Two-Dimensional Arrays of Colloidal Gold Particles - ACS Publications

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Langmuir 1996, 12, 2353-2361

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Two-Dimensional Arrays of Colloidal Gold Particles: A Flexible Approach to Macroscopic Metal Surfaces Katherine C. Grabar, Keith J. Allison, Bonnie E. Baker, Robin M. Bright, Kenneth R. Brown, R. Griffith Freeman,† Audrey P. Fox, Christine D. Keating, Michael D. Musick, and Michael J. Natan* Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802-6300 Received July 7, 1995. In Final Form: February 23, 1996X Covalent attachment of nanometer-scale colloidal Au particles to organosilane-coated substrates is a flexible and general approach to formation of macroscopic Au surfaces that have well-defined nanostructure. Variations in substrate (glass, metal, Al2O3), geometry (planar, cylindrical), functional group (-SH, -P(C6H5)2, -NH2, -CN), and particle diameter (2.5-120 nm) demonstrate that each component of these assemblies can be changed without adverse consequences. Information about particle coverage and interparticle spacing has been obtained using atomic force microscopy, field emission scanning electron microscopy, and quartz crystal microgravimetry. Bulk surface properties have been probed with UV-vis spectroscopy, cyclic voltammetry, and surface enhanced Raman scattering. Successful application of the latter two techniques indicates that these substrates may have value for Raman and electrochemical measurements. The assembly method described herein is compared with previous methods for controlling the nanoscale roughness of metal surfaces, and its potential applicability to the assembly of other colloidal materials is discussed.

Introduction Fabrication of structures with dimensions approaching those of molecules is playing an increasingly important role in the work of chemists1 and physicists.2 Reports describing physical properties of ensembles of nanometersized molecules, particles, films, or structures are now commonplace.3-7 Recent examples include giant-magnetoresistive films,5 conducting molecular wires,6 and nanomembranes with adjustable transport properties.7 * Author to whom all correspondence should be addressed. E-mail address: [email protected]. † Present address: Division of Science, Northeast Missouri State University, Kirksville, MO 63501. X Abstract published in Advance ACS Abstracts, April 15, 1996. (1) Mu¨ller, W. T.; Klein, D. L.; Lee, T.; Clarke, J.; McEuen, P. L.; Schultz, P. G. Science 1995, 268, 272-273. (b) Masuda, H.; Fukuda, K. Science 1995, 268, 1466-1468. (c) Martin, C. R. Science 1994, 266, 1961-1966. (d) Whitesides, G. M.; Mathias, J. P.; Seto, C. T. Science 1991, 254, 1312-1319. (e) Schnur, J. M. Science 1993, 262, 16691676. (2) (a) Kim, Y.; Lieber, M. Science 1992, 257, 375-377. (b) Douglas, K.; Devaud, G.; Clark, N. A. Science 1992, 257, 642-644. (c) Pease, R. F. W. J. Vac. Sci. Technol., B 1992, 10, 278-285. (3) (a) Boonekamp, E. P.; Kelly, J. J.; Fokkink, L. G. J. Langmuir 1994, 10, 4089-4094. (b) Meldrum, F. C.; Kotov, N. A.; Fendler, J. H. Langmuir 1994, 10, 2035-2040. (c) Gao, M.; Zhang, X.; Yang, B.; Shen, J. J. Chem. Soc., Chem. Commun. 