Site-specific immobilization of tetrazine-modified green fluorescent protein on trans-cyclooctene surfaces.

摘要

The ability to control the orientation of proteins attached to a surface, one of the goals of this work, has applications within areas such as microarrays, biomaterials, tissue engineering and biosensors. Non-covalent protein adsorption (physisorption), the easiest of all protein-attachment approaches, suffers from several drawbacks and was rejected for this work. Although the covalent attachment (chemisorption) of peptides and proteins to solid substrates has long been achieved, many of the procedures utilized are generally limited to schemes and reaction steps that can be detrimental to biological systems. Furthermore, while the attachment is covalent, the point(s) of attachment on the protein and its subsequent orientation at the surface remain uncontrolled, resulting in a distribution of protein orientations at the surface, and therefore a decrease in the effective bioactivity sought. The focus herein was to investigate the feasibility of control of attachment site(s) on the protein, and hence, the orientation of the protein at the surface. Specifically, we sought to functionalize a glass support substrate with one of several cyclooctene compounds (patterned through micro-contact printing, or un-patterned by simple immersion), followed by reaction in these functionalized areas with a site-specifically modified tetrazine-protein moiety, either through 'click chemistry' or non-specific binding (as a control for comparison purposes). While the strain-driven reactivity of cis- vs. trans-cyclooctene have been studied in homogenous solution systems, this is the first study of the reaction scheme at a surface. Time-of-flight secondary ion mass spectrometry (ToF-SIMS), fluorescence imaging, contact angle analysis, and principal component analysis were utilized to determine the outcome of each step in the reaction scheme. The covalent attachment of tetrazine-modified protein with trans-cyclooctene-modified surfaces was observed, as expected, whereas only non-specific absorption was observed on the cis-cyclooctene-modified surfaces.