Planar lipid bilayer reconstitution with a micro-fluidic system

A planar lipid bilayer which is widely used for the electrophysiological study of membrane proteins in laboratories is reconstituted using a micro-fluidic system, in a manner that is suitable for automated processing. We fabricated micro-channels on both sides of the substrate, which are connected through a 100-200 microm aperture, and showed that the bilayer can be formed at the aperture by flowing the lipid solution and buffer, alternately. Parylene coating is found to be suitable for both bilayer formation and electric noise reduction. Future applications include a high-sensitivity ion sensor chip and a high-throughput drug screening device.     

Interfacial tension of bilayer lipid membranes

Interfacial tension is an important characteristic of a biological membrane because it determines its rigidity, thus affecting its stability. It is affected by factors such as medium pH and by the presence of certain substances, for example cholesterol, other lipids, fatty acids, amines, amino acids, or proteins, incorporated in the lipid bilayer. Here, the effects of various parameters to on interfacial tension values of bilayer lipid membranes are discussed.     

Supramolecular chemistry in lipid bilayer membranes

Lipid bilayer membranes form compartments requisite for life. Interfacing supramolecular systems, including receptors, catalysts, signal transducers and ion transporters, enables the function of the membrane to be controlled in artificial and living cellular compartments. In this perspective, we take stock of the current state of the art of this rapidly expanding field, and discuss prospects for the future in both fundamental science and applications in biology and medicine.

This perspective provides an overview of the current state of the art in supramolecular chemistry in lipid bilayer membranes, including receptors, signal transducers, catalysts and transporters, and highlights prospects for the future.     

Super-resolution microscopy of lipid bilayer phases

Sub-diffraction optical imaging with nanometer resolution of lipid phase-separated regions is reported. Merocyanine 540, a probe whose fluorescence is sensitive to the lipid phase, is combined with super-resolution imaging to distinguish the liquid- and gel-phase nanoscale domains of lipid bilayers supported on glass. The monomer-dimer equilibrium of MC540 in membranes is deemed responsible for the population difference of single-molecule fluorescence bursts in the different phase regions. The extension of this method to other binary or ternary lipid models or natural systems provides a promising new super-resolution strategy.     

Elastic modulus of free-standing lipid bilayer

In the current study, molecular dynamics (MD), finite element (FE) method, and genetic algorithm are employed to compute Young’s modulus of free-standing DPPC lipid bilayer. MD method is utilized to simulate loading of a free-standing DPPC lipid bilayer under an indenter. Indentation experiment is also simulated with FE method where genetic algorithm controls value of Young’s modulus in FE simulation and finds the best value for it. The best value means the value results in a force–depth curve which agrees well with the curve obtained from MD simulation. While simulating indentation with MD method two distinct regimes are distinguished in force–depth curve before rupture of the bilayer. The first regime shows elastic response of the bilayer to indentation and it is shown that force–depth curve can be fitted with a cubic polynomial in this regime. The second regime starts at the point which the force–depth curve changes from convex to concave. This point is an inflection point and would be regarded as yield point of the bilayer. Slope of the curve decreases with indentation depth in this regime which shows changes in internal structure of the bilayer. Also we investigate effects of indenter’s shape and indentation speed on computed Young’s modulus and show rate-dependent behavior of free-standing lipid bilayer.     

Curvature-modulated phase separation in lipid bilayer membranes

Cellular membranes exhibit a variety of controlled curvatures, with filopodia, microvilli, and mitotic cleavage furrows being only a few of many examples. Coupling between local curvature and chemical composition in membranes could provide a means of mechanically controlling the spatial organization of membrane components. Although this concept has surfaced repeatedly over the years, controlled experimental investigations have proven elusive. Here, we introduce an experimental platform, in which microfabricated surfaces impose specific curvature patterns onto lipid bilayers, that allows quantification of mechanochemical couplings in membranes. We find that, beyond a critical curvature value, membrane geometry governs the spatial ordering of phase-separated domain structures in membranes composed of cholesterol and phospholipids. The curvature-controlled ordering, a consequence of the distinct mechanical properties of the lipid phases, makes possible a determination of the bending rigidity difference between cholesterol-rich and cholesterol-poor lipid domains. These observations point to a strong coupling between mechanical bending and chemical organization that should have wide-reaching consequences for biological membranes. Curvature-mediated patterning may also be useful in controlling complex fluids other than biomembranes.     