1994, 2229-2230. (d) Giersig, M.; Mulvaney, P. Langmuir 1993, 9, 3408-3413. (e) Giersig, M.; Mulvaney, P. J. Phys. Chem. 1993, 97, 6334-6336. (f) Doron, A.; Katz, E.; Willner, I. Langmuir 1995, 11, 1313-1317. (4) (a) Colvin, V. L.; Goldstein, A. N.; Alivisatos, A. P. J. Am. Chem. Soc. 1992, 114, 5221-5230. (b) Bowen Katari, J. E.; Colvin, V. L.; Alivisatos, A. P. J. Phys. Chem. 1994, 98, 4109-4117. (c) Chang, S.-Y.; Liu, L.; Asher, S. A. J. Am. Chem. Soc. 1994, 116, 6739-6744. (d) Brus, L. E.; Szajowski, P. F.; Wilson, W. L.; Harris, T. D.; Schuppler, S.; Citrin, P. H. J. Am. Chem. Soc. 1995, 117, 2915-2922. (e) Leon, R.; Petroff, P. M.; Leonard, D.; Fafard, S. Science 1995, 267, 1966-1968. (f) Kotov, N. A.; Meldrum, F. C.; Wu, C.; Fendler, J. H. J. Phys. Chem. 1994, 98, 2735-2738. (g) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993, 115, 8706-8715. (h) Facci, P.; Erokhin, V.; Tronin, A.; Nicolini, C. J. Phys. Chem. 1994, 98, 13323-13327. (5) (a) Wang, J.-Q.; Xiao, G. Phys. Rev. B 1995, 51, 5863-5867. (b) Hua, S. Z.; Lashmore, D. S.; Salamanca-Riba, L.; Schwarzacher, W.; Swartzenruber, L. J.; McMichael, R. D.; Bennett, L. H.; Hart, R. J. Appl. Phys. 1994, 76, 6519-6521. (6) (a) Huber, C. A.; Huber, T. E.; Sadoqi, M.; Lubin, J. A.; Manalis, S.; Prater, C. B. Science 1994, 263, 800-802. (b) Wu, C.-G.; Bein, T. Science 1994, 266, 1013-1015. (c) Pascual, J. I.; Me´ndez, J.; Go´mezHerrero, J.; Baro´, A. M.; Garcia, N.; Landman, U.; Luedtke, W. D.; Bogachek, E. N.; Cheng, H.-P. Science 1995, 267, 1793-1795.

The common experimental objectives linking nearly all these efforts are nanometer-level control of feature size and interfeature spacing, parameters that typically govern bulk physical properties. For noble metal surfaces, the correlation between nanoscale structure and bulk optical behavior is particularly well established. Both surfaceenhanced Raman scattering (SERS)8-9 and surface plasmon resonance10 rely on Au or Ag surfaces with nanometerscale roughness. We have developed a new approach to generation of macroscopic metal surfaces that is based upon selfassembly of nanometer-scale colloidal Au and Ag particles from solution onto polymer-coated substrates.11 Compared to previous routes to nanostructured Au or Ag interfaces,12 this approach has several distinguishing features. (i) Aqueous solutions of colloidal Au are easy to prepare and are kinetically stable for long periods of time.13 (ii) Extensive literature is available on physical and optical properties of isolated and aggregated colloidal Au particles.14 (iii) Colloidal Au can be prepared with a wide (7) Nishizawa, M.; Menon, V. P.; Martin, C. R. Science 1995, 268, 700-702. (8) (a) Laserna, J. J. Anal. Chim. Acta 1993, 283, 607-622. (b) Brandt, E. S.; Cotton, T. M. In Investigations of Surfaces and InterfacesPart B, 2nd ed.; Rossiter, B. W., Baetzold, R. C., Eds.; John Wiley & Sons: New York, 1993; Vol. IXB, pp 633-717. (c) Birke, R. L.; Lu, T.; Lombardi, J. R. In Techniques for Characterization of Electrodes and Electrochemical Processes; Varma, R., Selman, J. R., Eds.; John Wiley & Sons: New York 1991; pp 211-277. (d) Garrell, R. L. Anal. Chem. 1989, 61, 400A-411A. (e) Otto, A.; Mrozek, I.; Grabhorn, H.; Akemann, W. J. Phys. Condens. Matter 1992, 4, 1143-1212. (f) Moskovits, M. Rev. Mod. Phys. 1985, 57, 