Encapsulated Hydrogels by E-beam Lithography and Their Use in Enzyme Cascade Reactions

Abstract

Electron beam (e-beam) lithography was employed to prepare one protein encapsulated inside another by first fabricating protein-reactive hydrogels of orthogonal reactivity and subsequently conjugating the biomolecules. Exposure of thin films of eight arm star poly(ethylene glycol) (PEG) functionalized with biotin (Biotin-PEG), alkyne (Alkyne-PEG) or aminooxy (AO-PEG) end-groups to e-beam radiation resulted in cross-linked hydrogels with the respective functionality. It was determined via confocal microscopy that a nominal size exclusion effect exists for streptavidin immobilized on Biotin-PEG hydrogels of feature sizes ranging from 5 to 40 μm. AO-PEG was subsequently patterned as an encapsulated core inside a contiguous outer shell of Biotin-PEG. Similarly, Alkyne-PEG was patterned as a core inside an AO-PEG shell. The hydrogel reactive end-groups were conjugated to dyes or proteins of complimentary reactivity, and the 3-D spatial orientation was determined for both configurations using confocal microscopy. The enzyme glucose oxidase (GOX) was immobilized in the core of the encapsulated Alkyne-PEG core/AO-PEG shell architecture, and horseradish peroxidase (HRP) was conjugated to the shell periphery. Bioactivity for the HRP-GOX enzyme pair was observed in this encapsulated configuration by demonstrating that the enzyme pair was capable of enzyme cascade reactions.

Functional Changes during Electron-Beam Lithography of Biotinylated Poly(ethylene glycol) Thin Films

“…Patterning surfaces at nano- and microlength scales with multiple functionalities is important to an array of biointeractive applications including the control of cell signaling , and cooperative reaction cascades in detection and diagnostics. − Notably, methods based on electron-beam (e-beam) lithography are of particular interest. Despite the fact that it is a serial process and is best suited for relatively small and planar surfaces, e-beam lithography brings differentiating advantages relative to photo and/or soft lithography.…”

“…While the usefulness of PEG and other biorelevant polymers as additive surface-patterning materials has been amply demonstrated, ,,− relatively little is known about the underlying radiation chemistry driven by e-beam patterning. The problem is complicated by the fact that the patterned structures typically have microscale or nanoscale dimensions and do not lend themselves well to traditional wide-area characterization methods such as light-based spectroscopies or NMR.…”

Micro- to Nanoscale Bio-Hybrid Hydrogels Engineered by Ionizing Radiation

“…The first microscopic hydrogel patterns were created using spatially resolved electron beams from scanning electron microscopes (SEM) and solvent-free polyethylene glycol (PEG or PEO) films as e-beam resist [23,24]. Several further reports describe the fabrication of micro-/nano-gel patterns by EBL using other polymers, such as poly (vinyl methyl ether) [25], poly (vinyl pyrrolidone) (PVP) [26], polyamidoamine [27], oligo(ethylene glycol) methacrylate [28], and star PEGs with functional end groups [29].…”

“…Micro-/nanostructured hydrogel patterns have been developed as part of biosensing platforms [38,39] and as advanced substrates for cell cultures [40][41][42] and (bio)chemical syntheses [29]. In the development of protein biochips designed to study protein-protein or protein-cell interactions, patterning properties are important because they allow immobilization of active proteins and/or cells with optimal density and proximity.…”

Chemical Orthogonality in Surface-Patterned Poly(ethylene glycol) Microgels

“…PEG patterning has been extended to address a range of chemical functionalities including amine, biotin, alkyne, maleimide, and aminooxy, among others. Notably, Maynard et al have created PEG-patterned surfaces that exhibit not simply a single chemical functionality but also ones with two and three orthogonal functionalities. − In their approach, multiple orthogonal functionality was achieved by successive lithography steps where a PEG film with a first functionality was patterned, and then, using fiducial alignment marks to very precisely locate the same area, a next film with an orthogonal functionality was patterned on the first or in immediate proximity to it. The process can be repeated to introduce additional functionalities within the pattern and is thus able to position multiple molecular species at discrete locations within an otherwise antifouling PEG matrix.…”