Multiscale Fabrication of Multiple Proteins and Topographical

Feb 24, 2011 - University Program Division of Integrative Biosciences and Biotechnology (IBB), Pohang University of Science and Technology. (POSTECH) ...
3 downloads 0 Views 3MB Size
Download PDF
LETTER pubs.acs.org/Langmuir

Multiscale Fabrication of Multiple Proteins and Topographical Structures by Combining Capillary Force Lithography and Microscope Projection Photolithography Keon Woo Kwon,†,§ Jong-Cheol Choi,§ Kahp-Yang Suh,*,†,‡ and Junsang Doh*,§,|| School of Mechanical and Aerospace Engineering and ‡World Class University Program on Multiscale Mechanical Design, Seoul National University, Seoul 151-742, Korea § Department of Mechanical Engineering and School of Interdisciplinary Bioscience and Bioengineering (I-Bio) and World Class University Program Division of Integrative Biosciences and Biotechnology (IBB), Pohang University of Science and Technology (POSTECH) San 31, Hyoja-dong, Nam-Gu, Pohang, Gyeongbuk 790-784, Korea )

†

bS Supporting Information ABSTRACT: We present new methods that enable the fabrication of multiscale, multicomponent protein-patterned surfaces and multiscale topologically structured surfaces by exploiting the merits of two well-established techniques: capillary force lithography (CFL) and microscope projection photolithography (MPP) based on a protein-friendly photoresist. We further demonstrate that, when hierarchically organized micro- and nanostructures were used as a cell culture platform, human colon cancer cells (cell line SW480) preferentially adhere and migrate onto the area with nanoscale topography over the one with microscale topography. These methods will provide many exciting opportunities for the study of cellular responses to multiscale physicochemical cues.

’ INTRODUCTION Surfaces containing multiscale features, from nano- to microlength scales, are useful for many biological,1-8 biomimetic,9,10 and optical applications.11,12 In particular, the multiscale fabrication of biomolecules and topographical features would be extremely useful in studying cellular behavior in complex microenvironments because cells are known to respond to multiscale biochemical and topographical cues present in the body.1-8,13 Accordingly, there have been many fabrication techniques developed to create either micro-14-16 or nanoscale patterns of multiple biomolecules17-19 or multiscale topographical features,20-24 but few of them are versatile enough to be applied to the fabrication of multiscale, multicomponent bimolecular patterns as well as multiscale topographical features. To address the aforementioned issue, we combined capillary force lithography (CFL), a method specialized to the fabrication of nanoscale structures beyond the resolution of conventional photolithography with low cost,25,26 and microscope projection photolithography (MPP) based on a protein-friendly photoresist,16 a recently developed method for the fabrication of multicomponent protein micropatterned surfaces. By this novel combination, we successfully fabricated multiscale, multicomponent protein-patterned surfaces and multiscale topographically structured surfaces. Furthermore, we demonstrate that when hierarchically organized micro- and nanostructures were used as a cell culture platform, some cell types (e.g., human colon cancer cells) preferentially adhered and migrated onto the area with r 2011 American Chemical Society

nanoscale topography over the one with microscale topography. Techniques developed in this work will be useful in the study of various fundamental biological processes involving multiscale physicochemical signaling cues such as cell migration, differentiation, and cell-cell communication.1-8

’ RESULTS AND DISCUSSION A protein-friendly photoresist, poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP), was synthesized and used for the construction of spatially defined protein micropatterns. As described previously,16 PDMP has unique properties that are useful in biological applications. First, it is insoluble in water but becomes soluble in mild aqueous buffers such as phosphatebuffered saline (PBS; 10 mM sodium phosphate, 140 mM sodium chloride, pH 7.4) upon UV exposure. Second, it exhibits excellent resistance against protein and cell adhesions because of the presence of poly(ethylene glycol) (PEG) side chains. By harnessing these properties, we could successfully fabricate microscale multicomponent protein-patterned surfaces by MPP based on PDMP.16 However, this method cannot be extended to the submicrometer or nanoscale because of the diffraction limit of far-field optical fabrication. To overcome this Received: January 3, 2011 Revised: February 7, 2011 Published: February 24, 2011 3238

dx.doi.org/10.1021/la2000156 | Langmuir 2011, 27, 3238–3243

Langmuir

Figure 1. (a) Schematic diagram of the sequential fabrication of multiscale, multicomponent protein-patterned surfaces by combining CFL and MPP. (b) Fluorescence micrograph of TRITC-SVs line patterns with 350 nm width. (c) Fluorescence micrograph of FITC-SVs line patterns with 700 nm width inside a 16 μm square. (d) Overlay image of florescence micrographs of TRITC-SAv and FITC-SAv patterns.

