Langmuir 2004, 20, 3483-3486
3483
Notes Through-Mask Anodic Patterning of Copper Surfaces and Film Stability in Biological Media Haixia Dai,† Corrine K. Thai,† Mehmet Sarikaya,‡ Franc¸ ois Baneyx,† and Daniel T. Schwartz*,† Departments of Chemical Engineering and Materials Science and Engineering, University of Washington, Seattle, Washington 98195-1750 Received June 17, 2003. In Final Form: October 31, 2003
Introduction In recent years, combinatorial biology techniques have been used to identify inorganic-binding polypeptides for a number of different materials, including elemental semiconductors, gold, and silica.1-6 We are currently using cell surface display techniques to identify inorganicbinding polypeptides for cuprous oxide (Cu2O),7 a compound semiconductor with a direct band gap of 2.2 eV and four high-binding-energy exciton bands in the visible spectrum.8 The electronic and optical properties of Cu2O have sparked interest in the material for applications to solar energy,9,10 water splitting,11-13 and coherent exciton emitters.14 In contrast, the divalent forms of oxidized copper have much less interesting optical properties in the visible spectrum. To ensure selection of binders to the proper material, it is important to establish biological screening conditions where the cuprous oxide surface is chemically and electrochemically stable and cells remain viable. Moreover, we would like to screen for polypeptides that selectively bind just the material of interest (monovalent oxide), with little cross-binding affinity for higher valency films. * To whom correspondence should be addressed. E-mail: dts@ u.washington.edu. Tel.: 206 685 4815. Fax: 206 543 3778. † Department of Chemical Engineering, University of Washington. ‡ Department of Materials Science and Engineering, University of Washington. (1) Brown, S. Nat. Biotechnol. 1997, 15, 269. (2) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665. (3) Naik, R. R.; Brott, L. L.; Clarson, S. J.; Stone, M. O. J. Nanosci. Nanotechnol. 2002, 2, 95. (4) Brown, S.; Sarikaya, M.; Johnson, E. J. Mol. Biol. 2000, 299, 725. (5) Brown, S. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 8651. (6) Sarikaya, M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14183. (7) Thai, C. K.; Dai, H.; Sarikaya, M.; Schwartz, D. T.; Baneyx, F. Manuscript in preparation. (8) Hodby, J. W.; Jenkins, T. E.; Schwab, C.; Tamura, H.; Trivich, D. J. Phys. C: Solid State Phys. 1976, 9, 1429. (9) Smith, M.; Gotovac, V.; Aljinovic, L.; Luciclavcevic, M. Surf. Sci. 1995, 335, 171. (10) Fernando, C. A. N.; de Silva, P. H. C.; Wethasinha, S. K.; Dharmadasa, I. M.; Delsol, T.; Simmonds, M. C. Renewable Energy 2002, 26, 521. (11) de Jongh, P. E.; Vanmaekelbergh, D.; Kelly, J. J. Chem. Commun. 1999, 1069. (12) Hara, M.; Kondo, T.; Komoda, M.; Ikeda, S.; Shinohara, K.; Tanaka, A.; Kondo, J. N.; Domen, K. Chem. Commun. 1998, 357. (13) Ikeda, S.; Takata, T.; Kondo, T.; Hitoki, G.; Hara, M.; Kondo, J. N.; Domen, K.; Hosono, H.; Kawazoe, H.; Tanaka, A. Chem. Commun. 1998, 2185. (14) Snoke, D. Science 1996, 273, 1351.
