Limits...
High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition.

Watanabe R, Soga N, Yamanaka T, Noji H - Sci Rep (2014)

Bottom Line: We present a micro-device in which more than 10,000 asymmetric lipid bilayer membranes are formed at a time on micro-chamber arrays.The arrayed asymmetric lipid bilayers, where lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces.The lipid compositional asymmetry is an intrinsic property of bio-membranes, and therefore, this micro-device extends the versatility of artificial lipid-bilayer systems, which were previously limited to symmetric bilayer formation, and could contribute to the understanding of the role of lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan [2] PRESTO, JST, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
We present a micro-device in which more than 10,000 asymmetric lipid bilayer membranes are formed at a time on micro-chamber arrays. The arrayed asymmetric lipid bilayers, where lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces. The lipid compositional asymmetry is an intrinsic property of bio-membranes, and therefore, this micro-device extends the versatility of artificial lipid-bilayer systems, which were previously limited to symmetric bilayer formation, and could contribute to the understanding of the role of lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications.

No MeSH data available.


Related in: MedlinePlus

Flip-flop measurement using NBD-DPPE.(a) Quenching of NBD-DPPE resulting from reduction by dithionite. (b) Schematic illustration of NBD-reduction assay. First, micro-chambers and an upper flow channel are filled with buffer (pH 7). Second, the buffer in the flow channel is exchanged with buffer containing 50 mM dithionite. During these processes, we measured the fluorescence intensity of NBD-DPPE located in the bilayers. (c) Time course of fluorescence intensity of NBD-DPPE in bilayers. Green or grey plots represent the fluorescence intensity in the presence or absence of 50 mM dithionite after 40 min, respectively. The orange line represents the fitting curve based on a single-order reaction scheme with a time constant of 4.1 h. Insets show fluorescent images of NBD-DPPE in the representative micro-chamber.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4233334&req=5

f5: Flip-flop measurement using NBD-DPPE.(a) Quenching of NBD-DPPE resulting from reduction by dithionite. (b) Schematic illustration of NBD-reduction assay. First, micro-chambers and an upper flow channel are filled with buffer (pH 7). Second, the buffer in the flow channel is exchanged with buffer containing 50 mM dithionite. During these processes, we measured the fluorescence intensity of NBD-DPPE located in the bilayers. (c) Time course of fluorescence intensity of NBD-DPPE in bilayers. Green or grey plots represent the fluorescence intensity in the presence or absence of 50 mM dithionite after 40 min, respectively. The orange line represents the fitting curve based on a single-order reaction scheme with a time constant of 4.1 h. Insets show fluorescent images of NBD-DPPE in the representative micro-chamber.

Mentions: Instead of fluorescein-DHPE, we used NBD-DPPE to form asymmetric lipid bilayers, where NBD-DPPE is located in the inner leaflet of the bilayer. NBD-DPPE is a head-group-labelled fluorescent lipid that has been widely used as a model lipid for flip-flop measurement212223 (Fig. 5a). To visualize the flip-flop of NBD-DPPE, we performed the NBD-reduction assay using dithionite, which rapidly quenches the fluorescence of NBD21 (Supplementary Information). Initially, the micro-chambers and an upper flow channel were filled with buffer (MOPS buffer, pH 7.0). Then, the buffer in the flow channel was exchanged with the buffer containing 50 mM dithionite. Because dithionite does not permeate through lipid membranes on our system for 7 h (Supplementary Information), the fluorescence of NBD-DPPE will be quenched only when NBD-DPPE is exposed to upper flow channel upon flip-flop. After the buffer exchange, the fluorescent intensity of NBD-DPPE was gradually and exponentially decreased with a time constant of 4.1 h (Fig. 5b,c), which was similar to previous findings in liposomes21. These findings suggest that the fluidity of the lipid in our device, which affects the flip-flop rate, is almost the same as that in liposomes.


High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition.

Watanabe R, Soga N, Yamanaka T, Noji H - Sci Rep (2014)

Flip-flop measurement using NBD-DPPE.(a) Quenching of NBD-DPPE resulting from reduction by dithionite. (b) Schematic illustration of NBD-reduction assay. First, micro-chambers and an upper flow channel are filled with buffer (pH 7). Second, the buffer in the flow channel is exchanged with buffer containing 50 mM dithionite. During these processes, we measured the fluorescence intensity of NBD-DPPE located in the bilayers. (c) Time course of fluorescence intensity of NBD-DPPE in bilayers. Green or grey plots represent the fluorescence intensity in the presence or absence of 50 mM dithionite after 40 min, respectively. The orange line represents the fitting curve based on a single-order reaction scheme with a time constant of 4.1 h. Insets show fluorescent images of NBD-DPPE in the representative micro-chamber.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4233334&req=5

f5: Flip-flop measurement using NBD-DPPE.(a) Quenching of NBD-DPPE resulting from reduction by dithionite. (b) Schematic illustration of NBD-reduction assay. First, micro-chambers and an upper flow channel are filled with buffer (pH 7). Second, the buffer in the flow channel is exchanged with buffer containing 50 mM dithionite. During these processes, we measured the fluorescence intensity of NBD-DPPE located in the bilayers. (c) Time course of fluorescence intensity of NBD-DPPE in bilayers. Green or grey plots represent the fluorescence intensity in the presence or absence of 50 mM dithionite after 40 min, respectively. The orange line represents the fitting curve based on a single-order reaction scheme with a time constant of 4.1 h. Insets show fluorescent images of NBD-DPPE in the representative micro-chamber.
Mentions: Instead of fluorescein-DHPE, we used NBD-DPPE to form asymmetric lipid bilayers, where NBD-DPPE is located in the inner leaflet of the bilayer. NBD-DPPE is a head-group-labelled fluorescent lipid that has been widely used as a model lipid for flip-flop measurement212223 (Fig. 5a). To visualize the flip-flop of NBD-DPPE, we performed the NBD-reduction assay using dithionite, which rapidly quenches the fluorescence of NBD21 (Supplementary Information). Initially, the micro-chambers and an upper flow channel were filled with buffer (MOPS buffer, pH 7.0). Then, the buffer in the flow channel was exchanged with the buffer containing 50 mM dithionite. Because dithionite does not permeate through lipid membranes on our system for 7 h (Supplementary Information), the fluorescence of NBD-DPPE will be quenched only when NBD-DPPE is exposed to upper flow channel upon flip-flop. After the buffer exchange, the fluorescent intensity of NBD-DPPE was gradually and exponentially decreased with a time constant of 4.1 h (Fig. 5b,c), which was similar to previous findings in liposomes21. These findings suggest that the fluidity of the lipid in our device, which affects the flip-flop rate, is almost the same as that in liposomes.

Bottom Line: We present a micro-device in which more than 10,000 asymmetric lipid bilayer membranes are formed at a time on micro-chamber arrays.The arrayed asymmetric lipid bilayers, where lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces.The lipid compositional asymmetry is an intrinsic property of bio-membranes, and therefore, this micro-device extends the versatility of artificial lipid-bilayer systems, which were previously limited to symmetric bilayer formation, and could contribute to the understanding of the role of lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan [2] PRESTO, JST, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
We present a micro-device in which more than 10,000 asymmetric lipid bilayer membranes are formed at a time on micro-chamber arrays. The arrayed asymmetric lipid bilayers, where lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces. The lipid compositional asymmetry is an intrinsic property of bio-membranes, and therefore, this micro-device extends the versatility of artificial lipid-bilayer systems, which were previously limited to symmetric bilayer formation, and could contribute to the understanding of the role of lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications.

No MeSH data available.


Related in: MedlinePlus