Synthesis and electrochemical characterization of 4-thio pseudo-glycolipids as candidate tethers for lipid bilayer models (original) (raw)
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Nanostructural determination of a lipid bilayer tethered to a gold substrate
The European physical journal. E, Soft matter, 2016
Tethered lipid bilayer membranes (tBLM) are planar membranes composed of free lipids and molecules tethered to a solid planar substrate providing a useful model of biological membranes for a wide range of biophysical studies and biotechnological applications. The properties of the tBLM depend on the free lipids and on the chemistry of the tethering molecules. We present a nanoscale characterization of a tBLM composed of deuterated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (d-DMPC) free lipids, benzyl disulfide undecaethylene glycol phytanol (DLP) tethering molecules, and benzyl disulfiide tetraethylene glycol polar spacer molecules (PSM) used to control the areal density of tethering molecules through coadsorption. The use of selected isotopic substitution provides a way to distinguish the conformation and location of the tethered lipids from the free lipids and to elucidate how the two components influence the structure of the tBLM. These findings provide useful information to op...
Most of the model lipid membrane studies on gold involve the usage of various surface-modification strategies to rupture liposomes and induce lipid bilayer formation since liposomes with polar surfaces do not interact with bare, hydrophobic gold. In this study, a thiol-modified phospholipid, 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol (DPPTE) was incorporated into phosphatidylcholine (PC) based liposomes to form supported lipid bilayer (SLB) on gold surfaces without further modification. The binding kinet-ics of liposomes with different DPPTE ratio (0.01 to 100% mol/mol) and diameters were monitored by Quartz Crystal Microbalance with Dissipation (QCM-D). The dissipation change per frequency change, i.e. acoustic ratio, which is evaluated as a degree of the viscoelasticity, considerably decreased with the presence of DPPTE (from 162.3 GHz −1 for flattened PC liposomes to ca. 89.5 GHz −1 for 100% DPPTE lipo-somes) when compared to the results of two reference rigid monolayers and two viscoelastic layers. To assess the quality of SLB platform, the interpretation of QCM-D data was also complemented with Surface Plasmon Resonance. The optimum thiolated-lipid ratio (1%, lower thiol ratio and higher rigidity) was then used to determine the dry-lipid mass deposition, the water content and the thickness values of the SLB via viscoelastic modelling. Further surface characterization studies were performed by Atomic Force Microscopy with high spatial resolution. The results suggested that model membrane was almost continuous with minimum defects but showed more dissipative/soft nature compared to an ideal bilayer due to partially fused liposomes/overlapped lipid bilayers/multilayer islands. These local elevations distorted the planarity and led the increase of overall membrane thickness to ∼7.0 nm.
DPPTE Thiolipid Self-Assembled Monolayer: A Critical Assay
Langmuir, 2016
Supported lipid membranes represent an elegant way to design a fluid interface able to mimic the physico-chemical properties of biological membranes, with potential biotechnological applications. In this work, a diacyl phospholipid, the 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE), functionalized with a thiol group, was immobilized on a gold surface. In this molecule, the thiol group, responsible of the Au-S bond (55 kJ/mol) is located on the phospholipid polar head, letting the hydrophobic chain protruding from the film. This system is widely used in the literature, but not of less challenging, since its characterization is not complete, as several discording data have been obtained. In this work, the film was characterized by cyclic voltammetry blocking experiments, to verify the SAM formation, and by reductive desorption measurements to estimate the molecular density of DPPTE on the gold surface. This value has been compared to that obtained
Tethered Lipid Bilayers on Electrolessly Deposited Gold for Bioelectronic Applications
Biomacromolecules, 2006
