However, the close proximity of SLBs to the substrate affects the diffusion of lipids and proteins, which is more than twice slower than in free-standing bilayers under the same conditions [67]. This limitation is due to the fact that the substrate exerts a greater influence on the behavior of the proximal leaflet than the distal leaflet of the SLBs, due to its closer proximity to the surface ^[40][68][69][40,68,69]. The roughness of the substrate and the complementarity between the surface and lipid charge will determine the magnitude of this surface influence [50]. Furthermore, SLBs suffer from the crucial drawback of not possessing a large hydration reservoir on both sides of the membranes, which limit examination of transmembrane proteins. The fundamental requirement for a membrane protein to function properly is to be surrounded by buffered-saline solution on both sides of membranes [70]. In SLBs, hydration layer is often not thick enough for proper folding of large extra-membranous domains of transmembrane proteins, which can extend to several tens of nanometers far out from the bilayer [18]. The limited membrane-substrate distance, which can lead to strong non-physiological interactions with the solid support ^[18][20][18,20], can cause both a loss of protein dynamics and a partial loss of its functionality, or even complete denaturation of the protein ^[71][72][73][71,72,73]. In addition, anionic substrates (such as quartz, mica, silica, silicon oxide) may hinder (in the absence of divalent cations) the formation of SLBs enriched in negatively charged lipids in the proximal leaflet, due to electrostatic repulsions ^[46][66][74][46,66,74]. However, negatively charged lipids, like phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipin (CL) or even lipopolysaccharide (LPS) in the Gram-negative bacteria membrane, are important signaling lipids which can trigger membrane protein association and affect membrane-regulated pathways. They are essential in membrane function, and studies of the membrane phenomenon regulated by these lipids are becoming crucial for a realistic understanding of membrane-related events. The use of mimetic sample systems with ever greater biological precision in lipid composition is now required.

For all these reasons, more advanced planar model membranes are currently in development with the aim to create more accurate biomimetic systems adapted for integral (trans-)membrane protein characterization, where the substrate interactions are minimized and large solution reservoirs on both sides of the bilayer are provided. In tBLMs, the lipid bilayer is separated from the surface of the substrate by insertion of a soft and flexible hydrophilic layer of “tethering” molecules that anchor the proximal leaflet ^{[31][34][71][72][75][76]}[31,34,71,72,75,76]. This layer solves the substrate proximity by lifting the membrane off the surface and provides a reservoir underneath the bilayer in which the membrane proteins can fold into a native-like conformation, while keeping the membrane anchored to the support (Figure 1).

Figure 1. Different types of tethered-Bilayer Lipid Membranes (or tBLMs) obtained by a “bottom-up” approach. : represents either anchorlipid (i.e., Telechelics) mainly 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-d,l-α-lipoic acid ester lipid (DPhyTL), lipopolymer tethers or thio(lipo)peptides; : Backfillers, mainly β-mercaptoethaonl (βME) or 6-mercaptohexanol (6-MH); : benzyl-disulfide (tetra-ethyleneglycol)_n=2 C20-phytanyl tether (or DLP); : ethyleneoxy-linked cholesterol (or EO₃C).

The large variety of assembling molecules capable of forming a tethering layer offers multiple possibilities for fine-tuning the properties of tBLMs [37]. Whatever its proper nature, the role of the anchor (spacer group) is multiple. It should at the same time (i) maintain bilayer fluidity and provide a sufficient well-hydrated sub-membrane space to accommodate incorporated proteins, (ii) cover small surface roughness features in order to reduce the hydrophobic influence of the metal surface and the unfavorable frictions to the support, (iii) provide a hydrated reservoir between the substrate and the membrane, and (iv) supply ample space to harbor membrane protein ectodomains. The different types of tBLMs vary mainly in the chemical structure of the tethers and in their density, two factors significantly influencing the structural characteristics of the bilayer as well as the functional reincorporation of membrane components [51]. Ideally, and in order to mimic a natural membrane, a tBLM should have a high electrical impedance and a low capacitance—to be sure that transport across the membrane is mainly due to the function of the embedded protein, as well as high fluidity and high sub-membrane hydration—to ensure protein function. However, increases in membrane hydration and fluidity are generally accompanied by a reduction of the electrical sealing properties, resulting from a higher defect density ^[77][78][77,78]. Subsequently, applying ultrasensitive surface imaging techniques allowing the direct characterization of all the steps of the tBLM formation with a high lateral resolution could lead to an optimization, step-by-step during the tBLM building, in order to reduce the number of defects.

