Encapsulated droplet interface bilayers as a platform for high-throughput membrane studies

Abstract

Whilst it is highly desirable to produce artificial lipid bilayer arrays allowing for systematic high-content screening of membrane conditions, it remains a challenge due to the combined requirements of scaled membrane production, simple measurement access, and independent control over individual bilayer experimental conditions. Here, droplet bilayers encapsulated within a hydrogel shell are output individually into multi-well plates for simple, arrayed quantitative measurements. The afforded experimental throughput is used to conduct a 2D concentration screen characterising the synergistic pore-forming peptides Magainin2 and PGLa. Maximal enhanced activity is revealed at equimolar peptide concentrations via a membrane dye leakage assay, a finding consistent with models proposed from NMR data. The versatility of the platform is demonstrated by performing in situ electrophysiology, revealing low conductance pore activity (∼15 to 20 pA with 4.5 pA sub-states). In conclusion, this array platform addresses the aforementioned challenges and provides new and flexible opportunities for high-throughput membrane studies. Furthermore, the ability to engineer droplet networks within each construct paves the way for "lab-in-a-capsule" approaches accommodating multiple assays per construct and allowing for communicative reaction pathways.

Fig. 1. Encapsulated droplet interface bilayers (eDIBs) are nested droplet structures of water droplets in oil within a hydrogel shell. (a) Microfluidic device employed to produce eDIBs. (b) (i) Image of eDIB containing 10 aqueous cores. These cores are dyed with sulphorhodamine B for contrast. (ii) Diagram of an eDIB containing quenched calcein loaded aqueous cores, showing the presence of droplet interface bilayers between the internal cores as well as between the internal cores and the hydrogel shell. Scale bar = 1 mm.

Fig. 2. Individually addressable encapsulated droplet interface bilayers (eDIBs) in multi-well plate arrays. (a) (i) eDIBs in the outlet tube of their microfluidic production device are output into individual wells of a multi-well plate (ii) side view (left) and top-down view (right) of an individual eDIB within a plate well. (b) eDIBs reside in aqueous buffer in the well which is in diffusive contact with the encapsulated lipid bilayers enabling direct experimental additions to each well to interact with the lipid bilayer. A mineral oil layer prevents evaporation and allows storage. Self-quenched calcein (orange) within the internal aqueous droplets of eDIBs fluoresces upon leakage. (c) eDIB array demonstrated with fluorescent response in the well (yellow) to SDS induced bilayer permeabilisation upon exposure to increased SDS concentrations.

Fig. 3. Fluorescence response from individual droplet interface bilayers (eDIBs) in arrayed multi-well plate measurements. (a) Arrayed eDIBs measured by quantitative fluorescent plate reader either by well scanning (imaging) or whole-well fluorescence providing spatial and temporal fluorescence data on calcein leakage and membrane integrity. (i) intact bilayers in eDIBs not exposed to SDS exhibit no appreciable change in fluorescence (n = 8). In eDIBs exposed to 8 μM SDS two types of fluorescent response are observed, either (ii) steady increase in fluorescence due to calcein leakage through membrane pores, or (iii) rapid increase in fluorescence due to calcein leakage on bilayer rupture, reflecting the stochastic nature of membrane pore formation and membrane failure (b) leakage of calcein from individual eDIBs into the aqueous volume within a well, when exposed to 8 μM SDS (red) and in the absence of SDS (black). The leaked calcein concentration is calculated from fluorescence intensity calibration (ESI†). (c) High content webcam simultaneous capture of individual eDIBs in wells of 6 × 96 well-plates imaged under blue LED illumination.

Fig. 4. Characterisation of the synergistic activity of the membrane pore forming peptides Magainin 2 and PGLa by a 2D-concentration screen array. (a) Example eDIB 2D array screen of increasing Magainin 2 (rows) and PGLa (columns) concentrations with increased visible fluorescence response observed due to rupture of the eDIB membranes containing quenched calcein and its subsequent dilution and dequenching in the external aqueous phase of the well. The peptide concentrations are as shown in (b). (b) Activity heatmap of peptide activity across the 2D combinatorial space. Heatmap reflects the proportion of eDIBs evidencing fluorescent increase associated with membrane rupture and calcein release (n = matrices of 64 conditions each, total 384 eDIBs, (a) constitutes one of such experiments). (c) Interpolated heatmap of relative activity with total peptide concentration (x-axis) and Magainin 2: PGLa ratio (y-axis). PGLa is found to be more active than Magainin 2 in the eDIB DPhPC membranes, with the two peptides displaying synergistic activity in combination, maximized at 1 : 1 ratio. (d) ED₅₀ plot depicting the effective total peptide concentration required to elicit 50% maximal response across peptide mixing ratios. Maximal synergy at equimolar concentrations is consistent with the proposed direct stoichiometric interaction between Magainin 2 and PGLa in their combined mechanism of membrane activity.

Fig. 5. Electrophysiology of Magainin 2 (1.75 μM) and PGLa (1.75 μM) pores in eDIB membranes. (a) Electrophysiology can be performed in situ on eDIBs in selected wells of the array. Custom Ag/AgCl electrodes are inserted into internal aqueous droplets of eDIBs and placed in the external well environment. (b) Top and side view of electrode insertion viewed in a transparent 96-well plate. (c) Resultant single channel recording of membrane spanning pores in eDIBs on exposure to Magainin 2 (1.75 μM) and PGLa (1.75 μM) under an applied potential of −30 mV. Major, long-lived, conductance states of ∼15 to 20 pA are observed alongside shortlived step-like fluctuations of lower magnitude (∼4.5 pA). Histogram analysis of measured conductance states over short and long timescales is provided in the ESI. Scale bars = 1 mm.