WO2013002339A1 - Substrate for forming lipid membrane and method for producing substrate - Google Patents

Substrate for forming lipid membrane and method for producing substrate 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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WIPO (PCT)
Prior art keywords
substrate
opening
lipid membrane
laser
lipid
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PCT/JP2012/066574
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French (fr)
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/en
Publication of WO2013002339A1 publication Critical patent/WO2013002339A1/en

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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

Provided is a substrate for forming a lipid membrane, characterized in that the substrate for forming a lipid membrane comprises a base having inside wall surfaces, a space contained inside the base and into which flows a liquid comprising a lipid, and microchambers contained inside the base and having openings that are disposed on an inside wall surface of the base that forms the space, and in that at least the portion of the base that forms the openings is formed from a single member.

Description

脂質膜を形成するための基体、及び前記基体の製造方法SUBSTRATE FOR FORMING LIPID MEMBRANE AND METHOD FOR PRODUCING THE SUBSTRATE
　本発明は、微小チャンバーを備えた基体、及び前記基体の製造方法に関する。より詳しくは、本発明は、脂質膜を形成する微小チャンバーを備えた基体、及び前記基体の製造方法に関する。本願は、２０１１年６月２８日に、日本に出願された特願２０１１－１４２９４６号に基づき優先権を主張し、その内容をここに援用する。 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.
　体を構成する細胞の細胞膜に存在する膜タンパク質は、医薬品が作用するターゲットであるために、産官学において激しい研究競争が展開されている。膜タンパク質は、複雑かつ精緻な立体構造を有するため、変性および分解しやすく、微量の試料しか得られないのが現状である。このため、微量の試料しか入手できない場合であっても実験を行うことができるマイクロ流体チップのバイオテクノロジーへの応用が近年注目されている。 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, and academia. 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.
　マイクロ流体チップは、実験系のスケールがマイクロリットル（μｌ）という微量であるため、実験に要する溶液や試薬が微量で足りる。例えば、特許文献１には、たて×よこ×高さが、１９μｍ×１７μｍ×７μｍのマイクロチャンバーを用いた、脂質膜の形成および前記脂質膜への膜タンパク質の導入方法が開示されている。 The microfluidic chip requires only a small amount of solution and reagent required for the experiment because the scale of the experimental system is a microliter (μl). For example, 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 μm × 17 μm × 7 μm.
　ところが、特許文献１のマイクロ流体チップはＰＤＭＳ（Polydimethyl siloxane）を材料として使用しているため、緩衝液等の水系溶媒や脂質を溶解した有機溶媒を流し込むことによって、マイクロチャンバーを構成するＰＤＭＳが溶媒を吸収して膨張または収縮する結果、脂質膜を形成するマイクロチャンバーの開口部の孔径が変化してしまい、最悪の場合は、開口部が閉じてしまうという問題がある。また、前記膨張または収縮は、マイクロチャンバーだけでなく、マイクロ流路の閉塞を引き起こす問題がある。このため、特許文献１では、前記膨張または収縮する程度を予め想定して、乾燥時（製造時）のマイクロチャンバーの大きさを設定している。 However, since 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. As a result of absorbing and expanding or contracting, the pore diameter of the opening of the microchamber forming the lipid membrane changes, and in the worst case, the opening is closed. In addition, the expansion or contraction has a problem that not only the microchamber but also the microchannel is blocked. For this reason, in patent document 1, the magnitude | size of the microchamber at the time of drying (at the time of manufacture) is set supposing the extent to which the said expansion | swelling or shrinkage | contraction is carried out beforehand.
　しかしながら、使用する溶媒の種類や使用する温度によってＰＤＭＳが膨張または収縮する割合は変化するため、使用条件を変更する度に、その使用条件に合わせて、マイクロチャンバーの大きさやマイクロ流体チップの仕様を変更して製造することは非現実的である。 However, since the rate at which PDMS expands or contracts changes depending on the type of solvent used and the temperature used, the size of the microchamber and the specifications of the microfluidic chip must be adjusted according to the usage conditions each time the usage conditions are changed. It is unrealistic to make changes.
　また、従来のマイクロチャンバーは、ＰＤＭＳの基板表面に溝を形成し、その溝の上にＰＤＭＳ若しくはガラス等の別の部材を被せるように貼り合わせて形成されている。このため、溶媒や温度の影響によってマイクロチャンバーが膨張または収縮すると、マイクロチャンバーの開口部の口径が変化するだけでなく、前記貼り合わせ面が剥離して、マイクロチャンバーが破損したり、脂質膜を形成できなくなる恐れがある。 In addition, 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.
特開２００９－１２８２０６号公報JP 2009-128206 A
　本発明は上記事情に鑑みてなされたものであり、脂質膜を形成する開口部が単一部材によって形成された、脂質膜を形成するための基体、及び前記基体の製造方法の提供を課題とする。 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.
　本発明の第一態様の脂質膜を形成するための基体は、脂質膜を形成するための基体であって、内壁面を有する基材と、前記基材に内在され、脂質を含む液体を流入させる空間と、前記基材に内在され、前記空間を構成する前記基材の内壁面に開口部を有する微小チャンバーと、を含み、前記基材のうち、少なくとも前記開口部を構成する部位は、単一の部材で構成されている。
　本発明の第一態様の基体においては、前記単一の部材が、波長０．１μｍ～１０μｍを有する光のうち少なくとも一部の波長を有する光を透過することが好ましい。
　本発明の第一態様の基体においては、前記単一の部材がシリコン、ガラス、石英、又はサファイアであることが好ましい。
　本発明の第一態様の基体においては、前記微小チャンバーが、前記基体にレーザーを照射して改質された部分を、さらにエッチング処理で除去して形成されたことが好ましい。
　本発明の第二態様の基体の製造方法は、第一態様の基体として前述した、脂質膜を形成するための基体の製造方法であって、ピコ秒オーダー以下のパルス時間幅を有するレーザーを、前記単一の部材の前記微小チャンバーを形成する領域に照射することによって、前記領域に改質部を形成し、前記単一の部材に、前記空間を形成し、前記単一の部材から前記改質部をエッチングによって除去すること、を少なくとも有する。
　本発明の第三態様の基体の製造方法は、第一態様の基体として前述した、脂質膜を形成するための基体の製造方法であって、前記単一の部材に、前記空間を形成し、ピコ秒オーダー以下のパルス時間幅を有するレーザーを、前記単一の部材の前記微小チャンバーを形成する領域に照射することによって、前記領域に改質部を形成し、前記単一の部材から前記改質部をエッチングによって除去すること、を少なくとも有する。 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 μm to 10 μm.
