Protein Arrays on Patterned Porous Gold Substrates Interrogated with

Methyl- and carboxy-terminated self-assembled monolayers (SAMs) were custom-patterned on porous gold substrates with equipment commonly used to print ...
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Anal. Chem. 2008, 80, 1448-1458

Protein Arrays on Patterned Porous Gold Substrates Interrogated with Mass Spectrometry: Detection of Peptides in Plasma Kenyon M. Evans-Nguyen, Sheng-Ce Tao, Heng Zhu, and Robert J. Cotter*

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205

Methyl- and carboxy-terminated self-assembled monolayers (SAMs) were custom-patterned on porous gold substrates with equipment commonly used to print protein arrays, without complex surface chemistry protocols. Proteins were covalently immobilized on hydrophilic carboxy-terminated SAM spots, while the remainder of the surface was superhydrophobic due to the roughened gold surface and the methyl-terminated SAM. The resistance of these patterns to biofouling and the effective containment of MALDI matrix solution within the hydrophilic spot made these surfaces amenable to analyzing proteinpeptide binding with mass spectrometry. A model system of the affinity peptides HA, cmyc, and V5 and their corresponding antibodies was used to demonstrate the utility of the patterned porous gold. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) matrixassisted laser desorption/ionization time-of-flight (MALDITOF) spectra and images obtained reflected the effective capture of the affinity peptides directly from spiked bovine plasma. Microarrays of proteins immobilized on solid substrates have become a powerful tool in biology as a sensitive, high-throughput method that requires little material.1 Protein-protein, proteinlipid, protein-DNA, protein-drug, and protein-peptide interactions have been effectively characterized and kinase substrates have been identified in a high-throughput manner using protein microarrays.2-6 Through immobilization of antibodies specific to known analytes, they have also been used in an analytical * To whom correspondence should be addressed. E-mail: [email protected]. (1) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7, 55-63. (2) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Science 2001, 293, 2101-2105. (3) Hall, D. A.; Zhu, H.; Zhu, X.; Royce, T.; Gerstein, M.; Snyder, M. Science 2004, 306, 482-484. (4) Huang, J.; Zhu, H.; Haggarty, S. J.; Spring, D. R.; Hwang, H.; Jin, F.; Snyder, M.; Schreiber, S. L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16594-16599. (5) Ptacek, J.; Devgan, G.; Michaud, G.; Zhu, H.; Zhu, X.; Fasolo, J.; Guo, H.; Jona, G.; Breitkreutz, A.; Sopko, R.; McCartney, R. R.; Schmidt, M. C.; Rachidi, N.; Lee, S.-J.; Mah, A. S.; Meng, L.; Stark, M. J. R.; Stern, D. F.; De Virgilio, C.; Tyers, M.; Andrews, B.; Gerstein, M.; Schweitzer, B.; Predki, P. F.; Snyder, M. Nature 2005, 438, 679-684. (6) Jones, R. B.; Gordus, A.; Krall, J. A.; MacBeath, G. Nature 2006, 439, 168174.

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capacity.7-9 Protein microarrays are typically printed on glass slides functionalized with surface coatings that promote covalent, ionic, or adsorptive immobilization of proteins. Printing is most often performed with contact pin printers or inkjet printers. Thousands of different protein species can be immobilized on a single slide using automated robotics, facilitating high-throughput. The complexity of solutions that slides can be probed with is currently limited by detection schemes, most often fluorescencebased, as well as problems with nonspecific adsorption/interactions. Although sensitive, fluorescence requires the use of a probe species tagged with a fluorophore or the use of tagged secondary antibodies. Because of this, microarrays are usually only probed with simple mixtures containing known species that are specifically labeled. A further complication in probing microarrays with complex mixtures is that untagged species could interfere with the binding of the tagged probe species; the data indicate whether or not something has bound to protein spots on the array, but not what has bound. Although complex mixtures have been used with antibody arrays, any cross-reactivity of antibodies or nonspecific binding that occurs goes undetected and can skew results.10 This is also an issue with many alternative tag-free techniques being developed for protein microarray interrogation, such as surface plasmon resonance.11-13 Protein microarray experiments relying on detection methods which are blind to chemical identity have to be carefully designed to ensure that the identity of the bound species can be deduced with some certainty. Mass spectrometry (MS) could be a powerful technique to detect binding at protein arrays immobilized on surfaces. Mass spectrometry can provide molecular information for bound species, which eliminates the need for tags while detecting and potentially characterizing (via tandem mass spectrometry (MS/MS)) unan(7) Haab, B. B.; Dunham, M. J.; Brown, P. O. Genome Biol. 2001, 2, 1-13. (8) Joos, T. O.; Schrenk, M.; Hopfl, P.; Kroger, K.; Chowdhury, U.; Stoll, D.; Schorner, D.; Durr, M.; Herick, K.; Rupp, S.; Sohn, K.; Hammerle, H. Electrophoresis 2000, 21, 2461-2650. (9) Robinson, W. H.; DiGennaro, C.; Hueber, W.; Haab, B. B.; Kamachi, M.; Dean, E. J.; Fournel, S.; Fong, D.; Genovese, M. C.; Neuman de Vegvar, H. E.; Skriner, K.; Hirschberg, D. L.; Morris, R. I.; Muller, S.; Pruijn, G. J.; van Venrooij, W. J.; Smolen, J. S.; Brown, P. O.; Steinman, L.; Utz, P. J. Nat. Med. 2002, 8, 295-301. (10) Phizicky, E.; Bastiaens, P. I. H.; Zhu, H.; Snyder, M.; Fields, S. Nature 2003, 422, 208-215. (11) Lee, H. J.; Nedelkov, D.; Corn, R. M. Anal. Chem. 2006, 78, 6504-6510. (12) Nedelkov, D. Anal. Chem. 2007, 79, 5987-5990. (13) Kanoh, N.; Kyo, M.; Inamori, K.; Ando, A.; Asami, A.; Nakao, A.; Osada, H. Anal. Chem. 2006, 78, 2226-2230. 10.1021/ac701800h CCC: $40.75

