Surface Plasmon Resonance on Gold and Silver Films Coated with

Surface Plasmon Resonance on Gold and Silver Films Coated with Thin Layers of Amorphous Silicon Carbon Alloys ... Fax: +33 3 20 19 78 84. ... Ag/a-Si1...
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Surface Plasmon Resonance on Gold and Silver Films Coated with Thin Layers of Amorphous Silicon-Carbon Alloys )

Larbi Touahir,† Joanna Niedzioel ka-J€onsson,‡,§ Elisabeth Galopin,‡,§ Rabah Boukherroub,‡,§ Anne Chantal Gouget-Laemmel,† Ionel Solomon,† Mikhail Petukhov, Jean-No€el Chazalviel,† Franc-ois Ozanam,*,† and Sabine Szunerits*,‡,§

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† Physique de la Mati ere Condens ee, Ecole Polytechnique, CNRS, 91128 Palaiseau, France, ‡Institut de Recherche Interdisciplinaire (IRI, USR 3078), Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq, France, §Institut d’Electronique, de Micro electronique et de Nanotechnologie (IEMN, UMR 8520), Cit e Scientifique, Avenue Poincar e, BP 60069, 59652 Villeneuve d’Ascq, France, and Universit e des Sciences et Technologies de Lille (UCCS-UMR CNRS 8181), Cit e Scientifique, B^ atiment C3, 59655 Villeneuve d’Ascq Cedex, France

Received October 14, 2009. Revised Manuscript Received January 6, 2010 The paper reports on a novel surface plasmon resonance (SPR) substrate architecture based on the coating of a gold (Au) or silver (Ag) substrate with 5 nm thin amorphous silicon-carbon alloy films. Ag/a-Si1-xCx:H and Au/a-Si1-xCx: H multilayers are found to provide a significant advantage in terms of sensitivity over both Ag and Au for SPR refractive index sensing. The possibility for the subsequent linking of stable organic monolayers through Si-C bonds is demonstrated. In a proof-of-principle experiment that this structure can be used for real-time biosensing experiments, amine terminated biotin was covalently linked to the acid-terminated SPR surface and the specific streptavidin-biotin interaction recorded.

1. Introduction The use of surface-based analytical platforms for the rapid, sensitive, selective, and label-free monitoring of biomolecular interactions has become increasingly popular and has revolutionized molecular biology. Surface plasmon resonance (SPR)-based techniques have in this respect become very important instrumentations for the detection of biomolecular interactions in a label-free manner.1 The choice of the metal film where plasmon waves are generated is critical for sensitive SPR sensing.2 Gold substrates are most commonly used as they possess stable optical and chemical properties. Since most investigations in SPR use gold substrates, thiol attachment chemistry is routinely used to anchor functional groups and ligands to the surface.3-5 Even though Au-S bonds are relatively strong, they are susceptible to oxidation and thermal desorption.6 Other widely used surface functionalization techniques in SPR are based on conducting polymers7,8 or on a layer of functionalized dextran.9 In many biosensor applications, silane coupling chemistry on silicon dioxide substrates is often used for immobilization of biomolecules such *To whom correspondence should be addressed. (S.S.) E-mail: sabine. [email protected]. Telephone: þ33 3 20 19 79 87. Fax: þ33 3 20 19 78 84. (F.O.) E-mail: [email protected]. Telephone: þ33 1 69 33 47 04. Fax: þ33 1 69 33 47 99. (1) Homola, J. Surface Plasmon Resonance Based Sensors; Springer-Verlag: Berlin, 2006. (2) Lecaruyer, P.; Canva, M.; Rolland, J. Appl. Opt. 2007, 46, 2361–2369. (3) Smith, E. A.; Wanat, M. J.; Cheng, Y.; Barreira, S. V. P.; Frutos, A. G.; Corn, R. M. Langmuir 2001, 17, 2502–2507. (4) Smith, E. A.; Thomas, W. D.; Kiessling, L. L.; Corn, R. M. J. Am. Chem. Soc. 2003, 125, 6140–6148. (5) Damos, F. S.; Luz, R. C. S.; Kubota, L. T. Langmuir 2005, 21, 602–609. (6) Ulman, A. Chem. Rev. 1996, 96, 1533–1554. (7) Guedon, P.; Livache, T.; Martin, F.; Lesbre, F.; Roget, A.; Bidan, G.; Levy, Y. Anal. Chem. 2000, 72, 6003–6009. (8) Szunerits, S.; Knorr, N.; Calemczuk, R.; Livache, T. Langmuir 2004, 20, 9236–9241. (9) Thiel, A. J.; Frutos, A. G.; Jordan, C. E.; Corn, R. M.; Smith, L. M. Anal. Chem. 1997, 69, 4948–4956.