In the substrate according to the first aspect of the present invention, the single member is preferably silicon, glass, quartz, or sapphire.
In the substrate of the first aspect of the present invention, it is preferable that 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. By irradiating the region of the single member where the microchamber is formed, a modified portion is formed in the region, the space is formed in the single member, and the modification is performed from the single member. Removing the mass portion by etching.
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.
　本発明の脂質膜を形成するための基体（第一態様）によれば、脂質を含む液体を流入させる空間に露呈する、微小チャンバーの開口部において、脂質膜を形成することができる。前記開口部は、単一の部材で構成されているため、継ぎ目や貼り合わせ面がなく、段差を実質的に無くすことができる。このため、形成した脂質膜を安定に維持できる。したがって、形成した脂質膜の観察が容易であり、脂質膜にイオンチャネルやイオンポンプ等の膜タンパク質を導入することにより、前記膜タンパク質の高精度の機能解析が可能である。 According to the substrate for forming a lipid membrane of the present invention (first aspect), 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.
　前記単一の部材が、波長０．１μｍ～１０μｍを有する光のうち、一般に使用される加工用レーザー光線の波長（０．１μｍ～１０μｍ）の少なくとも一部に対して透明である（一部を透過可能である）場合、前記レーザー光線を照射して、前記単一の部材を改質して加工することができる。また、前記単一の部材が、前記波長の光のうち少なくとも一部の可視光線（０．３６μｍ～０．８３μｍ）に対して透明である（一部の可視光線を透過可能である）場合には、前記単一の部材を通して、前記形成した脂質膜を、顕微鏡等の光学装置によって容易に観察することができる。さらには、前記単一の部材が、前記波長の光のうち少なくとも一部の紫外光あるいは可視光線（０．１μｍ～０．８３μｍ）に対して透明である（一部の紫外光あるいは可視光線を透過可能である）場合には、蛍光色素によって標識された膜タンパク質等の生体分子の前記脂質膜中における挙動を蛍光観察することができる。
　ここで、「前記単一の部材が、特定波長の光に対して透明である」場合、前記部材に入射された光の一部が前記部材に吸収されたとしても、残りの光（光の残部）が前記部材を透過することが可能である。 The single member is transparent (partially transmits) at least a part of the wavelength (0.1 μm to 10 μm) of a commonly used processing laser beam among lights having a wavelength of 0.1 μm to 10 μm. 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 μm to 0.83 μm) 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. Further, the single member is transparent to at least a part of ultraviolet light or visible light (0.1 μm to 0.83 μm) of the light having the wavelength (part of the ultraviolet light or visible light is transmitted). When it is possible to observe the behavior of a biomolecule such as a membrane protein labeled with a fluorescent dye in the lipid membrane, the fluorescence can be observed.
Here, when “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.
　前記単一の部材がシリコン、ガラス、石英、又はサファイアで構成される部材である場合には、前記微小チャンバーの加工精度をさらに高めることができ、前記開口部をナノオーダー（１ｎｍ以上１μｍ未満）の口径で形成することが可能である。また、これらの部材は溶媒を吸収しないため、脂質膜形成の際に、開口部の口径が変化したり、微小チャンバーの体積が変化することは、ほとんど起こらない。このため、前記形成した脂質膜（例えば、膜厚）のサイズが実験毎に変化することなく、再現性の高い実験が可能である。 When 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 μm). Can be formed. Moreover, since 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.
　前記微小チャンバーを形成する方法が、レーザー照射による基材の改質及びエッチング処理による改質部の除去を行う方法である場合、微小チャンバーが精度よく、且つ高密度で配置された基体を得ることができる。 When 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.
　本発明の脂質膜を形成するための基体の製造方法（第二態様および第三態様）によれば、単一の部材に、微小チャンバーを形成し、前記微小チャンバーの端部を、当該基体に内在する空間に開口させることができる。また、前記微小チャンバーの開口部を、ナノオーダーの口径サイズで形成することができる。 According to the method for producing a substrate for forming a lipid membrane of the present invention (second embodiment and third embodiment), 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.
本発明にかかる脂質膜を形成するための基体の一例を示す模式的な斜視図である。It is a typical perspective view which shows an example of the base | substrate for forming the lipid film concerning this invention. 図１のＡ－Ａ線に沿う断面図である。FIG. 2 is a cross-sectional view taken along line AA in FIG. 本発明にかかる基体における脂質膜の形成方法の一例を示す模式的な断面図である。It is typical sectional drawing which shows an example of the formation method of the lipid film in the base | substrate concerning this invention. 本発明にかかる基体における脂質膜の形成方法の一例を示す模式的な断面図である。It is typical sectional drawing which shows an example of the formation method of the lipid film in the base | substrate concerning this invention. 本発明にかかる基体における脂質膜の形成方法の一例を示す模式的な断面図である。It is typical sectional drawing which shows an example of the formation method of the lipid film in the base | substrate concerning this invention. 本発明にかかる基体における脂質膜の形成方法の一例を示す模式的な断面図である。It is typical sectional drawing which shows an example of the formation method of the lipid film in the base | substrate concerning this invention. 本発明にかかる基体における脂質膜の形成方法の一例を示す模式的な断面図である。It is typical sectional drawing which shows an example of the formation method of the lipid film in the base | substrate concerning this invention. 本発明にかかる基体における脂質膜の形成方法の一例を示す模式的な断面図である。It is typical sectional drawing which shows an example of the formation method of the lipid film in the base | substrate concerning this invention. 本発明にかかる基体において、ベシクルが形成された状態を示す模式的な断面図である。In the base | substrate concerning this invention, it is typical sectional drawing which shows the state in which the vesicle was formed. 本発明の基体における微小チャンバーの断面の一例を示す模式図である。It is a schematic diagram which shows an example of the cross section of the micro chamber in the base | substrate of this invention. 本発明の基体における微小チャンバーの断面の一例を示す模式図である。It is a schematic diagram which shows an example of the cross section of the micro chamber in the base | substrate of this invention. 本発明の基体における微小チャンバーの断面の一例を示す模式図である。It is a schematic diagram which shows an example of the cross section of the micro chamber in the base | substrate of this invention. 本発明にかかる脂質膜を形成するための基体の一例を示す模式的な斜視図である。It is a typical perspective view which shows an example of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の一例を示す模式的な斜視図である。It is a typical perspective view which shows an example of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の一例を示す概略上面図である。It is a schematic top view which shows an example of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の一例を示す概略上面図である。It is a schematic top view which shows an example of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の一例を示す概略上面図である。It is a schematic top view which shows an example of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の製造方法の一例を示す概略断面図である。It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の製造方法の一例を示す概略断面図である。It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の製造方法の一例を示す概略断面図である。It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の製造方法の一例を示す概略断面図である。It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate for forming the lipid film concerning this invention. レーザー照射方法Ｓを示す模式的な斜視図である。2 is a schematic perspective view showing a laser irradiation method S. FIG. レーザー照射エネルギーと形成される改質部（酸素欠乏部）との関係を模式的に示す図である。It is a figure which shows typically the relationship between the laser irradiation energy and the modification part (oxygen deficient part) formed. レーザー照射エネルギーと形成される改質部（酸素欠乏部）との関係を模式的に示す図である。It is a figure which shows typically the relationship between the laser irradiation energy and the modification part (oxygen deficient part) formed. 本発明にかかる脂質膜を形成するための基体の製造方法の一例を示す概略断面図である。It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の製造方法の一例を示す概略断面図である。It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の製造方法の一例を示す概略断面図である。It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の製造方法の一例を示す概略断面図である。It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate for forming the lipid film concerning this invention. 本発明にかかる脂質膜を形成するための基体の製造方法の一例を示す概略断面図である。It is a schematic sectional drawing which shows an example of the manufacturing method of the base | substrate for forming the lipid film concerning this invention.
