The immobilization of the peptides on the SiNWs was demonstrated by synthesizing peptides with an Nterminal cysteine residue and labeled with tetramethylrhodamine or trifluoromethyl groups that were detected by fluorescence and X-ray photoelectron spectroscopy, respectively. The peptides labeled with tetramethylrhodamine or trifluoromethyl groups for fluorescence or X-ray photoelectron spectroscopy (XPS) detection studies were synthesized with an N-terminal cysteine residue. N-Terminal seryl peptides and carboxy-terminated SiNWs were used as controls to demonstrate the chemoselectivity of the peptide immobilization.

I. INTRODUCTION The integration of nanotechnology with biology has received increasing attention in recent years1 largely because of the desire to use biomolecular recognition to aid nanoscale assembly2−7 or to create sensitive biosensors based on nanoscale devices.8,9 Silicon nanowires (SiNWs) are particularly attractive because of their high surface to volume ratio and the semiconducting properties of silicon. For example, several studies have shown that the presence of charged biological molecules such as DNA near the surface of SiNWs induces a field effect9−11 similar to that observed on planar silicon surfaces.12 This field effect, which can be detected by monitoring the electronic properties of the nanowires, is typically exploited for designing biosensors. However, to use silicon nanowires for applications such as biosensing, it is necessary to have well-defined chemical attachment schemes that will provide the desired biomolecular recognition proper© 2012 American Chemical Society

ties, chemical stability, and good interfacial electrical properties. In this context, the development of methods allowing the controlled immobilization of biomolecules on SiNWs is an important field of research and, not surprisingly, a subject of considerable interest.9−11 Usually, the immobilization of biomolecules on surfaces first requires the functionalization of the solid substrate with an appropriate functional layer. The surface chemistries of silicon surfaces are well known and fully described in the literature. The surface functionalization of silicon surface can be achieved in two major ways: silanization or hydrosilylation. The silanization reaction involves linking molecules through the intermediate oxide sheath that typically surrounds air-exposed Received: July 26, 2012 Revised: August 27, 2012 Published: August 29, 2012 13336

dx.doi.org/10.1021/la3030217 | Langmuir 2012, 28, 13336−13344

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Figure 1. NCL between a free cysteine residue and a thioester group in which a transthioesterification step is followed by a rapid intramolecular S,Nacyl shift resulting in the formation of a stable amide bond.

methods such as oxime 32−35 or α-oxo semicarbazone ligations,36−39 the Staudinger ligation,40,41 the Diels−Alder reaction,42 “click” chemistry,43 and native chemical ligation (NCL).44−48 NCL (Figure 1), which is the reaction between a free cysteine residue and a thioester group, proceeds through a transthioesterification step and the formation of a transient thioester-linked intermediate. This reaction is followed by a rapid, spontaneous intramolecular S,N-acyl shift resulting in the formation of an amide bond. The formation of the thioesterlinked intermediate is reversible, but the subsequent rearrangement to the amide is irreversible, thereby allowing the displacement of the equilibrium toward the target amide bond ligated product.49 NCL has several features that make it uniquely attractive as a potential methodology for grafting molecules to surfaces.49−51 Indeed, NCL generates a stable amide bond in water under physiological conditions52 and is chemo- and regioselective, which are properties that have been extensively used in solution to connect small peptide segments to generate larger polypeptides.53,54 Moreover, free cysteine residue can easily be incorporated into peptides or other nonpeptidic biomolecules using solution- or solid-phase synthesis methods, whereas N-terminal Cys proteins can be produced using recombinant techniques. However, to date only a few examples of NCL reactions performed on surfaces have been described in the literature. Lesaicherre and co-workers44 reported the immobilization of a cysteine-fluorescein conjugate onto a thioester-functionalized glass slide surface. More recently, Anderson has used different thioester silylchlorides (S-phenyl-11-(chlorodimethylsilyl)-undecanethiolate and the benzyl analogue) to introduce the thioester group onto a glass slide and followed the NCL reaction with a cysteine-lissamine derivative by UV/vis absorption spectroscopy.55 The cysteine-lissamine derivative was used to interrogate these surfaces and to provide good evidence for fast, controllable NCL on the surface. Camarero and co-workers reported the inverse procedure in which a fluorescent thioester protein conjugate was immobilized on a cysteine-functionalized surface.47 More recently, Helms and coworkers have immobilized polypeptides on surface plasmon resonance (SPR) chips using NCL and used the SPR response to monitor both the immobilization of the probe and its binding to a model antibody.52 More seldom is the use of NCL on nanostructured surfaces. To the best of our knowledge, only