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as DNA, carbohydrates, or proteins. This can be done for SPR spectroscopy by producing silicate or silicon oxide films on gold SPR substrates as demonstrated by several groups.10-14 However, gold substrates are not the best candidates for achieving high-sensitivity SPR sensing. Theoretical modeling of SPR in conducting metal oxide thin films has been performed by Franzen et al.15,16 and suggests that ITO could be a better suited substrate. However, this would require excitation and detection in the infrared range. In the conventional visible range, silver substrates appear to be the most appealing, because plasmon coupling exhibits a sharper angular resonance as compared to that on gold, yielding an increased sensitivity.17-19 Silver substrates can be functionalized similarly to gold ones, by using thiol or disulfide molecules which adsorb from solution and self-assemble into densely packed monolayers.20,21 However, silver suffers from a poor chemical stability which hampers its wide use for SPR sensing. One strategy to circumvent this limitation is to use bimetallic layers, with a gold coating on top of the silver layer.22 In this case, the usual (10) Phillips, K. S.; Han, J.-H.; Martinez, M.; Wang, Z.; Carter, D.; Cheng, Q. Anal. Chem. 2006, 78, 596–603. (11) Tawa, K.; Morigaki, K. Biophys. J. 2005, 89, 2750–2758. (12) Reimhult, E.; Z€ach, M.; H€oo€k, F.; Kasemo, B. Langmuir 2006, 22, 3313– 3319. (13) Szunerits, S.; Boukherroub, R. Langmuir 2006, 22, 1660–1663. (14) Szunerits, S.; Coffinier, Y.; Janel, S.; Boukherroub, R. Langmuir 2006, 22, 10716–10722. (15) Rhodes, C.; Franzen, S.; Maria, J.-P.; Losego, M.; Leonard, D. N.; Laughlin, B.; Duscher, G.; Weibel, S. J. Appl. Phys. 2006, 100, 054905. (16) Franzen, S. J. Phys. Chem. C 2008, 112, 6027–6032. (17) Szunerits, S.; Castel, X.; Boukherroub, R. J. Phys. Chem. C 2008, 112, 15813–15817. (18) Chah, S.; Hutter, E.; Roy, D.; Fendler, J. H.; Yi, J. Chem. Phys. 2001, 272, 127–136. (19) Yuan, X.-C.; Ong, B. H.; Tan, Y. G.; Zhang, D. W.; Irawan, R.; Tjin, S. C. J. Opt. A: Pure Appl. Opt. 2006, 8, 959–963. (20) Laibinis, P. E.; Whitesides, G. M. J. Am. Chem. Soc. 1992, 114, 1990–1995. (21) Dubois, L. H.; Nuzzo, R. G. Annu. Rev. Phys. Chem. 1992, 43, 437–463. (22) Zynio, S. A.; Samoylov, A. V.; Surovtseva, E. R.; Mirsky, V. M.; Shirshov, Y. M. Sensors 2002, 2, 62–70.

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thiol-on-gold chemistry can be used for coupling probes to the sensor surface. Alternately, Szunerits and Boukherroub have successfully prepared SPR substrates coated with thin layers of various dielectrics (SiOx, ITO, or SnO2) to protect the underlying silver film and attach ligands to the surface hydroxyl groups using silane chemistry.17,23-25 In a related approach, Lockett et al. recently adapted their previous work on gold films26 by depositing a thin layer of amorphous carbon onto surface-plasmon active silver films.27 In this case, a very stable covalent chemistry through C-C bond formation can be used for probe binding. SPR imaging was successfully performed on such highly robust substrates, but the sensitivity remained lower than that reached on bare gold, which pointed to the need for an overlayer which does not absorb light in the visible range. Amorphous silicon-carbon alloys (abbreviated as a-Si1-xCx:H) could be an interesting alternative for the fabrication of multilayer SPR structures. They can be deposited as thin films,28,29 and changing the carbon content of the film allows for fine-tuning of the material properties. In particular, increasing the carbon content allows for enlarging the optical bandgap and obtaining a transparent material, and decreasing the refractive index,28,29 which could improve the sensitivity of the SPR structure. Surface hydrogenated a-Si1-xCx:H can be conveniently functionalized by stable organic layers through robust Si-C covalent bonds in a similar way as crystalline silicon.30-35 It is the purpose of the present work to examine whether a thin layer of amorphous silicon-carbon alloy could be the best compromise for protecting SPR-active silver layers, allowing for a robust covalent functionalization and providing high sensitivity and stability. In order to make clear the specific advantages brought by the use of a-Si1-xCx:H coatings onto SPR-active layers, the SPR results obtained on silver/ a-Si1-xCx:H structures will be systematically compared to those obtained on gold/ a-Si1-xCx:H.

