*3.1. Experimental Results*

The lipid composition *cBLM*, measured by the chosen two methods, was not statistically different (Table 1). Moreover, also the reproducibility of both measuring methods were comparable. Our results, thus, confirm that these two measuring methods are equivalent.


**Table 1.** Specific capacitance of planar lipid bilayers (*cBLM*) measured by discharge and capacitance to a period converting method. The values are given as mean ± standard deviation (number of measurements).

\* *p* > 0.05.

Voltage breakdown for each lipid composition was measured by means of linearly rising electrical signal. Fast transmembrane voltage build-up was obtained by voltagecontrolled experiments, while for a slow rise of transmembrane voltage current-controlled measurements were used. In Tables 2–4, *Ubr* values are presented for fast and slow rise of transmembrane voltage, for all three lipid compositions, POPC, POPS, and POPC:POPS mixture, respectively. *Ubr* values, measured using fast rise of transmembrane voltage, follow Equation (2) (Figure 2, left part), while *Ubr* values measured using slow rise of transmembrane voltage exhibit high variability (Figure 2, right part). Therefore, we assumed that *Ubr* did not depend on the slope of linearly rising current anymore and simply calculated the mean value of breakdown voltages *Ubr I* for each lipid composition, regardless of the linearly rising current slope. The values of *Ubr I* were 0.21 ± 0.04 V, 0.38 ± 0.11 V, and 0.46 ± 0.20 V for POPC, POPS, and POPC:POPS mixture, respectively. At these conditions, the *Ubr I* values for planar lipid bilayers of different compositions are not statistically different. At slow rise of transmembrane voltage, the breakdown of planar lipid bilayer occurred in the range of seconds, which is 10<sup>4</sup> times slower in comparison with experiments using fast rise of transmembrane voltage (Figure 2). For all lipid compositions *Ubr I* was lower than *Ubrmin*, which was obtained by fitting Equation (2) to the results from experiments with fast rise of transmembrane voltage. The obtained values of *Ubrmin* were 0.42 ± 0.01 V, 0.54 ± 0.02 V, and 0.38 ± 0.01 V for POPC, POPS, and POPC:POPS mixture, respectively, while values of *t<sup>c</sup>* were 10.4 µs, 4.5 µs, 13.1 µs, presented in the same order.

**Table 2.** Results for planar lipid bilayers composed of POPC. In the upper part of the table, the results of voltage-controlled measurements with seven different slopes *k<sup>u</sup>* are gathered while the results of current-controlled measurements with five different slopes *k<sup>i</sup>* are presented in the lower part of the table. For each slope of linearly rising signal, the planar lipid breakdown voltage *Ubr* and lifetime *<sup>t</sup>br* are presented. Values are given as means <sup>±</sup> standard deviations. *<sup>A</sup>wat A* is a calculated fraction of the planar lipid bilayer that is occupied by pores. *N* is the number of experiments in each experimental group.


**Table 3.** Results for planar lipid bilayers composed of POPS. In the upper part of the table, the results of voltage-controlled measurements with seven different slopes *k<sup>u</sup>* are gathered while the results of current-controlled measurements with five different slopes *k<sup>i</sup>* are presented in the lower part of the table. For each slope of linearly rising signal the planar lipid breakdown voltage *Ubr* and lifetime *<sup>t</sup>br* are presented. Values are given as means <sup>±</sup> standard deviations. *<sup>A</sup>wat A* is a calculated fraction of the planar lipid bilayer that is occupied by pores. *N* is the number of experiments in each experimental group.


**Table 4.** Results for planar lipid bilayers composed of POPC:POPS. In the upper part of the table, the results of voltage-controlled measurements with seven different slopes *k<sup>u</sup>* are gathered while the results of current-controlled measurements with five different slopes *k<sup>i</sup>* are presented in the lower part of the table. For each slope of linearly rising signal, the planar lipid breakdown voltage *Ubr* and lifetime *<sup>t</sup>br* are presented. Values are given as means <sup>±</sup> standard deviations. *<sup>A</sup>wat A* is a calculated fraction of the planar lipid bilayer that is occupied by pores. *N* is the number of experiments in each experimental group.


**Figure 2.** Breakdown voltages of specific lipid compositions measured during voltage-controlled (**left** side) and current-controlled (**right** side) experiments. The gray lines show different slopes (*ku*—**left** side, *ki*—**right** side) of linearly rising signal. Colored curves (**left** side) represent two-parameter strength-duration curves (Equation (2)) fitted to the data obtained by voltage-controlled measurements on POPS, POPC, and POPC:POPS mixture, respectively. Colored lines (**right** side) represent the average values of measured *Ubr I* for POPC, POPS, and POPC:POPS mixture, respectively.