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ticipated or unknown binding partners. Additionally, mass spectrometry could detect binding of multiple species from solution to a single spot. For example, the binding of multiple truncated or post-translationally modified versions of a peptide or protein in solution binding to a single immobilized protein can be detected.14 Another powerful application of interrogation of microarrays with mass spectrometry is probing arrays with mixtures of small molecules, such as drugs, which are difficult to label efficiently. Recently, several methods have been reported which couple matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry with proteins arrayed on macroporous silicon,15 methacrylate polymers,16 and self-assembled monolayers (SAMs) on gold.12,17 Alkanethiol SAMs on gold are particularly amenable to MALDI-TOF detection of binding to immobilized proteins.18-21 Self-assembled monolayers provide a simple and convenient method for specifying interfacial properties on a conductive gold substrate.22-24 Additionally, SAMs terminated with reactive functional groups can also be used to carry out reactions at the surface. In particular, carboxy-terminated SAMs yield a hydrophilic surface to which proteins can be covalently linked in a straightforward manner.25,26 Patrie and Mrksich have successfully used a SAM surface with several antibodies bound to simultaneously detect multiple proteins in plasma, while avoiding nonspecific binding through the use of polyethylene glycol (PEG)based SAMs which are inert to protein adsorption.21 Nedelkov also used SAMs on gold to effectively analyze a protein microarray with both surface plasmon resonance imaging and MALDI-TOF.12 The most promising aspect of using mass spectrometry with microarrays is the ability to use complex solutions. Ideally, thousands of different proteins printed on a single microarray could be exposed to numerous untagged probe species in a biological media, such as plasma, and meaningful data about the interactions that occur could be derived. MALDI is a logical ionization technique for coupling mass spectrometry and protein microarrays because it has been proven effective for molecular imaging applications, primarily tissue imaging.27-29 Further, (14) Brockman, A. H.; Orlando, R. Anal. Chem. 1995, 67, 4581-4585. (15) Finnskog, D.; Ressine, A.; Laurell, T.; Marko-Varga, G. J. Proteome Res. 2004, 3, 988-994. (16) Gavin, I. M.; Kukhtin, A.; Glesne, D.; Schabacker, D.; Chandler, D. P. BioTechniques 2005, 39, 99-107. (17) Min, D.-H.; Tang, W.-J.; Mrksich, M. Nat. Biotechnol. 2004, 22, 717-723. (18) Min, D.-H.; Su, J.; Mrksich, M. Angew. Chem., Int. Ed. 2004, 43, 59735977. (19) Min, D.-H.; Yeo, W.-S.; Mrksich, M. Anal. Chem. 2004, 76, 3923-3929. (20) Su, J.; Rajapaksha Tharinda, W.; Peter Marcus, E.; Mrksich, M. Anal. Chem. 2006, 78, 4945-4951. (21) Patrie, S. M.; Mrksich, M. Anal. Chem. 2007, 79, 5878-5887. (22) Bain, C. D.; Troughton, E. B.; Tao, Y. T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335. (23) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1103-1169. (24) Schaeferling, M.; Schiller, S.; Paul, H.; Kruschina, M.; Pavlickova, P.; Meerkamp, M.; Giammasi, C.; Kambhampati, D. Electrophoresis 2002, 23, 3097-3105. (25) Fung, Y. S.; Wong, Y. Y. Anal. Chem. 2001, 73, 5302-5309. (26) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999, 71, 777-790. (27) Chaurand, P.; Norris, J. L.; Cornett, D. S.; Mobley, J. A.; Caprioli, R. M. J. Proteome Res. 2006, 5, 2889-2900. (28) Rubakhin, S. S.; Jurchen, J. C.; Monroe, E. B.; Sweedler, J. V. Drug Discovery Today 2005, 10, 823-837. (29) Schwartz, S. A.; Reyzer, M. L.; Caprioli, R. M. J. Mass Spectrom. 2003, 38, 699-708.

MALDI is a surface-based technique that has been widely used for analysis of surface-captured analytes and has also been used for high-throughput immunoaffinity mass spectrometry.30-32 Two major challenges with MALDI-TOF detection of arrays are sensitivity and the required application of organic matrix. Mass spectrometry is less sensitive than fluorescence and is adversely affected by the salts and surfactants often present in biochemical experiments. Furthermore, matrixes in organic solvents spread significantly when deposited on most surfaces, potentially resulting in cross-contamination of spots in a microarray. A third problem that must be considered with any array that is exposed to complex solutions is nonspecific adsorption to the substrate. To effectively couple mass spectrometry to arrays probed with complex solutions, issues of sensitivity, cross-contamination due to matrix addition, and nonspecific adsorption must be overcome. A potential solution to these problems is the marriage of two surface modification techniques, deposition of porous gold, also referred to as roughened gold or nanostructured gold, and patterned SAMs. Porous gold can be electrochemically deposited on gold substrates by immersing the surface in a gold salt solution and applying a negative potential.13,33-37 The structure of the deposited gold is dependent on the potential applied and the solution conditions.36 Porous gold surfaces have been used for protein binding assays with several detection techniques amenable to gold surfaces, such as electrochemistry,34 the quartz crystal microbalance,33 and ellipsometry.37 The surface area of a gold substrate can be significantly enhanced by porous gold deposition, which increases the surface density of immobilized ligands/ biomolecules and thereby amplifies the signal in surface binding assays. The use of porous gold for SAM-based protein arrays should amplify the signal when mass spectrometry is used for detection, compensating for reduced sensitivity in comparison with fluorescence. In addition to the convenient surface functionalization SAMs allow, they can also be easily patterned onto surfaces using traditional surface patterning strategies, such as photolithography,38,39 or “inking”-based approaches, such as stamping40 and inkjet printing.41,42 Patterning can be used to create wettability contrasts on surfaces, such as discrete hydrophilic spots sur(30) Nelson, R. W.; Krone, J. R.; Bieber, A. L.; Williams, P. Anal. Chem. 1995, 67, 1153-1158. (31) Kiernan, U. A.; Tubbs, K. A.; Gruber, K.; Nedelkov, D.; Niederkofler, E. E.; Williams, P.; Nelson, R. W. Anal. Biochem. 2002, 301, 49-56. (32) Kiernan, U. A.; Tubbs, K. A.; Nedelkov, D.; Niederkofler, E. E.; Nelson, R. W. FEBS Lett. 2003, 537, 166-170. (33) Bonroy, K.; Friedt, J.-M.; Frederix, F.; Laureyn, W.; Langerock, S.; Campitelli, A.; Sara, M.; Borghs, G.; Goddeeris, B.; Declerck, P. Anal. Chem. 2004, 76, 4299-4306. (34) Imamura, M.; Haruyama, T.; Kobatake, E.; Ikariyama, Y.; Aizawa, M. Sens. Actuators, B 1995, B24, 113-116. (35) Notsu, H.; Kubo, W.; Shitanda, I.; Tatsuma, T. J. Mater. Chem. 2005, 15, 1523-1527. (36) Tian, Y.; Liu, H.; Zhao, G.; Tatsuma, T. J. Phys. Chem. B 2006, 110, 2347823481. (37) Van Noort, D.; Mandenius, C.-F. Biosens. Bioelectron. 2000, 15, 203-209. (38) Huang, J.; Dahlgren, D. A.; Hemminger, J. C. Langmuir 1994, 10, 626628. (39) Tarlov, M. J.; Burgess, D. R. F., Jr.; Gillen, G. J. Am. Chem. Soc. 1993, 115, 5305-5306. (40) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 14981511. (41) Bietsch, A.; Hegner, M.; Lang, H. P.; Gerber, C. Langmuir 2004, 20, 51195122. (42) Pardo, L.; Wilson, W. C., Jr.; Boland, T. Langmuir 2003, 19, 1462-1466.

