JP5770615B2 - Method for producing lipid bilayer membrane substrate - Google Patents

Description

  The present invention relates to a lipid bilayer membrane substrate, a method for producing a lipid bilayer membrane substrate, and a hydrogel substrate.

  Biochips that have proteins, nucleic acids, sugar chains, lipids, cells, etc. on substrates are actively used in drug discovery and drug discovery. Among proteins, a membrane protein that functions as a receptor or a channel in a cell membrane has received much attention as a drug discovery target. Of the top 20 pharmaceutical sales in the United States in FY 2000, about 50% of the drugs target membrane proteins (including GPCRs and ion channels) (see Non-Patent Document 1). The importance of membrane proteins will continue to increase in the future, and the development of experimental systems and biochips capable of measuring the functions of various membrane proteins is very important.

Conventionally known is a method for analyzing the function of a membrane protein contained in a cell membrane constituting a living cell without taking out the membrane protein from the cell, for example, a patch clamp method (see Non-Patent Document 2). . However, since the cell membrane used as a sample contains various membrane proteins, there is a problem that the obtained data contains a lot of noise. For this reason, it is desirable to use a sample containing a specific membrane protein at a specific concentration.
However, a membrane protein that is a drug discovery target has a huge and complicated structure, and there is a technical difficulty that it is easily denatured when purified from a cell membrane. For this reason, technological development for incorporating a membrane protein into an in vitro measurement system such as a biochip while maintaining a normal function is underway.

As an example of this measurement system, a black film is formed by dissolving lipid molecules in an organic solvent such as n-decane, and applying the organic solvent to a small hole provided in a plastic plate or the like in an aqueous solution. There is a technique in which a measurement system in which a membrane protein is fused with a membrane is prepared, and in this measurement system, a change in ion permeability in an electrolyte solution of the membrane protein is measured as a change in current (for example, Non-Patent Document 3). .
However, the above black film forming method has the following problems.
1) The organic solvent in which lipid molecules are dissolved has a high boiling point, and therefore remains after film formation.
2) In order to rely on spontaneous film formation of lipid solution applied to the small holes, the formed film is a mixed state of the lipid bilayer membrane and the annular bulk layer surrounding the periphery, and the entire surface of the small hole is a uniform lipid bilayer membrane It is difficult to cover with.
3) Since it is necessary to manually create a small hole for each measurement, the diameter of the hole is non-uniform and the measurement results vary.

  The entire surface of the cell membrane is formed of a lipid bilayer membrane, in which the membrane protein functions. The heterogeneity of residual organic solvents and lipid bilayers may affect the function and morphology of membrane proteins. Therefore, in order to accurately measure the function of the membrane protein, it is necessary to measure in a state that mimics the cell membrane, that is, a system in which the entire surface is covered with a lipid bilayer membrane. Moreover, in order to eliminate the variation in results for each measurement, it is desirable to produce a plurality of small holes having a uniform hole diameter. As a result, multipoint measurement can be performed by one measurement, and variations in measurement results can be averaged.

Accordingly, the present inventors have a substrate in which a lipid bilayer membrane having a membrane protein is developed on an upper portion of a substrate having an artificially prepared hole portion and an opening portion of the hole portion being an overhang shape. Proposed a technique for improving the above problem (for example, Non-Patent Document 4).
In this substrate, a huge vesicle made of a uniform lipid bilayer is used to form a lipid bilayer covering the opening of the microhole, so that the problem of residual solvent and non-uniformity of the membrane is solved. ing. In addition, since a plurality of minute holes are arrayed on the substrate, the measurement results can be averaged, and measurement can be performed quickly and with high sensitivity. Such a lipid bilayer membrane substrate (membrane protein-immobilized substrate) that has artificially prepared micropores and on which purified membrane proteins can be placed realizes an in vitro measurement system under conditions similar to those in vivo This is expected to be performed (for example, Patent Document 1). In addition, a structure having a hole (micro hole) artificially formed on a solid substrate is expected to be applied to a biosensor using the function of a membrane protein (for example, Non-Patent Document 5).

JP 2008-275481 A

Drews J. Science, 287: 1960-1964 (2000) E. Neher and B. Sakaman, Nature 260, 799 (1976) "New Patch Clamp Experiment Method" edited by Yasunobu Okada (2001, Yoshioka Shoten) K. Sumitomo et al. Applied Physics Express Vol. 3, 107001 (2010) K. Sumitomo et al. NTT Technical Review Vol. 4, No.9 pp40-47

  In order to quantitatively measure the function of a small amount of membrane protein with high sensitivity, it is necessary to stably cover the micropores with a lipid bilayer membrane. However, when the membrane tension, stress, osmotic pressure inside and outside the hole, temperature, and the like change during the measurement, there is a problem that the lipid bilayer membrane easily falls into the hole or is broken.

  In view of the above circumstances, the present invention provides a lipid bilayer substrate with improved stability of the lipid bilayer membrane, a method for producing the lipid bilayer membrane substrate, and a hydrogel substrate used in the production method. It is aimed.

The lipid bilayer membrane substrate related to the present invention is a lipid bilayer membrane substrate in which a hole is provided in the substrate, and the opening of the hole is covered with the lipid bilayer membrane, It is characterized by being filled with hydrogel.
According to the lipid bilayer membrane substrate related to the present invention , the hydrogel filled in the holes supports the lipid bilayer membrane, whereby the stability of the lipid bilayer membrane can be improved. As a result, it becomes difficult to be affected by changes in osmotic pressure and temperature. Further, it is difficult to be affected by vibrations or the like when transporting the substrate. As a result, it is possible to prevent the lipid bilayer membrane from falling into the hole or torn.

