直流偏压对于在玻碳电极上双层类脂膜成膜过程的影响

应用循环伏安法和电化学阻抗谱研究了直流偏压对卵磷脂在玻碳电极表面自组装成膜过程及其结构的影响.实验发现:无论在正偏压还是负偏压条件下,卵磷脂在玻碳电极上均可组装成膜.施加正偏压时,由于玻碳电极表面所带的正电荷与卵磷脂端基之间的静电作用,使得卵磷脂在电极表面倾向于形成双层的类脂膜,并在0.4V偏压下电极阻抗达到最大值.继续增大电极正向偏压,s-BLM缺陷增加,以至趋于被击穿.提出了适宜的等效电路,并据此非线性拟合电极过程,求得部分阻抗的模型参数.研究发现:膜电容和电荷传递电阻呈现良好的互补效应.     

Combined electrochemistry and surface-enhanced infrared absorption spectroscopy of gramicidin a incorporated into tethered bilayer lipid membranes

Support from the support: Tethered bilayer lipid membranes containing the cation-channel-forming peptide gramicidin?A were assembled on nanostructured Au films. The combination of surface-enhanced infrared absorption (SEIRA) and electrochemical impedance spectroscopy (EIS) was used for the in situ structural and functional characterization of gramicidin?A in the same device.     

The Role of Amyloid β-Biomembrane Interactions in the Pathogenesis of Alzheimer's Disease: Insights from Liposomes as Membrane Models

The aggregation and deposition of amyloid β (Aβ) peptide onto neuronal cells, with consequent cellular membrane perturbation, are central to the pathogenesis of Alzheimer's disease (AD). Substantial evidence reveals that biological membranes play a key role in this process. Thus, elucidating the mechanisms by which Aβ interacts with biomembranes and becomes neurotoxic is fundamental to developing effective therapies for this devastating progressive disease. However, the structural basis behind such interactions is not fully understood, largely due to the complexity of natural membranes. In this context, lipid biomembrane models provide a simplified way to mimic the characteristics and composition of membranes. Aβ-biomembrane interactions have been extensively investigated applying artificial membrane models to elucidate the molecular mechanisms underlying the AD pathogenesis. This review summarizes the latest findings on this field using liposomes as biomembrane model, as they are considered the most promising 3D model. The current challenges and future directions are discussed.     

Ceramide-enriched microdomains in planar membranes

Ceramide has a large effect on the properties of biological membranes, increasing lipid order and promoting lateral phase separation, and plays an important role in cell signaling. This review provides an overview of recent studies of the effects of direct ceramide incorporation and enzymatic ceramide generation on planar supported membranes, including lipid monolayers and supported lipid bilayers. Recent studies have focused on understanding the nucleation, growth and morphology of ceramide gel domains, characterizing the properties of ceramide-rich membrane phases and investigating the effects of ceramide on phase-separated membranes with co-existing liquid-ordered and fluid phases, as models for cellular membranes.     

双层类脂膜及其在电化学生物传感器中的应用

详细评述了各种双层类脂膜包括传统的双层类脂膜（ＢＬＭ）、固体载体支撑的自组双层类脂膜（ｓ－ＢＬＭ）、固体载体支撑的混合双层类脂膜（ｅ－ＢＬＭ）的制备方法和特性,比较了其优缺点。介绍了双层类脂膜在电化学生物传感器中的应用,并展望了发展前景。     

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。     

Agar-supported lipid bilayers — basic structures for biosensor design. Electrical and mechanical properties

The lipid bilayer is widely accepted as the basic structure of all biological membranes. Known as BLM (bilayer lipid membrane), it can be prepared artificially. Suitably modified, the BLM serves as a very appropriate model for biological membranes. Recent investigations have verified the high analytical potential of artificial lipid membranes. With a structure and composition almost identical to the lipid moiety of biomembranes, the BLM may serve as an ideal host for receptor molecules of biological origin, thus becoming a transducer which could “see” the environment the way the living cell does. For the construction of lipid bilayer based biosensors; however, stable, easy to prepare and long-lasting lipid membranes are required. With this aim in mind, we have prepared lipid bilayer membranes which use an agar gel as support. This as-BLM (agar-supported BLM) has been shown to possess the same electrical, mechanical and dynamic properties the conventional BLM is famous for, along with the benefits of long-term stability and considerably elevated breakdown voltages. Its preparation on the tip of an agar-filled Teflon tube of 0.5 mm diameter is easy and can be performed even by less-skilled personnel.