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rounded by a hydrophobic background. Hydrophobic/hydrophilic patterned surfaces have been used extensively in MALDI mass spectrometry. The hydrophilic spots act as “anchor points”, which contain and/or focus matrix solutions to small spots sizes.43,44 Recently, these patterns have also been used to generate protein microarrays with improved spot uniformity and enhanced reproducibility.45-47 Arrays based on hydrophobic/hydrophilic patterns with proteins immobilized in the hydrophilic spots could also make MALDI-TOF analyses more feasible by inhibiting crosscontamination between spots when matrix is added to the surface. However, most hydrophobic/hydrophilic patterns lose their integrity in complex solutions because nonspecific adsorption occurs readily at the hydrophobic background. When both the hydrophobic and hydrophilic regions are coated in protein, the wettability contrast between the spots and the background is lost and the pattern is ruined. Care must be taken to avoid bringing the hydrophobic regions of these patterns into contact with biofouling media, such as protein solutions. Consequently, the usefulness of conventional hydrophobic/hydrophilic patterns for protein arrays is limited.47 In order to fully leverage the advantages of mass spectrometry for interrogating protein microarrays, the hydrophobic/hydrophilic patterns used should maintain their integrity when probed with complex biofouling solutions. The most common method for preventing nonspecific adsorption is blocking the background regions between spots with an inert protein or polymer such as bovine serum album (BSA) or PEG. In SAM-based mass spectrometry assays, PEG-based SAMs have been frequently employed. Such blocking methods are not amenable to maintaining hydrophobic/hydrophilic patterns. In the current work, we have used a superhydrophobic background of porous gold functionalized with a methyl-terminated SAM to prevent the biofouling of the hydrophobic/hydrophilic pattern. In addition to amplifying the signal for surface-based assays, the enhanced surface area of porous gold influences the surface wettability, since wettability is a function of both the composition and the topography of the surface layer.48 Increased surface roughness amplifies the intrinsic contact angle of materials. As a result, an intrinsically hydrophobic surface can become a superhydrophobic surface (contact angle greater than 150°) when the surface roughness is increased.49 When the rough surfaces of porous gold are modified with methyl-terminated SAMs, the surface becomes superhydrophobic.13,35,50 Superhydrophobic surfaces should be resistant to nonspecific adsorption because liquid droplets exposed to these surfaces tend to ride on a pocket of trapped air and make minimal contact with the underlying surface, according to Marmur’s theoretical studies.49,51 Therefore, protein adsorption from biological solutions at superhydrophobic surfaces (43) Schuerenberg, M.; Leubbert, C.; Eickhoff, H.; Kalkum, M.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2000, 72, 3436-3442. (44) McLauchlin, M. L.; Yang, D.; Aella, P.; Garcia, A. A.; Picraux, S. T.; Hayes, M. A. Langmuir 2007, 23, 4871-4877. (45) Grote, J.; Dankbar, N.; Gedig, E.; Koenig, S. Anal. Chem. 2005, 77, 11571162. (46) Moran-Mirabal, J. M.; Tan, C. P.; Orth, R. N.; Williams, E. O.; Craighead, H. G.; Lin, D. M. Anal. Chem. 2007, 79, 1109-1114. (47) Zhang, H.; Lee, Y. Y.; Leck, K. J.; Kim, N. Y.; Ying, J. Y. Langmuir 2007, 23, 4728-4731. (48) Cassie, A. B. D.; Baxter, S. Trans. Faraday Soc. 1944, 40, 546-551. (49) Marmur, A. Biofouling 2006, 22, 107-115. (50) Bietsch, A.; Zhang, J.; Hegner, M.; Lang, H. P.; Gerber, C. Nanotechnology 2004, 15, 873-880. (51) Marmur, A. Langmuir 2006, 22, 1400-1402.

