WO2013002339A1 - Substrat destiné à la formation d'une membrane lipidique et méthode de production du substrat - Google Patents

Substrat destiné à la formation d'une membrane lipidique et méthode de production du substrat Download PDF

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Publication number
WO2013002339A1
WO2013002339A1 PCT/JP2012/066574 JP2012066574W WO2013002339A1 WO 2013002339 A1 WO2013002339 A1 WO 2013002339A1 JP 2012066574 W JP2012066574 W JP 2012066574W WO 2013002339 A1 WO2013002339 A1 WO 2013002339A1
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Prior art keywords
substrate
opening
lipid membrane
laser
lipid
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PCT/JP2012/066574
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English (en)
Japanese (ja)
Inventor
理 額賀
山本　敏
和仁 田端
正和 杉山
昌治 竹内
Original Assignee
株式会社フジクラ
技術研究組合Ｂｅａｎｓ研究所
国立大学法人東京大学
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Application filed by 株式会社フジクラ, 技術研究組合Ｂｅａｎｓ研究所, 国立大学法人東京大学 filed Critical 株式会社フジクラ
Priority to JP2013522950A priority Critical patent/JP5938039B2/ja
Publication of WO2013002339A1 publication Critical patent/WO2013002339A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23K—SOLDERING OR UNSOLDERING; WELDING; CLADDING OR PLATING BY SOLDERING OR WELDING; CUTTING BY APPLYING HEAT LOCALLY, e.g. FLAME CUTTING; WORKING BY LASER BEAM
    • B23K26/00—Working by laser beam, e.g. welding, cutting or boring
    • B23K26/36—Removing material
    • B23K26/361—Removing material for deburring or mechanical trimming

Definitions

  • the present invention relates to a substrate having a microchamber and a method for manufacturing the substrate. More specifically, the present invention relates to a substrate provided with a microchamber for forming a lipid membrane, and a method for producing the substrate.
  • This application claims priority based on Japanese Patent Application No. 2011-142946 filed in Japan on June 28, 2011, the contents of which are incorporated herein by reference.
  • Membrane proteins present in the cell membranes of the cells that make up the body are the targets on which pharmaceuticals act, so intense research competition has been developed in industry, government, andTECH. Since the membrane protein has a complicated and precise three-dimensional structure, it is easily denatured and decomposed and only a very small amount of sample can be obtained. For this reason, in recent years, attention has been paid to the application of microfluidic chips to biotechnology, which can perform experiments even when only a small amount of sample is available.
  • Patent Document 1 discloses a method for forming a lipid membrane and introducing a membrane protein into the lipid membrane using a microchamber having a length ⁇ width ⁇ height of 19 ⁇ m ⁇ 17 ⁇ m ⁇ 7 ⁇ m.
  • the microfluidic chip of Patent Document 1 uses PDMS (Polydimethyl siloxane) as a material
  • the PDMS constituting the microchamber is made into a solvent by pouring an aqueous solvent such as a buffer or an organic solvent in which a lipid is dissolved.
  • an aqueous solvent such as a buffer or an organic solvent in which a lipid is dissolved.
  • the pore diameter of the opening of the microchamber forming the lipid membrane changes, and in the worst case, the opening is closed.
  • the expansion or contraction has a problem that not only the microchamber but also the microchannel is blocked.
  • size of the microchamber at the time of drying (at the time of manufacture) is set supposing the extent to which the said expansion
  • the conventional micro chamber is formed by forming a groove on the surface of the PDMS substrate and attaching another member such as PDMS or glass on the groove. For this reason, when the microchamber expands or contracts due to the influence of the solvent or temperature, not only the diameter of the opening of the microchamber changes, but also the bonded surface peels off, the microchamber is damaged, or the lipid membrane is removed. There is a risk that it cannot be formed.
  • the present invention has been made in view of the above circumstances, and it is an object to provide a base for forming a lipid film, in which an opening for forming a lipid film is formed by a single member, and a method for manufacturing the base. To do.
  • the substrate for forming a lipid membrane according to the first aspect of the present invention is a substrate for forming a lipid membrane, and includes a base material having an inner wall surface and a liquid containing lipid contained in the base material. And a micro-chamber having an opening in the inner wall surface of the base material that is included in the base material and that constitutes the space, and at least a part of the base material that forms the opening portion includes: It is composed of a single member. In the substrate according to the first aspect of the present invention, it is preferable that the single member transmits light having at least some wavelengths among light having wavelengths of 0.1 ⁇ m to 10 ⁇ m.
  • the single member is preferably silicon, glass, quartz, or sapphire.
  • the micro chamber is formed by further removing a portion modified by irradiating the substrate with a laser by an etching process.
  • the method for producing a substrate according to the second aspect of the present invention is a method for producing a substrate for forming a lipid film as described above as the substrate according to the first aspect, wherein a laser having a pulse time width of picosecond order or less is used.
  • the method for producing a substrate according to the third aspect of the present invention is a method for producing a substrate for forming a lipid film described above as the substrate according to the first aspect, wherein the space is formed in the single member, By irradiating a laser having a pulse time width on the order of picoseconds or less to a region where the micro chamber of the single member is formed, a modified portion is formed in the region, and the modification is performed from the single member. Removing the mass portion by etching.
  • a lipid membrane can be formed at the opening of a microchamber exposed to a space into which a liquid containing lipid flows. Since the opening is composed of a single member, there is no seam or bonding surface, and the step can be substantially eliminated. For this reason, the formed lipid membrane can be stably maintained. Therefore, it is easy to observe the formed lipid membrane, and by introducing a membrane protein such as an ion channel or an ion pump into the lipid membrane, highly accurate functional analysis of the membrane protein is possible.
  • the single member is transparent (partially transmits) at least a part of the wavelength (0.1 ⁇ m to 10 ⁇ m) of a commonly used processing laser beam among lights having a wavelength of 0.1 ⁇ m to 10 ⁇ m. If possible, the single member can be modified and processed by irradiating the laser beam. In addition, when the single member is transparent to at least a part of visible light (0.36 ⁇ m to 0.83 ⁇ m) of the light having the wavelength (can transmit a part of visible light). Can easily observe the formed lipid membrane through an optical device such as a microscope through the single member.