Cooperative binding at lipid bilayer membrane surfaces

The binding of copper(II) ions to membrane-bound synthetic receptors has been investigated. Complexation fitted a 4:1 receptor:copper(II) model, and the observed binding constants are significantly enhanced at the membrane relative to solution; these effects can be explained by the lower polarity of the membrane-water interface and the concentrating effect of the membrane, with no observed contribution from receptor preorganization. The stoichiometry of the complex formed is very sensitive to the concentration of the receptor in the membrane, and at low concentrations, binding is reduced relative to solution controls. This implies that by increasing or decreasing the number of receptors in their membranes, cells can finely tune biological responses such as chemotaxis that depend on the size of the receptor-ligand clusters formed.     

Tethered bilayer lipid membranes with giga-ohm resistances

The successful reconstitution of a tethered BLM on μ-electrodes ranging from 4000 μm to 8 μm is shown in this article. The increase in membrane resistance with decreasing electrode size and the dependency of the membrane capacitance on the electrode size was studied. Furthermore the functional incorporation of α-hemolysin from Staphylococcus aureus into a tBLM situated on μ-electrodes was achieved.     

Migration of poly-L-lysine through a lipid bilayer

When a giant vesicle, composed of neutral and anionic lipid (90:10 mol %), comes into contact with various poly-l-lysines (MW 500-29 300), ropelike structures form within the vesicle interior. By using fluorescence lipids and epi-fluorescence microscopy, we have shown that both neutral and anionic lipids are constituents of the ropes. Evidence that the ropes are also comprised of poly-l-lysine comes from two experiments: (a) direct microinjection of poly(acrylic acid) into rope-containing vesicles causes the ropes to contract into small particles, an observation consistent with a polycation/polyanion interaction; and (b) direct microinjection of fluorescein isothiocyanate (a compound that covalently labels poly-l-lysine with a fluorescent moiety) into rope-containing vesicles leads to fluorescent ropes. The results may be explained by a model in which poly-l-lysine binds to the vesicle exterior, forms a domain, and enters the vesicle through defects or at the domain boundary. The model helps explain the ability of poly-l-lysine to mediate the permeation of a cancer drug, doxorubicine, into the vesicle interior.     

AFM-based force-clamp monitors lipid bilayer failure kinetics

The lipid bilayer rupture phenomenon is here explored by means of atomic force microscopy (AFM)-based force clamp, for the first time to our knowledge, to evaluate how lipid membranes respond when compressed under an external constant force, in the range of nanonewtons. Using this method, we were able to directly quantify the kinetics of the membrane rupture event and the associated energy barriers, for both single supported bilayers and multibilayers, in contradistinction to the classic studies performed at constant velocity. Moreover, the affected area of the membrane during the rupture process was calculated using an elastic deformation model. The elucidated information not only contributes to a better understanding of such relevant process, but also proves the suitability of AFM-based force clamp to study model structures as lipid bilayers. These findings on the kinetics of lipid bilayers rupture could be extended and applied to the study of other molecular thin films. Furthermore, systems of higher complexity such as models mimicking cell membranes could be studied by means of AFM-based force-clamp technique.     

Reconstituted olfactory receptors in bilayer lipid membranes

Bullfrog olfactory receptors were reconstituted in bilayer lipid membranes (BLMs). Three odorants were presented to the reconstituted system. The three structurally related odorants were diethylsulfide (DES), thiophene (THP) and diethanolsulfide (DOS). The ordorants were presented in pairs. DOS induced a response in the presence of either of the other two odorants. DES and THP did not induce a response in the presence of either of the other two odorants. These observations suggest that there are two substructures, one common to the three odorants and one that is unique to DOS. The results support the notion that olfactory receptors detect certain molecular segments of odorants.     