Copper can form mono- and divalent surface films by either thermal or electrochemical oxidation, though patterning a surface with mixed monovalent and divalent regions is difficult using thermal oxidation. In contrast, electrochemical methods are widely used to create functional films and surfaces whose composition and structure can be readily tuned, are easily patterned, and grow in aqueous media under ambient conditions.15-21 These attributes make electrochemically patterned inorganic films well-suited for polypeptide screening studies, provided the specific films produced are sufficiently stable in the relevant biological media. The electrochemistry of copper oxidation in alkaline media is well-studied, with the structure and composition of surface films known.22-25 For the most part, the oxidation of copper follows the predicted thermodynamic patterns in alkaline media. A cuprous oxide Cu2O layer forms via the oxidation reaction 2Cu + 2OH- f Cu2O + H2O + 2e- near the potential predicted by the Nernst equation: ECu/Cu2O (mV) ) 229.5 - 59.1pH (potential vs saturated calomel electrode, SCE, 298 K).26 Thermodynamically, the formation of the divalent oxide CuO is slightly favored over the hydroxide Cu(OH)2, but experiments show the hydroxide is the predominant form in alkaline media,25 with the oxidation reaction Cu2O + 2OH- + H2O f 2Cu(OH)2 + 2e- occurring near the reversible potential given by ECu2O/Cu(OH)2 (mV) ) 505.5 59.1pH(potential vs SCE, 298 K).26 The reduction of these oxides, however, is highly irreversible owing to the presence of an electron-deficient p-type semiconducting oxide underlayer that blocks the reverse-bias current unless illuminated.25 In this paper, we focus on the electrochemical growth and patterning of copper surfaces with regions of monoand divalent films. Raman and XPS is used to verify the phase purity and compositional stability of these surfaces when used in IMC (induction media with casamino acids) buffer, a biological media of relevance here. In addition, we describe an electrochemical microfabrication scheme that takes advantage of the different oxidation potentials for the mono- and divalent films to make patterned surfaces that can be used to assess polypeptide crossbinding among related compounds. (15) Datta, M.; Landolt, D. Electrochim. Acta 2000, 45, 2535. (16) Romankiw, L. T. Electrochim. Acta 1997, 42, 2985. (17) International ASM Handbook: surface engineering; ASM International: Materials Park, OH, 1994; Vol. 5. (18) Nakagawa, K.; Arao, K.; Iwasaki, Y. U.S. Patent 6,258,702, 2001. (19) Ruszczyk, S.; Ferrier, D.; Larson, G.; Gallegos, D.; Castaldi, S. U.S. Patent 4,761,303, 1988. (20) Oh, J.; Jung, Y.; Lee, J.; Tak, Y. Nanotechnology in Mesostructured Materials, 2003, 146, 205. (21) Jagminas, A.; Kuzmarskyte, J.; Niaura, G. Appl. Surf. Sci. 2002, 201, 129. (22) Kang, M. C.; Gewirth, A. A. J. Phys. Chem. B 2002, 106, 12211. (23) Mayer, S. T.; Muller, R. H. J. Electrochem. Soc. 1992, 139, 426. (24) Kunze, J.; Maurice, V.; Klein, L. H.; Strehblow, H. H.; Marcus, P. J. Phys. Chem. B 2001, 105, 4263. (25) Schwartz, D. T.; Muller, R. H. Surf. Sci. 1991, 248, 349. (26) De Zoubov, N.; Vanleugenhaghe, C.; Pourbaix, M. Atlas of Electrochemical Equlibria in Aqueous Solution; Pergamon Press: New York, 1966; section 14.1.
10.1021/la0350711 CCC: $27.50 © 2004 American Chemical Society Published on Web 12/16/2003
3484
Langmuir, Vol. 20, No. 8, 2004
Notes
Experimental Section Cu2O and Cu(OH)2 Thin-Film Growth. A Princeton Applied Research (Princeton, NJ) 263 potentiostat/galvanostat was used in all electrochemical experiments. A copper foil 10 mm × 10 mm × 0.127 mm (Alfa Aesar, 99.99%) working electrode was immersed in 1 M KOH alkaline solutions; a platinum wire was used as the counter electrode. All potentials were recorded versus a SCE. Cuprous oxide Cu2O films were formed by sweeping the potential from -1000 to -450 mV versus SCE, while cupric hydroxide Cu(OH)2 films were formed in the potential range from -400 to -125 mV versus SCE. The scan rate was 1 mV/s. All films were grown at room temperature. The deposited films were then thoroughly rinsed with deionized water and dried in the air. The deposition charge for either material was determined from the integrated peak in the linear sweep voltammogram used to deposit the specific film. Electrochemical Patterning Process. The surface was patterned by using a tape-mask method.27 An adhesive-backed Mylar tape was used to make the mask. The tape-mask pattern was first designed with Adobe Illustrator, and then the Mylar tape was placed in a Universal Laser System CO2 laser-cutting tool; finally, the Windows print driver was used to direct the cutting of the pattern in the tape. The cut mask was cleaned of cutting debris and then adhered to the substrate to be patterned. Growth of the desired material occurred by electrochemical