  • the single member is transparent to at least a part of ultraviolet light or visible light (0.1 ⁇ m to 0.83 ⁇ m) of the light having the wavelength (part of the ultraviolet light or visible light is transmitted).
  • the fluorescence can be observed.
  • the single member is transparent to light of a specific wavelength”, even if a part of the light incident on the member is absorbed by the member, the remaining light (light The remaining part) can penetrate the member.
  • the single member is a member made of silicon, glass, quartz, or sapphire
  • the processing accuracy of the micro chamber can be further increased, and the opening is nano-order (1 nm or more and less than 1 ⁇ m). Can be formed.
  • these members do not absorb the solvent, the diameter of the opening or the volume of the microchamber hardly changes during the formation of the lipid film. For this reason, experiments with high reproducibility are possible without the size of the formed lipid membrane (for example, film thickness) changing from experiment to experiment.
  • the method of forming the microchamber is a method of modifying the base material by laser irradiation and removing the modified portion by etching, obtain a substrate in which the microchamber is arranged with high accuracy and high density. Can do.
  • a microchamber is formed in a single member, and an end of the microchamber is formed on the substrate. It can be opened to an inherent space. Moreover, the opening part of the said micro chamber can be formed with the aperture size of nano order.
  • FIG. 2 is a cross-sectional view taken along line AA in FIG.
  • FIG. 2 is a schematic perspective view showing a laser irradiation method S.
  • FIG. It is a figure which shows typically the relationship between the laser irradiation energy and the modification part (oxygen deficient part) formed. It is a figure which shows typically the relationship between the laser irradiation energy and the modification part (oxygen deficient part) formed. It is a schematic sectional drawing which shows an example of the manufacturing method of the base
  • FIG. 1 is a perspective view of a base body 10A that is a first embodiment of a base body (hereinafter simply referred to as “base body”) for forming a lipid membrane according to the present invention.
  • FIG. 2 is a schematic diagram showing a cross section taken along line AA of FIG.
  • the substrate 10A is a substrate provided with a micro chamber 1 used for forming a lipid film.
  • the base body 10A has a space (well 2) that is contained in the base material 4 and allows the liquid P containing lipid to flow in, and an inner wall surface of the base material 4 that is contained in the base material 4 and constitutes the space (well 2).
  • the micro chamber 1 having the opening U is included, and at least a portion of the base material 4 constituting the opening U is formed of a single member.
  • “flowing the liquid P containing lipid into the space” means putting the liquid P into the space from the outside of the space.
  • the liquid P that has flowed in may stay in the space and stay (or be completely stationary), or may flow out of the space.
  • the flow of the liquid P is generated in the space by continuously flowing the liquid P into the space.
  • the well 2 is provided on the upper surface side (upper surface) of the substrate 4.
  • the well 2 constitutes a space into which the liquid P containing the lipid flows.
  • an opening U is formed through which the end 1 a of the micro chamber 1 is exposed.
  • An opening position of the well 2 (an opening that is flush with the upper surface of the substrate 4) and at least a part of the lower surface 2b are opened so that the lipid membrane M formed in the opening U can be optically observed. Or it is covered with a transparent member (not shown).
  • a portion of the base body 10A that constitutes at least the opening U is formed of a single member.
  • the lipid membrane M formed in the opening U may be observed from an oblique angle with respect to the side surface 2a.
  • the lipid membrane M formed in the opening U can be easily observed from the front side of the membrane surface. Or you may observe from the upper direction of the base material 4, ie, the direction parallel to the side surface 2a.
  • the pressure difference between the well 2 and the microchamber 1 or the vicinity of the opening U (position close to the opening U, area close to the opening U, close to the opening U)
  • a technique is adopted in which the lipid membrane M is deformed into a concave shape or a convex shape using the fluid pressure of the fluid P in the space, and the formed lipid membrane M is observed.
  • the structure (structure) which opens the micro chamber 1 in the bottom face 2b of the well 2 may be sufficient. In this case, when observing from the upper surface of the substrate, the surface is observed from the front of the opening U, and the surface state of the lipid membrane M formed in the opening U can be easily observed.
  • the opening U is composed of a single base material, there is no seam or bonding surface, and the step at the site where the lipid membrane is stretched can be substantially eliminated. For this reason, the formed lipid membrane can be stably maintained.
  • the lipid contained in the liquid P is not particularly limited.
  • phospholipids that are lipids contained in cell membranes are preferred lipids.
  • the phospholipid include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and the like.
  • a lipid membrane M having a lipid bilayer structure can be formed in the opening U of the microchamber 1. At this time, when cholesterol is added as a lipid component, the strength of the lipid membrane may be increased.
  • the solvent for dissolving the lipid contained in the liquid P is not particularly limited as long as it is a solvent that can dissolve the lipid.
  • a solvent that is not miscible with water is used.
  • examples of such a solvent include organic solvents such as hexadecane and fats and oils such as squalene (squalene).
  • the single member constitutes not only the microchamber 1 but also the entire base material 4.
  • the material for the single member include silicon, glass, quartz, and sapphire. These materials are preferable because they are excellent in workability of the micro chamber 1. Among these, it is preferable that the material is amorphous so that it is not easily affected by processing anisotropy due to crystal orientation.
  • the lipid membrane M in the opening U is observed with an optical device such as a microscope, it is more preferable to use glass, quartz, or sapphire as the single member. Since these members are transparent to visible light (wavelength: 0.36 ⁇ m to 0.83 ⁇ m), the lipid membrane M can be easily observed.
  • the opening U of the micro chamber 1 is formed in nanoscale units (nm) (for example, a size of 1 nm or more and less than 1 ⁇ m).
  • nm nanoscale units
  • the nanoscale opening U formed in the member that absorbs the solvent is easily closed when it absorbs the solvent and expands. Since the shape and size of the opening U do not change, an experiment with high reproducibility is possible without changing the size of the formed lipid membrane for each experiment.