Amphiphile bilayer films from DPPC: bilayer lipid membranes and Newton black films

Amphiphile bilayer films are obtained from 1,2 dipalmitoyl-glycero-3-phosphocholine (DPPC): bilayer lipid membranes (BLM) and Newton black films (NBF), through thinning of the respective thin liquid films, thus allowing for a very precise determination of the moment of their formation. Stability (or rupture) and formation of BLM and NBF are considered from a unified point of view with the microscopic theory of Kashchiev–Exerowa [J. Colloid Interface Sci., 77 (1980) 501–511], based on the formation of nanoscopic holes in them. BLM and NBF are obtained and studied with the microinterferometric method of Scheludko–Exerowa in its contemporary version. The equivalent thickness of both BLM (in benzene solution between two water phases with 0.1 M NaCl) and NBF in aqueous DPPC solution (in the presence of 0.1 M NaCl) is determined as being h_w = 7.0 nm for BLM and h_w = 7.8 nm for NBF. By means of the dependences: BLM lifetime versus DPPC concentration and probability for BLM formation versus DPPC concentration, it is established that there exist metastable BLM and stable NBF. The good fit between the experimental results of τ(C) dependence and theory in the case of BLM allow to determine the three constants: pre-exponential factor A = 1.5 × 10⁻³ s, related to the process kinetics; constant B = 20.2 ± 0.2, related to the specific hole energy γ = 1.7 × 10⁻¹¹ J/m and the equilibrium concentration C_e = 6 × 10⁻⁴ ± 7.2 × 10⁻⁶ m/l. The specific hole linear energy γ = 1.7 × 10⁻¹¹ J/m determined as well as the binding energy Q between first neighbor molecules in the bilayers Q = 1.48 × 10⁻¹⁹ J (36 kT) are lower than the ones determined for DPPC foam bilayer in gel state γ = 9.1 × 10⁻¹¹ J/m and Q = 55 kT. This means that interaction is weaker in the case of BLM. The critical concentration C_c at which bilayer formation starts is: for BLM C_c = 30 μg/ml and for NBF C_c = 70 μg/ml. This concentration characterizes quantitatively the formation of the amphiphile bilayer and is a very useful parameter that can be used for various purposes.     

Planar bilayer studies reveal multiple conductance states for synthetic anion transporters

Compounds of the general type R(1)(2)NCOCH(2)OCH(2)CO-(Gly)(3)-Pro-(Gly)(3)-OCH(2)Ph insert in phospholipid bilayers and conduct ions. Different levels of activity were observed when R(1) was either decyl or octadecyl, as judged either by Cl(-) release, detected by ion selective electrodes, or carboxyfluorescein dequenching, detected by fluorescence. Either method reports average behavior for all ionophores over all liposomes. These methods also show that at least two ionophores are involved in the formation of each pore. Planar bilayer experiments reported here confirm pore formation by these compounds but identify more than one conductance state for each. The pseudo-dimer, in which two molecules of the type shown above are covalently linked, shows only two conductance states, of which one is dominant. This state has been characterized by use of a current-voltage plot.     

The lipid bilayer concept and its experimental realization: from soap bubbles,kitchen sink,to bilayer lipid membranes

The inspiration for lipid bilayer research, without question, comes from the biological world. Although self-assembled bilayer lipid membranes (BLMs) in vitro, were first reported in 1961, experimental scientists have been dealing with BLM-type interfacial adsorption phenomena since Robert Hooke’s time (1672). BLMs (of planar lipid bilayers) have been used in a number of applications ranging from basic membrane biophysics including transport, practical AIDS research, and ‘microchips’ studies, to the conversion of solar energy via water photolysis, to biosensor development using supported bilayer lipid membranes (s-BLMs), and to photobiology comprising apoptosis and photodynamic therapy. This paper presents an overview of the origin of the lipid bilayer concept and its experimental realization, as well as the studies of our laboratory and recent research of others on the use of BLMs as models of certain biomembranes. In addition, we describe briefly our present work on supported BLMs as biosensors and molecular devices; the experiments carried out in close collaboration with colleagues on s-BLMs are delineated.     