Lipid bilayer substrate related to the present invention, and the lipid bilayer and said hydrogel, characterized in that the contact in the opening.
According to this configuration, the lipid bilayer membrane at the opening can be maintained more stably. Further, the lipid bilayer membrane can be brought close to a state (physiological state) close to the lipid bilayer membrane constituting the cell membrane. This is because the properties of the hydrogel are close to the cytoplasm constituting the cells. As a result, the function of the membrane protein arranged in the lipid bilayer membrane can be analyzed in a physiological state.

Lipid bilayer substrate related to the present invention is characterized in that the overhang portion extending in a direction to narrow the opening to the opening of the hole portion.
According to this configuration, the lipid bilayer membrane at the opening can be maintained more stably. Furthermore, it becomes easier to arrange the lipid bilayer so as to cover the opening during the production of the substrate.

Lipid bilayer substrate related to the present invention, the portion covering the opening in the lipid bilayer, wherein the membrane protein is located.
According to this configuration, the function of the membrane protein can be analyzed in a lipid bilayer membrane with improved membrane stability.

Lipid bilayer substrate related to the present invention is characterized in that it contains the fluorescent molecules inside the hydrogel.
According to this configuration, the state inside the hole sealed with the lipid bilayer membrane can be examined by the fluorescent molecule. Moreover, it can suppress that a fluorescent molecule flows out of a hole.

The method of manufacturing the lipid bilayer substrate of claim 1 of the present invention, pouring a solution containing a hydrogel precursor on the upper surface of the substrate, the solution in the hole portion provided in the upper surface of the substrate A filling step of filling, adding a giant vesicle to the solution above the substrate, allowing the giant vesicle to settle, developing the giant vesicle in the vicinity of the hole, and opening the hole to the lipid bimolecular The step of forming a lipid bilayer film covered with a membrane and gelling the hydrogel precursor in the solution inside and outside the hole without removing the solution containing the hydrogel precursor outside the hole And a gelation step for forming a hydrogel.
The method for producing a lipid bilayer membrane substrate according to claim 2 of the present invention further comprises the step of removing hydrogel outside the hole so as not to damage the lipid bilayer membrane according to claim 1. It is characterized by.

  According to the production method of the present invention, the solution containing the hydrogel precursor filled in the hole and the lipid bilayer membrane can be arranged in contact with each other at the opening of the hole. By gelling the solution in this state, the hydrogel formed in the hole and the lipid bilayer can be arranged in contact with each other at the opening of the hole. In this contact state, since the hydrogel in the hole can sufficiently support the lipid bilayer membrane, a lipid bilayer membrane substrate with further improved stability of the lipid bilayer membrane is produced. be able to. Further, the lipid bilayer membrane can be made closer to a physiological state.

The method for producing a lipid bilayer membrane substrate according to claim 3 of the present invention uses the substrate according to claim 1 or 2 , wherein an overhang portion extending in a direction of narrowing the opening is provided in the opening of the hole portion. It is characterized by.
According to this configuration, when the opening of the hole filled with the solution not yet gelled is covered with the lipid bilayer in the lipid bilayer formation step, the lipid bilayer is in the hole. Depressing can be suppressed. As a result, a lipid bilayer membrane substrate can be produced with high yield.
Moreover, in the manufactured lipid bilayer membrane substrate, the stability of the lipid bilayer membrane can be further improved.

The hydrogel substrate related to the present invention is a hydrogel substrate for producing the lipid bilayer membrane substrate, wherein the substrate is provided with a hole, and the hole is filled with the hydrogel. It is characterized by.
By using the hydrogel substrate related to the present invention , the lipid bilayer membrane substrate can be easily obtained. This is because when the lipid bimolecular membrane is disposed on the upper surface of the hydrogel substrate, the lipid bimolecular membrane can be prevented from falling into the hole.

  According to the lipid bilayer membrane substrate of the present invention, the hydrogel filled in the holes supports the lipid bilayer membrane, whereby the stability of the lipid bilayer membrane can be improved. As a result, it becomes difficult to be affected by changes in osmotic pressure and temperature. In addition, the lipid bilayer membrane can be prevented from falling into the hole or tearing. Furthermore, by arranging, for example, a membrane protein on a lipid bilayer membrane with improved membrane stability, it becomes easier to analyze the function of the membrane protein, and highly reliable data can be obtained. Moreover, since the stability of the lipid bilayer membrane is improved, resistance to physical impact such as vibration is also improved. As a result, the substrate can be transported more easily.

  According to the method for producing a lipid bilayer membrane substrate of the present invention, a lipid bilayer membrane substrate with further improved stability of the lipid bilayer membrane can be produced. Further, the lipid bilayer membrane can be made closer to a physiological state.

  By using the hydrogel substrate of the present invention, the lipid bilayer membrane substrate can be easily obtained.

It is typical sectional drawing of 1st embodiment of the lipid bilayer membrane substrate concerning this invention. It is typical sectional drawing of 2nd embodiment of the lipid bilayer membrane substrate concerning this invention. It is typical sectional drawing of 3rd embodiment of the lipid bilayer membrane substrate concerning this invention. 2 is a fluorescence observation image of the upper surface of a lipid bilayer substrate produced in Example 1. FIG. It is the result of investigating the tolerance with respect to the osmotic pressure change of the lipid bilayer membrane substrate produced in Comparative Example 1. It is the result of having observed the hydrogel board concerning this invention by AFM. It is the electron microscope image which observed the opening of the hole part in which the overhang part was provided from upper direction. (A) The electron micrograph which observed the opening of the hole part in which another overhang part was provided from upper direction; (b) The electron micrograph of the cross section.

Hereinafter, the present invention will be described with reference to the drawings based on preferred embodiments, but the present invention is not limited to such embodiments.
<First embodiment>
As shown in FIG. 1, the lipid bilayer membrane substrate 1 (first embodiment) of the present invention is provided with a hole 20 in the substrate 10, and an opening 21 of the hole is covered with a lipid bilayer membrane 30. Furthermore, the hole 20 is filled with the hydrogel 22.

