Fractional Polymerization of a Suspended Planar Bilayer Creates a Fluid, Highly Stable Membrane for Ion Channel Recordings

Heitz, Benjamin A.; Jones, Ian W.; Hall, H. K.; Aspinwall, Craig A.; Saavedra, S. Scott

doi:10.1021/ja100245d

Cited by 28 publications

(66 citation statements)

References 62 publications

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“…55 Following further accumulation of monomers, voltage-gated activity (Figure 6C) was observed, which agreed well with previous observations. 56 The observation of voltage-gated alamethicin activity further supported the presence of a lipid bilayer, and demonstrated that the bilayer was sufficiently fluid to allow normal alamethicin activity. These results suggest that the physiology of bilayers formed on planar SU-8 apertures will prove suitable for studies of other dynamic transmembrane protein systems, and will facilitate the development of new ligand-gated ion channel-based sensing technologies.…”

Section: Resultsmentioning

confidence: 82%

Photolithographic Fabrication of Microapertures with Well-Defined, Three-Dimensional Geometries for Suspended Lipid Membrane Studies

2013

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Robust and high-density biosensors incorporating suspended lipid membranes require microfabricated apertures that can be readily integrated into complex analysis systems. Apertures with well-defined, three-dimensional geometries enable the formation of suspended lipid membranes, and facilitate reduced aperture size compared to vertical-walled apertures. Unfortunately, existing methods of producing apertures with well-defined, three-dimensional geometries are based on complex and expensive fabrication procedures, some of which yield apertures in excessively fragile thin-film materials. Here, we describe a microfabrication method utilizing incline and rotate lithography that achieves sloped-wall microapertures in SU-8 polymer substrates with precision control of aperture diameter, substrate thickness, and wall angle. This approach is simple, low cost, and readily scaled up to allow highly reproducible parallel fabrication. The effect of incident angle of UV exposure and the size of photomask features on aperture geometry were investigated, yielding aperture diameters as small as 7 µm, and aperture wall angles ranging from 8° to 36° measured from the normal axis. Black lipid membranes were suspended across the apertures and showed normalized conductance values of 0.02 to 0.05 pS µm−2 and break down voltages of 400 to 600 mV. The functionality of the resulting sloped-wall microapertures was validated via measurement of reconstituted α-hemolysin activity and the voltage-gated channel activity of alamethicin.

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Section: Resultsmentioning

confidence: 82%

Photolithographic Fabrication of Microapertures with Well-Defined, Three-Dimensional Geometries for Suspended Lipid Membrane Studies

2013

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“…However mixtures of poly(bis-DenPC) and a nonpolymerizable lipid such as diphytanoyl phosphatidylcholine are sufficiently fluid to support alamethicin activity. 31 …”

Section: Resultsmentioning

confidence: 99%

Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity

et al. 2016

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Polymerization of substrate-supported bilayers composed of dienoyl phosphatidylcholine (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 inter-leaflet bonding. The D values measured after polymerization were 0.1 to 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.

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“…Pipet fabrication and modification were described previously 23, 24. Briefly, borosilicate capillaries were pulled to a sharp tip using a micropipet puller, then broken to an opening of ca.…”

Section: Methodsmentioning

confidence: 99%

Polymerized Planar Suspended Lipid Bilayers for Single Ion Channel Recordings: Comparison of Several Dienoyl Lipids

Heitz

Jones

et al. 2011

Langmuir

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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.

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Fractional Polymerization of a Suspended Planar Bilayer Creates a Fluid, Highly Stable Membrane for Ion Channel Recordings

Cited by 28 publications

References 62 publications

Photolithographic Fabrication of Microapertures with Well-Defined, Three-Dimensional Geometries for Suspended Lipid Membrane Studies

Photolithographic Fabrication of Microapertures with Well-Defined, Three-Dimensional Geometries for Suspended Lipid Membrane Studies

Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity

Polymerized Planar Suspended Lipid Bilayers for Single Ion Channel Recordings: Comparison of Several Dienoyl Lipids

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