A Novel Approach to Produce Protein Nanopatterns by Combining

Sep 21, 2004 - Rue Jaquet-Droz 1, CH-2007 Neuchaˆtel, Switzerland. Received July 6, 2004; Revised Manuscript Received August 25, 2004. ABSTRACT...
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NANO LETTERS

A Novel Approach to Produce Protein Nanopatterns by Combining Nanoimprint Lithography and Molecular Self-Assembly

2004 Vol. 4, No. 10 1909-1914

Didier Falconnet,† Daniela Pasqui,†,§ Sunggook Park,‡ Rolf Eckert,| Helmut Schift,‡ Jens Gobrecht,‡ Rolando Barbucci,§ and Marcus Textor*,† BiointerfaceGroup, Laboratory for Surface Science and Technology, Department of Materials, Swiss Federal Institute of Technology (ETH) Zu¨rich, CH-8093 Zurich, Switzerland, Laboratory for Micro- and Nanotechnology, Paul Scherrer Institut (PSI), CH-5232 Villigen, Switzerland, Department of Chemical and Biosystems Science and Technologies, UniVersity of Siena, Via A. Moro, 2, 53100 Siena, Italy, and Centre Suisse d’Electronique et de Microtechnique (CSEM), Rue Jaquet-Droz 1, CH-2007 Neuchaˆ tel, Switzerland Received July 6, 2004; Revised Manuscript Received August 25, 2004

ABSTRACT We describe a novel parallel method for the patterning of proteins with nanoscale resolution. Combining nanoimprint lithography (NIL) and molecular assembly patterning by lift-off (MAPL), we produced streptavidin patterns with feature sizes in the order of 100 nm. A stamp is imprinted into a heated PMMA film followed by a dry etching step that converts the topography into a PMMA/Nb2O5 contrast. A biotin functionalized copolymer, poly(L-lysine)-graft-poly(ethylene glycol)-biotin (PLL-g-PEG/PEG-biotin), spontaneously adsorbs on the oxide surfaces. After PMMA lift-off, the background is backfilled with protein-resistant PLL-g-PEG. We show that streptavidin selectively adsorbs on the biotin areas and thus can be used as a universal platform for immobilization of biotin-tagged molecules. This novel process is a parallel patterning method that is fast, reproducible, and economic. The PEG-copolymer can be functionalized with a variety of bioactive groups and thus allows a great flexibility in terms of surface chemistry.

The ability to immobilize proteins on sub-micro- to nanometric sized areas has become a major challenge for the development of bioengineered surfaces. The ongoing technological advances are partially driven by the aim for broadening the understanding of a variety of surfacemediated biological recognition events. Micropatterned surfaces are known to influence cell function through surfacetriggered interactions.1 However, little is known on how spatially controlled arrays of single (or few) surface ligands affect surface-attached living cells.2,3 Such biochemically nanopatterned surfaces are believed to provide, for example, a better insight into integrin-mediated intracellular pathways. Also, the ability to spatially control the immobilization of a low number of molecules, down to single protein or DNA molecules,4 will provide better platforms for the study of single molecular events, e.g., by AFM force measurements.5 * Corresponding author. E-mail: [email protected]; Tel: ++411-632 64 51. Fax: ++41-1-633 10 27. † Swiss Federal Institute of Technology. ‡ Paul Scherrer Institut. § University of Siena. | Centre Suisse d’Electronique et de Microtechnique. 10.1021/nl0489438 CCC: $27.50 Published on Web 09/21/2004

© 2004 American Chemical Society

Production of arrays of single nanoreactors such as micelles or vesicles again depends on the availability of chemical nanopatterns. Loaded vesicles containing femtoliters of reagents arranged in an array format constitute a promising surface platform for combinatorial chemistry.6 Immobilized vesicles are also considered to have the built-in potential to act as protein carriers for transmembrane or water-soluble proteins.7 Several patterning strategies have been developed to produce biologically relevant patterns on the nanometer scale.8 The techniques are generally categorized as serial and parallel methods. The most commonly used serial methods are electron-beam writing,9 focused ion beam10,11 and scanning probe based lithography.12,13 Their main disadvantages are high cost and long writing time (a number of writing techniques such as dip-pen lithography using multiple inkers in parallel are, however, more efficient).14 On the other hand, they are advantageous in terms of their flexibility in generating any type of pattern shape, from highly periodic patterns to heteroclite motifs.12 Examples of parallel techniques producing more or less random pattern distributions