In the present invention, the lipid bilayer membrane 30 and the hydrogel 22 may be in contact with each other at the opening 21 or may not be in contact with each other.
When they are in contact with each other, the lipid bilayer membrane 30 can always be supported, so that the membrane stability can be further improved. In addition, the lipid bilayer can be brought close to a physiological state.
When not in contact, an appropriate solvent, for example, an aqueous solution such as a buffer suitable for functional analysis of the membrane protein 31, is preferably disposed between the lipid bilayer membrane 30 and the upper surface of the hydrogel. Even when it is not in contact, when the lipid bilayer 30 falls into the hole 20, before the lipid bilayer 30 reaches the bottom of the hole 20, the drop is stopped halfway. Can do.
In the present invention, it is preferable that the lipid bilayer membrane 30 and the hydrogel 22 are in contact with each other from the viewpoint of more preferable effects.

  In addition, the upper surface of the hydrogel 22 may be flat in the opening 21, or may not be flat but may bulge upward, for example. Although the upper surface of the hydrogel 22 may swell or dent due to the influence of surface tension or the like, there is no problem if the degree is slight.

In the present invention, the shape of the opening 21 (opening 21) of the hole 20 is not particularly limited.
As the shape when the opening 21 is viewed from the upper surface, for example, a shape such as a circle, an ellipse, a triangle, a rectangle, and a polygon can be used. Moreover, the shape when the cross section of the hole 20 including the opening 21 is seen can be, for example, a simple concave shape or an overhang shape (eave shape) as shown in FIG. Here, the opening portion 21 having an overhang shape is referred to as an overhang portion 11a. The overhang portion 11a is provided in the opening 21 of the hole portion 20 so as to extend in a direction in which the opening 21 is narrowed.

In this embodiment, the overhang portion 11 a is formed by the thin film layer 11 provided on the upper surface 10 a of the substrate 10 projecting from the four corners of the opening 21 so as to narrow the opening 21 of the hole portion 20. The photograph which observed this state with the electron microscope is shown in FIG.
In the present embodiment, the overhang portions 11a are provided at the four corners (four corners) of the hole portion 20. However, the present invention is not limited to this configuration. For example, as shown in FIG. The overhang portion 11 a projecting in a circular shape toward the center of 21 can also be used. That is, in the present invention, the shape of the region surrounded by the overhang portion 11a from above the substrate 10 can be, for example, a shape such as a circle, an ellipse, a triangle, a rectangle, or a polygon.

The diameter of the opening 21 of the hole 20 or one side of the polygon forming the opening 21 is preferably 100 nm to 10 μm.
By setting it to be equal to or higher than the lower limit of the above range, a sufficient amount of membrane protein 31 can be arranged in the lipid bilayer membrane 30. By setting it to the upper limit of the above range or less, the opening 21 can contribute as a part of the force that prevents the lipid bilayer membrane 30 from falling into the hole 20 and maintains it stably.
The depth of the hole 20 is not particularly limited as long as the lipid bilayer 30 does not contact the bottom surface, and may be designed according to the diameter of the opening. The depth can be, for example, 0.1 μm to 10 μm.

The material of the substrate body 10 is not particularly limited, and examples thereof include silicon, glass, and plastic. Moreover, the thickness and shape of the substrate body 10 are appropriately adjusted according to the application.
The number of holes 20 provided in the substrate body 10 is not particularly limited, and may be, for example, 1 to 10,000.

  As a method for forming the hole 20 in the substrate body 10, for example, a fine processing technique such as a photolithography method, an electron beam lithography method, or a dry etching method can be applied.

  The type of lipid molecule of the lipid bilayer membrane 30 is not particularly limited as long as it can form a lipid bilayer membrane (lipid bilayer membrane). For example, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine ( PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidic acid (PA), phosphatidylglycerol (PG), sphingolipid and the like. These lipids may be used alone or in combination of two or more.

Examples of the method of forming the lipid bilayer membrane 30 include a method of developing a giant lipid vesicle having a diameter of 10 μm or more on a substrate.
Typical methods for forming vesicles include a static hydration method and an electro-swelling method (electrolytic formation method). The method for producing the vesicle is not particularly limited, but it is preferable to employ the electrolytic forming method from the viewpoint that it is easy to produce a huge vesicle and the reaction time and reaction process are simple. The electrolytic formation method is a method of forming a huge vesicle in an aqueous solution by applying an alternating electric field after forming a thin film of phospholipid on an electrode such as indium tin oxide (ITO). In order to obtain vesicles of uniform size, it is preferable to form a uniform phospholipid molecule film with a thickness of several tens of nm to several μm on the ITO substrate, and the alternating electric field is about several hundred mV to 2V. Application conditions are preferred.

  The hydrogel 22 filled in the hole 20 is not particularly limited as long as it does not affect the formation of the lipid bilayer membrane 30. For example, one type or two or more types of polymers such as agarose gel and polyamino acid are used. What is formed from the mixed solution of these is mentioned. Also, acrylamide, N, N′-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, N-methylolacrylamide, N-vinylpyrrolidone, hydroxyethyl methacrylate, (meth) acrylic acid, Hydrogel 22 is obtained by copolymerizing one or more monomers such as allylextrin and a polyfunctional monomer such as methylenebis (meth) acrylamide, polyethylene glycol di (meth) acrylate and the like. Can be used as Moreover, you may comprise the hydrogel 22 using the derivative | guide_body of the said monomer.