783-826. (g) Storey, J. M. E.; Barber, T. E.; Shelton, R. D.; Wachter, E. A. Spectroscopy 1995, 10, 20-25. (g) Brandt, E. S.; Cotton, T. M. Surface-Enhanced Raman Scattering. In Investigations of Surfaces and InterfacessPart B; Rossiter, B. W., Baetzold, R. C., Eds.; John Wiley and Sons: New York, 1993; pp 633-718. (9) Recent examples: (a) Crane, L. G.; Wang, D. X.; Sears, L. M.; Heyns, B.; Carron, K. Anal. Chem. 1995, 67, 360-364. (b) Barber, T. E.; List, M. S.; Haas, I., J. W.; Wachter, E. A. Appl. Spectrosc. 1994, 48, 1423-1427. (c) Roth, E.; Kiefer, W. Appl. Spectrosc. 1994, 48, 11931195. (d) Tsen, M.; Sun, L. Anal. Chim. Acta 1995, 307, 333-340. (e) Bercegol, H.; Boerio, F. J. Langmuir 1994, 10, 3684-3692. (f) Van Duyne, R. P.; Hulteen, J. C.; Treichel, D. A. J. Chem. Phys. 1993, 99, 2101-2115. (g) Roark, S. E.; Rowlen, K. L. Anal. Chem. 1994, 66, 261-270. (10) (a) Lo¨fas, S.; Johnsson, B. J. Chem. Soc., Chem. Commun. 1990, 1527-1528. (b) Salamon, Z.; Wang, Y.; Brown, M. F.; Macleod, H. A.; Tollin, G. Biochemistry 1994, 33, 13706-13711. (c) Jordan, C. E.; Frey, B. L.; Kornguth, S.; Corn, R. M. Langmuir 1994, 10, 3642-3648.

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range of diameters (2.5-120 nm) with relatively high monodispersity.13,15 As a result, surfaces made from colloidal Au have a (tunable) nanometer-scale roughness defined solely by the particle diameter, a feature that greatly simplifies the relationship between nanostructure and bulk properties. Making use of this property, two other groups have issued recent reports describing SERS behavior and electrochemical response of colloidal Au films.16 However, like our initial reports, these papers have focused on a single size of particle, a single crosslinking agent, and a single substrate. Establishing the scope of this strategy is thus important for the field of nanoparticle assembly. The types of substrates that can be used, viable polymer functional groups, preferred methods for characterization, and the stability of the resulting surfaces will ultimately define their practical value. Moreover, the extent to which this approach can be applied to the assembly of structures from other nanoparticles is also significant. These issues comprise the focus of this paper. It is shown herein that Au colloid self-assembly is a very general approach to macroscopic Au surfaces with well-defined nanostructure. These studies employ a variety of particle sizes, crosslinking chemistries, substrate compositions, and substrate geometries, and benefit from substrate characterization using two nanoscale imaging methods (atomic force microscopy (AFM) and field emission scanning electron microscopy (FE-SEM)), as well as four bulk interrogation methods (SERS, UV-vis, electrochemistry and in situ quartz crystal microgravimetry (QCM)). The data show that submonolayers of Au particles constitute new interfaces for Raman and electrochemical measurements. In a related paper, a detailed study of the kinetics of surface formation using 12-15 nm diameter Au particles is described, addressing issues such as the measurement and control of interparticle spacing, particle sticking probability, and reproducibility of surface formation.17 Together, these papers show that colloid self-assembly is an extremely