3. Characterization of Tethered-Bilayer Lipid Membranes

Because they are firmly held in place, tBLMs are considerably more robust than supported lipid bilayers such as black or bilayer lipid membranes (BLMs) ^[79][80][79,80], also renamed free-standing lipid layer or suspended-lipid bilayers, which are originally formed across on a microsized-aperture, and more recently, on nanopores ^[26][27][28][26,27,28]. Generally speaking, tBLMs typically show a high robustness and long-term stability and hence, they are accessible to a portfolio of different analytical tools operating at a bulk interface [42]. They include imaging techniques, like atomic force microscopy (AFM) ^{[30][81][82][83][84][85][86][87]}[30,81,82,83,84,85,86,87] and fluorescence microscopy (FM) ^{[34][87][88][89]}[34,87,88,89], fluorescence recovering after photobleaching (FRAP) ^{[20][35][81][87][90][91][92][93]}[20,35,81,87,90,91,92,93], neutron reflectometry (NR) ^{[77][94][95][96][97][98][99][100][101]}[77,94,95,96,97,98,99,100,101] and X-ray photoelectron spectroscopy (XPS) ^{[20][82][86][87][102][103]}[20,82,86,87,102,103], spectroscopic techniques such as ellipsometry ^[94][104][94,104], infrared reflection absorption spectroscopy (IRRAS) ^{[96][105][106]}[96,105,106] or surface-enhanced infrared absorption spectroscopy (SEIRAS) ^{[107][108][109][110]}[107,108,109,110], surface plasmon resonance (SPR) ^{[30][34][35][37][39][111][112][113]}[30,34,35,37,39,111,112,113] or quartz crystal micro-balance with dissipation monitoring (QCM-D) ^{[30][39][85][112]}[30,39,85,112], as well as electrochemical methods such as electrochemical impedance spectroscopy (EIS) ^{[24][32][41][88][94][108][109][114][115][116][117]}[24,32,41,88,94,108,109,114,115,116,117] and current-voltage (CV) analysis ^{[118][119][120][121][122][123]}[118,119,120,121,122,123]. These techniques, sensitive to net changes in packing or interfacial mass (QCM-D or ellipsometry), bilayer morphology (AFM, FM), the presence of chemical groups (IR, XPS), the structure and composition (NR), have been used so far to evaluate the full picture of the lipid membranes (i.e., structure, composition and functional properties) and represent a very powerful combination to unravel the mechanism of biomolecular interactions.

While SPR and QCM-D allow real-time monitoring of the tBLM formation in a label-free format, fluorescence microscopy (FM) and FRAP investigate domain morphology and membrane dynamics with the measurement of the lateral diffusion of lipids, respectively. AFM has been used to gather surface details in terms of occurrence of peculiar structure and defects. One of the unique features of this latter technique is that it can measure surface forces with a nanometer lateral resolution. Recently, single-molecule force spectroscopy (FS) measurements have provided in-depth insight to assess the orientation of reconstituted transmembrane proteins in tBLMs [86]. NR also provides high resolution structural information on lipid bilayer stacking and internal distribution of components after interaction between intrinsic proteins and disordered membrane [124]. EIS is an excellent tool to characterize the electric properties of membrane including resistance and capacitance.

This large panel allows a fine characterization of tBLMs during and after their formation, in terms of structure, (optical or acoustic) thickness, fluidity and sealing [125]. It shows that the chemical nature of the sub-membrane space has a significant impact on both the structure of the lipid bilayer and the functional incorporation of membrane components [51]. The possible combination of multiple complementary measurements with biologically accurate samples is key for a realistic understanding of membrane related phenomena. Only through the use of complementary techniques, such as the ones hereby mentioned, does it become a realistic aim to resolve the relative position, orientation and distribution of the membrane components to obtain detailed information on molecular mechanisms by which peptides, proteins or other chemical compounds (e.g., drugs) interact with biomembranes. Table 1, adapted from Rossi and Chopineau [126], Sondhi et al. [70], Clifton et al. [40], presents a synoptic of the characteristics of all the techniques useful for the study of supported planar membrane models, including tBLMs. For more details, see the recent review by Clifton et al. [40], which presents the main information that can be deduced from model membranes due to the different surface-sensitive techniques listed above.

Table 1. Characteristics of main surface-sensitive analytical techniques useful for investigation of supported planar model membranes included tBLMs (adapted from ^{[40][70][126]}[40,70,126]).