Cysteine-tagged chimeric avidin forms high binding capacity layers directly on gold

Inger Vikholm-Lundin (Corresponding Author), Sanna Auer, M. Paakkunainen, J.A.E. Määttä, Tony Munter, J. Leppiniemi, V.P. Hytönen, Kirsi Tappura

    Research output: Contribution to journalArticleScientificpeer-review

    8 Citations (Scopus)

    Abstract

    Cysteine-tagged, genetically engineered avidin named ChiAvd-Cys and wild-type avidin form monolayers or bilayer structures when immobilised directly on gold. Non-specific binding can be reduced by a post-treatment of the avidin layers with a N-[tris(hydroxymethyl)methyl]-acrylamide (pTHMMAA) polymer. ChiAvd-Cys showed excellent activity when immobilised on gold. About 70% of the ChiAvd-Cys molecules were able to bind two biotinylated green fluorescent proteins (per avidin tetramer). Amino-biotinylated antibody F(ab′)2 fragments could be bound to every 4th and 8th ChiAvd-Cys and wild-type avidin molecule, respectively, whereas on average one thiol-biotinylated antibody Fab′-fragment was bound to every ChiAvd-Cys. Antigen binding to the thiol-biotinylated Fab′-fragment bound to the ChiAvd-Cys/pTHMMAA layer was almost twice compared to that of the amino-biotinylated F(ab′)2-fragments. The high antigen binding was due to a site-directed orientation of the thiol-biotinylated fragments. The ChiAvd-Cys/pTHMMAA layers offer high capacity that may be used to couple biotinylated compounds on biosensor surfaces.
    Original languageEnglish
    Pages (from-to)440-448
    Number of pages8
    JournalSensors and Actuators B: Chemical
    Volume171-172
    DOIs
    Publication statusPublished - 2012
    MoE publication typeA1 Journal article-refereed

    Keywords

    • Avidin
    • biotin
    • cysteine tagged
    • non-specific binding
    • self-assembled monolayer

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