Langmuir 2004, 20, 3495-3497
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Site-Directed Molecular Assembly on Templates Structured with Electron-Beam Lithography D. Stamou,*,† C. Musil,‡ W.-P. Ulrich,† K. Leufgen,† C. Padeste,‡ C. David,‡ J. Gobrecht,‡ C. Duschl,†,§ and H. Vogel† Institut de Science Biomole´ culaire, ISB-VO, Ecole Polytechnique Fe´ de´ rale de Lausanne, CH-1015 Lausanne, Switzerland, and Laboratory for Micro- and Nanotechnology, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland Received January 6, 2004 We describe a simple method for patterning biomolecular films on surfaces with high resolution. A conventional polymeric resist is structured by electron-beam lithography. The exposed and developed patterns are then used for the directed self-assembly (SA) of a first molecule from solution. Removal of the remaining resist allows the SA of a second species. We illustrate the potential of the approach by assembling on gold (Au) substrates two alkanethiols of contrasting terminal functionality. The patterns have dimensions from the micrometer range down to 40 nm and an edge resolution of 3.5 nm.
Introduction Biochemical functionality can be introduced on surfaces in a uniform or in a laterally resolved manner. In the latter case, the patterned surface can serve as a template for the directed immobilization of compounds,1 biomolecular assemblies,2,3 or organic/inorganic nanostructures.4-6 It is due to microstructured immobilization that diagnostics and high-throughput screening in a microarray format became feasible. One way to create such patterns is the site-selective modification of a homogeneous molecular film, for example, by lithographic (UV,7 electron beam8,9) or scanning probe methods.10,11 Though of great utility to materials processing, direct patterning on an organic monolayer often exposes the remaining areas to conditions that compromise biological function (ultrahigh vacuum,8,9,11 UV ozone,12 contamination,10 etc.). The demand for biocompatible nanopatterning methods led to the development * Author to whom correspondence should be addressed. E-mail:
[email protected]. † Institut de Science Biomole ´ culaire. ‡ Paul Scherrer Institut. § Present address: Fraunhofer Institute, Biomedical Technology (AMBT), Invalidenstrasse 42, D-10115 Berlin, Germany. (1) Bernard, A.; Michel, B.; Delamarche, E. Anal. Chem. 2001, 73, 8-12. (2) Stamou, D.; Duschl, C.; Delamarche, E.; Vogel, H. Angew. Chem., Int. Ed. 2003, 42, 5580-5583. (3) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nat. Biotechnol. 1999, 17, 1105-1108. (4) Demers, L. M.; Ginger, D. S.; Park, S. J.; Li, Z.; Chung, S. W.; Mirkin, C. A. Science 2002, 296, 1836-1838. (5) Lee, I.; Zheng, H. P.; Rubner, M. F.; Hammond, P. T. Adv. Mater. 2002, 14, 572-577. (6) Huang, Y.; Duan, X. F.; Wei, Q. Q.; Lieber, C. M. Science 2001, 291, 630-633. (7) Tamchang, S. W.; Biebuyck, H. A.; Whitesides, G. M.; Jeon, N.; Nuzzo, R. G. Langmuir 1995, 11, 4371-4382. (8) Geyer, W.; Stadler, V.; Eck, W.; Golzhauser, A.; Grunze, M.; Sauer, M.; Weimann, T.; Hinze, P. J. Vac. Sci. Technol., B 2001, 19, 27322735. (9) Kuller, A.; Eck, W.; Stadler, V.; Geyer, W.; Golzhauser, A. Appl. Phys. Lett. 2003, 82, 3776-3778. (10) Delamarche, E.; Hoole, A. C. F.; Michel, B.; Wilkes, S.; Despont, M.; Welland, M. E.; Biebuyck, H. J. Phys. Chem. B 1997, 101, 92639269. (11) Kleineberg, U.; Brechling, A.; Sundermann, M.; Heinzmann, U. Adv. Funct. Mater. 2001, 11, 208-212. (12) Harnett, C. K.; Satyalakshmi, K. M.; Craighead, H. G. Langmuir 2001, 17, 178-182.