silicon,10,11 with the molecule being anchored via siloxane bonds. This method has been used previously to provide colloidal nanoparticles12 and Si nanowires10,11,13 with biomolecular recognition capability. The hydrosilylation reaction consists of the use of organic molecular layers bearing unsaturated CC or CC bonds (alkene or alkyne) that can be linked directly to hydrogen-terminated silicon under photo, thermal, or peroxide activation, leading to the formation of strong Si−C bonds without an intervening oxide. The method yields surfaces with improved stability and higher modification reproducibility.14−22 Both strategies for flat silicon surface modification can be easily transferred to silicon nanostructures as shown by Streifer et al.23 Indeed, they used hydrosilylation under photochemical conditions to immobilize DNA covalently on hydrogenated SiNWs in order to achieve biomolecular recognition. Another important parameter is the chemical nature of the bonding between the biomolecules and the sensing substrate. The noncovalent strategy allows to immobilize proteins or other biomolecules on surfaces reversibly in an oriented manner while maintaining their function for investigating receptor−ligand interactions. For example, Schmid et al. reported the immobilization of polyhistidine-tagged proteins by using a metal ion affinity chromatography (IMAC) technique.24,25 Alternately, the covalent immobilization strategy often consists of the formation of a stable amide bond between the biomolecules and the sensing substrate. Amide-bondforming reactions are often based on the use of surfaces presenting active esters, such as N-hydroxysuccinimide ester, which react efficiently with nucleophilic primary amino groups, typically α or ε amino groups within proteins.26 However, this chemistry leads to the random immobilization of peptides that usually present several primary amino groups and also other competing nucleophiles such as thiols or hydroxyl groups.27 Other non-site-specific immobilization strategies exist such as the reaction of surface-bound silyl chlorides and alcohols,28 the reaction between diazobenzylidene-functionalized glass and heteroatoms with acidic protons,29 and/or the Michael reaction between a maleimide-functionalized surface and thiols.30 But in all of these cases, the active sites of a substantial population of immobilized peptide molecules are not accessible to targets in the solution phase.31 For this and to ensure a specific orientation on the surface, various covalent chemoselective and site-specific immobilization strategies have been developed. Among them, we can mention Schiff-base site-specific ligation 13337


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Figure 2. Chemical structure of the four synthesized peptides bearing either tetramethylrhodamine or −CF3 moieties. resistivity) were used as substrates. The surface was first degreased in acetone and 2-propanol, rinsed with Milli-Q water, and then cleaned in a piranha solution (3/1 concentrated H2SO4/30% H2O2) for 15 min at 80 °C followed by copious rinsing with Milli-Q water. A thin layer of 4 nm of gold was thermally evaporated on the Si clean surface, and then the metallized substrate was placed in a quartz tube, which was heated in a tube furnace at 500 °C. Gold nanoparticles with a broad size distribution were obtained as a result of metal dewetting on the surface. Exposing the gold-coated surface to silane gas led to the growth of SiNWs. Safety Considerations. The H2SO4/H2O2 mixture (piranha solution) is a strong oxidant. It reacts violently with organic materials and can cause severe skin burns. It must be handled with extreme care in a well-ventilated fume hood while wearing appropriate chemical safety protection. HF is a hazardous acid that can result in serious tissue damage if burns are not appropriately treated. Silicon etching should be performed in a well-ventilated fume hood with appropriate safety considerations: face shield and double-layered nitrile gloves. SiNW Characterization. Scanning Electron Microscopy (SEM). SEM images were obtained using an electron microscope ULTRA 55 (Zeiss) equipped with a thermal field emission emitter and a highefficiency In-lens SE and energy-selective backscattering (EsB) detectors. Scanning Transmission Electron Microscopy (STEM) and HighAngle Annular Dark Field (HAADF). The scanning transmission electron microscope (STEM) used in this study is a Jeol 2200FS microscope equipped with a spherical aberration (Cs) corrector for probe formation. The convergence half angle of the electron beam was 30 mrad with a current probe of about 140 pA. The inner and outer half angles of detection for the upper annular dark field detector (uADF) were 100 and 170 mrad, respectively (as is, we obtained highangle annular dark field (HAADF) images under so-called “Z contrast” conditions). Chemical Functionalization of SiNWs and Peptide Immobilization via NCL. Carboxylic Acid Termination. The SiNW interfaces were first immersed in 50% HF for 2 to 3 min, resulting in hydrogenterminated surfaces. Then, the hydrogenated interfaces were placed in a Schlenk tube containing previously deoxygenated neat undecylenic acid (UA) and then held at 150 °C for 6 h. This thermal reaction was carried out under nitrogen gas bubbling to minimize the oxidation of the Si−H bonds. Then, the reaction was stopped by washing the surfaces twice in dichloromethane and twice in ethanol (5 min each)