are polymer demixing15 and colloidal lithography.16 The most widely accepted, parallel techniques for producing submicrometer patterns in a regular manner are microcontact printing,17,18 “controlled” colloidal lithography,19 X-ray interference lithography,20 and nanoimprint lithography (NIL).21-23 Applications can be found, for example, in the area of locally addressable patterns of recognition molecules for high-throughput analysis in genomics and proteomics; in the field of cell-surface interactions, where the ability to vary the separation between single integrin ligands in a controlled manner may contribute to an improved fundamental understanding of integrin clustering, focal contact formation, and induced cellular signaling.3 Independent of the technique used to create the biomolecular nanopatterns, a critical requirement is the ability to avoid nonspecific binding of proteins (or biomolecules in general). The uncontrolled presence of proteins on an array will most likely affect the outcome of the assay and thus bias the results. Therefore, the noninteractive areas of the pattern require special attention through designed surface chemistry in order to ensure a very low background signal. The method presented in this contribution combines a “topdown” and a “bottom-up” approach through NIL and the molecular assembly patterning by lift-off (MAPL). NIL consists of imprinting a hard master with desired features into a resist layer supported by a flat substrate which is heated above its glass transition temperature.21,22,24 Sub-10 nm resolution patterns have been demonstrated using NIL and metal lift-off.25,26 Furthermore, NIL is a low-cost and highthroughput technique, able to replicate a 100 mm wafer containing representative areas with feature sizes down to 50 nm within a few minutes with high reproducibility and pattern fidelity.27 The MAPL technique converts a prestructured resist film into a pattern of biointeractive chemistry in a noninteractive background using spontaneous, monomolecular assembly of biofunctional moieties. MAPL has been introduced recently28 as a technique to produce micropatterns via conventional photolithography and assembly of biofunctional PEG-graft polyelectrolyte copolymers, such as cationic poly(L-lysine)graft-poly(ethylene glycol) (PLL-g-PEG) on transparent, negatively charged niobium-oxide-coated glass slides. A particularly attractive feature of MAPL is its ability to vary and control quantitatively the surface density of bioactive molecules in the biointeractive patches of the pattern. This is achieved by diluting in solution the functionalized copolymer PLL-g-PEG/PEG-Y (Y ) a biospecific ligand such as biotin or peptides) with nonfunctionalized PLL-gPEG in any desired ratio and subsequently co-assembling a mixed adlayer of defined composition. This task is difficult to achieve with any of the established biochemical patterning techniques, but it is highly relevant for both the bioaffinity sensing and tissue-engineering field. Furthermore, the interaction of the immobilized biomolecules on the MAPL patterns with the analyte has been shown to be highly specific as a consequence of very low nonspecific interaction with the dense PEG-brush background (less than 2 ng/cm2 from full serum).28 1910

In the present study we combined NIL with MAPL to produce submicrometer-sized patterns of streptavidin biospecifically immobilized on monomolecular adlayers of biotinylated-PLL-g-PEG in a background of nonfunctionalized PLL-g-PEG. Schematics of the NIL process are presented in Figure 1a. Silicon wafers (Wafernet) and Pyrex plates (SensorPrep; 0.5 mm thick) sputter-coated with 12 nm transparent niobium oxide (Nb2O5) were used as substrates. These substrates were then spin-coated with a thin film of 125 nm of poly(methyl methacrylate) (PMMA, molecular weight ) 25 kg/mol), followed by a soft baking for 1 min at 170 °C. When micrometer featured stamps were used the spin-coated PMMA layer was 330 nm. For NIL, various stamps containing micro- and nanopatterns of lines, dots, and stars were fabricated using a Leica Lion LV1 electron-beam writer with subsequent reactive ion etching (RIE) of silicon. The stamps were coated with an anti-adhesive layer and then replicated by imprinting into the PMMA-coated substrates at a temperature of 180 °C and pressure of 50 bar. The size of both stamps and substrates was 2 × 2 cm2 with the nanostructure located in the central 1 × 1 cm2 of the stamp area. The PMMA layer that remained on the bottom of the imprinted structure (Figure 1a, stage III) was subsequently removed by O2 RIE operated at a flow of 21 sccm, a chamber pressure of 20 mTorr and an RF power of 21 W. This resulted in the creation of a nanostructured surface with PMMA/Nb2O5 contrast (Figure 1a, stage IV).26 The PMMA/Nb2O5 contrast produced by NIL was then converted into a biologically relevant pattern using the MAPL technique,28 as illustrated in Figure 1b. The sample was dipped into an aqueous solution of PLL-g-PEG/PEGbiotin.29 Due to the negative surface charge of Nb2O5 at neutral pH, the functionalized copolymer adsorbed on the Nb2O5 patches via electrostatic interaction with the positively charged amino-terminated side chains. The polymer also adsorbs on the PMMA regions (Figure 1b, stage V). The synthesis and the properties of this copolymer have been described extensively.30,31 In brief, PEG chains with and without bioactive terminal groups (e.g., biotin) are grafted, in solution, onto the PLL backbone (PLL ) 20 kDa). For the polymer used in this study, approximately every fourth lysine unit had a grafted PEG chain and 50% of the total PEG chains were biotinylated. After stripping the PMMA resist with an organic solvent (acetone, 40 °C, 30 min) in an ultrasonic bath (stage VI), the freshly created, bare niobia background was passivated with protein-resistant PLL-g-PEG to form a nonfouling background thanks to the formation of a dense PEG brush (stage VII). Finally, the substrate was dipped into a buffered solution (160 mM HEPES buffer solution (10 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid with 150 mM NaCl, adjusted to pH 7.4 with 6 M NaOH solution) with fluorescently labeled streptavidin (Fluoro Alexa-488 or Alexa-546 streptavidin, Molecular Probes, 20 µg/mL) and rinsed with 160 mM HEPES buffer and ultrapure water (stage VIII). The stepwise fabrication of protein nanopatterns (Figure 1a, b) was characterized at Nano Lett., Vol. 4, No. 10, 2004

