Sequential patterning of two fluorescent streptavidins assisted by photoactivatable biotin on an aminodextran-coated surface

Sequential patterning of two fluorescent streptavidins (SAvs) was carried out using photopatterning of photoactivatable biotin (photobiotin) on an aminodextran surface, which was crucial for the minimization of non-specific binding. Photobiotin was bound by photoreaction to the amine groups of aminodextran. Water contact angle at each step during the preparation of the aminodextran surface was measured to investigate the hydrophilicity of the surfaces. The specific and nonspecific binding of a fluorescent SAv was investigated for the aminodextran surface and the amine–silane surface. The aminodextran surface almost entirely prevented nonspecific binding of a fluorescent SAv and was successfully used for sequential patterning of two fluorescent SAvs. The addition of ethanolamine (40 mM) in the photobiotin solution diminished blurring of pattern shape. To decrease pattern size, the UV light was focused on the aminodextran surface in an inverted microscope system. Under optimized conditions, two fluorescent SAvs array of approximately 25 μm size was obtained using a shadow mask of 100 μm hole size in the inverted microscope system.

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► Sequential patterning of two fluorescent streptavidins.
► Photopatterning of photobiotin on an aminodextran-coated surface.
► Enhancement of spot resolution by adding ethanolamine in photobiotin solution.
► Comparison of nonspecific binding between the APTMS and aminodextran surface.