2. Experimental Part 2.1. Materials. Hydrofluoric acid (HF) and acetic acid were purchased from Carlo Erba and were of VLSI grade. Undecylenic acid (99%) was supplied by Acros Organics. Potassium ferrocyanide K4Fe(CN)6, N-Hydroxysuccinimide (NHS), N-ethylN0 -(3-(dimethylamino)propyl) carbodiimide (EDC), PBS, potassium chloride, ethanol, hexane, 1-butanol, 2-propanol, 1-hexanol, 1,3-propanediol, and 1-pentanol were obtained from Aldrich and were used without further purification, N-(2-aminoethyl)biotinamide hydrobromide was obtained from Invitrogen. Ultrapure water (Milli-Q, 18 MΩ cm) was used for the preparation of the solutions and for all rinses. (23) Szunerits, S.; Castel, X.; Boukherroub, R. J. Phys. Chem. C 2008, 112, 10883–10888. (24) Manesse, M.; Sanjines, R.; Stambouli, V.; Jorel, C.; Pelissier, B.; Pisarek, M.; Boukherroub, R.; Szunerits, S. Langmuir 2009, 25, 8036–8041. (25) Manesse, M.; Sanjines, R.; Stambouli, V.; Boukherroub, R.; Szunerits, S. Electrochem. Commun. 2008, 10, 1041–1043. (26) Lockett, M. R.; Weibel, S. C.; Phillips, M. F.; Shortreed, M. R.; Sun, B.; Corn, R. M.; Hamers, R. J.; Cerrina, F.; Smith, L. M. J. Am. Chem. Soc. 2008, 130, 8611–8613. (27) Lockett, M. R.; Smith, L. M. Anal. Chem. 2009, 81, 6429–6437. (28) Solomon, I.; Schmidt, M. P.; Senemaud, C.; Driss Khodja, M. Phys. Rev. B 1988, 38, 13263–13270. (29) Solomon, I.; Schmidt, M. P.; Tran-Quoc Phys. Rev. B 1988, 38, 9895–9901. (30) Boukherroub, R. Curr. Opin. Solid Stat. Mater. Sci. 2005, 9, 66–72. (31) Voicu, R.; Boukherroub, R.; Bartzoka, V.; Ward, T.; Wojtyk, J. T. C.; Wayner, D. D. M. Langmuir 2004, 20, 11713–11720. (32) Faucheux, A.; Gouget-Laemmel, A. C.; Henry de Villeneuve, C.; Boukherroub, R.; Ozanam, F.; Allongue, P.; Chazalviel, J.-N. Langmuir 2006, 22, 153–162. (33) Rosso, M.; Arafat, A.; Schro€en, K.; Giesbers, M.; Roper, C. S.; Maboudian, R.; Zuilhof, H. Langmuir 2008, 24, 4007–4012. (34) Linford, M. R.; Fenter, P.; Eisenberger, P. M.; Chidsey, C. E. D. J. Am. Chem. Soc. 1995, 117, 3145–3155. (35) Strother, T.; Cai, W.; Xhao, X. S.; Hamers, R. J.; Smith, L. R. J. Am. Chem. Soc. 2000, 122, 1205–1209.

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2.2. Preparation of the SPR Structures. Ti/Au films were prepared by thermal evaporation of 5 nm of titanium and 50 nm of gold onto cleaned glass slides. Titanium is used as an adhesion layer between the glass surface and the metal layer. Ti/Ag films were prepared by thermal evaporation of 5 nm of titanium and 38 nm of silver onto cleaned glass slides. Amorphous siliconcarbon alloy layers were deposited onto Ti/Au and Ti/Ag films using plasma-enhanced chemical vapor deposition (PECVD) in a “low-power” regime.28 The following parameters were used: pressure =35 mTorr, temperature =250 °C, power density = 0.06 W cm-2, gas flow rate = 20 cm3 min-1. By varying the methane ratio in the gas mixture [CH4]/([SiH4] þ [CH4]), the final carbon (C) content in the material and thus the optical properties can be adjusted. For the deposition of a thin film with the following stoichiometry: a-Si0.63C0.37:H, 94 at.% of [CH4] were used, while for an a-Si0.80C0.20:H film, 81 at.% were necessary. The carbon content in the film has been found to be governed by the methane ratio in the gas mixture only, as long as deposition remains performed in the low-power regime. The correspondence between the carbon content x in the a-Si1-xCx:H film and the methane ratio has been determined by using a combination of various techniques including electron spectroscopies and elemental analysis.29 The heating of the silver layer at 250 °C prior to a-Si0.63C0.37:H deposition might lead to oxidation of the silver film. Thus, on silver, a pretreatment with a hydrogen plasma (150 mT, 0.1 W/cm2) for 5 min has been applied to regenerate the silver surface just before turning on the silane/methane plasma for the deposition of the amorphous silicon-carbon films. The thicknesses of the different layers were adjusted by controlling the deposition time and afterward determined using ellipsometry on five different areas of the sample. The thicknesses varied by (0.5 nm from the targeted one. WinSpall 2.0 software (Max-Planck-Institute for Polymer Research, Mainz, Germany) was used to calculate the SPR curves and approximate the experimental dependences with an optical model within the framework of Maxwell macroscopic approach.36 2.3. Monolayer Formation on Amorphous Silicon-Carbon Alloys. Acid-Terminated Surface. The surface of the Ti/Ag/ a-Si0.63C0.37:H structure was first etched with HF vapor for 15 s. The hydrogen-terminated surface was placed at room temperature in a Schlenk tube containing previously deoxygenated neat undecylenic acid solution and irradiated at 312 nm for 3 h. The excess of unreacted and physisorbed reagent was removed by a final rinse in hot acetic acid for 30 min. Then the sample was dried under nitrogen flow. NHS-Functionalized Surface. The conversion of the acid function to succinimidyl ester was accomplished as follows: the acid-terminated surface was covered with 10 mL of an aqueous solution of NHS (5 mM) and EDC (5 mM) and allowed to react for 90 min at 15 °C. The resulting surface was copiously rinsed with deionized water and dried under a stream of argon. Biotin-Terminated Surface. The NHS-terminated surface was reacted with N-(2-aminoethyl)biotinamide hydrobromide (1 mg in 1 mL PBS) for 2 h at room temperature. The resulting surface was copiously rinsed with deionized water and dried under a stream of argon. Safety Considerations. Caution! HF is a hazardous acid which can result in serious tissue damage if burns are not appropriately treated. Etching of silicon should be performed in a well-ventilated fume hood with appropriate safety considerations: face shield and double layered nitrile gloves.

