Raman Enhancement of Azobenzene Monolayers on Substrates

Sep 12, 2008 - Nicolas Marquestaut,‡ Amanda Martin,† David Talaga,‡ Laurent Servant,‡ Serge Ravaine,§. Stéphane Reculusa,§ Dario M. Bassani...
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Langmuir 2008, 24, 11313-11321

11313

Raman Enhancement of Azobenzene Monolayers on Substrates Prepared by Langmuir-Blodgett Deposition and Electron-Beam Lithography Techniques Nicolas Marquestaut,‡ Amanda Martin,† David Talaga,‡ Laurent Servant,‡ Serge Ravaine,§ Ste´phane Reculusa,§ Dario M. Bassani,‡ Elizabeth Gillies,† and Franc¸ois Lagugne´-Labarthet*,†,‡ Department of Chemistry, UniVersity of Western Ontario, 1151 Richmond Street, London, On, N6A5B7, Canada, Institut des Sciences Mole´culaires, UMR 5255 CNRS, UniVersite´ Bordeaux 1, 351 Cours de la Libe´ration, 33405 Talence, France, and Centre de Recherche Paul Pascal, UPR 8641 CNRS, 33600 Pessac, France ReceiVed June 2, 2008. ReVised Manuscript ReceiVed August 6, 2008 Nanostructured metallic platforms for Raman enhancement were fabricated using Langmuir-Blodgett and electron beam (e-beam) lithography techniques. The gold platforms were inscribed on thin glass slides with the purpose of using them in a transmission geometry experimental setup under a confocal microscope. The plasmon frequency of the gold nanostructures was determined in the visible-near-infrared range for various pattern sizes prepared by Langmuir-Blodgett transfer and e-beam lithography. The surface Raman enhancement factors were determined for a monolayer of azobenzene molecules adsorbed on gold through thiol bonding and compared for both LB transfer and e-beam samples for nanostructures of comparable geometries.

Introduction Optical detection of very dilute systems or monolayers is of interest in many applications where in situ or in ViVo measurements in complex environments are required.1,2 In physiological investigations, intrusive measurements must be minimized, and endoscopic optical irradiation and localized measurements are quite effective methods to probe complex biological phenomena based on the detection and analysis of optically transmitted images. In more advanced designs, optical microscopy combined with spectroscopic methods such as single photon or multiphoton fluorescence can provide rapid identification of labeled domains through the spectral analysis of endogenous or exogeneous fluorophores.3 Raman vibrational microscopy combines all these advantages. It can be coupled to microscopy measurements with an inherent spatial resolution limited to about λ/2, and the irradiation wavelength can be tuned from the UV to the near-infrared (NIR) range allowing one to access specific tissue with a better penetration depth, avoid damage, and minimize fluorescence effects.2 More interestingly, it also offers a sensitivity and specificity that can be of great interest for biosensors and for protein or DNA/RNA detection and identification through the analysis of their molecular fingerprints.4,5 However, the main drawback of Raman measurements is the poor Raman scattering cross section of about 10-30 cm2/molecule/steradian in ordinary conditions, which makes signals difficult to detect with low laser * Author to whom correspondence should be addressed. E-mail: flagugne@ uwo.ca. † University of Western Ontario. ‡ UMR 5255 CNRS. § UPR 8641 CNRS. (1) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. ReV. 1999, 99, 2957–2975. (2) Yonzon, C. R.; Haynes, C. L.; Zhand, X.; Walsh, J. T.; Van Duyne, R. P. Anal. Chem. 2004, 76, 78–85. (3) Xu, C.; Webb, W. W. J. Opt. Soc. Am. B 1996, 13, 481–491. (4) Wang, Y.; Wei, H.; Li, B.; Ren, W.; Guo, S.; Dong, S.; Wang, E. Chem. Commun. 2007, 5220, 5222. (5) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 5586.

irradiance power and short acquisition times.6 Despite being inherently weak, Raman signals can be enhanced in two ways. The first approach involves working either in electronic resonance or preresonance conditions, tuning the input excitation light in the vicinity of an electronic absorption band of the molecular species. The second possibility is to benefit from local surface enhancements when using a rough metallic surface, metallic dimers or clusters,7,8 underneath the molecules of interest. The surface enhancement Raman scattering (SERS) effect can provide an enhancement of the field by 4 to 8 orders of magnitude, which brings the scattering cross section of the SERS signal comparable to the fluorescence signal, making it much easier to detect. When both electronic resonance and surface enhancement effects are combined, the surface enhanced resonance Raman scattering (SERRS) enhancement factor can reach values up to the single molecule detection level with enhancements exceeding 1014.9-12 It is thus a major issue of significance to be able to design adapted metallic nanostructures for maximum SERS, that can be produced easily and routinely. Two phenomena contribute to the SERS effect: an electromagnetic phenomenon that relies upon the excitation of localized surface plasmons of the rough metal underneath the probed molecule, and a chemical effect originating from the formation of charge-transfer complexes. The electromagnetic effect results in an amplification of the incident and Raman-scattered optical fields driven by the size, shape and dielectric environment of the metallic nanostructure supporting (6) Campion, A.; Kambhampati, P. Chem. Soc. ReV. 1998, 27, 241–250. (7) Talley, C. E.; Jackson, J. B.; Oubre, C.; Grady, N. K.; Hollars, C. W.; Lane, S. M.; Huser, T. R.; Nordlander, P.; Halas, N. J. Nano Lett. 2005, 5, 1569–1574. (8) Moskovits, M. J. Raman Spectrosc. 2005, 36, 485–496. (9) Pettinger, B.; Picardi, G.; Schuster, R.; Ertl, G. Single Mol. 2002, 5, 285– 294. (10) Nie, S.; Emory, D. R. Science 1997, 275, 1102–1106. (11) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. ReV. Lett. 1997, 1667–1670. (12) Qin, L.; Zou, S.; Xue, C.; Atkinson, A.; Schatz, G.; Mirkin, C. A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13300–13303.

