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Abstract Patterning biomolecules in synthetic hydrogels offers routes to visualize and learn how spatially‐encoded cues modulate cell behavior (e.g., proliferation, differentiation, migration, and apoptosis). However, investigating the role of multiple, spatially defined biochemical cues within a single hydrogel matrix remains challenging because of the limited number of orthogonal bioconjugation reactions available for patterning. Herein, a method to pattern multiple oligonucleotide sequences in hydrogels using thiol‐yne photochemistry is introduced. Rapid hydrogel photopatterning of hydrogels with micron resolution DNA features (≈1.5 µm) and control over DNA density are achieved over centimeter‐scale areas using mask‐free digital photolithography. Sequence‐specific DNA interactions are then used to reversibly tether biomolecules to patterned regions, demonstrating chemical control over individual patterned domains. Last, localized cell signaling is shown using patterned protein–DNA conjugates to selectively activate cells on patterned areas. Overall, this work introduces a synthetic method to achieve multiplexed micron resolution patterns of biomolecules onto hydrogel scaffolds, providing a platform to study complex spatially‐encoded cellular signaling environments. 

Abstract A cantilever‐free scanning probe lithography (CF‐SPL)‐based method for the rapid polymerization of nanoscale features on a surface via crosslinking and thiol‐acrylate photoreactions is described, wherein the nanoscale position, height, and diameter of each feature can be finely and independently tuned. With precise spatiotemporal control over the illumination pattern, beam pen lithography (BPL) allows for the photo‐crosslinking of polymers into ultrahigh resolution features over centimeter‐scale areas using massively parallel >160 000 pen arrays of individually addressable pens that guide and focus light onto the surface with sub‐diffraction resolution. The photoinduced crosslinking reaction of the ink material, which is composed of photoinitiator, diphenyl(2,4,6‐trimethylbenzoyl) phosphine oxide, poly(ethylene glycol) diacrylate, and thiol‐modified functional binding molecules (i.e., thiol‐PEG‐biotin or 16‐mercaptohexanoic acid), proceeds to ≈80% conversion with UV exposure (72 mW cm⁻²) for short time periods (0.5 s). Such polymer patterns are further reacted with proteins (streptavidin and fibronectin) to yield protein arrays with feature arrangements at high resolution and densities controlled by local UV exposure. This platform, which combines polymer photochemistry and massive arrays of scanning probes, constitutes a new approach to making biomolecular microarrays in a high‐throughput and high‐yielding manner, opening new routes for biochip synthesis, bioscreening, and cell biology research.