2.1. Chemicals and Microarray
All chemicals were American Chemical Society grade or higher and commercially available unless noted otherwise. We previously provided details on the microarray slide, which uses N‑hydroxysuccinimide modified polyethylene glycol (PEG-NHS) for both conjugation of potential ligands and presentation of ligands far from the glass surface [20]. All ligands were engineered to contain a single nucleophilic group (i.e., primary amine) and were dissolved as stock solutions in 70%/30% (V/V) glycerol:dimethyl sulfoxide (DMSO, Sigma, St Louis, MO, USA) at a concentration of 0.1–1.0 mM. The pH was adjusted to be ≈ 9.0 using diisopropylethylamine (DIEA, Sigma) to promote nucleophilic substitution using the primary amine. Polyallrylamine (PAAm, Sigma) was used as a positive control for cell binding at 10 mg/mL in 100 mM sodium bicarbonate buffer (pH 9.0). Cyclo Arg-Gly-Asp-D-Tyr-Lys (cRGDyk) peptide was purchased from AnaSpec, Inc. (Fremont, CA, USA). 2[(3-amino-3-carboxypropyl)(hydroxy)(phosphinyl)-methyl]pentane-1,5-dioic acid (GPI), 2-(3-(5-amino-1-carboxypentyl)ureido)pentanedioic acid (KUE), beta Ala-Gly (β-AG) and all trimeric ligands were in house compounds, which were synthesized as previously described [22,23]. The ligand solution was distributed into a 384-well plate and printed on the NHS functionalized slide surface [20] using a microarray robot (OmniGrid Accent, DigiLab, Inc., Holliston, MA, USA) mounted with SMP11 pins (Telechem, Sunnyvale, CA, USA). The spreading of spots was optimized to be 300 ± 10 μm in diameter, with a center-to-center spacing of 500 μm (i.e., 200-μm of clear space between any two spots). After incubating for 3 h at room temperature in air, the slides were immersed in deionized water to deactivate unreacted NHS groups. After washing with ethanol, the slides were dried under a nitrogen stream and stored in a dust free environment before use.