Langmuir 2009, 25, 1905-1907
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Nanoparticle-Based Solution Deposition of Gold Films Supporting Bioresistant SAMs Bartlomiej Kowalczyk,†,‡ Marta Byrska,† Goher Mahmud,† Sabil Huda,† Kristiana Kandere-Grzybowska,†,‡ and Bartosz A. Grzybowski*,†,‡ Department of Chemical and Biological Engineering and Department of Chemistry, Northwestern UniVersity, 2145 Sheridan Road, EVanston, Illinois 60208 ReceiVed October 6, 2008. ReVised Manuscript ReceiVed December 4, 2008 Thin films of gold on glass are prepared by solution deposition of functionalized gold nanoparticles followed by thermal treatment. The processed films adhere strongly to glass without any adhesion layers and can be micropatterned/ microetched without delamination from the substrate. The formation of self-assembled monolayers (SAMs) of oligo(ethylene glycol) alkane thiols (EG SAMs) renders the films resistant to cell adhesion and allows for cell patterning.
Metal films (∼10 nm to several µm) are important in applications including microelectronics,1 soft-lithographic patterning,2 substrates for corrosion protection layers,3 and biology.4 Typically, such films are deposited by chemical vapor deposition (CVD) and sputtering, e-beam or thermal evaporation, cathodic arc technology, or galvanic methods.5 Once prepared, the films can be further processed/patterned by reactive ion etching, ion milling, wet etching combined with self-assembled monolayer (SAM) protection,6 or reaction-diffusion.7 In the vast majority of applications, these methods give metal films of satisfactory quality using equipment that is nowadays considered to be standard in materials science or chemistry departments. This, however, is not necessarily the case in biological applications where micropatterned thin metal films (notably, gold) supporting oligo(ethylene glycol) EG SAMs are used to control cell shape and function.4 In these applications, the degree of ultimate bioresistance of Au/EG SAM is very sensitive to the gold deposition parameters and to the cleanliness of the metal source. For this reason, deposition instruments from open-use facilities are often inadequate, and to ensure reliable fabrication of bioresistant films, a dedicated evaporator station is usually needed. In addition, although gold-coated glass slides are commercially available from several suppliers and well suited for surface plasmon resonance or for general-purpose microcontact printing procedures, we found (through multiple rounds of tests) that * Corresponding author. E-mail:
[email protected]. † Department of Chemical and Biological Engineering. ‡ Department of Chemistry. (1) (a) Leskela, M.; Ritala, M. Thin Solid Films 2002, 409, 138. (b) Hantschel, T.; Wong, L.; Chua, C. L.; Fork, D. K. Microelectron. Eng. 2003, 67-68, 690. (c) Inberg, A.; Shacham-Diamand, Y.; Rabinovich, E.; Golan, G.; Croitoru, N. J. Electron. Mater. 2001, 30, 355. (2) Xia, Y. N.; Whitesides, G. M. Angew. Chem., Int. Ed 1998, 37, 551. (3) (a) Gray, J. E.; Luan, B. J. Alloys Compd. 2002, 336, 88. (4) (a) Kandere-Grzybowska, K.; Campbell, C. J.; Mahmud, G.; Komarova, Y.; Soh, S.; Grzybowski, B. A. Soft Matter 2007, 3, 672. (b) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingberg, D. E. Science 1997, 276, 1425. (5) (a) Hendricks, J. H.; Aquino, M. I.; Maslar, J. E.; Zachariah, M. R. Chem. Mater. 1998, 10, 2221. (b) Hultman, L.; Sundgren, J.-E.; Greene, J. E.; Bergstrom, D. B.; Pertov, I. J. Appl. Phys. 1995, 78, 5395. (c) Brown, I. G. Annu. ReV. Mater. Sci. 1998, 28, 243. (d) Sondag-Huethorst, J. A. M.; Fokkink, L. G. J. Langmuir 1995, 11, 4823. (e) Kim, Y.; Miyauchi, K.; Ohmi, S.; Tsutsui, K.; Iwai, H. Microelectron. J. 2005, 36, 41. (6) Kumar, A.; Whitesides, G. M. Appl. Phys. Lett. 1993, 63, 2002. (7) (a) Smoukov, S. K.; Bishop, K. J. M.; Klajn, R.; Campbell, C. J.; Grzybowski, B. A. AdV. Mater. 2005, 17, 1361. (b) Campbell, C. J.; Smoukov, S. K.; Bishop, K. J. M.; Baker, E.; Grzybowski, B. A. AdV. Mater. 2006, 18, 2004. (c) Smoukov, S. K.; Grzybowski, B. A. Chem. Mater. 2006, 18, 4722. (d) Grzybowski, B. A.; Bishop, K. J. M.; Campbell, C. J.; Fialkowski, M.; Smoukov, S. K. Soft Matter 2005, 1, 114.
