Characterization of Silane-Modified Immobilized Gold Colloids as a

A Practical Method to Fabricate Gold Substrates for Surface-Enhanced Raman Spectroscopy. Ratna Tantra , Richard J. C. Brown , Martin J. T. Milton , Di...
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Anal. Chem. 2001, 73, 4268-4276

Characterization of Silane-Modified Immobilized Gold Colloids as a Substrate for Surface-Enhanced Raman Spectroscopy Lydia G. Olson, Yu-Shui Lo, Thomas P. Beebe, Jr., and Joel M. Harris*

Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850

Immobilized gold colloid particles coated with a C-18 alkylsilane layer have been characterized as a substrate for surface-enhanced Raman scattering (SERS) studies of adsorption onto hydrophobic surfaces. Atomic force microscopy images, optical extinction spectra, and SERS measurements are reported as a function of accumulation of gold colloid on glass. As the metal particles become increasingly aggregated on the surface, the SERS enhancement increases until the plasmon resonance shifts to wavelengths longer than the excitation laser. The gold colloid substrates are stable and exhibit reproducible SERS enhancement. When octadecyltrimethoxysilane is self-assembled over the gold, the metal surface is protected from exposure to solution-phase species, as evidenced by the inhibition of chemisorption of a disulfide reagent to the overcoated gold surface. The results show that interactions with gold can be blocked by a silane layer so as not to significantly influence physisorption of molecules at the C-18/solution interface. The SERS enhancement from these C-18-overcoated gold substrates is reproducible for different films prepared from the same colloidal suspension; the substrates are also stable with time and upon exposure to laser irradiation. The ability to monitor adsorption of molecules onto solid surfaces from liquids is critical to understanding separation processes, heterogeneous catalysis, environmental transport, and other interfacial phenomena. The low concentration of an adsorbate layer compared with the overlaying bulk solvent concentration makes in situ observation of adsorption a challenging problem. One potential tool for overcoming this challenge is surface-enhanced Raman spectroscopy (SERS).1-3 When this phenomenon is combined with efficient collection optics and sensitive multichannel detectors, submonolayer coverages of adsorbate can be detected in observation times of less than 1 s.4 A variety of colloidal and solid support-based substrates have been developed for SERS.5 Requirements for a strong electromagnetic enhancement5 are typically met with silver using excitation in the green region of the spectrum or with gold when the excitation is in the red or near-infrared.6 (1) Fleischmann, M.; Hendra, P. J.; McQuillan, A. J. Chem. Phys. Lett. 1974, 26, 163-166. (2) Jeanmaire, D. L.; Van Duyne, R. P. J. Electroanal. Chem. 1977, 84, 1-20. (3) Albrecht, M. G.; Creighten, A. S. J. Am. Chem. Soc. 1977, 99, 5215-5218. (4) Lacy, W. B.; Williams, J. M.; Wenzler, L. A.; Beebe, T. P.; Harris, J. M. Anal. Chem. 1996, 68, 1003-1011.

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Metal substrates require controlled-size nanoscale roughness features to allow excitation of a plasmon resonance. Suspended metal colloids, with a high specific surface area, can be advantageous to use in observing SERS from low concentrations of adsorbate; colloidal suspensions, however, will often aggregate spontaneously depending on the composition of an added sample, which can change their enhancement.7 To avoid variation in enhancement due to changes in aggregation, most SERS investigations employ solid support-based SERS substrates. Some substrates developed for SERS are as follows: metal island films,4,8,9 electrochemically roughened metals,10 chemically etched metal foils,11,12 sol-gel silicate glass with trapped gold particles,13 and chemically reduced silver on alumina-coated glass slides14 or solid-phase extraction membranes.15 Silver has been thermally evaporated over a number of rough surfaces such as aluminacovered glass,16,17 TiO2-coated glass,18 filter paper,19,20 and surfacebound polystyrene latex particles.21 Silver has also been lithographically etched over an array of silica posts or a polymer sheet ablated by O2 sputter-etched posts22,23 and abrasively roughened optical fibers.24 A significant problem with many of these substrates, however, is that they do not provide reproducible enhancement; their response can be unstable over time or change with exposure to an adsorbate solution or laser irradiation. Silver can oxidize upon (5) Garrell, R. L. Anal. Chem. 1989, 61, 401A-411A. (6) Ruperez, A.; Laserna, J. J. In Modern Techniques in Raman Spectroscopy; Laserna, Ed.; John Wiley & Sons Ltd.: New York, 1996; Chapter 6. (7) Rodger, C.; Rutherford, V.; White, P. C.; Smith, W. E. J. Raman Spectrosc. 1998, 29, 601-606. (8) Schlegel, V. L.; Cotton, T. M. Anal. Chem. 1991, 63, 241-247. (9) Walls, D. J.; Bohn, P. W. J. Phys. Chem. 1989, 93, 2976-2982. (10) Kruszewski, S. Surf. Interface Anal. 1994, 21, 830-838. (11) Carron, K.; Peitersen, L.; Lewis, M. Environ. Sci. Technol. 1992, 26, 19501954. (12) Xue, G.; Dong, J. Anal. Chem. 1991, 63, 2393-2397. (13) Akbarain, F.; Dunn, B. S.; Zink, J. I. J. Raman Spectrosc. 1996, 27, 775783. (14) Li. Y. S.; Wang, Y. Appl. Spectrosc. 1992, 46, 142-146. (15) Szabo, N. J.; Winefordner, J. D. Appl. Spectrosc. 1998, 52, 500-512. (16) Vo-Dihn, T.; Stokes, D. L. Appl. Spectrosc. 1993, 47, 1728-1732. (17) Bello, J.; Stokes, D. L.; Vo-Dinh, T. Appl. Spectrosc. 1989, 43, 1325-1330. (18) Bello, J.; Stokes, D. L.; Vo-Dinh, T. Anal. Chem. 1989, 61, 1779-1783. (19) Cabalin, L. M.; Laserna, J. J. Anal. Chim. Acta 1995, 310, 337-345. (20) Lee, A. S. L.; Li, Y. S. J. Raman Spectrosc. 1994, 25, 209-214. (21) Schueler, P. A.; Ives, J. T.; DeLaCroix, F.; Lacy, W. B.; Becker, P. A.; Li, J.; Caldwell, K. D.; Drake, B.; Harris, J. M. Anal. Chem. 1993, 65, 31773186. (22) Wachter, E. A.; Storey, J. M. E.; Sharp, S. L.; Carron, K. T.; Jiang, Y. Appl. Spectrosc. 1995, 49, 193-199. (23) Szabo, N. J.; Winefordner, J. D. Anal. Chem. 1997, 69, 2418-2425. (24) Mullen, K. I.; Carron, K. T. Anal. Chem. 1991, 63, 2196-2199. 10.1021/ac000873b CCC: $20.00

