Irdye 800cw

Milli-Q water (18.2 MΩ/cm, Millipore Co., Bedford, MA, USA) was used for the preparation of aqueous solutions. Trypan blue solution 0.4% (w/v) and infrared dye (IRDye 800CW) were purchased from Sigma Aldrich and LI-COR (Lincoln, NE, USA), respectively. Polydimethylsiloxane (PDMS, Sylgard 184) was obtained from Dow Corning (Midland, MI, USA). 10 mM phosphate buffer (7.7 mM Na₂HPO₄, 2.3 mM NaH₂PO₄, pH 7.4) was prepared in the laboratory. Hyaluronan (sodium hyaluronate, average Mw was 150 kDa) was purchased from Lifecore Biomedical (Chaska, MN, USA). Vinylpolysiloxanes A-silicone (Elite Double 32a Normal) was purchased from the Zhermack Group (Badia Polesine, Italy) and two-component epoxy glue from Bison International B.V. (Goes, The Netherlands). 4 × 4 silicon microneedle arrays were obtained from Tyndall National Institute (Cork, Ireland), 24 × 24 silicon microneedle arrays were gifted from Bosch GmbH (Stuttgart, Germany), and ceramic alumina microneedle arrays (MLT-200 and MLT-475) were obtained from MyLife Technologies (MLT, Leiden BioScience Park, Leiden, The Netherlands). Conventional transparent tape Avery 440 Gloss Transparent Removable was obtained from Avery Dennison (Glendale, CA, USA).

Cetuximab (Erbitux, Ely Lily; 2 mgml^-1, 145781.6 g/mol as per FASTA sequence analysis) was conjugated to IRDye800CW (IRDye800CW-NHS Ester; Li-COR, 1166.20 g/mol) by amide coupling. A stock solution of IRDye800CW-NHS Ester (10 mg·ml^-1) was prepared in anhydrous Dimethyl Sulfoxide (DMSO; Sigma-Aldrich) and added to Cetuximab (2 mg·ml^-1 in phosphate buffer) at a quantity corresponding to a 10-fold molar excess of IRDye800CW to each molecule of Cetuximab. The mixture was thoroughly mixed using orbital rotation for 24h at room temperature. After conjugation, the Cet-IRDye800 was purified by removing unconjugated dye using PD-10 Desalting Columns (Cytivia) pre-equilibrated with sterile 1X DPBS (Corning). Purified Cet-IRDye800 was collected and concentrated in a 30kDa ultrafiltration tube (EMD Millipore) by centrifugation at 2,500 xg for 20 min at 4°C. The conjugated Cet-IRDye800CW was then stored at 4°C in the dark.