Black lipid membranes (BLMs) are widely used for recording the activity of incorporated ion channel proteins. However, BLMs are inherently unstable structures that typically rupture within a few hours after formation. Here, stabilized BLMs were formed using the polymerizable lipid bis-dienoyl phosphatidylcholine (bis-DenPC) on glass pipettes of ∼10 μm (I.D.). After polymerization, these BLMs maintained steady conductance values for several weeks, as compared to a few hours for unpolymerized membranes. The activity of an ion channel, α-hemolysin, incorporated into bis-DenPC BLMs prior to polymerization, was maintained for 1 week after BLM formation and polymerization. These lifetimes are a substantial improvement over those achievable with conventional BLM technologies. Polymerized BLMs containing functional ion channels may represent an enabling technology for development of robust biosensors and drug screening devices.
Suspended planar lipid membranes (or black lipid membranes (BLMs)) are widely used for studying reconstituted ion channels, although they lack the chemical and mechanical stability needed for incorporation into high-throughput biosensors and biochips. Lipid polymerization enhances BLM stability but is incompatible with ion channel function when membrane fluidity is required. Here we demonstrate the preparation of a highly stable BLM that retains significant fluidity by using a mixture of polymerizable and nonpolymerizable phospholipids. Alamethicin, a voltage-gated peptide channel for which membrane fluidity is required for activity, was reconstituted into mixed BLMs prepared using bis-dienoyl phosphatidylcholine (bis-DenPC) and diphytanoyl phosphatidylcholine (DPhPC)). Polymerization yielded BLMs that retain the fluidity required for alamethicin activity yet are stable for several days as compared to a few hours prior to polymerization. Thus these polymerized, binary composition BLMs feature both fluidity and long-term stability.
The stabilization of suspended planar lipid membranes, or black lipid membranes (BLMs), through polymerization of mono- and bis-functionalized dienoyl lipids was investigated. Electrical properties, including capacitance, conductance, and dielectric breakdown voltage, were determined for BLMs composed of mono-DenPC, bis-DenPC, mono-SorbPC, and bis-SorbPC both prior to and following photopolymerization, with diphytanoyl phosphocholine (DPhPC) serving as a control. Poly(lipid) BLMs exhibited significantly longer lifetimes and increased the stability to air-water transfers. BLM stability followed the order: bis-DenPC > mono-DenPC ≈ mono-SorbPC > bis-SorbPC. The conductance of bis-SorbPC BLMs was significantly higher than that of the other lipids, which is attributed to a high density of hydrophilic pores, resulting in relatively unstable membranes. The use of poly(lipid) BLMs as matrices for supporting the activity of an ion channel protein (IC) was explored using α – hemolysin (α-HL), a model IC. Characteristic i-V plots of α-HL were maintained following photopolymerization of bis-DenPC, mono-DenPC, and mono-SorbPC, demonstrating the utility of these materials for preparing more durable BLMs for single channel recordings of reconstituted ICs.
Phosphatidyl choline (PC) based materials have been found to be resistant to non-specific protein adhesion in vitro. In this study, a PC based planar supported phospholipid bilayer composed of 1,2-bis[10-(2′,4′-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphocholine (bis-SorbPC or BSPC) was generated on piranha treated silicon wafers by vesicle deposition. The bilayer was polymerized with redox initiation forming a stable 4 nm thick coating. Polymerized lipid bilayers (PLBs) were characterized and tested for uniformity, with ellipsometry and contact angle. Cellular adhesion and morphological changes in RAW 264.7 macrophages were investigated in vitro on PLBs and compared to bare silicon controls. Fluorescent and scanning electron microscopy were used to observe changes in cellular morphology. The PLBs showed much lower cellular adhesion than bare silicon controls. Of the cells that attached to the PLBs, a very low percentage showed the same morphological expressions seen on the controls. It is hypothesized that proteins adsorb to the defects in the PLBs, caused by incomplete polymerization, and this mediates the observed minimal cellular attachment and morphological changes.
Membrane active peptides represent a class of soluble proteins that interact and disrupt the plasma membrane. Examples of these include antimicrobial peptides, cancer therapeutics, and cell-penetrating peptides. These peptides are amphipathic and, in a concentration dependent manner, can self assemble to destabilize the lipid bilayer. These peptides are rich in positively charged lysine and arginine residues and thus have a strong preference for negatively charged bilayers. In order to study the insertion mechanism and kinetics of these peptides, we have designed a negatively charged, supported bilayer platform on silicon. The negative charge serves to electrostatically drive peptides to bind to the lipid bilayer interface. Furthermore, this platform is electrically addressable through electrochemical impedance spectroscopy, which yields bilayer resistance, thickness, and structural heterogeneity data. This platform consists of an asymmetrical bilayer with 10 mol% negatively charged POPS, cholesterol, and POPC in the upper leaflet and DPhPC lipids in the lower leaflet, all supported by a PEG cushion on a silicon wafer. Resistances up to 2*10 4 Ohm cm 2 and capacitances of 0.8 yF cm À2 have been measured for the platform. The high resistance allows for high accuracy in the detection of the activity of membrane active peptides of interest.
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