Monodisperse Liposomes with Femtoliter Volume Enable Quantitative Digital Bioassays of Membrane Transporters and Cell-Free Gene Expression

Digital bioassays have emerged as a new category of bioanalysis. However, digital bioassays for membrane transporter proteins have not been well established yet despite high demands in molecular physiology and molecular pharmacology due to the lack of biologically functional monodisperse liposomes with femtoliter volumes. Here, we established a simple and robust method to produce femtoliter-sized liposomes (femto-liposomes). We prepared 10⁶ monodispersed water-in-oil droplets stabilized by a lipid monolayer using a polyethylene glycol-coated femtoliter reactor array device. Droplets were subjected to the optimized emulsion transfer process for femto-liposome production. Liposomes were monodispersed (coefficient of variation = 5–15%) and had suitable diameter (0.6–5.3 μm) and uniform volumes of subfemtoliter or a few femtoliters; thus, they were termed uniform femto-liposomes. The unilamellarity of uniform femto-liposomes allowed quantitative single-molecule analysis of passive and active transporter proteins: α-hemolysin and F_oF₁-ATPase. Digital gene expression in uniform femto-liposomes (cell-free transcription and translation from single DNA molecules) was also demonstrated, showing the versatility of digital assays for membrane transporter proteins and cell-free synthetic biology.