In an attempt of further miniaturization the concept of the as-BLM was applied to thin-film micro-systems manufactured by standard micro-electronic techniques. The result is a lipid bilayer system, which, while preserving all the essential properties of the bilayer lipid membrane, can serve as a basic building block for cheap, disposable biosensoric systems.     

Molecular Dynamics Simulation of Small Molecules Interacting with Biological Membranes

Cell membranes protect and compartmentalise cells and their organelles. The semi-permeable nature of these membranes controls the exchange of solutes across their structure. Characterising the interaction of small molecules with biological membranes is critical to understanding of physiological processes, drug action and permeation, and many biotechnological applications. This review provides an overview of how molecular simulations are used to study the interaction of small molecules with biological membranes, with a particular focus on the interactions of water, organic compounds, drugs and short peptides with models of plasma cell membrane and stratum corneum lipid bilayers. This review will not delve on other types of membranes which might have different composition and arrangement, such as thylakoid or mitochondrial membranes. The application of unbiased molecular dynamics simulations and enhanced sampling methods such as umbrella sampling, metadynamics and replica exchange are described using key examples. This review demonstrates how state-of-the-art molecular simulations have been used successfully to describe the mechanism of binding and permeation of small molecules with biological membranes, as well as associated changes to the structure and dynamics of these membranes. The review concludes with an outlook on future directions in this field.     

Glyco-acrylate copolymers for bilayer tethering on benzophenone-modified substrates

Model biological membranes are becoming increasingly important for studying fundamental biophysical phenomena and developing membrane-based devices. To address the anticipated problem of non-physiological interactions between membrane proteins and substrates seen in “solid-supported lipid bilayers” that are formed directly on hydrophilic substrates, we have developed a polymer-tethered lipid bilayer system based on a random copolymer with multiple lipid analogue anchors and a glyco-acrylate backbone. This system is targeted at applications that, most importantly, require stability and robustness since each copolymer has multiple lipid analogues that insert into the bilayer. We have combined this copolymer with a flexible photochemical coupling scheme that covalently attaches the copolymer to the substrate. The Langmuir isotherms of mixed copolymer/free lipid monolayers measured at the air–water interface indicate that the alkyl chains of the copolymer lipid analogues and the free lipids dominate the film behavior. In addition, no significant phase transitions are seen in the isotherms, while hysteresis experiments confirm that no irreversible states are formed during the monolayer compression. Isobaric creep experiments at the air–water interface and AFM experiments of the transferred monolayer are used to guide processing parameters for creating a fluid, homogeneous bilayer. Bilayer homogeneity and fluidity are monitored using fluorescence microscopy. Continuous bilayers with lateral diffusion coefficients of 0.6 μm²/s for both leaflets of the bilayer are observed for a 5% copolymer system.     

The Impact of an Anchoring Layer on the Formation of Tethered Bilayer Lipid Membranes on Silver Substrates

