The analytical potential of chemoreception at bilayer lipid membranes

The potential use of the bilayer lipid membrane as an electrochemical sensor is discussed through a study of model systems known to cause increased membrane conductance. The limit of detection for amphotericin B, a molecule capable of forming membrane pores, is in the region of 1O^-9 M. The current—time profile is discussed in terms of a mechanism which involves micelle formation in the aqueous and lipid phases. Unlike previous experiments, two current maxima with time are observed for valinomycin response (limit of detection 1O^-11 M). The first transient signal is attributed to increased membrane permeability caused by a conformational change in valinomycin in the “surface” volume of the bilayer. Selective interactions at membranes and the nature of membrane responses are discussed in terms of analytical parameters.     

349 - Membrane potential and ion permeability of lipid bilayer membranes

In order to clarify the components consisting of transmembrane potential of membranes, measurements of surface potentials of phospholipid monolayers and concentration potentials of phospholipid bilayer membranes measurements were carried out with respect to salt concentrations.It was concluded that, for a highly charged membranes symmetrical with respect to the surface charge density, the observed transmembrane potential is due mostly to the difference between the two surface potentials on both sides of the membrane. On the other hand, for an uncharged membrane, the transmembrane potential is due to the ion diffusion potential through the membrane.Some discussion concerning ion permeabilities through the membranes is also made in relation to the observed transmembrane potentials.     

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.     

Stabilization of bilayer lipid membranes on solid supports by trehalose

Using the electrostriction method the effect of the glucose and trehalose on the elasticity modulus perpendicular to the membrane plane, E_⊥, and the electrical capacitance, C, of supported bilayer lipid membranes (s-BLM) formed on the freshly cut tip of Teflon-coated Ag wire was studied. Addition of saccharides into the electrolyte resulted in a decrease in the elasticity modulus of the s-BLM formed from the soybean phosphatidylcholine in n-hexadecane, while the capacitance was increased. In addition, the trehalose has a considerable stabilizing effect on the above parameters of the s-BLM. Treatment of the s-BLM in an electrolyte containing 0.3 M of the trehalose allowed storage of the s-BLM under dry conditions and under refrigeration, with the subsequent recovery of membrane parameters after the wire had been dipped into the electrolyte.     

Electrostriction of biotin-streptavidin-modified bilayer lipid membranes on a metal support

Using the electrostriction method we have studied the elasticity modulus perpendicular to the membrane plane, E⊥, electrical capacitance, C, coefficient of dynamic viscosity, η, and membrane potential difference δф_m of supported bilayer lipid membranes (s-BLM) modified by biotin-streptavidin, as a function of d.c. voltage applied to the membrane. Binding of streptavidin to biotin-modified s-BLM resulted in a slight decrease of membrane capacitance, increase of E_⊥ and increase of η, while δф_m did not change. The val of E_⊥ of unmodified membranes was found to change considerably with increasing d.c. voltage and the rate of voltage change. Modification of s-BLM by streptavidin leads to reduced changes of E_⊥ with the rate of d.c. voltage change, and it made s-BLM extremely stable even at an external d.c. voltage of 2 V. Our results indicate that streptavidin considerably stabilized s-BLM by means of the formation of a complex with biotin-modified phospholipids.     

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.     

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.     

Triggered release of molecules across droplet interface bilayer lipid membranes using photopolymerizable lipids

A combination of nonpolymerizable phospholipids (DPPC or DPhPC) and a smaller amount of cross-linking photopolymerizable phospholipids (23:2 DiynePC) is incorporated in an unsupported artificial lipid bilayer formed using the droplet interface bilayer (DIB) approach. The DIB is formed by contacting lipid monolayer-coated aqueous droplets against each other in a dodecane-lipid medium. Cross-linking of the photopolymerizable lipids incorporated in the DIB was obtained by exposure to UV-C radiation (254 nm), resulting in pore formation. The effect of cross-linking on the DIB properties was characterized optically by measuring the diffusion of selectively encapsulated dye molecules (calcein) from one droplet of the DIB to the other droplet. Changes in DIB conductivity due to UV-C exposure were investigated using current-voltage (I-V) measurements. The leakage of dye molecules across the DIB and the increase in DIB conductivity after UV-C exposure indicates the formation of membrane pores. The results indicate that the DIB approach offers a simple and flexible platform for studying phototriggered drug delivery systems in vitro.     

