Self-assembly of bilayer lipid membrane at multiwalled carbon nanotubes towards the development of photo-switched functional device

Bilayer lipid membrane (BLM) was self-assembled on a uniquely fabricated hydrophilic surface, containing N atoms from the carbon source of ethylene amine, of the multi-walled carbon nanotubes (MWNTs) to form the BLM/MWNTs nanocomposites. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and photoelectric experiments were taken to study the properties of the BLM/MWNTs nanocomposites. The thickness of the BLM, which was calculated from the CV data obtained at BLM/MWNTs electrode, turned out to be 4.38 nm, suggesting that the lipid self-assembled at the nanotubes surface was consistent with a bilayer structure. C₆₀-incorporated BLM could also be self-assembled at the nanotubes surface (C₆₀-BLM/MWNTs). The formation of BLM on the MWNTs surface blocked the diffusion of [Fe(CN)₆]^3/4− redox ions across BLM to the MWNTs electrode as no redox current was observed by CV measurement, whereas the incorporation of the electron mediator, C_60, resumed a pair of redox peaks at C₆₀-BLMs/MWNTs electrode. Moreover, the incorporation of C₆₀ led to a four order of magnitude reduction of the resistance of C₆₀-BLM/MWNTs (369.3 Ω) than that of BLM/MWNTs (3.238 × 10⁶ Ω). MWNTs electrode exhibited an intrinsic cathodic photocurrent (166 μA cm⁻²) while BLM/MWNTs electrode blocked photocurrent response of the MWNTs. Interestingly, C₆₀-BLM/MWNTs electrode resumed partial photoelectric properties (photo current: 65 μA cm⁻²) due to the electron mediation effect of C₆₀ incorporated into the lipid membrane. As a result, the novel self-assembled BLM/MWNTs nanocomposites provided a simple yet useful model to study the C₆₀-mediated photoelectric properties of the BLM/MWNTs which may be applicable to develop new biosensors and molecular devices.     

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 C60) 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 C60 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.     

Self-assembled BLMs: biomembrane models and biosensor applications

In the last few years, there have been a number of research papers on self-assemblies of molecules as ‘advanced’ or ‘smart’ materials. The inspiration for this exciting research, without question, comes from the biological world, where, for example, the lipid bilayer of the cell membrane is the most important self-assembling system. Although the first report on self-assembled bilayer lipid membranes (BLMs) in vitro was published in 1962, interface science, including surface and colloid science, has been dealing with these interfacial self-assemblies of amphiphilic molecules since Robert Hooke's time (1672). BLMs have been used in a number of applications, ranging from basic membrane biophysics studies to the conversion of solar energy via water photolysis, and to biosensor development using supported bilayer lipid membranes (s-BLMs and sb-BLMs). This paper briefly summarizes the past research on the use of BLMs as models of biological membranes and describes some details of our current work on supported BLMs as practical biosensors. Additionally, experiments carried out in close collaboration with others on s-BLMs and sb-BLMs are presented.     

An electrochemical approach tunes the electric property of benzoylferrocene-modified supported lipid membrane

A benzoylferrocene (BFc) supported 3-sn-phosphatidylcholine (PC) film electrode was prepared by casting the solution of BFc and PC in chloroform onto the surface of platinum (Pt). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) results showed that BFc, retained in the biological membrane, acted as a shuttle for electron transfer across the supported bilayer lipid membranes (s-BLMs). Doping of BFc increased membrane conductivity, while electrochemical oxidation of BFc greatly changed the membrane conductivity, the membrane impedance characterized by charge transfer resistance (R_ct) dramatically increased about 400 times (from 10.32 to 3919.67 kΩ). Interestingly, the electrochemical oxidized BFc buried in the membranes could be reduced by applying a low potential, and this led to recurrent of a conductive membrane. The conductivity of the s-BLMs could be controlled by the redox status of embedded BFc molecules. The approach provided a facile and novel way to electrochemically control the membrane conductance of s-BLMs by embedding BFc as a switchable redox mediator.     

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.     

Receptor–ligand interactions in a reconstituted bilayer lipid membrane

A simple method is described to reconstitute membrane receptors into bilayer lipid membranes (BLMs). After reconstitution, the receptor still retains its ligand activity. Furthermore, the relationship between receptor–ligand interactions and electrical properties of reconstituted BLMs such as membrane capacitance (C_m) and membrane resistance (R_m) was studied. When glycophorin in erythrocyte and asialoglycoprotein in hepatocyte were taken as examples, it was found that the resistance of reconstituted BLM decreased when adding blood type monoclonal antibody or the solutions of galactose, respectively, and the decrease is ligand-concentration dependent; however, the membrane capacitance was not influenced. This provides a simple, practical approach to determining the interactions between the receptor and its ligand.     

