Supported Bilayer Lipid Membranes Research Articles

Cyclic voltammetry study of glucose and insulin interactions with supported lipid membrane

The effect of d-glucose and insulin on conducting properties of supported bilayer lipid membranes (s-BLM) modified by anthraquinone-2-sulphonic acid (AQS) at the presence of potassium ferricyanide was studied by means of cyclic voltammetry (CV). Both the oxidation and the reduction current peaks were found to decrease at the presence of glucose in concentration range varying from 10 to 320 mM. The influence of insulin on membrane properties is ambiguous. While the pretreatment of membrane with 20 mU l −1 of insulin evoked slight increase of the current with unchanged course of the dependence of peak current on glucose, the decrease of conductance was observed above 10 5 mU l −1 of insulin.

High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast.

Total internal reflection fluorescence microscopy is widely used to confine the excitation of a complex fluorescent sample very close to the material on which it is supported. By working with high refractive index solid supports, it is possible to confine even further the evanescent field, and by varying the angle of incidence, to obtain quantitative information on the distance of the fluorescent object from the surface. We report the fabrication of hybrid surfaces consisting of nm layers of SiO(2) on lithium niobate (LiNbO(3), n = 2.3). Supported lipid bilayer membranes can be assembled and patterned on these hybrid surfaces as on conventional glass. By varying the angle of incidence of the excitation light, we are able to obtain fluorescent contrast between 40-nm fluorescent beads tethered to a supported bilayer and fluorescently labeled protein printed on the surface, which differ in vertical position by only tens of nm. Preliminary experiments that test theoretical models for the fluorescence-collection factor near a high refractive index surface are presented, and this factor is incorporated into a semiquantitative model used to predict the contrast of the 40-nm bead/protein system. These results demonstrate that it should be possible to profile the vertical location of fluorophores on the nm distance scale in real time, opening the possibility of many experiments at the interface between supported membranes and living cells. Improvements in materials and optical techniques are outlined.

The Studies on Hemin as Mimetic Peroxidase in Salt Bridge Supported Bilayer Lipid Membrane System

The Studies on Hemin as Mimetic Peroxidase in Salt Bridge Supported Bilayer Lipid Membrane System

The Studies on Hemin as Mimetic Peroxidase in Salt Bridge Supported Bilayer Lipid Membrane System

Hemin exhibits mimetic peroxidase activity at pH 7–8 in a salt bridge supported bilayer lipid membrane (sb-BLM) system. sb-BLM was modified with anionic site-lauric acid anions (LA), then, hemin was also adsorbed and incorporated in sb-BLM by electrostatic force between positively charged hemin and negatively charged LA, which results in the enhancement of the electron transfer between hemin and the electrode surface. An irreversible cyclic voltammetry (CV) behavior of hemin was observed even in the absence of mediators (hydrogen donor). A novel and biological compatible sb-BLM biosensor was constructed. The electrocatalytically kinetic behavior of hemin was investigated with this kind of biosensor. The cathodic peak current (IPc) of hemin was increased in the presence of hydrogen peroxide, the intensity of which is in proportion to the concentration of hydrogen peroxide. The effects of ionic strength and pH on the response of the biosensor were also investigated for optimum analytical performance.

Fullerene modified supported lipid membrane as sensitive element of sensor for odorants

A supported lipid bilayer membrane (s-BLMs) formed on a freshly cleaved metallic surface by the Tien method was applied for the design of an electrochemical sensor for detection of neutral odorant molecules. The lipid bilayer was modified by saturation with fullerene C 60, which possesses electron mediator properties and facilitates a redox reaction occurring at the border of the lipid membrane and metal surface. I 2/I − and ferrocenyl trimethyl bromide were used as electroactive marker ions. The smell compounds adsorb on the surface of the lipid layer and change its structure. As a consequence the ratio of marker ion penetration to the lipid membrane is altered. The magnitude of these changes depends on the amount and chemical structure of adsorbed molecules. The research presented was carried out by cyclic voltammetry. The magnitude of the electrochemical signal generated by smell compounds was correlated with other parameters describing their molecular properties such as: octanol/water partition coefficients and dipole moments.

Ion-channel sensing of ferricyanide anion based on a supported bilayer lipid membrane.