Biophysical aspects of agar-gel supported bilayer lipid nembranes: a new method for forming and studying planar bilayer lipid membranes

Conventional bilayer lipid membranes (BLMs), formed by either the painting method or the Langmuir-Blodgett technique, lack the desired stability. This paper presents a simple method for forming long-lived BLMs on agar-gel supports. The supported BLM reported has a greatly improved mechanical stability and also has desirable dynamic properties. These self-assembled BLMs are of significant interest, in view of their similarity of biological membranes, their molecular dimension and their spontaneous formation.     

Specific binding structures of dendrimers on lipid bilayer membranes

Dissipative particle dynamics simulations are used to study the specific binding structures of polyamidoamine (PAMAM) dendrimers on amphiphilic membranes and the permeation mechanisms. Mutually consistent coarse-grained (CG) models both for PAMAM dendrimers and for dimyristoylphosphatidylcholine (DMPC) lipid molecules are constructed. The PAMAM CG model describes correctly the conformational behavior of the dendrimers, and the DMPC CG model can properly give the surface tension of the amphiphilic membrane. A series of systematic simulations is performed to investigate the binding structures of the dendrimers on membranes with varied length of the hydrophobic tails of amphiphiles. The permeability of dendrimers across membranes is enhanced upon increasing the dendrimer size (generation). The length of the hydrophobic tails of amphiphiles in turn affects the dendrimer conformation, as well as the binding structure of the dendrimer-membrane complexes. The negative curvature of the membrane formed in the dendrimer-membrane complexes is related to dendrimer concentration. Higher dendrimer concentration together with increased dendrimer generation is observed to enhance the permeability of dendrimers across the amphiphilic membranes.     

Neuronal activation by GPI-linked neuroligin-1 displayed in synthetic lipid bilayer membranes

We have characterized, in vitro, interactions between hippocampal neuronal cells and silica microbeads coated with synthetic, fluid, lipid bilayer membranes containing the glycosylphosphatidyl inositol (GPI)-linked extracellular domain of the postsynaptic membrane protein neuroligin-1. These bilayer-neuroligin-1 beads activated neuronal cells to form presynaptic nerve terminals at the point of contact in a manner similar to that observed for live PC12 cells, ectopically expressing the full length neuroligin-1. The synthetic membranes exhibited biological activity at neuroligin-1 densities of approximately 1 to 6 proteins/microm(2). Polyolycarbonate beads with neuroligin-1 covalently attached to the surface failed to activate neurons despite the fact that neuroligin-1 binding activity is preserved. This implies that a lipid membrane environment is likely to be essential for neuroligin-1 activity. This technique allows the study of isolated proteins in an environment that has physical properties resembling those of a cell surface; proteins can diffuse freely within the membrane, retain their in vivo orientations, and are in a nondenatured state. In addition, the synthetic membrane environment affords control over both lipid and protein composition. This technology is easily implemented and can be applied to a wide variety of cellular studies.     

Peculiarities of the DNA hybridization on the surface of bilayer lipid membranes

The method of electrostriction was applied to study the peculiarities of interaction of short oligonucleotides with free standing (BLM) and supported lipid membranes (sBLM) and of the duplex formation between complementary oligonucleotides on a membrane surface. The 15-mer single stranded DNA (pentadecathymidylate-(dT)(15)) was modified either with cholesterol (CH(dT)(15)) or by palmitoyl chain (C16(dT)(15)). The interaction of CH(dT)(15) with free standing BLM or with BLM formed on an agar or gold support was accompanied by sharp increase of elasticity modulus in direction perpendicular to the membrane plane ,E( perpendicular), and by increase of surface potential. In contrast, C16(dT)(15) did not induce substantial changes of elasticity modulus, however, the surface potential was changed in a similar manner as for CH(dT)(15). Hybridization of DNA following addition of complementary chain (dA)(15) has been accompanied by a small decrease of elasticity modulus and by a slight increase of surface potential. Both the incorporation of chemically modified oligonucleotides into the lipid bilayer as well as hybridization of DNA are not cooperative processes as has been demonstrated by analysis using Scatchard plot of corresponding values.     