oxidation through the mask openings. After forming the chosen anodic film, the mask was peeled off and the patterned substrate went through a three-step rinse cycle (acetone, then methanol, then deionized water) prior to drying in air. Raman Spectroscopy. Raman spectroscopy of copper oxide/ hydroxide surfaces was acquired using the 488-nm line of an argon laser with 250-mW output power. The spectrograph was calibrated using Ar+ ion laser plasma lines as references. Each spectrum was acquired for 60 s. The details regarding lineimaging Raman spectroscopy can be found in previous literature.28 X-ray Photoelectron Spectroscopy (XPS). XPS analysis was performed at the National ESCA and Surface Analysis Center for biomedical problems (NESA/BIO), University of Washington. All XPS spectra were taken on a SSI X-probe spectrometer (Surface Science Instruments, Mountain View, CA) with a monochromatized Al KR1,2 X-ray source and a low-energy electron flood gun for charge neutralization. High-resolution spectra of the peaks were acquired at the takeoff angle of 55°. A program developed by Service Physics, Bend, OR, was used for data acquisition and reduction. The binding-energy scale for all highresolution spectra was calibrated by assigning the hydrocarbon component of the C(1s) peak fit a binding energy of 285.0 eV.
Results and Discussion Copper Oxide/Hydroxide Thin-Film Formation. Cyclic voltammetry of Cu in 1 M KOH is well-studied.23,25 There are two major peaks during the anodic sweep and one major composite peak in the cathodic sweep. A thin Cu2O layer forms around -450 mV versus SCE, and a bilayer film of Cu(OH)2 over Cu2O forms at about -125 mV versus SCE. The reduction of these surface films occurs quite irreversibly, beginning near -700 mV versus SCE, which is well below the equilibrium potentials for any of the relevant surface films. This electrochemical irreversibility imparts a large electrochemical window where the valence state of Cu2O or Cu(OH)2 should remain stable, implying that the surfaces will be (electrochemically) robust to biological media with a fairly wide range of oxidation-reduction potentials (ORPs). Figure 1 shows baseline-subtracted Raman spectra of electrochemically grown Cu2O and Cu(OH)2 films made in the manner described in the Experimental Section. Reference standards23,25 in previous work identified the (27) Wang, W. H.; Holl, M. R.; Schwartz, D. T. J. Electrochem. Soc. 2001, 148, C363. (28) Schwartz, D. T.; Haight, S. M. Colloids Surf., A 2000, 174, 209.
Figure 1. Raman spectra of electrochemically formed Cu2O (top) and Cu(OH)2 (bottom) thin films. Separate spectral windows are shown to highlight Cu-O modes (lower wavenumbers) and O-H modes (higher wavenumbers). Table 1. Properties of the IMC Buffer that Are Relevant to the Room Temperature Electrochemical Stability of Films on Copper pH
ORP
components29
8.0
200 mV versus SCE
Na2HPO4, KH2PO4, NaCl, NH4Cl, MgCl2, CaCl2, glucose, casamino acids
633 cm-1 peak as a Cu2O optical phonon mode, whereas the 488 cm-1 peak was attributed to Cu(OH)2. Looking at the O-H stretching region of the spectra, one observes a 3578 cm-1 peak in Cu(OH)2 but not in Cu2O. These spectra suggest we are forming quite phase-pure thin films. Visibly, the cuprous oxide surface appears brown-red and smooth, whereas the cupric hydroxide surface looks light blue and has a matte finish. The deposition charge for the Cu2O film shown in Figure 1 was 10 mC/cm2, giving an estimated film thickness of 13 nm, assuming 100% current efficiency and a dense film. Similarly, the Cu(OH)2 film had a deposition charge of 515 mC/cm2 and a corresponding film thickness of 659 nm under the same assumptions. Stability of the Cu2O Surface in IMC Buffer. Table 1 shows the relevant physical properties and electrolyte components in IMC buffer. IMC buffer is the main media applied to the substrates during the incubation and screening via cell surface display combinatorial libraries.29 The ORP of the IMC buffer is +200 mV versus SCE, and the pH of the solution is about 8.0. The ORP of IMC buffer is considerably more positive than the Cu2O/Cu(OH)2 equilibrium potential at this pH (33 mV versus SCE), which suggests that a driving force exists for the spontaneous oxidation of Cu2O to Cu(OH)2. Raman spectroscopy and XPS were used to assess the stability of Cu2O films under the relevant conditions. Spectra of the Cu2O film were acquired before and after incubation in IMC buffer. Figure 2a shows that the Cu2O film possesses the characteristic spectra of Cu2O both before and after a 30min IMC incubation. Because Raman probes the entire film thickness, these results indicate that the film as a whole does not convert to Cu(OH)2 over the incubation period. Cu(2p3/2) X-ray photoelectron spectra of Cu2O can (29) Tripp, B. C.; Lu, Z. J.; Bourque, K.; Sookdeo, H.; McCoy, J. M. Protein Eng. 2001, 14, 367.
