Reversible biofunctionalization of surfaces with a switchable mutant of avidin

Pollheimer, Philipp and Taskinen, Barbara and Scherfler, Andreas and Gusenkov, Sergey and Creus, Marc and Wiesauer, Philipp and Zauner, Dominik and Schöfberger, Wolfgang and Schwarzinger, Clemens and Ebner, Andreas and Tampé, Robert and Stutz, Hanno and Hytönen, Vesa P. and Gruber, Hermann J.. (2013) Reversible biofunctionalization of surfaces with a switchable mutant of avidin. Bioconjugate chemistry, Vol. 24, H. 10. pp. 1656-1668.

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Official URL: http://edoc.unibas.ch/dok/A6223369

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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.
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie
UniBasel Contributors:Creus, Marc
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:American Chemical Society
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:27 Feb 2014 15:46
Deposited On:27 Feb 2014 15:46

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