RESUMO
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.
Assuntos
DNA/química , Fator de Crescimento Epidérmico/química , Procedimentos Analíticos em Microchip , Oligonucleotídeos/química , Proteínas/química , Biotinilação , Biologia Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Vidro/química , Proteínas de Fluorescência Verde/química , Humanos , Ligantes , Células MCF-7 , Teste de Materiais , Nanotecnologia , Análise de Sequência com Séries de Oligonucleotídeos , Propriedades de SuperfícieAssuntos
Proteínas/metabolismo , Animais , Anticorpos Imobilizados/imunologia , Células COS , Chlorocebus aethiops , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Transferência Ressonante de Energia de Fluorescência , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Análise Serial de Proteínas , Mapas de Interação de Proteínas , Proteínas/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
A modular system for the DNA-directed immobilization of antibodies was applied to capture living cells on microstructured DNA surfaces. It is demonstrated in two different set-ups, static incubation and hydrodynamic flow, that this approach is well suited for specific capture and selection of cells from culture medium. The adhered cells show intact morphology and they can be cultivated to grow to dense monolayers, restricted to the lateral dimensions of DNA spots on the surface. Owing to the modularity of surface biofunctionalization, the system can readily be configured to serve as a matrix for adhesion and growth of different cells, as demonstrated by specific binding of human embryonic kidney cells (HEK293) and Hodgkin lymphoma L540cy cells onto patches bearing appropriate recognition moieties inside a microfluidic channel. We therefore anticipate that the systems described here should be useful for fundamental research in cell biology or applications in biomedical diagnostics, drug screening, and nanobiotechnology.