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exposure to air, which will change the metal film thickness and alter the enhancement. Evaporated metal island films can exhibit thickness variations across a substrate caused by different distances between the substrate surface and the metal vapor source. Electrochemically roughened SERS surfaces can also change enhancement over time, and the signal is not generally reproducible if the surface is roughened in the presence of the analyte.25 To avoid the stability and reproducibility problems with evaporated and electrochemically roughened films and with metal colloid suspensions, Natan and co-workers26-29 have shown that gold colloids can be formed in solution and then immobilized onto a solid support for use as a SERS substrate. Use of an amine- or mercapto-functionalized glass surface to capture the metal particles from solution allows the particle size, spacing, and aggregation to be controlled to a greater degree than many other enhancing surfaces.26-32 The method of colloid preparation allows the metal particle size to be controlled;33 citrate reduction gives monodispersed colloids from 10 to 40 nm but is irreproducible with colloids outside this size range. To make larger colloids with high monodispersity, Natan and co-workers34 have shown that small gold seed colloids can be added to a reaction solution and grown to sizes as large as 125-nm diameter. The SERS substrate is formed by capturing these colloids onto a surface by adsorption from a homogeneous suspension; this procedure allows for a large number of substrates to be prepared simultaneously to provide a uniform batch of SERS-active surfaces having the same enhancement characteristics. The average gold particle spacing on the surface can be controlled by the coverage of surface functional groups used to capture the colloids and by the presence of adsorbates29,35 that can shield electrostatic repulsion between particles.36 Since the phenomenon was first observed,1 SERS has been employed to study adsorption of molecules onto metal surfaces. Using SERS to study molecules adsorbed to dielectric surfaces, however, is less common. Walls and Bohn,9 Lacy et al.,4 and Hill et al.37 observed molecules adsorbed onto SiO2-overcoated Ag island films while Parry and Dendramis38 and Quagliano et al.39 coated an oxidized silicon or GaAs substrate with analyte and evaporated an Ag island film on top. Alkanethiols have been (25) Kudelski, A.; Bukowska, J. Vib. Spectrosc. 1996, 10, 335-339. (26) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735-743. (27) Freeman, R. G.; Grabar, K. C.; Allison, K. J.; Bright, R. M.; Davis, J. A.; Guthrie, A. P.; Hommer, M. B.; Jackson, M. A.; Smith, P. C.; Walter, D. G.; Natan, M. J. Science 1995, 267, 1629-1632. (28) Bright, R. M.; Walter, D. G.; Musick, M. D.; Jackson, M. A.; Allison, K. J.; Natan, M. J. Langmuir 1996, 12, 810-817. (29) Grabar, K. C.; Smith, P. C.; Musick, M. D.; Davis, J. A.; Walter, D. G.; Jackson, M. A.; Guthrie, A. P.; Natan, M. J. J. Am. Chem. Soc. 1996, 118, 1148-1153. (30) Doron, A.; Katz, E.; Willner, I. Langmuir 1995, 11, 1313-1317. (31) Chumanov, G.; Sokolov, K.; Gregory, B. W.; Cotton, T. M. J. Phys. Chem. 1995, 99, 9466-9471. (32) Chumanov, G.; Sokolov, K.; Cotton, T. M. J. Phys. Chem. 1996, 100, 51665168. (33) Frens, G. Nat. Phys. Sci. 1973, 241, 20-22. (34) Grabar, K. C.; Allison, K. J.; Baker, B. E.; Bright, R. M.; Brown, K. R.; Freeman, R. G.; Fox, A. P.; Keating, C. D.; Musick, M. D.; Natan, M. J. Langmuir 1996, 12, 2353-2361. (35) Musick, M. D.; Keating, C. D.; Keefe, M. H.; Natan, M. J. Chem. Mater. 1997, 9, 1499-1501. (36) Inoue, M.; Ohtaka, K. J. Phys. Soc. Jpn. 1983, 52, 3853-4864. (37) Hill, W.; Rogalla, D.; Klockow, D. Anal. Methods Instrum. 1993, 1, 89-96.