  • the material which comprises the opening part U is a material which absorbs a solvent and expand
  • the opening U of the micro chamber 1 according to the embodiment of the present invention is composed of a single member without a bonding surface. That is, the periphery of the opening U is composed of members having the same linear expansion coefficient. For this reason, a sudden temperature change occurs in the vicinity of the opening U, which may occur when the operating temperature of the base body 10A is changed or a liquid having a temperature different from the temperature of the base body 10A flows into the space 2 (well 2). Even in this case, there is no possibility that the opening U is broken from the bonding surface. Furthermore, since there is no bonding surface in the opening part U, the chemical resistance with respect to the chemical
  • the bonding surface may reflect light and interfere with observation. Since the opening U of the micro chamber 1 according to the embodiment of the present invention is composed of a single member that does not have a bonding surface, there is no possibility that reflected light that interferes with observation occurs.
  • the material of the single member is transparent to light having at least a part of light having a wavelength of 0.1 ⁇ m to 10 ⁇ m (can transmit light having at least a part of wavelength). Is preferred). Specifically, it is preferably transparent to at least a part of general light (wavelength of 0.1 ⁇ m to 10 ⁇ m) used as a processing laser. By being transparent to such laser light, the modified portion can be formed on the member by laser irradiation as will be described later. Further, the material of the single member is transparent to light in the visible light region (wavelength of about 0.36 ⁇ m to about 0.83 ⁇ m) (can transmit light in the visible light region), More preferred.
  • the formed lipid membrane M can be easily observed through an optical technique such as an optical microscope through the single member.
  • transparent refers to all states in which light is incident on the member and transmitted light is obtained from the member.
  • the single member which comprises the base material 4 is a transparent glass substrate.
  • the micro chamber 1 has an opening U that opens to the side surface 1 a that is the inner wall surface of the well 2.
  • Examples of the method for forming the lipid membrane M in the opening U in the substrate according to the embodiment of the present invention include the method described below. Please refer to FIG. 3A to FIG. 3F.
  • a buffer solution 5 such as physiological saline or a pH buffer solution is placed in the well 2 (space 2), and the buffer solution 5 is caused to flow into the micro chamber 1 by capillary action (FIG. 3A).
  • the buffer solution 5 is removed from the well 2 using a pipette or the like (not shown).
  • the buffer solution 5 in the microchamber 1 is kept in the microchamber 1 and the water surface of the buffer solution 5 is exposed to the inside of the well 2 using the surface tension in the opening U (FIG. 3B).
  • the liquid P containing the lipid flows into the well 2, and the liquid P and the water surface of the buffer liquid 5 are brought into contact with each other at the opening U.
  • lipid molecules contained in the liquid P adhere to the water surface of the buffer solution 5 with the polar part in the molecule facing the buffer solution 5 side.
  • the water surface of the buffer solution 5 is covered with lipid molecules (FIG. 3C).
  • a lipid membrane M composed of the attached lipid molecules is formed on the water surface of the buffer liquid 5 remaining in the opening U (FIG. 3D).
  • the buffer solution 5 in the well 2 and the buffer solution 5 in the micro chamber 1 are separated from each other by the lipid membrane M in the opening U (FIG. 3E).
  • the lipid membrane M in this state can take at least one of a lipid bilayer structure composed of the lipid molecules or a monolayer lipid membrane structure composed of the lipid molecules.
  • a pressure operation to apply pressure to the well 2 and the micro chamber 1 or to reduce the pressure (FIG. 3F).
  • the well 2 is sealed and a gas or buffer solution 5 is injected into the well 2 while being pressurized, or the well 2 is sealed and the buffer solution 5 is sucked from the well 2. Can be done by.
  • vesicle V liposome
  • the micro chamber 1 is formed in a single glass substrate 4 and is a micro space without a joint or a bonding surface. Naturally, no seam or bonding surface exists for the opening U at the end of the micro chamber. For this reason, the adhesive force between the lipid membrane M and the opening U can be sufficiently enhanced. Further, even when the glass substrate 4 provided with the micro chamber 1 is deformed or when the opening U is damaged by the chemical solution, the bonding surface does not exist in the opening U. No peeling or breakage. Therefore, even if heat sterilization or chemical sterilization is repeatedly performed on the glass substrate 4 provided with the micro chamber 1, the glass substrate 4 is not damaged. This is a particularly excellent feature for substrates that form membrane lipids that are routinely subjected to heat disinfection and chemical disinfection.
  • the “opening portion U” refers to a region on the side surface 2 a of the well 2 where the end portion 1 a of the micro chamber 1 is opened and the lipid membrane M is formed.
  • the shape of the hole in the side surface 2a of the well 2 of the end 1a of the micro chamber 1 constituting the opening U may be any shape, for example, a circle, a substantially circle, an ellipse, a substantially ellipse, a rectangle, or a triangle , And the like.
  • the diameter or major axis is preferably in the range of 0.02 ⁇ m to 5 ⁇ m. Among these, the range of 0.02 to 3 ⁇ m in diameter is more preferable, and the range of 0.02 to 1 ⁇ m is more preferable.
  • the lower limit that is 0.02 ⁇ m
  • the area of the opening U is too small, and the possibility that the lipid membrane M is not appropriately formed increases.
  • the upper limit of the above range ie, 5 ⁇ m
  • the sealing property between the opening U and the lipid membrane M is lowered, and the possibility that a long-term stable lipid membrane cannot be formed increases.
  • the shape of the holes constituting the opening U that is, the shape of the opening diameter where the lipid membrane M is formed is preferably a shape close to a perfect circle (substantially circular).
  • the lipid membrane M When the lipid membrane M is observed from above the glass substrate 4, that is, from a direction parallel to the side surface 2 a of the well 2, the lipid membrane M becomes difficult to observe due to the difference in refractive index between the glass substrate 4 and the buffer solution 5. Can be prevented.
  • the microchamber 1 preferably has a smaller hole diameter (inner diameter) as it goes from the opening U through which the buffer solution 5 flows into the inner part (inner part). That is, it is preferable that the opening diameter is the maximum outer diameter H1 of the micro chamber 1, and the opening diameter is larger than the innermost inner diameter H3. With this structure, the capillary force increases as the depth of the micro chamber 1 increases, so that the buffer solution 5 can easily enter the depth.