  Examples of the polymerization reaction for polymerizing the monomer (monomer) to form the hydrogel 22 include radical polymerization, cationic polymerization, and anionic polymerization. These polymerization methods are not particularly limited, and examples thereof include addition of a polymerization initiator. Examples of the polymerization initiator and its polymerization method include, for example, a method of mixing ammonium persulfate (hereinafter referred to as APS) and N, N, N ′, N′-tetramethylethylenediamine (hereinafter referred to as TEMED) in an aqueous monomer solution, azobisisobutronitrile (hereinafter referred to as AIBN). The method by thermal decomposition, etc., and the method by light irradiation to photoreactive polymerization initiators, such as Irgacure, are mentioned. Moreover, in the case of an agarose gel, it can be made to gelatinize by filling the hole part 20 with the heated solution, and cooling the said solution after that.

  The method for filling the hole 20 with the hydrogel 22 is not particularly limited. For example, the hole 20 is filled with a solution containing a hydrogel precursor before gelation, for example, an aqueous solution containing the monomer, If necessary, a polymerization initiator is added, and immediately after that (before gelation), the opening 21 of the hole 20 is sealed with the lipid bilayer membrane 30, and an excess hydrogel precursor outside the hole 20. A method of selectively forming the hydrogel 22 only inside the hole 20 by replacing the solution containing the body with water or an aqueous electrolyte solution or the like can be mentioned. As another method, the lipid bilayer membrane 30 is not damaged after the hydrogel 22 is formed inside and outside the hole 20 without removing the solution containing the excess hydrogel precursor outside the hole 20. Thus, a method of removing only the hydrogel 22 outside the hole 20 can also be exemplified.

  The hardness and water content of the hydrogel 22 are appropriately adjusted according to the application, for example, according to the type of the membrane protein 31 to be functionally analyzed. By examining the concentration of the precursor constituting the hydrogel 22 and the type of polymerization method, the hydrogel 22 having the optimum hardness and water content can be obtained. For example, when agarose gel is filled, the concentration can be 0.01 wt% to 5.0 wt%. Preferably it is 0.05 wt%-5 wt%, More preferably, it is 0.5 wt%-3 wt%. For example, when a polyacrylamide gel is filled, the concentration can be set to 1 wt% to 30 wt%. It is preferably 3 wt% to 20 wt%, more preferably 5 wt% to 15 wt%.

  The water contained in the hydrogel 22 may contain a pH buffer, various ions, an antioxidant, a surfactant and the like as long as the lipid bilayer membrane 30 is stably maintained. It is preferable to contain components necessary for optimal function of the membrane protein 31.

  In the present embodiment, the membrane protein 31 is disposed on the lipid bilayer membrane 30 covering the opening 21 of the hole 20, and the lipid bilayer membrane substrate 1 is the membrane protein-immobilized substrate 1. The lipid bilayer membrane 30 is covered with the external solution L. For example, by adding a drug candidate substance or the like to the external solution L, the function of the membrane protein 31 can be analyzed.

A known method can be used as a method for reconstituting the membrane protein 31 to be a function measurement target in the lipid bilayer membrane 30, and examples thereof include a vesicle fusion method. Specifically, by adding a vesicle containing a membrane protein called proteoliposome on the lipid bilayer, the proteoliposome and the lipid bilayer are fused, and the membrane protein is reconstituted in the lipid bilayer. Is done.
The membrane protein 31 fused to the lipid bilayer membrane 30 is preferably retained in the lipid bilayer membrane 30 and changes the ion concentration inside and outside the pore with the lipid bilayer membrane 30 as a boundary. Examples of the membrane protein 31 include an ion channel type receptor.

  Examples of ion channel receptors include cell-to-cell information transmission and temperature sensing, TRP (transient receptor potential) channels involved in inflammation and pain, cell-to-cell information transmission and pain-related ATP receptors, and cell-to-cell information transmission. Serotonin receptor involved in cell and emotion, NMDA receptor involved in intercellular communication and excitatory neurotransmission, AMPA receptor involved in intercellular communication and excitatory neurotransmission, intercellular communication and excitatory neurotransmission And the kainate receptor involved in the cell, the GABA receptor involved in intercellular signal transmission and inhibitory neurotransmission, and the like.

When the electrolyte solution is arranged inside and outside the hole, the electrolyte solution is preferably one in which the lipid bilayer membrane 30 in which the membrane protein 31 is embedded is stably held. For example, 100 mM NaCl, 5 mM KCl, 2 mM CaCl ₂ , Examples include 10 mM Tris-HCl (pH 7.4). The composition of the electrolyte solution inside and outside the hole may be the same composition or different compositions.

By the way, in the cytoplasm covered with a cell membrane constituting cells, a fiber network structure is formed by cytoskeleton molecules such as actin filaments, intermediate filaments, and microtubules in order to maintain the cell morphology. For this reason, the effect that the property of the molecule | numerator in the solvent called molecular crowding changes is seen. This effect (due to the high density of cytoskeletal molecules and other intracellular macromolecules in the cell) increases the activity of the molecules in the cytoplasm, thus allowing the diffusion and activity of proteins to occur in vitro (simple aqueous solutions It is known that it is different from the middle). Therefore, in order to more accurately measure protein functions in vivo, a measurement system that mimics not only the cell membrane but also the environment in the cytoplasm is required. However, few examples of protein function measurement in the state of mimicking the skeletal structure in the cytoplasm have been reported so far.
One of the reasons why the present invention is extremely useful is that the function of the membrane protein can be analyzed in a state close to in vivo. In the lipid bilayer membrane substrate (membrane protein-immobilized substrate) of the present invention, the lipid bilayer membrane 30 corresponding to the cell membrane is in contact with the hydrogel 22 corresponding to the cytoplasm. The function of the membrane protein 31 arranged on the membrane 30 can be measured in a state close to in vivo.