chemisorbed onto metal substrates for hydrophobic SERS substrates for adsorption studies.11,40 Similarly, a disulfide modified 4-(2-pyridylazo)resorcinol overlayer was used for detection of Pb2+, Cd2+, and Cu2+ ions.41 These studies demonstrate that SERS is not limited to investigating molecules directly adsorbed onto metal surfaces. A variety of SERS-active structures can be prepared for monitoring molecular adsorption to hydrophilic or hydrophobic surfaces. The surface modifications of the metal must be thin, ranging from a single molecular layer to no more than several hundred angstroms to avoid loss of electromagnetic enhancement.9,42 While the electromagnetic SERS enhancement can persist over these distances, an insulating layer over the metal will remove the possibility of chemical or charge-transfer enhancement; this can make the enhancement less sensitive to changes in solution composition that could modify the charge-transfer characteristics of the metal surface. In the present work, a trimethoxysilane with a C-18 alkane chain is self-assembled and cross-linked on an immobilized gold colloid surface to produce a stable, hydrophobic SERS substrate. The surface hydrophobicity is controlled by the time in silane reagent solution; the multiple reactive (methoxysilane) functional groups self-assembled onto a surface from a dry solvent allow the silane to polymerize laterally across the surface to form a robust monolayer.43,44 The structure and spectroscopy of silane-overcoated gold colloid substrates are investigated using AFM, UVvisible spectroscopy, and SERS measurements. The effects of the gold colloid aggregation on the plasmon resonance and the wavelength dependence of the SERS enhancement are compared. Using the C-18-modified substrate, the stability and reproducibility of the SERS response are characterized, and the integrity of the silane layer and its ability to block molecular contact with the underlying gold surface are tested. EXPERIMENTAL SECTION Reagents. Hydrogen tetrachloroaurate(III) trihydrate (reagent grade), 2-pyrrolidinone (99%), (n-octadecyl)trimethoxysilane (OTMS, 95%), dibenzyl disulfide (DBDS, 98%), and trisodium citrate (99%) were obtained from Aldrich; sodium borohydride and HCl came from EM Science; (3-aminopropyl)trimethoxysilane (APTMS, 95%) came from Fluka. Methanol (Optima grade), benzoic acid, nheptane (spectrophotometric grade), 30% H2O2, and glass microscope slides were acquired from Fisher. Concentrated HNO3 and H2SO4 (reagent grade) were purchased from Mallinckrodt. Cetylpyridinium chloride (CPC, 99%) was obtained from Janssen. Heptane was dried over sodium; all other chemicals were used without further purification. Water was quartz distilled and then filtered through a Barnstead Nanopure II system to give a resistivity of 18 MΩ‚cm. (38) Parry, D. B.; Dendramis, A. L. Appl. Spectrosc. 1986, 40, 656-661. (39) Quagliano, L. G.; Jusserand, B.; Ladan, F. R.; Izrael, A. J. Electron Spectrosc. Relat. Phenom. 1993, 64, 177-182. (40) Bercegol, H.; Foerio, F. J. Langmuir 1994, 10, 3684-3692. (41) Crane, L. G.; Wang, D. X.; Sears, L. M.; Heyns, B. Carron, K. Anal. Chem. 1995, 67, 360-364. (42) Kovacs, G. J.; Loufty, R. O.; Vincett, P. S.; Jennings, C.; Aroca, R. Langmuir 1986, 2, 689-694. (43) Wirth, M. J.; Fatunmbi, H. O. Anal. Chem. 1992, 64, 2783-2786. (44) Finklea, H. O.; Robinson, L. R.; Blackburn, A.; Richter, B. Langmuir 1986, 2, 239-244.

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Gold Colloid Preparation. Gold colloid suspension was prepared by following procedures developed by Natan and coworkers.26-29,34 The colloid size was controlled by the amount of citrate added to reduce the AuCl4-, where higher citrate concentration yields smaller colloids.33 All glassware was cleaned using freshly prepared aqua regia (3 parts of HCl to 1 part of HNO3). To produce 32-nm-diameter colloids, 1.0 mM aqueous HAuCl4 was brought to a rolling boil while stirring; 17.5 mL of aqueous trisodium citrate (38.8 mM) was then added, and boiling was continued for 10 min. After cooling the colloidal suspension while stirring, the solution was filtered through a 0.8-µm Gelman membrane filter. For the formation of larger colloids, seed colloids were added to aid in obtaining a smaller dispersion in size and shape. For the seed colloids, 1 mL of a 1% HAuCl4 solution was mixed with 100 mL of H2O; after stirring for 1 min, 1 mL of 1% citrate solution was added and the mixture was stirred for 1 min. One milliliter of 0.075% NaBH4 was then added; the colloidal solution was stirred for 5 min. Colloids ∼60 nm in diameter were made by refluxing 1 mL of 1% HAuCl4 in 225 mL of water and then adding 150 µL of seed colloids and 1 mL of a 1% citrate solution; the mixture was refluxed for 10 min and then allowed to cool while being stirred.34 Individual particle sizes were determined from averaging the heights of AFM images of 20 different colloids immobilized on glass at low surface coverages (see below). The lateral dimensions were convoluted with the AFM tip shape and thus are not an accurate measure of colloid size. From the height measurements, the smaller colloids exhibited an average diameter of 32 nm with a standard deviation of 6 nm while the larger colloids had an average diameter of 60 nm with a larger standard deviation of 17 nm. SERS Substrate Preparation. Glass slides were cleaned for 10 min in piranha solution (4 parts of H2SO4 to 1 part of H2O2 at 60 °C) and rinsed with methanol. The slides were oven-dried and cooled in a desiccator to remove excess water from the surface. They were then placed in 0.5% APTMS in n-heptane for 2 h with stirring, rinsed in n-heptane, and cured for 32 min at 110 °C. The slides were rinsed profusely in both methanol and water to remove physisorbed silane and then immersed into the gold colloid suspension to immobilize gold particles onto the surface. The slides were immersed alternately in the colloid and in a 1 mM aqueous solution of 2-pyrrolidinone. Molecules such as pyrrolidinone can reduce the electrostatic repulsion between the negatively charged gold colloid particles thus allowing greater surface aggregation.29,35 The slides were then oven-dried, cooled in a desiccator, and placed in a 1 mM solution of OTMS in n-heptane for 2-24 h to adsorb a hydrophobic C-18 layer over the immobilized gold colloid surface. After being rinsed in heptane, the slides were returned to the oven for 30 min, rinsed with acetone and methanol, and stored in methanol until use. The measured contact angle for a 2-h silane adsorption was found to be 93( 1 °C; due to surface roughness, the true contact angle should be slightly lower (∼1 °C) than the measured angle.45 Substrate Characterization. Optical transmission measurements were performed using a Hitachi U-3000 UV-visible spectrophotometer at a scan rate of 600 nm/min. Coated microscope (45) Andrade, J. D.; Gregonis, D. E.; Smith, L. M. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum Press: New York, 1985; Vol. 1, Chapter 2.

