Immobilization of Histidine-Tagged Recombinant Proteins onto Micropatterned Surfaces for Cell-Based Functional Assays

Kato, Koichi; Sato, Hideki; Iwata, Hiroo

doi:10.1021/la050893e

Cited by 62 publications

(58 citation statements)

References 28 publications

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“…The amine-reactive alkanethiol, 11-mercaptoundecanoyl-N-hydroxysuccinimide ester (MU-NHS), was tethered to the gold substrate in the first step. As alkanethiols self-assemble on metals like gold, forming a highly ordered monolayer, they facilitate the decoration of surfaces with a variety of linker molecules [30][31][32] . The SAM shields biomolecules from direct contact with solid surface, thereby minimizing the risk of denaturation 32 .…”

Section: Discussionmentioning

confidence: 99%

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Pohl

Schwab

Cavalcanti‐Adam

2013

JoVE

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Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short-and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Pohl

Schwab

Cavalcanti‐Adam

2013

JoVE

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“…The patterned immobilization of peptides and proteins on material surfaces is important for various bioanalytical and biomedical applications, such as the analysis of ligand-analyte interactions [1] and high-throughput discovery of engineered culture surfaces for stem cell maintenance [2,3] or selective differentiation [4]. Moreover, functionalized substrates that capture or expose a cell-adhesive peptide on their surfaces in response to an external stimulus, such as heat [5], voltage [6][7][8] or light [9][10][11], are useful for the dynamic control of cell adhesion.…”

Section: Introductionmentioning

confidence: 99%

Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

Nakanishi

Nakayama

Yamaguchi

et al. 2011

Science and Technology of Advanced Materials

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The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG 7 ). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni 2+ -ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG 7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment ) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII 7−10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

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“…The controlled and stable immobilization of one or multiple types of (bio)molecules to a surface has been identified as one of the critical challenges in several emerging research fields, such as the regulation of cell shape, [1] the development of advanced biological assays [2,3] and scaffolds for regenerative medicine, [4,5] or the fabrication of increasingly complex micrototal analytical systems (lTAS). [6,7] This is partly motivated by the need for defined surface architectures to simultaneously present multiple biological entities in controlled ratios.…”

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confidence: 99%