of techniques such as microcontact printing,13,14 microfluidic networks,15 or dip-pen nanolithography16 that can site-selectively deposit molecules on surfaces. Here, we present a method that integrates the concept of site-selective molecular deposition17 with electron-beam lithography (EBL)18 to define high-resolution patterns of organic molecules on surfaces. As illustrated in Figure 1, we decouple the patterning step (a-c) from the biochemical modification of the surface (d-f) by exposing to the electron beam not the organic self-assembled monolayer (SAM) but a conventional polymeric resist.19 The selective assembly of molecules on the surface becomes then a subsequent independent process that takes place from solution and is guided by the lithographically defined pattern. Assembly from solution in the case of SAMs allows the molecules to reorganize and heal any defects; in the case of biomolecules it may protect then from denaturing conditions (e.g., drying steps). After the first self-assembly (SA) step (Figure 1d), the sample is treated with a solvent to dissolve the remaining resist. Therefore, one has to ensure that the first selfassembled layer is solvent-compatible. Suitable molecules for this are, for example, alkanethiols, silanes, DNA oligomers, peptides, or organic ligands such as biotin. After the removal of the resist (Figure 1e), biological macromolecules can be assembled from water solutions under physiological conditions in the remaining areas (Figure 1f) or on the patterns functionalized with the first SA. In the present paper, we will use a model SA system (alkanethiols of contrasting terminal functionality) and focus on characterizing (i) with what fidelity we can define their assembly in patterns and (ii) what is the resolution limit of the method. (13) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 551-575. (14) Bernard, A.; Fitzli, D.; Sonderegger, P.; Delamarche, E.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Nat. Biotechnol. 2001, 19, 866-869. (15) Delamarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. Science 1997, 276, 779-781. (16) Ginger, D. S.; Zhang, H.; Mirkin, C. A. Angew. Chem., Int. Ed. 2004, 43, 30-45. (17) Michel, R.; Lussi, J. W.; Csucs, G.; Reviakine, I.; Danuser, G.; Ketterer, B.; Hubbell, J. A.; Textor, M.; Spencer, N. D. Langmuir 2002, 18, 3281-3287. (18) Marrian, C. R. K.; Tennant, D. M. J. Vac. Sci. Technol., A 2003, 21, S207-S215. (19) Healy, K. E.; Thomas, C. H.; Rezania, A.; Kim, J. E.; McKeown, P. J.; Lom, B.; Hockberger, P. E. Biomaterials 1996, 17, 195-208.
10.1021/la049954j CCC: $27.50 © 2004 American Chemical Society Published on Web 04/01/2004
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Langmuir, Vol. 20, No. 9, 2004
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Figure 1. Schematic representation of directed biomolecular assembly on surfaces prepatterned with EBL. PMMA layers spin-coated on Au substrates (a) are exposed with an electron beam (b) and then developed in 1:3 MiBK/IPA (c). The first three handling steps, though necessary for defining an initial chemical contrast on the substrate, expose the sample to ultrahigh vacuum and an oxygen plasma etch. Once the pattern is defined, however, the subsequent placement of functional (bio)molecules can be done under physiological conditions by SA from solution (steps d and f).