　以下、好適な実施の形態に基づき、図面を参照して本発明を説明する。
《第一態様》
＜脂質膜を形成するための基体＞
［基体１０Ａ］
　図１は、本発明にかかる、脂質膜を形成するための基体（以下では、単に「基体」と呼ぶことがある。）の第一実施形態である基体１０Ａの斜視図である。図２は、図１のＡ－Ａ線に沿う断面を示す模式図である。 The present invention will be described below based on preferred embodiments with reference to the drawings.
<< First aspect >>
<Substrate for forming lipid membrane>
[Substrate 10A]
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.
　基体１０Ａは脂質膜を形成するために用いられる微小チャンバー１を備えた基体である。基体１０Ａには、基材４に内在され、脂質を含む液体Ｐを流入させる空間（ウェル２）と、基材４に内在され、前記空間（ウェル２）を構成する基材４の内壁面に開口部Ｕを有する微小チャンバー１とを含み、基材４のうち、少なくとも開口部Ｕを構成する部位は、単一の部材で構成される。 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.
　ここで「前記空間に脂質を含む液体Ｐを流入させる」とは、前記空間の外部から前記空間に液体Ｐを入れることを意味する。流入された液体Ｐは、前記空間に留まって滞留（又は完全に静止）してもよいし、前記空間の外へ流出してもよい。液体Ｐを空間の外へ流出させる場合、連続的に液体Ｐを前記空間に流入させることによって、液体Ｐの流れが前記空間において生じる。このように液体Ｐの流れが前記空間において生じる作用は、本発明にかかる基体の全てに適用される。 Here, “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. When the liquid P flows out of the space, the flow of the liquid P is generated in the space by continuously flowing the liquid P into the space. Thus, the effect | action which the flow of the liquid P produces in the said space is applied to all the base | substrates concerning this invention.
　基体１０Ａでは、基材４の上面側（上面）にウェル２が設けられている。前記ウェル２が、前記脂質を含む液体Ｐを流入させる空間を構成している。
　ウェル２の側面２ａに、微小チャンバー１の端部１ａが露呈する開口部Ｕが形成されている。ウェル２の開口位置（基材４の上面と同一平面である開口部）及び下面２ｂの少なくとも一部分は、前記開口部Ｕに形成された脂質膜Ｍを光学的に観察可能なように、開口するか或いは透明な部材（不図示）で覆われている。基体１０Ａの、少なくとも前記開口部Ｕを構成する部位は、単一の部材で構成されている。 In the substrate 10A, 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.
On the side surface 2 a of the well 2, 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.
　このようにウェル２の側面２ａに微小チャンバー１が開口している場合は、前記側面２ａに対して斜めの角度から、開口部Ｕに形成した脂質膜Ｍを観察すればよい。側面２ａに対して斜めの角度から観察することによって、開口部Ｕに形成される脂質膜Ｍを膜面の正面側から容易に観察することができる。あるいは、基材４の上方から、すなわち、側面２ａに対して平行な方向から観察してもよい。一般的に、脂質膜Ｍを観察するには、ウェル２と微小チャンバー１との間の圧力差や開口部Ｕ近傍（開口部Ｕに近い位置、開口部Ｕに近い領域、開口部Ｕに近い空間）における流体Ｐの流体圧を利用して、脂質膜Ｍを凹状又は凸状に変形させ、その形成した脂質膜Ｍを観察する手法が採用される。
　なお、ウェル２の底面２ｂに微小チャンバー１を開口させる構成（構造）であっても良い。この場合、基体の上面から観察すると、開口部Ｕの正面から観察することになり、開口部Ｕに形成された脂質膜Ｍの表面状態を容易に観察することができる。 In this way, when the micro chamber 1 is opened on the side surface 2a of the well 2, the lipid membrane M formed in the opening U may be observed from an oblique angle with respect to the side surface 2a. By observing the side surface 2a from an oblique angle, 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. In general, in order to observe the lipid membrane M, 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.
In addition, 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.
　開口部Ｕは単一の基材で構成されているため、継ぎ目や貼り合わせ面がなく、脂質膜が張る部位の段差を実質的に無くすことができる。このため、形成した脂質膜を安定に維持できる。 Since 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.
　本発明の実施形態において、液体Ｐに含まれる脂質は特に制限されない。例えば、細胞膜に含まれる脂質であるリン脂質が好ましい脂質として挙げられる。リン脂質としては、例えば、ホスファチジルコリン、ホスファチジルセリン、ホスファチジルエタノールアミン等が挙げられる。これらの両親媒性の脂質を用いると、微小チャンバー１の開口部Ｕに、脂質二重膜構造を有する脂質膜Ｍを形成することができる。この際、脂質成分として、コレステロールを添加すると、脂質膜の強度を高められることがある。 In the embodiment of the present invention, the lipid contained in the liquid P is not particularly limited. For example, phospholipids that are lipids contained in cell membranes are preferred lipids. Examples of the phospholipid include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and the like. When these amphiphilic lipids are used, 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.