<Second embodiment>
As shown in FIG. 2, the lipid bilayer membrane substrate 2 (second embodiment) of the present invention includes a fluorescent molecule 23 (fluorescent substance) inside the hydrogel 22 in addition to the configuration of the first embodiment. ing.
Except for this feature, it is the same as the lipid bilayer membrane substrate 1 (membrane protein-immobilized substrate 1) of the first embodiment described above.
Below, the point which the lipid bilayer membrane 2 of 2nd embodiment differs from the lipid bilayer membrane substrate 1 of 1st embodiment is demonstrated.

The fluorescent molecule 23 is preferably a water-soluble molecule that emits fluorescence when a change in the state of the inside of the hole portion 20 occurs, and more preferably, the fluorescent molecule 23 changes in fluorescence intensity as the ion concentration inside the hole portion 20 changes. Specifically, for example, Fluo4, Fura-2, and Fluo-3 capable of detecting a change in calcium ion concentration can be exemplified.
By using a suitable fluorescent molecule, for example, when an ion-permeable membrane protein 31 is arranged in the lipid bilayer membrane 30 and ions flow into or out of the hole 20 through the membrane protein, the hydrogel It can be detected by a fluorescence microscope or the like that the fluorescence intensity of the fluorescent molecules 23 in 22 has changed. Such a detection system is useful in the functional analysis of the membrane protein 31.

<Third embodiment>
As shown in FIG. 3, the lipid bilayer membrane substrate 3 (third embodiment) of the present invention includes an electrode 40 in the hole 20 in addition to the configuration of the first embodiment, and the outside of the hole 20. A counter electrode 41 is provided in the solution L.
Except for this feature, it is the same as the lipid bilayer membrane substrate 1 (membrane protein-immobilized substrate 1) of the first embodiment described above.
Below, the point which the lipid bilayer membrane 3 of 3rd embodiment differs from the lipid bilayer membrane substrate 1 of 1st embodiment is demonstrated.

The lipid bilayer membrane substrate 3 according to the third embodiment is an embodiment as a membrane protein-immobilized substrate 3 suitable for measuring the function of the membrane protein 31 using an electrophysiological technique.
In measuring the function of the membrane protein 31, it is preferable to dispose the counter electrode 41 in the external solution L in the hole 20. The electrode 40 and the counter electrode 41 are preferably used by being connected to a patch clamp measuring device.
For example, when an ion channel protein is arranged as the membrane protein 31, the function of the membrane protein 31 can be measured by measuring an ionic current that has passed through the ion channel protein with an electric meter.

As a method for arranging the electrode 40, for example, when the lipid bilayer membrane substrate 3 is manufactured, a metal layer is formed by a film forming method such as sputtering, and the metal layer is appropriately patterned by etching or the like to insulate the formed electrode. By covering with a layer, the holes 20 can be embedded and arranged. Here, when the electrode 40 is embedded in the substrate body 10, the substrate body 10 needs to be insulative. In this case, as the material of the substrate body 10, silicon oxide, silicon nitride, alumina, tantalum oxide, a resist film, or the like can be used.
Examples of the material of the electrode 40 include silver / silver chloride, gold, and platinum.

<Method for producing lipid bilayer membrane substrate>
A preferred method for producing the lipid bilayer substrate of the present invention will be described with reference to FIG.
As the production method, a method of performing a filling step, a lipid bilayer formation step, and a gelation step in this order is preferable. In addition, as long as it does not deviate from the meaning of this invention, you may add another process other than the three processes mentioned here.

[Filling process]
The filling step is a step of pouring a solution containing the precursor of the hydrogel 22 on the upper surface 10a of the substrate 10 and filling the hole 20 provided in the upper surface 10a of the substrate with the solution.
The precursor of the hydrogel 22 is the monomer before polymerization as described above. The solvent for dissolving or dispersing the precursor is not particularly limited, and an aqueous solvent is preferable. The concentration of the precursor is appropriately adjusted according to the hardness and water content of the hydrogel 22.
The amount of the solution poured onto the upper surface 10a of the substrate 10 is not particularly limited as long as the hole 20 can be filled, but the amount that can overflow the upper surface 10a, that is, the solution above the upper surface 10a. It is preferable to pour an amount that can be accumulated.
The method for pouring the solution onto the upper surface 10a of the substrate 10 is not particularly limited, and after dropping the solution onto the upper surface 10a with a pipette or the like, by subjecting the substrate and the solution to ultrasonic treatment or vacuum treatment as necessary, It is possible to discharge bubbles from the hole 20 and fill the hole 20 with the solution.

[Lipid bilayer formation process]
In the lipid bilayer process, a giant vesicle is added to the solution above the substrate 10, the giant vesicle is allowed to settle on the upper surface 10 a of the substrate 10 or the upper surface of the thin film layer 11, and the giant vesicle is near the hole 20. And the opening 21 of the hole 20 is covered with the lipid bilayer membrane 30.
As the giant vesicle, those prepared by a known method as described above can be used. The size (diameter) of the giant vesicle is preferably sufficiently larger than the opening 21 and can be, for example, 10 μm or more. For example, a giant vesicle having a size of 10 μm to 100 μm can be used, but the size is not limited to this range, and other sizes may be used.
The method for adding the giant vesicle to the solution above the upper surface of the substrate 10 is not particularly limited, and examples thereof include a method of preparing a vesicle dispersion containing the giant vesicle and adding it.
As a standard of the concentration of the giant vesicle in the vesicle dispersion, the concentration of the giant vesicle added is such that it occupies a wide area of the upper surface 10a of the substrate 10, for example, 80% or more of the upper surface 10a. preferable. For example, it can be used at a concentration of 0.1 mM to 10 mM, but is not limited to this concentration range, and other concentrations may be used.