Wojtyk and co-workers have shown the preparation of thioethyl ester-functionalized porous silicon (pSi) and the subsequent reaction with cysteine. All chemical modification steps were followed by diffuse reflectance Fourier transform infrared spectroscopy (FT-IR).48 Here, we report the site-specific immobilization of model Cys peptides on thioester-derivatized SiNWs using NCL. First, we present an efficient method for installing benzyl thioester groups on hydrogenated SiNWs using the hydrosilylation strategy. NCL immobilization was studied using model cysteine (Cys) peptides presenting either fluorescent tetramethylrhodamine (R) or trifluoromethyl (F) moieties. Tetramethylrhodamine-labeled peptides allowed the detection of immobilized peptides using fluorescence spectroscopy, whereas the successful capture of fluorinated peptides was probed with Xray photoelectron spectroscopy (XPS) measurements. Serine (Ser) peptides were used as controls for probing the chemoselectivity of the immobilization process. Finally, we have examined the importance of thiol−thioester exchange occurring on the surface in the reactivity of the thioesterfunctionalized SiNWs.

II. MATERIALS AND METHODS Reagents. All cleaning and etching reagents were VLSI grade. Acetic acid, sulfuric acid (96%), hydrofluoric acid (50%), and hydrogen peroxide (30%) were purchased from Carlo Erba. Nitric acid (65%) and hydrochloric acid (37%) were purchased from Merck. All other chemicals were reagent grade or higher and were used as received unless otherwise specified. Sodium hydroxide (NaOH), acetone, isopropyl alcohol (iPrOH), dichloromethane (DCM), undecylenic acid (UA), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCCI), triethylamine, dimethylsulfoxide (DMSO), dimethylformamide (DMF), benzyl mercaptan (BM), tris(2carboxyethyl)phosphine (TCEP) hydrochloride, 4-mercaptophenylacetic acid (MPAA), 4-dimethylaminopyridine (DMAP), and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich. Surface Preparation. Silicon Nanowire Growth via a Vapor− Liquid−Solid (VLS) Mechanism. Single-side-polished silicon (100)oriented p-type wafers (Siltronix) (boron-doped, 0.001−0.0009 Ω cm 13338