　本発明の実施形態において、液体Ｐに含まれる脂質を溶解する溶媒としては、脂質を溶解できる溶媒であれば特に制限されないが、脂質膜Ｍをより容易に形成できるので、水と混和しない溶媒が好ましい。このような溶媒としては、例えば、ヘキサデカン等の有機溶媒、スクアレン（スクワレン）等の油脂が挙げられる。 In the embodiment of the present invention, 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. However, since the lipid membrane M can be formed more easily, a solvent that is not miscible with water is used. preferable. Examples of such a solvent include organic solvents such as hexadecane and fats and oils such as squalene (squalene).
　図１に示す基体１０Ａにおいては、前記単一の部材は、微小チャンバー１を構成するだけでなく、基材４全体を構成している。
　前記単一の部材の材料としては、例えばシリコン、ガラス、石英、サファイアなどが挙げられる。これらの材料は、微小チャンバー１の加工性に優れるので好ましい。なかでも、結晶方位による加工異方性の影響を受けにくい非結晶質である方が好ましい。
　更に、顕微鏡等の光学装置によって、開口部Ｕの脂質膜Ｍを観察する場合には、前記単一の部材として、ガラス、石英、又はサファイアを用いることがより好ましい。これらの部材は、可視光線（波長０．３６μｍ～０．８３μｍ）に対して透明であるため、脂質膜Ｍを容易に観察することができる。 In the base body 10 </ b> A shown in FIG. 1, the single member constitutes not only the microchamber 1 but also the entire base material 4.
Examples of 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.
Furthermore, when 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 μm to 0.83 μm), the lipid membrane M can be easily observed.
　これらの部材（材料）は溶媒を吸収しないため、脂質膜形成の際に、開口部の口径が変化したり、微小チャンバーの体積が変化したりすることは、ほとんど起こらない。このことは、微小チャンバー１の開口部Ｕがナノスケールの単位（ｎｍ）（例えば、１ｎｍ以上１μｍ未満の大きさ）で形成されている場合には、決定的に重要な要素である。溶媒を吸収する部材に形成されたナノスケールの開口部Ｕは、溶媒を吸収して膨張すると容易に閉じてしまう。開口部Ｕの形状や大きさが変化しないことによって、形成した脂質膜のサイズが実験毎に変化することなく、再現性の高い実験が可能となる。
　なお、開口部Ｕを構成する材料が溶媒を吸収して膨張する材料である場合、ナノスケールの開口部Ｕを形成することは困難である。しかしマイクロスケールの単位（μｍ）（例えば、１μｍ以上１ｍｍ未満の大きさ）で形成することは可能である。 Since these members (materials) do not absorb the solvent, the diameter of the opening and the volume of the microchamber hardly change during the formation of the lipid film. This is a critical factor when 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 μm). 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.
In addition, when the material which comprises the opening part U is a material which absorbs a solvent and expand | swells, it is difficult to form the nanoscale opening part U. However, it can be formed in microscale units (μm) (for example, a size of 1 μm or more and less than 1 mm).
　本発明の実施形態の微小チャンバー１の開口部Ｕは貼り合わせ面の無い単一の部材で構成されている。つまり、開口部Ｕの周囲は線膨張係数の等しい部材で構成されている。このため、基体１０Ａの使用温度が変わったり、基体１０Ａの温度とは異なる温度の液体を空間２（ウェル２）に流入した際などに生じ得る、開口部Ｕ近傍の急激な温度変化が起きた場合にも、開口部Ｕが貼りあわせ面から破断する恐れが無い。さらに、開口部Ｕに貼り合せ面がないので、開口部Ｕの薬液に対する化学耐性も高い。このため、貼り合せ面を侵食するような薬液であっても使用することができる。 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 | medical solution of the opening part U is also high. For this reason, even if it is a chemical | medical solution which erodes a bonding surface, it can be used.
　また、形成した脂質膜を光学顕微鏡等で観察する場合、開口部Ｕに貼り合せ面があると、前記貼り合せ面が光を反射して観察の邪魔になる場合がある。本発明の実施形態の微小チャンバー１の開口部Ｕは貼り合せ面を持たない単一の部材で構成されるため、観察の邪魔になる反射光が生じる恐れが無い。 Further, when the formed lipid film is observed with an optical microscope or the like, if there is a bonding surface in the opening U, 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.
　また、前記単一の部材の材料は、波長０．１μｍ～１０μｍを有する光のうち少なくとも一部の波長を有する光に対して透明であること（少なくとも一部の波長を有する光を透過可能であること）が好ましい。
　具体的には、加工用レーザーとして使用される一般的な光（波長０．１μｍ～１０μｍ）の、少なくとも一部に対して透明であることが好ましい。このようなレーザー光に対して透明であることによって、後述するように、レーザー照射することによって前記部材に改質部を形成することができる。
　また、前記単一の部材の材料は、可視光領域（波長約０．３６μｍ～約０．８３μｍ）の光に対して透明であること（可視光領域の光を透過可能であること）が、より好ましい。可視光領域の光に対して透明であることによって、形成した脂質膜Ｍを、前記単一部材を透して光学顕微鏡等の光学的手法を用いて容易に観察することができる。
　なお、本発明における「透明」とは、前記部材に光を入射して、前記部材から透過光が得られる状態の全てをいう。
　図１では、基材４を構成する単一の部材は透明なガラス基板である。 The material of the single member is transparent to light having at least a part of light having a wavelength of 0.1 μm to 10 μm (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 μm to 10 μm) 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 μm to about 0.83 μm) (can transmit light in the visible light region), More preferred. By being transparent to light in the visible light region, the formed lipid membrane M can be easily observed through an optical technique such as an optical microscope through the single member.
In the present invention, “transparent” refers to all states in which light is incident on the member and transmitted light is obtained from the member.
In FIG. 1, the single member which comprises the base material 4 is a transparent glass substrate.
　図２に示すように、微小チャンバー１はウェル２の内壁面である側面１ａに開口する、開口部Ｕを有する。 As shown in FIG. 2, 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.
