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Assembly of Oriented 2-D Protein and Virus Ordered Arrays by Chemo-Selective Ligation Methods and Dip-pen Nanolithography

Julio A. Camarero, Chin Li Cheung, Tianwei Lin, John E. Johnson, Brandon L. Weeks, Alex Noy, and Jim J. de YoreoDe Yoreo
Biosecurity Support Laboratory, Lawrence Livermore National Laboratory, US

Keywords: Dip-pen nanolithography, chemoselctive ligation, protein chip

Many experimental techniques in biology and biophysics, and applications in diagnosis and drug discovery, require proteins immobilized on solid substrates. In fact, the concept of arrays of proteins attached to a solid support has attracted increasing attention over the last three years due to the sequencing of several genomes, including the human genome. Protein arrays can be used easily for the parallel analysis of whole proteomes. Another powerful application employs ordered nanometric arrays of proteins as nucleation templates for protein crystallization. Recent advances in nanoprinting techniques have allowed the creation of sub-micrometer arrays of proteins [1,2]. All these applications demonstrate the use of protein arrays and also highlight the need for methods able to attach proteins in a well defined and ordered way onto a solid supports. Various methods are available for attaching proteins to solid surfaces. Most rely on non-specific adsorption, or on the random cross-linking of proteins to chemically reactive surfaces. In both cases the protein is attached to the surface in random orientations. The use of recombinant affinity tags addresses the orientation issue. However, in most cases the interactions of the tags are reversible and therefore not stable over the course of subsequent assays or require large mediator proteins. Covalent attachment and orientation of a protein to a solid support requires two unique and mutually reactive groups on the protein and the support surface. The reaction between these two groups should be highly chemoselective, thus behaving like a molecular ‘velcro’. Here, we will present our ongoing effort towards the creation of nano-scaled ordered arrays of protein and virus covalently bonded to site-specific chemical linkers patterned by dip-pen nanolithography (DPN) . We will first discuss a new and efficient solid-phase approach for the synthesis of chemically modified long alkyl-thiols . These compounds can been used to introduce chemoselective reacting groups onto gold and silicon-based surfaces. Furthermore, these modified thiols can be used to create nanometric patterns by using DPN. These patterns can react chemoselectively with proteins and/or virus which have been chemically or recombinantly modified to contain complementary chemical groups at specific positions thus resulting in the ordered attachment of the protein or virus to the surface. We will also show preliminary results showing the chemoselectivity of these linkers with genetically modified proteins and virus on chemically patterned templates fabricated by micro-contact printing and DPN. Future directions and applications of these patterns for protein and virus crystallization will also be discussed.

NSTI Nanotech 2003 Conference Technical Program Abstract

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