10.1021/la801697u CCC: $40.75  2008 American Chemical Society Published on Web 09/12/2008

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the probe molecules.8,13 Efficient SERS surfaces have been designed and fabricated by numerous approaches including electrochemical roughening,14 direct rough metal deposition,15 sol-gel encapsulated metal colloids,16 and using coated samples from nature,17 but the associated preparation methods often have little sample-to-sample reproducibility. This is a major drawback, especially for analytical science, where reproducibility and normalization is a prerequisite. More reproducible sample preparation methods such as nanosphere lithography (NSL),18,19 templating from organized colloidal surfaces20,21 or use of periodic arrays of optical fibers coated with metal22,23 are recent elegant ways to fabricate SERS platforms on large surfaces and with minimized defects. On the other hand, advanced lithographic techniques such as electronic-beam lithography and focused ion beam milling allow one to tailor the SERS efficiency of the structured surface by finely tuning the architecture of the metallic structures as well as the gap between the adjacent structures with a 10-20 nm resolution.24,25 However, they present the disadvantage of being technologically demanding and costly.26-30 In the present experimental study, two approaches have been used to make SERS platforms of comparable sizes (features in the 90-400 nm range) and geometries. In the first series of experiments, a modified NSL method based on the LangmuirBlodgett (LB) deposition technique was used to control the deposition of a single layer of functionalized silica nanospheres with a good reproducibility and quality over a large surface area exceeding other deposition methods such as nanosphere selfassembly onto surfaces31 or blade coating of colloidal solution.32 In comparison with traditional methods such as spin-coating33 or evaporation of colloidal solutions, the LB technique is a very efficient approach to prepare two-dimensional (2D) colloidal crystals of silica particles having a well-defined architecture over large dimensions (in the centimeter square range) and minimizing (13) Otto, A.; Mrozek, I.; Grabhorn, H.; Akemann, W. J. Phys. Condens. Matter 1992, 4, 1143–1212. (14) Jeanmaire, D. L.; Van Duyne, R. P. J. Electroanal. Chem. 1977, 84, 1–20. (15) Park, H. K.; Yoon, J. K.; Kim, K. Langmuir 2006, 22, 1626–1629. (16) Murphy, T.; Lucht, S.; Schmidt, H.; Kronfeldt, H.-D. J. Raman Spectrosc. 2000, 31, 943–948. (17) Stoddart, P. R.; Cadush, P. J.; Boyce, T. M.; Erasmus, R. M.; Comins, J. D. Nanotechnology 2006, 17, 680–686. (18) Hulteen, J. C.; Duyne, R. P. V. J. Vac. Sci. Technol. A 1994, 13, 1553– 1558. (19) Dieringer, J. A.; McFarland, A. D.; Shah, N. C.; Stuart, D. A.; Whitney, A. V.; Yonzon, C. R.; Young, M. A.; Zhang, X.; Duyne, R. P. V. Faraday Discussions 2006, 132, 9–26. (20) Bartlett, P. N.; Baumberg, J. J.; Coyle, S.; Abdelsalam, M. Faraday Discuss. 2003, 125, 19. (21) Baumberg, J. J.; Kelf, T. A.; Sugawara, Y.; Cintra, S.; Abdelsalam, M. E.; Bartlett, P. N.; Russell, A. E. Nano Lett. 2005, 5, 2262–2267. (22) White, D. J.; Mazzoloni, A. P.; Stoddart, P. R. J. Raman Spectrosc. 2007, 38, 377–382. (23) Guieu, V.; Lagugne´-Labarthet, F.; Servant, L.; Talaga, D.; Sojic, N. Small 2008, 4, 96–99. (24) Cui, B.; Clime, L.; Li, K.; Veres, T. Nanotechnology 2008, 19, 145216. (25) Li, K.; Clime, L.; Cui, B.; Veres, T. Nanotechnology 2008, 19, 145517. (26) Brolo, A.; Arctander, E.; Gordon, R.; Leathem, B.; Kavanagh, K. Nano Lett. 2004, 4, 2015–2018. (27) De Jesus, M. A.; Gielsfeldt, K. S.; Oran, J. M.; Abu-Hatab, N. A.; Lavrilk, N. V.; Sepaniak, M. J. Appl. Spectrosc. 2005, 59, 1501–1507. (28) Cialla, D.; Hubner, U.; Schneidewind, H.; Moller, R.; Popp, J. Chem. Phys. Chem. 2008, 9, 258–762. (29) Felidj, N.; Truong, S. L.; Aubard, J.; Le´vi, G.; Krenn, J. R.; Hohenau, A.; Leitner, A.; Aussenegg, F. R. J. Chem. Phys. 2004, 120, 7141–7146. (30) Fromm, D. P.; Sundaramurthy, A.; Schuck, P. J.; Kino, G.; Moerner, W. E. Nano Lett. 2004, 4, 957–961. (31) Haynes, C. L.; McFarland, A. D.; Duyne, R. P. V. Anal. Chem. 2005, A, 338–346. (32) Ormonde, A.; Hicks, E. C. M.; Castillon, J.; Van Duyne, R. P. Langmuir 2004, 20, 6927–6931. (33) Ma, W.; Hesse, D.; Go¨sele, U. Small 2005, 1, 837–841.

