Free-standing Lipid Membranes Research Articles

Hydrogels for in situ encapsulation of biomimetic membrane arrays

AbstractHydrogels are hydrophilic, porous polymer networks that can absorb up to thousands of times their own weight in water. They have many potential applications, one of which is the encapsulation of freestanding black lipid membranes (BLMs) for novel separation technologies or biosensor applications. We investigated gels for in situ encapsulation of multiple BLMs formed across apertures in a hydrophobic ethylene tetrafluoroethylene (ETFE) support. The encapsulation gels consisted of networks of poly(ethylene glycol)‐dimethacrylate or poly(ethylene glycol)‐diacrylate polymerized using either a chemical initiator or a photoinitiator. The hydrogels were studied with regards to volumetric stability, porosity, and water permeability. All hydrogels had pore sizes around 7 nm with volumetric changes >2% upon crosslinking. Photoinitiated hydrogels had a lower hydraulic water permeability compared to chemically initiated hydrogels; however, for all hydrogels the permeability was several‐fold higher than the water permeability of conventional reverse osmosis (RO) membranes. Lifetimes of freestanding BLM arrays in gel precursor solutions were short compared to arrays formed in buffer. However, polymerizing (crosslinking) the gel stabilized the membranes and resulted in BLM arrays that remained intact for days. This is a substantial improvement over lifetimes for freestanding BLM arrays. Optical images of the membranes and single channel activity of incorporated gramicidin ion channels showed that the lipid membranes retained their integrity and functionality after encapsulation with hydrogel. Our results show that hydrogel encapsulation is a potential means to provide stability for biomimetic devices based on functional proteins reconstituted in biomimetic membrane arrays. Copyright © 2010 John Wiley & Sons, Ltd.

Hydrogel for in Situ Encapsulation of Multiple Black Lipid Membranes

Hydrogels are hydrophilic, porous polymer networks that can absorb water up to thousands of times their own weight. They have many applications, one of which is the encapsulation of free-standing black lipid membranes (BLMs) for novel separation technologies or biosensor applications. We investigated gels for in situ encapsulation of multiple black lipid membranes across apertures in a hydrophobic ethylene tetrafluoroethylene (ETFE) support. These gels consisted of networks of poly(ethylene glycol)-dimethacrylate or poly(ethylene glycol)-diacrylate polymerized using either a chemical initiator or a photoinitiator. The hydrogels were studied with regard to their material properties such as chemical resistance, swelling behaviour, water permeability and porosity. We found that lifetimes of membranes in gel precursor solutions were short compared to lifetimes in buffer. However, crosslinking the gel stabilized the membranes and increased BLM longevity substantially over lifetimes in buffer. Optical images of the membranes and incorporation of the transmembrane peptide gramicidin A showed that the lipid membranes retained their integrity after encapsulation with hydrogel.

Electrical characterizations of biomimetic molecular layers on gold and silicon substrates

Electrical impedance technology was used to characterize DNA recognition in a monolayer containing single-stranded DNA probes immobilized on a gold substrate using thiol self-assembly chemistry. Recognition of targeted complementary DNA was principally correlated with an eight-fold increase in the conductance of the monolayer and attributed to electron conduction through double helices formed upon the binding of the DNA targets to the probes. The high recognitive sensitivity was possible without the use of the redox labels or large bias voltages required for recognition using cyclic and Osteryoung square wave voltammetry. The impedance technology also provided atomic resolution of a hybrid bimolecular lipid membrane formed by deposition of a phospholipid:cholesterol monolayer onto a hydrophobic alkyl monolayer covalently attached to a silicon substrate via silicon–carbon bonds. Atomic resolution was achieved through preparation of membranes on surfaces approaching atomic flatness and the performance of impedance measurements over precisely defined areas of the surface in contact with solutions. Principally capacitive properties distinguished between the immobilized (octadecyl) and more fluidic (lipid:cholesterol) leaflets of the hybrid membrane. The lipid:cholesterol leaflets were structurally similar to those leaflets in free-standing bimolecular lipid membranes. The hybrid membrane therefore provides a highly stable and physiologically relevant surface for studying biomolecular interactions with membrane surfaces.

Native supported membranes: Creation of two-dimensional cell membranes on polymer supports (Review)

In nature, membranes serve as fundamental platforms for many important biological processes, such as i hormone transduction and amplification through generation of second messengers, ii biosynthesis by membrane associated ribosomes at the endoplasmic reticulum, iii control of cell adhesion via protein-protein recognition, and iv lateral segregation of cell-surface receptors and the associated reorganization of the membrane-coupled cytoskeleton. Phospholipid bilayers deposited onto solid substrates called solid-supported membranes have been the simplest and most commonly used experimental model systems that allow us to gain insights into structure-function relationships in biological membranes. Supported membranes retain the intrinsic “fluid” nature to self-heal local defects and achieve an excellent mechanical stability. These properties offer distinct advantages over either freestanding black lipid membranes or spherical lipid vesicle suspensions: they can be subjected to various surface-sensitive physical characterization techniques such as ATR-FTIR, surface plasmon resonance, quartz crystal micro balance, and neutron reflectivity. However, in spite of great scientific achievements through solid-supported membranes, such systems have some fundamental drawbacks. Solid-supported membranes are confined at a deep potential minimum governed by van der Waals interactions and stay in close proximity of solid substrates. The water reservoir between the membrane and the underlying solid substrate, which is typically 5–20 A, is not thick enough to prevent proteins coming into direct contact with the bare substrate. In fact, this can cause a serious problem in dealing with transmembrane proteins, such as cell adhesion receptors, whose functional extracellular domains can extend to several tens of nanometers. In fact, the spreading of proteoliposomes doped with human platelet integrin IIb 3 and ATP synthase on quartz or glass substrates results in inhomogeneous patches of “pinned” proteins Fig. 1 a . Such problems can be overcome by separating the membrane from the solid substrate using soft polymeric spacer layers, whose thickness is less than 100 nm. Here, the membranes are separated from underlying solids via i polymer cushions or ii polymer/oligomer tethers. This

