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Reversible biofunctionalization of surfaces with a switchable mutant of avidin

Research output: Contribution to journalArticleScientificpeer-review


Original languageEnglish
Pages (from-to)1656-1668
Number of pages13
JournalBioconjugate Chemistry
Issue number10
Publication statusPublished - 16 Oct 2013
Publication typeA1 Journal article-refereed


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.