From the viewpoint of forming the lipid bilayer membrane 30 with good yield, it is preferable to use the substrate 10 in which the overhang portion 11a extending in the direction of narrowing the opening is provided in the opening 21 of the hole portion 20 in the manufacturing method of the present invention.
When the overhang portion 11a is present, the lipid bilayer 30 falls into the hole 20 when the lipid bilayer 30 covers the opening 21 of the hole 20 filled with the solution that has not yet been gelled. Can be suppressed.

[Gelling process]
The gelation step is a step of forming a hydrogel by gelling the hydrogel precursor in the solution.
The method for polymerizing the precursor is as described above. After the filling step, when the solution containing the hydrogel precursor is outside the hole 20, it may be removed and the hydrogel precursor may be gelled only inside the hole 20, or The hydrogel precursor may be gelled inside and outside the hole 20 without removing the solution containing the hydrogel precursor outside the hole 20. In the former case, there is a method of gelling after replacing the external solution of the hole 20 with, for example, a physiological salt solution or the like. In the latter case, the hydrogel that has gelled above the hole 20 can be removed by an appropriate means, for example, a method such as slowly peeling so as not to damage the lipid bilayer membrane. When the lipid bilayer membrane 30 is exposed to the air, the structure and function of the lipid bilayer membrane 30 may be impaired. Therefore, the hydrogel can be removed with an appropriate external solution L such as a physiological salt solution. It is preferable to carry out in a covered state.

<Hydrogel substrate>
The hydrogel substrate according to the present invention is used for producing the above-described lipid bilayer membrane substrate according to the present invention. A hole 20 is provided in the substrate 10, and the hydrogel is formed in the hole 20. 22 is filled.
Examples of the method for producing a hydrogel substrate include the method performed in the same manner as the filling step in the method for producing a lipid bilayer membrane substrate described above.
The substrate 10 provided with the hole 20 is prepared, a solution containing a hydrogel precursor is poured onto the upper surface 10a, the solution is filled into the hole 20, and then the gel is formed. The hydrogel gelled outside the hole 20 can be removed by scraping with a spatula or simply peeling off. At this time, since the hydrogel in the hole 20 is held by the hole 20, it can be left without being removed.
Further, as another method for producing a hydrogel substrate, a method of producing the aforementioned lipid bilayer membrane substrate and then removing the lipid bilayer membrane on the substrate using a surfactant or the like can also be exemplified. .
Regardless of the hydrogel substrate manufactured by either method, the lipid bilayer substrate (membrane protein-immobilized substrate) is produced by the user performing a process of covering the opening of the hole with the lipid bilayer membrane at the time of use. be able to.

[Example 1]
A production example of the lipid bilayer membrane substrate 2 (membrane protein-immobilized substrate 2) of the second embodiment schematically represented in FIG. 2 will be specifically described.
<Formation of hole>
A silicon substrate was used as the substrate body 10. A silicon oxide film layer (thin film layer) 11 having a thickness of 120 nm was formed on the upper surface 10a of the substrate body 10 by a thermal oxidation method. Further, a hole 20 having a circular opening 21 (4 μm × 4 μm) was formed by using a photolithography method and a dry etching method. The depth was 1 μm. Further, the substrate body 10 under the silicon oxide film layer 11 is selectively etched with a potassium hydroxide solution (10 wt%) to form overhang portions 11 a at the four corners of the opening 21 of the hole 20. did.

<Preparation of huge vesicle>
The lipid bilayer membrane 30 was formed as follows. A mixed chloroform solution (concentration 2.5 mM) of diphytanyl phosphatidylcholine (DPhPC) (80 mol%) and cholesterol (20 mol%) was prepared. Subsequently, 200 μL of the chloroform solution was uniformly applied on an indium tin oxide (ITO) substrate (a substrate in which ITO having a thickness of 100 nm was thinned on SiO ₂ , size 40 × 40 mm, 50 to 100 Ω / cm). . The substrate was dried under reduced pressure at room temperature for 2 hours to completely remove the chloroform solvent, thereby forming a uniform phospholipid thin film on the ITO substrate. On top of that, a silicone rubber having a window portion (having a window portion in which a silicone rubber having an outer dimension of 30 × 30 mm and a thickness of 1 mm is cut in a size of 20 × 20 mm) is closely attached, and 200 mM of the window portion is placed. 400 μL of a sucrose aqueous solution was dropped. Further, an ITO substrate was placed on the top, and the solution in the silicone rubber window was sandwiched between the ITO substrates and sealed. Subsequently, a clip electrode was joined to the ITO substrate, and an alternating electric field (sine wave, 1 V, 10 Hz) was applied on a hot plate at 60 ° C. for 2 hours, thereby producing a huge vesicle by an electric field forming method.

<Formation of hydrogel>
A monomer aqueous solution containing 7% acrylamide as an unpolymerized monomer constituting hydrogel 22 and 1/19% by weight of bisacrylamide with respect to acrylamide, fluorescent molecule 23 (100 μM calcein), and an aqueous solution containing sodium chloride, An aqueous solution for filling was prepared by adding APS and TEMED to the mixed aqueous solution. This filling aqueous solution was dropped on the upper surface 10a of the substrate 10 to fill the inside of the hole and sufficiently overflowed above the upper surface 10a. Immediately after the monomer filling, the giant vesicle is added to the filling aqueous solution overflowing the upper surface 10a of the substrate body 10, the giant vesicle is allowed to settle, and the giant vesicle is developed on the silicon oxide film layer 11 on the upper face 10a. Thus, the lipid bilayer membrane 30 was formed. Thereafter, the mixture is allowed to stand for 15 minutes or more to form a hydrogel 22 in which the monomer has gelled, and then a sufficient amount of 200 mM glucose solution containing sodium chloride is added to remove excess hydrogel on the lipid bilayer membrane 30. It was.
By the above method, a lipid bilayer membrane substrate having the hole 20 shown in FIG. 2 was produced. In addition, by arranging the membrane protein 31 on the lipid bilayer membrane 30, the lipid bilayer membrane according to the present invention can be used as a membrane protein-immobilized substrate. As a method of arranging the membrane protein 31 on the lipid bilayer membrane 30, a known method can be applied.