Marquestaut et al.

defects, particularly on the edges of the substrate.34 In addition, in the LB approach, a smaller quantity of colloidal solution is needed for the particle monolayer fabrication. Although previous work on the LB transfer of silver nanoparticles (20 nm in diameter) has been demonstrated for the fabrication of SERS substrates,35 this represents the first application of the LB technique to transfer functionalized silica spheres of large diameter (290-980 nm) used as a template for the preparation of SERS platforms. The second approach involves state-of-the-art high-resolution electron beam (e-beam) lithography methods that present the tremendous advantage of allowing the fully controlled design of 2D structures with a 20-30 nm resolution.27,36 Because of the quasi-perfect reproducibility of the e-beam lithography method, the absorption band of the plasmonic devices can be tuned very precisely by adjusting the size of the features and associated gaps. The two approaches were used to fabricate nanostructures with comparable shapes, i.e., a regular arrangement of Fischer’s patterns.37 After gold deposition, optical transmission measurements were performed to locate the plasmon band for the various samples prepared by the LB technique and e-beam lithography. Using these SERS platforms, we have specifically investigated the adsorption of azobenzene monolayers organized at the surface of nanostructured gold. Azobenzene mono-38 and multilayers39 have attracted significant attention during the past decade because of their interest as a surface command owing to the reversible E-Z isomerization of the azobenzene units.40,41 The efficiency and the reversibility of the photoinduced isomerization followed by the angular reorientation of the azobenzene units allow a photoswitching of the azobenzene moiety that can trigger changes in the orientation of a surrounding ensemble of molecules, such as in liquid crystalline materials, making such monolayers applicable as active surface command units.42-44 The same process has been investigated to generate mechanical changes in polymer shape,45 and photoinduced forces have been precisely measured by atomic force microscopy (AFM).46 Probing such a monolayer with vibrational techniques is of tremendous interest since the molecular fingerprint of the molecule can be probed selectively.47 In the more specific case of azobenzene molecules, changes of conformation could be followed through the change of signal intensity or shifting of some peaks, and indications of the molecular orientation and interactions could be given through polarized measurements.47,48 In this study, we have investigated azobenzene monolayers that have been covalently adsorbed on nanostructured gold surfaces and probed using Raman microscopy. The fabrication methods for the gold patterns on glass slides as well as the (34) Reculusa, S.; Ravaine, S. Chem. Mater. 2003, 15, 598–605. (35) Lu, Y.; Liu, G. L.; Lee, L. P. Nano Lett. 2005, 5, 5–9. (36) Fe´lidj, N.; Aubard, J.; Le´vi, G.; Krenn, J. R.; Salerno, M.; Schider, G.; Lamprecht, B.; Leitner, A.; Aussenegg, F. R. Phys. ReV. B 2002, 65, 0754191-9. (37) Fischer, U. C.; Zingsheim, H. P. J. Vac. Sci. Technol. 1981, 19, 881–885. (38) Ye, Q.; Fang, J.; Sun, L. J. Phys. Chem. B 1997, 101, 8221–8224. (39) Yu, H.-Z.; Zhqng, J.; Zhqng, H.-L.; Liu, Z.-F. Langmuir 1999, 15, 16–19. (40) Delaire, J.; Nakatani, K. Chem. ReV. 2000, 100, 1817–1845. (41) Natansohn, A.; Rochon, P. Chem. ReV. 2002, 102, 4139–4176. (42) Buchel, M.; Sekkat, Z.; Paul, S.; Weichart, B.; Menzel, H.; Knoll, W. Langmuir 1995, 11, 4460–4466. (43) Yokoyama, S.; Yamada, T.; Kajikawa, K.; Kakimoto, M.-a.; Imai, Y.; Takezoe, H.; Fukuda, A. Langmuir 1994, 10, 4599–4605. (44) Shishido, A.; Tsutsumi, O.; Kanazawa, A.; Shiono, T.; Ikeda, T.; Tamai, N. J. Am. Chem. Soc. 1997, 119, 7791–7796. (45) Yu, H.; Asaoka, S.; Shishido, A.; Iyoda, T.; Ikeda, T. Small 2007, 3, 768–771. (46) Hugel, T.; Holland, N. B.; Cattani, A.; Moroder, L.; Seitz, M.; Gaub, H. E. Science 2002, 296, 1103. (47) Buffeteau, T.; Natansohn, A.; Rochon, P.; Pe´zolet, M. Macromolecules 1996, 29, 8783–8790. (48) Sekkat, Z.; Wood, J.; Geerts, Y.; Knoll, W. Langmuir 1995, 11, 2856– 2859.

Raman Enhancement of Azobenzene Monolayers

synthesis of a thiol functionalized azobenzene material are reported. Surface Raman enhancement factors (REFs) are then precisely quantified and compared for both types of platforms for the first time.