  • the microchamber 1 has a convex portion E protruding at the center of the hole diameter in the opening U, and the minimum inner diameter H2 of the opening U where the convex portion E is formed is very small. It is preferable that the inner diameter of the chamber 1 is smaller than the maximum inner diameter H1. With the opening U having such a structure, the lipid membrane M can be formed more stably.
  • the micro chamber 1 has a convex portion E that protrudes from the center of the hole diameter in the opening U, and the minimum inner diameter H2 of the opening U in which the convex portion E is formed has a micro chamber. It is preferable that the innermost inner diameter H1 is smaller than the innermost inner diameter H1, and the innermost inner diameter H3 of the micro chamber 1 is less than 50% of the maximum inner diameter H1.
  • the microchamber 1 having such a structure facilitates the introduction of the buffer solution 5 into the microchamber 1 by the action of capillary force, and allows the lipid membrane M to be more formed in the opening U where the convex portion E is formed. It can be formed easily.
  • the inner diameter H3 of the microchamber 1 is the innermost line on the virtual line corresponding to the total length L in the depth direction of the microchamber 1. The distance is measured from the back position, and the inner diameter at the position of (1/20) ⁇ L is considered.
  • the height E1 of the convex portion E is defined as a height difference between a portion having the maximum inner diameter H1 and a portion having the minimum inner diameter H2 of the opening U, and the height E1 is preferably 20 nm to 200 nm. If it is less than the lower limit (that is, 20 nm) of this range, the effect of the convex portion E can hardly be expected, and if it exceeds the upper limit (that is, 200 nm), the buffer solution 5 can be introduced into the micro chamber 1. It can be difficult.
  • the width E2 of the convex portion E is defined as the distance from the position at which the micro chamber 1 opens to the side surface 2a to the position at which the inner diameter starts to expand, and the width E2 in the depth direction of the micro chamber 1 It is preferably less than 20% of the total length L. With such a structure, the buffer solution 5 can be more easily introduced into the micro chamber 1.
  • the lipid membrane M can be more stably formed by performing a surface treatment so that the vicinity of the opening U of the micro chamber 1 becomes hydrophobic.
  • the vicinity of the opening U means a region in contact with the lipid membrane M and its periphery.
  • the surface treatment include a treatment for bonding an alkyl group to the surface.
  • the surface is glass, it can be alkylated using a silane coupling agent.
  • the length of one side thereof is preferably 0.1 ⁇ m to 30 ⁇ m.
  • the lipid membrane M can be easily formed in the opening U, and the vesicle V can be formed.
  • the volume of the micro chamber 1 is preferably about the same as that of microorganisms such as cells.
  • microorganisms When microorganisms are caught inside the micro chamber 1 and a lipid membrane M is formed in the opening U in that state, the microorganisms are confined inside the micro chamber 1. Then, by irradiating the captured microorganism with a laser to destroy the cell membrane of the microorganism, it is possible to observe how the destroyed cell membrane is regenerated. In general, it is said that microorganisms can be regenerated if the components constituting the microorganisms are present in the vicinity, but there is no example in which the state is directly observed. By using the apparatus of the embodiment of the present invention, there is a possibility that the state of regeneration of microorganisms can be confirmed.
  • the hole diameter of the hole is processed with high accuracy in nano order (for example, a size of 1 nm or more and less than 1 ⁇ m), it is possible to form a lipid membrane having a smaller size than the conventional one.
  • the function of a membrane protein that has been conventionally observed as an aggregate of a plurality of molecules can be observed as a single molecule by using the substrate according to the embodiment of the present invention. That is, the substrate according to the embodiment of the present invention can be used as an apparatus for confirming various molecular properties of membrane proteins that have not been elucidated so far.
  • the diameter of the opening of the hole is reduced to the order of nano order, a stable sealing property between the lipid membrane and the opening is realized, so that experiments such as functional analysis can be performed stably.
  • the substrate of the embodiment of the present invention it is possible to analyze the characteristics of the lipid membrane M formed in the opening U of the micro chamber 1 with high accuracy.
  • an ionic substance is confined inside the micro chamber 1 in which the lipid membrane M is formed in the opening U, and this ionic substance is transported to the outside of the micro chamber 1 through the ion channel of the lipid membrane M. Status and the transportation speed can be checked. In order to examine the transport rate, it is necessary to observe the state of permeation through the lipid membrane M over a long period of time, so that the microchamber 1 having a large capacity is preferable.
  • the micro chamber 1 is formed so as to be substantially perpendicular to the side surface 2 a of the well 2. However, it is not necessarily required to be substantially vertical, and can be freely arranged on the single glass substrate 4 in accordance with the design of the base body 10A. Furthermore, the opening diameter of the opening U located at the end 1a of the micro chamber 1 can be processed by slightly widening the opening diameter slightly inward. That is, the opening U can be processed into a funnel-like shape. When processed in this way, the lipid membrane M can be formed in a state where a part of the lipid membrane M enters the end of the microchamber 1.
  • the lipid membrane M Since part of the lipid membrane M is inside the end portion, even when there is a flow in the well 2, the lipid membrane M may be more stably maintained for a long time.
  • the case where the well 2 is used is described here, the same description is applied to the case where the channel 3 is used in the base body 10B described later.
  • a plurality of micro chambers 1 may be formed on the base body 10A. Since each microchamber 1 has an opening U, a plurality of lipid membranes M can be formed.
  • the substrate 4 is made of silicon, glass, quartz, sapphire, or the like, the processing accuracy is high, so that the plurality of micro chambers 1 can be densely arranged.
  • the openings U of the plurality of micro chambers 1 can be arranged at a pitch of 0.1 ⁇ m to 6 ⁇ m.
  • the lower surface 2b of the well 2 is formed of a base material 4 formed of a glass substrate.
  • the opening position of the well 2 at the position facing the lower surface 2b (the opening portion which is the same plane as the upper surface of the substrate 4) is opened and has no lid.
  • the lipid membrane M formed in the opening U can be observed by an optical observation device such as a microscope.