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slides were supported in a water-filled cuvette. Water contact angle measurements were performed using a goniometer with a RameHart microscope (model A-100), averaging results from six sessile drops. Atomic force microscopy (AFM) imaging of the substrates was carried out in contact mode in air using a TMX 2000 Explorer (TopoMetrix, Santa Clara, CA). The force used during imaging was small (∼2 nN), and the scan rate was 5 µm/s for the 300 × 300 pixel images. The Raman spectrometer used for SERS measurements has previously been described in detail.4 The microscope slide-based substrate was tilted at 68° relative to the incident laser beam (150 mW) which was focused to a line on the sample surface using a cylindrical lens. Scattered radiation was collected at 90° relative to the excitation beam, filtered, focused into a 0.5-m spectrograph, and detected with a TE-cooled CCD. Scatter from the 647.1-nm line of a krypton ion laser (Coherent Innova 90) was filtered with a holographic notch filter (Kaiser Optics), while scatter from the 752.5-nm line was filtered with two 5-mm RG 780 optical glass filters (Schott Glass). RESULTS AND DISCUSSION Characterization of Gold Colloid SERS Substrates. The enhancement of Raman scattering from most metal colloid substrates derives principally from aggregates, which greatly increase the amplitude of the plasmon resonance and corresponding electromagnetic enhancement compared to isolated metal particles.46-48 For example, SERS from an isolated metal particle ∼1 µm in diameter was observed to exhibit an enhancement on the order of 104 while a cluster of the same size particles provides an additional 100-fold increase in the enhancement.49 To determine how the aggregation of captured colloids influences their optical properties and Raman-scattering enhancement, SERS substrates were prepared by first binding an amine-terminated silane (APTMS) onto the surface of a glass slide, which then captures gold colloids onto the glass surface upon exposure to a gold colloid suspension. Additional layers of gold particles are deposited by alternate immersion of the substrate in the metal colloid suspension and a 1 mM aqueous solution of pyrrolidinone to reduce electrostatic repulsion between gold particles and to allow aggregation of a multilayer on the surface.25,29 To determine the trend of SERS enhancement with the aggregation of immobilized gold colloids on the surface, samples were made with an increasing number of immersions in 32-nm colloid suspension and then 1 mM pyrrolidinone (30 min each). Figure 1 shows the optical extinction spectra of the 32-nm gold colloid substrates for 1-19 deposition steps. At low coverages, the Mie resonance of the isolated gold colloid particles appears as a weak band at 528 nm; this maximum does not change as particle aggregation increases on the surface. The wavelength of maximum extinction and the plasmon resonance bandwidth are dependent on the size and shape of the metal particles or aggregates.50,51 As more colloid is bound to the surface, the band (46) Mabuchi, M.; Takenaka, T. Fujiyoshi, Y.; Uyeda, N. Surf. Sci. 1982, 119, 150-158. (47) Blatchford, C. G.; Campbell, J. R.; Creighton, J. A. Surf. Sci. 1982, 120, 435-455. (48) Ahern, A. M.; Garrell, R. L. Langmuir 1995, 7, 254-261. (49) Xiao, T,; Ye, Q.; Sun, L. J. Phys. Chem. B 1997, 101, 632-638. (50) Goodman, S. L.; Hodges, G. M.; Trejdosiewicz, L. K.; Livingston, D. C. J. Microsc. 1981, 123, 201-213.