Experimental Section Azobenzene Experimental Details. 2-[4-(4-Decycloxylphenylazo)-phenyl]ethyl Alcohol (3). A mixture of 4-[4-(2-hydroxyethyl)phenylazo]phenol49 (2) (1.2 g, 5.0 mmol, 1.0 equiv), 1-bromodecane (1.2 g, 5.5mmol, 1.1 equiv), KI (0.17 g, 1.0 mmol, 0.2 equiv.), 18-crown-6 (0.13 g, 0.50 mmol, 0.1 equiv), and K2CO3 (0.76 g, 5.5 mmol, 1.1 equiv) in anhydrous tetrahydrofuran (THF; 60 mL) was heated at reflux under a nitrogen atmosphere for 48 h. The reaction was monitored by thin layer chromatography (TLC) and additional 18-crown-6 was added as required. The solution was diluted with CH2Cl2, filtered, and the filtrate was concentrated to give an orange solid. Recrystallization from ethanol gave 1.68 g (88%) of the product as an orange solid. 1H NMR (400 MHz, CDCl3): δ 7.90 (d, 2H, J ) 8.7 Hz), 7.84 (d, 2H, J ) 8.2 Hz), 7.37 (d, 2H, J ) 8.2 Hz), 7.01 (d, 2H, J ) 8.9 Hz), 4.05 (t, 2H, J ) 6.6 Hz), 3.92 (t, 2H, J ) 6.3 Hz), 2.96 (t, 2H, J ) 6.5 Hz), 1.86-1.79 (m, 2H), 1.56-1.29 (m, 16H), 0.89 (t, 3H, J ) 6.8 Hz); 13C NMR (100 MHz, CDCl3): δ 161.6, 151.6, 146.9, 141.1, 129.7, 124.6, 122.8, 114.7, 68.4, 39.1, 31.9, 29.6, 29.4, 29.2, 26.0, 22.7, 14.1. MS: calcd [M]+ (C24H34N2O2): 382.3. Found: (ES+) 382.3. IR (cm-1, thin film from CH2Cl2): 3260, 2954, 2917, 2849, 1602, 1583, 1498. 2-[4-(4-Decycloxylphenylazo)-phenyl]ethyl Bromide (4). A solution of PPh3 (0.21 g, 0.78 mmol, 1.5 equiv) in anhydrous CH2Cl2 (2 mL) was added to a suspension of 3 (0.20 g, 0.50 mmol, 1.0 equiv) and CBr4 (0.21 g, 0.63 mmol, 1.2 equiv) in anhydrous CH2Cl2 (8 mL), and the reaction mixture was stirred for two hours at room temperature under a nitrogen atmosphere. The resulting solution was diluted with CH2Cl2, filtered, and concentrated under reduced pressure. Pure 4 was obtained by column chromatography (95:5, hexanes/EtOAc), providing 0.18 g (77%) as an orange solid. 1H NMR (400 MHz, CDCl3): δ 7.92 (d, 2H, J ) 9.0 Hz), 7.86 (d, 2H, J ) 8.4 Hz), 7.34 (d, 2H, J ) 8.2 Hz), 7.01 (d, 2H, J ) 9.0 Hz), 4.30 (t, 2H, J ) 6.6 Hz), 3.61 (t, 2H, J ) 7.6 Hz), 3.24 (t, 2H, J ) 7.5 Hz), 1.86-1.79 (m, 2H), 1.53-1.31 (m, 16H), 0.92 (t, 3H, J ) 6.6 Hz); 13C NMR (100 MHz, CDCl3): δ 161.6, 151.7, 146.7, 141.1, 129.3, 124.7, 122.7, 114.6, 68.3, 39.1, 32.5, 31.9, 29.5, 29.3, 29.1, 26.0, 22.7, 14.1. MS: calcd [M+H]+ (C24H34BrN2O): 445.2. Found: (ES+) 446.0. IR (cm-1, thin film from CH2Cl2): 3494, 2959, 2920, 2851, 1645, 1602, 1583, 1500. 2-[4-(4-Decycloxylphenylazo)-phenyl]ethyl Thiol (5). In the dark, a solution of 4 (0.18 g, 0.40 mmol, 1.0 equiv) and thiourea (30 mg, 0.40 mmol, 1.0 equiv) in ethanol (5 mL) was heated at reflux overnight. The reaction was monitored by TLC, and additional thiourea was added as required. Upon completion, 10% NaOH was added (5 mL), and the solution was heated at reflux for 2 h. The solvents were removed under reduced pressure, and then the residue was dissolved in a mixture of CH2Cl2 and water. The phases were separated, and the aqueous phase was extracted with two additional portions of CH2Cl2. The organic phases were combined and dried with magnesium sulfate, filtered, and concentrated under reduced pressure to yield 0.15 g (83%) of the pure product as an orange solid. 1H NMR (400 MHz, CDCl ): δ 7.92-7.81 (m, 4H), 7.33 (d, 2H, 3 J ) 8.5), 7.02-6.99 (m, 2H), 4.05-4.02 (m, 2H), 3.09-2.95 (m, 3H), 2.86-2.82 (m, 2H), 1.86-1.79 (m, 2H), 1.52-1.27 (m, 16H), 0.90 (t, 3H, J ) 6.64 Hz); 13C NMR (100 MHz, CDCl3): δ 161.9, 151.8, 147.2, 142.8, 129.6, 125.0, 123.1, 115.0, 68.7, 40.3, 36.5, 35.9, 32.2, 29.9, 29.7, 29.6, 29.6, 29.5, 26.3, 23.0, 14.4. MS: calcd [M]+ (C24H34N2OS): 398.2. Found: (ES+) 398.3. IR (cm-1, thin film from CH2Cl2): 3442, 2954, 2920, 2850, 2085, 1644, 1603, 1584, 1499. Preparation of Silica Particles and Functionalization. Monodisperse silica spheres with diameters ranging between 290 and 980 nm were prepared by hydrolysis of tetraethyl orthosilicate in an (49) Lynch, D. E.; Forkam, M. G.; Miller, L. S.; Parsons, S. Aust. J. Chem. 1998, 51, 1053–1056.

