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

• Published in: Sensors and actuators. B, Chemical Chemical, 2012-08, Vol.171-172, p.440-448
• Main Authors: Vikholm-Lundin, Inger, Auer, Sanna, Paakkunainen, Maija, Määttä, Juha A.E., Munter, Tony, Leppiniemi, Jenni, Hytönen, Vesa P., Tappura, Kirsi
• Format: Article
• Language: English
• Subjects: antigens; Avidin; binding capacity; biosensors; Biotin; chimerism; Cysteine tagged; gold; green fluorescent protein; Non-specific binding; polymers; Self-assembled monolayer
• ISSN: 0925-4005; 1873-3077
• DOI: 10.1016/j.snb.2012.05.008

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.