2.4. Surface Characterization. X-ray Photoelectron Spectroscopy. X-ray photoelectron spectroscopy (XPS) measurements were performed with an ESCALAB 220 XL spectrometer (36) Raether, H. Surface Plasmons on Smooth and Rough Surfaces and on Gratings; Springer-Verlag: Berlin, 1988.

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Figure 1. (A) Scheme of our surface plasmon resonance architecture, consisting of a multilayer structure in which a thin metal (gold or silver) film was coated with an a-Si1-xCx:H silicon carbon thin film, (B) Surface hydrogenation of a-Si1-xCx:H thin film and subsequent functionalization with undecylenic acid. from Vacuum Generators featuring a monochromatic Al KR X-ray source (1486.6 eV) and a spherical energy analyzer operated in the CAE (constant analyzer energy) mode (CAE=100 eV for survey spectra and CAE = 40 eV for high-resolution spectra), using the electromagnetic lens mode. No flood gun source was needed due to the conducting character of the substrates. The angle between the incident X-rays and the analyzer is 58°. The detection angle of the photoelectrons is 30°, as referenced to the sample surface. The XPS spectra were corrected according to the binding energies of Au 4f7/2, equal to 80.0 eV, and Ag 3d5/2, equal to 368.3 eV.

Attenuated Total Reflection Fourier-Transform Infrared (ATR-FTIR). For infrared characterization of the functionalized layer, Fourier-transform infrared spectroscopy in the attenuated total reflection geometry (ATR-FTIR) was chosen owing to its high sensitivity. For that purpose, 20 nm a-Si0.63C0.37:H thin films were deposited onto a ∼45° crystalline silicon prism used as the ATR element. The dimensions of the prism (typically 15  15  0.5 mm3) were limiting the infrared path length in silicon, providing access to the vibrations down to 1000 cm-1. The spectra were recorded on a Bruker Equinox FTIR spectrometer coupled to a homemade, nitrogen-gas purged external ATR compartment. The spectra, taken with a 4 cm-1 resolution, result from the averaging of 100 scans. They are displayed as absorbance per reflection (computed using natural logarithm) by using the spectrum recorded prior to surface modification as the reference, and by normalizing the spectrum with the actual number of reflections determined by measuring the exact prism dimensions and angle. Contact Angle Measurements. Water contact angles were measured using deionized water. We used a remote-computer controlled goniometer system (DIGIDROP by GBX, France) for measuring the contact angles. The accuracy is (2°. All measurements were made in ambient atmosphere at room temperature. Ellipsometry. Spectroscopic ellipsometry data in the visible range were obtained using a UVISEL Jobin Yvon Horiba Spectroscopic Ellipsometer equipped with DeltaPsi 2 data analysis software. The system acquired a spectrum ranging from 2 to 4.5 eV (corresponding to 300-750 nm) with 0.05 eV (or 7.5 nm) intervals. Data were taken using an angle of incidence of 70°, and the compensator was set at 45°. Data were fitted by regression analysis to a film-on-substrate model as described by the film thicknesses and the two complex refractive indices. Optical Transmission. Film thickness and refractive index of a-Si1-xCx:H were more routinely determined by optical transmission spectroscopy on thin layers deposited on bare glass. Spectroscopy was performed in the range of a-Si1-xCx:H 6060 DOI: 10.1021/la903896m

transparency, in which the spectrum exhibits oscillations due to optical interferences in the thin film. The optical bandgap, complex refractive index and film thickness were extracted from the fit of the data to an optical model based on the shape of the refractive index dispersion below the absorption edge.37 Thicknesses and refractive indexes determined using this procedure were found to be in close agreement with those determined by ellipsometry. Electrochemical Measurements. Electrochemical experiments were performed using an Autolab potentiostat 20 (Eco Chemie, Utrecht, The Netherlands). The electrode cell is the commercially available electrochemical cell of the Autolab SPRINGLE Instrument (Eco Chemie, Utrecht, The Netherlands), allowing simultaneous Surface Plasmon Resonance and electrochemical measurements. The configuration of this equipment is described elsewhere.38,39 In short, polarized laser light (λ = 670 nm) is directed to the bottom side of the sensor disk via a hemispherical lens placed on a prism (BK7 having a refractive index of n = 1.52) and the reflected light is detected using a photodiode. The angle of incidence is varied using a vibrating mirror with a frequency of 44 Hz. SPR curves were scanned on the forward and backward movement of the mirror and the minima in reflectance determined and averaged. Sheet Resistance Measurements. The sheet resistance R of the samples is measured using a homemade four-point probe configuration at room temperature.

