Affinity‐Based Protein Surface Pattern Formation by Ligand Self‐Selection from Mixed Protein Solutions

Abstract

Photolithographically prepared surface patterns of two affinity ligands (biotin and chloroalkane) specific for two proteins (streptavidin and HaloTag®, respectively) are used to spontaneously form high-fidelity surface patterns of the two proteins from their mixed solution. High affinity protein-surface self-selection onto patterned ligands on surfaces exhibiting low non-specific adsorption rapidly yields the patterned protein surfaces. Fluorescence images after protein immobilization show high specificity of the target proteins to their respective surface patterned ligands. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging further supports the chemical specificity of streptavidin and HaloTag® for their surface patterned ligands from mixed protein solutions. However, ToF-SIMS did detect some non-specific adsorption of bovine serum albumin, a masking protein present in excess in the adsorbing solutions, on the patterned surfaces. Protein amino acid composition, surface coverage, density and orientation are important parameters that determine the relative ToF-SIMS fragmentation pattern yields. ToF-SIMS amino acid-derived ion fragment yields summed to produce surface images can reliably determine which patterned surface regions contain bound proteins, but do not readily discriminate between different co-planar protein regions. Principal component analysis (PCA) of these ToF-SIMS data, however, improves discrimination of ions specific to each protein, facilitating surface pattern discrimination and contrast.

Microcup Arrays Featuring Multiple Chemical Regions Patterned with Nanoscale Precision

“…[1][2][3] Considerable improvements in the methodologies for micro-and nanoscale surface patterning have occurred recently, as highlighted in a number of topical reviews on the different fabrication techniques. [4][5][6][7] Some of the notable examples include dip pen nanolithography (DPN), [ 8 ] polymer pen lithography (PPL), [ 9 ] beam pen lithography (BPL), [ 10 ] electron beam lithography, [ 11 ] photolithography, [ 12 ] inkjet printing, [ 13 ] and soft-lithography-based techniques. [ 14 ] These techniques can position single and multiple chemical and biological moieties in micrometer to nanometer features with extreme accuracy.…”

Immobilization of Antibodies and Enzymes on 3-Aminopropyltriethoxysilane-Functionalized Bioanalytical Platforms for Biosensors and Diagnostics

“…98 Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is another technique to analzye immobilized biomolecules. 99,100 The disulfide bond signal in ToF-SIMS is directly related to the activity of adsorbed protein coatings. A pertinent model with combined information from AFM, ARXPS, and ToF-SIMS can provide for insight into the vertical structure with respect to the surface density of biomolecules.…”

Protein adsorption and surface patterning

“…More specific reaction routes, utilizing proteins with highly selective and well-defined interactions with low-molecular weight ligands, were also used to achieve parallel indirect patterning in that study. Another example of this approach was described by Dubey et al [73], who prepared patterns of biotin and chloroalkane ligands in patterns on a PEG-based protein resistant substrate. When the surface was exposed to a mixture of the proteins streptavidin and HaloTag, the proteins self-organized on the surface, binding to their respective ligands only.…”