2. Experimental Section

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.

2.2. Cell Culture and Adhesion Assay
Melanoma cell lines including M21, M21-L, and B16 were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin/streptomycin (P/S) under 5% CO2 at 37 °C. Human prostate cancer cells, LNCaP and PC3, were cultured in RPMI supplemented with 10% FBS and 100 units/mL P/S under 10% CO2 at 37 °C. All cells were plated into 100-mm culture dishes (Corning, Tewksbury, MA, USA) in 10 mL of pre-warmed culture media. When the cells reached 70%–80% confluence at a density of 2–5 × 106 cells/dish, either ESNF10 (700 nm NIR fluorophore) [24] or IR786 (800 nm NIR fluorophore) [21] was added to the dish at 2 µM in media. After 20 min incubation at 37 °C, the cells were washed twice with media and the NIR fluorescence signals were observed under a multi-channel fluorescence microscope (see below). The cells were then trypsinized and seeded onto the ligand-bearing surface in DMEM containing 10% FBS at 37 °C. Incubation parameters, including the effect of rocking (0 vs. 30 rpm), incubation time (30–180 min), presented ligand concentration at the time of spotting (0.1–1.0 mM), and applied cell density (0.2 × 106−8 × 106 cells), were systematically optimized. After incubation, the slides were gently washed with cell culture media before scoring.

2.3. Fluorescence Microscopy and Software
Living cells bound to chemical spots were imaged using a Nikon TE2000 epifluorescence microscope equipped with a 75 W Xenon light source and an Orca-ER (Hamamatsu, Bridgewater, NJ, USA) camera [25,26]. Two custom filter sets (Chroma Technology Corporation, Brattleboro, VT, USA) composed of 650 ± 22 nm and 750 ± 25 nm excitation filters, 675 nm and 785 nm dichroic mirrors, and 710 ± 25 nm and 810 ± 20 nm emission filters were, respectively, used to detect ESNF10 (700 nm, pseudo-colored in red) and IR786 (800 nm, pseudo-colored in lime green) emission. For high-throughput imaging of microarrays, we have previously developed an automated microscope stage and software [21]. The complete scanning time for one microarray slide containing 5076 spots was approximately 2 h (1 s per spot plus stage movement time) using the automated microscope. IPLab 3.6 software (Nikon Inc., Melville, NY, USA) and ImageJ 1.45q (NIH, Bethesda, MD, USA) were used for normalization and autosegmentation of the fluorescence intensity of each spot. Sequential procedures for scoring were defined through region-of-interest (ROI) selection, static thresholding, binary image, and auto-counting. Data plotting was performed using Prism version 4.0a software (GraphPad, San Diego, CA, USA) and Microsoft Excel (Redmond, WA, USA).