powerful approach to fabrication of func(11) (a) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735-743. (b) 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. (c) Bright, R. M.; Walter, D. G.; Musick, M. D.; Jackson, M. A.; Allison, K. J.; Natan, M. J. Langmuir 1996, 12, 810817. (12) (a) Schlegel, V. L.; Cotton, T. M. Anal. Chem. 1991, 63, 241247. (b) Yogev, D.; Efrima, S. J. Phys. Chem. 1988, 92, 5754-5760. (c) Yogev, D.; Efrima, S. J. Phys. Chem. 1988, 92, 5761-5765. (d) AlObaidi, A. H. R.; Rigby, S. J.; McGarvey, J. J.; Walmsley, D. G.; Smith, K. W.; Hellemans, L.; Snauwaert, J. J. Phys. Chem. 1994, 98, 1116311168. (e) Barnickel, P.; Wokaun, A. Mol. Phys. 1989, 67, 1355-1372. (f) Sutherland, W. S.; Winefordner, J. D. J. Colloid Interface Sci. 1992, 148, 129-141. (g) Wachter, E. A.; Moore, A. K.; Haas, I., J. W. Vib. Spectrosc. 1992, 3, 73-78. (h) Dawson, P.; Alexander, K. B.; Thompson, J. R.; Haas, J. W., III; Ferrell, T. L. Phys. Rev., B: Condens. Matter 1991, 44, 6372-6381. (i) Goudonnet, J. P.; Begun, G. M.; Arakawa, E. T. Chem. Phys. Lett. 1982, 92, 197-201. (j) Ni, F.; Cotton, T. M. Anal. Chem. 1986, 58, 3159-3163. (13) Colloidal Gold: Principles, Methods, and Applications; Hayat, M. A., Ed.; Academic Press: San Diego, Ca, 1989; Vols. 1-2. (14) (a) Siiman, O.; Bumm, L. A.; Callaghan, R.; Blatchford, C. G.; Kerker, M. J. Phys. Chem. 1983, 87, 1014. (b) Arai, M.; Mitsui, M.; Ozaki, J.-I.; Nishiyama, Y. J. Colloid Interface Sci. 1994, 168, 473477. (c) Bloemer, M. J.; Haus, J. W.; Ashley, P. R. J. Opt. Soc. Am., B 1990, 7, 790-795. (d) Wiesner, J.; Wokaun, A. Chem. Phys. Lett. 1989, 157, 569-575. (e) Thompson, D. W.; Collins, I. R. J. Colloid Interface Sci. 1994, 163, 347-354. (15) (a) Frens, G. Nature, Phys. Sci. 1973, 241, 20-22. (b) Goodman, S. L.; Hodges, G. M.; Trejdosiewicz, L. K.; Livingston, D. C. J. Microscopy 1981, 123, 201-213. (16) (a) Chumanov, G.; Sokolov, K.; Gregory, B. W.; Cotton, T. M. J. Phys. Chem. 1995, 99, 9466-9471. (b) Doron, A.; Katz, E.; Willner, I. Langmuir 1995, 11, 1313-1317. (17) Grabar, K. C.; Smith, P. C.; Davis, J. A.; Musick, M. D.; Jackson, M. A.; Walter, D. G.; Guthrie, A. P.; Natan, M. J. J. Am. Chem. Soc. 1996, 118, 1148-1153.

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tional macroscopic surfaces of well-defined and controllable nanostructure. Experimental Section Reagents and Materials. The following organosilanes were obtained from Hu¨ls America and used as received: (3-mercaptopropyl)trimethoxysilane (MPTMS), (3-mercaptopropyl)methyldimethoxysilane (MPMDMS), (mercaptomethyl)dimethylethoxysilane (MMDMES), (mercaptomethyl)methyldiethoxysilane (MMMDES), (3-aminopropyl)trimethoxysilane (APTMS), (3aminopropyl)methyldiethoxysilane (APMDES), (3-cyanopropyl)dimethylmethoxysilane (CPDMMS), (3-cyanopropyl)triethoxysilane (CPTES), (2-pyridylethyl)trimethoxysilane (PETMS), and (2-(diphenylphosphino)ethyl)triethoxysilane (DPPETES) (Chart 1). Na3[Fe(CN)6], pyridine, HAuCl4‚3H2O, KCl, p-nitrosodimethylaniline (p-NDMA), Na3citrate dihydrate, AgNO3, NaBH4, Na2SO4, ethylenediaminetetraacetic acid (EDTA), spectrophotometric grade n-C6H14 (hexane), and trans-1,2-bis(4-pyridyl)ethylene (BPE) were purchased from Aldrich. [Ru(NH3)6]Cl3 was purchased from Alfa. Absolute C2H5OH was obtained from