Figure 1. UV-visible spectra of immobilized 32-nm gold colloids with increasing surface coverage. The spectra range from 1 to 19 deposition steps, which increase the optical extinction and shift the multiparticle plasmon resonance to longer wavelengths (arrow).

at 528 nm in the extinction spectrum appears as a shoulder at longer wavelengths on a much larger band. This band between 600 and 700 nm is from the aggregate plasmon resonance and exists only when the colloids become close enough together to interact electronically. As the number of dips into the colloid suspension is increased, the extinction of the multiple-particle resonance grows and shifts to longer wavelengths as the characteristic aggregate size increases. This trend is also observed in the optical spectra of metal island films; as the average thickness of the evaporated film increases, the average size of the islands increases while the distance between them decreases, causing the maximum of the extinction spectra to shift to longer wavelengths,52 To address the question of how the gold colloid particle size affects the properties of the immobilized film, samples were also prepared by alternate exposures of the substrate to a suspension of 60-nm gold particles (1 h) and 1 mM pyrrolidinone (30 min). The time in colloid solution and also the number of dips were increased for the 60-nm colloids to partially offset the 16 times lower particle concentration (0.14 versus 2.3 nM). The larger colloids did not bind as readily to the amine-terminated surfaces as did the 32-nm colloids and were not as stable in suspension. The maximum extinction is less than one-third of that of the 32nm colloid following similar exposure to the colloid suspension as shown in Figure 2. Due to the larger particle size and corresponding lower frequency plasmon resonance, the singleparticle extinction for the 60-nm colloids is shifted to longer wavelength by 24 nm to a maximum at 552 nm, as shown in Figure 2. The multiparticle resonance is also shifted even farther to the red and is broader for the 60-nm colloid substrate, with a final maximum wavelength of 850 nm compared to 690 nm for the 32nm particles. Note that the peak from isolated-particle plasmon resonances persists even at high colloid coverages; this response is not due to regions of the glass surface that are sparsely coated since the AFM results (see below) indicate that the substrate is well covered at high coverages. Most likely the isolated-particle resonance derives from colloids that protrude from the surface (51) Anno, E.; Hoshino, R. J. Phys. Soc. Jpn. 1982, 51, 1185-1192. (52) Gotschy, W.; Vonmetz, K.; Leitner, A.; Aussenegg, F. R. Appl. Phys. B 1996, 63, 381-384.

Figure 2. UV-visible spectra of immobilized 60-nm gold colloids with increasing surface coverage. Spectra are plotted for 1, 2, 3, 4, 7, 13, 16, 19, 22, and 25 deposition steps indicated by the arrow.

of the aggregated structure and are oriented such that the incident electric field does not lead to efficient coupling with neighboring particles; evidence of particles are shown in the AFM results (see below). Persistent isolated-particle resonances have been observed in the extinction spectra of aggregated gold particles in solution;53 TEM micrographs showed no individual particles in the dispersion, but isolated-particle resonances were still observed, probably arising from edges of the aggregated structures. To learn how the optical properties of the immobilized colloids relate to the physical structure of these films, AFM images were collected for both the 32- and 60-nm gold substrates for several different surface coverages, and example results are presented in Figure 3. Individual colloids could be readily observed in the lowest coverage samples (Figure 3 b,d). The sizes (height and diameter) of the isolated particles are equivalent to the colloids aggregated into clusters, which shows the growth of clusters from individual particles in the assembly of the films. In aggregates of particles, the spacing appears to be close-packed within the AFM resolution (spacing less than 0.2 particle diameter), which is consistent with their strong optical coupling predicted theoretically54 and demonstrated previously with isolated two-particle silver clusters.55 Several particles in the low-coverage panels are truncated on an edge, which could be due to their movement at low coverages by the shear force exerted from the AFM tip during scanning. This effect was not observed in images of higher coverage, aggregated films. These results likely indicate that the aggregated films are more stable structures due to multiple points of contact and adhesion. The occasional perturbation of the lateral dimension in the low-coverage images would not, however, significantly affect the particle size or surface roughness measurements (see below) since these parameters are determined from height data and not the lateral information in the image. It has been suggested that characterization of surface roughness can be effectively done with four parameters having low statistical correlation: arithmetic mean roughness, Ra; bearing ratio, Tb; radius of asperity, r; and mean slope, ∆a, as a subset of the more than 30 parameters describing surface roughness.56 (53) Blatchford, C. G.; Campbell, J. R.; Creighton, J. A. Surf. Sci. 1982, 102, 435-455. (54) Gersten, J. I.; Nitzan, A. Surf. Sci. 1985, 158, 165-189. (55) Xiao, T.; Yi, Q.; Sun, L. J. Phys. Chem. B 1997, 101, 632-638.

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Figure 3. AFM images of immobilized colloids: (a) APTMS-derivatized slide (b) APTMS-derivatized slide exposed to 32-nm gold colloid suspension for 30 min. (c) APTMS-derivatized slide following 18 deposition steps of 32-nm gold colloids. (d) APTMS-derivatized slide exposed to 60-nm gold colloid suspension for 1 h. (e) APTMS-derivatized slide following 24 deposition steps of 60-nm gold colloids.

Since r and ∆a describe surface feature shape (and since we have bound spherical colloids to the surface), the two parameters Ra and Tb are most relevant to describe the colloidal film structure in the present system. (56) Nowicki, B. Wear 1985, 102, 161-176.

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The arithmetic mean roughness describes the pixel-wise deviation of the surface height from its average, Ra ) (1/N)∑|zi - zav|. The glass support is flat and smooth on the scale of these measurements (Ra ) 0.15 nm) and does not contribute to the topography of the colloid-coated substrate. An AFM image taken

