Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.
Total time
~4–6 hours per experiment (including liposome preparation, bilayer formation, and stimulation trials)
Steps
1
Prepare liposomes containing mechanosensitive channels
Prepare liposomal suspensions incorporating bacterial mechanosensitive ion channels (e.g., MscL) through standard extrusion or rehydration methods. Verify liposome size and channel incorporation before proceeding.
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2
Manufacture oil reservoir and prepare electrodes
Prepare the oil phase (mineral oil or equivalent) and assemble electrode apparatus for electrical measurements across the forming bilayer. Calibrate electrode positioning and impedance.
▶ 02:00
3
Set up experiment with micropipettes and imaging
Position micropipettes, oil reservoir, and recording electrodes under microscopy. Calibrate mechanical stimulation apparatus and verify electrical connectivity before bilayer formation.
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4
Form lipid bilayer at droplet interface
Use micropipettes to bring liposome-containing aqueous droplets into contact within the oil reservoir, forming a planar lipid bilayer. Optimize contact geometry for stable bilayer formation.
▶ 06:22
5
Apply harmonic mechanical stimulation and record channel activity
Deliver calibrated harmonic mechanical stimuli via micropipette displacement while recording ion channel currents. Measure mechanosensitive channel opening and gating kinetics in response to mechanical stress.
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