A Novel Approach To Produce Biologically Relevant Chemical Patterns at the Nanometer Scale: Selective Molecular Assembly Patterning Combined with Colloidal Lithography Roger Michel, † Ilya Reviakine, †,‡ Duncan Sutherland, § Christian Fokas, | Gabor Csucs, ⊥ Gaudenz Danuser, ⊥ Nicholas D. Spencer, † and Marcus Textor* ,† Laboratory for Surface Science and Technology, Department of Materials, ETH Zurich, Schlieren, Switzerland, Department of Applied Physics, Chalmers University of Technology, Go ¨ teborg, Sweden, Laboratory for Organic Chemistry, Department of Chemistry, ETH Zurich, Schlieren, Switzerland, and BioMicroMetricsGroup, Department for Mechanical and Process Engineering, ETH Zurich, Schlieren, Switzerland Received April 10, 2002. In Final Form: August 6, 2002 A novel patterning technique that combines colloidal patterning with selective adsorption of organic molecules has been used to chemically pattern metal oxide surfaces at length scales down to 50 nm. Lithographic nanofabrication using surface-assembled colloids as etch masks (“colloidal lithography”) was used to create nanopillars of TiO2 (50-90 nm in diameter, ∼20 nm in height) on whole oxidized silicon or quartz wafer substrates. These nanopillars were then rendered hydrophobic by the selective self- assembly of an organophosphate, whereas a poly(ethylene glycol)-grafted copolymer was adsorbed onto the surrounding SiO 2 rendering it protein resistant. This resulted in a two-component chemical pattern, displaying contrast with respect to protein adsorption (protein-adhesive pillars on nonadsorbing background). This property allows for efficient translation of the lithographic pattern into a surface protein pattern by two simple dip-and-rinse processes in aqueous solutions. The feasibility of the method and its quality were tested by adsorbing fluorescently labeled streptavidin and biotinylated phospholipid vesicles. The sequential adsorption steps were monitored by fluorescence microscopy, atomic force microscopy, and scanning near- field optical microscopy. These techniques conclusively demonstrated the utility of the described approach for chemical patterning surfaces on the nanometer scale over large areas. 1. Introduction The biomaterials used today for the fabrication of biomedical devices such as implants are usually not engineered to induce specific biological responses. In particular, the biochemical processes taking place at the interface between the artificial material surface and the bioenvironment are rarely addressed in the surface design. As a consequence, a large variety of proteins and other extracellular matrix components can adsorb to the bio- material surface in different conformations and orienta- tions. It has been hypothesized that such undefined biofilms are uncommon to nature’s biological recognition and immune system, with the effect that the body reacts toward the synthetic material as toward a foreign body. 1 A frequent response of the body to biomaterials is encapsulation and isolation of the implanted device from the blood stream, causing a cascade of reactions that may adversely affect the healing process and the functionality of the device. It is well-known that morphological and topographical features of the biomaterial surface influence the interaction between implant and tissue, most likely through their effect on the proteinacious surface film. 2-6 Improved control over the functional organization of the adsorbed protein layers is therefore one possible approach to improve the ability of the implant to integrate in the host tissue without encapsulation. A key feature of such a strategy would be the elimination or reduction of nonspecific adsorption while at the same time providing chemically and structurally designed interactive sites for the attachment of desirable proteins (such as cell-adhesive proteins) in active conformations. Ultimately, this would imply complete control over the material surface properties on the scale of individual protein molecules. While a variety of standard techniques is available for engineering of surfaces on the micrometer scale, important advances in nanofabrication technology have only recently opened up new technical solutions to surface engineering on the sub-micrometer scale. Electron-beam lithography, for example, has been successfully used to manufacture nanometer structures with applications ranging from model catalysts 7 to optically active substrates for surface- enhanced Raman spectroscopy (SERS). 8 Such sequential * To whom correspondence may be addressed at: ETH Zurich, Oberfla ¨ chentechnik, Wagistrasse 2, CH-8952 Schlieren, Switzer- land. E-mail: textor@surface.mat.ethz.ch. Fax: ++41 1 633 10 48. † Laboratory for Surface Science and Technology, Department of Materials, ETH Zurich. ‡ Current address: Department of Chemical Engineering, Uni- versity of Houston, Houston, TX. § Department of Applied Physics, Chalmers University of Technology. | Laboratory for Organic Chemistry, Department of Chemistry, ETH Zurich. ⊥ BioMicroMetricsGroup, Department for Mechanical and Pro- cess Engineering, ETH Zurich. (1) Ratner, B. D. In Titanium in Medicine; Brunette, D., Tengvall, P., Textor, M., Tompson, P., Eds.; Springer: Berlin, 2001, pp 2-12. (2) Brunette, D. M. In Surface Characterization of Biomaterials; Ratner, B. D., Ed.; Elsevier Science Publishers B.V.: Amsterdam, 1988; pp 203-217. (3) von Recum, A. F.; van Kooten, T. G. J. Biomater. Sci., Polym. Ed. 1995, 7, 181-198. (4) Norde, W.; Lyklema, J. J. Biomater. Sci., Polym. Ed. 1991, 2, 183-202. (5) Horbett, T. A. In Proteins At Interfaces; American Chemical Society: Washington, 1995; Vol. 602, pp 1-23. (6) Malmsten, M. J. Colloid Interface Sci. 1998, 207, 186-199. 8580 Langmuir 2002, 18, 8580-8586 10.1021/la0258244 CCC: $22.00 © 2002 American Chemical Society Published on Web 10/05/2002