3. Results and Discussion We have investigated the hybrid structure depicted in Figure 1. A thin film of a-Si1-xCx:H is deposited onto a SPR-active metal layer. The thickness of the metal layer (Ag: 38 nm, Au: 50 nm) was calculated in order to benefit from an optimum SPR sensitivity. The sensitivity of the structure has been measured first, as a function of the carbon content in the a-Si1-xCx:H layer. Then, an optimized structure has been characterized in more depth, with special care to its stability and its functionalization, in order to demonstrate the capability of the structure for biosensing. 3.1. Influence of the Carbon Content on the SPR Signal. Amorphous silicon-carbon alloy films between 0 and 10 nm with carbon concentration between 20 and 37% were deposited onto (37) Solomon, I. Philos. Mag. B 1997, 76, 273–280. (38) Wink, T.; Van Zuilen, S. J.; Bult, A.; Van Bennekom, W. P. Anal. Chem. 1998, 70, 827–832. (39) Kooyman, R. P. H.; Lenferink, A. T. M.; Eenink, R. G.; Greve, J. Anal. Chem. 1991, 63, 83–85.

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Figure 2. Scanning angle reflectivity curves of gold-based SPR substrates coated with 10 nm (A) and 5 nm (B) of a-Si1-xCx films: uncoated gold (black), a-Si0.80C0.20 (green), a-Si0.67C0.33 (red), a-Si0.63C0.37 (blue) in water; experimentally obtained values (circles) were compared to theoretically calculated SPR curves (lines) using WinSpall 2.0. (C) List of refractive indexes at 670 nm determined from SPR and optical reflectivity measurements.

metal films using plasma-enhanced chemical vapor deposition (PECVD) in a “low-power” regime.28 Figure 2 shows the experimentally measured SPR curves together with Fresnel fits in the case of gold films. The differences in the SPR curves referring to structures of equal film thickness are due to the change in refractive index. A higher carbon content induces a decrease of the refractive index from n = 2.51 - i0.002 (a-Si0.80C0.20:H) to n = 1.95 - i0.03 (a-Si0.67C0.33:H) and n = 1.81 - i0.04 (a-Si0.63C0.37: H), respectively.29 A too-low carbon content is associated with an exceedingly high refractive index which, in spite of the low optical absorption of the material, results in a large angular shift, but also a significant broadening of the resonance. Increasing the carbon content up to 37% widens the band gap, but also increases the density of states in the band tails, resulting in an increase of the imaginary part of the refractive index. As a matter of fact, the absorption is higher due to the defects in the band gap. Note that higher n00 values are obtained from the SPR fits (where a-Si1-xCx:H is deposited on the metal) than from optical transmission fits (where a-Si1-xCx:H is directly deposited on optical glass). The best structure in terms of SPR signal was obtained using a gold-based SPR interface coated with a 5 nm film of a-Si0.63C0.37:H. Compared to naked gold, only a slight broadening of the SPR curves is seen. The overall sensitivity of SPR structures to a change in the refractive index n of the adjacent medium is a complex function of the SPR curve parameters, especially sensitive to the steepness of the SPR dip measured by its full width at half-maximum (fwhm). A figure of merit (FOM) can be defined to quantitatively evaluate the refractive-index sensitivity of optical interfaces:40 FOM ¼

m ðdegree RIU -1 Þ fwhm ðdegreeÞ

ð1Þ

(40) Sherry, L. J.; Chang, S.-H.; Schatz, G. C.; Van Duyne, R. P. Nano Lett. 2005, 5, 2034–2038.

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Figure 3. Change in the ratio ΔΘSPR/fwhm with increasing refractive index n of the outer dielectric medium: Au (blue), Ag (green), Au/a-Si0.63C0.37 (5 nm) (red), Ag/a-Si0.63C0.37 (5 nm)(black).

where m is the linear regression slope for the curve giving the angle corresponding to the minimum of reflectivity associated with SPR (ΘSPR) as a function of refractive index units (RIU) of n and fwhm is the full angular width at half-maximum of the resonance in the scanning-angle reflectivity curves. Figure 3 shows a plot of the ratio of the change in ΘSPR (as compared to water) to fwmh as a function of n for various structures in contact of various liquids. A linear relationship is observed whose slope gives the FOM value defined above for each structure. FOM values of 35 RIU-1 for gold and 74 RIU-1 for Au/ a-Si0.63C0.37:H are found. Therefore, the presence of the a-Si0.63C0.37:H coating results in an overall increase in sensitivity. This result is in marked contrast with that obtained with a carbon film.26 This enhancement is even larger than previously observed for Ag/ITO where a FOM of 47 RIU-1 was determined.17 As expected, a higher sensitivity can even be obtained by replacing gold by silver. A major contribution to this enhancement DOI: 10.1021/la903896m

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Figure 4. Scanning angle reflectivity curves of silver-based SPR substrates (black) coated with 5 nm a-Si0.63C0.37 (blue) and Au/ 5 nm a-Si0.63C0.37 (red) in water; experimentally obtained values (circles) were compared to theoretically calculated SPR curves (lines) using WinSpall 2.0.