Experimental Section Gold surfaces. We fabricated Au surfaces in two different ways. The first approach involved simple evaporation on clean glass substrates of an adhesive layer (5 nm of Ti) and then a 20-nm-thick film of Au. The Au surfaces produced in this manner have a root-mean-square (RMS) roughness on the order of several nanometers. They were used for etching experiments (see the following) but were not suitable for imaging patterned SAMs with atomic force microscopy (AFM). AFM images of monolayers were acquired on flat Au surfaces prepared by template stripping according to a previously published procedure.20 EBL. For the electron beam writing, a 100-nm-thick layer of poly(methyl methacrylate) (PMMA) was spun on the Au-coated glass wafer and exposed at an energy of 2.5 keV. The optimized patterning time was 4 min/mm2 for exposing roughly 12% of the surface. The samples were developed in 1:3 4-methyl-2-pentanone/2-propanol (MiBK/IPA) for 60 s and rinsed in IPA. They were then exposed to an oxygen plasma for 30 s, during which time about 30 nm of the PMMA was removed. SA. The patterned PMMA surfaces were incubated in a 1 mM solution of octadecane thiol (ODT) in heptane for 18 h (during which time a SAM formed on the uncovered areas) and then removed, rinsed with heptane, and blown dry with nitrogen (N2). The remaining unexposed PMMA was removed by first soaking the samples for 30 min in acetone and then rinsing the samples with acetone and IPA for 30 s each. After removing the nonexposed parts of the resist, the sample was incubated with a second alkanethiol 11-mercaptoundecanoic acid (MUA), 1 mM in EtOH for 1 h. The sample was then dipped and sprayed with EtOH and finally blown dry with N2. AFM Measurements. Topography and friction measurements were performed with a commercially available instrument (Bioscope, 100-µm scanner, Digital Instruments, Veeco Instruments) operated under ambient conditions. We used triangular Si3N4 cantilevers with a nominal spring constant of 0.06 N/m. The surfaces were scanned at rates of 1-2 Hz per line and applied loads of 8-10 nN. Images are flattened (x and y) to remove the bow effect. Selective Gold Etching. The ODT monolayer was used as a mask against a ferricyanide Au etch consisting of 0.01 M K3(20) Stamou, D.; Gourdon, D.; Liley, M.; Burnham, N. A.; Kulik, A.; Vogel, H.; Duschl, C. Langmuir 1997, 13, 2425-2428.
Figure 2. Example of molecular assembly on EBL-defined structures. Domains of ODT, 240 nm in diameter, are surrounded by the surface reacted with MUA. Patterns were imaged with AFM under ambient conditions. (a and b) Friction images of the array at different magnifications. The white line in part b indicates the edge of the domain. (c) The line edge recorded from part b is subtracted from a perfect circle with a radius of 240 nm. The deviation is then plotted along the circumference. (d) Topography image at the edge of a domain. (e) Plot of the cross section marked on part d by an arrow. Fe(CN)6, 0.001 M K4Fe(CN)6, 0.1 M K2S2O3, and 1 M KOH. An etching time of 10 min under mild agitation resulted in the optimal definition of the pattern with an etched depth of 20 nm in the non-SAM-covered areas and negligible etching in the SAMcovered areas. Image Analysis. Prior to image analysis, typical AFM artifacts that appeared as white stripes in the image had to be edited manually where the stripes crossed the domain border. Image analysis was performed using the program IGOR Pro (WaveMetrics, Lake Oswego, U.S.A.). The treated image was thresholded to remove gray levels. Fitting a circle to the thresholded image yielded the radius and the center of the domain. The pixel positions of the domain perimeter were extracted by the built-in particle analysis function. Finally, the RMS differences between the circle and the domain perimeter were calculated.