330 - Determination of surface charge of bilayer lipid membranes

The determination of the membrane surface charge is based on the measurement of the surface potential difference at both sides of the bilayer lipid membrane (BLM) connected with the asymmetrical concentration of the electrolyte in both solutions. In the short-circuit regime the intramembrane potential jump is caused by the difference in the two surface potentials. In order to find the intramembrane potential jump the BLM capacitance dependence on voltage was used. In some range of electrolyte concentrations a dependence of the potential jump on the surface charge was found. The charge density was calculated by applying the Gouy-Chapman theory of the diffuse double layer. Surface charges were determined for BLM of common bovine brain lipids, phosphatidylethanolamine, dioleyllecithin and azolectine.     

Real-time QCM-D monitoring of electrostatically driven lipid transfer between two lipid bilayer membranes

The lipid exchange/transfer between lipid membranes is important for many biological functions. To learn more about how the dynamics of such processes can be studied, we have investigated the interaction of positively and negatively charged lipid vesicles with supported lipid bilayers (SLBs) of opposite charge. The vesicle-SLB interaction leads initially to adsorption of lipid vesicles on the SLB, as deduced from the mass uptake kinetics and the concerted increase in dissipation, monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. Eventually, however, vesicles (and possibly other lipid structures) desorb from the SLB surface, as judged from the mass loss and the dissipation decrease. The mass loss is approximately as large as the initial mass increase; i.e., at the end of the process the mass load is that of a SLB. We interpret this interesting kinetics in terms of initial strong electrostatic attraction between the added vesicles and the SLB, forming a structure where lipid transfer between the two bilayers occurs on a time scale of 10-40 min. We suggest that this lipid transfer causes a charge equilibration with an accompanying weakening of the attraction, and eventually repulsion, between the SLB and vesicles, leading to desorption of vesicles from the SLB. The composition of the latter has thus been modified compared to the initial one, although no net mass increase or decrease has occurred. Direct evidence for the lipid exchange was obtained by sequential experiments with alternating positive and negative vesicles, as well as by using fluorescently labeled lipids and FRAP. The above interpretation was further strengthened by combined QCM-D and optical reflectometry measurements.     

Melting and mechanical properties of polymer grafted lipid bilayer membranes

The influence of polymer grafting on the phase behavior and elastic properties of two tail lipid bilayers have been investigated using dissipative particle dynamics simulations. For the range of polymer lengths studied, the L(c) to L(α) transition temperature is not significantly affected for grafting fractions, G(f) between 0.16 and 0.25. A decrease in the transition temperature is observed at a relatively high grafting fraction, G(f) = 0.36. At low temperatures, a small increase in the area per head group, a(h), at high G(f) leads to an increase in the chain tilt, inducing order in the bilayer and the solvent. The onset of the phase transition occurs with the nucleation of small patches of thinned membrane which grow and form continuous domains as the temperature increases. This region is the co-existence region between the L(β)(thick) and the L(α)(thin) phases. The simulation results for the membrane area expansion as a function of the grafting density conform extremely well to the scalings predicted by self-consistent mean field theories. We find that the bending modulus shows a small decrease for short polymers (number of beads, N(p) = 10) and low G(f), where the influence of polymer is reduced when compared to the effect of the increased a(h). For longer polymers (N(p) > 15), the bending modulus increases monotonically with increase in grafted polymer. Using the results from mean field theory, we partition the contributions to the bending modulus from the membrane and the polymer and show that the dominant contribution to the increased bending modulus arises from the grafted polymer.     

Determination of lipid phase transition temperatures in hybrid bilayer membranes

The main gel-to-liquid-crystal (LC) phase transition temperature, T(m), of the lipid monolayer in hybrid bilayer membranes (HBMs) was investigated using vibrational sum frequency spectroscopy (VSFS). In the gel phase, the acyl chains of the lipid molecules assume an ordered, all-trans configuration, whereas in the LC phase, the acyl chains exhibit a significant number of disordered gauche conformers. VSFS has unique sensitivity to the order/disorder transitions in the acyl chains and was used to determine T(m) for a series of saturated phosphatidylcholine lipids on octadecanethiolate self-assembled monolayers (SAMs). The values obtained for T(m) for all lipids studied are significantly higher than for the corresponding lipids in vesicles in solution. Additionally, the transition widths are broader for the lipids in HBMs. The underlying SAM clearly influences the phase behavior of the overlying lipid monolayer.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: detection by antibiotic membrane probes.

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylcholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmetrically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.