*2.2. Experimental Setups*

Planar lipid bilayers were formed by the Montal–Muller method [28] across a circular hole 117 µm in diameter (d). The hole was in a thin Teflon sheet sandwiched between two reservoirs in the Teflon chamber. The glass window in the Teflon chamber allowed easy viewing of the hole during the formation of the planar lipid bilayer using stereo microscope with led illumination. Both sides of the hole in the Teflon sheet were first treated with 1 µL of the lipid solution. After the lipid solvent had completely evaporated, the hole was treated on each side with 1.5 µL of the hexadecane and pentane mixture. Both reservoirs were filled with electrolyte just below the hole. A drop of 2 µL of the lipid solution was carefully applied to the electrolyte surface in each reservoir and allowed to spread for at least 15 min. To form a planar lipid bilayer, the electrolyte level in both reservoirs was raised to the same height just above the hole (Figure 1A). After a planar lipid bilayer was ruptured, a new one was formed by lowering the electrolyte levels below the hole, and then raising them again. All experiments were performed at room temperature (24 °C).

The voltage-controlled measurement system [29] consisted of a signal generator, two Ag-AgCl electrodes (E-205, IVM, Healdsburg, CA, USA) immersed in the electrolyte, one on each side of the planar lipid bilayer, and amplifiers connected to an oscilloscope for measuring transmembrane current and voltage. The electrodes were used to apply the transmembrane voltage and measure the transmembrane current. The transmembrane voltage was measured using the LeCroy differential amplifier 1822 and custom made ampere meter was used for transmembrane current measurements. Both signals were stored in Matlab format using LeCroy Waverunner-2 354M oscilloscope and analyzed afterwards.

The current-controlled experiments were performed using a potentiostat-galvanostat measurement system controlled by a computer as described in [20]. Four Ag-AgCl electrodes (E-205, IVM, Healdsburg, CA, USA) immersed in the electrolyte were used, two on each side of the planar lipid bilayer. One pair of electrodes was used to apply current and the other was used as a reference electrodes. The transmembrane voltage measurements were stored on a computer in a raw file and later imported into Matlab for further analysis.

**Figure 1.** The measurement protocol consisted of formation of the planar lipid bilayer (**A**), capacitance measurement (**B** or **C**), and measurement of the breakdown voltage *Ubr* of the planar lipid bilayer (**D** or **E**).
