Fabrication of Assembled Virus Nanostructures on Templates of Chemoselective Linkers Formed by Scanning Probe Nanolithography Chin Li Cheung, † Julio A. Camarero,* ,† Bruce W. Woods, † Tianwei Lin, ‡ John E. Johnson, ‡ and Jim J. De Yoreo † Chemistry and Materials Science Directorate, Lawrence LiVermore National Laboratory, 7000 East AVenue, LiVermore, California, 94551, and Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037 Received February 3, 2003; E-mail: camarero1@llnl.gov In recent years, methods for fabricating nanometric biomolecular arrays have attracted intense interest due to their great potential in numerous applications including protein chips for proteomic analysis, DNA chips for genomic analysis, and 2D or 3D crystalline arrays for determination of protein structure. 1 Because supramo- lecular assemblies of macromolecules, such as viruses, are often on the order of tens of nanometers in size and can be produced with atomic precision, they are ideal monodisperse, nanometric building blocks for the study of molecularly directed assembly of biomolecular arrays. Here we present a multistep approach to biomolecular assembly, which combines scanning probe nanolithography (SPN) with chemoselective protein-to-surface linkers to create nanometric chemical templates for fabricating virus arrays. The strategy for the assembly of these arrays is to introduce unique target chemical groups on the virus surface and to design a chemoselective linker and reaction scheme to covalently bind the virus onto a patterned chemical template created by SPN. Our model system consists of gold substrates, functionalized alkanethiols as the linkers, and genetically modified cow pea mosaic virus (Cys-CPMV) 2 as the adsorbate. The virus was genetically engineered to present Cys residues at geometrically equivalent positions on the viral capsomer as shown in the inset to Figure 2a. 2 Long aliphatic thiols are well-known to form high-density self- assembled monolayers 3 and are well suited to fabrication of nano- metric patterns on gold surfaces by SPN. Moreover, the reaction between sulfhydryl groups on the virus and maleimides is highly chemoselective. 4 Thus, we have devised a sulfhydryl-maleimide chemoselective reaction scheme with functional thiols for linking the mutated virus onto chemical templates where triethylene glycol- terminated thiols are applied as “protein resist” to the surrounding regions for prevention of nonspecific adsorption 5 (Scheme 1). The synthesis of chemically modified long alkanethiol linkers was accomplished by a new and efficient solid-phase approach developed by our group 4 (Scheme 2). Briefly, 11-mercaptounde- canoic acid was first immobilized on a trityl chloride resin through the selective formation of thioether bond to the solid-support. The free carboxylic function was acylated with mono-Fmoc-1,4- diaminobutane and converted into a more versatile amino group. Afterward, this group was acylated with various activated acids to yield the two different thiols used in this work. In both cases, the final products were fully deprotected and cleaved from the solid support by acidolytic cleavage. The whole synthetic process was extremely efficient in both cases with overall yields of ca. 90% and thus required minimal purification. Nanometric chemical templates of the amine terminated al- kanethiol 4 6 with dimensions close to the diameter of the mutated virus (ca. 30 nm) were created by two SPN techniques: dip-pen nanolithography (DPN) 7 and nanografting. 8 To generate chemical templates by DPN an atomic force microscope (AFM) probe coated with thiol 4 was used to deposit the “ink” onto the gold “paper” by operating an AFM in contact mode. The suitability of this ink for DPN at the 10-100-nm-length scale is demonstrated by Figure 1a. A variation of conventional nanografting 8 was also used to create large area line patterns. An AFM probe was used to displace the “protein resist” thiol 5 precoated on flat gold substrates under high load and high scan speed to create linear “trenches”. The samples were subsequently functionalized with thiol linker 4, thus yielding a functionalized pattern of parallel lines. Figure 1b shows that the resulting sample has patterns of ca. 30-nm-width lines filled with an organic film layer of thickness of 0.5-2.0 nm above the background. Using the highly selective thiol-maleimide reaction, Cys-CPMV virus was chemoselectively attached on chemical templates con- taining the maleimido functionality according to Scheme 1. First, the Cys-CPMV sample deposited on poly-Lys-coated mica sur- face was characterized by AFM. The positions of the virions as well as their relative orientation to the surface can be readily resolved by AFM (Figure 2a). To demonstrate a proof of prin- ciple for the virus assembly scheme, we fabricated a micrometer- † Lawrence Livermore National Laboratory. ‡ The Scripps Research Institute. Scheme 1 Scheme 2 Published on Web 05/15/2003 6848 9 J. AM. CHEM. SOC. 2003, 125, 6848-6849 10.1021/ja034479h CCC: $25.00 © 2003 American Chemical Society