Tethered bilayer lipid membranes (tBLMs) have been known as stable and versatile experimental platforms for protein–membrane interaction studies. In this work, the assembly of functional tBLMs on silver substrates and the effect of the molecular chain-length of backfiller molecules on their properties were investigated. The following backfillers 3-mercapto-1-propanol (3M1P), 4-mercapto-1-butanol (4M1B), 6-mercapto-1-hexanol (6M1H), and 9-mercapto-1-nonanol (9M1N) mixed with the molecular anchor WC14 (20-tetradecyloxy-3,6,9,12,15,18,22 heptaoxahexatricontane-1-thiol) were used to form self-assembled monolayers (SAMs) on silver, which influenced a fusion of multilamellar vesicles and the formation of tBLMs. Spectroscopic analysis by SERS and RAIRS has shown that by using different-length backfiller molecules, it is possible to control WC14 anchor molecules orientation on the surface. An introduction of increasingly longer surface backfillers in the mixed SAM may be related to the increasing SAMs molecular order and more vertical orientation of WC14 at both the hydrophilic ethylenoxide segment and the hydrophobic lipid bilayer anchoring alkane chains. Since no clustering of WC14 alkane chains, which is deleterious for tBLM integrity, was observed on dry samples, the suitability of mixed-component SAMs for subsequent tBLM formation was further interrogated by electrochemical impedance spectroscopy (EIS). EIS showed the arrangement of well-insulating tBLMs if 3M1P was used as a backfiller. An increase in the length of the backfiller led to increased defectiveness of tBLMs. Despite variable defectiveness, all tBLMs responded to the pore-forming cholesterol-dependent cytolysin, vaginolysin in a manner consistent with the functional reconstitution of the toxin into phospholipid bilayer. This experiment demonstrates the biological relevance of tBLMs assembled on silver surfaces and indicates their utility as biosensing elements for the detection of pore-forming toxins in liquid samples.     

双层磷脂膜的电化学性质及其在生物传感器中的应用

由于双层磷脂膜（BLM）可模仿自然界的生物细胞膜的生物相容性，成为物分子的天然固定化材料，因此生物传感器的研制领域显示出广泛的应用前景，本文介绍了BLM、s-BLM的电化学性质，制备技术，并评述了其在生物传感器的应用研究进展。     

Structural, dynamic, and electrostatic properties of fully hydrated DMPC bilayers from molecular dynamics simulations accelerated with graphical processing units (GPUs)

We present results of molecular dynamics simulations of fully hydrated DMPC bilayers performed on graphics processing units (GPUs) using current state-of-the-art non-polarizable force fields and a local GPU-enabled molecular dynamics code named FEN ZI. We treat the conditionally convergent electrostatic interaction energy exactly using the particle mesh Ewald method (PME) for solution of Poisson's Equation for the electrostatic potential under periodic boundary conditions. We discuss elements of our implementation of the PME algorithm on GPUs as well as pertinent performance issues. We proceed to show results of simulations of extended lipid bilayer systems using our program, FEN ZI. We performed simulations of DMPC bilayer systems consisting of 17,004, 68,484, and 273,936 atoms in explicit solvent. We present bilayer structural properties (atomic number densities, electron density profiles), deuterium order parameters (S(CD)), electrostatic properties (dipole potential, water dipole moments), and orientational properties of water. Predicted properties demonstrate excellent agreement with experiment and previous all-atom molecular dynamics simulations. We observe no statistically significant differences in calculated structural or electrostatic properties for different system sizes, suggesting the small bilayer simulations (less than 100 lipid molecules) provide equivalent representation of structural and electrostatic properties associated with significantly larger systems (over 1000 lipid molecules). We stress that the three system size representations will have differences in other properties such as surface capillary wave dynamics or surface tension related effects that are not probed in the current study. The latter properties are inherently dependent on system size. This contribution suggests the suitability of applying emerging GPU technologies to studies of an important class of biological environments, that of lipid bilayers and their associated integral membrane proteins. We envision that this technology will push the boundaries of fully atomic-resolution modeling of these biological systems, thus enabling unprecedented exploration of meso-scale phenomena (mechanisms, kinetics, energetics) with atomic detail at commodity hardware prices.     

The Effect of Bacterial Signal Indole on the Electrical Properties of Lipid Membranes

Indole is an important biological signalling molecule produced by many Gram positive and Gram negative bacterial species, including Escherichia coli. Here we study the effect of indole on the electrical properties of lipid membranes. Using electrophysiology, we show that two indole molecules act cooperatively to transport charge across the hydrophobic core of the lipid membrane. To enhance charge transport, induced by indole across the lipid membrane, we use an indole derivative, 4 fluoro‐indole. We demonstrate parallels between charge transport through artificial lipid membranes and the function of complex eukaryotic membrane systems by showing that physiological indole concentrations increase the rate of mitochondrial oxygen consumption. Our data provide a biophysical explanation for how indole may link the metabolism of bacterial and eukaryotic cells.     