　本発明の実施形態の基体における開口部Ｕに脂質膜Ｍを形成する方法としては、次に説明する方法が例示できる。図３Ａ～図３Ｆを参照する。
　まず、ウェル２（空間２）に生理学的食塩水等やｐＨ緩衝液等のバッファ液５を入れて、微小チャンバー１内にバッファ液５を、毛細管現象によって流入させる（図３Ａ）。つづいて、ウェル２から、ピペット等（不図示）を使用してバッファ液５を除去する。
　この際、微小チャンバー１内のバッファ液５を微小チャンバー１内に留めて、開口部Ｕに、表面張力を利用して、バッファ液５の水面をウェル２の内部に露呈させる（図３Ｂ）。 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.
First, 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). Subsequently, the buffer solution 5 is removed from the well 2 using a pipette or the like (not shown).
At this time, 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).
　つぎに、ウェル２に前記脂質を含む液体Ｐを流入し、開口部Ｕにおいて、液体Ｐとバッファ液５の水面を接触させる。この際、液体Ｐに含まれる脂質分子が、分子中の極性部をバッファ液５側に向けて、バッファ液５の水面に付着する。これにより、バッファ液５の水面は、脂質分子によって覆われる（図３Ｃ）。
　その後、ウェル２から、ピペット等を使用して液体Ｐを除去すると、開口部Ｕに留まるバッファ液５の水面には、前記付着した脂質分子で構成される脂質膜Ｍが形成される（図３Ｄ）。
　さらに、ウェル２内に、バッファ液５を流入させると、ウェル２内のバッファ液５と微小チャンバー１内のバッファ液５とが、開口部Ｕの脂質膜Ｍによって隔たれた状態となる（図３Ｅ）。 Next, 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. At this time, 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. Thereby, the water surface of the buffer solution 5 is covered with lipid molecules (FIG. 3C).
Thereafter, when the liquid P is removed from the well 2 using a pipette or the like, 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). ).
Further, when the buffer solution 5 is caused to flow into the well 2, 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). ).
　この状態における脂質膜Ｍは、前記脂質分子で構成される脂質二重膜構造又は前記脂質分子で構成される単層の脂質膜構造のうち、少なくとも何れかの膜構造をとりうる。この状態において、ウェル２及び微小チャンバー１に圧力を加える又は圧力を減じる圧力操作を行うことによって、脂質膜Ｍの厚さを制御し、脂質二重膜構造を形成することも可能である（図３Ｆ）。この圧力操作の方法としては、例えばウェル２を密閉して、ウェル２にガスやバッファ液５を加圧しながら注入する方法や、ウェル２を密閉して、ウェル２からバッファ液５を吸引する方法によって行うことができる。 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. In this state, it is possible to control the thickness of the lipid membrane M and form a lipid bilayer membrane structure by performing a pressure operation to apply pressure to the well 2 and the micro chamber 1 or to reduce the pressure (FIG. 3F). As a method of this pressure operation, for example, 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.
　また、脂質膜Ｍを構成する脂質分子の種類や微小チャンバー１の大きさにもよるが、前記圧力操作において加圧する程度を調整すると、脂質膜Ｍが微小チャンバー１内に陥入して変形し、最終的にはベシクルＶ（リポソーム）を形成することも可能である（図４）。 Also, depending on the type of lipid molecules constituting the lipid membrane M and the size of the microchamber 1, adjusting the degree of pressurization in the pressure operation will cause the lipid membrane M to intrude into the microchamber 1 and deform. Finally, vesicle V (liposome) can be formed (FIG. 4).
　微小チャンバー１は、単一のガラス基板４に形成されており、継ぎ目又は貼り合わせ面がない微小な空間である。当然に、微小チャンバーの端部における開口部Ｕについても、継ぎ目や貼り合わせ面は存在しない。このため、脂質膜Ｍと開口部Ｕとの密着力を十分に高められる。また、微小チャンバー１を備えたガラス基板４が変形した場合や、開口部Ｕが薬液によるダメージを受けた場合においても、開口部Ｕには貼り合せ面が存在しないため、貼り合わせ面を起点とする剥離や破損が生じない。よって、微小チャンバー１を備えたガラス基板４に対して、加熱消毒や薬液消毒を繰り返して行ったとしても、ガラス基板４が破損することがない。これは、日常的に加熱消毒や薬液消毒を行う、膜脂質を形成する基体にとって、特に優れた特徴である。更には、貼り合せ面が存在しない微小チャンバー１の開口部Ｕの近傍においては、貼り合せ面に起因する光の屈折率差が生じないので、開口部Ｕからの光を光学顕微鏡等の光学装置へ容易に集光させることができる。そのため、形成された脂質膜Ｍを、容易に観察することができる。ここで「開口部Ｕ」とは、ウェル２の側面２ａにおいて、微小チャンバー１の端部１ａが開口し、脂質膜Ｍが形成される領域をいう。 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. Furthermore, in the vicinity of the opening U of the micro chamber 1 where the bonding surface does not exist, there is no difference in the refractive index of the light due to the bonding surface, so that the light from the opening U is optical devices such as an optical microscope. Can be easily condensed. Therefore, the formed lipid membrane M can be easily observed. Here, 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.
　開口部Ｕを構成する、微小チャンバー１の端部１ａの、ウェル２の側面２ａにおける孔の形状は、どの様な形状でもよく、例えば、円、略円、楕円、略楕円、矩形、又は三角形、等の形状にすることができる。孔の形状が円又は略円の場合には、その直径又は長径が０．０２μｍ～５μｍの範囲であることが好ましい。その中でも、直径が０．０２～３μｍの範囲がより好ましく、さらには直径が０．０２～１μｍの範囲がより好ましい。このように小さい面積の口径を有する開口部において脂質膜Ｍを形成することによって、脂質膜Ｍを用いた実験をより精密に行うことができる。上記範囲の下限値（即ち、０．０２μｍ）未満であると、開口部Ｕの面積が小さ過ぎて、適切に脂質膜Ｍが形成されない可能性が高まる。また、上記範囲の上限値（即ち、５μｍ）を超えると、開口部Ｕと脂質膜Ｍとのシール性が低下し、長時間の安定した脂質膜形成が出来ない可能性が高まる。 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. When the shape of the hole is a circle or a substantially circle, the diameter or major axis is preferably in the range of 0.02 μm to 5 μm. Among these, the range of 0.02 to 3 μm in diameter is more preferable, and the range of 0.02 to 1 μm is more preferable. By forming the lipid membrane M in the opening having a small diameter as described above, an experiment using the lipid membrane M can be performed more precisely. If it is less than the lower limit (that is, 0.02 μm) of the above range, the area of the opening U is too small, and the possibility that the lipid membrane M is not appropriately formed increases. If the upper limit of the above range (ie, 5 μm) is exceeded, 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.