Notes
Langmuir, Vol. 20, No. 8, 2004 3485
Figure 3. Process flow for electrochemical patterning of Cu2O and Cu(OH)2 surfaces using CO2 laser-cut Mylar tape masks. The effect of each process step is illustrated schematically using a top view (left) and side view (right) of the copper substrate. Step 1: grow the Cu2O film by scanning potentials from -1000 to -450 mV versus SCE. Step 2: mask the Cu2O surface. Step 3: grow the Cu(OH)2 film through the mask by scanning potentials from -400 to -125 mV versus SCE. Step 4: remove the mask to reveal alternating Cu2O/Cu(OH)2 stripes.
Figure 2. (a) Raman spectra of a Cu2O film before and after incubation for 30 min in IMC buffer. (b) High-resolution XPS Cu(2p3/2) spectra of a Cu2O film before and after incubation for 30 min in IMC buffer.
be used to distinguish changes in the oxidation state of the top surface region of the film, provided one takes necessary precautions to avoid spontaneous reduction of the Cu(II) compound during measurement. In this case, our use of a monochromatic X-ray source is generally considered an adequate precaution for the accurate interpretation of copper valency.30,31 Figure 2b shows Cu(2p3/2) XPS spectra of the Cu2O film before and after a 30-min IMC incubation. No appreciable growth in the divalent copper peak at Eb ) 934.6 eV is observed nor is the monovalent copper peak at Eb ) 932.2 eV attenuated. Thus, we conclude that the Cu2O surface remains sufficiently stable for biological screening in IMC buffer. Surface Patterning. The native copper surface can be easily patterned with Cu2O or Cu(OH)2 by using a Mylar tape mask. In this method, a laser-cut, adhesive-backed Mylar mask is adhered to the copper foil; the masked foil is immersed in 1 M KOH and electrochemically oxidized to Cu2O or Cu(OH)2 by scanning to the appropriate potential. The mask is then peeled off, revealing the Cu foil patterned with either Cu2O or Cu(OH)2. We have used (30) Klein, J. C.; Chung, P. L.; Hercules, D. M.; Black, J. F. Appl. Spectrosc. 1984, 38, 729. (31) Iijima, Y.; Niimura, N.; Hiraoka, K. Surf. Interface Anal. 1996, 24, 193.
Figure 4. Cu2O/Cu(OH)2 alternating stripe pattern on a Cu surface. (a) Schematic of alternating horizontal Cu2O/Cu(OH)2 stripes on the surface of a Cu foil. Laser illumination (vertical blue line) is orthogonal to the stripes. (b) Low wavenumber Raman line image of the Cu2O/Cu(OH)2 stripe pattern. The vertical image axis denotes the distance along the laser illumination line. (c) High wavenumber Raman line image. The color table for the Raman images denotes the background intensity with blue and maximum intensity with red.