Figure 1. (a) The NIL process: I. Spin coating of PMMA and prebaking; II. Imprinting the PMMA layer using the silicon stamp; III. Demolding of the stamp from the substrate; IV. Anisotropic O2 RIE for the “window opening” down to the niobium oxide. (b) the MAPL process: V. The prepatterned sample is dipped into an aqueous solution of PLL-g-PEG/PEG-biotin; VI. Lift-off of the residual PMMA in acetone; VII. The background is filled with nonfunctionalized PLL-g-PEG that resists adsorption of proteins; VIII. Streptavidin specifically binds to the biotinylated areas.

each process step in order to ensure highly defined surface chemistry and pattern geometry. The initial step of the NIL process, after imprinting, is the “window opening” of the pattern trenches down to the Nb2O5 substrate via O2 RIE (Figure 1a, stage IV). For smaller feature sizes and larger patterned areas, accurate control over the anisotropic etching process becomes increasingly important in order to achieve homogeneous etch depth and window opening across the whole patterned areas. Figure 2 shows atomic/lateral force microscopy (AFM/LFM) images in contact mode after the window opening. The AFM image shows that the oxide stripes had a width comparable to the initially imprinted stripes (prior to etching) indicating that the etching was truly anisotropic and did not significantly modify the lateral dimension of the imprinted features. LFM was used (Figure 2b) to visualize the chemical contrast between the oxide and the PMMA areas. A relative increase in the friction contrast was revealed after the oxide window opening. The higher friction contrast (185 mV) suggests that the desired chemical contrast of PMMA and Nb2O5 was indeed achieved, confirming the window opening. In comNano Lett., Vol. 4, No. 10, 2004

parison, the friction contrast at stage III of Figure 1a was only 27 mV (image not shown); the fact that this value was not zero is likely a consequence of the stripe topography. In the subsequent MAPL process (Figure 1b) the PMMA/ Nb2O5 patterned surface was dipped into an aqueous solution of PLL-g-PEG/PEG-biotin, resulting in the adsorption of the polymer on both the Nb2O5 and PMMA areas (Figure 1b, stage V). In step VI, the PMMA resist was selectively removed by dipping the sample into a sonicated bath of hot acetone. This is the critical step in the MAPL process as the organic solvent must not desorb or otherwise deteriorate the PLL-g-PEG/PEG-biotin adlayer attached to the Nb2O5 substrate. AFM was used again to study the quality of the pattern after the PMMA lift-off (Figure 3a). The stripes of immobilized PLL-g-PEG/PEG-biotin in the Nb2O5 background are well visible, and their width 100(10 nm and period of 408(8 nm correspond with the dimensions of the nanostructured stamp used in the nanoimprinting step. The average step height between oxide and PLL-g-PEG/PEGbiotin (1.5(0.7 nm) is in the range of the thickness of a PLL-g-PEG monolayer as determined by ellipsometry.32 The 1911

Figure 2. (a) AFM image in contact mode of the PMMA/oxide topography after O2 RIE for the window opening (stage IV in Figure 1a). The imprinted stamp had stripes of 100 nm in width and a period of 400 nm (300 nm spacings). (b) LFM image after the etching step. The friction contrast has increased from 27 mV (before etching) to 185 mV due to the exposure of oxide stripes by the window opening.