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Figure 1. Schematic illustration and SEM images of evaporated gold films (A) without and (B) with porous gold deposited on the surface at (B1) 3500×, (B2) 1000×, and (B3) 10000× magnification and (C) after patterning with mercaptoundecanoic acid and dodecanethiol SAMs. The white bar in each image represents 10 µm.

should be minimized since there is very little contact between the surface and the solution. As illustrated in Figure 1, we deposited porous gold onto a bare gold surface and subsequently

patterned the substrate with methyl and carboxy-terminated SAMs, yielding a superhydrophobic background and well-defined hydrophilic spots. The surfaces were patterned using the same equipment that is routinely used to print protein microarrays and without the use of lithography or complex surface modifications. Since both the pattern generation and protein printing could be done on the same equipment, the protein printing could be aligned with the pattern easily, allowing for the future production of custom-made protein microarrays. We used a model system of antibodies to several peptides (HA, cmyc, and V5) with goat immunoglobulin G (IgG) controls to demonstrate that porous gold surfaces with patterned alkanethiol SAMs facilitate MALDI-TOF detection of protein arrays. Figure 2 illustrates how proteins were covalently immobilized in the carboxy-terminated SAM spots, exposed to plasma spiked with the antigen peptides, and finally detected with mass spectrometry. In previous literature reports, MALDI-TOF mass spectra were obtained by manually aligning the laser with individual spots on a microarray. In addition to obtaining spectra of individual spots, we have used mass spectrometry imaging to interrogate entire arrays, both eliminating the need for manual alignment and verifying the integrity of the spots. EXPERIMENTAL METHODS Materials. Gold-coated microscope slides (50 nm Cr adhesion layer, 100 nm gold) were purchased from EMF Corp. (Ithaca, NY) and used as the gold substrates in all experiments. Hydrogen tetrachloroaurate(III) was purchased from Acros Organics (Geel, Belgium). Mercaptoundecanoic acid and dodecanethiol were obtained from Sigma (St. Louis, MO). 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Pierce Biotechnology (Rockford, IL). Affinity peptides (HA, V5, and cmyc) were acquired from Anaspec (San Jose, CA), and the corresponding goat polyclonal antibodies were obtained from QED Biosciences (San Diego, CA); the goat IgG used for controls was purchased from Sigma. R-Cyano-4-hydroxycinnamic acid (CHCA) was used as the MALDI matrix for all experiments and was obtained from Sigma. HPLC grade water was used for all solutions as well as for washing and rinsing steps. Bovine plasma was obtained from bovine whole blood purchased from Ruppersberger slaughterhouse (Baltimore, MD). Whole blood was collected from a single cow into a container with 4 mg/mL EDTA as anticoagulant. The blood was centrifuged at low speeds to remove the cells without rupturing them. The supernatant was collected and centrifuged again at moderate speeds to deplete the platelets without rupturing them. The platelet-poor plasma supernatant was collected and used for all plasma experiments. Porous Gold Deposition. First, gold substrates were cleaned in piranha solution (3:1 H2SO4, 30% H2O2) for approximately 30 min. After rinsing thoroughly with water and ethanol, the slides were blown off under a stream of nitrogen, mounted in a Teflon deposition cell, and immersed in a 3 mg/mL hydrogen tetrachloroaurate solution in 0.5 M H2SO4. Under constant stirring conditions, a potential of -400 mV versus a Ag/AgCl reference electrode was applied, using a Pt mesh counter electrode with a solution-exposed surface area larger than that of the gold-coated microscope slide. The potentiostat circuit used was constructed based on a previously published design52 and could sustain

Figure 2. Schematic illustrating (A) exposure of protein-functionalized porous gold patterns to a complex mixture, (B) species from the solution bound to the immobilized proteins after rinsing, and the subsequent MALDI (C) image and (D) spectra of the bound species. The MS image shown in frame C illustrates three different images, each corresponding to the m/z of the molecular ion for the dominant binding peptide, overlaid as the three different colors shown in the legend. The spectra shown in frame D are meant to illustrate the ability of mass spectrometry to recognize multiple species, as shown in the middle spectrum, as well as recognize several variant ligands, as shown in the bottom spectrum, which are simultaneously bound to a single array element.

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currents up to approximately 200 mA. The deposition was stopped when 175 C of charge had accumulated. To conserve gold, the gold solutions were reused for multiple porous gold depositions. This resulted in a gradual depletion of gold salts from solution and a decrease in steady-state current after multiple depositions. Therefore, the stopping point for porous gold deposition was based on a fixed charge rather than a fixed time. After the deposition, the substrates were removed from the cell and recleaned with piranha solution as described above. After cleaning, the substrates were rinsed thoroughly, blown off under a nitrogen stream, and stored dry. Patterning of Self-Assembled Monolayers. Patterns of SAMs were generated using a microarray pin printer (VersArray ChipWriter; Hercules, CA), a chemical inkjet printer (CHIP 1000, Shimadzu Biotech; Columbia, MD), or an Epson Stylus Photo R220 (Epson America, Inc.; Long Beach, CA). For printing with the Epson printer, the CD-printing tray was fitted with a mask to hold a microscope slide and a roller was removed to avoid roller contact with the surface. Empty ink cartridges were purchased from CompuBiz Inkjet (Wheatland, WY) and were filled with appropriate thiol solutions. For initial experiments testing the signal as a function of peptide concentration, slides were printed using the Epson inkjet. An array consisting of five columns and three rows of carboxyterminated SAM spots 2000 µm in diameter was printed from ∼2 mM solutions of mercaptoundecanoic acid. For experiments testing antibody arrays in bovine plasma, a pattern of three arrays was printed. Each array consisted of four columns and three rows and contained either large, medium, or small spot sizes. To verify the integrity of the printed carboxy-terminated SAM spots, a patterned test slide was immersed in a mild gold etchant solution41 until all the unprotected gold regions were removed. To create a methyl-terminated SAM background, the remaining slides were immersed in ∼2 mM solutions of ethanolic dodecanethiol for approximately 10 s. Once the slides were removed, they were rinsed thoroughly with ethanol and water and then blown dry under a stream of nitrogen. The successful formation of the hydrophobic/hydrophilic pattern was monitored by immersion of the slides in solutions of ultrapure water. For acquiring camera and microscope images of hydrophobic/hydrophilic patterns, the slides were immersed into a 1:1 glycerol/water solution to prevent rapid evaporation from smaller spots. Protein Immobilization and Peptide Capture Experiments. The carboxy groups on the hydrophilic SAM spots were activated for covalent protein immobilization by immersion in a freshly made solution of 4 mM EDC/10 mM NHS for 1 h.25,26 Antibody and control IgG or BSA solutions were then manually spotted onto the NHS ester functionalized hydrophilic spots using a microliter pipet. The slides were incubated for 2 h in a humidity chamber and then rinsed thoroughly with water. Once the slides were functionalized with the proteins, they were not allowed to dry at any point until just before CHCA matrix was added. In initial experiments, anti-V5-functionalized surfaces were exposed to varying concentrations of the V5 peptide antigen in purified water. In experiments testing the multiple-antibody arrays, the slides were exposed to bovine plasma spiked to a concentration of 2 (52) Kirkup, L.; Bell, J. M.; Green, D. C.; Smith, G. B.; MacDonald, K. A. Rev. Sci. Instrum. 1992, 63, 2328-2329.