appears on the experimentally measured SPR curves for silverbased SPR interfaces coated with a-Si0.63C0.37:H, which are shown together with Fresnel fits in Figure 4. For comparison, the SPR curve for the Au/a-Si0.63C0.37:H (5 nm) structure is included. The resonance width fwhm of the Ag/a-Si0.63C0.37:H interface is largely decreased as compared to Au/a-Si0.63C0.37:H, due to the favorable optical properties. The sensitivity of the structure was found (Figure 3) to be 101 RIU-1, which favorably compares to the value of 51 RIU-1 measured on bare Ag. It is therefore 2 times and 2.8 times as large as that observed on silver- and gold-based SPR structures, respectively. We clearly see that the coating provides good properties concerning the sensitivity, as the a-Si0.63C0.37:H/ gold structure is more sensitive than the uncoated silver structure. The optical transparency of the coating appears of prime importance, as shown by the comparison with the results obtained with amorphous carbon.26,27 The refractive index of the coating material should be high enough to strengthen the plasmon mode at the solid surface, but not too much in order to avoid the broadening of the resonance and the associated loss in sensitivity. Finally, the coating thickness should be limited in order to avoid a similar broadening of the plasmon resonance. The Ag/a-Si0.63C0.37:H (5 nm) structure appears to satisfactorily meet these requirements. It should now be checked that this structure provides an efficient means for obtaining a stable immobilization of biological probes at the surface, and therefore allows for convenient biosensing. 3.2. Stability of Ag/a-Si0.63C0.37:H (5 nm). The chemical stability of the surface was studied by immersion for 6 h into 0.1 M H2SO4 and 0.1 M NaOH. No change in the SPR signal was recorded after these treatments, indicating that a 5 nm thick a-Si0.63C0.37:H film efficiently passivates the metal surface and will withstand further chemical functionalization steps (SPR characteristics would be dramatically affected in case of Ag oxidation). Immersion of the Ag/a-Si0.63C0.37:H (5 nm) structures into water or PBS buffer showed degradation of the interfaces after about a month only. This is most likely due to pinholes in the a-Si0.63C0.37: H layer which allow for water penetration over time and oxidation of the underlying silver film. However, for most bioassays, the time scale of the study of analyte-ligand interactions is in the hour to day range, where the interface showed no chemical degradation. When storing in air, no optical degradation was observed after 1 month. 3.3. Electrical and Electrochemical Characterization. Electrical and electrochemical measurements (cyclic voltammetry) were performed on the different structures. When measuring 6062 DOI: 10.1021/la903896m

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in-plane conductivity, a multilayer structure such as Ti/Ag/a-Si1-xCx can be described by three resistances in parallel and the sheet resistance R of the whole structure can be described by: 1/R = 1/RTi þ 1/RAu þ 1/Ra-Si1-xCx. Figure 5A displays the evolution of the resistivity of the hybrid structures as a function of the thickness for three different carbon contents. The resistivity decreases with increasing carbon content and increases with the thickness of the layer reaching a limit when the layer is about 20 nm thick. This result suggests that the global resistivity is that of the silver layer and that the conductivity of the coating is negligible. Since the resistivity of thin metal layers is known to be governed by grain-boundary scattering,41 the increase in layer resistivity can be attributed to dissolution of a small amount of silicon into the silver film during the deposition and its segregation at the grain boundaries upon cooling. The electrochemical properties of the modified surfaces were investigated by cyclic voltammetry experiments using Fe(CN)64- as a redox probe (Figure 5B). Silver coated with 5 nm a-Si0.63C0.37:H shows a charge transfer kinetics similar to that of bare silver, while coating with 5 nm of a-Si0.67C0.33:H or a-Si0.80C0.20:H results in partially blocked electrode transfer kinetics, the blocking being stronger in the case of a-Si0.80C0.20:H. This blocking effect is attributable to the semiconducting character of a-Si1-xCx:H and the associated interface barrier rather than to the series resistance of the layer (negligible for a 5 nm thick layer). In addition, we investigated the electrochemical properties of the Ti/Ag/a-Si1-xCx structures after exposure for 6 h into 0.1 M H2SO4 and 0.1 M NaOH. No significant changes could be observed after the immersion into base and acid. In the case of a-Si0.63C0.37:H, the low blocking effect might be ascribed to the presence of numerous pinholes in the amorphous layer. This possibility appears in conflict with the stability results presented in the previous section, which suggests the presence of a limited amount of such defects. As a matter of fact, the increase in electrical resistivity of the structure observed upon a-Si1-xCx:H deposition suggests that some intermixing takes place between silver and a-Si1-xCx:H. In agreement with this possibility, the analysis of the XPS survey of an a-Si0.63C0.33 structure (see Supporting Information) reveals that a limited amount of silver is present close to the surface (apparent Ag/Si ratio of 0.37 after correction of atomic sensitivity factors, whereas a complete attenuation of the Ag 3d signal would be expected in the case of a continuous, silver-free, 5 nm thick film by taking into account the photoelectron escape depth). In this context, the lower blocking effect observed for higher carbon concentrations is plausibly associated with the higher bandgap density of states, which increases with x,28,29 but may also be increased by intermixing with Ag. Finally, the idea of intermixing is given further support from the higher n00 values obtained from the SPR fits (a-Si1-xCx:H deposited on Ag as compared to a-Si1-xCx:H deposited on glass, see Figure 2). 3.4. Introduction of Carboxylic Acid Groups onto the Surface of Ag/a-Si0.63C0.37:H Structures. Surface-hydrogenated a-Si0.63C0.37:H films were immersed into undecylenic acid and submitted to UV irradiation in order to form an organic monolayer covalently bonded to the surface through Si-C bonds (Figure 1B). X-ray photoelectron spectroscopy (XPS) and contact angle measurements were used to analyze the chemical composition and the nature of the chemical bonding on the a-Si0.63C0.37:H surface before and after modification with undecylenic acid. A change in the contact angle from 95 ( 1° for as-deposited a-Si0.63C0.37:H to 75 ( 1° for the carboxydecylmodified a-Si0.63C0.37:H indicates that a reaction took place. (41) Mayadas, A. F.; Shatzkes, M. Phys. Rev. B 1970, 1, 1382–1389.