Aaper Alcohol and Chemical Company. Polyethylene glycol (PEG, MW ) 20 000), horseradish peroxidase and horse heart cytochrome c were obtained from Sigma. Spectrophotometric grade CH3OH was obtained from EM Science. H2O (18 MΩ) was obtained from a Barnstead Nanopure water purification system. With the exception of BPE (which was recrystallized from CH3OH/H2O) and cytochrome c (which was purified according to literature protocols18), all reagents were used as received. Substrates were obtained as follows: Al2O3 ceramic from Akzo Chemical Co., glass capillaries (50 µm i.d., 350 µm o.d.) from Polymicron Technologies, In-doped SnO2 (30 Ω cm2) from PPG Industries, Sb-doped SnO2 (100 Ω cm2) from Delta Technology, 5 mm glass NMR tubes were from Wilmad, quartz microscope slides from Technical Glass Products, 9.0-MHz polished AT-cut quartz crystals (surface features ≈ 0.02 µm in depth)19 with Au electrodes from International Crystal Manufacturing (Oklahoma City), Formvar-coated Cu TEM grids from Ted Pella, Inc., and glass microscope slides from Fisher Scientific. Teflon tape (76 µm thick, 1 in. wide) was purchased from Berghof/America. Single component Ag epoxy was obtained from Epoxy Technology, Inc., and insulating epoxy was obtained from Dexter Corp. Preparation of Colloidal Particles. All glassware used in the following procedures was cleaned in a bath of freshly prepared 3:1 HCl:HNO3 and rinsed thoroughly in H2O prior to use. Preparations for colloidal Ag,20 12 nm diameter colloidal Au,11 and Ag-clad Au particles21 using citrate as a reductant are described elsewhere, as are those for Ag and Au particles with NaBH422 or EDTA23 as the reductant. Au “Seed Colloid” Solution. A solution of 2.6 nm-diameter particles (321 particles analyzed; major axis ) 2.6 ( 0.05 nm, minor axis ) 2.3 ( 0.05 nm) was prepared by adding 1 mL of 1% aqueous HAuCl4‚3H2O to 100 mL of H2O with vigorous stirring, followed one min later by addition of 1 mL of 1% aqueous Na3citrate. After an additional minute, 1 mL of 0.075% NaBH4 in 1% Na3citrate was added. The solution was stirred for 5 min and then stored at 4 °C until needed. 60 nm Au Particles. Elliptical particles of major axis 61 ( 10 nm and minor axis 49 ( 7.5 nm were prepared by adding 2 mL of 1% Na3citrate and 300 µL of Au seed colloid to a refluxing solution of 455 mL of 0.01% HAuCl4‚3H2O. Refluxing was continued for 15 min, at which time the sol was allowed to cool to room temperature and then stored in a dark bottle. 125 nm Au Particles. Elliptical particles with 125 ( 28 nm major axes and 85 ( 15 nm minor axes were obtained by heating 100 mL of 0.01% HAuCl4 to a boil with vigorous stirring. Rapid addition of 0.4 mL of 1% Na3citrate and 15 µL of Au seed colloid resulted in a slow color change, from muddy brown to orange. (18) Brautgan, D. L.; Ferguson-Miller, S.; Margoliash, E. Methods Enzymol. 1978, 53, 128-164. (19) Yang, M.; Thompson, M. Langmuir 1993, 49, 1990-1994. (20) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391-3395. (21) Freeman, R. G.; Hommer, M. B.; Grabar, K. C.; Jackson, M. A.; Natan, M. J. J. Phys. Chem. 1996, 100, 718-724. (22) Sun, L. Ph.D. Thesis, Northwestern University, June 1990. (23) Lee, N.-S.; Sheng, R.-S.; Morris, M. D.; Schopfer, L. M. J. Am. Chem. Soc. 1986, 108, 6179-6182.