Results and Discussion One of the patterns we exposed in the resist was an array of dots separated by 1 µm. The exposed parts of the resist were developed and ODT (a hydrophobic alkanethiol terminated with CH3) was self-assembled in the uncovered areas of Au. In the next step, the rest of the resist was removed and the background was reacted with MUA (a hydrophilic alkanethiol terminated with COOH). The hydrophobic/hydrophilic contrast allowed visualization of the patterns with an AFM in friction mode. A lowmagnification image on a representative part of the sample (Figure 2a) shows the homogeneity of the array. The ODT
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domains showed a lower friction, as has been reported previously.21 In Figure 2b, zooming on one of the domains allows us to identify more clearly its morphology. The domain extends over several atomically flat Au terraces and has an average diameter of 240 nm, well in agreement with the prestructured resist template. By using the friction contrast as a guide, we can digitally trace the domain border, as indicated by the white line in the right-hand part of Figure 2b. We can then subtract this trace from a perfect circle and obtain the edge-line irregularity plotted along the circumference. This is shown in Figure 2c, and it is a measure of the resolution by which we can define a given feature. The standard deviation (SD) in the edge line is (3.4 nm. We can approximate a lower limit of 4 × SD ≈ 14 nm to the size of attainable features one would expect to obtain with this technique. Any smaller feature would be hard to distinguish keeping in mind the 7-nm (2 × SD) irregularities in the definition of an edge. The two alkanethiols we used, apart from different terminal functionalities, also have a difference of seven carbon atoms in length. This is enough to give a topography signal between the two phases (Figure 2d). A trace along the edge of the domain (dotted arrow) is plotted in Figure 2e and reveals a height difference of 7 ( 2 Å, which correlates well with the difference in molecular lengths. The AFM data (both friction and topography) show qualitatively the contrast one would expect between the two regions. Because they are obtained at low resolution, however, they do not contain information on the molecular order of these SAMs. Molecular films that are well-ordered over large areas (µm2) indicate that the Au areas revealed after patterning have uniform chemical properties. One efficient way to assess this is to use the monolayer as a mask against etching solutions that would otherwise attack the underlying substrate.22 For this purpose, we skip the second step of SA (Figure 1e) and leave the background areas of Au unprotected. The ODT patterns are then exposed to a ferricyanide etch solution. The etching conditions (time, concentration, etc.) were optimized for etching through the total thickness of the sample, that is, ∼20 nm. An advantage of defining patterns with EBL is the ability to control in a precise manner the shape, characteristic dimension, and surface density of features in different parts of the same sample. Figure 3a,b shows optical microscopy images of Au domains, 2.5 × 2.5 µm2 wide, that are spaced on a square lattice of 12- and 1-µm separation, respectively. A series of test features of decreasing size allowed us to assess the potential of the method for creating structures of nanometer dimensions. The domain of Figure 3c has an average diameter of 40 nm, equivalent to about 80 ODT molecules. As Zhang et al.23 recently demonstrated, patterning smaller features in thin Au films with wet-chemical etching is possible but rather irreproducible, mainly due to fundamental limitations of the process like nonuniform etching or polycrys(21) Vezenov, D. V.; Noy, A.; Rozsnyai, L. F.; Lieber, C. M. J. Am. Chem. Soc. 1997, 119, 2006-2015. (22) Zhao, X. M.; Wilbur, J. L.; Whitesides, G. M. Langmuir 1996, 12, 3257-3264. (23) Zhang, H.; Chung, S. W.; Mirkin, C. A. Nano Lett. 2003, 3, 4345.
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Figure 3. Laterally structured ODT SAMs were used as a mask for protecting a 20-nm-thick Au film against a ferricyanide etch solution. In all images, bright regions correspond to Au domains that were not etched. (a and b) Optical images of 2.5 × 2.5 µm2 boxes arrayed every 12 and 1 µm, respectively. (c and d) Scanning electron microscopy images of etched Au features with characteristic dimensions of less than 50 nm. Note that in part d the contrast is inverted because we have defined a trench.
tallinity of the Au film. We did not attempt to further reduce the patterning dimensions; however, from the results of Figure 3c we would suggest that 40 nm is roughly the resolution limit of this method. The excellent etch resistance of the ODT monolayer allowed us in addition to reverse the patterning contrast. This is illustrated in Figure 3d, which shows the definition of a trench, 50 nm in width. As before, the bright areas represent the raised Au features, while the thin gray line is the nonprotected part of the Au film that was attacked by the etchant. The good etch selectivity testifies for the high molecular integrity of the ODT monolayer, that is, the successful completion of the SA process. Conclusions In this letter, we have presented a method that allows one to assemble biomolecules on surfaces, in patterns with dimensions as small as 40 nm, at a resolution of about 3 nm. We illustrate the proof of principle by directing the assembly of two different alkanethiols on Au substrates. Contrary to previously published approaches, we do not expose the thin molecular films to the electron beam. This fundamental difference should allow the method to be applied to the patterning of proteins or supramolecular assemblies (membrane fragments, lipid bilayers, vesicles, and so forth) that can only be placed on the surface by assembly from solution. Acknowledgment. This work was supported by the board of the Swiss Federal Institutes of Technology and the Swiss National Science Foundation. LA049954J