of the APTMS-derivatized slide (Figure 3a) exhibits a roughness that is slightly greater than the glass substrate, Ra ) 0.7 nm, indicating some variation in silane thickness. The AFM images from substrates prepared by a single exposure to gold colloid (Figure 3b,d) exhibit a relatively smooth surface as indicated by a surface roughness Ra ) 6 ((3) and 22 ((6) nm, for 32- and 60-nm colloids, respectively, where the average deviation is much less than the particle size due to the limited coverage of the surface by the particles where the uncovered, smooth substrate contributes significantly to the average. This result is consistent with extinction spectra of the plasmon resonance in Figures 1 and 2, which showed that the surface optical properties are dominated by the isolated particle response. For the slides with films derived from multiple deposition steps, the AFM images are similar in appearance and exhibit indistinguishable surface roughness after several depositions have been made; average roughness for the two different colloids are Ra ) 11 ((4) nm for the 32-nm gold colloids and Ra ) 263 ((10) nm for the 60-nm colloids. The average roughness of the 32-nm colloid surface is comparable to the particle radius, which indicates a well-aggregated film with few protrusions; the much larger roughness of the 60-nm colloids (4 times the particle diameter) exhibits many more protruding particles, which is consistent with the optical extinction spectrum that exhibits significant isolated-particle plasmon resonance structure even at high coverages. The bearing ratio of a surface, given by Tb ) (100/L)∑Zi(b), is the percentage of the surface between the highest peak and a variable depth b relative to the profile length L; Zi(b) is the amount of the surface that falls above b over the given length. The shape of a Tb curve is an indication of the topography of the surface.56,57 The bearing ratio for slides with 1, 4, 7, 12, and 18 depositions of 32-nm colloid is plotted in Figure 4a, that for slides with 1, 7, 19, and 27 depositions of 60-nm colloid is given in Figure 4b. Both of these plots show that as the number of depositions increases, the fraction of bare surface decreases and a more uniform height film (flat top of the bearing ratio curve) is produced as the colloid particles aggregate on the surface. The greater uniformity of the aggregated 32-nm colloid film is also apparent in these results, where at the highest coverages the bearing ratio falls rapidly at large values of b, whereas the 60-nm colloid film shows a long decay of the bearing ratio with b, which is consistent with greater height variation and more particles from the film surface. The Raman scattering enhancement provided by the immobilized gold colloid films was assessed by exposing each of the samples to a solution of DBDS, which has been shown to chemisorb onto metal surfaces through dissociation of the disulfide bond and binding of benzylthiolate groups to the metal.58 SERS spectra of DBDS bound to the gold colloid substrates (see details below, Figure 7a) allow the relative enhancement of the different substrates to be compared. The intensity of the symmetric ring-breathing mode of DBDS (1001 cm-1) from excitation at 647.1 and 752.5 nm is plotted against the number of colloid depositions in Figures 5 and 6. All cases show a similar trend where the strength of the Raman scattering is observed initially to increase with the number of depositions of colloid, followed by a decrease with further depositions. For a 32-nm gold colloid (57) Westra, K. L.; Thomson, D. J. J. Vac. Sci. Technol. 1995, 13, 344-349. (58) Sandroff, C. J.; Herschbach, D. R. J. Phys. Chem. 1982, 86, 3277-3279.

Figure 4. Bearing ratio, Tb, of the colloid surfaces versus depth into the film, b: (a) 32-nm colloid surfaces with 1 (b), 4 (9), 7 (2), 12 ([), and 18 (X) depositions; (b) 60-nm colloid surfaces with 1 (b), 7 (9), 19 (2), and 27 ([) depositions.

Figure 5. Maximum SERS intensity at 1001 cm-1 (left axis, b) and the wavelength of the multiparticle extinction maximum (right axis, 9) vs number of 32-nm colloid depositions: (a) excitation at 647.1 nm; (b) excitation at 752.5 nm. The lines through the points are only to guide the eye.

substrate (Figure 5), the SERS intensity of DBDS with excitation at 647.1 nm increases for the first five depositions; the intensity then falls off to a level that is independent of further accumulation Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

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Figure 6. Maximum SERS intensity at 1001 cm-1 (left axis, b) and the wavelength of the multiparticle extinction maximum (right axis, 9) vs number of 60-nm colloid depositions: (a) excitation at 647.1 nm; (b) excitation at 752.5 nm. The lines through the points are only to guide the eye.

of colloid. When an excitation wavelength of 752.5 nm is used, the SERS intensities versus deposition steps continue to increase for more than 10 steps and then decrease only slightly. The amplitude of the optical extinction of the films at the two excitation wavelengths (which does not change with exposure to DBDS) cannot account for the difference in these two results. Extinction intensities at both 647.1 and 752.5 nm continue to increase with deposition of colloid, indicating an increase in accumulated gold with each deposition step. Comparing the trends in the SERS intensity from DBDS with the wavelength of the extinction maximum from the multiparticle resonance shows an interesting correlation. For 32-nm colloids (Figure 5), the wavelength of the extinction maximum shifts to the red by nearly 100 nm and then becomes constant near 690 nm as more colloid is deposited onto the slide. The SERS data taken with 647.1-nm excitation show that when the plasmon resonance is at wavelengths shorter than the excitation, the SERS intensity increases with further deposition of colloid. When the resonance maximum shifts to a wavelength longer than that of the excitation, the SERS intensity falls off. It has been shown previously that the excitation wavelength for maximum SERS intensity is generally coincident with the wavelength of the extinction maximum of the aggregate excitation band.59,60 For 752.5-nm excitation, the plasmon resonance maximum of the 32nm colloids does not exceed the excitation wavelength, and the SERS intensity continues to increase with continued deposition of colloid with only a slight decrease at the highest coverages. A correlation between SERS intensities from DBDS and the wavelength of the plasmon resonance is also observed for the (59) Fornasiero, D.; Greiser, F. J. Chem. Phys. 1987, 5, 3213-3217. (60) Creighton, J. A.; Blatchford, C. G.; Albrecht, M. G. J Chem. Soc., Faraday Trans. 2 1979, 75, 790-798.