Figure 1. Scheme of the deposition procedure. The thickness of the films heated to 160 °C is ca. 70% of that of the original nanoparticle film before heating.
these films are unsuitable for cell patterning, where they often delaminate during slide precleaning or cell plating (Supporting Information). Altogether, these complications limit the popularity of cell micropatterning. Here, we present a straightforward experimental method in which the solution-based deposition of gold nanoparticles (AuNPs) combined with thermal treatment yields thin, glass-supported gold films (Figure 1) compatible with cell-patterning techniques. We demonstrate that with the proper optimization of thermal processing the solution-deposited films allow for the formation of high-quality EG SAMs whose dense packing is evidenced8 by their ability to block cell adhesion efficiently. Although the films have no adhesion-promoting layer, they do not delaminate from the glass substrates during micropatterning/etching or cell-culturing procedures. The formation of metal films from metal NP precursors has recently been demonstrated using several methods including e-beam writing,9a-c laser-assisted local thermal decomposition,9d micropatterning and thermal decomposition,9e or a combination of UV exposure and thermal treatment.9f Although the films prepared using these methods were continuous over the patterned regions and were generally stable against delamination from the substrate, the ability of the processed films to support new SAMs has not been investigated in quantitative detail. In this context, the removal of the organics initially stabilizing the NPs have (8) Witt, D.; Klajn, R.; Barski, P.; Grzybowski, B. A. Curr. Org. Chem. 2004, 8, 1763. (9) (a) Bedson, T. R.; Nellist, P. D.; Palmer, R. E.; Wilcoxon, J. P. Microelectron. Eng. 2000, 53, 187. (b) Griffith, S.; Mondol, M.; Kong, D. S.; Jacobson, J. M. J. Vac. Sci. Technol., B 2002, 20, 2768. (c) Klajn, R.; Gray, T. P.; Wesson, P. J.; Myers, B. D.; Dravid, V. P.; Smoukov, S. K.; Grzybowski, B. A. AdV. Funct. Mater. 2008, 18, 2763. (d) Tonneau, D.; Bouree, J. E.; Correia, A.; Roche, G.; Pelous, G.; Verdeyme, S. J. Appl. Phys. 1995, 78, 5139. (e) Ko, S. H.; Park, I.; Pan, H.; Grigoropoulos, C. P.; Pisano, A. P.; Luscombe, C. K.; Frechet, J. M. J. Nano Lett. 2007, 7, 1869. (f) Lu, C. H.; Wei, F.; Wu, N. Z.; Huang, L.; Zhao, X. S.; Jiao, X. M.; Luo, C. Q.; Cao, W. X. Langmuir 2004, 20, 974.