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under stirring. Because the formation of multilayers of UA can occur as a result of van der Waals forces and hydrogen bonding, the surfaces were immersed in boiling acetic acid for 5 min in order to obtain a carboxylic acid monolayer on the surface. The resulting surfaces were rinsed twice with deionized water to remove traces of acetic acid. This step was followed by an ethanol rinsing step under stirring, and the surfaces were finally dried under a gentle stream of nitrogen. Benzyl Thioester Formation on SiNWs. Benzyl mercaptan (70 μL) was added dropwise to a solution of DMAP (8.2 mg)/DCC (1.28 g) in DMSO (10 mL). The SiNW substrate was immersed overnight in this solution at room temperature. Then, the surface was rinsed twice with DMSO (5 min) under orbital stirring followed by two baths of dichloromethane (5 min) and ethanol (5 min) and finally dried under a gentle stream of nitrogen. Peptide Synthesis. Figure 2 details the structure of the four peptides used in this study. Peptides Cys-pep-R and Ser-pep-R feature a tetramethylrhodamine dye on an internal lysine side chain for fluorescence studies. Peptides Cys-pep-F and Ser-pep-F feature a 4trifluorophenyl acetyl group on the lysine side chain to enable XPS detection. The protocols used to synthesize these peptides are reported in detail in the Supporting Information. Immobilization of N-Cysteine Peptide. We report herein the chemoselective and regioselective reaction of Cys-pep-R or Cys-pep-F peptides on thioester-terminated SiNWs in the presence of 4mercaptophenylacetic acid (MPAA) and tris(2-carboxyethyl)phosphine (TCEP). This reaction takes place in either one or two steps in aqueous buffer at pH 7 and leads to the formation of a native peptide bond in good yield. Two-Step Procedure. Step 1 consisted of the incubation of thioester-terminated SiNWs in PBS buffer (200 mM) with 80 mM MPAA, 20 mM TCEP, and 0.1% of Tween 20, with a final pH of 7.6 overnight at 37 °C. After the reaction, the SiNW surface was rinsed with PBS (200 mM) and 0.1% Tween 20 (pH 7) for 20 min. Then, a second rinsing step was performed by using deionized water/DMF 9/ 1 v/v for 5 min, followed by rinsing with ethanol. The surface was finally dried with a gentle stream of nitrogen. Step 2 consisted of the immobilization of Cys-pep-R or Cys-pep-F peptides using NCL. For that, the activated SiNW surface was immersed in a solution containing either Cys-pep-R or Cys-pep-F at 3 mM in PBS (200 mM) with 80 mM MPAA, 20 mM TCEP, and 0.1% Tween 20 (pH 7.6) for 3 h at 37 °C. After the reaction, the surface was rinsed with PBS (200 mM) and 0.1% Tween 20 (pH 7) for 1 h. Then, a second rinsing step was performed by using deionized water/DMF 9/1 v/v for 20 min, followed by rinsing with ethanol (10 min). The surface was finally dried with a gentle stream of nitrogen. One-Step Procedure. The one-step strategy consisted of the immobilization of peptides Cys-pep-R and Cys-pep-F following step 2 of the protocol described above (i.e., the incubation of thioesterterminated SiNWs in a solution containing either Cys-pep-R or Cyspep-F at 3 mM in PBS (200 mM) with 80 mM MPAA, 20 mM TCEP, and 0.1% Tween 20 (pH 7.6) for 3 h at 37 °C. After the reaction, the surface was rinsed with PBS (200 mM) and 0.1% Tween 20 (pH 7) for 1 h. Then, a second rinsing step was performed by using deionized water/DMF 9/1 v/v for 20 min followed by rinsing with ethanol (10 min). The surface was finally dried with a gentle stream of nitrogen. Surface Characterization. X-ray Photoelectron Spectroscopy (XPS). XPS measurements were performed with an ESCALAB 220 XL spectrometer from Vacuum Generators. A monochromatic Al Kα Xray source (1486.6 eV) was operated in constant analyzer energy (CAE) mode (CAE = 100 eV for survey spectra and CAE = 40 eV for high-resolution spectra) using the electromagnetic lens mode. The angle between the incident X-rays and the analyzer is 58°. The detection angle of the photoelectrons is 90° with respect to the sample surface. Fluorescence Measurements. The surfaces were scanned using the Cy3-channel of an Affymetrix 418 array scanner at a resolution of 10 μm, and the fluorescence was quantified using Scan Array Express software (Perkin-Elmer).

