Nitrilotriacetic Acid Functionalized Graft Copolymers: A Polymeric Interface for Selective and Reversible Binding of Histidine-Tagged Proteins

Authors


  • We thank Verena Eggli for her help in preparing the proteins and Doris Suter (Department of Chemistry and Applied Biosciences, ETH Zürich, Switzerland) for the NMR measurements. Financial support from the Swiss National Science Foundation (Swiss National Science Foundation, National Research Program NRP-47 “Supramolecular Functional Materials”, Project No. 4047-057548/1) is gratefully acknowledged.

Abstract

A series of novel graft copolymers, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) with 28, 46, and 96 % of the PEG chains carrying a terminal nitrilotriacetic acid (NTA) group, have been synthesized. Through electrostatic interactions, these polycationic graft copolymers assemble spontaneously from aqueous solution onto negatively charged surfaces, forming polymeric monolayers that present NTA groups at controlled surface densities. Such NTA-presenting surfaces on a highly PEGylated background are attractive sensor platforms for the reversible binding, through Ni2+ ions, of histidine (His)-tagged biomolecules such as proteins and antibodies. With these three polymers, NTA-ligand surface densities of 16, 23, and 37 pmol cm–2, respectively, have been obtained. The surface assembly of the polymers, as well as their sensing performance, has been monitored quantitatively using in-situ optical-waveguide light-mode spectroscopy. The NTA-functionalized PLL-g-PEG surfaces prove to be highly resistant to non-specific adsorption in contact with human serum, while allowing the specific and reversible surface binding of the 6 × His-tagged green fluorescent protein (GFP)uv-6His in its native conformation. The amount of GFPuv-6His immobilized on the polymeric surface increases with increasing NTA surface density. Furthermore, micropatterns consisting of NTA-functionalized PLL-g-PEG in a background of PLL-g-PEG are produced using the “molecular assembly patterning by lift-off” technique. Exposure to Ni2+ and GFPuv-6His results in a protein pattern of excellent contrast, as judged by fluorescence microscopy. The NTA-functionalized PLL-g-PEG surface is considered to be a promising sensor platform for binding 6 × His-tagged proteins, thanks to the simplicity and cost-effectiveness of the surface modification protocol, the high specificity and nearly quantitative reversibility of the protein binding, and the potential for fabricating microarrays of multiple capture molecules.

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