limitation, we first fabricated PDMP nanostructures using CFL with assistance from a small amount of hydraulic pressure as shown in Figure 1a(i-iii). A thin film of PDMP was prepared by spin coating a PDMP solution (10 w/v % in 1,4-dioxane) onto a biotinylated glass coverslip. Then, a polyurethane acrylate (PUA) mold was placed on the PDMP film with a pressure of ∼0.01 bar while heating in a vacuum oven for 2 h at 90 °C, and the PUA mold was peeled off at 70 °C to minimize the ripping of structures (Figure 1a(ii)).27 This lithographic step using PUA molds can be termed rigiflex lithography because PUA molds are not only sufficiently rigid for fabricating nanoscale structures but also flexible for conformal contact over a large area.28,29 When we fabricated 700 nm ridge/350 nm groove structures with 500 nm height (measured by cross-sectional SEM) using PDMP thin films spin coated from a 10% PDMP solution, biotin molecules under 350 nm groove regions were exposed, presumably because of the dewetting of PDMP on the biotinylated glass surfaces (Figure 1a(iii)). The metastable conditions of PDMP thin films were confirmed independently by a simple dewetting experiment: when PDMP thin films were prepared using concentrations of PDMP solution lower than 8%, disconnected ridges were

LETTER

formed after CFL (Supporting Information Figure 1), suggesting that the bottom-exposure mechanism is mediated by a disjoining pressure under contact regions of PDMP films.30 The exposure of the biotin surface could be further verified by performing a fluorophore-labeled streptavidin adsorption experiment: the patterned PDMP surface was immersed in a tetramethyl rhodamine isothiocyanate-conjugated streptavidin (TRITC-SAv) solution in PBS for 30 min and rinsed with PBS, and adsorbed TRITC-SAv was visualized by fluorescence microscopy. As shown in Figure 1b, TRITC-SAv was selectively attached to the exposed biotinylated region of 350 nm groove surfaces (Figure 1a(iv)), and nonspecific binding of SAv on PDMP ridges turned out to be minimal because PDMP surfaces were shown to repel protein adsorption.16 In the next step, the TRITC-SAvpatterned surface immersed in PBS was mounted on a microscope stage and MPP was performed by illuminating the surface with 365 nm UV through a photomask containing an array of 16 μm squares (Figure 1a(v)). PDMP 700 nm ridges in the region defined by the photomask were spontaneously dissolved in PBS, resulting in additional exposure of the biotin surface located underneath (Figure 1a(v)). Subsequently, fluorescein isothiocyanate (FITC)-conjugated SAv was attached to the freshly exposed biotinylated region (Figure 1a(vi)). Fluorescence micrographs acquired from such a two-step fabrication method schematized in Figure 1a are shown in Figure 1b-d. The fluorescence intensity of TRITC in the square regions was slightly lower (∼10%) than that of the outside (Figure 1b), in part because of the photobleaching of TRITC in the square regions by selective UV exposure through the photomask during the MPP process (Figure 1a(v)). In this way, we could obtain multiscale two-component SAv-patterned surfaces. As demonstrated previously,2,16 this method can be readily extended to the patterning of multiple distinct biotinylated proteins, which are either commercially available or easily prepared in the laboratory using commercially available reagents, by simply adding incubation steps with biotinylated proteins after each SAv deposition. Remarkably, all of the processes were performed without using any clean room facilities, and patterned proteins were not exposed to any harsh conditions that otherwise degrade the activity of proteins throughout the processes. It is noted that the multiscale, multicomponent proteinpatterned surfaces presented here also contain multiscale topographical structures as shown in Figure 1a(v). These multiscale PDMP structures, if necessary, can be readily removed by flood exposure to UV followed by PBS development, which gives rise to spatially defined multiscale patterns of different proteins. Resistance to protein adsorption due to PEG side chains of PDMP is essential to the patterning of multiple proteins by minimizing nonspecific background binding but may not be desirable for cell studies because they also prevent cells from adhering to the surfaces.16 Multiscale topographical surfaces with identical surface chemistry would be useful in studying how cells response to multiscale topographical cues. To achieve this goal, we replicated multiscale topographical structures formed on PDMP with a different UV-curable polymer (PUA) by utilizing a self-replication characteristic.29,31 For cell adhesion studies, PUA has many advantages compared to PDMP. First, it forms chemically cross-linked polymer networks by UV cross-linking. Thus, topographical structures on PUA surfaces are much more stable than those on PDMP surfaces. Second, its surfaces can be readily modified by plasma treatment to enable extensive protein adsorption.32 Third, it can be 3239