Langmuir, Vol. 24, No. 19, 2008 11315 alcoholic medium in the presence of water and ammonia.34,50,51 The polydispersities of the silica spheres were approximately 1.05. The spheres were functionalized with vinyl or amine groups using the coupling agents allyltrimethoxysilane and aminopropyltriethoxysilane, respectively. The amount of coupling agent was around 10 times greater than the amount necessary to cover the inorganic surface with a monolayer (a molecule occupies ∼ 0.5 nm2). The resulting amphiphilic silica spheres could be organized to form a stable Langmuir film at the air-water interface. The hydrophobic character gives particles smaller than 1 µm diameter the ability to stay at the water surface. As pointed out by Ravaine et al.,34,50 a subtle balance between hydrophilic and hydrophobic character of the surface of the particles must be obtained to avoid aggregation in the solution (too hydrophilic) or at the air-water interface (too hydrophobic). In such extreme cases, it is not possible to reach a high degree of organization of the particles to form colloidal arrays at the interface.52 Allyltrimethoxysilane and aminopropyltriethoxysilane were good candidates to avoid these problems, allowing researchers to achieve a good balance of the particle-particle and particle-subphase interactions, leading to the formation of a cohesive layer on the water surface.53,54 In order to eliminate the remaining reagents, all the suspensions were dialyzed against water several times or submitted to several cycles of washing and centrifugation. Preparation of LB SERS Platforms. Built-up films have been obtained by the vertical lifting method using a homemade LB trough at room temperature under a continuous dried nitrogen flow. In this rectangular Teflon trough (250 × 550 × 3 mm3), the subphase level was maintained constant during the experiment by adding some water (Millipore Q grade water with a resistivity higher than 18 MΩ · cm-1) when needed. Prior to LB deposition, the glass slides were cleaned with chloroform and then rinsed with distilled water and dried. A diluted suspension of functionalized silica particles in a 80%/20% (v/v) mixture of chloroform and ethanol was prepared. After spreading on a pure water subphase (pH ) 5.5), a stepwise compression of the 2D particles at the water surface was carried out until a surface pressure of 6 mN · m-1 was reached. The hydrophilic glass slides were then immersed quickly in the subphase (10 cm · min-1) and then slowly pulled out of the water (0.1 cm · min-1). In these optimized conditions, for all sizes of particles, the deposition on the substrate only occurred during the upstroke with a transfer ratio close to unity, with only one cycle to obtain a monolayer of hexagonally packed silica particles. Gold was deposited onto the colloidal crystal by a sputtering method with a Balzers SCD 030 coater. A current of 30 mA was applied between the target and sample, with a distance of 5 cm. For all substrates, the gold thickness varies between 30 and 40 nm. Following metallization, samples were sonicated in a chloroform solution for 1 min to remove the silica spheres, leaving behind gold metal triangles nanostructures that had been deposited in the interstices of the adjoining spheres. Finally, the substrates were rinsed with chloroform and dried. Preparation of Electron-Beam SERS Platforms. The substrates were prepared using a conventional e-beam lithography approach on glass substrates. ZEP 520 photoresist was used in conjunction with a LEO 1530 SEM microscope equipped with e-beam lithography capabilities. A standard development procedure was used. Patterns were generated using NPGS software. After the developing step, a thin layer of gold (40 nm) was deposited using e-beam evaporation. Functionalization of the SERS Platforms. The SERS substrates were functionalized with azobenzene thiol molecules. Samples were immersed in a 10-4 M solution of compound 5 in chloroform for 12 h. Subsequent washing of the surface with chloroform was conducted to remove any excess of azobenzene molecules or aggregates that were noncovalently adsorbed onto gold. (50) Reculusa, S.; Ravaine, S. Appl. Surf. Sci. 2005, 246, 409–414. (51) Masse´, P.; Reculusa, S.; Ravaine, S. Colloids Surf. 2006, A, 229–233. (52) Lee, Y.-L.; Du, Z.-C.; Lin, W. X.; Yang, Y.-M. J. Colloid Interface Sci. 2006, 296, 233–241. (53) Horvolgyi, Z.; Ne´meth, S.; Fendler, J. H. Langmuir 1996, 12, 997–1004. (54) Binks, B. P. Curr. Opin. Colloid Interface Sci. 2002, 7, 21–41.

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Marquestaut et al. Scheme 1

Characterization Techniques for the SERS Platforms. For large-scale platforms, the extinction maximum, λmax, of each sample was monitored and recorded with a Cary Varian V5 UV-visible-NIR Spectrometer. All macroscopic measurements were performed in standard transmission geometry within the 300-1300 nm spectral range. Scanning electron microscopy (SEM) images were collected with a LEO 1530 field emission scanning electron microscope. Raman measurements were collected by a Horiba Jobin-Yvon HR800 spectrometer with a liquid nitrogen CCD detector coupled to an Olympus inverted confocal microscope. The excitation wavelength that we used in this work was 647 nm from an Ar-Kr laser with an average intensity of 200 µW. Under such irradiation conditions, the recorded spectra were stable over time even after long acquisition times, therefore excluding any thermal damage to the monolayer or the patterned surface. The objective was either a 60×, 0.7 N.A. for measurements in solution or a 100×, 0.9 N.A. for surface measurements.

Results and Discussion Azobenzene Materials. The target azobenzene derivative was prepared in four steps from commercially available materials. First, as shown in Scheme 1, 4-(2-hydroxyethyl)phenylamine (1) was coupled with phenol via the diazonium salt to provide 2 as previously reported.49 A decyl chain was then introduced by reaction of 2 with bromodecane in the presence of K2CO3 and 18-crown-6 in THF to provide 3. The alcohol in 3 was then converted to the bromide 4 using tetrabromomethane and triphenylphosphine. Finally, the target thiol 5 was obtained by reaction of 4 with thiourea followed by basic hydrolysis. Preparation of SERS Platforms by LB Transfer. NSL has been revealed as an easy and efficient method to fabricate SERS platforms. Following the pioneering work of Fischer37 and later Van Duyne for SERS applications,31 different methods such as dip coating,55 spin coating,33 blade coating32 or dynamic thin laminar flow of latex spheres56 have been used to fabricate nanosphere monolayers in a desire to fabricate larger areas and minimize packing defects. However, the above methods often need large amounts of monodisperse colloidal solution during (55) Jensen, T. R.; Malinsky, M. D.; Haynes, C. L.; Duyne, R. P. V. J. Phys. Chem. B 2000, 104, 10549–10556. (56) Picard, G. Langmuir 1998, 14, 3710–3715.