  • the upper surface is not necessarily open, and may be covered with a lid made of a member such as plastic, resin, or glass (not shown). This can be performed while observing the formation of the lipid membrane M in the opening U.
  • Substrate 10B As a second embodiment of the base for forming the lipid membrane of the present invention, there is a base 10B shown in FIG. In the base body 10 ⁇ / b> B, the flow path 3 is provided on the upper surface side of the base material 4. The flow path 3 constitutes a space into which the liquid P containing the lipid flows. Compared with the well 2 in the substrate 10A, a large amount of liquid P can be introduced and circulated by using the structure in which the channel 3 is provided in the substrate 10B. Moreover, there exists an advantage that the exchange of the solution in the flow path 3 (space 2) is easy.
  • the operation of sequentially bringing a plurality of solutions such as the buffer solution 5 and the liquid P containing the lipid into contact with the opening U can be performed more easily.
  • Other configurations of the base body 10B are the same as those of the base body 10A.
  • the micro chambers 1 can be arranged at arbitrary positions, and a plurality of micro chambers 1 can be arbitrarily arranged.
  • FIG. 10 is a schematic top view (perspective view seen from above) of a base body 20A, which is an example (third embodiment) of a base body for forming a lipid membrane according to the present invention.
  • the arrangement configuration of the well 12 and the micro chamber 11 in the top view is applicable to the above-described base body 10A.
  • a plurality of micro chambers 11 may be arranged in one well 12.
  • FIG. 11 is a schematic top view (perspective view seen from above) of a base body 30A which is an example of a base body (fourth embodiment) for forming a lipid membrane according to the present invention.
  • the arrangement configuration of the flow path 22 and the micro chamber 21 in the top view is applicable to the above-described base body 10B.
  • the glass substrate 24 In the glass substrate 24, four sets of flow paths 22 each having a plurality of minute chambers 21 arranged on the inner wall surface are arranged.
  • the micro chamber 21 is opened on the inner wall surface of the flow path 22.
  • the position of the opening U is indicated by an “X” mark.
  • the liquid P flows from the upstream side F1 of the flow path 22 and flows to the downstream side F2 of the flow path 22.
  • FIG. 12 is a schematic top view (perspective view seen from above) of a base body 30B which is an example of the base body (fifth embodiment) for forming a lipid membrane according to the present invention.
  • the arrangement configuration of the flow path 22, the micro chamber 21, and the second flow path 29 in the top view is applicable to the above-described base body 10B.
  • the glass substrate 24 is provided with a flow path 22 in which a plurality of micro chambers 21 are arranged on the inner wall surface. As described above, the micro chamber 21 is opened on the inner wall surface of the flow path 22. The position of the opening U is indicated by an “X” mark.
  • the liquid P flows from the upstream side F5 of the flow path 22 and flows to the downstream side F6 of the flow path 22.
  • the second flow path 29 arranged on the glass substrate 24 communicates with the flow path 22.
  • the chemical liquid or gas can also be diffused into the flow path 22. That is, the chemical solution or gas can be brought into contact with the lipid membrane M formed in the opening U.
  • the place where the second flow path 29 communicates with the flow path 22 and the shape of the second flow path 29 are not particularly limited. Therefore, it is also possible to arrange the second flow path 29 in the vicinity of the micro chamber 21 (position close to the micro chamber 21), and the upstream side (F5 side) of the second flow path 29 and the flow path 22 has their respective functions. Can be substituted. In addition, a plurality of second flow paths 29 or a plurality of second flow paths 29 branched into the plurality of flow paths 22 can be disposed.
  • Step A1 for forming the modified portion 51 in the region
  • Step A2 for forming the flow path 57 or the through-hole forming the space in the single member 59
  • Step A3 for removing the modified portion 51 from the one member 59 by etching.
  • the laser L is preferably a laser beam having a pulse width of a pulse time width of the order of picoseconds or less, for example, 1 femtosecond or more and less than 10 picoseconds.
  • a titanium sapphire laser, a fiber laser having the pulse width, or the like can be used.
  • the laser L laser light L
  • the member 59 which is a workpiece.
  • the modified portion 51 can be formed on the member 59.
  • the “modified portion” means a portion that has low etching resistance and is selectively or preferentially removed by etching.
  • Examples of the material of the member 59 include silicon, glass, quartz, and sapphire. These materials are preferable because they are excellent in workability of the micro chamber 55. Among these, it is preferable that the material is amorphous so that it is not easily affected by processing anisotropy due to crystal orientation. Furthermore, when observing with an optical apparatus such as a microscope, it is more preferable to use glass, quartz, sapphire, or the like that is transparent to visible light (wavelength 0.36 ⁇ m to 0.83 ⁇ m) as the material. preferable.
  • the material of the member 59 is preferably transparent (transmits light) to light having at least a part of the light having a wavelength of 0.1 ⁇ m to 10 ⁇ m. Specifically, it is preferably transparent to light in at least a part of the wavelength region of a general wavelength region (0.1 ⁇ m to 10 ⁇ m) used as a processing laser beam. Since the material of the member 59 is transparent to such laser light, the modified portion can be formed by irradiating the member with laser as described later. More preferably, it is transparent to light in the visible light region (wavelength of about 0.36 ⁇ m to about 0.83 ⁇ m). By being transparent to light in the visible light region, the formed lipid membrane M can be easily observed with the naked eye through the single member.
  • the single member 59 is a transparent glass substrate (hereinafter referred to as a glass substrate 59).
  • a glass substrate 59 the case where the member 59 is a glass substrate will be described, but the same can be performed even when the member 59 is another member, for example, silicon, quartz, or sapphire. Silicon, quartz, and glass are more suitable for the workability in step A2 to be described later.
  • the glass substrate 59 for example, a glass substrate composed of quartz, a glass mainly composed of silicate, a glass substrate composed of borosilicate glass, or the like can be used.
  • a glass substrate made of synthetic quartz is preferable because of good workability.
  • the thickness of the glass substrate 59 is not particularly limited.
  • the modified portion 51 in which the glass is modified is formed by irradiating the laser beam L so as to be focused and focused on the inside of the glass substrate 59 and scanning the focal point in the arrow direction.
  • the modified portion 51 having a desired shape can be formed.
