Fig. 1. Formation and patterning of poly(ethylene glycol) arrays (PEG arrays).

(A) Schematic representation of the procedure for forming PEG arrays. PEG arrays were crosslinked with SH-PEG-SH₃₄₀₀ and functionalized with pendant Cys-Arg-Gly-Asp-Ser (CRGDS) peptides for adhesion. Scrambled, non-bioactive CRDGS peptide was included to maintain constant pendant group concentration (4 mM CRGDS + CRDGS) when investigating adhesion-dependent motility. Patterning: A PEG thin film was first formed with 1:1 or 4:1 molar ratio SH-PEG-SH₃₄₀₀:PEG-NB and 0.5 - 4 mM CRGDS. The PEG hydrogel film was incubated in crosslinker solution at excess concentration overnight, followed by a second exposure to UV under a photomask to fully crosslink hydrogels in distinct regions (See also, Fig. 2) while maintaining the same RGD concentration. (B) A fluorescently tagged CRGDSK peptide was photopatterned into a PEG substrate to illustrate spatial control over polymer properties. (C) Elastic moduli for PEG hydrogels formed with different monomer concentrations, where reported values for wt% are based on the concentration of PEG-norbornene (8-arm, 20K MW, PEG-NB) in the monomer solution (e.g., 4 wt% = 40 mg/mL PEG-NB). Shear modulus was also tuned by changing the molar ratio of SH-PEG-SH₃₄₀₀ to PEG-NB molecules (1:1 or 4:1, equivalent to 25% or 100% crosslinker thiol to total norbornene groups in the monomer solution). (D) Illustration of CRGDS incorporation into PEG hydrogels (8 wt%; 1:1 SH-PEG-SH:PEG-NB) for the range of concentrations investigated (reported for [CRGDS] in monomer solution). Relative CRGDS concentration was determined by labeling the N-terminus with fluorescein (see Methods). (E) Mean fluorescence intensity for PEG array spots functionalized with fluorescently tagged CRGDS. Error bars = standard deviation for (C) and (E).