III. RESULTS AND DISCUSSION Preparation of SiNWs. The SiNW substrates used in this study were prepared using the VLS growth mechanism as described in our previous work.56−59 The fundamental process is based on the chemical decomposition of silane gas (SiH4) catalyzed by gold nanoparticles at high temperatures (440−540 °C). In this process, the diameter of the nanowires is determined by the diameter of the catalyst particles; therefore, the method provides an efficient way to obtain uniformly sized nanowires. The gold nanoparticles were obtained by the thermal evaporation of a 4-nm-thick thin film of gold on a clean Si(100) substrate. The samples were placed in a chemical vapor deposition (CVD) furnace within a quartz tube. Gold nanoparticles with a large size distribution were obtained as a result of metal dewetting at high temperatures on the surface prior to SiH4 gas injection. The SiNW width, length, and morphology obtained in this way depend on the duration, the pressure, and the temperature of the process as well as on the crystal orientation of the silicon substrate. The orientation is also influenced by the pressure prescribed during the VLS process.60 The orientation of our nanowires varies from about 30 to 90° with respect to the horizontal. The larger the pressure, the straighter the SiNWs grow (also with a narrower orientation distribution). In this study, the silicon nanowire growth was achieved using the following parameters: gas flow, 40 sccm of silane (SiH4); P = 0.532 mbar; T = 500 °C; and t = 20 min. Figure 3 shows an

Figure 3. SEM image of SiNWs grown on a Si substrate under the following conditions: 40 sccm of silane (SiH4); P = 0.532 mbar; T = 500 °C; and t = 20 min. (Inset) High-magnification SEM image of the top of the SiNWs showing the gold seed particles.

example of the silicon nanowires grown under these conditions. The diameter of the SiNWs is in the range of 50−100 nm with a length of ∼12 μm. The SiNW morphology comprises a mixture of short entangled SiNWs and straight SiNWs. Some of them even have a “hook” shape on their top end. The wide distribution of the nanowire size is related to the size of the gold nanoparticle catalyst formed during the dewetting of the Au film on the silicon at high temperatures. The gold catalysts can be easily seen in the inset of Figure 3 (bright part at the top of the wires). This behavior is common for Au nanoparticles prepared by a physical methodology, consisting of thermal evaporation of a thin Au film and its subsequent annealing. 13339


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SiNW Functionalization. SiNWs were next chemically modified to allow the immobilization of model Cys peptides on their surfaces using NCL. The support used for the initial growth process was considered to be a convenient way to functionalize, handle, and characterize the SiNWs, which can then be released into solution with well-characterized properties for further applications. Here, all chemical steps and subsequent characterizations were achieved on as-grown SiNWs (i.e., SiNWs on Si substrates). First, the direct reaction of the hydrogen-terminated SiNW surface with undecylenic acid (UA) under thermal conditions led to the formation of an organic layer covalently attached to the surface through Si−C bonds.61−65 Then, the carboxylic acid terminal group was converted to a benzylthiol ester group, allowing the immobilization of model Cys peptides using NCL (Figure 4). For this, carboxylic acid groups were activated with