dx.doi.org/10.1021/la2000156 |Langmuir 2011, 27, 3238–3243

Langmuir

Figure 2. (a) Schematic illustration of the fabrication of a PUA replica from PDMP multiscale structures using double-replication steps. (b) SEM images of various PUA multiscale structures (top left, 350 nm ridges/700 nm grooves with a 10 μm gap; top right, 350 nm pillars with 4 μm holes; bottom left, 350 nm pillars on 6 μm squares; bottom right, 350 nm pillars with 6 μm squares).

self-replicated in such a way that one can duplicate both positive and negative patterns from the same original masters.29,31 To replicate multiscale structures with PUA, PDMP multiscale structures fabricated by the sequential application of CFL and MPP (Figure 1a) were treated with 1H,1H,2H,2H-perfluorooctyl-trichlorosilane to minimize surface energy. Then, a PUA negative replica of PDMP multiscale structures was fabricated by replica molding followed by UV irradiation (Figure 2a(ii,iii)). By repeating the same process on the first-replicated PUA multiscale structure (Figure 2a(iv-vi)), one could obtain the identical multiscale structures of PUA to PDMP. Scanning electron microscope (SEM) images of doubly replicated PUA surfaces with various multiscale structures of lines/pillars/holes are shown in Figure 2b. Multiscale structures containing both micro- and nanoscale components are useful in many applications, thus a number of methods such as the growth of carbon nanotubes or nanowires,20 transfer printing,21 multistep molding,22 and two-step UV-assisted CFL23 were developed. Compared with the methods listed above, our method is highly advantageous when various microscale features need to be systematically incorporated into pre-existing nanostructures. MPP requires only high-resolution laser-printed transparent films, which can be prepared in a fast, low-cost manner, and once a multiscale replica of PUA is fabricated, we can produce multiscale

LETTER

PUA micro/nanostructures in a single step. Considering that the preparation steps for the original silicon master are most timeconsuming and expensive, the capability to transform pre-existing single-scale masters into new multiscale masters would provide many exciting opportunities, in particular, for the study of how cells response to multiscale topographical cues. For a proof-of-concept experiment, 350 nm ridge/700 nm groove PDMP nanostructures were prepared by CFL, and various microscale equally spaced ridge/groove structures (8, 16, and 32 μm) were incorporated by MPP either parallel or perpendicular to the nanostructures. Then, PUA-structured surfaces with identical dimensions to PDMP were fabricated by the dual-replication process as described earlier (Figure 2a). For comparison, PUA surfaces containing only microscale topographical structures were fabricated by performing MPP on flat PDMP spin-coated surfaces and dual replication. Representative SEM images of three different types of multiscale topographical features are shown in Figure 3a. Here, “perpendicular” (“parallel”) denotes nanogrooves on the ridges of microgrooves that are perpendicular (parallel) to the direction of microgrooves, and “flat” represents no nanostructure on the ridges of microgrooves. Then, colon cancer cells (cell line SW480) were cultured on PUA multiscale structured surfaces coated with 0.1% gelatin. When we examined the cells after 1 day of culturing, the majority of SW480 cells (80-90%) were selectively adhered to the ridges of multiscale structures, both in perpendicular and parallel configurations for all microgrooves used in the experiment (8, 16, and 32 μm), and most cells on flat ridge/groove micropatterns were randomly adhered without a particular affinity for microgroove regions (Figure 3b). This result suggests that nanoscale topographical cues, as compared to microscale topographical cues, have an ability to elicit the selective adhesion of SW480 cells. Also, the cells on multiscale ridges appeared to align along the direction of nanoscale ridge/groove structures (Figure 3c), revealing that the nanoscale features provide topographical guidance over cell elongation.33-35 Interestingly, when cells were placed on perpendicular nanoscale ridge/groove patterns with their diameter being close to or larger than the width of micropatterns (e.g., 8 and 16 μm ridge/groove), most cells exhibited round-shape morphology as shown in the first two rows of the left column of Figure 3c. It appears that some cells at the edges of microgrooves were also elongated along the grooves, suggesting that they might respond to microtopography. Nonetheless, their number was significantly lower and seems to be negligible as compared to other cell populations. It is noted that the cell response to multiscale topographical features presented here is not universal. For example, other cell types such as fibroblast cells (NIH 3T3) and cervical cancer cells (HeLa) were not selectively adhered to multiscale structures, suggesting that some specific cell types are predominantly regulated by nanoscale topographical cues. Given the fact that living cells are surrounded by different microenvironments with complex interactions with various biochemical/biophysical cues, it is not surprising to observe different cellular behaviors on multiscale topographical surfaces. To gain further insight into the mechanism of the selective adhesion of SW480 cells on nanoscale structures, the migration trajectory of cells was monitored in real time under a video microscope. As shown in Figure 3d and Supporting Information Video 1, when SW480 cells were located on a border between nanoscale structures (ridges) and flat surface (grooves), they preferentially migrated to nanoscale structures (Figure 3d, black and white arrows, and Supporting Information Video 1). Some 3240