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µM with HA, cmyc, and V5 peptides. The spiked plasma was either directly added as droplets to the hydrophilic spots with a pipet or the slide was briefly immersed in the spiked plasma. To avoid drying of the liquid retained on the hydrophilic spots, the slides were quickly placed into a humidity chamber and incubated for an hour. The slides were then rinsed with water, immersed in water three times for 10 min each time, rinsed again with water, and blown off under a stream of nitrogen. Once the spots were dry, 0.2 µL of either 5, 1, or 0.5 mg/mL of CHCA matrix in 70% acetonitrile, 30% trifluoroacetic acid (0.1%) was applied to the large, medium, or small spot size arrays, respectively. Mass Spectrometry. All MS and MS/MS spectra were acquired in the positive ion mode at an acceleration voltage of 20 kV using a Kratos Analytical (Manchester, U.K.) AXIMA-CFR Plus MALDI-TOF high-performance mass spectrometer capable of acquiring linear or reflectron spectra. External calibration was performed using a mixture of three calibrant peptides spotted in the center of each slide in a region that did not contain any arrayed spots. The peptides used in the binding experiments (HA, cmyc, V5) were never used as calibrant peptides. MS/MS spectra were collected without collision gas. MS images were acquired by dividing the imaged area into pixels of 200 µm × 200 µm. At each pixel, 20 spectra were acquired, averaged, and converted to ASCII files. The ASCII files were sorted and compiled into images using a LabVIEW program (written in-house) and MathCAD. The images are comprised of the signal intensities represented in false color, sorted spatially based on the pixel position, and were generated from spectra in two ways. The signal intensity for each m/z window of (7 was determined at each pixel by either summing all of the signal in the window or the window was searched for the point of maximum signal, and this was assigned as the pixel intensity. RESULTS AND DISCUSSION Preparation of Patterned Porous Gold Surfaces. Although the charge obtained from integration of the current was used as a qualitative indicator of the thickness of the deposited porous gold layer, we did not use these values to rigorously calculate thickness. Due to reaction conditions, it is likely that there were minor contributions to the current from side reactions, such as hydrogen gas evolution in solution and reduction at small solutionexposed portions of the Pt wire making the electrical connection with a gold surface. The color of the gold-coated substrates was visibly altered after the porous gold was deposited, turning from bright yellow to tan. Scanning electron microscope (SEM) images acquired after porous gold deposition show the nanostructured porous gold with significantly enhanced surface area (Figure 1). Self-assembled monolayers formed rapidly when either the pin printer or the chemical inkjet printer were used to deposit mercaptoundecanoic acid solutions onto the porous gold substrates. Self-assembled monolayer formation in the mercaptoundecanoic acid-printed regions was tested by immersion of a test slide in a gold etchant solution. While the underlying gold in the printed regions was protected, gold in the unprinted regions dissolved in the etchant solution (Supporting Information Figure S1).41 Immersion of mercaptoundecanoic acid-printed surfaces in dodecanethiol solution resulted in hydrophobic SAM formation in the unprinted, bare gold regions. These hydrophobic/hydrophilic patterns were readily apparent when the slides were

Figure 3. Digital photograph of a porous gold surface with a hydrophobic/hydrophilic SAM pattern (A) after brief immersion in and removal from a 1:1 aqueous glycerol solution and (B) while immersed in water, viewed from the front (B1) and side (B2).

immersed in aqueous solution (Figure 3). Spot sizes were dependent on the volume of mercaptoundecanoic acid deposited on the surface for both the pin-printed, where the volume is determined by the number of times the pin contacted the surface, and inkjet-printed surfaces. Numerous patterns with different layouts and spot sizes were easily printed using the Epson inkjet printer, the Shimadzu CHIP inkjet printer, and the pin printer. Additionally, patterns could be created manually by carefully spotting mercaptoundecanoic acid solution with a microliter pipet. The porous gold substrates could be reused, even after exposure to bovine plasma and matrix, by cleaning the surfaces with piranha solution twice. We have reused porous gold surfaces numerous times (>10 thus far) and have not observed any detrimental affects on the SAM patterns produced or the mass spectrometry signal for the antibody array assay. However, porous gold layers are easily scratched through contact with the surface, and scratches significantly affected the patterns produced. Comparison of MS and MS/MS Signals from Porous and Flat Gold Surfaces. When spots on porous gold were functionalized with anti-V5, exposed to V5 solution, rinsed, and exposed to matrix solution, reflectron MALDI-TOF spectra were obtained where the m/z of the base peak correlated with the [M + H]+ ion for V5 (Figure 4). Further, the bound V5 was de novo sequenced using the MS/MS spectra. Control spots functionalized with BSA yielded no significant peaks. The surface roughness does not appear to adversely affect the mass spectrometry, and the conductivity of the substrate likely yields improved signal relative to glass slides, since previous studies of mass spectrometry on glass-based affinity slides have noted reduced signal as well as

Figure 4. Representative reflectron MALDI-TOF (A) MS and (B) MS/MS spectra, annotated with the de novo sequencing of the peptide, of an anti-V5 spot on a porous gold surface probed with 100 nM V5 peptide in water.