increased after benzylmercaptan immobilization because of the additional benzyl moiety. From the XPS analysis, we can conclude that SiNWs are terminated by benzylthiol ester groups. NCL of Model Peptides on Thioester-Terminated SiNWs. Alkylthiol esters are known to be relatively unreactive toward Cys peptides in water at pH 7. Usually, NCL is carried out in the presence of an exogenous thiol catalyst such as thiophenol or 4-mercaptophenyl acetic acid (MPAA).51 These mildly acidic thiols allow the in situ formation of arylthioesters by thiol−thioester exchange, which react rapidly with the Cys peptide. The thiol−thioester exchange reaction leading to the arylthioester is known to be rate-limiting.51 The added thiol also maintains the N-terminal cysteine in reduced form and reverses any nonproductive thiolesters formed by thiol− thioester exchange with the thiol moieties of internal cysteine residues. The most common thiol catalysts used for NCL consists of a 1% benzylmercaptan/3% thiophenol mixture or MPAA for chemically synthesized peptide-thioesters51 or 2mercaptoethanesulfonate sodium salt (MESNA) for recombinant peptide-thioesters.66 In this study, we used MPAA, which is odorless, very water-soluble, and effective at catalyzing NCL. Two different NCL procedures were examined. In the first one, we carried out the thiol−thioester exchange between benzylthiol ester groups on the SiNWs and MPAA prior to the reaction with the Cys peptides. The first step consisted of the incubation of the benzylthiol ester-terminated SiNWs in PBS buffer (200 mM) containing 80 mM MPAA, 20 mM TCEP, and 0.1% Tween 20, with a final pH of 7.6 overnight at 37 °C to ensure the formation of more active MPAA thioesterterminated SiNWs. Then, the SiNWs were washed and incubated with Cys-peptides. In the second procedure, thiol− thioester exchange between benzylthiol ester groups on the SiNWs and MPAA activation were carried out in situ during the NCL. As controls, the same procedures were carried out with Ser-pep-R or Ser-pep-F peptides. XPS measurements results are summarized in Table 1 for the in situ activation strategy. The incubation of activated SiNWs in an aqueous solution containing 3 mM Cys-pep-F led to the detection of fluorine (F 1s) (−CF3 group) and the N 1s signal, proving that NCL was successfully achieved. However, in the control incubation (Ser-pep-F), traces of fluorine and nitrogen were also detected, probably because of the small amount of physisorbed peptide. The value of the C 1s/F 1s ratio, after incubation with Cys-pep-F peptide, is 23, which is very close to the theoretical value of 24 expected for full coverage of the surface by the peptide, whereas the value of the C 1s/F 1s ratio after incubation with Ser-pep-F peptide is 575. On the basis of these ratios, we can estimate that the amount of Cys-pep-F peptide immobilized on SiNWs is 24 times higher than the amount of Ser-pep-F peptide. From the Si 2p signal, we can clearly see that the Si 2p signal decreased after incubation with Cys-pep-F in comparison with that obtained for thioester- and acid-terminated SiNW surfaces. This observation was expected because peptides now cover the entire SiNW surface. For the control experiment (i.e., incubation with Ser-pep-F), the Si 2p signal was close to the values obtained for thioester-terminated SiNWs, proving once again that no peptide immobilization occurred. It has to be noted that a small amount of oxidized silicon (10% of the total signal of Si 2p) was also observed. Traces of sodium ion were also detected through the observation of the Na 1s peak. The sodium ion most likely results from sodium chloride, present in PBS, adsorption on the

Figure 4. Schematic representation of the chemical steps for SiNW functionalization with benzylthiol groups.

dicyclohexylcarbodiimide in the presence of benzylmercaptan and 4-dimethylaminopyridine (DMAP) used as an acylation catalyst. Each step in the chemical surface modification has been characterized by X-ray photoelectron spectroscopy (XPS). The percentages of elemental composition after grafting UA and its subsequent transformation to thioester are summarized in Table 1. We can clearly see that only C, O, and Si were Table 1. Percentage of Total Area for Each Atomic Species at Different Chemical Steps Performed on SiNWs Obtained by XPS Analysis for the One-Step Ligation Strategy Cys-pep-F Ser-pep-F thioester (benzylmercaptan) undecylenic acid (UA) a

Si 2p

O 1s

C 1s

S 2s

N 1s

F 1s

Na 1s

28 40.9 44.5

21.5 20 17

38.5 34.5 37

1.06 0.72 1.34

4 1.12

1.7 0.06

5.2 2.8

a

a

a

50

22

28

a

a

a

a

Not detected.

detected after the grafting of UA on H-terminated SiNWs. After the reaction of undecylenic acid-terminated SiNWs with benzylmercaptan, an S2s signal appeared, proving the successful formation of thioester groups on SiNWs. That was confirmed by the high-resolution spectra of C 1s as shown in Figure 5A1,A2 corresponding to UA grafting and benzylthiol ester formation, respectively. Figure 5A1 displays two peaks appearing at 285.37 and 290.09 eV, assigned to C−C* and HO−C*=O carbon atoms, respectively. In Figure 5A2, two peaks are also observed but at 285.19 and 288.66 eV attributed to C−C* and S−C*=O carbon atoms, respectively. The peak at 288.66 eV is in accordance with the formation of a thioester bond, as confirmed by the high-resolution spectrum of S 2s (inset in Figure 5A). Note that the C*−C peak intensity 13340