dx.doi.org/10.1021/la2000156 |Langmuir 2011, 27, 3238–3243

Langmuir

Figure 3. (a) SEM images of 16 μm ridge/groove PUA structures with perpendicular (top) or parallel (middle) 350 nm ridge/700 nm groove structures on ridges. Simple ridge/groove structures without nanogrooves are also shown (bottom). (b) Percentage of colon cancer cells (SW480) attached on the ridges of various structured substrates shown in a (***, p < 0.001). These values were averaged over four repetitions, and error bars represent the standard deviation. The average number of cells scored for each condition was about 50. (c) Bright-field optical microscope images of SW480 cells on various structured surfaces. (d) Time-lapse images of the migration and division of SW480 cells on multiscale ridge/groove structures (350 nm ridges/700 nm grooves are perpendicular to 16 μm ridges/groove). The elapsed time is represented as h:min. White arrows represent dividing cells and daughter cells. Black arrows represent cells migrating from the border.

cells underwent cell division during our 24 h time-lapse imaging (Figure 3d, white arrows). Interestingly, daughter cells also either quickly migrated onto the multiscale ridges or divided on the multiscale ridges. Therefore, most cells remained on the tops of ridges containing nanostructures even after cell division. In summary, we developed new techniques to fabricate multiscale, multicomponent protein-patterned surfaces and multiscale topological structures by exploiting dewetting-mediated CFL and MPP based on a protein-friendly photoresist, PDMP. These methods will provide many exciting opportunities for the study of cellular responses to multiscale physicochemical cues as well as multiscale topographical guidance.

’ EXPERIMENTAL SECTION Synthesis and Patterning of PDMP by CFL. Random terpolymer PDMP was synthesized and characterized as described

