A general methodology for patterning of multiple
protein ligands with lateral dimensions below those of single cells is described. It employs dip pen nanolithography (
DPN) patterning of
DNA oligonucleotides which are then used as capture strands for
DNA-directed immobilization (DDI) of
oligonucleotide-tagged
proteins. This study reports the development and optimization of PEG-based liquid ink, used as carrier for the immobilization of alkylamino-labeled
DNA oligomers on chemically activated glass surfaces. The resulting
DNA arrays have typical spot sizes of 4-5 μm with a pitch of 12 μm micrometer. It is demonstrated that the arrays can be further functionalized with covalent
DNA-
streptavidin (
DNA-STV) conjugates bearing
ligands recognized by cells. To this end,
biotinylated epidermal growth factor (
EGF) is coupled to the
DNA-STV conjugates, the resulting constructs are hybridized with the
DNA arrays and the resulting surfaces used for the culturing of MCF-7 (human breast
adenocarcinoma) cells. Owing to the lateral diffusion of transmembrane
proteins in the cell's plasma membrane, specific recruitment and concentration of
EGF receptor can be induced specifically at the sites where the
ligands are bound on the solid substrate. This is a clear demonstration that this method is suitable for precise functional manipulations of subcellular areas within living cells.