Proton transport into a tethered bilayer lipid membrane

Tethered bilayer lipid membranes (tBLMs) are increasingly used to study biological membranes, membrane proteins and a variety of related topics. A tBLM is formed by binding a lipid bilayer to a metal surface (usually gold) via a hydrophilic tether (usually an ethyleneoxy chain). In this report we present an electrochemical study on ubiquinone in a tBLM which has provided insights into the properties of this hydrophilic layer, which has a very limited capability of storing and releasing protons. It is concluded that the often observed decrease in tBLM resistance upon addition of ionophores (or protonophores) could be due to the penetration of ions (or protons) into the membrane rather than transport through the membrane.     

Examination of bilayer lipid membranes for 'pin-hole' character

BLM prepared on electrode substrates by supporting or tethering were tested for 'pin-hole' character, comparing data from cyclic voltammetry (CV), surface plasmon resonance (SPR) and rotating disc electrodes (RDE). 1-hexadecylamine tethered BLMs on SAM modified gold electrodes were compared with BLMs assembled on modified polyHEMA or sol-gel layers. BLM formation followed by SPR showed that the initial phase of the assembly was complete in 5-20 minutes and produced layers of thickness >5 nm, compared with the expected final BLM thickness of approximately 3 nm. The CVs of the K(3)[Fe(CN)(6)] couple were significantly suppressed irrespective of the method of BLM assembly, without major differences emerging for the different methods. However, data from the RDE distinguished the 'pin-hole' character of the different preparations. The data were consistent with incomplete initial (<1 h, SPR estimated BLM thickness >5 nm) vesicle fusion leaving 'pin-holes' of approximately 2 microm (HDA-11-mercaptoundecanoic acid (MUA) tethered BLM) to approximately 3 microm (tetraethylorthosilicate sol-gel supported BLM) followed by a slow maturation (>15 h; impedance spectroscopy estimated thickness approximately 3 nm) and lateral spreading and fusion, resulting in loss of 'pin-hole' character (<1 microm). The BLM could be used in conjunction with potentiometric measurement to observe the incorporation of nystatin into the BLM and the rate of incorporation adjusted according to original permeability of the BLM. The 'pin-hole-free' BLM construction with lowest permeability (TEOS supported, 4 x 10(-10) cm s(-1) compared with HDA-MUA, 3 x 10(-9) cm s(-1)) gave a potentiometric signal independent of bulk ion-concentration across 5 decades change in concentration. Formed on an ion-selective electrode, nystatin incorporation could be followed as a change in potential, over >2 h, whereas the TEOS supported BLM with permeability 1 x 10(-9) cm s(-1) shows nystatin incorporation within 1 h. In this instance, addition of ConA reduced the potential to the same value as prior to nystatin incorporation, consistent with nystatin channel closure.     

Adsorption of gentamicin onto a bilayer lipid membrane

Addition of the aminoglycoside antibiotic, gentamicin (GM), to one side of a bilayer lipid membrane (BLM) results in a potential difference across the membrane. Evidence is presented that the membrane potential is caused by the adsorption of GM, bearing four positive charges, on the BLM surface. The experimental results are subjected to a quantitative analysis based on the double-layer theory and the Langmuir adsorption isotherm. The adsorption is saturated (i.e., the BLM is fully covered) at the bulk GM concentration of about 80 μmol/1. At this point, the calculated GM-induced increase in the BLM surface charge density is σ = 0.0054 C m⁻², which is equivalent to one positive charge per 50 lipids or one molecule of GM per 200 lipids.     

Diffusion of liquid domains in lipid bilayer membranes

We report diffusion coefficients of micron-scale liquid domains in giant unilamellar vesicles of phospholipids and cholesterol. The trajectory of each domain is tracked, and the mean square displacement grows linearly in time, as expected for Brownian motion. We study domain diffusion as a function of composition and temperature and measure how diffusion depends on domain size. We find mechanisms of domain diffusion which are consistent with membrane-dominated drag in viscous L(o) phases and bulk-dominated drag for less viscous L(alpha) phases. Where applicable, we obtain the membrane viscosity and report activation energies of diffusion.