oxide background appears smooth in the AFM image of Figure 3a, with no remaining visible residues of PMMA. The mean roughness value Ra in the Nb2O5 areas was determined to be 0.14(0.01 nm, identical within experimental error to the value found for the O2 plasma cleaned Nb2O5 coated wafers before spinning the PMMA layer (Ra ) 0.11(0.05 nm). This suggests that the oxygen plasma removed efficiently the PMMA resist layer. To support this observation X-ray photoelectron spectroscopy (XPS) measurements were performed on homogeneous PMMA coated Nb2O5 surfaces before and after lift-off. The lift-off protocol was optimized to achieve the smallest C/Nb atomic ratio, i.e., lowest degree of remaining contamination. When the lift-off was performed in acetone at 40 °C for 30 min in an ultrasonic bath, the C/Nb ratio was found to be 0.05(0.01, close to the value of a Nb2O5 surface cleaned by O2 plasma (C/Nb ) 0.03(0.01). Potential desorption of the functionalized copolymer during lift-off was also evaluated by XPS based on the peak areas of C(1s, PEG) and Nb(3d, Nb2O5) on homogeneous model surfaces. The reduction in surface coverage was found to be 5% (data not shown). During the backfill with PLL-g-PEG, the defect sites were healed again, 1912

Figure 3. AFM scans (1.5 × 1.5 µm2) in tapping mode in air of 100 nm patterned stripes. (a) PLL-g-PEG/PEG-biotin stripes in an oxide background (after lift-off, stage VI in Figure 1b). (b) After PLL-g-PEG backfill the pattern is still visible due to the longer PEG chains supporting the biotin molecules (stage VII in Figure 1b).

the difference being that the bioligand density (e.g., biotin) is now smaller by 5%, a value comparable to the uncertainty in tailoring the ligand density by the mixed self-assembly process.29 To inhibit nonspecific protein adsorption in the background, the Nb2O5 areas were rendered nonfouling by spontaneous adsorption of the nonfunctionalized PLL-g-PEG from an aqueous solution (“backfilling” step, stage VII in Figure 1b). The residual step height after backfill (Figure 3b) can be attributed to the longer PEG chains (mol wt 3.4 kDa) used for the biotinylated poly(ethylene glycol) in comparison to the nonfunctionalized PEG (mol wt 2 kDa). Different PEG chain lengths are generally preferred in surface assembled molecular systems in order to improve the spatial availability and presentation of the bioligand (here biotin) at the interface and reduce the probability of the ligand being embedded in the PEG structure. Also, step height measurements of soft materials are very sensitive to the parameters used and therefore difficult to perform in a reproducible manner. In the final step (stage VIII), alexa-488 (or alexa546) conjugated streptavidin was adsorbed onto the biotinfunctionalized patterned surfaces. Scanning near-field optical microscopy (SNOM) was used to image (Figure 4) the fluorescently labeled alexa-546 streptavidin adsorbed onto the 100 nm PLL-g-PEG/PEGbiotin stripes. The regular line pattern of 400 nm periodicity Nano Lett., Vol. 4, No. 10, 2004

Figure 4. Scanning near field optical microscope (SNOM) analysis of 100 nm biotinylated-PLL-g-PEG stripes in a PLL-g-PEG background. The blurred fluorescent signal (alexa-546 conjugated streptavidin) from the nanolines is caused by the resolution limit of the technique. The 400 nm periodicity of the grating is respected.

Figure 5. Confocal laser scanning microscope (CLSM) image of 20 and 50 µm stripes of biotinylated-PLL-g-PEG in a PLL-g-PEG background. The fluorescent-labeled streptavidin binds specifically to the biotin areas.

can be clearly recognized, verifying that streptavidin adsorbed selectively on the PLL-g-PEG/PEG-biotin pattern. The fluorescent-labeled lines appear broader than 100 nm because the optical resolution of the SNOM is limited to 80-100 nm. Figure 5 is a laser scanning confocal microscope (CLSM) image of microlines (20 and 50 µm) produced in the exact same way (by NIL and MAPL) as the samples with the 100 nm lines, but using a microstructured stamp with lines of width ranging from 3 to 50 µm. The use of micrometersized lines allowed us to judge the pattern quality with the CLSM. The streptavidin lines displayed an excellent contrast between the biotinylated areas and the PLL-g-PEG background. No fluorescent signal could be detected on the PEG background. Figure 6 shows an AFM image of an array of dots of PLLg-PEG/PEG-biotin in a Nb2O5 background (stage VI in Figure 1b). The sample was again produced by NIL combined with MAPL, but this time a stamp containing pillars of 110 nm in diameter with a spacing of 800 nm was used. The depth of the stamp features was 100 nm. Although the AFM image presents dot features of approximately the Nano Lett., Vol. 4, No. 10, 2004