  • the irradiation intensity is set to a value close to the processing upper limit threshold value (a value close to the processing appropriate value) of the glass substrate 59 or less than the processing upper limit threshold value (less than the processing appropriate value), and the polarization of the laser light L It is preferable that the direction (electric field direction) be perpendicular to the scanning direction.
  • this laser irradiation method is referred to as a laser irradiation method S.
  • the laser irradiation method S will be described with reference to FIG.
  • the propagation direction of the laser light L is an arrow Z
  • the polarization direction (electric field direction) of the laser light L is an arrow Y.
  • the irradiation region of the laser light L is set within a plane 59a configured by the propagation direction of the laser light and a direction perpendicular to the polarization direction of the laser light.
  • the laser irradiation intensity is set to a value close to the processing upper limit threshold of the glass substrate 59 or less than the processing upper limit threshold.
  • the modified portion 51 having a nano-order (for example, 1 nm or more and less than 1 ⁇ m) diameter can be formed in the glass substrate 59.
  • the modified portion 51 having a substantially elliptical cross section with a minor axis of about 20 nm and a major axis of about 0.2 ⁇ m to 5 ⁇ m is obtained.
  • the direction along the laser propagation direction is the major axis
  • the direction along the laser electric field direction is the minor axis.
  • the cross section may be a shape close to a rectangle.
  • the obtained modified portion 51 may be formed with a periodic structure.
  • the processing upper limit threshold processing appropriate value
  • the formed periodic structure becomes a layer with weak etching resistance.
  • the oxygen-deficient layer and the oxygen-enriched layer are periodically arranged (FIG. 15B), and the etching resistance of the oxygen-deficient portion is weak. Can be formed. Such periodic recesses and protrusions are not necessary in the formation of the microchamber 55 described later.
  • the laser irradiation intensity is lower than the processing upper limit threshold of the glass substrate 59, and the lower limit value of the laser irradiation intensity that can modify the glass substrate 59 to reduce the etching resistance ( If it is equal to or higher than the processing lower limit threshold value, the periodic structure is not formed, and one oxygen-deficient portion (layer having low etching resistance) is formed by laser irradiation (FIG. 15A). When this etching is performed, one minute chamber 55 can be formed.
  • the shape of the micro chamber 55 can be made elliptical or substantially elliptical.
  • the minor axis can be controlled to a nano-order size by etching.
  • the shape of the micro chamber 55 is an ellipse or a substantially elliptical shape
  • the lipid membrane may be more easily formed by setting the minor axis to the nano-order size.
  • the capillary force increases as the chamber becomes finer. There is a case where a harmful effect that does not come out to the outside (the space) may occur.
  • the capillary force is suppressed even at a short diameter sufficient to form a lipid membrane, and the adverse effect that the liquid does not come out of the space or the like is suppressed. be able to.
  • modified part 51 Even when only one layer with low etching resistance (oxygen-deficient part in quartz or glass) is formed by laser irradiation (referred to as modified part 51 in this specification), the oxygen-deficient part is extremely etched. It becomes a layer with high selectivity (selectivity ratio). This has been found by the inventors' diligent study.
  • the processing upper limit threshold is defined as a lower limit value of laser pulse power at which the periodic structure can be formed (upper limit value in a range of laser pulse power at which the periodic structure is not formed).
  • the “lower limit value of laser irradiation intensity (processing lower limit threshold value) (threshold value) that can lower the etching resistance by modifying the glass substrate 59” can form the micro chamber 55 on the glass substrate 59 by the etching process. It is a limit value. If it is lower than this lower limit value, a layer with low etching resistance cannot be formed by laser irradiation, and the micro chamber 55 cannot be formed.
  • the “processing upper limit threshold (processing appropriate value)” refers to the mutual relationship between the base material and the laser light at the focal point (condensing area) of the laser light irradiated into the base material. Interference between the electron plasma wave generated by the action and the incident laser beam occurs, and means the lower limit value of the laser irradiation intensity at which the striped modified portion can be formed in a self-forming manner on the base material due to the interference. .
  • the “processing lower limit threshold (threshold value)” means a modified portion obtained by modifying the base material at the focal point (condensing area) of the laser light irradiated into the base material.
  • the lower limit value of the laser irradiation intensity that can be formed and can reduce the etching resistance of the modified portion to such an extent that it can be selectively or preferentially etched by the subsequent etching process.
  • a region irradiated with laser with a laser irradiation intensity lower than the lower limit value is difficult to be selectively or preferentially etched in the subsequent etching process. For this reason, in order to form a modified portion that becomes a fine hole after etching, it is preferable to set the laser irradiation intensity to be equal to or higher than the processing lower limit threshold.
  • the processing upper limit threshold and the processing lower limit threshold are generally determined by the wavelength of the laser beam, the material (material) of the substrate that is the target of laser irradiation, and the laser irradiation conditions.
  • the processing upper limit threshold and the processing lower limit threshold may be slightly different.
  • the processing upper limit threshold and the processing lower limit threshold may differ between when the scanning direction is perpendicular to the polarization direction and when the scanning direction is parallel to the polarization direction. Therefore, the processing upper limit threshold and the processing lower limit threshold when the relative relationship between the polarization direction of the laser light and the scanning direction is changed in the wavelength of the laser light to be used and the base material to be used are examined in advance. It is preferable.
  • the method of scanning the focal point of the laser beam L is not particularly limited, but the modified portion 51 that can be formed by one continuous scanning is a one-dimensional direction perpendicular to the polarization direction (arrow Y direction) and the propagation of the laser beam L. It is limited within the two-dimensional direction (plane 59a) in the direction (arrow Z direction). Within this two-dimensional direction, the modified portion can be formed in an arbitrary shape.
  • the laser transmittance of the modified part is different from the laser transmittance of the unmodified part, so it is normal to control the focal position of the laser beam that has passed through the modified part. Have difficulty. Therefore, it is desirable to form the modified portion first in the region located behind as viewed from the surface on the laser irradiation side.
  • the modified portion 51 may be formed by condensing the laser beam L using a lens 52 and irradiating it as described above.
