7 research outputs found
Field-effect detection using phospholipid membranes -Topical Review
The application of field-effect devices to biosensors has become an area of intense research interest. An attractive feature of field-effect sensing is that the binding or reaction of biomolecules can be directly detected from a change in electrical signals. The integration of such field-effect devices into cell membrane mimics may lead to the development of biosensors useful in clinical and biotechnological applications. This review summarizes recent studies on the fabrication and characterization of field-effect devices incorporating model membranes. The incorporation of black lipid membranes and supported lipid monolayers and bilayers into semiconductor devices is described
Induced Rupture of Vesicles Adsorbed on Glass by Pore Formation at the Surface–Bilayer Interface
Supported lipid bilayers (SLBs) are
often formed by spontaneous
vesicle rupture and fusion on a solid surface. A well-characterized
rupture mechanism for isolated vesicles is pore nucleation and expansion
in the solution-exposed nonadsorbed area. In contrast, pore formation
in the adsorbed bilayer region has not been investigated to date.
In this work, we studied the detailed mechanisms of asymmetric rupture
of giant unilamellar vesicles (GUVs) adsorbed on glass using fluorescence
microscopy. Asymmetric rupture is the pathway where a rupture pore
forms in a GUV near the edge of the glass–bilayer interface
with high curvature and then expansion of the pore yields a planar
bilayer patch. We show that asymmetric rupture occasionally resulted
in SLB patches bearing a defect pore. The defect formation probability
depended on lipid composition, salt concentration, and pH. Approximately
40% of negatively charged GUVs under physiological conditions formed
pore-containing SLB patches, while negatively charged GUVs at low
salt concentration or pH 4.0 and positively charged GUVs exhibited
a low probability of defect inclusion. The edge of the defect pore
was either in contact with (on-edge) or away from (off-edge) the edge
of the planar bilayer. On-edge pores were predominantly formed over
off-edge defects. Pores initially formed in the glass-adsorbed region
before rupture, most frequently in close contact with the edge of
the adsorbed region. When a pore formed near the edge of the adsorbed
area or when the edge of a pore reached that of the adsorbed area
by pore expansion, asymmetric rupture was induced from the defect
site. These induced rupture mechanisms yielded SLB patches with an
on-edge pore. In contrast, off-edge pores were produced when defect
pore generation and subsequent vesicle rupture were uncoupled. The
current results demonstrate that pore formation in the surface-adsorbed
region of GUVs is not a negligible event
Packing Density Changes of Supported Lipid Bilayers Observed by Fluorescence Microscopy and Quartz Crystal Microbalance-Dissipation
Various properties of supported lipid
bilayers such as diffusion
and lipid partitioning are well characterized. However, little attention
has been paid to their molecular packing density. In this work, the
adsorption of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) vesicles
on glass and silicon dioxide was investigated using fluorescence microscopy,
quartz crystal microbalance-dissipation (QCM-D), and atomic force
microscopy. Fluorescence recovery after photobleaching data showed
that the adsorption of large unilamellar vesicles (LUVs) on glass
yielded supported bilayers with full mobility under alkaline (pH 8.3)
and acidic (pH 3–4) conditions. These fluid bilayers exhibited
quite different diffusion constants; those at alkaline pH were ∼10
times larger than those at acidic pH. The reason for this pH dependence
was clarified by investigation of the rupture of giant unilamellar
vesicles (GUVs) on glass. Fluorescence data revealed that the area
of planar bilayer patches increased at alkaline pH. Thus, we conclude
that the rapid diffusion in alkaline solution arises from the decreased
molecular density. QCM-D data showed that dissipation increased in
a stepwise manner during vesicle fusion on silicon dioxide at alkaline
pH. We attribute this behavior to the decrease in packing density
of planar bilayers
Binding of Lipopolysaccharide and Cholesterol-Modified Gelatin on Supported Lipid Bilayers: Effect of Bilayer Area Confinement and Bilayer Edge Tension
Binding of amphiphilic molecules
to supported lipid bilayers (SLBs)
often results in lipid fibril extension from the SLBs. Previous studies
proposed that amphiphiles with large and flexible hydrophilic regions
trigger lipid fibril formation in SLBs by inducing membrane curvature
via their hydrophilic regions. However, no experimental studies have
verified this mechanism of fibril formation. In this work, we investigated
the binding of lipopolysaccharide (LPS) and cholesterol-modified gelatin
to SLBs using fluorescence microscopy. SLBs with restricted and unrestricted
bilayer areas were employed to identify the mechanism of fibril generation.