FIG. 4 shows fluorescence observation when calcein is filled as the fluorescent molecule 23 inside the hole 20 and acrylamide is filled as the hydrogel 22 and the lipid bilayer 30 containing rhodamine that exhibits red fluorescence is developed in the hole opening. Results are shown. From the hole 20a in which the hydrogel 22 is filled in the hole 20, since green fluorescence derived from calcein is observed, the fluorescent molecule 23 does not diffuse from the inside of the hole into the external solution and is stable in the hydrogel. You can see that it is trapped in Moreover, in the hole 20b, since green and red fluorescence overlap and yellow fluorescence is observed, the inside of the hole is filled with the hydrogel 22, and the opening is covered with the lipid bilayer membrane 30. I understand.
The membrane protein-immobilized substrate 1 can be produced by arranging the membrane protein on the substrate produced by the above method.

[Example 2]
Next, a production example of the lipid bilayer membrane substrate 2 (membrane protein-immobilized substrate 2) of another second embodiment will be specifically described.
In Example 2, a membrane protein-immobilized substrate 2 in which α-hemolysin that is a membrane protein 31 is arranged in a lipid bilayer membrane is used. This substrate 2 holds fluorescent molecules 23 whose fluorescence intensity changes due to the inflow of ions inside the hole. Since these features are the same as those of the first embodiment described above, the same parts are denoted by the same reference numerals and description thereof is omitted.
Hereinafter, the difference between the membrane protein-immobilized substrate 2 used in Example 2 and the membrane protein-immobilized substrate 2 of Example 1 will be described.
The inside of the hole 20 is filled with a hydrogel 22 made of acrylamide containing a fluorescent dye Fluo4 whose fluorescence intensity varies with the concentration of calcium ions so that the final concentration is 10 μM, and the lipid bilayer membrane 30 is opened in the hole. Expanded to the part 21, the lipid bilayer membrane substrate 2 was obtained.
A membrane protein-immobilized substrate in which a transmembrane channel (membrane protein 31) through which calcium ions permeate is formed by arranging α-hemolysin on the lipid bilayer membrane 30 using the obtained lipid bilayer membrane substrate 2 2 is obtained. By using this, the permeation of calcium ions through α-hemolysin is quantitatively detected by measuring the change in the fluorescence intensity of Fluo4 when the external aqueous solution in the hole 20 is replaced with a 1 mM calcium chloride aqueous solution. Can do.

[Example 3]
Next, a production example of the lipid bilayer membrane substrate 3 (membrane protein-immobilized substrate 3) of the third embodiment schematically represented in FIG. 3 will be described in detail.
In Example 3, in order to detect the inflow of ions by an electrophysiological technique, the membrane protein-immobilized substrate 3 having the electrodes 40 and 41 inside and outside the hole 20 is used. Except for this feature, the second embodiment is the same as the first embodiment and the second embodiment described above.

Below, the point which the membrane protein fixed board | substrate 3 used in Example 3 differs from the membrane protein fixed board | substrate of Example 1 and Example 2 is demonstrated.
First, on a Si wafer covered with an insulating layer made of a thermal oxide film having a thickness of 200 nm, a metal layer is deposited in the order of titanium, gold, and platinum and patterned by a photolithography method to obtain a thickness of about 60 nm. Electrode layer 40 (electrode 40) was formed. The electrode layer 40 is connected to a sufficiently large pad portion (300 nm square), and can be connected to an electrophysiological measurement device.
Next, 1 μm of a silicon nitride film was deposited as an insulating layer on the Si wafer on which the electrode layer 40 was formed by a plasma CVD method. On this insulating layer, as a thin film layer 11 (overhang shape forming layer 11) on which an overhang portion is formed, a silicon oxide film was deposited by sputtering to a thickness of 200 nm.
The hole 20 and the opening 21 were formed in the substrate body 10 thus formed by photolithography and dry etching. The etching for forming the hole was performed until it reached the electrode layer 40.
In the process of this dry etching, the overhang portion 11a was formed by utilizing the selectivity between the silicon oxide film (overhang shape forming layer 11) and the silicon nitride film (substrate body 10).
After the hole 20 and the opening 21 were formed, the resist film was washed and removed to obtain a substrate having the electrode 40 in the hole 20.

  In this embodiment, the number ratio between the hole 20 and the electrode 40 is preferably 1: 1. A plurality of holes 20 may be provided for one electrode 40, but in this configuration, it is necessary to cover all the holes 20 with the lipid bilayer membrane 30 when measuring with the one electrode 40. Therefore, measurement preparation and measurement conditions are complicated. Therefore, it is desirable to form one hole 20 in one electrode 40.

  In the same manner as in Example 1 described above, the hydrogel 22 was filled on the substrate provided with the electrode 40 in the hole portion 20 formed in this way, and the giant lipid membrane vesicle was developed, thereby providing a hole. The opening part 21 of the part 20 is covered with a lipid bilayer membrane 30. Further, as in Example 2, α-hemolysin is arranged as a membrane protein 31 on the lipid bilayer membrane 30, and when a voltage is applied between the electrode 40 and the counter electrode 41, the current flowing between them is measured. -Quantitative detection of ion permeation through hemolysin.

