Fabricating microarrays of functional proteins using affinity contact printing.

Supporting Information Proteins were from Sigma and used as received. Antibodies were labelled either with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC). Solutions of proteins and adsorption experiments were done in PBS or in PBS-containing 1% BSA. Bis(sulfosuccinimidyl)suberate (BS 3) was from Pierce, aminopropyltriethoxysilane (APTS) from Fluka. PDMS stamps were made of Sylgard 184 ™ (Dow Corning), cured against the bottom of polystyrene dishes (Falcon). Si structures for μFNs and subtractive printing were microfabricated. Microwells were made by photopatterning a resist, which had been spin-coated onto a Si(100) wafer, and by anisotropically etching Si with KOH using the resist as a mask. The resist was removed, and the top and bottom surfaces of the μwells were rendered hydrophobic by microcontact-printing perfluorodecyltrichlorosilane (Gelest). The μwells had an access area of 400×400 μm 2 , a contact area of 100×100 μm 2 with the substrate, and a density of 100 wells on a 5×5 mm 2 area. PDMS stamps were oxidized using an O 2 plasma for 10 s, placed in a 10% solution of APTS in water (acidified to pH 6 with acetic acid), and refluxed for 1 h at 80 °C. The stamps were then thoroughly rinsed with water, and covered with a 1 mM solution of BS 3 in water for 10 min. The stamps were rinsed with water once more, dried under a stream of N 2 , and used immediately. The μwells (Figure 2) were placed on an activated stamp, and filled with ~50 nL of a 1 mL g 200 − µ solution of capture proteins using a micropipetting robot (Probot, BAI GmbH). After 10 min, the μwells were carefully rinsed with water and separated from the stamp under a 1% solution of BSA in PBS. Unreacted BS 3 was quenched by leaving the solution of BSA for 10 min. Irrespective of the preparation method of the α-stamps, loosely bound capture proteins were removed in one print onto a polystyrene surface. Lines of proteins were deposited onto an activated stamp using μFNs (Figure 3) and 200 μg mL –1 solutions of proteins in PBS. The μFN was removed from the PDMS stamp under a 1% solution of BSA in PBS. The patterned PDMS stamp was then contacted with a flat activated stamp for 10 min. Both stamps were separated, and the activated stamp, which retained the proteins, was exposed to a 1% BSA solution for 10 …