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Figure 5. XPS spectra of C 1s of acid-terminated SiNWs (A2), benzylthiol ester-terminated SiNWs (A2), and after immobilization of Cys-pep-F (one-step strategy) (B). (Insets of A and B) XPS high-resolution of S 2s and F 1s, respectively.

surface. It is a potential counterion of negatively charged amino acid residues present within a peptide such as glutamic acid (E). In accordance with this hypothesis, a greater amount of sodium contamination was detected after the incubation of thioesterterminated SiNWs with Cys-pep-F. Figure 5B displays the high-resolution spectrum of C 1s after the immobilization of Cys-pep-F. The spectrum can be deconvoluted into four components with positions at 285.28, 286.57, 288.53, and 292.98 eV corresponding to C*−C, C*− NH, HN−C*=O (amide bonds), and C*F3 atom species, respectively. The inset in Figure 5B shows the high-resolution XPS spectrum of F 1s, confirming the Cys-pep-F immobilization on SiNWs. XPS analyses were also conducted after NCL of Cys-pep-F following the two-step procedure. A low value for the C 1s/F 1s ratio (11) was obtained whereas an increase in the SiNW oxidation (40% of the total signal of Si 2p) was observed (data not shown). From all of the results described above, we can conclude that NCL of model Cys-peptides on benzylthiol ester-functionalized SiNWs was chemoselective and site-specific. Moreover, the in situ activation strategy gave better results than the two-step procedure in terms of peptide ligation efficiency while preventing oxidation on the the SiNW surface. This surface oxidation can be troublesome for field effect transistor applications based on silicon nanowire sensors. Indeed, it was shown that nonoxidized silicon nanowires gave a higher sensitivity of detection with a 2 orders of magnitude improvement compared to that for the oxidized ones.67 The chemoselectivity of NCL ligation on SiNWs was also confirmed by fluorescence measurements as shown in Figure 6. The signal intensities after NCL of Cys-pep-R and Ser-pep-R peptides were 343 ± 20 and 23 ± 3, respectively, with a ratio of

Figure 6. Fluorescence measurements after the incubation of thioester-terminated SiNWs with Cys-pep-R and Ser-pep-R.

15 (ratio of fluorescence intensities of Cys-pep-R/Ser-pep-R), which is comparable to the value calculated from XPS measurements. Although the SiNW layer is thick, a potential concern deals with the chemical functionalization steps that might also modify the underlying substrate (i.e., the silicon wafer), limiting the ability to distinguish the chemistry of the nanoscale materials from that of their underlying support. To ensure that the NCL occurs mainly on the SiNW layer, we used GaAs substrates, instead of silicon substrates, to grow the SiNWs. For this purpose, the SiNWs were grown on GaAs by using the same growth conditions as described above (i.e., 40 sccm of silane (SiH4), P = 0.532 mbar, T = 500 °C, and t = 20 min). SiNWs 13341


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Table 2. Percentage of the Total Area for Each Atomic Species after Incubation (One-Step Strategy) on Carboxy-Terminated SiNWs Synthesized under Different Growth Conditions SiNW growth conditions Cys-pep-F Ser-pep-F a

XPS analysis (% of total surface area)

SiH4; H2(sccm)

P(mbar)

t(min)

T(°C)

partial pressure silane (mbar)

Si 2p

O 1s

C 1s

S 2s

N 1s

F 1s

40 260;3700 40 260;3700

0.532 15 0.532 15

20 20 20 20

500 430 500 430

0.532 1 0.532 1

39.5 39 39.3 40

48.5 47.3 46.2 45.5

12 13.2 14.5 14

a

a

a

a

a

a

0.5

a

a

a

a

a

a

a

0.5

Au 4f a

Not detected.

