Incorporation of EB into cellular membranes represents the basis for its action as an ionophore. Since Na + and K + are the most prevalent cations in the extracellular medium and K + is the cation that EB has the highest selectivity towards, we studied their influence on the EB incorporation mechanism. To analyse the effect, we constructed tBLMs using POPC as lipid. These model membranes were assembled on a phaseseparated mixed self-assembled monolayer (SAM) composed of 6-mercaptohexanol and the cholestanol-tethered WK3SH (Fig. ), and characterized using SEIRA and electrochemical impedance spectroscopy, similar to our previous studies. EB was added at a concentration of 100 µM into the supernatant solution containing 100 mM of Na + /K + that covered the tBLM. SEIRA spectra were recorded taking the spectrum of the tBLM with the same ions as reference. Due to the distance-dependent decay of the enhancement in SEIRA (decaying with d -6 ), bulk water does not or only weakly interferes with the spectrum. Therefore, almost exclusively spectral changes originating from EB (or lipids) are observed in the SEIRA spectrum in the 1850 -1500 cm -1 region. In certain cases, these spectral features may overlap with minor changes of water molecules at the membrane/solution interface occurring at ca. 1650 cm -1 (H2O bending mode). The correction of such an overlap is straightforward due to the broad width of this spectral feature compared to the much narrower bands of EB and lipids, and the intense H2O stretching modes at ca. 3400 cm -1 (Figure ). Figure shows SEIRA spectra monitoring the incorporation of EB into the tBLM in the presence of Na + or K + . In both cases, two bands are observed that can be readily assigned to the C=O stretching modes of the esters and amides (spectral position denoted as ν(COester)/ν(COamide) in the following), i.e. at 1746/1659 cm -1 for Na + and 1747/1665 cm -1 for K + (see DFT section below), showing evidence for association of EB with the membrane. In contrast to other "regular" peptides, in which amide I and amide II modes are characteristic marker bands in the IR spectrum, EB does not have an amide II mode (~1550 cm -1 ) due to the methylation of the amide nitrogen (Figure ). The differences in the exact positions of these modes will be discussed later in this work. A major contribution of lipid C=O modes (at ca. 1735 cm -1 ) to the EB ester mode can be excluded based on the narrow width of the EB-ester mode. In addition, we repeated this experiment in the absence of any ions, i.e. with water as supernatant solvent, but did not detect any spectral features specific to EB (Figure ).