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Covalent Surface Chemistry Gradients for Presenting Bioactive Peptides



Matt J. Kipper, Hynda K. Kleinman, Francis W. Wang


The activation of surfaces by covalent attachment of bioactive moieties is an important strategy for improving the performance of biomedical materials. Such techniques have also been used as tools to study cellular responses to particular chemistries of interest. The creation of gradients of covalently bound chemistries is a logical extension of this technique. Gradient surfaces may permit the rapid screening of a large range of concentrations in a single experiment. Additionally, the biological response to the gradient itself may provide new information on receptor requirements and cell signaling. This work describes a rapid, flexible technique for the covalent addition of bioactive peptide gradients to a surface or gel, and a simple fluorescence technique for assaying the gradient. In this technique, bioactive peptides with a terminal cysteine are bound via an heterobifunctional coupling agent to primary amine-containing surfaces and gels.A gradient in the coupling agent is created on the surfaces or gels by varying the residence time of the coupling agent across the surface or gel, and thereby controlling the extent of reaction. We demonstrate this technique using poly(L-lysine) coated glass surfaces and fibrin gels. Once the surface or gel has been activated by addition of the coupling agent gradient, the bioactive peptide is added. Quantitation of the gradient is achieved by measuring the reaction kinetics of the coupling agent with the surface or gel of interest. This can be done by either fluorescently labeling the coupling agent (in the case of surfaces) or by spectrophotometrically detecting the release of pyridine-2-thione, which is produced when the the thiol-reactive portion of the coupling agent reacts. Validation with changes in fibroblast cell migration behavior across a bioactive peptide gradient illustrates preservation of peptide function as well as the usefulness of this technique.
Analytical Biochemistry


cell migration, laminin, peptide gradient


Kipper, M. , Kleinman, H. and Wang, F. (2008), Covalent Surface Chemistry Gradients for Presenting Bioactive Peptides, Analytical Biochemistry (Accessed April 14, 2024)
Created October 16, 2008