Ferricyanide anion has usually been used as a marker of ion-channel sensors. In this work we first found that ferricyanide, itself, can act as a stimulus to regulate the permeability of sBLM prepared from didodecyldimethylammonium bromide (a kind of synthetic lipid) on a GC electrode. We used cyclic voltammetry and a.c. impedance to investigate this phenomenon. The interaction between sBLM and ferricyanide concerns time. Furthermore, we developed a sensor for ferricyanide anion. The ion-channel sensor is highly sensitive. It can detect ferricyanide concentration as low as 5 microM.

Nitric oxide enhances the capacitance of self-assembled, supported bilayer lipid membranes

The effect of nitric oxide (NO) at biologically relevant concentrations on the electrochemical features of the membrane was investigated by cyclic voltammetry (CV) at self-assembled, stainless steel supported lipid bilayer membranes (s-BLMs) using a three-electrode system. The results showed that the membrane capacitance ( C m) of s-BLMs was dramatically enhanced by the presence of increasing NO concentration from 0 to 70 μM. For comparison, fullerene C 60 doped s-BLMs (C 60@s-BLMs) was also studied. The C m of C 60@s-BLMs increased with NO concentration from 0 to 16 μM and gradually reached a plateau value when NO concentration was over 16 μM. We concluded that (i) NO accumulated inside lipid bilayer increases the C m of s-BLMs, and (ii) C 60 inside s-BLMs changes the dielectric constant of lipid bilayer, thus reducing the effect of NO on the C m of C 60@s-BLMs. This novel self-assembled lipid modified probe provides a simple yet interesting model to study the effect of NO on the electrical conductance of the membrane.

Study on interaction of hemin with pyronarine and analytical application in salt bridge supported bilayer lipid membrane system

Pyronarine (PY) is a novel antimalarial drug. The interaction of hemin with PY was investigated in salt bridge supported bilayer lipid membrane (Sb-BLM) system. Sb-BLM was modified with anionic site lauric acid anions (LA). Then, hemin was also adsorbed and incorporated in Sb-BLM by electrostatic force between hemin and LA. Hemin showed well cyclic voltammetry behavior. The cathodic peak current I Pc of hemin decreased in the presence of PY, which illustrated the interaction between hemin and PY. The I Pc decreases proportional to the increase of PY concentration from 2.75×10 −9 to 3.02×10 −5 mol −1, which is the basis for the determination of PY. The interacting mechanism was studied and the optimum conditions for assay of PY were also investigated.

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.

Studies on Electrocatalytical Kinetic Behavior of Horseradish Peroxidase and Assay for Hydrogen Peroxide at Salt Bridge Supported Bilayer Lipid Membrane

This article reports on the electrochemical behavior, relative to the Fe(III)-Fe(II) conversion, of horseradish peroxidase (HRP) incorporated in a salt bridge-supported bilayer lipid membrane (sb-BLM) modified with lauric acid (LA). The incorporation of HRP was achieved by electrostatic interaction between HRP and LA anion sites in the sb-BLM, which results in the enhancement of the electron exchange between the protein and the electrode surface. A quasireversible electron transfer was observed even in the absence of mediators. A novel and biological compatible biosensor was constructed. The electrocatalytically kinetic behavior of HRP was investigated with this biosensor. Furthermore, the cathodic peak current (IPc) of HRP was increased in the presence of hydrogen peroxide, the intensity of which is strictly dependent on the concentration of hydrogen peroxide. The relationship is ΔIPc(A)=[1.215 lnC(mmol L−1)+1.018]×10−7. The effects of the coexisting substances and the pH values on the response of the biosensor were also investigated for optimum analytical performance.

The study on the interaction of DNA with hemin and the detection of DNA using the salt bridge supported bilayer lipid membrane system

The interaction of DNA (calf thymus) with hemin was studied by means of cyclic voltammetry (CV) in a salt bridge supported bilayer lipid membrane (sb-BLM) system. Sb-BLM was modified with lauric acid (LA) dissolved in the membrane forming solution, then hemin molecules in electrolyte could also be embedded into the BLM by electrostatic interaction between hemin and LA. Hemin showed a well-defined CV behavior. The cathodic peak current ( I Pc,) of hemin decreased in the presence of DNA, which is consistent with the results of decreasing hemin concentration and illustrates the existence of interaction between hemin and DNA. The interacting mechanism was studied. The effect of pH on the interaction was also considered. The I Pc of hemin decreases in proportion to the DNA concentration from 8.44×10 −5 to 6.75×10 −4 mol l −1, which provided a possibility of detecting DNA with a natural biological substance in a biomimetic membrane system.