10.1021/la803287u CCC: $40.75 2009 American Chemical Society Published on Web 01/26/2009
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been verified by sensitive methods such as XPS,9c,10 but evidence for full ligand exchange and the formation of high-quality, tight SAMs on the processed films has been inconclusive and is usually based on indirect measures such as contact angles. Indeed, we have verified that the published exchange protocols11 applied to NP films fail to produce EG SAMs resistant to cell adhesion. Therefore, the motivation and challenge of the present work is to deposit gold films that combine structural integrity (including strong adhesion to large areas of the substrate and the ability to withstand chemical patterning/etching) with chemical processability suitable for cell-biological experiments. Figure 1 illustrates the procedure for the preparation of gold films from solutions of AuNP (average diameter 5.5 nm, s.d. 0.8 nm) functionalized with 11-mercaptoundecanol (MUO) and synthesized according to the previously published procedure.12 MUO thiol was chosen as a stabilizing molecule for two reasons. First, AuMUO NPs are soluble in methanol. Such methanolic solutions evaporate rapidly during the deposition of NPs on glass. Second, relatively long thiols reduce van der Waals attractions between metal cores13 so that the NPs are stable in solution for months (in contrast to shorter thiols, such as HS-(CH2)6-OH, for which the NPs aggregate and precipitate within hours). An as-prepared methanolic solution of AuMUO NPs was spread uniformly onto a glass slide (22 × 22 mm2; VWR cat. no. 48366227) and evaporated slowly while being covered with a Petri dish. After about 1 h, a thin nanoparticle film was formed that could still be washed off of the surface with methanol. The thickness of this film was controlled by the concentration of the NPs and the volume of the deposited solution and varied between 50 nm (e.g., from 0.1 mL of a 24.5 mM AuMUO solution) and 150 nm (0.3 mL of a 24.5 mM solution). Deposited films appeared to be metallic and shiny gold under reflected light, but under transmitted light, their color was purple because of the surface plasmon resonance (SPR) of individual NPs (Figure 2a, top). When the films were heated to 160 °C for 1.5 h, the individual NPs coalesced into larger, densely packed clusters of ∼100-200 nm in diameter. Although films still appeared shiny/metallic, their color in transmitted light changed from purple to dark blue, confirming the coalescence of NPs and reflecting the red shift of the SPR (Figure 2a, middle).14 These films did not delaminate when soaked/sonicated in either organic or inorganic solvents for prolonged periods of time. Films heated for longer times or to T > 160 °C were also stable against soaking/ sonication but were no longer continuous nor uniformly spread on the surface (Figure 2a, bottom). The color of such films in transmitted light was light blue because of the red shift of the SPR maximum from the visible to the near-IR. However, for T < 160 °C the films were continuous but still contained a considerable fraction of individual, noncoalesced NPs. Such films were similar to those of nonheated NPs (cf. Figure 2a) and delaminated easily when soaked in water or ethanol. The films prepared at 160 °C were optimal for the deposition of cell-resistant EG SAMs and for cell patterning. To pattern cells, we first used reaction-diffusion (RD) microetching7 initiated (10) Blondiaux, N.; Zu¨rcher, S.; Liley, M.; Spencer, N. D. Langmuir 2007, 23, 3489. (11) (a) Brewer, N. J.; Janusz, S.; Critchley, K.; Evans, S. D.; Leggett, G. J. J. Phys Chem. B. 2005, 109, 11247. (b) Kim, D. J.; Pitchimani, R.; Snow, D. E.; Hope-Weeks, L. J. Scanning 2008, 30, 118. (12) (a) Kalsin, A. M.; Fialkowski, M.; Paszewski, M.; Smoukov, S. K.; Bishop, K. J. M.; Grzybowski, B. A. Science 2006, 312, 420. (b) Kalsin, A. M.; Kowalczyk, B.; Smoukov, S. K.; Klajn, R.; Grzybowski, B. A. J. Am. Chem. Soc. 2006, 128, 15046. (13) Bishop, K. J. M.; Grzybowski, B. A. Chem. Phys. Chem. 2007, 8, 2171. (14) (a) Klajn, R.; Pinchuk, A. O.; Schatz, G. C.; Grzybowski, B. A. Angew. Chem., Int. Ed. 2007, 46, 8363. (b) Kalsin, A.; Pinchuk, A. O.; Smoukov, S. K.; Paszewski, M.; Schatz, G. C.; Grzybowski, B. A. Nano Lett. 2006, 6, 1896.
Letters
Figure 2. SEM and UV-vis spectra (i.e., absorbance as a function of wavelength, λ) of gold films on glass substrates: (a) Nanoparticle film before heating (top), after heating to 160 °C for 1.5 h (middle), and after heating to 170 °C for 1.5 h (bottom). Insets in the SEM images illustrate the color of slides (left) in transmitted light and (right) in reflected light. Scale bars ) 100 nm. (b) Gold film prepared by e-beam evaporation; scale bar ) 100 nm.