Biomembrane mimetic electrochemical sensors

Biomimetic approaches to the assembling of regular layers of electrochemical sensors make it possible to extend their performance because of the tuning shape and charge of the lipid analogs and implementation of artificial receptors in the lipid layers. The role of artificial components in modification of the properties of the surface layers and application of the nanopore devices are of particular interest. In review, recent trends in assembling modified lipid membranes and their artificial analogs with nanopores have been considered with particular emphasis on their use in electrochemical sensors.     

Investigation of interaction of Leu-enkephalin with lipid membranes

Enkephalins are peptides with morphine-like activity. To achieve their biological function, they must be transported from an aqueous phase to the lipid-rich environment of their membrane bound receptor proteins. In our study, zeta potential (ZP) method was used to detect the association of Leu-enkephalin and Leu-enkephalinamide with phospholipid liposomes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), cholesterol (Chol), sphingomyelin (SM) as well as soybean phospholipid (SBPL). Transfer of the peptides over lipid membranes was examined by electrophysiology technique (ET) and fluorescence spectroscopy (FS), and further confirmed using 4-fluoro-7-nitrobenzofurazan (NBD-F) labeled Leu-enkephalin (NBD-F-enkephalin) with confocal laser scanning microscopy method (CLSM). Results of zeta potential showed that enkephalinamide associated with lipid membranes and gradually saturated on the membranes either hydrophobically or electrostatically or both. Data from electrophysiology technique indicated that Leu-enkephalin could cause transmembrane currents, suggesting the transfer of peptides across lipid membranes. Transfer examined by fluorescence spectroscopy implied that it could be separated into three steps, adsorption, transportation and desorption, which was afterward reaffirmed by confocal laser scanning microscopy. Transfer efficiencies of enkephalin across SBPL, EPC/DOPE, EPC/DOPE/SM, EPC/SM and EPC/Chol lipid bilayer membranes were evaluated with ET and CLSM experiments. Results showed that the addition of either sphingomyelin or cholesterol, or negatively charged lipid in lipid membrane composition could lower the transfer efficiency.     

Biosensors for the Rapid Repetitive Detection of Adrenaline Using Stabilized Bilayer Lipid Membranes (BLMs) with Incorporated Calix[4]resorcinarene Receptor

This work uses lipid film based biosensors with incorporated calix[4]resorcinarene receptor (lipophilic macrocyclic host molecule) for the rapid electrochemical detection of adrenaline. Freely‐suspended and metal supported BLMs (composed of egg phosphatidylcholine (PC) and 35% (w/w) dipalmitoyl phosphatidic acid) modified with the resorcin[4]arene receptor were used as one shot sensors to rapidly detect this catecholamine. The interactions of this compound with freely‐suspended BLMs were found to be electrochemically transduced in the form of a transient current signal with duration of seconds, which reproducibly appeared about 14 s after exposure of the membranes to adrenaline. The response time for these BLMs without incorporated receptor for adrenaline was about 1.5 min. The magnitude of the transient current signal was related to the concentration of adrenaline in bulk solution in the micromolar range. Differential scanning calorimetric (DSC) experiments were performed to explore the mechanism of interactions of BLMs with incorporated receptor with adrenaline. The interactions of adrenaline with surface‐stabilized bilayer lipid membranes (sBLMs) with incorporated receptor produced electrochemical ion current increases, which reproducibly appeared within a few seconds after exposure of the membranes to the stimulant. The use of the receptor in sBLMs increased the sensitivity of the method 6‐fold. The current signal increases were related to the concentration of adrenaline in bulk solution in the micromolar range. Stabilized lipid membranes formed by polymerization on glass fiber microfilters were used as practical chemical biosensors for the rapid detection of adrenaline. The interactions of polymerized lipid films with adrenaline were also found to provide transient current signals similar to those of freely‐suspended BLMs. The magnitude of the transient current signal was also related to the concentration of the stimulating agent in bulk solution in the micromolar range and these stabilized lipid films can be used again after storage in air. No interferences from ascorbic acid were noticed because of the negatively charged lipids in membranes. The effect of other compounds such as proteins and other compounds closely related to adrenaline was also investigated. Results of recovery experiments using human urine have shown recoveries ranged between 94 to 105%, which shows no interferences from matrix effects due to the presence of urine constituents. The present sensor based on stabilized lipid films can be used for the rapid repetitive detection of this pharmaceutical substance and keep prospects for the selective determination of catecholamines in biofluids.     