Some Electrochemical Features of Supported Bilayer Lipid Membranes

The cyclic voltammogram and electrochemical impedance spectroscopy of self-assembled, stainless steel supported bilayer lipid membranes (s-BLMs) were investigated using a three-electrode system. The membrane resistance and capacitance calculated from the whole impedance spectrum rather than at certain frequency were close to the conventional BLMs (planar lipid bilayers). The time course of impedance under certain applied frequencies was investigated. The results showed that the membrane capacitance of s-BLM fluctuated under low frequency and gradually reached a constant value with the increase of the applied frequency. Fullerenes doped s-BLM were demonstrated to intensify this fluctuation behavior of membrane capacitance. A possible mechanism of this peculiar property of s-BLMs under low frequencies is discussed.

A facile approach to immobilize protein for biosensor: self-assembled supported bilayer lipid membranes on glassy carbon electrode

A kind of solid substrate, glassy carbon (GC) electrode, was selected to support self-assembled lipid layer membranes. On the surface of GC electrode, we made layers of dimyristoylphosphatidy1choline (DMPG, a kind of lipid). From electrochemical impedance experiments, we demonstrated that the lipid layers on the GC electrode were bilayer lipid membranes. We immobilized horseradish peroxidase (HRP) into the supported bilayer lipid membranes (s-BLM) to develop a kind of mediator-free biosensor for H 2O 2. The biosensor exhibited fine electrochemical response, stability and reproducibility due to the presence of the s-BLM. As a model of biological membrane, s-BLM could supply a biological environment for enzyme and maintain its activity. So s-BLM is an ideal choice to immobilize enzyme for constructing the mediator-free biosensor based on GC electrode.

Cell adhesion to protein-micropatterned-supported lipid bilayer membranes.

A new method for constructing controlled interfaces between cells and synthetic supported lipid bilayer membranes is reported. Microcontact printing is used to define squares and grid lines of fibronectin onto glass, which subsequently direct the self-assembly of fluid lipid bilayers onto the complementary, uncoated regions of the surface. Features of fibronectin as small as 5 microm effectively control the lateral organization of the lipid bilayers. These fibronectin barriers also facilitate the adhesion of endothelial cells, which exhibit minimal adhesion to fluid supported lipid bilayers alone. Cells selectively adhere to the features of fibronectin, spanning over and exposing the cells to the intervening regions of supported lipid bilayer. Cell spreading is correlated with both the geometry and dimensions of the fibronectin barriers. Importantly, lipids underlying adherent cells are laterally mobile, suggesting that, in contrast to the regions of fibronectin, cells were not in direct contact with the supported membrane. Protein micropatterning thus provides a valuable tool for controlling supported membranes and for juxtaposing anchorage-dependent cells with lipid bilayers. These systems should be generally useful for studying specific interactions between cells and biomolecules incorporated into supported membranes, and as an approach for integrating living cells with synthetic, laterally complex surfaces.

Fabrication and photoelectric properties of self-assembled bilayer lipid membranes on conducting glass

Supported bilayer lipid membranes (s-BLMs with and without the doping of fullerene C 60) self-assembled on indium–tin oxide (ITO) glass were fabricated and characterized by cyclic voltammetry and electrochemical impedance spectroscopy using a three-electrode system. The photoelectric properties of the ITO supported planar lipid bilayers were studied. Light intensity of irradiation, bias voltage, and concentration of donors have been found to be limiting factors of the transmembrane photocurrent. The facilitation effect of C 60 doping in s-BLMs on the photoinduced electron transfer across s-BLM is discussed. This novel self-assembled ITO/s-BLM system may provide a simple and mechanically stable model for the study of the photoelectric and photodynamic properties of biomembranes.