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Arrays of Colloidal Au Particles The solution was boiled for 10 min and then cooled and stored in a dark bottle. Preparation of Colloid-Based Surfaces. The general procedure for surface assembly has been described previously.11,24 Specific modifications to this procedure are listed below. Unless stated otherwise, all substrates were cleaned in a bath of 4:1 H2SO4/30% aqueous H2O2 at 60 °C (piranha solution) and rinsed in H2O and CH3OH prior to derivatization with organosilane. Experimental protocols for immobilization of colloidal Au using streptavidin-biotin binding affinity have been described elsewhere.24 FE-SEM Sample. A clean glass slide was derivatized in a solution of 1:3 APMDES/CH3OH for 24 h. The slide was rinsed in CH3OH and derivatized in a solution of 12 nm colloidal Au for 5 weeks. After rinsing in H2O and CH3OH, the slide was allowed to dry in air. Al2O3. Al2O3 surfaces were sonicated in CH3OH for 7 min using a Bransonic 2200 sonicator followed by cleaning in a bath of HCl/HNO3 (3:1). Surfaces were rinsed in H2O and CH3OH before derivatization in neat MPMDMS for 3 h. Surfaces were rinsed in CH3OH and H2O and placed in a solution of 12 nm colloidal Au for 12 h. Surfaces were then rinsed and stored in H2O. Near-Infrared SERS Samples. Clean glass slides were derivatized in a 1:10 mixture of APTMS/CH3OH for 2.5 days. After the slides were rinsed in CH3OH and H2O, surfaces were derivatized for 19.5 h in a solution of 60 nm diameter colloidal Au, which was stirred during the first 7.5 h of coating. Similarly, clean glass slides, which were immersed for 30 min in 3 M NaOH and then rinsed in H2O and CH3OH, were derivatized for 2.75 h in a solution of 1:10 MPTMS/CH3OH. These surfaces were then exposed to 12-nm colloidal Au for 1 week. QCM Crystals. CH3CH2OH was substituted for CH3OH in the initial cleaning procedure; samples were then dried with a stream of Ar. After a 40-min stabilization period in air, the fundamental frequency was determined. The Au electrode surface was derivatized in a solution of 1:5 APTMS/CH3OH for 60 min. Unbound APTMS was removed by two rinses each with CH3CH2OH and H2O. The crystal was dried with Ar and equilibrated in air before the new frequency was determined. Absorption of 12 nm diameter Au colloid onto the APTMS-coated electrode surface was monitored by taking frequency measurements every 6 s over a 6-h period. After rinses in 18 MΩ H2O and CH3CH2OH, the sample was dried with Ar, and the final frequency was recorded in air. The surface roughness of QCM electrodes was determined by cyclic voltammetry. From the area under the gold oxide reduction peak at 1.2 V, a surface roughness of 2.25 ( 0.25 was calculated. SnO2 Electrodes. Cu wires were affixed to the conducting surfaces of In- or Sb-doped SnO2 using Ag epoxy. The Cu wires were encased in glass, and any exposed Ag epoxy or Cu was insulated with Dexter Epoxi-Patch. Electrodes were cleaned by sequential sonications in acetone, soap, and distilled H2O (2×, 15 min each), and were stored in H2O after electrochemical characterization (cyclic voltammetry of 5 mM [Ru(NH3)]6Cl3 in 0.1 M aqueous Na2SO4). Au colloid monolayers were prepared on these electrodes as follows: After exposure to piranha solution for 5 min, electrodes were sonicated for 20 min each in H2O and acetone, followed by 15 min of sonication in H2O. The electrodes were then treated with 5 M NaOH for 5 h prior to rinsing with H2O and derivatization in an aqueous solution of 1% APMDES for 10-15 min. Substrates were rinsed in H2O and allowed to dry in air. Subsequent exposure to 12 nm diameter colloidal Au for 8 h resulted in colloid binding. Optical and Raman Instrumentation. A Coherent Mira900 Ti:sapphire laser, pumped by a 13-W Coherent I-400 Ar+ laser, provided continuous wave excitation at 755 nm for nearinfrared (near-IR) SERS experiments. The horizontally-polarized output from the Mira was converted to vertical polarization by passing the beam through a Fresnel rhomb and beamsplitting optics. Laser power was recorded with a Coherent Fieldmaster power/energy meter. Spectra were collected on a SPEX 1877 triple monochromator fitted with a UV-coated, back-thinned 1024 × 512 pixel Spectrum One CCD detector maintained at 140 K. (24) Grabar, K. C.; Deutsch, J. E.; Natan, M. J. Polym. Prepr. 1995, 69-70.

