Universal Chemical Gradient Platforms Using Poly(methyl methacrylate) Based on the Biotin−Streptavidin Interaction for Biological Applications

Lagunas, Anna; Comelles, Jordi; Martínez, Elena; Samitier, J.

doi:10.1021/la102640w

Cited by 38 publications

(38 citation statements)

References 47 publications

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“…13 Most of these reports demonstrate surface gradient immobilization of biotin for use as avidin or SAV binding platforms. 9,10,12,13 Plasma polymerization has also been employed for gradient fabrication. 14 It has shown utility in the preparation of gradient protein surfaces through its use as a generic coupling platform to make gradients of single proteins, 15,16 two proteins, 17 other bioactive agents such as heparin sulfate, 18 and nanoparticles.…”

Section: ■ Introductionmentioning

confidence: 99%

Immobilized Streptavidin Gradients as Bioconjugation Platforms

et al. 2012

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Surface density gradients of streptavidin (SAV) were created on solid surfaces and demonstrated functionality as a bioconjugation platform. The surface density of immobilized streptavidin steadily increased in one dimension from 0 to 235 ng cm(-2) over a distance of 10 mm. The density of coupled protein was controlled by its immobilization onto a polymer surface bearing a gradient of aldehyde group density, onto which SAV was covalently linked using spontaneous imine bond formation between surface aldehyde functional groups and primary amine groups on the protein. As a control, human serum albumin was immobilized in the same manner. The gradient density of aldehyde groups was created using a method of simultaneous plasma copolymerization of ethanol and propionaldehyde. Control over the surface density of aldehyde groups was achieved by manipulating the flow rates of these vapors while moving a mask across substrates during plasma discharge. Immobilized SAV was able to bind biotinylated probes, indicating that the protein retained its functionality after being immobilized. This plasma polymerization technique conveniently allows virtually any substrate to be equipped with tunable protein gradients and provides a widely applicable method for bioconjugation to study effects arising from controllable surface densities of proteins.

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Section: ■ Introductionmentioning

confidence: 99%

Immobilized Streptavidin Gradients as Bioconjugation Platforms

et al. 2012

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“…1 In order to experimentally parse out the detailed contributions of immobilized ligand density on cell behavior, substrates that present biomolecular gradients on surfaces are of fundamental interest for in vitro assays enabling the investigation of cell migration and polarization in response to immobilized protein concentration. 2–3 Of even greater interest are in vitro platforms that enable multi-parameter studies that systematically address the combined effects of multiple stimuli, such as the density of multiple biomolecules, topography, or shear stress, amongst others, on cell behavior. 4 …”

Section: Introductionmentioning

confidence: 99%

Probing dynamic cell–substrate interactions using photochemically generated surface-immobilized gradients: application to selectin-mediated leukocyte rolling

Herman

Potts

Michael

et al. 2011

Integrative Biology

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Model substrates presenting biochemical cues immobilized in a controlled and well-defined manner are of great interest for their applications in biointerface studies that elucidate the molecular basis of cell receptor-ligand interactions. Herein, we describe a direct, photochemical method to generate one-component surface-immobilized biomolecular gradients that are applied to the study of selectin-mediated leukocyte rolling. The technique employs benzophenone-modified glass substrates, which upon controlled exposure to UV light (350 – 365 nm) in the presence of protein-containing solutions facilitate the generation of covalently immobilized protein gradients. Conditions were optimized to generate gradient substrates presenting P-selectin and PSGL-1 (P-selectin Glycoprotein Ligand-1) immobilized at site densities over a 5- to 10-fold range (from as low as ~200 molecules/μm2 to as high as 6000 molecules/μm2). The resulting substrates were quantitatively characterized via fluorescence analysis and radioimmunoassays before their use in the leukocyte rolling assays. HL-60 promyelocytes and Jurkat T lymphocytes were assessed for their ability to tether to and roll on substrates presenting immobilized P-selectin and PSGL-1 under conditions of physiologically relevant shear stress. The results of these flow assays reveal the combined effect of immobilized protein site density and applied wall shear stress on cell rolling behavior. Two-component substrates presenting P-selectin and ICAM-1 (intercellular adhesion molecule-1) were also generated to assess the interplay between these two proteins and their effect on cell rolling and adhesion. These proof-of-principle studies verify that the described gradient generation approach yields well-defined gradient substrates that present immobilized proteins over a large range of site densities that are applicable for investigation of cell-materials interactions, including multi-parameter leukocyte flow studies. Future applications of this enabling methodology may lead to new insights into the biophysical phenomena and molecular mechanism underlying complex biological processes such as leukocyte recruitment and the inflammatory response.

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“…The biotin-streptavidin system has been extensively studied as a model biointeraction [50][51][52][53][54][55]. Streptavidin has an extraordinarily high affinity for biotin (the dissociation constant (K d ) of the biotinstreptavidin complex is on the order of $10 À14 mol/L) [50].…”

Section: Derivation Reaction Of Dopamine Monolayermentioning

confidence: 99%