Oriented protein immobilization using covalent and noncovalent chemistry on a thiol-reactive self-reporting surface

J Am Chem Soc. 2013 Feb 27;135(8):3104-11. doi: 10.1021/ja3102133. Epub 2013 Feb 19.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Proteins / chemistry*
  • Sulfhydryl Compounds / chemistry*
  • Surface Properties


  • Proteins
  • Sulfhydryl Compounds