Advanced microfluidic tool for rapid buffer exchange and in situ imaging of phase separation in immobilized giant unilamellar vesicles

Abstract

Semi-permeable Giant Unilamellar Vesicles (GUVs) are powerful tools for studying macromolecular assembly, notably liquid-liquid phase separation (LLPS). Here, we present a microfluidic protocol for GUVs production, immobilization and rapid buffer exchange. It enables real-time imaging of phase separation within isolated GUVs with various microscopy technics (optical, confocal and UV-autofluorescence). The principle of this device is based on the production of water-in-oil-in-water double emulsion droplets and their turning into GUVs by elimination of their oil phase. The immobilization of GUVs is provided by physical and chemical traps, using the strong biotin-streptavidin affinity. These vesicles become semi-permeable to small molecules (< 2000 g/mol) viathe α-hemolysin pore protein. Within minutes, the internal GUV phase can be changed using different external buffers. This provides a way to induce the assembly of macromolecules and monitor overtime the dynamic of phase separation. In particular, we show, on a specific example, the occurrence of LLPS through both nucleation and growth and spinodal decomposition. The protocol can be divided into two distinct phases: (1) the chip fabrication spans approximately two days but yields approximately 10 chips, and (2) the GUV production, immobilization, and buffer exchange, can be completed in just half an hour. This microfluidic tool represents a significant advance by offering a faster and more controlled buffer exchange as compared to alternative methods. It opens new routes to investigate the dynamics of macromolecular assembly, particularly in the context of LLPS.