  • a lens for example, a refractive objective lens or a refractive lens can be used, but it is also possible to irradiate by, for example, a Fresnel, reflective, oil immersion or water immersion method.
  • a cylindrical lens it is possible to irradiate a laser on a wide area of the glass substrate 59 at a time.
  • the laser beam L can be irradiated at once in a wide range in the vertical direction of the glass substrate 59.
  • the polarization of the laser light L needs to be horizontal with respect to the direction in which the lens has a curvature.
  • the laser irradiation condition S include the following various conditions.
  • a titanium sapphire laser laser using a crystal in which sapphire is doped with titanium as a laser medium
  • a pulse laser having a pulse time width of 1 fs or more and less than 10 picoseconds
  • the laser light to be irradiated for example, a wavelength of 800 nm and a repetition frequency of 200 kHz are used, and the laser light L is condensed and irradiated at a laser scanning speed of 1 mm / second.
  • These values of wavelength, repetition frequency, and scanning speed are examples, and the present invention is not limited to this, and can be arbitrarily changed.
  • the “pulse time width of the order of picoseconds or less” is preferably a pulse time width of 1 femtosecond or more and less than 1 nanosecond, and preferably 1 femtosecond or more and less than 10 picoseconds. More preferably, the pulse time width is 1 femtosecond or more and less than 3 picoseconds, more preferably 1 femtosecond or more and less than 2 picoseconds.
  • the pulse time width is less than the picosecond order, particularly less than 10 picoseconds, the electron temperature and ion temperature of the base material in the light condensing part are heated in a non-equilibrium state, and so-called non-thermal process proceeds. To do.
  • the thermal diffusion length is suppressed to the limit. Furthermore, since non-linear processing starting from multiphoton absorption becomes dominant, the shape obtained after processing can be changed from nanoscale to micro-order micropores.
  • a pulse laser having a relatively large pulse time width for example, laser light having a pulse time width of 10 picoseconds or more, the electron temperature and ion temperature of the base material in the condensing part are in an equilibrium state. Thermal processing becomes dominant. In thermal processing, the thermal diffusion length increases, and it may be difficult to perform nano to micro order scale processing. In this way, since the reaction time mechanism may be completely different at a pulse time width of about 1 to 10 picoseconds, it is preferable to use a pulse laser having a pulse time width of less than 10 picoseconds.
  • the pulse intensity is preferably set to a value close to the processing upper limit threshold or a value less than the processing upper limit threshold and close to the processing upper limit threshold.
  • a pulse time width of 300 fs, a repetition frequency of 200 kHz, a scanning speed of about 1 mm / s, and a pulse energy of about 80 nJ / pulse or less are set.
  • the irradiation intensity is preferably about 550 kW / cm 2 and the laser fluence per pulse is preferably about 2.7 J / cm 2 .
  • the irradiation intensity is equal to or higher than the processing upper limit threshold value or larger than the laser fluence (power) per pulse corresponding to the processing upper limit threshold value, a periodic structure is formed and they are connected by etching. It may be difficult to form the minute chamber 55, the diameter may be in the order of microns, or the periodic structure may be formed.
  • N A. Machining is possible even when set to ⁇ 0.7, but the spot size is smaller and the laser fluence per pulse is larger, so laser irradiation with a smaller pulse intensity (pulse energy) can be performed. Desired.
  • Laser fluence refers to the amount of energy per unit area and is expressed in J / cm 2 or W / cm 2 .
  • a resist 52 is patterned on the upper surface of the glass substrate 59 by, for example, photolithography. Subsequently, the region where the resist 52 is not formed on the upper surface of the glass substrate 59 is eroded and removed until it reaches a predetermined depth by a method such as dry etching, wet etching, or sand blasting (FIG. 13B). When the resist 52 that has become unnecessary is peeled off, a glass substrate 59 in which the flow path 57 is formed is obtained.
  • step A2 it is preferable to expose the cross section of the modified portion 51 formed in step A1 on the side surface of the flow path 57 to be formed. It becomes easier to form the micro chamber 55 by the etching process in the subsequent step A3.
  • a through hole may be formed as the flow path 57.
  • the flow path 57 may be formed by excavating a region to be a flow path in the glass substrate 59 from the surface of the glass substrate 59 with a micro drill (laser drill) or the like to form a through hole. This drilling method may be used in combination with various etching methods.
  • the modified portion 51 formed in step A1 is removed from the single glass substrate 59 by etching (FIG. 13C).
  • etching As an etching method, wet etching is preferable.
  • the modified portion 51 having a cross section exposed on the side surface of the flow path 57 (or the through hole) has low etching resistance, and can be selectively or preferentially etched.
  • the micro chamber 55 having a nano-order aperture can be formed at a predetermined position in the glass substrate 59 so as to open to the side surface of the flow path 57.
  • the opening of the formed microchamber 1 is appropriately masked, and the microchamber 1 is further etched to provide the opening U with the convex portion E as shown in FIGS.
  • the micro chamber 1 can be formed. Specifically, for example, in the opening U of the micro chamber 1, an etching mask is formed in a region where the convex portion E is to be formed using a CVD method or the like. Next, an etchant capable of selectively etching the glass substrate 59 is introduced into the inside of the micro chamber 1, and the inner wall of the inside of the micro chamber 1 (the inside of the inside) is etched, so that the inside of the micro chamber 1 can be etched. The inner diameter can be increased.
  • the micro chamber 1 provided with the opening part U which has an opening diameter smaller than the internal diameter in an inner depth by reworking the micro chamber 1 can be formed.
  • the method of bonding the member 56 and the upper surface of the glass substrate 59 may be performed by a known method according to the material of the member 56.
  • the material of the member 56 is not particularly limited, and a resin substrate such as PDMS or PMMA, or a glass substrate can be used.
  • the material of the member 56 may be transparent or opaque with respect to the light beam (for example, visible light) of the observation apparatus. That is, the material of the member 56 may be a material that transmits the light beam of the observation apparatus, or may be a material that does not transmit the light beam of the observation apparatus.
  • the purpose is only to form a lipid membrane, it is not necessarily transparent to the light beam of the observation apparatus.
  • a member that is transparent to the light beam of the observation device is preferable because observation by an optical method from above is possible.