LETTER

elsewhere.16 Glass coverslips were biotinylated as previously reported.36 Briefly, glass coverslips were functionalized with 3-aminopropyl triethoxysilane (Sigma), and poly(acrylic acid) (PAA, Sigma) was adsorbed to the silanized substrates. Subsequently, biotin was conjugated to the PAA-coated substrates by incubation of the coverslips in an aqueous solution of biotin-PEO-amine (Pierce, 500 μg/mL) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 5 mg/mL, Sigma) for 4 h. Biotinylated substrates were rinsed with deionized water and dried overnight in vacuo. PDMP dissolved in 1,4dioxane (10 w/v %) was spin coated onto the biotinylated coverslips at 2000 rpm for 60 s and dried in vacuo at 80 °C for 24 h. Then, a PUA mold was placed on the PDMP-coated surface using slight pressure (∼0.01 bar), and the temperature was raised to 90 °C for 2 h. Finally, the PUA mold was peeled off of the substrate at 70 °C to avoid ripping the structures.27 Fluorescence Microscopy. A modified Zeiss Axio Observer Z1 epifluorescence microscope with a 100 (Plan-Neofluar, NA = 1.30) objective lens and a Roper Scientific CoolSnap HQ CCD camera was used for imaging and micropatterning. An XBO 75 W/2 xenon lamp (75 W, Osram) and DAPI (EX. 365, BS 395, EMBP445/50), eGFP (EX BP 470/40, BS 495, EMBP 525/50), Cy3 (EX BP 550/25, BS 570, EMBP 605/70), and Cy5 (EX BP 620/60, BS 660, EMBP 770/75) filter sets were used for illumination and fluorescence imaging. The microscope was automatically controlled by Axiovision 4.6 (Carl Zeiss), and acquired images were analyzed and processed with Methamorph (Universal Imaging, Molecular Devices). Microscope Projection Photolithography (MPP). Photomasks were printed on transparent films using a high-resolution image setter with 40 000 dpi resolution (Microtech Co., Ltd.) and inserted at the field diaphragm of the microscope. PDMP-patterned substrates were loaded into a Chamlide magnetic chamber (Live Cell Instrument, Seoul, Korea) filled with PBS. Then, the magnetic chamber was loaded onto the microscope stage. For UV illumination, the xenon lamp and DAPI filter described above were used, and the UV exposure time was automatically controlled by a MAC 5000 computer-controlled shutter system (Ludl Electronics Products, Ltd.). Microscope projection lithography was performed by exposing PDMP films with plane-focused UV though photomasks. Feature sizes on the transparent film were reduced 40-fold when a 100 objective lense was used. For surfaces containing nanoscale ridge/groove structures, we first aligned nanolines on the surfaces horizontally by manually rotating circular chambers containing nanostructured surfaces while examining the orientation of nanolines with a microscope. Then, we inserted photomasks containing microscale lines into the field diaphragm in either a vertically or horizontally aligned manner. Self-Replication of Multiscale PDMP Structures. PDMPstructured substrates were dried in vacuo over 6 h, exposed to UV using a UV illuminator (Minuta Tech MT UV A01, Seoul, Korea, λ = 200400 nm, dose = 100 mJ/cm2) over 6 h, and treated with 1H,1H,2H,2Hperfluorooctyl-trichlorosilane (Sigma) by vacuum deposition. Then, the liquid PUA precursor (MINS 311RM, Minuta Tech) was drop dispensed onto PDMP-structured surfaces, and flexible transparent poly(ethylene terephthalate) (PET) films were brought into contact with the precursor surfaces. Subsequently, the mold was exposed to UV for 20 s through the transparent backplane. After UV curing, the mold was peeled off of the surface and additionally cured overnight to terminate the remaining active acrylate groups. To fabricate PUA structures on a glass surface, glass surfaces were first coated with an adhesion promoter (glass primer, Minuta Tech) and baked at 120 °C for 20 min. Then, the PUA precursor was drop dispensed on a glass surface, and the PUA mold was placed and exposed to UV light for 20 s. The PUA mold was removed after UV curing. Cell Culturing and Live Cell Imaging. SW480 human colon cancer cells (Korean Cell Line Bank, Korea) were maintained at 37 °C in 3241

dx.doi.org/10.1021/la2000156 |Langmuir 2011, 27, 3238–3243

Langmuir 5% CO2 in RPMI 1640 (Gibco) with 10% fetal bovine serum (FBS, Gibco) and 100 U/mL of penicillin and 100 μg/mL of streptomycin (Invitrogen). Cells were harvested from culture dishes by treating with 0.25% trypsin-EDTA (Invitrogen) and seeded onto various structured surfaces mounted on the microscope stage equipped with a Chamlide TC incubator system (at 37 °C in 5% CO2) for live cell imaging. Timelapse microscopy was immediately initiated with images recorded in 10 min intervals for 24 h.

