Department of Biomedical Engineering, Duke UniVersity, Box 90281, Durham, North Carolina 27708-0281, Department of Mechanical and Materials Science, Duke UniVersity, Box 90300, Durham, North Carolina 27708-0300, and Center for Biologically Inspired Materials and Materials Systems, Duke UniVersity Introduction. The spatially controlled immobilization of biomolecules on solid surfaces on the nanometer length scale is driven by the possibility of fabricating protein nanoarrays with well-defined feature size, shape, and spacing. Such structures are important for the fundamental study of the interactions between cells and surfaces 1,2 and have potential applications in the design of cell-based sensors and bioma- terials. 3,4 This paper describes how dip-pen nanolithography (DPN) 5-7 in combination with the high-affinity streptavidin- biotin protein-ligand system 8,9 provides a simple and versatile “bottom-up” approach to create nanoscale biomo- lecular structures readily in a stepwise fashion. Our method involves the fabrication of chemically reactive nanoscale features by patterning a self-assembled monolayer (SAM) of a COOH-terminated alkanethiol on a gold substrate by DPN, followed by covalent immobilization of a high-affinity small-molecule ligand (biotin) on the nanopatterned SAM and subsequent molecular recognition of its protein-binding partner (streptavidin) from solution. The resulting streptavidin nanopattern provides a universal platform for molecular recognition-mediated protein immobilization because of the ubiquity of biotin-tagged molecules. Typical periodic arrays of biotin-BSA fabricated by our method are shown in Figure 1. Experimental Section. Gold substrates with an average Au grain diameter of 30 nm were prepared by thermal evaporation of a chromium adhesion layer (100 Å), followed by gold deposition (1000 Å) onto a glass cover slide at 4 × 10 -7 Torr. Before deposition, the glass surface was cleaned in a 5:1:1 (v/v/v) mixture of H 2 O, H 2 O 2 , and NH 3 at 80 °C for 20 min. MHA (16-mercaptohexadecanoic acid) was patterned on the gold surface with DPN using an atomic force microscope (AFM) (MultiMode TM , Digital Instruments). For this purpose, an AFM cantilever (silicon nitride cantilever, 0.05 N/m, Digital Instruments) was incubated in a saturated solution of MHA in degassed acetonitrile for 1 min. The relative humidity during patterning ranged from 35 to 55%. Patterns were generated with writing speeds of up to 8 μm/s and resulted in feature widths of less than 100 nm that could be resolved with lateral force microscopy. MHA SAM arrays with periodic features (feature sizes ranging from 100 to 2000 nm) were routinely patterned by programming the XY motion of the AFM tube scanner through a customized nanolithog- raphy program (NanoScript TM , Digital Instruments). Patterned areas could be located accurately and repeatedly by pixel * Corresponding authors. E-mail: zauscher@duke.edu. chilkoti@duke.edu. † Department of Biomedical Engineering, Duke University. ‡ Department of Mechanical and Materials Science, Duke University. § Center for Biologically Inspired Materials and Materials Systems. NANO LETTERS 2002 Vol. 2, No. 11 1203-1207