Study on selectivity of permeating chiral complexes in bilayer lipid membranes (BLMs)

Electrical properties such as membrane potential (E_m) of planar bilayer lipid membranes (BLMs) are readily measured. Planar BLMs have been extensively used as models of biomembranes. In this paper we report BLMs formed in the solutions containing chiral complexes: d-K[Co(EDTA)], l-K[Co(EDTA)]; d-[Co(C₂O₄)(en)₂]I, and l-[Co(C₂O₄)(en)₂]I, whose E_m values display great differences, implying strong chiral selectivity. The permeability ratios of different chiral complexes calculated from E_m are the same as those obtained from human erythrocyte experiments. These results showed that chiral selectivity of cell uptake was mainly caused by the chirality of the membrane phospholipid itself. As a rapid and sensitive analytic tool, the BLM may be used to study permeating pathways and drug–membrane interactions. With further research, the BLM system may be developed into a useful method for drug screening.     

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.     

A minisensor for the rapid screening of atenolol in pharmaceutical preparations based on surface-stabilized bilayer lipid membranes with incorporated DNA

This work describes an electrochemical technique that is suitable for the rapid and sensitive screening of atenolol based on surface-stabilized bilayer lipid membranes (s-BLMs) composed from egg phosphatidylcholine (PC). The interactions of atenolol with s-BLMs produced electrochemical ion current increases that reproducible appeared within a few seconds after the exposure of the membranes to the drug. The current signal increase was related to the concentration of atenolol in bulk solution in the micromolar range. The present lipid film-based sensor provided fast response (i.e. on the order of a few seconds) to alterations of atenolol concentration (20 to 200 micro M) in electrolyte solution. ssDNA incorporated into s-BLMs can interact with atenolol, and decreased the detection limit of this drug by one order of magnitude. The oligomers used were single stranded deoxyribonucleic acids: thymidylic acid icosanucleotide terminated with a C-16 alkyl chain to assist incorporation into s-BLMs (5'-hexadecyl-deoxythymidylic acid icosanucleotide, dT(20)-C(16)). The electrochemical transduction of the interactions of atenolol with s-BLMs was applied in the determination of these compounds in pharmaceutical preparations by using the present minisensor.     

Forming Lipid Bilayer Membrane Arrays on Micropatterned Polyelectrolyte Film Surfaces

A novel method of forming lipid bilayer membrane arrays on micropatterned polyelectrolyte film surfaces is introduced. Polyelectrolyte films were fabricated by the layer‐by‐layer technique on a silicon oxide surface modified with a 3‐aminopropyltriethoxysilane (APTES) monolayer. The surface pK_a value of the APTES monolayer was determined by cyclic voltammetry to be approximately 5.61, on the basis of which a pH value of 2.0 was chosen for layer‐by‐layer assembly. Micropatterned polyelectrolyte films were obtained by deep‐UV (254 nm) photolysis though a mask. Absorbed fluorescent latex beads were used to visualize the patterned surfaces. Lipid bilayer arrays were fabricated on the micropatterned surfaces by immersing the patterned substrates into a solution containing egg phosphatidylcholine vesicles. Fluorescence recovery after photobleaching studies yielded a lateral diffusion coefficient for probe molecules of 1.31±0.17 μm² s^?1 in the bilayer region, and migration of the lipid NBD PE in bilayer lipid membrane arrays was observed in an electric field.     

Interaction of cholesterol with artificial bilayer lipid membrane system and development of an electrochemical sensor

Interaction of Cholesterol with the bilayer arrangement of phospholipid molecules was studied using electrochemical impedance spectroscopy in Sodium Chloride (NaCl) bath solutions. The membrane resistance (R_m) was decreased from 3.35 GΩ in 1.0 M NaCl bath to 0.756 GΩ in 0.01 M NaCl bath. The cholesterol molecules were found to penetrate into Bilayer Lipid Membrane (BLM) and fluidized the BLM phase. Due to fluidization, the membrane resistance was decreased. The fluidization effect of cholesterol was dependent on the concentration of bath solutions. In 1.0 M NaCl bath solution, the membrane was stable up to 200 µM concentration of cholesterol. With the addition of cholesterol in NaCl bath solutions, the membrane capacitance was increased. An impedimetric sensor was developed based on the membrane resistance in the presence of cholesterol at various concentrations. The detection limit of cholesterol by impedimetric sensor was dependent on the concentration of NaCl in the bath.     