Voltammetric study of gold-supported lipid membranes in the presence of perfluorooctanesulphonic acid

The effect of perfluorooctanesulphonic acid (PFOS) on lipid membranes was studied using supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer as the model membrane. Phospholipid bilayer was deposited on gold electrode using a combination of the Langmuir–Blodgett and Langmuir–Schaefer (LB/LS) techniques. Electrodes were modified with two different types of membranes: DMPC bilayers initially containing PFOS and pure DMPC bilayers later exposed to the PFOS solutions. Such approach allowed studying both the changes in membrane characteristic imposed by the perfluorinated compound present in the model membrane and the process of its incorporation into the membrane. Studies with anticancer drug doxorubicin revealed that PFOS inhibits drug transport through the phospholipid bilayer and its effect can be compared to that of cholesterol. Moreover, the different trends observed in the changes in electron transfer rate constant (k_s) calculated for ferricyanides and in peak current of hexaamineruthenium chloride showed that electrostatic interactions between electroactive probes and PFOS molecules incorporating into phospholipid bilayers play an important role and should be taken into account while explaining the interactions of perfluorooctanesulphonic acid with model biological membranes.     

基于色谱-质谱联用的脂质分析样品前处理技术研究进展

脂质作为细胞膜和亚细胞膜的主要结构成分,在能量来源、细胞信号传导等多种生物学过程中发挥着重要作用。近年来,脂质分析受到越来越多的关注,其中色谱-质谱联用技术在脂质分析中占据主导地位。由于样品基质复杂,样品前处理有富集痕量物质和减少基质干扰的作用,成为脂质分析中的一个关键步骤。该文综述了近年来基于色谱-质谱联用技术的脂质分析中样品前处理技术的研究进展和应用,对各种样品前处理技术进行了阐述和总结。基于液相的萃取方法有液-液萃取和单一有机溶剂萃取。基于固相的萃取方法包括固相萃取和固相微萃取。场辅助萃取方法包括超临界流体萃取、加压流体萃取、微波辅助萃取和超声辅助萃取。此外,还介绍了在线联用样品前处理方法和用于活体分析的样品前处理方法。最后,对基于色谱-质谱联用的脂质分析样品前处理技术存在的问题及发展趋势进行了探讨。样品前处理技术的发展将提高脂质分析的灵敏度、选择性和分析速度。     

Centerband‐only analysis of rotor‐unsynchronized spin echo for measurement of lipid 31P chemical shift anisotropy

Structural diversity and molecular flexibility of phospholipids are essential for biological membranes to play key roles in numerous cellular processes. Uncovering the behavior of individual lipids in membrane dynamics is crucial for understanding the molecular mechanisms underlying biological functions of cell membranes. In this paper, we introduce a simple method to investigate dynamics of lipid molecules in multi‐component systems by measuring the ³¹P chemical shift anisotropy (CSA) under magic angle spinning (MAS) conditions. For achieving both signal separation and CSA determination, we utilized a centerband‐only analysis of rotor‐unsynchronized spin echo (COARSE). This analysis is based on the curve fitting of periodic modulation of centerband intensity along the interpulse delay time in rotor‐unsynchronized spin‐echo experiments. The utility of COARSE was examined by using phospholipid vesicles, a three‐component lipid raft model system, and archaeal purple membranes. We found that the apparent advantages of this method are high resolution and high sensitivity given by the moderate MAS speed and the one‐dimensional acquisition with short spin‐echo delays. COARSE provides an alternative method for CSA measurement that is effective in the investigation of lipid polymorphologies. Copyright © 2015 John Wiley & Sons, Ltd.