Widely used in catalysis and biosensing applications, aluminum oxide has become popular for surface functionalization with biological macromolecules, including lipid bilayer coatings. However, it is difficult to form supported lipid bilayers on aluminum oxide, and current methods require covalent surface modification, which masks the interfacial properties of aluminum oxide, and/or complex fabrication techniques with specific conditions. Herein, we addressed this issue by identifying simple and robust strategies to form fluidic lipid bilayers on aluminum oxide. The fabrication of a single lipid bilayer coating was achieved by two methods, vesicle fusion under acidic conditions and solvent-assisted lipid bilayer (SALB) formation under near-physiological pH conditions. Importantly, quartz crystal microbalance with dissipation (QCM-D) monitoring measurements determined that the hydration layer of a supported lipid bilayer on aluminum oxide is appreciably thicker than that of a bilayer on silicon oxide. Fluorescence recovery after photobleaching (FRAP) analysis indicated that the diffusion coefficient of lateral lipid mobility was up to 3-fold greater on silicon oxide than on aluminum oxide. In spite of this hydrodynamic coupling, the diffusion coefficient on aluminum oxide, but not silicon oxide, was sensitive to the ionic strength condition. Extended-DLVO model calculations estimated the thermodynamics of lipid-substrate interactions on aluminum oxide and silicon oxide, and predict that the range of the repulsive hydration force is greater on aluminum oxide, which in turn leads to an increased equilibrium separation distance. Hence, while a strong hydration force likely contributes to the difficulty of bilayer fabrication on aluminum oxide, it also confers advantages by stabilizing lipid bilayers with thicker hydration layers due to confined interfacial water. Such knowledge provides the basis for improved surface functionalization strategies on aluminum oxide, underscoring the practical importance of surface hydration.

The fate of adsorbed lipid vesicles on solid supports depends on numerous experimental parameters and typically results in the formation of a supported lipid bilayer (SLB) or an adsorbed vesicle layer. One of the poorly understood questions relates to how divalent cations appear to promote SLB formation in some cases. The complexity arises from the multiple ways in which divalent cations affect vesicle-substrate and vesicle-vesicle interactions as well as vesicle properties. These interactions are reflected, e.g., in the degree of deformation of adsorbed vesicles (if they do not rupture). It is, however, experimentally challenging to measure the extent of vesicle deformation in real-time. Herein, we investigated the effect of divalent cations (Mg(2+), Ca(2+), Sr(2+)) on the adsorption of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles onto silicon oxide- and titanium oxide-coated substrates. The vesicle adsorption process was tracked using the quartz crystal microbalance-dissipation (QCM-D) and localized surface plasmon resonance (LSPR) measurement techniques. On silicon oxide, vesicle adsorption led to SLB formation in all cases, while vesicles adsorbed but did not rupture on titanium oxide. It was identified that divalent cations promote increased deformation of adsorbed vesicles on both substrates and enhanced rupture on silicon oxide in the order Ca(2+) > Mg(2+) > Sr(2+). The influence of divalent cations on different factors in these systems is discussed, clarifying experimental observations on both substrates. Taken together, the findings in this work offer insight into how divalent cations modulate the interfacial science of supported membrane systems.

With increasing temperature, biological macromolecules and nanometer-sized aggregates typically undergo complex and poorly understood reconfigurations, especially in the adsorbed state. Herein, we demonstrate the strong potential of using localized surface plasmon resonance (LSPR) sensors to address challenging questions related to this topic. By employing an LSPR-based gold nanodisk array platform, we have studied the adsorption of sub-100-nm diameter 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles on titanium oxide at two temperatures, 23 and 50 °C. Inside this temperature range, DPPC lipid vesicles undergo the gel-to-fluid phase transition accompanied by membrane area expansion, while DOPC lipid vesicles remain in the fluid-phase state. To interpret the corresponding measurement results, we have derived general equations describing the effect of deformation of adsorbed vesicles on the LSPR signal. At the two temperatures, the shape of adsorbed DPPC lipid vesicles on titanium oxide remains nearly equivalent, while DOPC lipid vesicles become less deformed at higher temperature. Adsorption and rupture of DPPC lipid vesicles on silicon oxide were also studied for comparison. In contrast to the results obtained on titanium oxide, adsorbed vesicles on silicon oxide become more deformed at higher temperature. Collectively, the findings demonstrate that increasing temperature may ultimately promote, hinder, or have negligible effect on the deformation of adsorbed vesicles. The physics behind these observations is discussed, and helps to clarify the interplay of various, often hidden, factors involved in adsorption of biological macromolecules at interfaces.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membranerelated processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Complement activation plays an important role in innate immune defense by triggering formation of the membrane attack complex (MAC), which is a biomacromolecular assembly that exhibits membrane-lytic activity against foreign invaders including various pathogens and biomaterials. Understanding the details of MAC structure and function has been the subject of extensive work involving bulk liposome and erythrocyte assays. However, it is difficult to characterize the mechanism of action of MAC inhibitor drug candidates using the conventional assays. To address this issue, we employ a biomimetic supported lipid bilayer platform to investigate how two MAC inhibitors, vitronectin and clusterin, interfere with MAC assembly in a sequential addition format, as monitored by the quartz crystal microbalance-dissipation (QCM-D) technique. Two experimental strategies based on modular assembly were selected, precincubation of inhibitor and C5b-7 complex before addition to the lipid bilayer or initial addition of inhibitor followed by the C5b-7 complex. The findings indicate that vitronectin inhibits membrane association of C5b-7 via a direct interaction with C5b-7 and via competitive membrane association onto the supported lipid bilayer. On the other hand, clusterin directly interacts with C5b-7 such that C5b-7 is still able to bind to the lipid bilayer, and clusterin affects the subsequent binding of other complement proteins involved in the MAC assembly. Taken together, the findings in this study outline a biomimetic approach based on supported lipid bilayers to explore the interactions between complement proteins and inhibitors, thereby offering insight into MAC assembly and regulation.