Buoyancy-driven sorting of synthetic cells for nanopore activity

Abstract

The development of sorting strategies that directly report on functional activity remains a bottleneck in synthetic cell research. Current methodologies typically rely on sequential label-dependent probing, which limits throughput. Here, we introduce a label-free, buoyancy-driven selection strategy in which the mode of separation and the mode of decision-making are intrinsically linked, coupling pore activity directly to the synthetic vesicles internal density in a one-pot assay. In this system, sorting emerges intrinsically: Giant unilamellar vesicles (GUVs) that contain a dense medium sediment by default, while only those with functional transmembrane pores undergo solute exchange, leading to density equilibration and flotation. We exploit this principle to separate pore-active from non-functional GUVs without external markers or imaging-based readouts. Using protein pores and DNA origami and DNA tile nanopores, we demonstrate that buoyancy-driven separation enables parallel functional assessment of heterogeneous populations and supports flow-based enrichment of highly active synthetic cells. By directly linking molecular transport performance to GUV buoyancy, this approach collapses decision-making into the physical separation process itself, providing a scalable platform for screening, sorting, and evolving membrane pores in synthetic cell systems.