with similar morphology and thickness (12 μm) were obtained as compared to those grown on a Si substrate. It is known that GaAs reacts with thiols or disulfide molecules, even if the link is not stable over time.68,69 From that, we cannot exclude the reaction between MPAA and/or Cys-pep-F molecules and the GaAs underlying substrate. The native chemical ligation XPS characterization results were identical to those obtained previously on the SiNW/Si surface for all chemical steps (Table S1 in the SI). XPS is a surfacesensitive technique with a probing depth ranging from 3 to 10 nm, depending on the materials. In our case, the SiNW layer was too thick, so no Ga or As signals have been obviously detected. Such a result proves that the XPS characterization probes only the SiNW layer. Even if we cannot exclude that some peptides are immobilized on the underlying substrate, they do not contribute to the XPS signals. Gold is known to diffuse along the SiNW sidewalls during the growth, depending on the growth conditions,70,71 whereas thiol molecules are known to react with noble metals such as Ag, Au, and Pt.72 In that case, gold clusters can potentially participate in the attachment of thiol groups in addition to the direct linkage of Cys-pep-F to nanowires via NCL. Nevertheless, an increase in the partial pressure of silane favors the adsorption of silane molecules on the nanowire sidewalls, and decreasing the growth temperature reduces the rate of silane dissociation and also the mobility of Au, as demonstrated by Hertog and co-workers.71 Therefore, to ensure that the Cyspep-F was immobilized on SiNWs only through NCL and not through links between the thiol group of cysteine and gold clusters, we performed control experiments. For that, we carried out the chemical treatments described above using SiNWs that were synthesized under two different growth conditions (Table 2). The first type of SiNWs was grown under experimental conditions of 260 sccm of silane (SiH4) diluted in 3700 sccm of H2, P = 15 mbar, and T = 430 °C corresponding to a partial pressure of silane of 1 mbar whereas the synthesis of the second type of SiNWs was achieved by using the same conditions as described above (i.e., 40 sccm of silane (SiH4), P = 0.532 mbar, and T = 500 °C corresponding to a silane partial pressure of 0.532 mbar). In the first case, no gold dewettting was observed as shown in Figure 7A (i.e., no gold clusters have been observed by high-angle annular dark field (HAADF) imaging using STEM). In contrast, the second type of SiNWs showed gold clusters formed as a result of the gold dewetting phenomenon (Figure 7B). XPS analysis was realized after incubating both types of carboxy-terminated SiNWs with either Cys-pep-F or Ser-pep-F peptides. The results are summarized in Table 2. Because in all cases no fluorine signal was detected, we have first confirmed the site-specific immobilization of Cys-pep-F peptide through NCL occurring with only surface-exposed benzylthiol ester groups. Second, we proved that gold clusters did not contribute

Figure 7. HAADF images (STEM) of SiNWs without (A) and with (B) gold nanoclusters. The diameter of the nanowire is ∼70 nm.

to the immobilization of Cys-pep-F via the thiolate−gold interaction. In addition, no S 2s signal was detected by XPS, showing that MPAA molecules did not react with gold seed particles and the Au-rich clusters adsorbed on the SiNW sidewalls.

IV. CONCLUSIONS We have demonstrated that native chemical ligation can be successfully achieved on a hydrogenated silicon nanowire substrate. NCL is based on the reaction of a thioester group with a cysteinyl peptide in which a first trans-thioesterification step is followed by an intramolecular S,N-acyl shift. This process results in the formation of a stable native peptide bond. The reaction occurred chemoselectively and efficiently in water under physiological conditions on benzylthiol ester-functionalized SiNWs. It was directly characterized by XPS and fluorescence measurements using tetramethylrhodamine- and trifluoromethyl-labeled peptides, respectively. The chemistry developed here should be applicable to a wide range of biomolecules for biosensing applications.

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ASSOCIATED CONTENT

S Supporting Information *

Peptide synthesis and additional XPS analyses. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: yannick.coffi[email protected]. Tel: +33 3 20 19 79 87. Fax: +33 3 20 19 78 84. 13342


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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported and granted by the French National Research Agency (ANR), ANR-06-NANO-001, Nanobiodetecteur. We also thank Gilles Patriarche from the Laboratory for Photonics and Nanostructures (LPN-CNRS) for STEM (HAADF) characterization.

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