reduced fragmentation in MS/MS spectra relative to metallic substrates.53 The mass accuracy in these experiments was slightly lower than in standard MALDI-TOF experiments done using a finely machined stainless steel plate with the same instrumentation. We attribute this to the way that the slides were mounted on the stainless steel plate, in depressions machined to match the height of the slides and secured with double-sided tape. This arrangement is not ideal because there the slide is not precisely level, resulting in minor drift from the initial m/z calibration across the length of the slide. These MS and MS/MS spectra for the individual spots demonstrate that patterned porous gold is an effective substrate for immunoaffinity MS experiments. To study the signal enhancement imparted by deposition of porous gold, five columns on an array of anti-V5 antibody were probed with 10 µL droplets of varying concentrations of V5. The V5 [M + H]+ peak for arrays on both porous gold and flat gold probed with 100, 50, 10, 5, and 1 nM V5 are shown in Figure 5. A signal-to-noise ratio of 5 was obtained from the porous gold substrate when probed with 1 nM V5, whereas the flat gold probed with peptide concentrations below 10 nM yielded no significant signal. An equivalent signal improvement was obtained for MS/ MS spectra of bound V5 peptide. Meaningful sequence data could be obtained from the MS/MS spectra from the spots on the porous gold probed with V5 concentrations as low as 10 nM, (53) Afonso, C.; Fenselau, C. Anal. Chem. 2003, 75, 694-697.

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Figure 5. Normalized signal for the V5 molecular ion obtained from reflectron spectra of 2000 µm diameter anti-V5 spots from patterned arrays on (left) porous and (right) flat gold probed with (A) 100, (B) 50, (C) 10, (D) 5, and (E) 1 nM V5 peptide solutions in water.

whereas 100 nM V5 was necessary to derive spectra useful for de novo sequencing when the experiment was done with the flat gold (Supporting Information Figure S2). The signal enhancement observed was anticipated based on previous studies. Electrochemical and quartz crystal microbalance experiments have previously shown that the enhanced surface area of porous gold nanostructures significantly increased the amount of protein immobilized on the surface, amplifying the signal obtained when species bound to immobilized protein.33,34,37 Exposure of the Patterned Slides to Potentially Fouling Solutions. In addition to increasing the amount of protein that could be immobilized at the surface, the use of porous gold also resulted in superhydrophobicity in the background regions functionalized with the methyl-terminated SAMs. When the patterned porous gold surfaces are immersed in water and viewed from the side, the trapped layer of air over the superhydrophobic regions appears as a silver color (Figure 3B2). The printed pattern can be visualized since the hydrophilic spots are wetted and thus do not have a layer of trapped air above them. Patterns generated on flat gold or surfaces with thinner porous gold layers did not display this behavior; therefore, the hydrophobic SAM as well as sufficient surface roughness were both required to achieve underwater superhydrophobicity. 1454

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As predicted by Marmur,49 trapped air seemed to inhibit nonspecific adsorption in the superhydrophobic regions. When hydrophobic/hydrophilic patterns were generated on flat gold surfaces, the pattern was apparent when the slides were immersed in water, similar to the patterned porous gold surfaces. However, when the patterned flat gold surfaces were briefly immersed in a 1 mg/mL BSA solution, the pattern was irreversibly ruined. That is, even solutions of pure water coated the entire surface and were no longer confined to the hydrophilic spots. In contrast, patterned porous gold slides could be left in a 1 mg/mL BSA overnight with no significant change in the pattern or spot size (Supporting Information Figure S3). Patterned porous gold surfaces could even be immersed in bovine platelet-poor plasma without ruining the pattern (Supporting Information, plasma immersion video). Furthermore, platelet-poor plasma droplets manually added to patterned porous gold surfaces were effectively pinned in the hydrophilic regions without apparent droplet spreading, which could occur if there was nonspecific adsorption to the surrounding hydrophobic surface (Supporting Information, plasma addition video). After repeated or prolonged immersion of these surfaces into platelet-poor plasma, a thin layer of liquid was visible over the hydrophobic regions. However, if the surface was rinsed and dried, the hydrophobic/hydrophilic pattern was restored. The extent of matrix solution spreading on hydrophilic, glassbased surfaces makes it difficult to obtain MALDI-TOF spectra for individual spots since matrix solution dissociates bound species from immobilized proteins in each spot. Therefore, if the matrix solution spreads over several different spots, the species bound to those spots will be mixed and the spatial information defining their immobilized binding partner is lost. Matrix solution spotted onto a glass slide spread to a diameter of ∼9 mm (Supporting Information Figure S4). In previous reports, matrix has been allowed to encompass several duplicate spots,15 the entire surface has been uniformly coated with a mixture of antibodies,21 matrix solution has been confined by three-dimensional (3D) polymer structures engineered with photolithography,16 or matrix has been carefully applied as a fine aerosol mist.12 In our experiments, matrix solution was effectively contained by the hydrophilic spots of the patterned porous gold (Supporting Information, matrix addition video), maintaining the integrity of individual array elements. Although matrix was manually added to the spots in these experiments, we are currently working toward more sophisticated methods of matrix application such as aerosol29 or inkjet-based54,55 deposition. We anticipate that our hydrophobic/ hydrophilic pattern will still prevent cross-contamination between spots and serve to focus deposited matrix when these methods are employed. MALDI-TOF Spectra of Peptides Bound to Arrays Exposed to Spiked Plasma. Figure 6 shows representative reflectron MALDI-TOF spectra from antibody-functionalized spots after exposure to spiked plasma, rinsing, and addition of matrix solution. In this experiment, the slide was patterned by inkjet printing (Shimadzu CHIP) and droplets of the spiked plasma were manually added to the surface. In the spectra obtained for each spot, the m/z of the base peak correlates with the [M + H]+ ion (54) Aerni, H.-R.; Cornett Dale, S.; Caprioli Richard, M. Anal. Chem. 2006, 78, 827-834. (55) Baluya, D. L.; Garrett, T. J.; Yost, R. A. Anal. Chem. 2007, 79, 6862-6867.