’ ASSOCIATED CONTENT

bS

Supporting Information. Optical microscope image of disconnected 700 nm ridges by dewetting. Time-lapse imaging of SW480 cells attached to 16 μm microgrooves with perpendicular nanogrooves. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*(K.-Y.S.) Tel: þ82-2-880-9103. Fax: þ82-2-883-0179. E-mail: [email protected]. (J.D.) Tel: þ82-54-279-2189. Fax: þ82-54279-5899. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (grant no. 2009-0073922). ’ REFERENCES (1) Bettinger, C. J.; Langer, R.; Borenstein, J. T. Engineering substrate topography at the micro- and nanoscale to control cell function. Angew. Chem., Int. Ed. 2009, 48, 5406–5415. (2) Doh, J.; Irvine, D. J. Immunological synapse arrays: Patterned protein surfaces that modulate immunological synapse structure formation in T cells. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 5700–5705. (3) Khademhosseini, A.; Langer, R.; Borenstein, J.; Vacanti, J. P. Microscale technologies for tissue engineering and biology. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2480–2487. (4) Sniadecki, N.; Desai, R. A.; Ruiz, S. A.; Chen, C. S. Nanotechnology for cell-substrate interactions. Ann. Biomed. Eng. 2006, 34, 59–74. (5) Suh, K. Y.; Park, M. C.; Kim, P. Capillary force lithography: a versatile tool for structured biomaterials interface towards cell and tissue engineering. Adv. Funct. Mater. 2009, 19, 2699–2712. (6) Torres, A. J.; Wu, M.; Holowka, D.; Baird, B. Nanobiotechnology and cell biology: micro- and nanofabricated surfaces to investigate receptor-mediated signaling. Ann. Rev. Biophys. 2008, 37, 265–288. (7) Khademhosseini, A.; Eng, G.; Yeh, J.; Kucharczyk, P. A.; Langer, R.; Vunjak-Novakovic, G.; Radisic, M. Microfluidic patterning for fabrication of contractile cardiac organoids. Biomed. Microdev. 2007, 9, 149–157. (8) Aubin, H.; Nichol, J. W.; Hutson, C. B.; Bae, H.; Sieminski, A. L.; Cropek, D. M.; Akhyari, P.; Khademhosseini, A. Directed 3D cell alignment and elongation in microengineered hydrogels. Biomaterials 2010, 31, 6941–6951. (9) Jeong, H. E.; Suh, K. Y. Nanohairs and nanotubes: efficient structural elements for gecko-inspired artificial dry adhesives. Nano Today 2009, 4, 335–346. (10) Xia, F.; Jiang, L. Bio-inspired, smart, multiscale interfacial materials. Adv. Mater. 2008, 20, 2842–2858. (11) Henzie, J.; Lee, M. H.; Odom, T. W. Multiscale patterning of plasmonic metamaterials. Nat. Nanotechnol. 2007, 2, 549–554. (12) Liang, G. Q.; Zhu, X. L.; Xu, Y. G.; Li, J.; Yang, S. Holographic design and fabrication of diamond symmetry photonic crystals via dualbeam quadruple exposure. Adv. Mater. 2010, 22, 4524–4529.

