title: Reversible biofunctionalization of surfaces with a switchable mutant of avidin.
abstract: Label-free biosensors detect binding of prey molecules (″analytes″) to immobile bait molecules on the sensing surface. Numerous methods are available for immobilization of bait molecules. A convenient option is binding of biotinylated bait molecules to streptavidin-functionalized surfaces, or to biotinylated surfaces via biotin-avidin-biotin bridges. The goal of this study was to find a rapid method for reversible immobilization of biotinylated bait molecules on biotinylated sensor chips. The task was to establish a biotin-avidin-biotin bridge which was easily cleaved when desired, yet perfectly stable under a wide range of measurement conditions. The problem was solved with the avidin mutant M96H which contains extra histidine residues at the subunit-subunit interfaces. This mutant was bound to a mixed self-assembled monolayer (SAM) containing biotin residues on 20% of the oligo(ethylene glycol)-terminated SAM components. Various biotinylated bait molecules were bound on top of the immobilized avidin mutant. The biotin-avidin-biotin bridge was stable at pH ≥3, and it was insensitive to sodium dodecyl sulfate (SDS) at neutral pH. Only the combination of citric acid (2.5%, pH 2) and SDS (0.25%) caused instantaneous cleavage of the biotin-avidin-biotin bridge. As a consequence, the biotinylated bait molecules could be immobilized and removed as often as desired, the only limit being the time span for reproducible chip function when kept in buffer (2-3 weeks at 25 °C). As expected, the high isolectric pH (pI) of the avidin mutant caused nonspecific adsorption of proteins. This problem was solved by acetylation of avidin (to pI < 5), or by optimization of SAM formation and passivation with biotin-BSA and BSA.
authors: Institute of Biophysics, Johannes Kepler University , Gruberstr. 40, 4020 Linz, Austria.
authors: Philipp
authors: P
authors: Pollheimer
authors: Barbara
authors: B
authors: Taskinen
authors: Andreas
authors: A
authors: Scherfler
authors: Sergey
authors: S
authors: Gusenkov
authors: Marc
authors: M
authors: Creus
authors: Philipp
authors: P
authors: Wiesauer
authors: Dominik
authors: D
authors: Zauner
authors: Wolfgang
authors: W
authors: Schöfberger
authors: Clemens
authors: C
authors: Schwarzinger
authors: Andreas
authors: A
authors: Ebner
authors: Robert
authors: R
authors: Tampé
authors: Hanno
authors: H
authors: Stutz
authors: Vesa P
authors: VP
authors: Hytönen
authors: Hermann J
authors: HJ
authors: Gruber
mesh_terms: D000818:Animals; D001360:Avidin / Q000737:chemistry* / Q000235:genetics; D015374:Biosensing Techniques / Q000295:instrumentation; D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation; D055767:Immobilized Proteins / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D013499:Surface Properties
chemical_list: D055767:Immobilized Proteins; D001360:Avidin; D001710:Biotin