This paper presents the formation of a novel biomimetic interface consisting of an electrolessly deposited gold film overlaid with a tethered bilayer lipid membrane (tBLM). Self-assembly of colloidal gold particles was used to create an electrolessly deposited gold film on a glass slide. The properties of the film were characterized using field-effect scanning electron microscopy, energy dispersive spectroscopy, and atomic force microscopy. Bilayer lipid membranes were then tethered to the gold film by first depositing an inner molecular leaflet using a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate], 1,2-di-O-phytanyl-snglycero-3-phosphoethanolamine (DPGP), and cystamine in ethanol onto a freshly prepared electrolessly deposited gold surface. The outer leaflet was then formed by the fusion of liposomes made from DPGP or 1,2-dioleoylsn-glycero-3-phosphocholine on the inner leaflet. To provide functionality, two membrane biomolecules were also incorporated into the tBLMs: the ionophore valinomycin and a segment of neuropathy target esterase containing the esterase domain. Electrochemical impedance spectroscopy, UV/visible spectroscopy, and fluorescence recovery after pattern photobleaching were used to characterize the resulting biomimetic interfaces and confirm the biomolecule activity of the membrane. Microcontact printing was used to form arrays of electrolessly deposited gold patterns on glass slides. Subsequent deposition of lipids yielded arrays of tBLMs. This approach can be extended to form functional biomimetic interfaces on a wide range of inexpensive materials, including plastics. Potential applications include high-throughput screening of drugs and chemicals that interact with cell membranes and for probing, and possibly controlling, interactions between living cells and synthetic membranes. In addition, the gold electrode provides the possibility of electrochemical applications, including biocatalysis, bio-fuel cells, and biosensors.
Langmuir, 2014
There is an increasing interest to express and study membrane proteins in vitro. New techniques to produce and insert functional membrane proteins into planar lipid bilayers have to be developed. In this work, we produce a tethered lipid bilayer membrane (tBLM) to provide sufficient space for the incorporation of the integral membrane protein (IMP) Aquaporin Z (AqpZ) between the tBLM and the surface of the sensor. We use a gold (Au)-coated sensor surface compatible with mechanical sensing using a quartz crystal microbalance with dissipation monitoring (QCM-D) or optical sensing using the surface plasmon resonance (SPR) method. tBLM is produced by vesicle fusion onto a thin gold film, using phospholipid-polyethylene glycol (PEG) as a spacer. Lipid vesicles are composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Npoly(ethyleneglycol)-2000-N-[3-(2-pyridyldithio)propionate], so-called DSPE-PEG-PDP, at different molar ratios (respectively, 99.5/0.5, 97.5/2.5, and 95/5 mol %), and tBLM formation is characterized using QCM-D, SPR, and atomic force technology (AFM). We demonstrate that tBLM can be produced on the gold surface after rupture of the vesicles using an α helical (AH) peptide, derived from hepatitis C virus NS5A protein, to assist the fusion process. A cell-free expression system producing the E. coli integral membrane protein Aquaporin Z (AqpZ) is directly incubated onto the tBLMs for expression and insertion of the IMP at the upper side of tBLMs. The incorporation of AqpZ into bilayers is monitored by QCM-D and compared to a control experiment (without plasmid in the cellfree expression system). We demonstrate that an IMP such as AqpZ, produced by a cell-free expression system without any protein purification, can be incorporated into an engineered tBLM preassembled at the surface of a gold-coated sensor.
Tethered lipid bilayers on ultraflat gold surfaces
2003
Tethered lipid bilayers (tBLMs) were obtained by the fusion of liposomes from diphytanoylphosphatidylcholine (DPhyPC) with self-assembled monolayers (SAMs) of a newly designed archaea analogue thiolipid, 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-D,L-R-lipoic acid ester (DPTL) on template stripped gold (TSG) films from silicon wafer as a template. SAMs, as characterized by reflection absorption infrared spectroscopy (RAIRS), show a mixture of different conformations of the tetraethylene segment in air, which appears to rearrange into the fully extended conformation when the SAM is immersed into an aqueous electrolyte solution, as deduced from thickness measurements by surface plasmon resonance spectroscopy (SPR). The fusion of liposomes was followed by SPR, quartz crystal microbalance (QCM), and fluorescence microscopy. Highly resistive tBLMs were obtained, as demonstrated by electrochemical impedance spectroscopy (EIS) results, which are equivalent to those for the BLM. This large resistivity is attributed to the ultraflat surface of TSG, as well as to the distinctive architecture of the newly designed molecule. The roughness of the TSG obtained from mica and silicon wafer as template was determined by AFM and compared to that of a Au(111) surface on mica. The largest roughness features of TSG are shown to be 0.5-1 nm, which is small compared to the vertical dimension of the DPTL molecules. This is regarded to be crucial for the self-assembly process, particularly in the case of amphiphilic molecules.
