Positioning Multiple Proteins at the Nanoscale with Electron Beam Cross-Linked Functional Polymers

Abstract

Constructing multicomponent protein structures that match the complexity of those found in Nature is essential for the next generation of medical materials. In this report, a versatile method to precisely arrange multicomponent protein nanopatterns in two-dimensional single-layer or three-dimensional multilayer formats using electron beam lithography is described. Eight arm poly(ethylene glycol)s were modified at the chain ends with either biotin, maleimide, aminooxy, or nitrilotriacetic acid. Analysis by 1H NMR spectroscopy revealed that the reactions were efficient and that end group conversions were 91-100%. The polymers were then cross-linked onto Si surfaces using electron beams to form micron sized patterns of the functional groups. Proteins with biotin binding sites, a free cysteine, an N-terminal α-oxoamide, and a histidine tag, respectively, were then incubated with the substrate in aqueous solutions without the addition of any other reagents. By fluorescence microscopy experiments it was determined that proteins reacted site-specifically with the exposed functional groups to form protein micropatterns. Multicomponent nanoscale protein patterns were then fabricated. Different PEGs with orthogonal reactivity were sequentially patterned on the same chip. Simultaneous assembly of two different proteins from a mixture of the biomolecules formed the multicomponent two dimensional patterns. Atomic force microscopy demonstrated that nanometer sized patterns of polymer were formed and fluorescence microscopy demonstrated that side-by-side patterns of the different proteins were obtained. Moreover, multilayer PEG fabrication produced micron and nanometer sized patterns of one functional group on top of the other. Precise three-dimensional arrangements of different proteins were then realized.

Controlling Protein Adsorption through Nanostructured Polymeric Surfaces

“…[85] Electron-beam lithography has been proven successful in the creation of well-defined nanopatterns via local modification of hydrophobicity or functionality of the polymer surfaces. [86,87] Polymer exposure to a focused e-beam enables the creation of protein-and cell-adhesive regions with sub-100 nm resolution. [88] In contrast to the above-mentioned techniques, e-beam lithography can pattern features with arbitrary shape.…”

Atomized Spray Plasma Deposition of Structurally Well-Defined Bioactive Coatings

“…Another noteworthy class of bioactive surfaces is protein-resistant coatings which are highly sought after for antifouling applications 25 and cell-resistant biomedical devices. 26 Some examples include poly(ethylene glycol), 27,28,29 polyacrylamide, 30 or polysaccharide 31,32,33 layers formed by physisorption, 34 graft polymerization, 35,36 plasmachemical deposition, 37,38,39 or selfassembled monolayers (SAMs). 27,40,41 Many of these preparative methods for bioactive surfaces tend to suffer from inherent limitations: physisorption is by its very nature reversible; graft polymerization requires an initiator layer 42 or surface functionalisation prior to commencing the grafting step; 17,35 whilst self-assembled monolayers are substrate-specific 43,44 and can be moisture sensitive (e.g.…”

Arrays of topographically and peptide-functionalized hydrogels for analysis of biomimetic extracellular matrix properties

“…7 Microcontact printing is a common technique to design chemical patterns on the order of the size of cells (microns) to study, for example, geometric restraints on cell apoptosis 8 or viral propagation. 9 Other techniques such as e-beam lithography, 10,11 dip pen lithography, 12 laser scanning lithography, 13 and colloidal lithography 14 have been used to pattern chemical cues on the length scales of focal adhesions (nanometers). Chemical patterns on the nanometer scale have been used to explore fundamental cell-substrate interactions, including the limits of focal adhesion spacing.…”

“…10 The chemical patterns have a) Electronic mail: nealey@engr.wisc.edu recently advanced to include techniques to spatially pattern multiple chemical cues. 11,13 This paper is not an additional demonstration of the ability to pattern biochemical cues in hydrogels, but rather, we use biomimetic length scale topographic patterns to enable the simultaneous study of biomimetic physical and biologically relevant chemical cues in a soft material platform.…”