Pdc 32g 2 plasma cleaner

Dual-labeled POR reconstituted in lipid nanodiscs was immobilized on a PLL-PEG functionalized surface. The surfaces were prepared according to established protocols^{31 (link),48 (link)}. In brief, glass coverslips were cleaned, dried under nitrogen flow and plasma etched (Harrick Plasma Cleaner PDC-32G-2) for 5–10 min at 60 Pa. Immediately after plasma etching, flow chambers were assembled using 6-channel sticky slides (Ibidi) and each chamber was incubated with a 1:100 mixture of PLL-PEG-biotin/PLL-PEG in HEPES buffer (pH 5.6) for at least 1 h. Chambers were flushed with 1 mL buffer to remove excess PLL-PEG and afterwards incubated with 0.1 g/L NeutrAvidin for at least 10 min. The chambers were stored at 5 °C until further use. Prior to each measurement, excess NeutrAvidin was removed by flushing each chamber with ~1 mL buffer followed by incubation with lipid nanodisc for at least 5 min to ensure immobilization. Excess nanodiscs were removed by flushing with buffer.

3 × 5 cm² polyethylene (PE) films were cut from KitKare Ltd Clear Polythene Plastic Bags (120 gauge, 30 μm). The films were then treated in a Harrick Plasma Cleaner PDC-32G-2 set to RF level HI for 4 minutes to produce oxidised PE (PE^ox). The PE^ox films were then clamped into a film stretcher built at the University of Warwick, with the clamps set at a separation of 2.5 cm and the film oriented with the manufacturing stretch direction aligned with the film stretcher stretching direction. In some experiments the film was stretched before adding the sample thus enabling the baseline to be collected on the same film. In others (where stretch was the variable being considered) the sample was added to the unstretched film and then stretched and the baseline was a different film stretched to the same extent. The dye was then dried under vacuum for a further 15 minutes. The standard stretch amount was 1.8× its original length, 4.5 cm, unless the effect of stretching was being studied. All samples were measured using a Jasco J-1500 Circular Dichroism Spectrometer (parameters for each dye are provided in the ESI†).

Microfluidic devices were constructed by pouring polydimethylsioxane (Sylgard™ 184 Silicone Elastomer Kit, Dowsil) over the dust-free SU-8 mold, using a 1:10 mixture of the base and curing agent, following the supplier’s guidelines. Then, to remove bubbles, the freshly poured microfluidic device was placed in a desiccator for 1 h, after which it was baked at 80 °C for another hour (for curing). The microfluidic device was carefully removed from the mold using a scalpel and holes at the channel inlets and outlets were punched by hand, using a biopsy puncher (0.75 mm; WPI, No. 504529). The surface of microfluidic device was cleaned by repeatedly applying and removing the Scotch tape, which takes away most dust particles. A round cover glass (diameter = 50 mm; Menzel-Gläser, Braunschweig, Germany) was bound to the cleaned surface after treating both the cover glass and microfluidic device with oxygen plasma for 30 s at full power (PDC-32G-2 Plasma Cleaner, Harrick Plasma, New York, USA). Finally, the microfluidic device was heated on a plate at 100 °C for 1 min to remove moisture that was released during the binding process.