the deposition process. In addition, while these methods can lead to homogeneous samples of a few square millimeters, larger surfaces often present dislocation defects and aggregates located on the side of the slide, preventing perfect sample-to-sample reproducibility. In contrast, the LB transfer of chemically functionalized silica spheres onto a glass slide allows one to have large-area 2D crystals on microscope slides on the order of 2.5 × 2.5 cm2. In our setup (Figure 1) involving a large Langmuir trough, several samples can be fabricated simultaneously with good reproducibility. For silica spheres with diameters ranging from 290 to 980 nm, the surface homogeneity was relatively good and no major defects were observed. As seen in SEM images (Figure 2), dislocations in the hexagonal arrangements were still observed, likely due to polydispersities of the silica nanospheres as well as by the vertical lifting through the floating silica sphere monolayer, but a high density of gold bow tie patterns were present. It is noteworthy that samples with large square centimeter surfaces were fabricated with good quality, which makes the LB transfer very effective and versatile, as shown in Figure 3. The ratio of the gold triangle sizes over the sphere projection on the substrate can be calculated considering a sphere compact arrangement. The theoretical surface of the gold triangle assemblies, ST, varies with the square of the radius of the particles or linearly with the surface of the sphere projections, SD, that corresponds to the voids on the substrate, leading to eq 1, which is calculated using an elemental site of the hexagonal surface lattice (Figure 1)

(

ST ) √3 ST )

(

π 2 r ) 1.1026r2 2

)

)

√3 1 - S ) 0.0513SD π 2 D

(1a) (1b)

The theoretical surface percentage of the gold curved triangles and voids are %ST ) 4.88% and %SD ) SD/(SD + ST) ) 95.12%, respectively, and are independent of the particle radius. We have compared these values with our samples after a thorough image analysis considering the significant deviation to perfect hexagonal compact arrangement. The image analysis using AFM software

Raman Enhancement of Azobenzene Monolayers

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Figure 1. Schematic representation of the preparation of the SERS platforms designed by the LB method. The Langmuir film of nanospheres (a) is compressed at the water surface (ab) and then transferred onto a glass substrate (c). A layer of gold (30-60 nm) is deposited onto the LB compact nanosphere layer (d), and the nanoparticles are finally removed by sonication (e). The sample is finally soaked in a solution containing the azobenzene chromophores (f).

(SPIP, Image Metrology, Table 1) shows that the percentage of gold area varies between 10 and 20% of the total sample surface. This parameter is important to evaluate the surface functionalization density and subsequently the REF. Preparation of SERS Platforms by E-Beam Lithography. E-beam lithography was used to reproduce Fischer’s pattern arrangement.21 As shown in Figure 4, the resulting patterns were obtained after deposition of 30 nm of gold. Depending on the irradiation flux of the e-beam used in the lithographic process, the size of the pattern could be varied precisely. Therefore, on the same coverslip we have inscribed a series of Fischer’s patterns where the sizes of individual triangles shown in Figure 4 a-c were varied from 145 to 385 nm and the gaps between the bow tie assemblies were 45 nm, 65, and 110 nm, respectively. The area of the individual pattern was generally (100 × 150) µm2 and was quasi-free of defects. The transmission spectra in the visible range were recorded on these samples using an optical fiber and collimating lens to homogeneously probe areas with diameters of about 50 µm, which correspond to large numbers of lattice sites. Spectroscopic Characterization. The transmission spectra of the samples were conducted in the visible-NIR range. Shown in Figure 5a are the absorption spectra of patterns obtained by the LB method. The transmission spectra showed a significant shift of the plasmon bands due to the coupling effects between the gold bow tie assemblies. The silica particle diameters were 290 (A), 390 (B), 590 (C), 770 (D), and 980 nm (E), and the normalized plasmon resonance varied from 630 to 993 nm, accordingly. The larger structures displayed a plasmon band shifted toward longer wavelengths. Shown in Figure 4b are the associated Raman spectra of an adsorbed azobenzene monolayer in the 800-1800 cm-1 spectral range. The Raman spectra exhibited intense signals at 1140 cm-1 (δ(CH) ring + ν9a), 1188 cm-1 (δ(CH) ring + ν9b), 1308 cm-1(νφ - N) assymmetric, 1408 cm-1 (νN ) N), 1456 cm-1 (ωCdC + δCH), and 1601 cm-1(ωCdC) characteristic of the E azobenzene. The absence of peak at 2590 cm-1 (not shown here) indicates the cleavage of