Raman spectroscopy to confirm this straightforward method for patterning a native copper surface with a film possessing a single valence state (not shown). More interesting, and challenging, is the creation of a patterned surface with different regions comprised of phase-pure Cu2O and Cu(OH)2 (a mixed valence pattern). Figure 3 shows a schematic of the electrochemical microfabrication process flow for creating such a surface. First, Cu2O is grown on a piece of as-purchased copper foil by scanning the potential from -1000 to -450 mV versus SCE in 1 M KOH electrolyte. Next, the Mylar tape mask is adhered to the Cu2O surface, and Cu(OH)2 is grown through the mask by scanning to -125 mV versus SCE. Finally, the mask is peeled off, and the patterned Cu2O/Cu(OH)2 surface is rinsed with acetone, methanol, and deionized water and then dried in air. Line-imaging Raman spectroscopy can be used to confirm the composition of the patterned surface. Figure 4 shows the Raman line image of a surface patterned with alternating Cu2O and Cu(OH)2 stripes. The stripes are oriented horizontally, and the Raman line image is taken vertically to resolve the periodic composition variation (shown schematically in Figure 4a). Figure 4b shows the low wavenumber region of the Raman line image for the alternating Cu2O and
3486
Langmuir, Vol. 20, No. 8, 2004
Figure 5. Fluorescence microscopy images of E. coli cells incubated with a striped Cu2O/Cu(OH)2 surface and then stained with a fluorescent dye. The right side of the image is Cu2O, and the left side (with a fluorescent background) is Cu(OH)2.
Cu(OH)2 stripes. Figure 4c shows the corresponding high wavenumber line image presenting the spatial distribution of O-H on the surface. Raman intensity is denoted by the color table, with blue indicating background and red indicating the maximum signal. The patterned surface clearly shows a spatially modulated Raman signal that alternates between 250-µm-wide cuprous oxide stripes (at 633-cm-1 signal) and 250-µm-wide cupric hydroxide stripes (the 488- and 3758-cm-1 signals). It is also seen that some of the small secondary peaks in Cu2O nearly overlap with the 488 cm-1 peak in Cu(OH)2, but the O-H region of the spectrum clearly distinguishes the hydroxide film. Selective Adhesion of Cells on Patterned Surfaces. The surfaces and patterns described here have been used to isolate polypeptides that selectively bind to Cu2O, starting from a large library of 12-residue-long random peptides displayed on the surface of Escherichia coli.32 Although the results of the bio-panning process will be published elsewhere,7 Figure 5 shows that electrodeposited metal oxides can be used to identify inorganic-binding (32) Lu, Z. J.; Murray, K. S.; Vancleave, V.; Lavallie, E. R.; Stahl, M. L.; McCoy, J. M. Biotechnology 1995, 13, 366.
Notes
polypeptides and assess their cross-specificity toward related compounds. In this experiment, E. coli variant CN48 (a Cu2O binder that we have isolated in our lab) labeled with the viable-cell fluorescent dye SYTO-9 (Molecular Probes, Eugene, OR) was contacted with a striped surface of Cu2O and Cu(OH)2. Shown in Figure 5 is a fluorescence microscopy image of the boundary between the two materials [Cu(OH)2 on the left, Cu2O on the right]. E. coli cells appear as bright rods approximately 2 µm in length. The Cu(OH)2 region in this image is easily distinguished as a result of the fact that it adsorbs free dye and exhibits a mild fluorescent background. Inspection of the micrograph reveals that CN48 cells adhere to the Cu2O stripes, with few cells present on the Cu(OH)2 stripes, indicating that the surface-displayed peptide exhibits a strong affinity for Cu2O and low cross-binding affinity for Cu(OH)2. Negative control E. coli cells do not adhere to either material (data not shown). These results further confirm that the surface layers remain chemically distinct, and this chemical distinction leads to differential adhesion properties that these engineered cells respond to. Moreover, the dye used is only fluorescent in viable cells, showing that the media used is compatible both with the surface and the cells. Implications and Concluding Remarks We have shown that a through-mask anodic deposition method can be used to create chemically patterned surfaces on copper. The method should be equally applicable to other metals that form interesting anodic thin films. Raman spectroscopy and XPS were used to assess the stability of the monovalent film when subjected to an oxidizing biological media (IMC buffer) and typical biological processing conditions. Despite a thermodynamic driving force for conversion to the divalent form, it was found that the monovalent surface film was sufficiently stable for these studies. In short, we have generally found it necessary to carefully assess the compatibility of inorganic surfaces with the various media used in combinatorial biology because simple thermodynamic criteria for surface stability (or instability) are rarely sufficient. Acknowledgment. The work is supported by the DURINT Program (Defense University Research Initiative in Nanotechnology) through Army Research Office (Dr. Robert Campbell) with ARO Grant DAAD19-01-1-0499. LA0350711
