Preparation of Protein Gradients through the Controlled Deposition of Protein−Nanoparticle Conjugates onto Functionalized Surfaces

Krämer, Stephan; Xie, Haibo; Gaff, John; Williamson, John; Ткаченко, А. Г.; Nouri, Navid; Feldheim, David A.; Feldheim, Daniel L.

doi:10.1021/ja031674n

Cited by 56 publications

(50 citation statements)

References 49 publications

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“…Other techniques have been used to gradually immobilize biomolecules, such as proteins, on surfaces, for example, microfluidics, 55-57 covalent coupling to an alkanethiol gradient, 58,59 covalent coupling by laser irradiation, 60 the use of stamping techniques and electrophoretics, 61 ink-jet printing, 62,63 drainage, 64 or by controlling the adsorption kinetics. 65 In this study we present the generation and characterization of PLL-g-PEG gradients prepared by means of an immersion process originally developed for alkanethiols ͓Fig. 1͑b͔͒.…”

Section: Introductionmentioning

confidence: 99%

Poly(l-lysine)-grafted-poly(ethylene glycol)-based surface-chemical gradients. Preparation, characterization, and first applications

et al. 2006

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A simple dipping process has been used to prepare PEGylated surface gradients from the polycationic polymer poly(l-lysine), grafted with poly(ethylene glycol) (PLL-g-PEG), on metal oxide substrates, such as TiO2 and Nb2O5. PLL-g-PEG coverage gradients were prepared during an initial, controlled immersion and characterized with variable angle spectroscopic ellipsometry and x-ray photoelectron spectroscopy. Gradients with a linear change in thickness and coverage were generated by the use of an immersion program based on an exponential function. These single-component gradients were used to study the adsorption of proteins of different sizes and shapes, namely, albumin, immunoglobulin G, and fibrinogen. The authors have shown that the density and size of defects in the PLL-g-PEG adlayer determine the amount of protein that is adsorbed at a certain adlayer thickness. In a second step, single-component gradients of functionalized PLL-g-PEG were backfilled with nonfunctionalized PLL-g-PEG to generate two-component gradients containing functional groups, such as biotin, in a protein-resistant background. Such gradients were combined with a patterning technique to generate individually addressable spots on a gradient surface. The surfaces generated in this way show promise as a useful and versatile biochemical screening tool and could readily be incorporated into a method for studying the behavior of cells on functionalized surfaces.

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Section: Introductionmentioning

confidence: 99%

Poly(l-lysine)-grafted-poly(ethylene glycol)-based surface-chemical gradients. Preparation, characterization, and first applications

et al. 2006

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“…Protein gradients have also been incapsulated in a microporous gel by creating a linear gradient in a mold before gelation (Kapur and Shoichet, 2004). There are many approaches for producing gradients on surfaces (Kramer et al, 2004;Ruardy et al, 1997) and recently an elegant method for generating complex gradient shapes in solution and on surfaces using micro fluidics has been demonstrated (Dertinger et al, 2001), but these technologies are not suitable for investigating many biological processes that occur in three-dimensional environments.…”

Section: Introductionmentioning

confidence: 99%

Generating controlled molecular gradients in 3D gels

Rosoff

McAllister

Esrick

et al. 2005

Biotech & Bioengineering

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A new method for producing molecular gradients of arbitrary shape in thin three dimensional gels is described. Patterns are produced on the surface of the gel by printing with a micropump that dispenses small droplets of solution at controlled rates. The molecules in the solution rapidly diffuse into the gel and create a smooth concentration profile that is independent of depth. The pattern is relatively stable for long times, and its evolution can be accurately described by finite element modeling of the diffusion equation. As a demonstration of the method, direct measurements of protein gradients are performed by quantitative fluorescence microscopy. A complementary technique for measuring diffusion coefficients is also presented. This rapid, flexible, contactless approach to gradient generation is ideally suited for cell culture experiments to investigate the role of gradients of diffusible substances in processes such as chemotaxis, morphogenesis, and pattern formation, as well as for high-throughput screening of system responses to a wide range of chemical concentrations.

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“…+ Both authors contributed equally to this work. 19 On the molecular length scale, these methods enable quantification of an average molecular concentration, which gradually varies along the adhesive surface; however, they are too "noisy" to quantify the densities themselves, and density variations capable of inducing cell polarization. …”

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Induction of Cell Polarization and Migration by a Gradient of Nanoscale Variations in Adhesive Ligand Spacing

et al. 2008

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Cell interactions with adhesive surfaces play a vital role in the regulation of cell proliferation, viability and differentiation, and affect multiple biological processes. Since cell adhesion depends mainly on the nature and density of the adhesive ligand molecules, spatial molecular patterning, which enables the modulation of adhesion receptor clustering, might affect both the structural and signalling activities of the adhesive interaction. We herein show that cells plated on surfaces that present a molecularly defined spacing gradient of an integrin RGD ligand, can sense small but consistent differences in adhesive ligand spacing of about 1 nm across the cell diameter, which is approximately 61 m when the spacing includes 70 nm. Consequently, these positional cues induce cell polarization, and initiate cell migration and signalling. We propose that differential positional clustering of the integrin transmembrane receptors is used by cells for exploring and interpreting their environment, at high spatial sensitivity.Adhesion of tissue cells to the extracellular matrix depends on the activation of specific transmembrane receptors; e.g. integrins, which leads to the assembly of specialized adhesion sites known as focal adhesions (FA). 1 Activation and spatial organization of integrins are mainly controlled by epitopes containing a RGD (R = arginine, G = glycine, D = aspartate) sequence, that are present on a variety of adhesive extracellular matrix (ECM) proteins. Recent studies indicated that beyond the chemical specificity of the adhesive epitope, 2 many physical features of the adhesive surface, including its topography, 3 rigidity, 4 and precise epitope spacing, 5 are critical for guiding receptor-mediated adhesion formation and signalling. Specifically, it was demonstrated that cyclic RGDfK peptides linked to nanogold particles 6 which were arranged in pattern on substrates and interspaced by 58 or 73 nm, influence cell adhesion, 5 spreading, focal adhesion assembly, and migration 7,8 in very different ways. In these studies, control experiments demonstrated that only the specific functionalization of nanogold particles with cyclic RGD induced integrin clustering and such focal adhesion formation upon interaction with cells such as 3T3-fibroblasts or -* Corresponding author: spatz@mf.mpg.de. + Both authors contributed equally to this work. 19 On the molecular length scale, these methods enable quantification of an average molecular concentration, which gradually varies along the adhesive surface; however, they are too "noisy" to quantify the densities themselves, and density variations capable of inducing cell polarization. NIH Public AccessWe herein demonstrate a novel surface patterning technique for the generation of nanoparticle spacing gradients, which is based on controlled modulation of the selfassembly of diblock copolymer micelles. Due to the self-assembly of macromolecules, large-scale surface areas are easily processed by this technique. After successful biofunctionalization of the nano...

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Preparation of Protein Gradients through the Controlled Deposition of Protein−Nanoparticle Conjugates onto Functionalized Surfaces

Cited by 56 publications

References 49 publications

Poly(l-lysine)-grafted-poly(ethylene glycol)-based surface-chemical gradients. Preparation, characterization, and first applications

Poly(l-lysine)-grafted-poly(ethylene glycol)-based surface-chemical gradients. Preparation, characterization, and first applications

Generating controlled molecular gradients in 3D gels

Induction of Cell Polarization and Migration by a Gradient of Nanoscale Variations in Adhesive Ligand Spacing

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