On-the-Fly Microfluidic Control of Giant Vesicle Compositions Validated by DNA Surface Charge Sensors

The specific lipid composition of cell membrane microenvironments plays a critical role in regulating a range of cellular processes such as integral and peripheral membrane protein function, cell morphology, and membrane potential. However, harnessing similar complex capabilities in artificial membrane mimics remains challenging. In large part, progress has been slow due to a scarcity of techniques for both (i) accurately quantifying composition-dependent properties of artificial cell models at the single-vesicle level and (ii) efficiently exploring large multidimensional composition spaces. Here, we address both challenges by first developing an assay for quantitatively sensing giant unilamellar vesicle (GUV) membrane surface potentials using a fluorescent cholesterol-labeled DNA duplex sensor. We then devised a microfluidic vesicle assembly line enabling the continuous, on-chip production of lipid vesicles with variable compositions. This enabled real-time, on-the-fly adjustment of membrane compositions and biophysical properties as vesicles were being produced, followed by membrane analysis using our assay. Analysis of the association of our DNA fluorescent probe with single vesicles reveals that we may quantify the surface potential of vesicle membranes <i>in situ</i> through quantification of the membrane-probe binding constant. Our work paves the way for the production and biophysical analysis of artificial cell libraries that can enable rational artificial cell engineering.