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Langmuir, Vol. 12, No. 10, 1996 2355 The Ti:sapphire wavelength was determined by steering the beam into the SPEX (which had been previously calibrated with Raman scattered photons from a sample of DMSO/DMSO-d6 using 647.1 nm excitation). SERS substrates were pressed up against the front face of 3 mL glass cuvettes; liquid samples were run in the same cuvettes without refocusing. Scattered photons were collected using a Melles Griot 75 mm diameter symmetric airspaced synthetic fused silica condenser and focused into the monochromator using a 76 mm diameter fused silica planoconvex lens from JML Direct Optics. Scattered radiation was not polarization-scrambled before entering the monochromator. The bandpass for experiments using this apparatus with two 600 grooves/mm gratings (400 nm blaze) in the filter and a 1200 grooves/mm grating (500 nm blaze) in the spectrograph was 2.7 cm-1. For each sample, 10 spectra were acquired with 15-s integrations and averaged using SPEX DM3000R data acquisition software. Other Raman instrumentation has been previously described.11 Optical spectra were recorded using an HP8452A diode array ultraviolet-visible spectrophotometer with 2-nm resolution and a 1-s integration time. A Teflon block (1/3 sample height) was used to keep samples upright in cuvettes. Electrochemical Instrumentation. All electrochemical measurements were carried out using either a Cypress Model CS-1090 potentiostat operated with Version 6 Software on a Swan Technologies 386SX IBM-compatible computer, a Bioanalytical Systems Model CV-27 potentiostat and Model RXY recorder, a PAR Model 273A potentiostat/galvanostat operated with Model 270 Software on a Gateway 486 IBM-compatible computer, or a PAR Model 173/175/379 potentiostat-galvanostat/programmer/ coulometer with a Model Se790 Goerz XY/XY-Yt-Recorder. QCM Instrumentation. A 5.5-V potential was delivered to homebuilt apparatus consisting of an oscillating circuit and a specially-designed power supply. A gain control circuit was added to the fundamental oscillating circuit to prevent overtone interference. Frequency changes were monitored with a Fluke high resolution programmable timer/counter (Model PM-6680B) equipped with a high-stability oven oscillator time base. Data were collected via an IEEE interface to a Gateway IBMcompatible computer. To reduce electrical interference, all experiments were conducted inside a Faraday cage. Further reduction of line noise was effected using a Cole-Parmer isolation transfomer (Model H-26102). The crystal holder was an integrated-circut plug-in design that allowed direct attachment to the circuit. Particle Size/Spacing Analysis. TEM analysis of colloidal particle sizes was performed on a JEOL 1200EXII instrument with an accelerating voltage of 80 kV. FE-SEM data were acquired on a JEOL USA, Inc., Model 6400F SEM operated at an accelerating voltage of 5 kV. Samples were sputter-coated with 1.5 nm of Pt prior to analysis. Data analyses on developed photographs were performed on a Macintosh IIci computer connected to a LaCie Silverscanner II (800 dots-per-inch (DPI) × 400 DPI resolution) using Adobe Photoshop and/or the public domain program NIH Image 1.55.25 Small Volume Flow Cell Construction. Teflon tape was attached to one side of two 1-in.2 glass microslides. A rectangular area (1.0 cm × 0.4 cm) was carved out of the center of the Teflon, along with 0.1 cm × 0.5 cm channels above and below the rectangle to allow for sample introduction and flow. One slide was then derivatized using APTMS and Ag colloid as previously described.11 The slides were then epoxied together under pressure at all common edges (except for input/output flow channels). Injection was accomplished using a Hamilton microsyringe, while ejection resulted from gravity-induced flow. The total cell volume was between 6 and 7 µL, verified by UV-vis measurements on K3[Fe(CN)6]. Capillary Flow Cell Construction. The polyimide coating on a quartz capillary (350 µm o.d., 50 µm i.d.) was removed to provide a window for spectral measurements. A bonded phase column rinse kit with He pressurization was utilized to establish flow through the column. The capillary was loaded with a 1:10 mixture of APTMS/CH3OH; the pressure was then decreased (25) Written by Wayne Rasband at the U.S. National Institutes of Health and available from the Internet by anonymous FTP from zippy.nimh.nig.gov or on floppy disk from NTIS, 5285 Port Royal Rd., Springfield, VA, 22161, part number PB93-504868.