Electrochimica Acta, 2019
In this work formation of a planar phospholipid bilayer of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine on an integrated porous cushion and its interaction with the ionic liquid 1-ethyl-3-methylimidazolium tosylate was studied. The integrated porous cushion was prepared by grafting chitosan covalently on porous 11-mercaptoundecanoic acid self-assembled monolayer on gold. Pores on the self-assembled monolayer were created by partial electro-oxidative desorption of the thiol molecules from the gold surface. Chitosan was grafted on the porous self-assembled monolayer by EDC/NSH activation procedure. Lipid bilayer was formed through solvent exchange method and evidenced with QCM-D, fluorescence microscopy, and electrochemical impedance spectroscopy. A lowering of QCM frequency of 26.4±1.5 Hz was observed during the bilayer formation. Impedance measurement showed that the lipid bilayer deposited integrated membrane can attain a high electrical resistance of 27 MUcm 2. The said ionic liquid created pores in the bilayer through interaction that was evidenced by significant decrease in charge transfer resistance in the impedance spectra and corroborated with fluorescence imaging. The work has indicated that the ionic liquid 1-ethyl-3-methylimidazolium tosylate can be cytotoxic. The new strategy for the formation of polymer cushion on a porous tether has shown new possibility of studying membrane attack interaction and ion diffusion in lipid bilayers through simple impedance analysis technique.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1996
One topic of this study is the comparison of different preparation techniques to build up solid supported lipid bilayers onto gold substrates. The deposited lipid bilayers were investigated by a.c. impedance spectroscopy. Three different strategies were applied: (1) The gold surface was initially covered with a chemisorbed monolayer of octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). The second monolayer consisting of phospholipids was then deposited onto this hydrophobic surface by (i) the Langmuir-Schaefer-technique, (ii) from lipid solution in n-decane/isobutanol, (iii) by the lipid/detergent dilution technique or (iv) by fusion of vesicles.
Langmuir, 2007
Highly oriented monolayers of bacteriorhodopsin (BR) in purple membrane (PM) form are obtained by the reaction of BR-Q3C, where a cysteine was introduced into the N-terminal region, with a gold surface. Single molecule force spectroscopy was used to show that about 50% of the BRs are covalently bound to the surface. The linkage between the cysteine and the gold causes an additional characteristic peak in the force-distance curves to appear. Because several thousand cysteine-gold bonds exist between each PM patch and the surface, the PM is irreversibly bound. Such oriented PM monolayers may serve as an interface between metal surfaces and biomaterials, which may be linked to the PM surface chemically. Photoelectric applications of BR will benefit from the high degree of orientation obtained by this method.
Formation of tethered bilayer lipid membranes probed by various surface sensitive techniques
Biointerphases, 2009
Tethered bilayer lipid membranes are promising biomimetic architectures. Their formation has been investigated using four different surface sensitive techniques, including optical, acoustic, and electrical methods. The lipid bilayers are built in a two-step procedure; the proximal layer is formed by self-assembly and is then completed to a bilayer by fusion with small vesicles. The different technical approaches revealed specific aspects of the layer formation processes, namely, first a fast adsorption process followed by a longer rearrangement period. Similar phenomena have been observed for the vesicle fusion process. The results allow for a more controlled assembly protocol for the preparation of highly insulating lipid membranes.