Ionic strength and composition govern the elasticity of biological membranes. A study of model DMPC bilayers by force- and transmission IR spectroscopy Suzana Šegota a, *, Danijela Vojta b , Galja Pletikapi  c a , Goran Baranovi  c b a Division for Marine and Environmental Research, Ruper Boškovi c Institute, POB 180, Zagreb, Croatia b Division of Organic Chemistry and Biochemistry, Ruper Boškovi c Institute, POB 180, Zagreb, Croatia A R T I C L E I N F O Article history: Received 30 April 2014 Received in revised form 5 November 2014 Accepted 11 November 2014 Available online 13 November 2014 Keywords: AFM Force spectroscopy Ion binding Ion mixture IR spectroscopy Lipid bilayers Nanomechanics Seawater A B S T R A C T Infrared (IR) spectroscopy was used to quantify the ion mixture effect of seawater (SW), particularly the contribution of Mg 2+ and Ca 2+ as dominant divalent cations, on the thermotropic phase behaviour of 1,2- dimyristoyl-sn-glycero-3-posphocholine (DMPC) bilayers. The changed character of the main transition at 24  C from sharp to gradual in films and the 1  C shift of the main transition temperature in dispersions reflect the interactions of lipid headgroups with the ions in SW. Force spectroscopy was used to quantify the nanomechanical hardness of a DMPC supported lipid bilayer (SLB). Considering the electrostatic and ion binding equilibrium contributions while systematically probing the SLB in various salt solutions, we showed that ionic strength had a decisive influence on its nanomechanics. The mechanical hardness of DMPC SLBs in the liquid crystalline phase linearly increases with the increasing fraction of all ion-bound lipids in a series of monovalent salt solutions. It also linearly increases in the gel phase but almost three times faster (the corresponding slopes are 4.9 nN/100 mM and 13.32 nN/100 mM, respectively). We also showed that in the presence of divalent ions (Ca 2+ and Mg 2+ ) the bilayer mechanical hardness was unproportionally increased, and that was accompanied with the decrease of Na + ion and increase of Cl  ion bound lipids. The underlying process is a cooperative and competitive ion binding in both the gel and the liquid crystalline phase. Bilayer hardness thus turned out to be very sensitive to ionic strength as well as to ionic composition of the surrounding medium. In particular, the indicated correlation helped us to emphasize the colligative properties of SW as a naturally occurring complex ion mixture. ã 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Biophysical behaviour of lipid membranes is of fundamental importance due to their role as flexible mechanical barrier, which enables controlled exchange of information, substances and energy between the cell interior and its environment. Since the scaffold of a cell membrane are lipid bilayers, they not only determine the structure of the cell membrane but also are involved in a variety of mechanical events such as cell adhesion, fusion, growth and migration. Due to the high complexity of cell membranes, model membranes such as liposomes and supported lipid bilayers (SLBs) have been extensively used. In liposomes, a bilayer of lipid molecules forms a spherical shell, separating intraliposomal liquid from the bulk solvent (Šegota and Težak, 2006). In case of SLB, the bilayer rests on a solid surface and the nature of interactions between them is determined by the properties of the lipid molecules and the supporting surface as well as the nature of the dispersing medium. The lipid composition of the membrane is one of the determining factors in membrane interactions with surrounding molecules or ions which further alter the structure, stability and functions of the membrane itself. Membrane properties such as the surface potential (Eisenberg et al., 1979), the dipole potential (Clarke and Lüpfert, 1999), structure and mechanical strength (Sachs et al., 2004; Miettinen et al., 2009) are tightly associated with ions that are present in the cell interior and its environment as well. Therefore, the study of interactions of ions with the lipid bilayer, e.g. ion binding to headgroups whereby lipid–ion networks are formed (Fukuma et al., 2007) is of considerable interest. It is also well established that the ion binding reduces the area per lipid for both negatively charged and zwitterionic phospho- lipids (Böckmann and Grubmüller, 2004; Mukhopadhyay et al., * Corresponding author. Tel.: +385 1 456 1128; fax: +385 1 468 0242. E-mail address: ssegota@irb.hr (S. Šegota). http://dx.doi.org/10.1016/j.chemphyslip.2014.11.001 0009-3084/ ã 2014 Elsevier Ireland Ltd. All rights reserved. Chemistry and Physics of Lipids 186 (2015) 17–29 Contents lists available at ScienceDirect Chemistry and Physics of Lipids journal homepa ge: www.elsev ier.com/locate /chemphyslip