Biochips for Cell Biology by Combined Dip‐Pen Nanolithography and DNA‐Directed Protein Immobilization

Abstract

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.

Spatiotemporally Controlled Reorganization of Signaling Complexes in the Plasma Membrane of Living Cells

“…[8][9][10] These techniques have been successfully applied for unraveling the role of spatial regulation in signaling processes, [11][12][13][14][15][16] but also for monitoring the assembly of signaling complexes as well as interactions of downstream effector proteins in a systematic and quantitative manner. [17][18][19][20][21][22] However, a fundamental drawback of currently available approaches is that protein capturing already occurs during adhering and culturing cells on surface-functionalized substrates, thus potentially altering cellular homoeostasis or even activating cellular signaling. [ 21 ] Here, we present a simple and robust methodology to temporally control spatial organization of signaling complexes in the plasma membrane of livings cells.…”

Nanotechnologies in Protein Microarrays

“…Therefore, substrates immobilization by micro-or nano-printing [68], or using of nanovessels or nanoliposomes [57,69] is favorable. The application of lipid nanostructure hybrids for nanobiotechnology was shown by Lee and coworkers [70].…”

“…Lectin-coated QDs (Poly)saccharide Protein glycosylation FRET between QDs-AuNPs [53] GNP (Poly)saccharide Lectin Fluorescence of FSNPs [54] NiNTA-DsRed FT Proteins Immobilization concept Fluorescence [55] Cys-DNA + recombinant fusion proteins Proteins Immobilization concept Fluorescence [56] DDI oligonucleotide-tagged proteins Proteins EGF Location of subcellular area of cells [57] Lipidic membrane + ganglioside receptors Proteins Cholera toxin b SPRi [72] SWNT + RA-specific (CCC) peptide Peptides Rheumatoid factor QCM [73] A-enolase peptides Peptides Sera-specific PDAC Abs QCM_D [74] Aptamer-AgNPs Nucleic acids/aptamers PDGF-BB SPR/fluorescence of modified AgNPs [58] ssDNA aptamer monolayer Nucleic acids/aptamers Thrombin Nanoparticles-enhanced SPRi [59] DNA bar code Nucleic acids/aptamers PSA NPs absorbance [75] DNA template Nucleic acids/aptamers BRIP1, Jun, ATF2 QCM_F + QCM_D [76] DNA template on anodic porous alumina Nucleic acids/aptamers CdK2, CDKN1A, p53, Jun AFM [77] Fluorescent SWNT + His-tagged proteins…”

“…Employing dip-pen nanolithography of DNA oligonucleotides, which are then used as capture strand for DNA-directed immobilization of oligonucleotidetagged proteins with spot-size below those of single cell (units of μm), was described. The methodology was tested to form a live cell array, where the biotinylated EGF was coupled to DNA-streptavidin conjugates and used for capturing and culturing of human breast adenocarcinoma cells [57].…”

Adsorption mechanisms of l-Glutathione on Au and controlled nano-patterning through Dip Pen Nanolithography

“…Through all these, dip-pen nanolithography (DPN) is a direct-write printing technique of lithography that involves the coating of an atomic force microscope (AFM) cantilever tip with the desired chemical ink and patterned on a variety of substrates for creating and functionalizing nanoscale devices [6,7]. This technique has evolved to embrace a wide range of tip-based nanofabrication applications, including new ink combinations from polymers [8], small molecules [9,10], biomolecules [11], dendrimers [12], solid state materials [13], DNA [14], proteins [15,16], colloidal particles [17], sols [18], fluorescent dyes [19] and new substrates ranging from metals [20][21][22] to semiconductors [23] and insulators [20] or organic thin films [24]. The various chemical approaches and the systems in which they have been applied are strongly dependent on the deposition processes, including the chemical properties of the ink molecules and the chemical nature of the tip/substrate [25][26][27][28][29][30][31].…”