the H-S bond and the formation of a self-assembled azobenzene monolayer on the nanostructured gold surface. The Raman experiments were performed using an excitation wavelength of 647 nm in preresonance conditions with plasmon bands (A) as shown in Figure 5a. Note that the use of this wavelength does not induce efficient photoisomerization of the azobenzene moieties since it is located outside the absorption band of the chromophores. The largest Raman enhancement was observed for the A pattern, revealing that even the preresonance condition with the surface plasmon frequency can induce efficient Raman enhancement as opposed to out-of-resonance measurements (samples C-E). The larger enhancement was obtained with the smallest particles (290 nm) and triangular sizes of about 85 nm sizes. Similar measurements were performed on e-beam platforms. Shown in Figure 6 are the transmission spectra performed for samples with triangle sizes varying from 120 to 380 nm. Similarly with the observations reported for the LB nanosphere films, the transmission spectra were red-shifted for patterns with larger triangular sizes. The Raman spectra were recorded on the e-beam samples, and significant enhancements were observed for samples with plasmon frequencies localized in resonance with the excitation wavelength. With the excitation laser wavelength (λ ) 647 nm) being in the middle of the plasmon bands of platforms F and G, the Raman spectra should be very similar for both samples before normalization to the gold surface coverage. However, in this set of samples, the Raman spectrum was slightly more intense for the F sample, and since the gold coverage of F was smaller than for G, this results in a higher Raman enhancement as shown in Table 2. More precisely, one has to consider that, in SERS, the enhancement factor at each molecule varies with the geometry, the size, and the nature of the metallic substrate but also depends on the matching of the electric field of both the excitation source and the Raman signal with the plasmon band.21,57 (57) Schatz, G. C.; Young, M. A.; Van Duyne, R. P. In Surface-Enhanced Raman Scattering; Kneipp, K., Moskovits, M.,Kneipp, H., Eds.; Springer-Verlag Berlin: Berlin/Heidelberg, 2006; Vol. 103, pp 19-46.

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The electromagnetic enhancement factor of SERS signal is thus given by57

F ) |E(νexcitation)|2|E(νRaman)|2

(2)

where E(νexcitation) and E(νRaman) are the local electric-field enhancement factors at the incident frequency (νexcitation) and at the Raman Stokes frequency(νRaman), respectively. However, since the plasmon width is large compared to the Raman stokes shift, both electromagnetic fields are often in resonance and/or preresonance with the plasmon band. Therefore, an approximation is to assume that E(νexcitation) and E(νRaman) are the same, leading to an enhancement factor proportional to F ) |E(νexcitation)|4. In such an approximation, the matching of the excitation laser line

Figure 3. Example of an optimized sample transferred by LB and followed by gold deposition and sonication for modified silica particles of 312 nm. The quality of the hexagonal arrangement is similar on the whole 2.5 × 2.5 cm2 area.

with the plasmon frequency is an essential condition to obtain large Raman surface enhancements. It is noteworthy that the SERS Raman features and frequencies of the peaks were very similar to those obtained in conventional NSL samples, but a significant background was superimposed on the spectra as shown in Figure 6b. This background is most likely due to residue of the photopolymer used for the lithography process. In order to establish the relationship between the nanostructured surface and the SERS activity of the platform, the enhancement factors have been determined. The determination of the REF in SERS is a prerequisite to quantify the enhancement factor of the Raman signal. The REF can be estimated by comparing the measured SERS intensities (ISERS) with the nonenhanced Raman scattering intensities (INE) using eq 1:29,36

REF )

Figure 2. SEM images of SERS platforms prepared by the LB technique using functionalized silica nanospheres of (a) 290 nm, (b) 390 nm, (c) 460 nm, (d) 590 nm, and (e) 980 nm. A layer of 30 nm of gold was deposited followed by sonication.

ISERS NNE INE NSERS

(1)

where NNE and NSERS are the number of molecules under laser illumination for the reference sample (liquid) and for the functionalized surface patterns. Despite an inherent experimental difficulty in precisely measuring the REF, it is necessary to have a good knowledge of the metallic surface area to estimate the number of scattering molecules that participate in the Raman signal. In the present approach we have considered the following points to precisely estimate the enhancement factors obtained with our optical configuration. (i) A Raman reference sample was first measured in solution. An azobenzene solution was prepared in chloroform and probed with our microscope apparatus using a 60×, 0.7 N.A. objective. The volume of the focal point in the solution was first measured by measuring the change of intensity of the 520 cm-1 band of a silicon substrate immersed in the same solvent. By varying the focus of the laser on the immersed silicon sample, the intensity variation of the Si peak shows a Gaussian-type variation as shown

Raman Enhancement of Azobenzene Monolayers

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Table 1. Geometric and Spectroscopic Parameters for the NSL Platformsa sample

A

B

C

D

E

silica spheres diameter D (nm) triangles side size (nm) extinction maximum λmax (nm) theoretical gold surface coverage theoretical enhancement factor (λexc)647nm) experimental gold surface coverage experimental enhancement factor (λexc)647nm)

290 85 645 4.88% 1.14 105 15% 3.70 × 104

390 100 704 4.88% 5.90 104 20% 1.44 × 104

460 150 740 4.88% 2.21 104 18% 5.99 × 103

590 180 840 4.88% 1.62 104 15% 5.28 × 103

980 315 996 4.88% 9.10 103 10% 4.44 × 103

a

Extinction maxima were recorded on three samples for each size by transmission UV-visible-NIR spectroscopy.

Figure 4. (a-c) SEM images of the gold patterns prepared by e-beam lithography. In this series, the sizes of the triangle sides are (a) 145 nm, (b) 185 nm, and (c) 385 nm. Similar samples have been used for Raman tests, with gaps of 45, 65, and 110 nm for triangles with similar sizes. (d) large size area showing the regularity of the e-beam substrates.

in Figure 7. The width at half-height gives the waist length of the focal volume in the considered liquid. The transverse dimension (radius of the spot) is determined with the numerical aperture of the objective used for a specific wavelength. For the liquid Raman reference measurement, the waist of the beam is about 14.5 µm (Figure 7), and the radius of the beam with a 0.7 N.A. objective is about 1.22 × λ/N.A. ) 1.13 µm, resulting in a cylindrical volume of ∼16 µm3. We however apply a (10%

