Oriented Protein Immobilization using Covalent and Noncovalent Chemistry on a Thiol-Reactive Self-Reporting Surface

D. Wasserberg, Carlo Nicosia, E.E. Tromp, Vinod Subramaniam, Jurriaan Huskens, Pascal Jonkheijm

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We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin.
Original languageUndefined
Pages (from-to)3104-3111
Number of pages8
JournalJournal of the American Chemical Society
Issue number8
Publication statusPublished - 2013


  • METIS-301589
  • IR-90132

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