3.3. Supported Lipid Bilayers (SLBs)

Although extensively used for the study of biological interactions at interfaces, SAMs resemble only remotely real biological membranes and lack some typical membrane properties, such as membrane fluidity, which is essential to mimic mobility and ligand reorganization occurring at cell membranes upon interactions.⁴³ In this regard, the formation of SLBs has emerged as a valid alternative method for the modification of surfaces for biological studies.⁴⁴ SLBs are two‐dimensional fluid platforms consisting of phospholipids retaining some of the relevant properties of cell membranes.⁴⁵ Phospholipid vesicles, under specific conditions, rupture on activated hydrophilic surfaces such as mica, glass and silicon, forming a notably stable lipid bilayer.^44a The presence of a thin water layer (approx. 10 Å thick)⁴⁶ between the formed lipid bilayer and the underlying surface allows the mobility of the lipids on the surface providing fluidity to SLBs. In particular, the fluidity of the system can be regulated by varying the chemical composition of the SLB.⁴⁷ The mobility of the SLB is an additional key feature in the modification of surfaces with ligands as, compared to SAMs, it allows dynamic clustering of receptors, which can effectively compensate for a low ligand density at the interface.

SLBs have been reported to present excellent antifouling properties, preventing the nonspecific adsorption of proteins and cells onto their surface.⁴⁸ Methods for the introduction of ligands/receptors of choice in the SLB have been reported in literature.⁴⁵ Briefly, lipids modified with a particular functional moiety can be added to the lipid mixture during the vesicle preparation and thus get displayed on the surface after the formation of the SLB. Subsequently, as in the case of SAMs, modified ligands that react or interact with these functional groups can be anchored onto the surface. In a different approach, lipids modified synthetically with ligands before SLB formation can be directly added to the lipid mixture before the formation of the SLB. In both approaches, controlling the molar fraction of functionalized lipids in the lipid mixture provides an exquisite method to control the surface density.

Several ligands and receptors have been anchored to SLBs with tunable densities by exploiting both non‐covalent and covalent interactions. Biotinylated lipids can be introduced in SLBs by mixing 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) or 1‐palmitoyl‐2‐oleoyl‐glycero‐3‐phosphocholine (POPC) lipids with biotinylated phosphatidylethanolamine (biotin−DOPE) during the preparation of vesicles.⁴⁹ Streptavidin (SAv) can, therefore, be used as a linker for the further functionalization of surfaces with biotinylated linkers by exploiting the strong non‐covalent biotin−SAv interaction.⁵⁰ Koçer and Jonkheijm, for example, varied the amounts of DOPE−biotin (between 0.01 and 1 mol%) in fluid DOPC and non‐fluid DPPC‐based SLBs in order to functionalize surfaces with varying RGD ligand densities.⁵¹ Alternatively, lipid molecules containing a nitrilotriacetic acid (NTA) head group can be doped into the SLB, and proteins modified with histidine tags can be chelated with the NTA lipids in the presence of Ni²⁺ ions.⁵² Multiple histidine are typically added to ensure, at the same time, the stability of the attachment of the proteins and their proper orientation at the interface. Lipids modified with two NTA moieties (bis‐NTA) were synthesized by Piehler and coworkers for the modification of SLBs with histidine‐tagged proteins.⁵³

In the case of covalent modification of SLBs, lipids containing reactive functional groups, as for example maleimide, can be incorporated into the bilayer for the binding of complementary, e. g. thiol, functionalized molecules or proteins containing cysteine residues. Thid et al. used vesicles doped with 0–5 % maleimide‐terminated lipids for the formation of SLBs on SiO₂ substrates, which were subsequently functionalized with IKVAV‐containing peptides.⁵⁴ A different approach consists of the direct modification of biomolecules with a lipid that can be inserted into the SLB. Control of the surface ligand density can be quantitatively achieved by adjusting the molar fraction of modified lipids in the lipid mixture during the preparation of the vesicle. Synthetic glycolipids have been used, for example, for the introduction of controlled densities of glycans in SLBs.²⁵, ⁵⁵, ⁵⁶

As a valid alternative to SLBs, supported lipid monolayers (SLMs) can be also employed for the modification of surfaces with ligands. Methods as Langmuir‐Blodgett or Langmuir‐Schaefer can be used for the formation of monolayers.⁵⁷ Alternatively, SLMs can be formed from the rupture of lipid vesicles on hydrophobic self‐assembled monolayers.⁵⁸ Octadecanethiol on gold or octadecyltrichlorosilane on glass surfaces are two typical examples of hydrophobic monolayers used for hybrid bilayer formation, owing to the possibility of forming highly ordered and well‐packed monolayers.⁵⁹

Kiessling and coworkers developed an SLM in order to control the mannose ligand density on surfaces (Figure 5).⁶⁰ In their studies, POPC liposomes containing different ratios of synthetic glycolipids bearing mannose groups where added on gold surfaces pre‐functionalized with alkanethiols. As in the case of SLBs, by tuning the molar ratio of synthetic glycolipids in the mixture with POPC during the formation of liposomes, it was possible to control the density of mannose exposed on the surface.

Scheme illustrating control over ligand density using supported lipid monolayers. Adapted with permission from ref. [60]. Copyright 1998 American Chemical Society.