　開口部Ｕを構成する孔の形状、すなわち脂質膜Ｍが形成される開口径の形状は、真円に近い形状（略円状）であることが好ましい。ガラス基板４の上方から、すなわちウェル２の側面２ａに対して平行な方向から脂質膜Ｍを観察する場合、ガラス基板４とバッファ液５との屈折率差によって脂質膜Ｍが観察しづらくなることを防ぐことができる。 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). 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.
　微小チャンバー１は、図５に示すように、バッファ液５を流入させる開口部Ｕから内奥へ（内部の奥へ）向かうほど、その孔径（内径）が小さくなっていることが好ましい。すなわち、開口径が微小チャンバー１の最大外径Ｈ１であり、前記開口径が最も内奥の内径Ｈ３よりも大きくなっていることが好ましい。この構造であると、微小チャンバー１の内奥へ向かうほど、毛細管力が大きく働くため、バッファ液５を容易に内奥へ侵入させられる。 As shown in FIG. 5, 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.
　また、微小チャンバー１は、図６に示すように、開口部Ｕにおいて、孔径の中心に張り出した凸部Ｅを有し、且つ、凸部Ｅが形成された開口部Ｕの最小内径Ｈ２が微小チャンバー１の内奥の最大内径Ｈ１より小さくなっていることが好ましい。このような構造を有する開口部Ｕであると、脂質膜Ｍをより安定に形成することができる。 Further, as shown in FIG. 6, 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.
　また、微小チャンバー１は、図７に示すように、開口部Ｕにおいて孔径の中心に張り出した凸部Ｅを有し、且つ、凸部Ｅが形成された開口部Ｕの最小内径Ｈ２が微小チャンバー１の内奥の最大内径Ｈ１より小さく、且つ、微小チャンバー１の最も内奥の内径Ｈ３が前記最大内径Ｈ１の５０％未満であることが好ましい。このような構造を有する微小チャンバー１であると、毛細管力の作用によりバッファ液５を微小チャンバー１の内部に導入しやすくなるとともに、凸部Ｅが形成された開口部Ｕにおいて脂質膜Ｍをより容易に形成することができる。 In addition, as shown in FIG. 7, 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.
　微小チャンバー１の断面を調べた際に、微小チャンバー１の最も内奥の内径Ｈ３を特定できない場合、前記内径Ｈ３は、微小チャンバー１の奥行き方向の全長Ｌに相当する仮想の線上において、最も内奥の位置から距離を測って（１／２０）×Ｌの位置における内径である、とみなす。 If the innermost inner diameter H3 of the microchamber 1 cannot be specified when the cross section of the microchamber 1 is examined, the inner diameter H3 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.
　前記凸部Ｅの高さＥ１は、最大内径Ｈ１となる部位と開口部Ｕの最小内径Ｈ２となる部位との高低差として定義され、その高さＥ１は２０ｎｍ～２００ｎｍであることが好ましい。この範囲の下限値（即ち、２０ｎｍ）未満であると凸部Ｅによる効果が殆ど期待できず、上限値（即ち、２００ｎｍ）を超えると、微小チャンバー１の内部にバッファ液５を導入することが困難になる場合がある。
　前記凸部Ｅの幅Ｅ２は、微小チャンバー１が側面２ａに開口する位置から、内奥へ進んで、内径が拡がり始める位置までの距離として定義され、その幅Ｅ２は微小チャンバー１の奥行き方向における全長Ｌの２０％未満であることが好ましい。このような構造であると、バッファ液５を微小チャンバー１へ、より一層容易に導入することができる。 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.
　本発明の実施形態において、微小チャンバー１の開口部Ｕの近傍が疎水性になるように、表面処理することによって、脂質膜Ｍをより安定して形成することができる。ここで、開口部Ｕの近傍とは、脂質膜Ｍが接する領域およびその周辺を意味する。前記表面処理としては、例えば表面にアルキル基を結合させる処理が挙げられる。当該表面がガラスである場合、シランカップリング剤を用いてアルキル化処理することができる。 In the embodiment of the present invention, 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. Here, the vicinity of the opening U means a region in contact with the lipid membrane M and its periphery. Examples of the surface treatment include a treatment for bonding an alkyl group to the surface. When the surface is glass, it can be alkylated using a silane coupling agent.
　微小チャンバー１を立方体状又は直方体状の空間であると近似した場合、その一辺の長さは、０．１μｍ～３０μｍであることが好ましい。
　上記範囲であると、脂質膜Ｍを開口部Ｕに容易に形成することが可能であり、ベシクルＶの形成も可能となる。 When the micro chamber 1 is approximated as a cubic or cuboid space, the length of one side thereof is preferably 0.1 μm to 30 μm.
Within the above range, the lipid membrane M can be easily formed in the opening U, and the vesicle V can be formed.
　また、微小チャンバー１の容積は、細胞などの微生物と同程度であることが好ましい。微小チャンバー１の内部に微生物を捉え、その状態で開口部Ｕに脂質膜Ｍを形成すると、微生物が微小チャンバー１の内部に閉じ込められる。そして、捉えた微生物にレーザーを照射して微生物の細胞膜を破壊することで、破壊された細胞膜が再生する様子を観察することができる。一般に微生物は、微生物を構成する成分が近くに存在すれば再生することができると言われているが、その様子を直接的に観察された事例はない。本発明の実施形態の装置を用いることで、微生物の再生の様子を確認することができる可能性がある。 The volume of the micro chamber 1 is preferably about the same as that of microorganisms such as cells. 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.
　前記孔の孔径をナノオーダー（例えば、１ｎｍ以上１μｍ未満の大きさ）で高精度に加工すると、従来よりも更にサイズの小さな脂質膜を形成することが可能である。この場合、従来、複数の分子の集合体として観察されていた膜タンパク質の機能を、本発明の実施形態に係る基体を用いることにより、単一分子で観察することが可能である。つまり、本発明の実施形態に係る基体は、今まで解明されていない膜タンパク質の様々な分子特性を確認する装置として使用することができる。さらに、孔の開口部の径をナノオーダー程度まで微小化すると、脂質膜と開口部との安定したシール性が実現されるため、機能解析等の実験を安定して行うことが可能となる。 When 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 μm), it is possible to form a lipid membrane having a smaller size than the conventional one. In this case, 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. Furthermore, when 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.