冠醚修饰的固体支撑双层类脂膜的形成及性能研究

用饱和了胆固醇和饰用冠醚的角鲨烷／氯仿溶液作成膜液，制备了冠醚修饰的固体支撑双层类脂分子膜。重点考察了成膜物种及技术对膜稳定性及电特生影响。其膜电势随接触水相中的变化呈现Ｎｅｒｎｓｔ响应，线性范围１０＾－４－１０＾－１ｍｏｌ／Ｌ。     

Partial stripping of Ag atoms from silver bilayer on a Au(111) surface accompanied with the reductive desorption of hexanethiol SAM

The interfacial structures of Ag bilayer prepared by underpotential deposition on Au(111) (Ag(2ML)/Au(111)) were determined by ex situ scanning tunneling microscopy and in situ surface X-ray scattering measurements before and after oxidative adsorption and after reductive desorption of a self-assembled monolayer (SAM) of hexanethiol (C₆SH) in alkaline ethanol solution. While no structural change was observed after oxidative formation of C₆SH SAM on the Ag(2ML)/Au(111) in an ethanol solution containing 20 mM KOH and 0.1 mM C₆SH, some of the Ag atoms in the bilayer were stripped when the SAM was reductively desorbed. Dedicated to Professor J. O’M. Bockris on the occasion of his 85th birthday.     

Vagaries of artificial bilayers and gating modes of the SCl channel from the sarcoplasmic reticulum of skeletal muscle

The elucidation of the structure-function relation of reconstituted ion channel proteins into artificial planar bilayers is expected to be greatly influenced by the physical properties of these bilayers. The dependence of the gating mode of a voltage- and calcium-dependent small chloride channel (SCl channel) on the bilayer properties, as deduced from the specific bilayer capacitance, C_b, was investigated. In bilayers having a C_b value of 0.42 μF/cm² the SCl channel was in the “burst mode”. At a C_b value of 0.28 μF/cm² the “burst mode” gave way to a “flickering mode”. At C_b values between 0.24 and 0.28 μF/cm² channel bursts were often infrequent and brief. No ion channel activities were observed at C_b values lower than 0.24 μF/cm². These results suggest that changes in bilayer thickness, mediated via n-decane, contribute to changes in channel gating mode, i.e. “gear shift”. Standardizing C_b and its time-independence are important in comparative and detailed investigations of ion channel characteristics.     

Effect of DODAB Nano-Sized Cationic Bilayer Fragments against Leishmania amazonensis

The dioctadecyldimethylammonium bromide (DODAB) is a double-chained cationic lipid with potent bactericide and fungistatic activities; however, its toxicity on protozoan parasites is still unknown. Here, we show the antileishmanial activity of DODAB nano-sized cationic bilayer fragments on stationary-phase promastigotes and amastigotes of Leishmania amazonensis, the causative agent of cutaneous leishmaniasis. Upon treatment with DODAB, we analyzed the parasite surface zeta-potential, parasite viability, cellular structural modifications, and intracellular proliferation. The DODAB cytotoxic effect was dose-dependent, with a median effective concentration (EC₅₀) of 25 µM for both life-cycle stages, comparable to the reported data for bacteria and fungi. The treatment with DODAB changed the membrane zeta-potential from negative to positive, compromised the parasite’s morphology, affected the cell size regulation, caused a loss of intracellular organelles, and probably dysregulated the plasma membrane permeability without membrane disruption. Moreover, the parasites that survived after treatment induced small parasitophorous vacuoles and failed to proliferate inside macrophages. In conclusion, DODAB displayed antileishmanial activity, and it remains to be elucidated how DODAB acts on the protozoan membrane. Understanding this mechanism can provide insights into the development of new parasite-control strategies.     

Interaction between Potassium Phosphate Bu er Solution and Modeling Cell Membrane Investigated by Sum Frequency Generation Vibrational Spectroscopy (cited: 1)

Potassium phosphate buffer solution has been widely used in the biological experiments, which represents an important process of the interaction between ions and biomolecules, yet the in fluences of potassium phosphate on biomolecules such as the cell membrane are still poorly understood at the molecular level. In this work, we have applied sum frequency generation vibrational spectroscopy and carried out a detailed study on the interaction between potassium phosphate buffer solution (PBS) and negative 1,2-dimyristoyl-d₅₄-sn-glycero-3-[phospho-rac-(1-glycerol)] (d₅₄-DMPG) lipid bilayer in real time. The PBS-induced dynamic change in the molecular structure of d₅₄-DMPG lipid bilayer was monitored using the spectral features of CD₂, CD₃, lipid head phosphate, and carbonyl groups for the first time. It is found that K⁺ can bind to the cell membrane and cause the signal change of CD₂, CD₃, lipid head phosphate, and carbonyl groups quickly. Potassium PBS interacts with lipid bilayers most likely by formation of toroidal pores inside the bilayer matrix. This result can provide a molecular basis for the interpretation of the effect of PBS on the ion-assisted transport of protein across the membrane.     