LETTER

(13) Salaita, K.; Nair, P. M.; Petit, R. S.; Neve, R. M.; Das, D.; Gray, J. W.; Groves, J. T. Restriction of receptor movement alters cellular response: physical force sensing by EphA2. Science 2010, 327, 1380–1385. (14) Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006, 27, 3044–3063. (15) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Patterning proteins and cells using soft lithography. Biomaterials 1999, 20, 2363–2376. (16) Kim, M.; Choi, J. C.; Jung, H. R.; Katz, J. S.; Kim, M. G.; Doh, J. Addressable micropatterning of multiple proteins and cells by microscope projection photolithography based on a protein friendly photoresist. Langmuir 2010, 26, 12112–12118. (17) Christman, K. L.; Enriquez-Rios, V. D.; Maynard, H. D. Nanopatterning proteins and peptides. Soft Matter 2006, 2, 928–939. (18) Christman, K. L.; Schopf, E.; Broyer, R. M.; Li, R. C.; Chen, Y.; Maynard, H. D. Positioning multiple proteins at the nanoscale with electron beam cross-linked functional polymers. J. Am. Chem. Soc. 2009, 131, 521–527. (19) Lee, K. B.; Lim, J. H.; Mirkin, C. A. Protein nanostructures formed via direct-write dip-pen nanolithography. J. Am. Chem. Soc. 2003, 125, 5588–5589. (20) Meng, G. W.; Jung, Y. J.; Cao, A. Y.; Vajtai, R.; Ajayan, P. M. Controlled fabrication of hierarchically branched nanopores, nanotubes, and nanowires. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7074–7078. (21) Zaumseil, J.; Meitl, M. A.; Hsu, J. W. P.; Acharya, B. R.; Baldwin, K. W.; Loo, Y. L.; Rogers, J. A. Three-dimensional and multilayer nanostructures formed by nanotransfer printing. Nano Lett. 2003, 3, 1223–1227. (22) Bao, L. R.; Cheng, X.; Huang, X. D.; Guo, L. J.; Pang, S. W.; Yee, A. F. Nanoimprinting over topography and multilayer three-dimensional printing. J. Vac. Sci. Technol., B 2002, 20, 2881–2886. (23) Jeong, H. E.; Lee, J. K.; Kim, H. N.; Moon, S. H.; Suh, K. Y. A nontransferring dry adhesive with hierarchical polymer nanohairs. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5639–5644. (24) Zhang, Y.; Lin, C. T.; Yang, S. Fabrication of hierarchical pillar arrays from thermoplastic and photosensitive SU-8. Small 2010, 6, 768–775. (25) Suh, K. Y.; Lee, H. H. Capillary force lithography: large-area patterning, self-organization, and anisotropic dewetting. Adv. Funct. Mater. 2002, 12, 405–413. (26) Yoon, H.; Kim, T. I.; Choi, S. J.; Suh, K. Y.; Kim, M. J.; Lee, H. H. Capillary force lithography with impermeable molds. Appl. Phys. Lett. 2006, 88, 254104. (27) Park, S.; Song, Z. C.; Brumfield, L.; Amirsadeghi, A.; Lee, J. Demolding temperature in thermal nanoimprint lithography. Appl. Phys. A 2009, 97, 395–402. (28) Kim, P.; Suh, K. Y. Rigiflex, spontaneously wettable polymeric mold for forming reversibly bonded nanocapillaries. Langmuir 2007, 23, 4549–4553. (29) Suh, D.; Choi, S. J.; Lee, H. H. Rigiflex lithography for nanostructure transfer. Adv. Mater. 2005, 17, 1554–1560. (30) Kim, P.; Lee, B. K.; Lee, H. Y.; Kawai, T.; Suh, K. Y. Molded nanowell electrodes for site-selective single liposome arrays. Adv. Mater. 2008, 20, 31–36. (31) Choi, S. J.; Yoo, P. J.; Baek, S. J.; Kim, T. W.; Lee, H. H. An ultraviolet-curable mold for sub-100-nm lithography. J. Am. Chem. Soc. 2004, 126, 7744–7745. (32) Lee, M. R.; Kwon, K. W.; Jung, H.; Kim, H. N.; Suh, K. Y.; Kim, K.; Kim, K. S. Direct differentiation of human embryonic stem cells into selective neurons on nanoscale ridge/groove pattern arrays. Biomaterials 2010, 31, 4360–4366. (33) Kim, D. H.; Han, K.; Gupta, K.; Kwon, K. W.; Suh, K. Y.; Levchenko, A. Mechanosensitivity of fibroblast cell shape and movement to anisotropic substratum topography gradients. Biomaterials 2009, 30, 5433–5444. 3242

dx.doi.org/10.1021/la2000156 |Langmuir 2011, 27, 3238–3243

Langmuir

LETTER

(34) Kim, D. H.; Lipke, E. A.; Kim, P.; Cheong, R.; Thompson, S.; Delannoy, M.; Suh, K. Y.; Tung, L.; Levchenko, A. Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 565–570. (35) Kim, D. H.; Seo, C. H.; Han, K.; Kwon, K. W.; Levchenko, A.; Suh, K. Y. Guided cell migration on microtextured substrates with variable local density and anisotropy. Adv. Funct. Mater. 2009, 19, 1579–1586. (36) Katz, J. S.; Doh, J.; Irvine, D. J. Composition-tunable properties of amphiphilic comb copolymers containing protected methacrylic acid groups for multicomponent protein patterning. Langmuir 2006, 22, 353–359.

3243

dx.doi.org/10.1021/la2000156 |Langmuir 2011, 27, 3238–3243