Figure 6. Representative MALDI-TOF spectra of individual spots with (A) anti-HA antibody, (B) anti-cmyc antibody, (C) anti-V5 antibody, and (D) control IgG immobilized on the surface after exposure to spiked plasma, rinsing, and matrix addition. The intensities of the spectra are normalized to the intensity of the base peak for each spectra: 294, 137, 483, and 9 mV for the spectra in A, B, C, and D, respectively. The insets display the isotopic resolution achieved for each major peak.

for the peptide antigen corresponding with the antibody immobilized at that spot. The bound peptides were also de novo sequenced using the MS/MS spectra (Figure 7). No nonspecific binding of the peptides was observed in the control IgG spectra, and the intensity of the peaks observed were 2 orders of magnitude lower than the intensity of the peaks for the captured peptides. The data also serendipitously emphasizes an advantage of using mass spectrometry for detection of antibody arrays. The peak at m/z 1365 in Figure 6 corresponds to a degradation product of the V5 peptide, the loss of the N-terminus glycine residue. This truncated V5 peptide was also captured by the immobilized antiV5 antibody and would be recognized as intact V5 peptide by a fluorescence or surface plasmon resonance detection scheme. Whereas antibody specificity can be difficult to determine using other detection methods, the specificity of the immobilized antibodies was readily apparent with mass spectrometry. In future work, we will be looking at protein interactions not as well-defined or specific as antibody-antigen binding and the ability to detect several different species bound to the same immobilize protein will be critical.

Figure 7. MS/MS identification of the major peaks observed for individual spots with (A) anti-HA antibody, (B) anti-cmyc antibody, and (C) anti-V5 antibody immobilized on the surface after exposure to spiked plasma, rinsing, and matrix addition.

The spectra shown in Figures 6 and 7 were obtained from the largest spot sizes, approximately 1500 µm in diameter, patterned using the Shimadzu CHIP inkjet printer. For all patterns and slides studied, the anti-V5 spots yielded the highest peak intensity, the anti-HA spots yielded an intermediate intensity, and the anti-cmyc spots had the lowest peak intensities. Whether this is due to differences in ionization efficiency between the peptides, differences in affinity between the antibodies, or a convolution of these two is unknown. As the spot sizes decreased, adjustments had to be made to obtain spectra for each spot. For the large spot sizes, high-quality spectra were obtained for each bound peptide in reflectron mode. For intermediate spots sizes, approximately 900 µm in diameter, high-quality spectra were obtained for each bound peptide in linear mode. However, in the less sensitive reflectron Analytical Chemistry, Vol. 80, No. 5, March 1, 2008

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Figure 9. Overlaid MALDI-TOF images of the surface shown in Figure 8A after exposure to spiked plasma, rinsing, and matrix addition for the HA parent ion (m/z 1102sgreen), the cmyc parent ion (m/z 1203sblue), and the V5 peak (m/z 1422sred). The z-axis intensity is in arbitrary units, and the yellow-brown background is set at a threshold of 20 units. The grid in the xy plane indicates the 200 µm2 pixels.

Figure 8. (A) Digital image of protein solutions incubating on a patterned porous gold surface and the subsequent MALDI-TOF images of (B) a CHCA matrix peak (m/z 378), (C) the HA parent ion (m/z 1102), (D) the cmyc parent ion (m/z 1203), and (E) the V5 parent ion (m/z 1422) after exposure to spiked plasma, rinsing, and matrix addition. The column labels in (A) correspond to the antibody or control immobilized on spots in that column: HA ) anti-HA antibody, cmyc ) anti-cmyc antibody, V5 ) anti-V5 antibody, IgG ) IgG control. The false color scheme represents peak intensity, where red is the highest intensity in the image, violet is a weak signal, and black is no signal. 1456 Analytical Chemistry, Vol. 80, No. 5, March 1, 2008

mode, only the V5 and HA peptides were detected. For the smallest spot sizes, approximately 700 µm in diameter, only the V5 peptide was detected, and only in linear mode. Increased smoothing and baseline subtraction of the spectra enhances the detection of the bound peptides, but at the sacrifice of isotopic resolution. As would be expected, the effectiveness of mass spectrometry detection of arrays decreases with decreasing spot size. Current work is focused on improving higher resolution detection and MS/MS analyses of smaller spots. It is likely that significant amounts of bound peptide dissociated from immobilized antibodies when the slides were vigorously rinsed with pure water prior to the addition of matrix. It is also likely that residual salts from the plasma suppress the MS signal, resulting in a situation where too much rinsing can dissociate bound peptides and decrease the signal, whereas too little rinsing results in suppression and also decreases the signal. The competition between these two factors makes optimization of rinsing procedure a critical step to maximize the signal obtained from spots on the array. One strategy being employed is the use of ammonium salt solutions in rinsing steps. Ammonium buffers are more likely to sustain noncovalent biological interactions while also inhibiting suppression by residual sodium and potassium. Previous work by Kiernan et al. has successfully employed ammonium salts during the rinsing steps in affinity-MS experiments extracting proteins from plasma.32 MALDI-TOF Imaging of Slides after Peptide Capture. Image data in a format similar to traditional protein array data was generated by dividing the slides into 200 µm2 pixels and compiling the spectra collected for each pixel into images. MALDITOF images were generated for the slide discussed in the previous section, which was patterned with an inkjet printer and had plasma manually added to the surface as well as for an experiment where the slide was patterned using a pin printer and later exposed to plasma by brief immersion.

Figure 10. (A) Digital image of protein solutions incubating on a patterned porous gold surface and the subsequent MALDI-TOF images of (B) a CHCA matrix parent ion (m/z 378), (C) the HA parent ion (m/z 1102), (D) the cmyc parent ion (m/z 1203), and (E) the V5 parent ion (m/z 1422) after immersion in spiked plasma, rinsing, and matrix addition. The column labels in A correspond to the antibody or control immobilized on spots in that column: HA ) anti-HA antibody, cmyc ) anti-cmyc antibody, V5 ) anti-V5 antibody, IgG ) IgG control. The false color scheme represents peak intensity, where red is the highest intensity in the image, violet is a weak signal, and black is no signal. The image show in (F) is the overlaid MALDI-TOF images for the HA parent ion (m/z 1102sgreen), the cmyc parent ion (m/z 1203sblue), and the V5 parent ion (m/z 1422sred). The z-axis intensity is in arbitrary units, and the yellow-brown background is set at a threshold of 20 units. The grid in the xy plane indicates the 200 µm2 pixels.