　本発明の実施形態の基体を用いることによって、微小チャンバー１の開口部Ｕに形成した脂質膜Ｍの特性を高精度に機能解析することができる。例えば、開口部Ｕに脂質膜Ｍが形成された微小チャンバー１の内部に、イオン性物質が閉じ込められた状態をつくり、このイオン性物質が脂質膜Ｍのイオンチャネルを通して微小チャンバー１の外部に輸送される状態や、その輸送速度を調べることができる。輸送速度を調べるためには、脂質膜Ｍを透過する様子を長時間に渡って観測する必要があるため、容量の大きな微小チャンバー１であるとよい。 By using 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. For example, 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.
　図２において、微小チャンバー１は、ウェル２の側面２ａに対して略垂直となるように形成されている。しかし、必ずしも略垂直である必要はなく、基体１０Ａの設計に合わせて、単一のガラス基板４において自由に配置することが可能である。
　更には、微小チャンバー１の端部１ａに位置する開口部Ｕの開口径を、少し内奥へ進んだ口径よりもわずかに広げて加工することも可能である。つまり、開口部Ｕを漏斗の様な形状に加工することが可能である。このように加工した場合、脂質膜Ｍの一部が微小チャンバー１の端部に入り込んだ状態で、脂質膜Ｍを形成することが可能になる。この脂質膜Ｍの一部は、端部の内側にあるため、ウェル２に流れがあるときにおいても、より安定して長時間の脂質膜Ｍの維持が可能になる場合がある。ここではウェル２を使用した場合について説明しているが、後述する基体１０Ｂにおいて、流路３を使用した場合についても同様の説明が適用される。 In FIG. 2, 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. 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. Although 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.
　基体１０Ａには、複数の微小チャンバー１が形成されていてもよい。各々の微小チャンバー１に対して開口部Ｕが各々備わるため、複数の脂質膜Ｍを形成できる。
　基材４がシリコン、ガラス、石英、又はサファイアなどであると、加工精度が高いので、複数の微小チャンバー１を密集させて配置することが可能である。 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.
When 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.
　本発明の実施形態によれば、後で説明するように、微小領域にレーザー光を集光させ、その集光領域の位置を高精度に制御することが可能な加工方法を用いているため、複数の微小チャンバー１を高密度に配置することが可能である。例えば、複数の微小チャンバー１の開口部Ｕを、０．１μｍ～６μｍのピッチで配置することができる。 According to the embodiment of the present invention, as will be described later, since a laser beam is focused on a minute area and a processing method capable of controlling the position of the focused area with high accuracy is used, It is possible to arrange a plurality of micro chambers 1 with high density. For example, the openings U of the plurality of micro chambers 1 can be arranged at a pitch of 0.1 μm to 6 μm.
　基体１０Ａでは、ウェル２の下面２ｂはガラス基板で構成される基材４で構成されている。
　下面２ｂに対向する位置における、ウェル２の開口位置（基材４の上面と同一平面である開口部）は開口されて無蓋となっている。この下面２ｂ又は上面のうち少なくとも一方から、顕微鏡等の光学的観察装置によって、開口部Ｕに形成された脂質膜Ｍを観察することができる。なお、前記上面は必ずしも無蓋である必要はなく、プラスチック、樹脂やガラス等の部材で構成される蓋によって、覆われていてもよい（不図示）。開口部Ｕにおける脂質膜Ｍの形成を観察しながら行うことが可能となる。 In the base body 10A, 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. From at least one of the lower surface 2b and the upper surface, the lipid membrane M formed in the opening U can be observed by an optical observation device such as a microscope. Note that 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.
［基体１０Ｂ］
　本発明の脂質膜を形成するための基体の第二実施形態として、図８に示す基体１０Ｂが挙げられる。基体１０Ｂでは、基材４の上面側に流路３が設けられている。前記流路３が、前記脂質を含む液体Ｐを流入させる空間を構成している。
　基体１０Ａにおけるウェル２と比べて、基体１０Ｂに流路３が設けられている構造を用いることによって大量の液体Ｐを流入及び流通させることができる。また、流路３（空間２）における溶液の交換が行い易いという利点がある。前述の脂質膜Ｍの形成方法で説明したように、バッファ液５、前記脂質を含む液体Ｐ等の複数の溶液を、開口部Ｕに順次接触させる操作をより容易に行うことができる。
　基体１０Ｂの他の構成については、基体１０Ａと同様である。 [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. As described in the above-described method for forming the lipid membrane M, 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.
　また、図９に示すように、流路３やウェル２の側面において、複数の微小チャンバー１を各々対向させて配置することも可能である。本発明の実施形態の基体においては、微小チャンバー１を任意の位置に配置し、複数の微小チャンバー１を任意に配列させることが可能である。 Moreover, as shown in FIG. 9, it is also possible to arrange a plurality of micro chambers 1 so as to face each other on the side surfaces of the flow path 3 and the well 2. In the substrate of the embodiment of the present invention, the micro chambers 1 can be arranged at arbitrary positions, and a plurality of micro chambers 1 can be arbitrarily arranged.
　図１０は、本発明にかかる脂質膜を形成するための基体の一例（第三実施形態）である基体２０Ａの模式的な上面図（上面から見た透視図）である。この上面図におけるウェル１２、微小チャンバー１１の配置構成は、前述の基体１０Ａに適用可能である。
　ガラス基板１４には、ウェル１２および微小チャンバー１１がそれぞれ６セット配置されている。ウェル１２の内壁面に微小チャンバー１１が開口していることは、前述の通りである。開口部Ｕの位置は、「Ｘ」の印で示してある。
　一つのウェル１２に複数の微小チャンバー１１が配置されていてもよい。 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.
On the glass substrate 14, six sets of wells 12 and micro chambers 11 are arranged. As described above, the micro chamber 11 is opened on the inner wall surface of the well 12. The position of the opening U is indicated by an “X” mark.
A plurality of micro chambers 11 may be arranged in one well 12.