Figure 5. (a) Absorption spectra of the gold pattern inscribed on glass using the NSL method. The size of the triangle sides was varied from 85 nm (A) to 315 nm (E) for corresponding nanospheres of 290 to 980 nm, respectively. (b) Raman spectra of an azobenzene-thiol functionalized at the surface of the SERS platform. The excitation wavelength was fixed at 647 nm, and irradiance was similar for the five samples.

uncertainty to this estimation because of the noncylindrical shape of the waist. (ii) Knowing the concentration of the reference solution (C ) 10-2 M), we can estimate the number of molecules located at the focus that generate the bulk Raman signal, NNE, which is about 9.84 × 107 molecules. (iii) For the SERS platforms, one needs to assume that our gold patterns are homogeneously covered with a monolayer of azobenzene moieties with molecular area of 0.3 nm2 using the

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Figure 7. (a) Set up to measure the longitudinal dimension of the beam waist in a liquid sample. (b) Variation of the Raman signal of silicon (520 cm-1) as as function of the Z position of the objective. The full width at half-maximum (fwhm) is ∼14.5 µm.

Figure 6. (a) Microabsorption spectra of Fischer patterns inscribed on glass by e-beam lithography. The sizes of the triangles are (F) 145 nm (G), 185 nm and (H) 385 nm with corresponding λmax at 600, 650, and 800 nm, respectively. (b) Maximum Raman enhancement was obtained for the e-beam pattern with the smallest structures and with plasmon frequency maximum located at λ ) 600 nm. The irradiation source was λ ) 647 nm with intensity of 220 µW measured at the sample. Spectra were averaged over 10 different spots on the patterns. Table 2. Geometric and Spectroscopic Parameters of the Substrates As Prepared by E-Beam Lithographya sample

F

G

H

triangles size /nm maximum extinction /nm gold coverage enhancement Factor

145 622 16% 1.07 × 105

185 667 25% 3.93 × 104

385 813 57% 1.49 × 103

a Extinction maxima and gold coverage were recorded over three samples for each size.

average estimated from Langmuir compression measurements.58 We used the same molecular area for gold funtionalization with azobenzene thiols, although we can consider that our estimation is within an error of (5%. The surface of gold can be measured with image analysis software from the SEM images of our patterns. Such surfaces vary with the size of the inscribed features. (58) Engelking, J.; Witteman, M.; Rehahn, M.; Menzel, H. Langmuir 2000, 16, 3407–3413.

Assuming that a monolayer is formed on gold, and that we know the total gold surface as well as the molecular area, one can determine the number of scattering moieties under the focalized beam. (iv) INE and ISERS are the Raman intensities of a given vibrational mode of the azobenzene chromophore. Intensities of the laser irradiation source as well as acquisition times were normalized for comparison of the Raman intensities. Summarized in Tables 1 and 2 are the enhancement factors calculated for the 1140 cm-1 Raman peak (δ(CH) ring + ν9a) for the LB platforms as well as for the e-beam substrates. For all the samples, it is noteworthy that the magnitude of the enhancement varies between 103 and 105. The maximum enhancements were observed for measurements where the excitation source matched the plasmon frequency of the array. When such conditions were fulfilled, the REFs were about 3.7 × 104 and 1.7 × 105 for LB and e-beam platforms, respectively. These values are of the same order of magnitude, indicating that for the preparation of SERS platforms involving large surface areas, the LB method is quite effective, while at the same time being rapid and economical. In addition, the background signal was very low in comparison with that associated with the e-beam substrates. Nevertheless, the e-beam substrates are more versatile since the gap and the size of the structures are well controlled. Even in simple structures such as the Fischer patterns, this permits one to precisely tune the plasmon frequency of the surface according to the Raman excitation line. In the case of a functionalization with a self-assembled monolayer, the Raman spectra can be recorded in short times (seconds) while no signal was recorded on a flat gold surface as prepared by

Raman Enhancement of Azobenzene Monolayers

e-beam evaporation. This emphasizes the tremendous interest of developing e-beam patterns on transparent substrates (glass coverslips, fused silica) for Raman measurements in a transmission geometry setup.

Conclusions In the present study we have compared two series of SERS platforms made on glass coverslip substrates. In the first series of experiments, we have investigated for the first time the ability of the LB technique to fabricate large sample areas of compact monolayers of silica particles that were used as a template to make gold triangle arrangements. This method was originally an effective approach for preparing large quantities of 2D colloidal crystals, and we have now demonstrated that these colloidal crystals can be used as templates to prepare SERS platforms with large dimensions. In the second series of experiments, e-beam lithography was used to reproduce defect-free samples with similar geometries. In both types of samples, Raman enhancement was observed, allowing one to record the signal of a monolayer within seconds of acquisition under low irradiances. In each case, our experiments showed that maximum Raman enhancement was observed when the Raman excitation frequency was close to the

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surface plasmon resonance frequency of the substrates. The larger enhancements were obtained with e-beam substrates although perfect matching of the excitation laser light with the plasmon frequency was not exactly fulfilled. Good enhancements were also observed for the LB substrates, considering their ease of preparation and the large surfaces available through this fabrication method. Choice of platform should therefore be considered on a case-by-case basis depending on the specific requirements of the application. In general, such metallic patterns inscribed on optically transparent samples could be incorporated in glass microfluidic channels for in situ measurements to detect low level concentrations using vibrational Raman spectroscopy. Acknowledgment. The authors wish to thank Be´atrice Agricole, CRPP, for the preparation of the LB nanosphere platforms. E.G. and F.L.-L. wish to thank NSERC discovery grants (Canada) and the Canadian Research Chair program. The authors are grateful to the CNRS and to the conseil regional d’Aquitaine (France) for financial support including a Ph.D. fellowship (N.M.). LA801697U