[Comparative Example 1]
A lipid bilayer membrane substrate identical to that of Example 1 was prepared except that the hydrogel 22 was not provided inside the hole 20. FIG. 5 shows a fluorescent image (a) on the upper surface of the substrate and a schematic diagram (c) of a cross section of the substrate.
The main point different from the manufacturing method of Example 1 is that an aqueous solution Q containing no fluorescent molecules and containing fluorescent molecules is dropped onto the upper surface of the substrate to fill the holes 20. Example 1 is that the opening 21 is covered with a lipid bilayer 30 using a giant vesicle, and then the aqueous solution Q containing fluorescent molecules outside the hole is replaced with an appropriate aqueous solution to remove the fluorescent molecules outside the hole. It is the same.

<Evaluation of membrane strength by osmotic pressure change>
Using the lipid bilayer membrane substrate of Comparative Example 1, the membrane strength was evaluated by examining the membrane state when an osmotic pressure change occurred. This result will be described with reference to FIG.
FIG. 5 (a) is a fluorescence image on the upper surface of the substrate when the osmotic pressure inside and outside the hole is the same. FIG. 5B is a fluorescence image on the upper surface of the substrate when the ion concentration inside the hole is 200 mM and the ion concentration outside the hole is 600 mM. FIG. 5C is a schematic diagram of the substrate cross section of FIG. 5A, and FIG. 5D is a schematic diagram of the substrate cross section of FIG. 5B.

When the osmotic pressure is higher in the outside of the hole than in the inside of the hole, water flows out from the inside of the hole toward the outside, and thus a force for pulling the lipid bilayer membrane into the inside of the hole is generated.
FIG. 5 shows this result. Initially, the lipid bilayer membrane sealed the plurality of holes, and light emission from the encapsulated fluorescent molecules was observed (FIG. 5 (a)). However, after changing the osmotic pressure, much fluorescence intensity was not observed. This is because the lipid bilayer falls into the hole or is broken due to the force accompanying the osmotic pressure change. Some of the fluorescence intensity changed partially by falling into the hole (a hole surrounded by a broken line in FIG. B). As shown in the schematic diagram of FIG. (D), this shows that the lipid bilayer has fallen into the hole.

As described above, the lipid bilayer membrane of Comparative Example 1 sometimes breaks due to changes in osmotic pressure.
On the other hand, when the same osmotic pressure change was applied to the lipid bilayer membrane substrate 2 of Example 1, light emission from the fluorescent molecules was observed in almost all the holes without change. That is, the lipid bilayer membrane 30 covering the opening 21 was stably maintained before and after the osmotic pressure change.
From these results, it is clear that the lipid bilayer membrane substrate according to the present invention is hardly affected by changes in osmotic pressure, and the stability (membrane strength) of the membrane is improved as compared with the conventional one.

<Observation of gel inside hole by AFM>
Except not forming the lipid bilayer membrane 30, it carried out like Example 1 and the board | substrate (hydrogel board | substrate) with which the hydrogel 22 was filled in the hole part 20 was prepared. At this time, the hydrogel formed outside the hole 20 was peeled off and removed.
A fluorescent image of the upper surface of the hydrogel substrate is shown in FIG. Luminescence from calcein contained in the hydrogel 22 was observed from the three holes. The same region on the upper surface of the hydrogel substrate is observed with an AFM (atomic force microscope), and the results are shown in FIGS. From these results, it was confirmed that the gel was buried in the three holes where luminescence was observed.

<Evaluation of film strength by AFM>
When the lipid bilayer membrane 30 in the lipid bilayer membrane substrate 2 of Example 1 was observed with AFM, it was confirmed that the lipid bilayer membrane 30 was stretched on the opening 21. This is probably because the lipid bilayer 30 is supported by the hydrogel 22 and thus the membrane strength is improved to such an extent that the AFM probe can detect the lipid bilayer.
On the other hand, we tried to observe the lipid bilayer membrane on the lipid bilayer membrane substrate of Comparative Example 1 shown in FIG. 5 (c) with AFM, but the lipid bilayer membrane in the opening was stable and reliable. Could not get. This is considered to indicate that the membrane strength of the lipid bilayer membrane is weaker than that of Example 1.

  From the above results, it is clear that the lipid bilayer membrane substrate according to the present invention is hardly affected by osmotic pressure change, temperature change, etc., and has improved membrane stability (membrane strength) than before. .

1-4 ... Lipid bilayer substrate (membrane protein-immobilized substrate),
DESCRIPTION OF SYMBOLS 10 ... Substrate (substrate body), 10a ... Upper surface of substrate, 11 ... Thin film layer (silicon oxide film, overhang forming layer), 11a ... Overhang part, 20 ... Hole part, 20a ... Covered with lipid bilayer Non-perforated hole, 20b ... hole covered with lipid bilayer, 21 ... opening (opening), 22 ... hydrogel, 23 ... fluorescent molecule (fluorescent substance), 30 ... lipid bilayer (lipid bilayer) Heavy film), 31 ... membrane protein, 40 ... electrode, 41 ... counter electrode, L ... external solution, Q ... aqueous solution containing fluorescent molecules.

Claims (3)

Pouring a solution containing a hydrogel precursor on the upper surface of the substrate, and filling the solution into a hole provided in the upper surface of the substrate;
Giant vesicles are added to the solution above the substrate, the giant vesicles are allowed to settle, the giant vesicles are developed in the vicinity of the hole, and the lipid bilayer covering the opening of the hole with a lipid bilayer membrane A film forming step;
A gelation step of forming a hydrogel by gelling the hydrogel precursor in the solution inside and outside the hole without removing the solution containing the hydrogel precursor outside the hole. A method for producing a lipid bilayer membrane substrate, comprising: The method for producing a lipid bilayer membrane substrate according to claim 1, further comprising a step of removing a hydrogel outside the hole so as not to damage the lipid bilayer membrane. The method for producing a lipid bilayer substrate according to claim 1 or 2 , wherein a substrate in which an overhang portion extending in a direction of narrowing the opening is provided in the opening of the hole portion.