1. Enhanced long-term stability for single ion channel recordings using suspended poly(lipid) bilayers
Benjamin A Heitz, Juhua Xu, Henry K Hall Jr, Craig A Aspinwall, S Scott Saavedra J Am Chem Soc. 2009 May 20;131(19):6662-3. doi: 10.1021/ja901442t.
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 approximately 10 microm (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, alpha-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.
2. Polymerized planar suspended lipid bilayers for single ion channel recordings: comparison of several dienoyl lipids
Benjamin A Heitz, Juhua Xu, Ian W Jones, John P Keogh, Troy J Comi, Henry K Hall Jr, Craig A Aspinwall, S Scott Saavedra Langmuir. 2011 Mar 1;27(5):1882-90. doi: 10.1021/la1025944. Epub 2011 Jan 12.
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 of 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.
3. Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity
Kristina S Orosz, Ian W Jones, John P Keogh, Christopher M Smith, Kaitlyn R Griffin, Juhua Xu, Troy J Comi, H K Hall Jr, S Scott Saavedra Langmuir. 2016 Feb 16;32(6):1577-84. doi: 10.1021/acs.langmuir.5b03437. Epub 2016 Feb 2.
Polymerization of substrate-supported bilayers composed of dienoylphosphatidylcholine (PC) lipids is known to greatly enhance their chemical and mechanical stability; however, the effects of polymerization on membrane fluidity have not been investigated. Here planar supported lipid bilayers (PSLBs) composed of dienoyl PCs on glass substrates were examined to assess the degree to which UV-initiated polymerization affects lateral lipid mobility. Fluorescence recovery after photobleaching (FRAP) was used to measure the diffusion coefficients (D) and mobile fractions of rhodamine-DOPE in unpolymerized and polymerized PSLBs composed of bis-sorbyl phosphatidylcholine (bis-SorbPC), mono-sorbyl-phosphatidylcholine (mono-SorbPC), bis-dienoyl-phosphatidylcholine (bis-DenPC), and mono-dienoyl phosphatidylcholine (mono-DenPC). Polymerization was performed in both the Lα and Lβ phase for each lipid. In all cases, polymerization reduced membrane fluidity; however, measurable lateral diffusion was retained which is attributed to a low degree of polymerization. The D values for sorbyl lipids were less than those of the denoyl lipids; this may be a consequence of the distal location of polymerizable group in the sorbyl lipids which may facilitate interleaflet bonding. The D values measured after polymerization were 0.1-0.8 of those measured before polymerization, a range that corresponds to fluidity intermediate between that of a Lα phase and a Lβ phase. This D range is comparable to ratios of D values reported for liquid-disordered (Ld) and liquid-ordered (Lo) lipid phases and indicates that the effect of UV polymerization on lateral diffusion in a dienoyl PSLB is similar to the transition from a Ld phase to a Lo phase. The partial retention of fluidity in UV-polymerized PSLBs, their enhanced stability, and the activity of incorporated transmembrane proteins and peptides is discussed.
