What is known about flux through these proteins, together with their structure and function, is discussed-illustrated with reference to particularly significant pathways. The important part played by the red blood cell as a paradigm for membrane transport is explained. Existing gaps in our understanding of transport across membranes are emphasized. In addition, the nature of the residual or "leak" permeability, which remains when all such pathways are inhibited, is unclear.
The residual permeability of biological membranes appears to be about 2 orders of magnitude greater than that of artificial lipid bilayers. An important caveat here is the existence of unknown pathways yet to be described. Future discoveries will add to our understanding of membrane permeability. It is likely that the red blood cell will play an important part in this new chapter.
BLM Analyzer: a software tool for experiments on planar lipid bilayers
Bloom, E. Evans, O. Non-Patent Document 4: B.
Alberts et al. Non-Patent Document 5: T. Ide, and T. Non-Patent Document 6: R. Hemmier, G. Bose, E.
Wagner, and R. Non-Patent Document 7: N. Malmstadt, M. Nash, R. Purnell, and J. Non-Patent Document 8: M. Mayer, J. Kriebel, M.
Tosteson, and G. As described above, a simple method and a device for the same having the above-mentioned advantages have not existed yet. In view of the above-described problems, the present invention is intended to provide a planar lipid bilayer array formed by microfluidic technique and a method of analysis using the planar lipid bilayers having the advantages such as portability, decreased analysis time, a smaller amount of required reagents, and parallel automation with high reproducibility.
In order to achieve the object described above, the present invention provides the following:. A planar lipid bilayer array formed by microfluidic technique according to the present invention is the planar lipid bilayer array formed by microfluidic technique saturated with water by preliminarily immersing in water, comprising microchannels connected to an inlet of a microfluidic channel and arranged in parallel, and microchambers having apertures on both sides of the microchannel.
Hereinbelow, embodiments of the present invention will be described in detail. In these figures, reference numeral 1 denotes a PDMS Polydimethylsiloxane device, 2 denotes a microchannel formed in the PDMS device 1 , and 3 denotes a microchamber having an aperture alternately arranged on both sides of the microchannel 2. Although the both sides of the microchannel 2 are exemplified as side walls of the microchannel herein, the both sides of the microchannel 2 may refer to top and bottom walls of the microchannel.
Here, although the smaller membrane area of a planar lipid bilayer 5 formed on the aperture of the microchamber 3 aids the stability of itself, as described later, the excess specific area may cause a problem that the PDMS device 1 swells significantly by absorbing an organic solvent. For solving this problem, the microchambers having the apertures are alternately arranged on the both sides of the microchannel 2 , so that PDMS pillars 4 are aligned in the middle of the main microchannels, imparting a robust structure to the PDMS device 1 to prevent the microchannels from structurally collapsing.
Moreover, the PDMS device 1 can set a vast number of microchambers arranged in parallel.
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Here, while PDMS is the preferred material for the array described above, it is not limited thereto and inorganic or plastic materials such as glass, SU-8, silicon, acrylic resin, and PET may be used. This device can attain portability, decreased analysis time, a smaller amount of required reagents, and parallel automation with high reproducibility. Here, the organic solvent, hexadecane, mentioned above can be replaced by another organic solvent, squalene, to extend the applications.
First, as shown in FIG.
This is for preventing water from absorbing into the PDMS device 13 during the operation. Next, as shown in FIG. Thereby, as shown in FIGS. Note that FIG. In this manner, according to the present invention, the planar lipid bilayers 19 are readily fabricated, that allows for rapid and accurate analyses of the membrane proteins.
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In this manner, the membrane proteins such as ion channels, pumps, receptors, and transporters can be manipulated. When a flow rate of the buffer solution 18 containing no dissolved materials, shown in FIG. In order to prevent this, as shown in FIG. With the decrease in the flow rate, the dynamic stress is decreased which is applied by the flow of the buffer solution 18 to the membranes of the organic solvent containing lipid.
Thereby, the probability of the planar lipid bilayers to be broken before fabrication can be reduced. However, there has been a problem that, if syringes are exchanged per solution, a large quantity of micellar contamination is introduced due to the entrapment of air or the like. In the present invention, each process can be performed without air entrapment between the solutions by using a single syringe to sequentially load a single tube with the solutions.
Note that although a syringe is preferred for liquid feeding in the above embodiment, it is not limited thereto and the liquid feeding can be performed manually by using other tools such as a pump or a pipette. Department of Microbial Pathogenesis W. Baltimore St. Room Baltimore, MD Electrophysiology is generally used to monitor the transport rates of ions across membranes through ion channels, where the rate of transport is measured as an ionic current [units of picoamps pA ].
This phenomenon arises simply due to the fact that ions carry charges, and potentials, or voltages, [units of millivolts mV ] can be applied across membanes.watch
Publications - Lipid Nanostructures Laboratory
Planar lipid bilayer apparatus. Each compartment contains a buffer with an electrolyte, typically mM KCl. Electrodes are connected to each compartment via salt bridges. The membrane potential is controlled clamped and the current is amplified using a voltage-clamp amplifier.
A channel-forming preparation is added to the cis side, and channel formation is monitored by step-wise increases in the current due to the insertion of individual channels into the membrane. An interesting analogy can be drawn between biological ion channels and electronic circuits. In simple bilayer electrophysiology setups, the membrane capacitance C m is on the order of picofarads pF.
Planar lipid bilayers are used for functional studies of ion channel proteins using electrophysiological techniques. A lipid solution is then formed into a planar bilayer membrane in the hole. Ion channels can then be incorporated into the planar bilayer by directly applying the channel protein or fusing liposomes containing the channel of interest. A voltage-clamp amplifier Fig. Single-channel planar bilayer setup. Single-channel recordings of ion channels have deepened our understanding of channel function, helping to elucidate mechanisms of membrane transport and cellular physiology.
Patch clamping is a typical approach used to make single-channel recordings. For example, patch-clamped membranes allow for the study of the ion channel in its native surroundings. However, planar bilayer electrophysiology provides several distinct advantages, including control of the constituents on either side of the membrane, manipulation of lipid composition, and the ability to study rare or challenging channels inaccessible to patch-clamp methods. One major drawback is that planar lipid bilayers can have larger capacitances, because they have wider diameters than excised patches.
These larger capacitances cause for a slow voltage response time due to the known resistor-capacitor- RC- filtering effect. All that said, each method offers complementary advantages and disadvantages for studying single ion channels. We have three Axon Axoclamp B voltage-clamp amplifiers Fig. They interface with a custom bilayer perfusion cell Fig.
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