Method for Fabricating Supported Bilayer Lipid Membranes on Gold

The paper describes the synthesis of surfaces based on self-assembled monolayers (SAMs) of alkanethiolates on gold that were designed to support the adsorption of bilayer lipid membranes, and the feasibility of using these surfaces for making high-density arrays (HDAs) of membranes. The synthesis involved (i) the formation of SAMs of 16-mercaptohexadecanoic acid (MHA) on gold; (ii) the activation of the carboxylic acid groups to interchain anhydrides; (iii) the treatment of the reactive surfaces with Brij-76 (C18H37(OCH2CH2)n-10OH) (1) or Brij-76-amine (2) (C18H37(OCH2CH2)n-10NH2); and (iv) the formation of supported membranes by incubation or printing of the lipids. The oligo(ethylene glycol) moiety of the anchor lipid provides a hydrophilic spacer between the surface and the adsorbed lipid to enable the incorporation of membrane-spanning proteins with extramembrane domains. Data from infrared spectroscopy confirmed the coupling of 2 to the surface through the formation of peptide bonds. Ellipsometric measurements showed an increase of ∼15 Å in the thickness of the SAM after coupling to 2; this observation suggests that ∼25% of the carboxylic acid groups of the MHA-SAM are derivatized with Brij groups. The yields for coupling of 1 were ∼40% of that observed for 2. Experiments using surface plasmon resonance (SPR) were consistent with the binding of lipid bilayers to the Brij-derivatized surface, although alternative structures for the supported lipids cannot be ruled out; by contrast, SAMs of hexadecanethiolate on gold were observed to bind lipid monolayers. Biospecific binding of neutravidin was observed on supported membranes incorporating biotinylated lipids. HDAs of lipids were made by printing lipids onto the Brij-derivatized surfaces using a quill-pin printer. Fluorescence microscopy indicated that the printed lipids remained confined to the printed areas; these observations demonstrate the applicability of using commercial HDA printers for generating high-density membrane arrays on the Brij-modified surfaces.

Electrochemical biosensor based on supported planar lipid bilayers for fast detection of pathogenic bacteria

This paper presents a new ion-channel biosensor based on supported bilayer lipid membrane for direct and fast detection of Campylobacter species. The sensing element of a biosensor is composed of a stainless-steel working electrode, which is covered by artificial bilayer lipid membrane (BLM). Antibodies to bacteria embedded into the BLM are used as channel forming proteins. The biosensor has a strong signal amplification effect, which is defined as the total number of ions transported across the BLM. The total number of (univalent) ions flowing through the channels is 10 10 ions s −1. The biosensor showed a very good sensitivity and selectivity to Campylobacter species.

Photoelectric measurements of s-BLM/nucleoli: a new technique for studying apoptosis

A new method based on photoelectric measurement for analyzing apoptosis of cell-free MCF-7 nucleoli is reported. Supported bilayer lipid membrane (s-BLM) was used to enclose nucleoli in biological environment. The s-BLM was self-assembled on the wall of a super-thin cell. During the apoptosis induced by Taxol, the photoelectric current of the self-assembled s-BLM/nucleoli was found decreasing with time, suggesting the degradation of nucleus DNA. Electron transfer along the DNA double helix and along nuclear skeleton is assumed in the interpretation. This novel photoelectric analytical method may provide a rapid and sensitive technique to evaluate apoptosis.

Patterning Barriers to Lateral Diffusion in Supported Lipid Bilayer Membranes by Blotting and Stamping

Two new methods are introduced for patterning fluid lipid bilayer membranes on solid supports. These methods are based on the observation that supported membranes undergo self-limiting lateral expansion when bilayer material is removed from the surface or when it is deposited in a pattern on a surface. Spatially selective (patterned) removal of bilayer material can be achieved by using a poly(dimethylsiloxane) (PDMS) stamp. Following slight lateral expansion into the bilayer-free region created by this blotting process, stable barriers to lateral diffusion are formed. Inspection of the barrier regions indicates that nearly all of the bilayer material is removed, implying that it has been transferred to the stamp. As a consequence, it also proves possible to transfer the lifted material from the stamp onto a fresh surface. The transferred material retains the original pattern from the stamp and is also laterally mobile, and the mobility is confined to the printed region. Alternatively, bilayers assembled o...

Salt-Bridge Supported Bilayer Lipid Membrane Modified with Calix[n]arenes as Alkali Cation Sensors

ABSTRACT Potentiometric behaviors of a salt-bridge supported bilayer lipid membrane (Sb-BLM) modified with Calix[n]arene (n=4, 6, 8) derivatives are described for some alkali metal ions. The modified Sb-BLM was used as an alkali cation sensor. The membrane potentials were observed to generate Nernstian responses to the concentration of alkali metal ions in electrolyte. The Sb-BLM modified with the calix[n]arenes show high selectivity for individual alkali metal ions: Calix[8]arenes for K+, calix[6]arenes for Cs+ Calix[4]arenes show no selectivity for any alkali ions. The interacting mechanism is also discussed.