Paper BI+AS+NS-ThA9
Formation of Protein Surface Patterns by Ligand Self-Selection from Mixed Protein Solutions

Thursday, November 12, 2009, 4:40 pm, Room K

Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) has been shown to be very sensitive for surface analysis and has been very useful for trace element detection. With the advent of improved analyzers, imaging ToF-SIMS provides spatial distribution of different species and helps in plotting the surface reactivity maps. Use of multivariate analysis, especially Principal Component Analysis (PCA) makes this technique even more powerful by differentiating regions with different chemistries. ToF-SIMS and PCA has been used in this work to study a very important two component patterned chemistry, which can have applications in bio-chips and cell-based biosensors. The chemistry is based on n-hydroxysuccinimide (NHS) esters; these molecules are widely used as leaving groups to activate covalent coupling of amine-containing biomolecules onto surfaces. The present work utilizes our knowledge of a previously studied model system, where NHS molecules were self-assembled on a gold substrate, and XPS and ToF-SIMS was used to characterize and understand the effect of hydrolysis and regeneration. We have demonstrated the extension of this chemistry to a commercial poly(ethylene glycol) (PEG)-based polymer films coated on glass slides. NHS and methoxy-capped regions were co-patterned onto these slides using photolithographic methods; then imaged with ToF-SIMS/PCA. NHS surface reactive zones are clearly resolved at high sensitivity despite the complexity of the matrix chemistry. Surface-specific protein coupling was observed by surface-selective reaction of streptavidin with the NHS patterns. The next step involved the preparation of photolithographic patterns of two affinity ligands (biotin and chloroalkane) for the specific immobilization of two different proteins (Streptavidin and HaloTag®). Spontaneous formation of high-fidelity surface patterns of the two proteins from their mixed solution was observed and characterized. In addition to Streptavidin and HaloTag®, ToF-SIMS detected the presence of non-specific BSA adsorption, a masking protein present in excess in the protein solutions, onto the patterned surfaces. 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. However PCA of the ToF-SIMS data, improves discrimination of ions specific to each protein, facilitating surface pattern discrimination based on protein type. Also, ToF-SIMS imaging detected regions where residue from incompletely removed UV-exposed photoresist was present and its influence on protein adsorption.