  • wet etching or dry etching can be applied.
  • wet etching for example, 1% or less of hydrofluoric acid is most preferably used, but other acid or basic etchants may be used.
  • isotropic etching methods include various dry etching methods such as barrel type plasma etching, parallel plate type plasma etching, and downflow type chemical dry etching.
  • anisotropic dry etching method for example, as a method using reactive ion etching (hereinafter referred to as RIE), for example, parallel plate type RIE, magnetron type RIE, ICP type RIE, NLD type RIE, or the like can be applied. In addition, for example, etching using a neutral particle beam can be applied.
  • RIE reactive ion etching
  • a process close to isotropic etching can be performed by shortening the mean free path of ions by a method such as increasing the process pressure.
  • the gases used are mainly gases that can chemically etch materials such as fluorocarbon, SF, CHF 3 , fluorine gas, and chlorine gas, and other gases such as oxygen, argon, and helium are mixed as appropriate. And can be used. Also, processing by other dry etching methods is possible.
  • step A2 more preferable etching is anisotropic etching, and in step A3, more preferable etching is isotropic etching.
  • the material of the member 69 is preferably transparent to light having at least a part of light having a wavelength of 0.1 ⁇ m to 10 ⁇ m. Specifically, it is preferably transparent to at least a part of light in a general wavelength region (0.1 ⁇ m to 10 ⁇ m) used as a processing laser beam. By being transparent to such laser light, the modified portion can be formed by irradiating the member with laser as described later. More preferably, it is transparent to light in the visible light region (about 0.36 ⁇ m to about 0.83 ⁇ m). By being transparent to light in the visible light region, the formed lipid membrane can be easily observed with the naked eye through the single member.
  • the single member 69 is a transparent glass substrate (hereinafter referred to as a glass substrate 69).
  • a micro chamber 65 having a nano-order aperture can be formed at a predetermined position in the glass substrate 69 so as to open to the side surface of the flow path 67.
  • the method of bonding the member 66 and the upper surface of the glass substrate 69 may be performed by a known method according to the material of the member 66.
  • the material of the member 66 is not particularly limited, and a resin substrate such as PDMS or PMMA, or a glass substrate can be used.
  • the material of the member 66 may be transparent or opaque with respect to the light beam (for example, visible light) of the observation apparatus. That is, the material of the member 56 may be a material that transmits the light beam of the observation apparatus, or may be a material that does not transmit the light beam of the observation apparatus.
  • the purpose is only to form a lipid membrane, it is not necessarily transparent to the light beam of the observation apparatus.
  • a member that is transparent to the light beam of the observation device is preferable because observation by an optical method from above is possible.
  • a substrate for forming a lipid membrane of the present invention and a method for producing the substrate include the use of a microfluidic device or the like for performing various observations, analyzes, and measurements by forming a minute lipid membrane on a substrate. Can be widely used for manufacturing.

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Abstract

L'invention concerne un substrat destiné à la formation d'une membrane lipidique, caractérisée par le fait que ce substrat se compose d'une base possédant des parois intérieures, un espace contenu à l'intérieur de cette base et dans lequel s'écoule un liquide contenant un lipide, et des micro-chambres disposées à l'intérieur de cette base et présentant des ouvertures pratiquées sur la surface d'une paroi intérieure de la base formant cet espace, au moins la partie de la base formant ces ouvertures étant composée d'un seul élément.
PCT/JP2012/066574 2011-06-28 2012-06-28 Substrat destiné à la formation d'une membrane lipidique et méthode de production du substrat WO2013002339A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2021090900A1 (fr) * 2019-11-07 2021-05-14 株式会社エンプラス Stratifié, puce à microcanaux et son procédé de fabrication
WO2021090899A1 (fr) * 2019-11-07 2021-05-14 株式会社エンプラス Plaque de verre ayant un motif de trou traversant formé à l'intérieur de celle-ci, son procédé de fabrication et tube à micro-canal

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JP2005144622A (ja) * 2003-11-18 2005-06-09 Seiko Epson Corp 構造体の製造方法、液滴吐出ヘッド、液滴吐出装置
WO2005071405A1 (fr) * 2004-01-21 2005-08-04 Japan Science And Technology Agency Methode permettant de former une membrane lipidique double plane a des fins d'analyse proteique membranaire
JP2008194573A (ja) * 2007-02-09 2008-08-28 Matsushita Electric Ind Co Ltd 脂質二重膜形成方法
JP2009128206A (ja) * 2007-11-26 2009-06-11 Univ Of Tokyo マイクロ流体による平面脂質二重膜アレイ及びその平面脂質二重膜を用いた分析方法
WO2010023848A1 (fr) * 2008-08-26 2010-03-04 パナソニック株式会社 Procédé de production d'une membrane lipidique artificielle et appareil de production d'une membrane lipidique artificielle

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005144622A (ja) * 2003-11-18 2005-06-09 Seiko Epson Corp 構造体の製造方法、液滴吐出ヘッド、液滴吐出装置
WO2005071405A1 (fr) * 2004-01-21 2005-08-04 Japan Science And Technology Agency Methode permettant de former une membrane lipidique double plane a des fins d'analyse proteique membranaire
JP2008194573A (ja) * 2007-02-09 2008-08-28 Matsushita Electric Ind Co Ltd 脂質二重膜形成方法
JP2009128206A (ja) * 2007-11-26 2009-06-11 Univ Of Tokyo マイクロ流体による平面脂質二重膜アレイ及びその平面脂質二重膜を用いた分析方法
WO2010023848A1 (fr) * 2008-08-26 2010-03-04 パナソニック株式会社 Procédé de production d'une membrane lipidique artificielle et appareil de production d'une membrane lipidique artificielle

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021090900A1 (fr) * 2019-11-07 2021-05-14 株式会社エンプラス Stratifié, puce à microcanaux et son procédé de fabrication
WO2021090899A1 (fr) * 2019-11-07 2021-05-14 株式会社エンプラス Plaque de verre ayant un motif de trou traversant formé à l'intérieur de celle-ci, son procédé de fabrication et tube à micro-canal

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