Figure 6. AFM scan (4 × 4 µm2) of PLL-g-PEG/PEG-biotin nanodots of approximately 100 nm in diameter and about 800 nm separation. The background is bare Nb2O5 (after PMMA lift-off, stage VI).

expected size, some islands are missing, which is likely a consequence of a fraction of the stamp pillars having broken away during imprinting. Reproducible, homogeneous window opening during the RIE step is a critical step that is facilitated by a high aspect ratio of the imprinted photoresist structure; therefore, it is advantageous to use NIL stamps with high aspect ratio features. The drawback, however, is a reduced mechanical stability of the stamp and increased risk of stamp damage during the imprinting step. Another approach to overcome this is to use a thinner initial resist layer resulting in a thinner residual layer in the embossed area. In this case, however, the flow of resist will be dramatically restricted during imprinting, requiring a much longer imprint time. Furthermore, wafer bending might occur due to inhomogeneous resist flow at the edges of the structures, potentially resulting in mechanical damages of the stamp structures. Optimization of NIL relies therefore on a critical balance between stamp design, the initial resist thickness, and the process parameters during imprinting. Nevertheless, Figure 6 demonstrates that it is basically feasible to create arrays of nanometric biofunctionalized islands by combining the NIL and MAPL techniques. Since MAPL has been successful in creating micron-sized patterns with controlled (bio)ligand density (through co-assembly of mixed adlayers of PLL-g-PEG/PEG-biotin and PLL-g-PEG), it seems realistic to produce nanopatterns with few to single biological moieties per patch. In summary, we have demonstrated a novel versatile process for the patterning of surfaces with controlled density of biomolecules in the micrometer down to the 100-nm range. The combination of a “top-down” approach via NIL and a “bottom-up” approach through MAPL is presented as an economical alternative to other nanopatterning techniques such as dip-pen nanolithography12 or colloidal lithography.16,19 We presented the successful, reproducible immobilization of streptavidin molecules on 100 nm-wide lines with immobilized biotinylated polymer, while the reproducible production of dots and other small features needs further optimization. The streptavidin patterns can be used as a platform for subsequent immobilization of biotin-tagged 1913

proteins or vesicles at controlled surface densities through tailored biotin surface concentrations in the adhesive nanosized patches. The functionalization of the PLL-g-PEG is not limited to biotin. Other molecules such as RGD peptides have been used as bioactive groups covalently linked to PLLg-PEG and shown to specifically bind to integrin cell receptors while preserving its protein repelling properties. Functionalizing the polymer with nitrilotriacetic acid (NTA) is another linkage strategy compatible with MAPL that allows the binding of Ni2+ ions and 6× histidine-tagged proteins in a reversible manner and with controlled protein orientation and density.33 The advantage of this particular approach is that the immobilization of complex and delicate biomolecules such as proteins is carried out as the last step of the surface functionalization procedure without exposing them to the organic solvent or other harsh conditions. By decreasing the island size further we believe in the capability to produce single arrays of single peptide and also single complex biomolecules. The NIL/MAPL nanopatterning technique is therefore expected to be useful for investigating integrin clustering/focal adhesions formation as a function of patch size, peptide ligand density and ligand separation. Finally, by decreasing the island size further, production of arrays consisting of single molecules would likely become feasible. The lower feature size achievable with this system is probably limited by the size of the PLLg-PEG molecule used in this work, which has a footprint of approximately 70 nm2. Acknowledgment. The authors gratefully acknowledge Konrad Vogelsang for support with the nanoimprinting and Michael Horisberger for the Nb2O5 coatings (PSI, Switzerland). We thank Dr. Fabiano Assi for AFM support, Ste´phanie Pasche for providing the polymers, and Laurent Feuz for XPS support (ETH Zurich). This work was financially supported by the Swiss funding program on nanotechnology, Top Nano21 (Projects 4597.1 and 6209.1), EPF Lausanne and ETH Zurich. References (1) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Biotechnol. Prog. 1998, 14, 356-363. (2) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702-1705. (3) Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Blummel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. ChemPhysChem. 2004, 5, 383-388. (4) Seo, Y. S.; Luo, H.; Samuilov, V. A.; Rafailovich, M. H.; Sokolov, J.; Gersappe, D.; Chu, B. Nano Lett. 2004, 4, 12734-12735.

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Nano Lett., Vol. 4, No. 10, 2004