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Figure 5. (A) Sheet resistance measurements and (B) Cyclic voltammetry curves in Fe(CN)64- (10 mM)/PBS (0.1 M) aqueous solution for a 38 nm Ag film deposited on glass with 5 nm Ti adhesion layer uncoated (black line) and coated with 5 nm of a-Si0.63C0.37 (blue), a-Si0.67C0.33 (red) and a-Si0.80C0.20 (green), scan rate: 0.05 V s-1, electrode area: 0.07 cm2.

Figure 6. High-resolution XPS spectra of C 1s of as-deposited a-Si0.63C0.37 before (a) and after modification with undecylenic acid (b).

These figures are somewhat larger that those found on crystalline silicon, an expected feature indeed in view of the presence of methyl groups in the amorphous material. The high resolution XPS of the C 1s band is shown in Figure 6a. The main peak is centered at 283.9 eV (64%) and is characteristic for C-Si bonding. This is the expected signal for a low-power deposited a-Si1-xCx:H material in which carbon incorporates as methyl groups.28 The band at 284.9 eV (26%) is characteristic of C-C(H) bonding and of carbon contaminations, usually observed in most of the active surfaces due to ambient hydrocarbon adsorption. The signals at 286.4 and 287.6 eV correspond to C-O (6%) and CdO (4%) species. The signals point out the presence of an organic contamination at the analyzed surface. The presence of this unwanted contamination can be accounted for by the large amount of time elapsed between the deposition and the transfer into the XPS chamber (a few days). The high resolution XPS spectrum of the C 1s after chemical functionalization with undecylenic acid is displayed in Figure 6b. It can be deconvoluted into five different components. The main peak centered at 284.7 eV (43%) is characteristic of the CH2 groups of the alkyl chain, C-C(H) structures and carbon contaminations, while the other peaks at 284.0 (37%), 286.1 (10%), and 287.3 eV (5%), correspond to C-Si, C-O, and CdO functions. Strikingly, the 284.7 eV signal is now overcoming the C-Si contribution. Since the former peak contains indiscernible contributions from adventitious contamination and alkyl-chain backbone, its increase relative to the bulk a-Si1-xCx:H signal is a consistent indication of the functionalization of the surface with alkyl-based molecular groups. Furthermore, an additional band at 288.6 eV (5%), absent in the spectrum Langmuir 2010, 26(8), 6058–6065

of the as-deposited sample, is characteristic of O-CdO linkage. It is a strong indication of the successful functionalization of the surface with undecylenic acid. In addition to the XPS we performed ATR-FTIR measurement on a-Si0.63C0.37:H films. Figure 7a shows the ATR-FTIR spectrum of a thin film of a-Si0.63C0.37:H deposited on a silicon ATR prism. The intense peak at 2100 cm-1 confirms the presence of a large amount of silicon-hydrogen bonds in the bulk of the material. The bands at 2890 and 2953 cm-1 and between 1250 and 1500 cm-1 suggest that the carbon in the film is essentially in the form of CH3, as expected for a material deposited in the lowpower regime.28 After hydrosilylation of undecylenic acid by photochemical irradiation the vibrational bands of CdO at 1711 cm-1 and CH2 at 2855 and 2930 cm-1 are clearly seen (Figure 7b). This indicates a successful transfer of the carboxydecyl groups onto the a-Si0.63C0.37:H film. By making a quantitative analysis of the infrared data, as described in detail by Faucheux et al.,32 integration of the peak area of the CdO band for s- and p-polarization enables one to determine the molecular density of linked carboxydecyl groups, which is found to be N ∼ 2  1013 mol cm-2. This value is lower than that on crystalline silicon,32,42 as expected for a material incorporating a significant amount of methyl groups. The acid function was further converted into an activated ester group (Figure 7b middle curve). The complete disappearance of the acid peak at 1711 cm-1 and the new peaks at 1744, 1788, and 1816 cm-1 due to the stretching modes of the carbonyl functions (42) Boukherroub, R.; Morin, S.; Bensebaa, F.; Wayner, D. D. M. Langmuir 1999, 15, 3831–3835.

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Figure 8. Schematic illustration of linking biotin-NH2 to an acidterminated surface via EDC/NHS.

Figure 9. (A) Scanning angle reflectivity curves of a silver substrate (black) coated with 5 nm acid-terminated a-Si0.63C0.37 (blue) and modified with biotin (red) in PBS; experimentally obtained values (dotted line) were compared to theoretically calculated SPR curves (lines) using WinSpall 2.0. (B) SPR binding of streptavidin (10 μg/mL) to biotin-modified Ag/a-Si0.63C0.37 (black), and to nonmodified Ag/a-Si0.63C0.37 (gray).