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larger (60-nm) immobilized colloids, which are compared in Figure 6 at two excitation wavelengths. With 647.1-nm excitation (Figure 6a), the SERS intensity increases rapidly and then levels out after seven depositions as the peak of the multiparticle plasmon band has shifted to longer wavelengths than the excitation; a slight SERS increase with further depositions is observed followed by a distinct drop in scattering intensity. With 752.5-nm excitation (Figure 6b), the rate of increase per deposition rises continuously until a maximum at ∼20 deposition steps. This point is where the wavelength of the plasmon resonance maximum is closest to the laser excitation at 752.5 nm; further depositions lead to longer wavelengths for the plasmon resonance and a decrease in SERS intensity consistent with earlier findings.59,60 The SERS enhancement at 647.1 nm for these films was estimated for a 32-nm colloid film prepared by five deposition steps; the enhancement was determined by comparing the scattering intensity of the symmetric ring-breathing mode of DBDS bound to the surface with the intensity from a 0.1 mM solution of DBDS. Within the laser beam volume that is projected onto the slit, there is 1.5 µL of solution containing 1.5 × 10-10 mol of DBDS. The surface coverage of DBDS chemisorbed to the gold surface is estimated from previous work61 to be ∼3.0 µmol/m2. The illuminated sample area (∼1.0 × 10-3 cm2) must be corrected for the area gained by having spherical protrusions above the flat surface. A correction factor was developed using line scan data from the AFM images of the surface; the gain in surface area is taken as the square of the ratio of total distance the tip moved over the colloidal surface compared to the lateral distance of the AFM scan. This correction does not account for the convolution of the AFM tip with small surface features; therefore, the estimated surface area may be smaller than the actual area and the reported SERS enhancement should be considered an upper bound. In the corrected sample area, there are 3.9 × 10-13 mol of DBDS chemisorbed to the surface. The DBDS Raman scattering intensities from the SERS substrate was 440 times greater than the solution-phase sample; this result corresponds to a SERS enhancement for the 32-nm gold colloid film of 2 × 105. Comparing the enhancements of the different sizes of immobilized colloid is complicated by the films having different surface areas, colloid densities, and extinction maximums. Although extinction measurements showed that the surface density of the 60-nm colloids was lower than that of the 32-nm particles, the larger particle substrates exhibited somewhat greater SERS enhancement at both excitation wavelengths. The advantage of the smaller colloids, however, lies in their greater stability in suspension as well as their more organized binding to an amineterminated surface. The reproducibility and photolytic stability of the SERS enhancement of these films was investigated using adsorbates deposited onto a C18-modified gold colloid substrate, as discussed in the following section. C-18 Modified Gold Colloid SERS Substrate. A major goal of this work was to prepare an alkane-terminated SERS substrate that could be useful for investigating the adsorption of hydrophobic molecules from aqueous solutions. The ideal characteristics of such a substrate would be chemical stability, spectroscopic reproducibility, and the ability to block access by adsorbates in solution to the underlying metal surface. The octadecylsilane(61) Soriaga, M. P.; Hubbard, A. T. J. Am. Chem. Soc. 1982, 104, 2735-2742.

Figure 7. Test of integrity of the C-18 film. SERS spectra of substrate after exposure to DBDS: (a) DBDS adsorbed directly to the gold surface; (b) DBDS on the C-18-coated gold surface.

modified SERS substrates were prepared by self-assembly of octadecyltrimethoxysilane (OTMS) reagent from n-heptane solution, which should provide a well-ordered monolayer by adsorption from the dry, nonpolar solvent62,63 that is stabilized by subsequent lateral polymerization across the surface.43 While most silane monolayers are formed on oxide substrates to which they can directly bind, it has been shown that insoluble monolayers of trifunctional octadecylsilanes can also be assembled onto gold surfaces,44 the stability of which should depend on lateral crosslinking and polymerization and not direct binding to the gold substrate. Depending on the time allowed for adsorption of the silane to the gold colloid substrate, the contact angle could be increased to values as high as 115°, indicative of a highly ordered, wellpacked monolayer.64 Adsorption of hydrophobic molecules to this tightly packed C-18 layer was limited, probably due to the ordering of the alkyl chains that prevents intercalation of molecules within the hydrophobic film.43,65 Adsorption increased for C-18 films exhibiting a smaller contact angle; for example, a 2-h adsorption of the octadecylsilane (OTMS) produced a stable contact angle of 93((1)° and strong adsorption of hydrophobic solutes from aqueous solution. A smaller contact angle, however, could be accompanied by pinholes or other defects that would allow access by adsorbates to the underlying gold surface.4 It is important to ascertain whether the interactions between a probe adsorbate and the surface are dominated by alkyl chains or whether there are significant interactions of the probe with the underlying metal. DBDS undergoes disulfide bond cleavage and binds to noble metals58 but experiences only weak physisorption to an alkane surface. To test whether there is significant gold surface that is available for adsorption and binding, DBDS was allowed to adsorb from a methanol solution onto a bare gold colloid substrate and onto the C-18-overcoated gold colloid substrate to test for defects in the C-18 silane film. Both slides were left in 5 mM DBDS for 10 min and then rinsed well with methanol to remove any physisorbed DBDS. Figure 7 shows the SERS spectra of DBDS of the slides. The underivatized gold slide (Figure 7a) shows a strong signal from chemisorbed DBDS, whereas the OTMS-coated (62) Moaz, R.; Sagiv, J. J. Colloid Interface Sci. 1984, 100, 465-496. (63) Moaz, R.; Sagiv, J. Langmuir 1987, 3, 1034-1044. (64) Golander, C. G.; Lin, Y. S.; Hlady, V.; Andrade, J. D. Colloids Surf. 1990, 49, 289-302. (65) Dorsey, J. G.; Dill, K. A. Chem. Rev. 1989, 89, 331-346.

