The polymers 1,2-polybutadiene-b-poly(ethylene oxide) (1,2-PBd-PEO) of Mw= 10 kg/mol (PDI= 1.15) and wEO= 0.40, and poly(isobutylene) (PIB) of Mw= 0.9 kg/mol (PDI= 1.3) were obtained from Polymer Source, Inc. (Canada), and were used as received. Bovine serum albumin (BSA) was purchased from Sigma and used as received. Glass coverslips were purchased from Fisher. Trypsin-EDTA solution, and Dulbecco's modified Eagle's medium (DMEM) were obtained from ATCC.
Synthesis of Poly(butylene)-b-poly(ethylene oxide) (PB-PEO)
PB-PEO of Mw= 10.2 kg/mol (PDI= 1.14) and wEO= 0.39 was produced by homogeneous hydrogenation of 1,2-PBd-PEO following the procedure of Hahn et al. . The PB-b-PEO polymer was modified to the amine reactive succinimidyl ester NHS-ester derivative (PB-b-PEO-NHS) by treating with disuccinimidyl carbonate and base as described in Kourouklis et al..The chemical structures of the used polymers are also presented in supplemental information S3.
Fabrication of supported block copolymer films
PB-PEO block copolymer films were assembled at the air/water interface by means of Langmuir trough (MicrotroughXS, Kibron Inc., Finland). Langmuir-Blodgett (LB) technique was employed to transfer the polymer film on a glass coverslip that is elevated across (Figure 1a) the polymer film. The glass coverslip was previously rinsed with ethanol (200 proof) and undergone UV O2 plasma irradiation (Harrick, 200 millitorr, 10 min). Langmuir-Schaefer (LS) technique was used to transfer a second monolayer through horizontal contact between the supported and the interfacial PB-PEO block copolymer films (Fig. 1B). To ensure efficient microcontact printing of fibronectin (FN) on supported bilayer films, the top monolayer contained 5 n/n% of NHS-ester modified PEO blocks that conjugate with the free amines of fibronectin. To strengthen the fluid character of the polymer film, trace amount (1 n/n%) of polyisobutylene (PIB) homopolymer was mixed with block copolymers during the LB fabrication step; such films are hereafter referred to as “doped” (3.5×10-10cm2/s). Films without polyisobutylene show lower lateral mobility of polymer chains and are referred to as “neat” (1×10-10cm2/s).
PDMS stamp preparation and contact printing
Microcontact printing was employed to create well defined protein patterns on supported polymer films. Polydimethylsiloxane stamp (PDMS, Sylgard 184, Dow Corning GmbH) created by casting over a silicon master mold (12 h at 65°C) has a post diameter of 18 μm (l), an edge-to-edge distance between the posts (f) of 9 μm and a post height (h) of 8 μm. The aspect ratio (l/h) was selected to be ≥0.2 in order to diminish defects on the printed patterns due to the mechanical distortions of the stamp. Stamps were treated with oxygen plasma in a Trion Phantom III ICP/ RIE etcher (10 s, 20 mT, 30 sccm O2 and ICP and RIE power of 20 and 30 W respectively) to control the wetting and adsorption of fibronectin solution.
FN solution (HiLyte 488, Cytoskeleton Inc., 50 ul of 100 ug/mL) in phosphate buffer saline (PBS) was added over the PDMS stamp for 5 min (Fig. 1C). Excess FN solution was removed by stamp inversion and wicking followed by air-drying. The FN-coated PDMS stamp (1.0×0.5×0.5 cm3) was then slowly released over the supported polymer film without any compressive forces (Fig. 1D). In the absence of strong adhesion forces due to the hydrophylic (PEO) outer surface of the polymer film, the transfer of the FN pattern depends on the chemical conjugation between NHS-esters and FN free-amines. Under these conditions and a stamp aspect ratio of f/h ≤ 20, FN stamping onto the polymer film resulted to the formation of film-conjugated FN dots without significant sagging effects (Fig. 2B).