Droplet microfluidics for assembly of endomembrane multicompartments

” Assembly of dsGUVs by acidification from the oil–surfactant phase via the bulk shaking method or microfluidics. (A) Representative confocal fluorescence images of pH-sensitive SUVs [DOBAQ/DOPG/DOPC/Liss Rhod B labeled-DOPE (60/20/19.5/0.5 mol %)] within W/O droplets stabilized by 1.4 wt % PEG-based fluorosurfactant and 10 mM Krytox in HFE-7500 oil that contains various concentrations of acetic acid (4, 8, 18, and 36 mM). Droplets were produced by the bulk shaking method and the aqueous phase consisted of 1.5 mM SUVs in 50 mM KH₂PO₄/K₂HPO₄, 75 mM KCl, pH 7.4. Scale bars, 50 μm. (B) Following droplet production by a bulk shaking method (i), the oil–surfactant mixture was exchanged by the acidic oil–surfactant mix supplemented with 36 mM acetic acid, thus provoking the rapid assembly of a supported lipid bilayer at the droplet periphery (ii). Scale bar, 50 μm. (C) Sequential assembly of dsGUVs via a microfluidic mechanical splitting module by entrapping SUVs in a droplet with an oil–surfactant mix without supplementing acetic acid (i) and following the substitution of the oil phase by an acidic oil containing 36 mM acetic acid (ii). Alternatively, assembly of dsGUVs produced by microfluidics splitting can be achieved through the direct usage of an acidic oil containing 36 mM acetic acid as the continuous phase (iii). Scale bars, 25 μm. To minimize droplet coalescence under acidic oil conditions, the oil phase contained 3 wt % PEG-based fluorosurfactant and 10 mM Krytox and 35 mM acetic acid in HFE-7500. (D) Assembly of multicompartment dsGUVs by microfluidic mechanical splitting through post-production acidification. Droplets encapsulated two SUV populations: 1.5 mM SUVs composed of DOBAQ/DOPG/DOPC/Liss Rhod B labeled-DOPE (60/20/19.5/0.5 mol %) and 1 mM of Q_paDOPE/ATTO 488-labeled DOPE (99.5/0.5 mol %), both in 50 mM KH₂PO₄/K₂HPO₄, 75 mM KCl, pH 7.4. The oil–surfactant mixture was composed of 3 wt % PEG-based fluorosurfactant and 10 mM Krytox in HFE-7500 (Top). Following the production and collection of the dsGUVs, the oil phase was substituted by an acidic oil, initiating the rapid and selective fusion of DOBAQ SUVs to the droplet periphery (bottom). Scale bars, 25 μm.” Reproduced under Creative Commons Attribution 4.0 International License from Lussier, F., Schroter, M. et al. pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells. ACS Synth. Biol., (2021). DOI: 10.1021/acssynbio.1c00472.