Abstract
In this work, a collimated helium beam was used to activate a thiol-poly(ethylene glycol) (SH-PEG) monolayer on gold to selectively capture proteins in the exposed regions. Protein patterns were formed at high throughput by exposing a stencil mask placed in proximity to the PEG-coated surface to a broad beam of helium particles, followed by incubation in a protein solution. Attenuated Total Reflectance–Fourier Transform Infrared Spectroscopy (ATR–FTIR) spectra showed that SH-PEG molecules remain attached to gold after exposure to beam doses of 1.5–60 µC/cm2 and incubation in PBS buffer for one hour, as evidenced by the presence of characteristic ether and methoxy peaks at 1120 cm−1 and 2870 cm−1, respectively. X-ray Photoelectron Spectroscopy (XPS) spectra showed that increasing beam doses destroy ether (C–O) bonds in PEG molecules as evidenced by the decrease in carbon C1s peak at 286.6 eV and increased alkyl (C–C) signal at 284.6 eV. XPS spectra also demonstrated protein capture on beam-exposed PEG regions through the appearance of a nitrogen N1s peak at 400 eV and carbon C1s peak at 288 eV binding energies, while the unexposed PEG areas remained protein-free. The characteristic activities of avidin and horseradish peroxidase were preserved after attachment on beam-exposed regions. Protein patterns created using a 35 µm mesh mask were visualized by localized formation of insoluble diformazan precipitates by alkaline phosphatase conversion of its substrate bromochloroindoyl phosphate-nitroblue tetrazolium (BCIP-NBT) and by avidin binding of biotinylated antibodies conjugated on 100 nm gold nanoparticles (AuNP). Patterns created using a mask with smaller 300 nm openings were detected by specific binding of 40 nm AuNP probes and by localized HRP-mediated deposition of silver nanoparticles. Corresponding BSA-passivated negative controls showed very few bound AuNP probes and little to no enzymatic formation of diformazan precipitates or silver nanoparticles.
Highlights
Surface patterning of biomolecules is important in the study of cell adhesion, in tissue engineering, and in the development of diagnostics and biomedical assays such as protein nanoarrays [1,2,3,4]
Photolithography is a high-throughput lithographic process but its resolution is diffraction-limited below the micron-scale, and it is expensive to use for low-volume manufacturing
We explored the use of helium beam proximity lithography to create micron and nanoscale patterns of proteins on grafted poly(ethylene glycol) (PEG) in a high-throughput manner
Summary
Surface patterning of biomolecules is important in the study of cell adhesion, in tissue engineering, and in the development of diagnostics and biomedical assays such as protein nanoarrays [1,2,3,4]. Controlled attachment of biomolecules can be achieved by approaches generally categorized as ‘‘top-down’’ (e.g., photolithography, focused ion beam lithography, electron beam lithography, nanografting), or ‘‘bottom-up’’ (e.g., self-assembly of monolayers, dip-pen nanolithography, micro/nano contact printing or stamping), or combinations of these techniques. Photolithography is a high-throughput lithographic process but its resolution is diffraction-limited below the micron-scale, and it is expensive to use for low-volume manufacturing. Electron beam and focused ion beam lithography have very high resolution but very low throughput. Bottom-up techniques take advantage of the spontaneous organization of molecules to produce a more complex and functional patterned material. These methods can allow patterning below the current resolution of lithographic techniques, but often involve high costs (for large areas) and imperfect patterning. Current approaches to biomolecule patterning, often employ combinations of top-down and bottom-up techniques [4,5,6]
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