Biology

Benzylguanine Thiol Self-Assembled Monolayers for the Immobilization of SNAP-tag Proteins on Microcontact-Printed Surface Structures

Abstract

The site-selective, oriented, covalent immobilization of proteins on surfaces is an important issue in the establishment of microarrays, biosensors, biocatalysts, and cell assays. Here we describe the preparation of self-assembled monolayers consisting of benzylguanine thiols (BGT) to which SNAP-tag fusion proteins can be covalently linked. The SNAP-tag, a modified O6-alkylguanine-DNA alkyltransferase (AGT), reacts with the headgroup of BGT and becomes covalently bound upon the release of guanine. Bacterially produced recombinant His-tag-SNAP-tag-GFP was used to demonstrate the site-specific immobilization on BGT surface patterns created by microcontact printing (μCP). With this versatile method, any SNAP-tag protein can be coupled to a surface.

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Benzylguanine Thiol Self-Assembled Monolayers for the Immobilization of SNAP-tag Proteins on Microcontact-Printed Surface Structures

Antifouling and Antimicrobial Mechanism of Tethered Quaternary Ammonium Salts in a Cross-linked Poly(dimethylsiloxane) Matrix Studied Using Sum Frequency Generation Vibrational Spectroscopy

Abstract

Poly(dimethylsiloxane) (PDMS) materials containing chemically bound (‘‘tethered’’) quaternary ammonium salt (QAS) moieties are being developed as new contact-active antimicrobial coatings. Such coatings are designed to inhibit the growth of microorganisms on surfaces for a variety of applications which include ship hulls and biomedical devices. The antimicrobial activity of these coatings is a function of the molecular surface structure generated during film formation. Sum frequency generation (SFG) vibrational spectroscopy has been demonstrated to be a powerful technique to study polymer surface structures at the molecular level in different chemical environments. SFG was successfully used to characterize the surface structures of PDMS coatings containing tethered QAS moieties that possess systematic variations in QAS chemical composition in air, in water, and in a nutrient growth medium. The results indicated that the surface structure was largely dependent on the length of the alkyl chain attached to the nitrogen atom of the QAS moiety as well as the length of alkyl chain spanning between the nitrogen atom and silicon atom of the QAS moiety. The SFG results correlated well with the antimicrobial activity, providing a molecular interpretation of the activity. This research showed that SFG can be effectively used to aid in the development of new antimicrobial coating technologies by correlating the chemical structure of a coating surface to its antimicrobial activity.

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Antifouling and Antimicrobial Mechanism of Tethered Quaternary Ammonium Salts in a Cross-linked Poly(dimethylsiloxane) Matrix Studied Using Sum Frequency Generation Vibrational Spectroscopy

Stem cell plasticity, osteogenic differentiation and the third dimension

Abstract

Different cues present in the cellular environment control basic biological processes. A previously established 3D microwell array was used to study dimensionality-related effects on osteogenic differentiation and plasticity of marrow stromal cells. To enable long-term culture of single cells in the array a novel surface functionalization technique was developed, using the principle of subtractive micro contact printing of fibronectin and surface passivation with a triblock-copolymer. Immunohistochemical stainings showed that when cultivated in 3D microenvironments, marrow stromal cells can be maintained in the wells for up to 7 days and be induced to commit to the osteogenic lineage. In conclusion, this work shows the modification of a 3D microwell array allowing the long term study of single stem cell plasticity and fate in controlled microenvironments.

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Stem cell plasticity, osteogenic differentiation and the third dimension

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