Figure 7. ATR-FTIR spectra recorded on a 20 nm thick a-Si0.63C0.37 film: (a) as deposited; the reference spectrum is the initial hydrogenated prism of silicon; p polarization (blue), s polarization (red), (b) after modification with undecylenic acid (top, red), after further reaction with EDC/NHS (middle, blue) and after amidation with N-(2-aminoethyl)biotinamide hydrobromide (bottom, green). In all cases, the reference spectrum is the hydrogenated surface of the a-Si0.63C0.37:H layer.

of the activated ester are consistent with the formation of the activated ester. The amount of activated ester formed at the surface has also been determined by a quantitative analysis of the ester peaks, as developed by Moraillon et al.43 It is found to be N ∼ 1  1013 mol cm-2. Aminolysis of the ester groups with N-(2-aminoethyl)biotinamide hydrobromide results in the linking of the amine-terminated biotin to the interface (Figure 7b bottom curve). The peaks at 1651 and 1551 cm-1 assigned to the carbonyl function and the CNH vibration of the amide group unambiguously show that covalent linking has taken place. (43) Moraillon, A.; Gouget-Laemmel, A. C.; Ozanam, F.; Chazalviel, J.-N. J. Phys. Chem. C 2008, 112, 7158–7167.

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3.5. Detection of the Biotin/Streptavidin Interaction on Ag/a-Si0.63C0.37:H Structures. The carboxylic acid functional group is particularly useful for its chemical versatility and wetting properties.43,44 The interest in carboxyl-terminated SPR structures is that they can be readily employed for the linking of amineterminated ligands as outlined in Figure 8. The streptavidin-biotin system has often been used as a standard reaction to develop biosensor devices.45 In a proof-of-concept experiment that the interface can be used in real-time biosensing experiments, amineterminated biotin was covalently linked to the COOH groups through a two-step procedure, as explained in the previous section. The biotin-modified interface showed a contact angle of 70 ( 1°. For a comparison the contact angle for as-deposited a-Si0.63C0.37:H was 95 ( 1°, while it changed to 75 ( 1° for the carboxydecylmodified a-Si0.63C0.37. The stability of the modified interface when immersed in water was checked by comparing the ATR- FTIR spectra. After a month no significant changes were observed. From the scanning angle reflectivity curves (Figure 9A), linking biotin to the surface results in an angle shift of 0.12°, corresponding to an organic layer of 3.1 nm, which fits the molecular dimension of unmodified biotin (0.52 nm  l.00 nm  2.10 nm). The molecular recognition with streptavidin (5.4 nm  5.8 nm  4.8 nm)46,47 was monitored in a solution of 10 μg/mL and the (44) Blankespoor, R.; Limoges, B.; Sch€ollhorn, B.; Syssa-Magale, J.-L.; Yazidi, D. Langmuir 2005, 21, 3362–3375. (45) Wayment, J. R.; Harris, J. M. Anal. Chem. 2009, 81, 336–342. (46) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243, 85–88. (47) Hendrickson, W. A.; Paeller, A.; Smith, J. L.; Satow, Y.; Herritt, E. A.; Phizackorley, R. P. Proc. Nat. Acad. Sci. USA 1989, 86, 2190–2194.

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kinetics of the binding are shown in Figure 9B. As expected, a strong uptake (0.25° angle shift) was seen on the biotin modified interface while only a small increase was observed on the unmodified one. The 0.25° angle shift corresponds to a streptavidin layer thickness of ∼6.3 nm or 2.5 ng/mm2 of linked streptavidin as expected for a streptavidin monolayer.48 By repeating the experiment with various concentrations of streptavidin in solution, a detection limit of 6 ng/mL of streptavidin was estimated.

4. Conclusion A 5-nm-thick layer of amorphous silicon-carbon alloy deposited on an SPR-active gold or silver substrate can enhance the sensitivity of the substrate for SPR sensing significantly. For instance, the sensitivity of such a Ag/a-Si0.63C0.37:H structure is 2.8 times as high as that of naked gold and 2 times as high as that of naked silver. Reaching such a level of performance requires that the coating layer be quite thin and optically transparent with an appropriate refractive index. Amorphous silicon-carbon alloys can fulfill these requirements by adjusting the carbon (48) Knoll, W.; Zizlsperger, M.; Liebermann, T.; Arnold, S.; Badia, A.; Liley, M.; Piscevic, D.; Schmitt, F.-J.; Spinke, J. Colloid. Surf. A: Physicochem. Eng. Aspects 2000, 161, 115–137.

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content of the material. The resulting structure proves to be chemically resistant, a mandatory requirement for biosensing applications. The reduced thickness of the coating represents a practical limitation for obtaining an ideally continuous layer. In the deposition conditions used here, the layer cannot be considered to be perfectly homogeneous: the presence of a limited amount of pinholes and some intermixing between the amorphous alloy and the metal have been found. The grafting through Si-C bonds of stable organic monolayers on the amorphous coating has been demonstrated. Despite the limitation of the amount of Si surface sites by the presence of carbon in the alloy and intermixing with silver, attaching biomolecules with a surface concentration of 1013 cm-2 (i.e., in excess of those typical of probe concentration in biochips) has been achieved. In a proof of principle experiment, amine terminated biotin was covalently linked to the acid-terminated SPR interface and its specific interaction with streptavidin was monitored in real time. The high sensitivity reached for this assay demonstrates that the developed architecture expands the possibility of using SPR for the analysis of interfacial binding interactions. Supporting Information Available: Figure showing the XPS survey spectrum. This material is available free of charge via the Internet at http://pubs.acs.org.

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