X-ray Structure Analysis of Free-Standing Lipid Membranes Facilitated by Micromachined Apertures

Silicon and Teflon substrates have been structured by wet etching and a focused ion beam (FIB) to obtain very defined, clean apertures. Planar, free-standing lipid membranes (black lipid membranes (BLM)) with enhanced long-term stability have been prepared on these apertures by the methods of Montal and Müller(1,2) as well as Müller and Rudin.(3) The stability and geometric control enables the use of X-ray analysis of free-standing single bilayers. With the presented setup, simultaneous structural and electrophysiological measurements will become feasible.

Mechanical Properties of Pore-Spanning Lipid Bilayers Probed by Atomic Force Microscopy

We measure the elastic response of a free-standing lipid membrane to a local indentation by using an atomic force microscope. Starting point is a planar gold-coated alumina substrate with a chemisorbed 3-mercaptopropionic acid monolayer displaying circular pores of very well defined and tunable size, over which bilayers composed of N, N,-dimethyl- N, N,-dioctadecylammonium bromide or 1,2-dioleoyl-3-trimethylammonium-propane chloride were spread. Centrally indenting these “nanodrums” with an atomic force microscope tip yields force-indentation curves, which we quantitatively analyze by solving the corresponding shape equations of continuum curvature elasticity. Since the measured response depends in a known way on the system geometry (pore size, tip radius) and on material parameters (bending modulus, lateral tension), this opens the possibility to monitor local elastic properties of lipid membranes in a well-controlled setting.

Mechanical studies of hydrogel encapsulated membranes

ABSTRACTWe have encapsulated lipid bilayer membranes within a polyethylene glycol dimethacrylate hydrogel (PEG-DMA). These hydrogel encapsulated membranes (HEMs) are significantly longer-lived and more mechanically stable than traditional lipid membranes. Over 50 attempts, HEMs usually remained intact for over 48 hours, and some lasted up to 5 days. The electrical characteristics of the HEMs were consistently stable over this period of time. The approximate thickness of the HEM was measured to be 4.7±0.5 nm (n=25), consistent with a lipid bilayer. The resistance of the HEM remained over 10 GΩ over the period of electrical measurement. Simultaneous electrical and optical measurements showed that HEMs have unusual mechanical stability, whereas free-standing lipid membranes are typically susceptible to mechanical perturbation. The HEMs could withstand much greater applied pressures than unsupported membranes. In situ electrical and optical monitoring of the HEMs showed that the gel made intimate contact with the membrane, suggesting that direct mechanical support of the bilayer is the mechanism of membrane stabilization. Single channels of alpha-hemolysin, were incorporated into HEMs and continuously measured for over 4 days. Finally, combination of the HEM with an automated membrane microfluidic formation process is proposed as a prototype platform for high throughput drug screening or small molecule sensing.

Hydrogel-Encapsulated Lipid Membranes

We have encapsulated free-standing planar lipid membranes in a polymer hydrogel via in situ photopolymerization. These encapsulated membranes were measured to last at least 5 days and provided a stable environment for single-molecule measurements of incorporated alpha-hemolysin channel proteins. The translocation of single-stranded DNA through alpha-hemolysin incorporated into these encapsulated membranes is slowed over 100 times relative to that of the gel-free state. These stabilized membranes, therefore, have application to the single-molecule analysis of DNA as well as broader application to biomolecular sensors, membrane protein biophysical studies, and drug discovery.

Deposition of highly resistive lipid bilayer on silicon–silicon dioxide electrode and incorporation of gramicidin studied by ac impedance spectroscopy

Highly resistive planar supported lipid membranes were deposited onto highly doped p-type silicon–silicon dioxide electrodes. Physical parameters of the substrates (e.g. dopant, doping ratio and oxide layer thickness) were optimized by a combined study using ellipsometry and ac impedance spectroscopy. Lipid bilayer was deposited by fusion of small unilamellar vesicles, and the self-assembling of the homogeneous bilayer could be monitored as a function of time. Impedance spectroscopy over a wide frequency range (from 20 kHz to 10 mHz) enables separating membrane resistance and capacitance from the background signals. Membrane resistance amounted to 1.0 MΩ cm 2, and the capacitance was around 0.7 μF cm −2. The resistance obtained here is comparable to that of the freestanding black lipid membrane. Although the area of the supported membrane (0.5 cm 2) is much larger than that of the black lipid membrane (∼0.002 cm 2), the electrical properties were stable for more than a week. Gramicidin D was inserted into the membrane from trifluoroethanol solution, and activity of the channels was checked in terms of membrane conductance and ion selectivity. Functional incorporation of ion channels into the supported membrane demonstrated here suggested that the gramicidin monomers could diffuse over the membranes to form transmembrane pores.