Zepto plasma cleaner

We fabricated the microfluidic chips following standard soft lithography techniques. More specifically, for the 24- and 48-h long biofilm experiments, we designed 1-cm long, 500 or 250-μm wide channels in Autodesk AutoCAD 2018 and printed them on a soft plastic photomask. We then coated silicon wafers with photoresist (SU8 2025, Microchem), with varying thicknesses (25, 50, and 90 μm) to allow a wider range of mean flow velocities for identical flow rate settings. The wafer was exposed to UV light through the mask and developed in PGMEA (Sigma-Aldrich) in order to produce a mold. PDMS (Sylgard 184, Dow Corning) was subsequently casted on the mold and cured at 80 °C for about 1 h 30 min. After cutting out the chips, we punched 1 mm inlet and outlet ports. We finally bonded the PDMS chips to glass coverslips (Marienfeld 1.5) in a ZEPTO plasma cleaner (Diener electronic). To fabricate channels for the 6-day long biofilm experiments, we followed a similar procedure, but adjusted the dimensions of the channel to leave more space for large 3D structures to form. More precisely, the channel was 2 mm wide, 110 μm high.

PDMS (Sylgard 184, Dow Corning) was casted on the mold and cured at 60°C for about 1 h and 30 min. We then used a scalpel to cut out each chip individually and carefully remove it from the mold. In parallel, we prepared PDMS rods to pattern the lumens according to a published protocol [34 (link)]. In short, we filled gauge 14 needles (Sterican 2.1 × 80 mm, B. Braun) with PDMS and cured it as described above. We then used pliers to break the needles and extract the PDMS rods; their diameter was approximately 1.6 mm (i.e., inner diameter of the needle). We used a scalpel to cut them into 8–mm long pieces. Then, PDMS chips and rods were briefly immersed in isopropanol, left to dry, and cleaned using tape. Afterwards, the rods were inserted into the chips using tweezers; the assembled devices were subsequently autoclaved. They were then plasma bonded to either glass–bottom dishes or glass–bottom 6–well plates (1.5 coverslip, glass diameter 20 mm, MatTek) in a ZEPTO plasma cleaner (Diener electronic). Note that the chips contained thin PDMS membranes at the bottom of their inlet reservoirs (obtained owing to a shallow cavity in the 3D printed mold), so that the rod was not in direct contact with the underlying coverslip. Finally, we exposed the chips to 2 cycles of UV sterilization in a biosafety cabinet.

Glass coverslips (22 × 22 mm, Marienfeld) were cleaned with a Zepto plasma cleaner (Diener Electronic) and incubated in acetone containing 2 % (v/v) 3-aminopropyltriethoxysilane for 5 min. Silanized coverslips were rinsed with ddH₂O, dried and baked at 110°C for 30 min. Coverslips were then covered with a fresh solution of 0.4 % (w/v) Biotin-PEG-Succinimidyl Carbonate (MW 5,000) and 15 % (w/v) mPEG-Succinimidyl Carbonate (MW 5,000) in fresh 0.1 M NaHCO₃ and incubated overnight at room temperature. Coverslips were rinsed with ddH₂O, dried and incubated again with in a fresh Biotin-PEG/mPEG solution as described above. Functionalized PEG-Biotin microscope slides were again washed and dried and finally stored under vacuum.