We show that the main cause of lipid fibril formation is an approximately
20% expansion in the bilayer area rather than increased membrane curvature.
The data indicate that bilayer area confinement plays a critical role
in morphological changes of SLBs even when bound amphiphilic molecules
have a large hydrophilic domain. We also show that bilayer area change
after LPS insertion is dependent on the patch shape of the SLB. When
an SLB patch consists of a broad bilayer segment connected to a long
thin streak, bilayer area expansion mainly occurs within the bilayer
streak. The results indicate that LPS insertion causes net lipid flow
from the broad bilayer region to the streak area. The differential
increase in area is explained by the instability of planar bilayer
streaks that originate from the large energetic contribution of line
tension arising along the bilayer edge
Induction of Intermembrane Adhesion by Incorporation of Synthetic Adhesive Molecules into Cell Membranes
Modulation
of cell adhesion by synthetic materials is useful for
a wide range of biomedical applications. Here, we characterized cell
adhesion mediated by a semisynthetic molecule, cholesteryl-modified
gelatin (chol-gelatin). We found that this hybrid molecule facilitated
cell adhesion by connecting two apposed membranes via multiple cholesterol
moieties on the gelatin molecules, whereas unmodified gelatin did
not bind to cell membranes. Analyses revealed that the rate of the
formation of cell adhesions was increased by displaying more cholesterol
moieties on the cell membrane. In contrast, the area of the cell adhesion
site was unchanged by increasing the number of cholesterol molecules,
suggesting that chol-gelatin may suppress cell spreading. Such restriction
was not observed in cell adhesion mediated by the mutant of physiological
adhesion protein CD2, which lacked its cytoplasmic domain and was
unable to connect to cytoplasmic actin filaments, but had a similar
affinity for its ligand compared with the chol-gelatin–cell
membrane interaction. Further analysis suggested the restriction of
cell spreading by chol-gelatin was largely independent of the modulation
of the surface force, and thus we hypothesize that the restriction
could be in part due to the modulation of cell membrane mechanics
by membrane-incorporated chol-gelatin. Our study dissected the two
roles of the hybrid molecule in cell adhesion, namely the formation
of a molecular connection and the restriction of spreading, and may
be useful for designing other novel synthetic agents to modulate various
types of cell adhesions
Effect of Average Phospholipid Curvature on Supported Bilayer Formation on Glass by Vesicle Fusion
The adsorption of large unilamellar vesicles composed of various combinations of phosphatidylcholine, phosphatidylethanolamine (PE), monomethyl PE, and dimethyl PE (PE-Me(2)) onto a glass surface was studied using fluorescence microscopy. The average lipid geometry within the vesicles, described mathematically by the average intrinsic curvature, C(0,ave), was methodically altered by changing the lipid ratios to determine the effect of intrinsic curvature on the ability of vesicles to rupture and form a supported lipid bilayer. We show that the ability of vesicles to create fluid planar bilayers is dependent on C(0,ave) and independent of the identity of the component lipids. When the C(0,ave) was ∼−0.1 nm(−1), the vesicles readily formed supported lipid bilayers with almost full mobility. In contrast, when the C(0,ave) ranged from ∼−0.2 to ∼−0.3 nm(−1), the adsorbed vesicles remained intact upon the surface. The results indicate that the average shape of lipid molecules within a vesicle (C(0,ave)) is essential for determining kinetically viable reactions that are responsible for global geometric changes