Towards a perfusion system for functional study of membrane proteins with independent control of the electrical and chemical transmembrane potential.

The main motivation of this work was to address the challenge of single-molecule functional study of membrane proteins under stable and independently controlled electrical and chemical membrane potentials. Although transmembrane potential is often essential for the function of membrane proteins, current in vitro systems provide only limited options for studying them under biologically relevant conditions. Our experimental assay is based on the droplet-on-hydrogel bilayer technique (Leptihn et al. Nat Protoc 8:1048-1057, 2013), where a lipid bilayer forms between a sub-millimetre water droplet and a thin hydrogel layer on a glass cover slip, enabling high-resolution microscopy in total internal reflection mode. To extend the application of this assay beyond channels to other membrane proteins, we introduce a custom-built, electronically controlled perfusion system that is designed to directly connect to the droplet above the lipid bilayer. This system can supply a stable voltage to the bilayer and is suitable for delivery of fragile membrane proteins embedded in proteoliposomes via charged fusion (Ishmukhametov et al. Nat Commun 7:13025, 2016), introducing changes of chemical potentials, and timed introduction of labels or substrate into the droplet. This work represents one of the steps towards single-molecule functional study of F Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-025-01344-4.