The image of the signal intensity at m/z 378, a peak routinely observed when CHCA matrix is used, displays the effectiveness

of the inkjet-printed hydrophobic/hydrophilic pattern in constraining the matrix solution to the hydrophilic spots (Figure 8B). Analytical Chemistry, Vol. 80, No. 5, March 1, 2008

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Images C-E in Figure 8 illustrate the specific capture of the appropriate peptides by the immobilized antibodies. The hydrophobic areas retained no matrix resulting in an intensity of zero for all m/z ranges, displayed as a black background. Because they contain matrix, there is some chemical noise across the entire m/z range of the mass spectra in the hydrophilic spots. In Figure 8, the images are normalized to the highest intensity for the m/z range selected and the chemical noise background can be seen as dim violet spots, particularly in the spots not corresponding to the peptide of interest. The signal-to-noise ratios can be visualized as the contrast and intensity between the violet chemical noise in the spots which do not correspond to the peptide of interest and the multicolored spots corresponding to the peptide of interest. The contrast between the V5 spots and the chemical noise spots is so high in the V5 image (Figure 8E) that the violet chemical noise spots are indistinguishable from the black background, whereas the lower signal-to-noise ratio in the cmyc image makes the violet spots visible (Figure 8D). The HA image had an intermediate signal-to-noise ratio. Figure 9 demonstrates an alternative method for viewing the MALDI-TOF image, where the three m/z regions, corresponding to the molecular ion for each of the three peptides, are overlaid against a yellow background. The z-axis is fixed, and the relative intensities of the signals for the HA, cmyc, and V5 peptides are apparent. The mixed colors and low intensity of the IgG control spots demonstrate that the chemical noise from each overlaid m/z region resulted in the small signal visible in the image. Figure 10 shows MALDI-TOF images generated from a slide in which the surface was first patterned using a pin printer (rather than an inkjet printer) and then briefly immersed in spiked plasma instead of droplets being manually added. This slide consisted of three arrays with decreasing spot diameters of approximately 1500, 1100, and 850 µm, respectively. The signal-to-noise ratios for preliminary spectra taken before the slide was imaged were lower than for the previously discussed slide, where spiked plasma droplets were manually added (data not shown). The increased amount of chemical noise and salt adducts observed in the spectra from the immersed slide suggest an increased amount of residual salt after rinsing. To compensate for the lower signal-to-noise, the spectrum for each pixel in the image was acquired in linear mode with extensive smoothing and baseline subtraction. Therefore, isotopic resolution and the potential for MS/MS were lost. In the images generated under these conditions, the spots correlating to the HA and V5 peptides are apparent at each spot size (Figure 10, parts C, E, and F). As in the previous experiment, the signal for the cmyc peptide is comparably lower (Figure 10, parts D and F). For the smallest spot size, the cmyc signal is difficult to discern from the background. Although smoothing, baseline subtraction, and linear mode were required to enhance the signal in these plasma “dipping” experiments, this approach is more convenient and more amenable to high-throughput analyses. CONCLUSIONS These experiments have shown that patterned porous gold substrates are well-suited as platforms for printing protein microarrays for detection with MALDI-TOF mass spectrometry. Porous gold was deposited on gold-coated microscope slides in a straightforward procedure to yield reusable substrates with very high surface area. The substitution of porous gold for flat gold in 1458

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V5 antibody array experiments significantly amplified the signal when the array was interrogated with both MS and MS/MS, resulting in an order of magnitude improvement in the limit of detection. The superhydrophobic background resulting from the use of porous gold prevented biofouling of the pattern even when immersed in bovine plasma. Since the pattern was maintained when exposed to plasma and because of the strong wettability contrast between the hydrophilic protein spots and the hydrophobic background, the matrix solutions (70% acetonitrile, 30% trifluoroacetic acid (0.1%)) were effectively contained within the spots. These factors facilitated MALDI-TOF spectral and imaging analysis of peptide binding to the protein arrays. Additionally, the patterns were generated in a straightforward approach using equipment commonly available in protein microarray facilities. MALDI-TOF detection of protein microarrays adds a new dimension of data to protein microarray analysis and eliminates the need for fluorescent tags. While work is ongoing to increase the spot density on the surfaces and improve the incorporation of the surfaces into high-throughput methodologies, this successful MALDI-TOF imaging of an antibody array immersed in plasma supports the use of this approach in routine, high-throughput analyses of protein arrays with mass spectrometry. Thus far, experiments coupling microarrays with mass spectrometry have almost exclusively been done using antibody-based model systems probed with simple solutions. Although such welldefined systems are excellent for proof-of-concept experiments, our goal is to develop a system that is not dependent on antibody specificity and yet is still reliable when probed with complex solutions. We are working toward a system where microarrays of proteins can be exposed to complex solutions, such as plasma or cell lysates, and the resulting myriad of protein interactions could be catalogued using mass spectrometry. Such a system, capable of high-throughput open-ended discovery, would be a powerful biochemistry technique for applications such as drug and biomarker discovery. ACKNOWLEDGMENT Funding for this work was provided by a National Heart, Lung, and Blood Institute contract N01HV28180 and an NIH Roadmap Grant U5RR020839. K.M.E.-N. gratefully acknowledges a postdoctoral research fellowship from the NIH/Johns Hopkins School of Medicine Anti-Cancer Drug Development Training Program (CA 09243). The authors thank Joe Fox and Shimadzu Biotech for assistance with the chemical inkjet printer (CHIP), Jae Ho Shin for assistance with initial porous gold deposition experiments, Alisa Wohlberg for discussions about handling bovine plasma, and Mike Delannoy for the SEM images. SUPPORTING INFORMATION AVAILABLE Videos illustrating the manual addition of plasma to, plasma immersion of, and addition of matrix to functionalized patterned porous gold substrates as well as figures noted in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.

Received for review August 26, 2007. Accepted November 28, 2007. AC701800H