Binding properties of a streptavidin layer formed on a biotinylated Langmuir–Schaefer film of unfolded protein

• Langmuir–Schaefer film of carbonic anhydrase (LSF-CA) was biotinylated.
• A densely packed streptavidin (SAv) layer was formed on the biotinylated LSF-CA.
• Biotinylated proteins were bound to the SAv layer at high density.
• Nonspecific adsorption of intact proteins to the SAv layer was weak.
• Atomic force microscopy showed the binding of proteins at molecular resolution.

A Langmuir monolayer of carbonic anhydrase (CA) unfolded at an air/water interface was transferred onto the hydrophobic surface of a silicon wafer by means of the Langmuir–Schaefer technique. The transferred CA film was biotinylated and was incubated in a streptavidin (SAv) solution to obtain a densely packed SAv layer by biotin–SAv linkage. Biotinylated proteins including ferritin, catalase, alcohol dehydrogenase, and carbonic anhydrase were incubated with the SAv layer and binding of these proteins was examined by atomic force microscopy. High-density binding of the biotinylated proteins was observed, whereas the amount of adsorbed non-biotinylated proteins was low or negligible. The SAv layer on the Langmuir–Schaefer film of unfolded protein could become a basic architecture for protein immobilization studies.