from micropatterned agarose stamps soaked in a 20% v/v aqueous solution of a gold etchant (TFA gold etchant, Transene Company, Danvers, MA) to etch transparent microislands into the gold films (Figure 3a). We have recently shown4a that this method is useful for studying cytoskeleton organization in geometrically confined cells and, owing to the transparency of the etched islands, is compatible with various cell-imaging modalities (e.g., live-cell fluorescence imaging, total internal reflection fluorescence microscopy, and confocal microscopy). Interestingly, when used in conjunction with thermally evaporated films, RD etching can sometimes cause gold delamination, even with Ti or Cr adhesion layer. For our “nanofilms”, such delamination is not observed, confirming strong bonding between heated AuNPs and glass (most likely due to the partial diffusion of gold atoms into glass15). One of the major advantages of RD microetching over other methods is that the unetched regions of gold do not have to be protected so that after etching is complete they can be functionalized with SAMs. We expected that this should also be the case with the thermally treated NP films baked at 160 °C, that is, well above the temperature at which MUO thiols start to (15) Darque-Ceretti, E.; Deram, V.; Aucouturier, M. Surf. Eng. 2008, 24, 103.
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Langmuir, Vol. 25, No. 4, 2009 1907
ProChimia, Poland), the unetched regions were covered with a SAM resistant to cell adhesion. This is illustrated in Figure 3c-e, where B16F1 cells17 were plated onto the micropatterned substrates. As seen, the cells localized exclusively onto the unprotected microislands or linear tracks, on which they fully spread/moved. The films were compatible with all standard cellbiological procedures, including cell fixation with fibronectin, immunostaining (Figure 3c), and live cell imaging (Figure 3d,e). In summary, we developed and optimized a nanoparticlebased method for the formation of thin gold films compatible with cell patterning. The most appealing features of this method are its reliability and simplicity because the films are deposited from readily available nanoparticle solutions and can be processed on a standard hot plate without the need for any specialized equipment. The films are durable and do not require any adhesionpromoting layers. Future research could investigate similar solution-based schemes for other types of metallic and nonmetallic nanoparticles.
Figure 3. (a) Scheme of the RD microetching technique used to pattern the islands. In the process, an agarose stamp patterned in bas relief delivers fresh etchant to the stamp-substrate interface (orange arrows) while removing the etching products from the interface to the stamp’s bulk (violet arrows). As a result, the stamp “cuts” into the gold film to produce transparent regions surrounded by opaque gold. For details of this process, see refs 4a and 7a. (b) Truncated-triangle microislands (scale bar ) 50 µm) etched into AuNP films. (c) B16F1 cells plated on triangular microislands such as those in Figure 3b. Lower portion of the image magnifies two representative cells. Green staining using fluorescent phalloidin shows actin filaments. Bright-field images of live B16F1 cells (d) immobilized onto circular islands and (e) moving on linear tracks. For further biological details, see ref 4a. All scale bars in a-c ) 50 µm.
desorb from the surface of gold (∼95 °C).9c,16 Indeed, when the etched films were soaked in a 5 mM solution of hexa(ethylene glycol)undecane-1-thiol (EG6, HS-(CH2)11-(OCH2CH2)6-OH, (16) Karpovich, D. S.; Blanchard, G. J. Langmuir 1994, 10, 3315.
Acknowledgment. This work was supported by the NIH/NCI Northwestern Center of Cancer Nanotechnology Excellence, award no. U54CA119341. B.A.G. gratefully acknowledges financial support from the Pew Scholars Program in the Biomedical Sciences. G.M. gratefully acknowledges funding from the Gates Fellowship. Supporting Information Available: Stability of commercial versus nanoparticle films during the precleaning procedure. This material is available free of charge via the Internet at http://pubs.acs.org. LA803287U (17) B16F1 is a murine malignant melanoma cell line used widely in cell motility and cell polarization studies (e.g., Chandrasekar, I.; Stradal, T. E.; Holt, M. R.; Entschladen, F.; Jockusch, B. M.; Ziegler, W. H. J. Cell Sci. 2005, 118, 1461. or Vicente-Manzanares, M.; Koach, M. A.; Whitmore, L.; Lamers, M. L.; Horwitz, A. F. J. Cell Biol. 2008, 183, 543) because of its highly migratory potential as well as in cancer research as one of the model cell lines for studying cancer metastasis (e.g., Hill, R. P.; Chambers, A. F.; Ling, V.; Harris, J. F. Science 1984, 224, 998 and Stackpole, C. W. Nature 1981, 289, 798).
