Al 1000 220

Cell lysates containing TK ranging from 20% to 28% (w/w) were loaded at a protein concentration of 14.8 ± 2.6 mg ml⁻¹ and 14.3 ± 3.1 mg ml⁻¹ for the 1-step and 3-step immobilised microfluidic devices, respectively. The lysates were pumped into the microchannels using a syringe pump AL 1000-220 (World Precision Instruments, USA) at a flow rate of 5 μl min⁻¹ at 4 °C for several channel volumes. After 1 h a solution of 50 mM Tris-HCl, pH 7.5 was pumped through the microchannels at 20 μl min⁻¹ to remove non-specifically bound enzyme. Samples were collected periodically and assayed for protein content. After operational stability studies, the bound enzyme was removed by treating the channel with EDTA elution buffer (50 mM EDTA; pH 8.0) at 20 μl min⁻¹ for at least 2 channel volumes. Collected samples were concentrated down to 75 μl volume using Amicon Ultra Centrifugal filters (30,000 NMWL).

A PMMA microfluidic device channel was functionalised and immobilised with His-tagged enzyme according to protocols adapted from Fixe et al. and Oshige et al. [[34] , [36] (link)]. Briefly, a channel in the PMMA microfluidic device was flushed and filled with isopropanol (99%) and incubated at 30 °C for 3 h. The microfluidic device channel was then rinsed thoroughly with Milli-Q water and incubated with 10% (v/v) hexamethylene-diamine (HMDA) in 100 mM borate buffer pH 11.5, for 2 h. The channel was then thoroughly flushed with Milli-Q water for several channel volumes. Afterwards, the channel was incubated with 1% (v/v) glutaraldehyde overnight at 37 °C. Another overnight incubation at 37 °C was done with a 0.05% (w/v) solution of N-(5-amino-1-carboxy-pentyl) iminodiacetic acid (AB-NTA) in 0.1 M HEPES buffer, pH 8.0. Then, the channel was flushed with Milli-Q water using syringe pump AL 1000-220 (World Precision Instruments, USA) at flow rates of 20 μl min⁻¹. Finally, a solution of 0.5 M NiCl₂ was pumped through the microchannel at a flow rate of 10 μl min⁻¹ for 1 h followed by a Milli-Q wash.

Once characterization of the SLB was complete, the sample surface was placed in the correct solution isotopic contrast (D₂O for h-proteins and H₂O for d-proteins) and ~6 ml of a Bax solution (0.1 mg/ml) was injected into the flow cell (the cell volume is 3 ml) either by hand (SURF) or using a syringe pump (OFFSPEC, AL1000-220, World Precision Instruments, Hitchin, UK). In most cases, the interaction of the protein with the SLB was monitored by NR with datasets collected continuously until an equilibrium interaction between the protein and the SLB was verified by no further changes in the data being observed against time. At this point, a final equilibrium dataset was collected, and, then, the excess protein was flushed from the cell and the structure of the surface protein-lipid complex was examined by NR under three solution isotopic contrast conditions (D₂O, Si-MW, and H₂O). It should be noted that no difference was found in any sample between the equilibrium Bax–bound data before and after flushing of the excess protein, suggesting the protein-lipid complexes formed at the sample surface were stable.