Figure 8. SERS reproducibility test: (a) SERS spectra of benzoic acid deposited onto a C-18 silane-coated gold slide at eight different locations on the same substrate; (b) SERS spectra of benzoic acid acquired from six different C-18-coated gold substrates prepared simultaneously.

gold slide (Figure 7b) exhibits less than 4% of this DBDS intensity. This result indicates that very little DBDS was able to penetrate the polymerized silane layer and reach the gold surface. This demonstrates that the surface available for adsorption is dominated by aliphatic hydrocarbon, with minimal access to the metal substrate that would influence probe molecule adsorption. It is interesting that scattering from pyrrolidinone used to aggregate the gold colloid on the surface is not detected in these or any other spectrum acquired. This observation would imply that residual pyrrolidionone in the film is buried within the conductive structure and is not subject to strong surface fields that lead to scattering from interfacial species. To employ a SERS substrate for optical sensing or for studies of adsorption, the optical response must be reproducible and stable. Lack of substrate reproducibility has been a major challenge for the application of SERS as a quantitative analytical technique. This problem is largely overcome with the C-18overcoated gold colloid substrates. The optical properties and SERS enhancement of the modified gold colloid films are uniform and stable over several months; each batch of substrates prepared simultaneously gives nearly identical extinction spectra and SERS enhancement. To test the SERS reproducibility, benzoic acid was deposited onto several C-18-modified substrates by adsorption from methanol solution; SERS spectra of the adsorbate were acquired in air and are plotted in Figure 8. The reproducibility at different locations across a single substrate was acquired by translating the substrate across the laser excitation spot and recording SERS spectra at eight different points (Figure 8a); the scattering intensity variation between spots averages ∼5%, which is close to the level of the baseline noise. Reproduciblilty between different substrates prepared simultaneously is shown in Figure 8b for six different slides, where the slide-to-slide intensity variation averages ∼15% or only a few times larger than the baseline noise. The variations in the SERS intensity of these spectra include not only irregularities in the enhancement of the substrate but also errors in sample positioning, fluctuations in laser intensity, and any variations in the amount of benzoic acid in the probed region caused by a nonuniform silane layer. Some of the Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

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remarkable SERS reproducibility of these substrates may be due to the fact that the alkylsilane layer insulates the adsorbate from direct contact with the metal. While this eliminates the “chemical” or “charge-transfer” contribution to the SERS enhancement, which lowers the sensitivity, it may contribute to greater reproducibility of the enhancement4 since the degree of charge-transfer interaction can vary significantly with surface potential, dielectric environment, or the presence of other species on the surface. As a final test of the utility of these C-18-coated gold colloid substrates for sensing or adsorption chemistry studies, they were tested to determine their ability to survive long-term exposure to laser excitation. Many SERS substrates are easily damaged by laser radiation; silver island films can undergo significant changes in their optical and SERS enhancement properties upon laser exposure.66 This damage is problematic if moderate laser powers are needed to follow a change in an analyte signal; the laserinduced changes in the SERS response could easily be confused with the variation in the amount of analyte at the surface. To test the effect of laser irradiation on the substrate, a long-term, in situ SERS observation was made of a C-18-modified surface in equilibrium with a 10 µM aqueous solution of CPC, a cationic surfactant. At this concentration, CPC adsorbs to the C-18 surface at ∼0.5 monolayer coverage.67 An in situ experiment was used in this case so that the photolytic stability of only the SERS substrate and not the adsorbate is tested; the experiment is less sensitive to the photolysis of the adsorbate since there is a continuous exchange with molecules in solution. The substrate was illuminated continuously in the same spot for 1 h with a cylindrically focused beam of 150 mW at 647.1 nm, and the SERS intensity of adsorbed CPC is compared before and after exposure. The (66) Semin, D. J.; Rowlen, K. L. Anal. Chem. 1994, 66, 4324-4331. (67) Olson, L. G. Ph.D. Dissertation, University of Utah, 1999; Chapter 3.

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scattering intensity after exposure to more than 500 J of laser energy is indistinguishable from the initial spectrum, within the 5% baseline noise. This result indicates that the substrates are stable to long-term laser exposure, allowing their use in timedependent monitoring applications. Summary. Immobilized gold colloids with a C-18 silane overlayer have been shown to be a stable and reproducible SERS substrate for detecting molecules adsorbed to hydrophobic surfaces. The optical properties and SERS enhancement of the substrates were found to vary systematically with aggregation of the immobilized gold colloid particles. The surface of the immobilized particles was modified by self-assembly of a C-18 silane overlayer that prevented significant contact between adsorbates and the underlying gold surface. SERS enhancement of the C-18modified surface is reproducible for different substrates that are prepared from the same colloidal suspension. The substrates are stable with respect to time and exposure to laser irradiation. Applications of these films to study kinetics of surfactant adsorption and to detect polycyclic aromatic hydrocarbons in aqueous solution are in progress. ACKNOWLEDGMENT The authors are grateful to M. J. Natan for helpful discussions concerning the preparation of the captured gold colloid films. This work was supported in part by grants from the U.S. Department of Energy (DE-FG03-93ER14333) and from the National Science Foundation (CHE 98-14477 to T.P.B.).

Received for review July 28, 2000. Accepted June 26, 2001. AC000873B