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Chart 1. Organosilanes Used for Colloid Immobilization

Figure 1. (left) SERS spectrum (12 nm diameter Au/APTMS/ capillary) and Raman spectrum (uncoated capillary) of 6 M pyridine. Acquisition parameters: 20 mW; 632.8 nm excitation; 5 cm-1 bandpass; 1 cm-1 step; 1 cm-1/s scan rate. (right) Raman and SERS spectra of 50 mM pyridine/0.1 M KCl in a 6 µL flow cell. For the Raman spectrum, both faces of the cell were glass; for the SERS spectrum, the back face was a Ag/APTMS/glass slide. Acquisition parameters: 20 mW; 632.8 nm excitation; 5 cm-1 bandpass; 1 cm-1 step; 1 cm-1/s scan rate.

until flow ceased, and derivatization of the static system proceeded for 12 h. CH3OH was then flushed through the capillary for 45 min. Finally, a solution of colloidal Au (12 nm) was flowed through capillary for 2.5 h; the capillary was then rinsed with H2O for 1 h.

Results and Discussion Generality and Flexibility. Preparation of macroscopic Au surfaces by covalent attachment of colloidal Au particles to substrate-confined organosilane polymer films is an extremely general process, encompassing wide variations in polymer precursor, in substrate, and in particle size and composition. Chart 1 shows the organosilanes used to tether colloidal Au to substrate surfaces. Surface derivatization with organosilanes can be carried out in a straightforward manner using mono-, di-, and trialkoxysilanes.26 Pendant thiol, amine, cyanide, and diphenylphosphine functional groups, by virtue of available electron lone pairs, bind tightly to colloidal Au and Ag particles; carboxylate terminal groups on alkanethiol self-assembled monolayers also bind Au and Ag.11 Polymers such as that derived from propyltrimethoxysilane do not interact with colloidal metal particles and consequently do not lead to particle immobilization. Each molecule in Chart 1 is commercially available; thus, metal colloid assemblies are easily prepared with off-the-shelf reagents. It should be noted that organosilanes with longer alkyl chain lengths or with other Au-binding groups (such as dithiolenes or dithiocarbamates)27 are available from known syntheses. The strong interactions between streptavidin and biotin28 can also be used to tether colloidal particles to glass surfaces.24 In this approach, biotin derivatives are covalently attached to surface-bound organosilanes. Re(26) Plueddemann, E. P. Silane Coupling Agents; Plenum Press: New York, 1982. (27) Greenwood, N. N.; Earnshaw, A. Chemistry of the Elements; Pergamon Press: Oxford, 1984.

action of these surfaces with streptavidin-Au conjugates results in irreversible immobilization of Au particles yielding surfaces with identical optical properties to those fabricated from covalent assembly strategies. Colloid self-assembly is also flexible with regard to the underlying solid support, in terms of both shape and surface composition. Since most oxide surfaces are very reactive toward alkoxysilanes, it has proven possible to assemble colloidal Au or Ag films on quartz, glass, Al2O3, doped SnO2, and Pt. Because polymers derived from alkoxysilanes are insoluble, covalent attachment between the polymer and surface is not required. As a result, nonoxide materials such as mica29 and Au foil have also been successfully derivatized. Common limitations in substrate size and shape are circumvented by this method, since both silanization and colloid binding are solution phase processes. Accordingly, Au colloid films can be assembled in geometries that are difficult to access by other means. For example, derivatization of the inner surface of conventional NMR tubes yields a SERS-active surface that is easily spun, a desirable but typically unattainable situation in SERS experiments.30 Likewise, small volumes pose no problem: SERS-active flow cells have been prepared in both thinlayer (76 µm slab) and capillary (50 µm i.d.) configurations (Figure 1). In the former case, the total cell volume is 6-7 µL, with 0.1 µL being interrogated by the laser beam; the capillary total volume is 0.6 µL, with