Suppression of nitric oxide production in mouse macrophages by soybean flavonoids accumulated in response to nitroprusside and fungal elicitation

Background

The anti-inflammatory properties of some flavonoids have been attributed to their ability to inhibit the production of NO by activated macrophages. Soybean cotyledons accumulate certain flavonoids following elicitation with an extract of the fungal pathogen Diaporthe phaseolorum f. sp. meridionalis (Dpm). Sodium nitroprusside (SNP), a nitric oxide donor, can substitute for Dpm in inducing flavonoid production. In this study, we investigated the effect of flavonoid-containing diffusates obtained from Dpm- and SNP-elicited soybean cotyledons on NO production by lipopolysaccharide (LPS)- and LPS plus interferon-γ (IFNγ)-activated murine macrophages.

Results

Significant inhibition of NO production, measured as nitrite formation, was observed when macrophages were activated in the presence of soybean diffusates from Dpm- or SNP-elicited cotyledons. This inhibition was dependent on the duration of exposure to the elicitor. Daidzein, genistein, luteolin and apigenin, the main flavonoids present in diffusates of elicited cotyledons, suppressed the NO production by LPS + IFNγ activated macrophages in a concentration-dependent manner, with IC₅₀ values of 81.4 μM, 34.5 μM, 38.6 μM and 10.4 μM respectively. For macrophages activated with LPS alone, the IC₅₀ values were 40.0 μM, 16.6 μM, 10.4 μM and 2.8 μM, respectively. Western blot analysis showed that iNOS expression was not affected by daidzein, was reduced by genistein, and was abolished by apigenin, luteolin and Dpm- and SNP-soybean diffusates at concentrations that significantly inhibited NO production by activated macrophages.

Conclusions

These results suggest that the suppressive effect of flavonoids on iNOS expression could account for the potent inhibitory effect of Dpm- and SNP-diffusates on NO production by activated macrophages. Since the physiological concentration of flavonoids in plants is normally low, the treatment of soybean tissues with SNP may provide a simple method for substantially increasing the concentration of metabolites that are beneficial for the treatment of chronic inflammatory diseases associated with NO production.

     

十二烷基磺基甜菜碱与铂电极支撑的磷脂双层膜作用的电化学研究

以铂电极支撑的磷脂双层膜(Supported Bilayer Lipid Membrane,s-BLM)作为生物膜的模型,以Fe(CN)36-和Fe(CN)64-为探针分子,利用循环伏安法(CV)和交流阻抗谱(EIS)研究两性表面活性剂十二烷基磺基甜菜碱(Dodecyl Sulfobetaine,DSB)对s-BLM相互作用。结果显示,DSB可以嵌入到s-BLM的疏水区,容易使其表面分子的排列发生变化,产生缺陷或孔洞,探针分子Fe(CN)63-和Fe(CN)64-可以通过这些微孔接近电极,产生氧化还原响应。并且作用时间、DSB的浓度以及胆固醇的存在与否对二者的相互作用有直接影响。此外作用后的双层膜在0.1mol/LKCl溶液中能够自我修复,这表明DSB与s-BLM的相互作用是可逆的。     

The effect of diffuse layer on adsorption potential shifts and differential capacitance in systems (In-Ga)/[N-methylformamide + mc KA + (1 - m)c KClO4], where KA is KCl, KBr and KI

By the regression analysis of dependences of the adsorption potential shift (E _ads) on the electrode charge in systems (In-Ga)/[NMF + 0.1m M KCl + 0.1(1 - m) M KClO₄, (In-Ga)/[NMF + 0.1m M KBr + 0.1(1 ? m) M KClO₄], and (In-Ga)/[NMF + 0.1m M KI + 0.1(1 - m) M KClO₄] with the following m fractions of the surface-active anion: 0.05, 0.1, 0.2, 0.5, and 1, the adsorption parameters are calculated in terms of two models both based on the Frumkin isotherm and considering the free adsorption energy as a function of the electrode charge, where one model takes into account the diffuse layer and the other ignores it. It is shown that for the studied electrode charges q ≤ 10 μC/cm², both models provide equal accuracy in calculating E _ads and the differential capacitance (C) in the systems under study. However, for determination of adsorption parameters, the regression analysis of E _ads vs. q curves has several advantages over the analogous analysis of C vs. q curves.