Site-Specific Immobilization and Micrometer and Nanometer Scale Photopatterning of Yellow Fluorescent Protein on Glass Surfaces

Abstract

A simple method is described for the site-specific attachment of yellow fluorescent protein (YFP) to glass surfaces on length scales ranging from tens of micrometers to ca. 200 nm. 3-Mercaptopropyl(triethoxy silane) is adsorbed onto a glass substrate and subsequently derivatized using a maleimide-functionalized oligomer of ethylene glycol. The resulting protein-resistant surface is patterned by exposure to UV light, causing photochemical degradation of the oligo(ethylene glycol) units to yield aldehyde groups in exposed regions. These are covalently bound to N-(5-amino-1-carboxypentyl)iminoacetic acid, yielding a nitrilotriacetic acid (NTA)-functionalized surface, which following complexation with Ni(2+), is coupled to His-tagged YFP. Using scanning near-field photolithography, in which a UV laser coupled to a scanning near-field optical microscope is utilized as the light source for photolithography, it is possible to fabricate lines of protein smaller than 200 nm, in which the biomolecules remain strongly optically active, facilitating the acquisition of diffraction-limited fluorescence images by confocal microscopy.

Photocatalytic Nanolithography of Self-Assembled Monolayers and Proteins

“…One example is the use of near-field photodegradation of poly(ethylene glycol)-functionalized SAMs on glass by Reynolds et al , who derivatized the patterned features with nitrilotriacetic acid and, after complexation with nickel, bound histidine-tagged yellow-fluorescent protein to the surface. 57 …”

Modifying biomaterial surfaces to control stem cell growth and differentiation

“…Most approaches to presenting proteins on surfaces involve simple adsorption; however, there are plenty of ways of covalently attaching them via either their amino or carboxy termini, or side chains. There are also methods to attach the molecules in a site-specific manner in order to ensure that access to the cell binding sites is not restricted by changes in protein conformation due to physical interactions with the underlying surface (Reynolds, 2009, Christman, 2007.…”

Chemoselective Immobilization of Proteins by Microcontact Printing and Bio‐orthogonal Click Reactions

“…Biomolecule patterns have been made by using various techniques [35] including microcontact printing, [36] dip-pen lithography (DPN), [37,38] nanografting, [39] imprint lithography, [40] photolithography, [41] and electron beam lithography. [17,42] Microcontact printing, developed by Whitesides and co-workers, has Herein, a combination of microcontact printing of functionalized alkanethiols and site-specific modification of proteins is utilized to chemoselectively immobilize proteins onto gold surfaces, either by oxime-or copper-catalyzed alkyne-azide click chemistry.…”