　図１１は、本発明にかかる脂質膜を形成するための基体の一例（第四実施形態）である基体３０Ａの模式的な上面図（上面から見た透視図）である。この上面図における流路２２、および微小チャンバー２１の配置構成は、前述の基体１０Ｂに適用可能である。
　ガラス基板２４には、複数の微小チャンバー２１が内壁面に配された流路２２が、４セット配置されている。流路２２の内壁面に微小チャンバー２１が開口していることは、前述の通りである。開口部Ｕの位置は、「Ｘ」の印で示してある。
　流路２２の上流側Ｆ１から、液体Ｐが流入されて、流路２２の下流側Ｆ２へ流通する。 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.
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. 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 F1 of the flow path 22 and flows to the downstream side F2 of the flow path 22.
　図１２は、本発明にかかる脂質膜を形成するための基体の一例（第五実施形態）である基体３０Ｂの模式的な上面図（上面から見た透視図）である。この上面図における流路２２、微小チャンバー２１、および第二流路２９の配置構成は、前述の基体１０Ｂに適用可能である。
　ガラス基板２４には、複数の微小チャンバー２１が内壁面に配された流路２２が、配置されている。流路２２の内壁面に微小チャンバー２１が開口していることは、前述の通りである。開口部Ｕの位置は、「Ｘ」の印で示してある。
　流路２２の上流側Ｆ５から、液体Ｐが流入されて、流路２２の下流側Ｆ６へ流通する。 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.
　また、ガラス基板２４に配された第二流路２９は、流路２２と連通している。第二流路２９から流路２２へ、所望の薬液又はガスを流通させることによって、流路２２内にも前記薬液又はガスを拡散流入させることができる。つまり、前記薬液又はガスを、開口部Ｕに形成された脂質膜Ｍへ接触させることができる。 Further, the second flow path 29 arranged on the glass substrate 24 communicates with the flow path 22. By flowing a desired chemical liquid or gas from the second flow path 29 to 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.
　第二流路２９が流路２２に連通する場所や第二流路２９の形状は特に限定されない。そのため、微小チャンバー２１の近傍（微小チャンバー２１に近い位置）に第二流路２９を配置することも可能であり、第二流路２９と流路２２の上流側（Ｆ５側）はそれぞれの機能を代替することが可能である。また複数の第二流路２９や複数に分岐した第二流路２９が流路２２に連通するように配置することも可能である。 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.
《第二態様》
　次に、本発明にかかる脂質膜を形成するための基体の製造方法を説明する。
＜脂質膜を形成するための基体の製造方法（第二態様）＞
　本発明の第二態様である製造方法の一例を、前述の第二実施形態の基体１０Ｂを例にとって説明する。この場合、前記製造方法は、図１３Ａ～図１３Ｄで示すように、ピコオーダー秒以下のパルス時間幅を有するレーザーＬを、単一の部材５９において、微小チャンバー５５を形成する領域に照射することによって、前記領域に改質部５１を形成する工程Ａ１（図１３Ａ）と、単一の部材５９に、前記空間を構成する流路５７若しくは貫通孔を形成する工程Ａ２（図１３Ｂ）と、単一の部材５９から改質部５１をエッチングによって除去する工程Ａ３（図１３Ｃ）と、を少なくとも有する。 << Second aspect >>
Next, the manufacturing method of the base | substrate for forming the lipid membrane concerning this invention is demonstrated.
<Method for producing substrate for forming lipid membrane (second embodiment)>
An example of the manufacturing method according to the second aspect of the present invention will be described by taking the substrate 10B of the second embodiment as an example. In this case, as shown in FIGS. 13A to 13D, the manufacturing method irradiates a region where the microchamber 55 is formed on a single member 59 with a laser L having a pulse time width of pico-order seconds or less. Step A1 (FIG. 13A) for forming the modified portion 51 in the region, Step A2 (FIG. 13B) for forming the flow path 57 or the through-hole forming the space in the single member 59, Step A3 (FIG. 13C) for removing the modified portion 51 from the one member 59 by etching.
［工程Ａ１］
　レーザーＬ（レーザー光Ｌ）は、パルス時間幅がピコ秒オーダー以下、例えば１フェムト秒以上１０ピコ秒未満、のパルス幅を有するレーザー光を用いることが好ましい。例えばチタンサファイアレーザー、前記パルス幅を有するファイバーレーザーなどを用いることができる。ただし部材５９に対して透明な波長を使用することが必要である。より具体的には、部材５９に対する透過率が６０％以上のレーザー光であることが好ましい。　 [Step A1]
The laser L (laser beam 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. For example, a titanium sapphire laser, a fiber laser having the pulse width, or the like can be used. However, it is necessary to use a transparent wavelength for the member 59. More specifically, a laser beam having a transmittance of 60% or more with respect to the member 59 is preferable.
　前記レーザーＬ（レーザー光Ｌ）は、加工用レーザーとして使用される一般的な波長領域（０．１～１０ｕｍ）の光を適用することができる。その中でも、被加工部材である部材５９に対して透明である必要がある。部材５９に対して透明な波長のレーザー光を適用することによって、部材５９に対して改質部５１を形成することができる。
　ここで、本明細書及び特許請求の範囲において、「改質部」とは、エッチング耐性が低くなり、エッチングによって選択的に又は優先的に除去される部分」を意味する。 As the laser L (laser light L), light in a general wavelength region (0.1 to 10 μm) used as a processing laser can be applied. Among these, it is necessary to be transparent with respect to the member 59 which is a workpiece. By applying a laser beam having a transparent wavelength to the member 59, the modified portion 51 can be formed on the member 59.
Here, in the present specification and claims, the “modified portion” means a portion that has low etching resistance and is selectively or preferentially removed by etching.
　部材５９の材料は、例えばシリコン、ガラス、石英、サファイアなどが挙げられる。これらの材料は、微小チャンバー５５の加工性に優れるので好ましい。なかでも、結晶方位による加工異方性の影響を受けにくい非結晶質である方が好ましい。
　更には、顕微鏡等の光学装置によって観察する場合には、前記材料として、可視光線（波長０．３６μｍ～０．８３μｍ）に対して透明であるガラス、石英、又はサファイア等を用いることが、より好ましい。 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 μm to 0.83 μm) as the material. preferable.
　また、部材５９の材料は、波長０．１μｍ～１０μｍを有する光のうち少なくとも一部の波長を有する光に対して透明である（光を透過する）ことが好ましい。
　具体的には、加工用レーザー光として使用される一般的な波長領域（０．１μｍ～１０μｍ）の、少なくとも一部の波長領域の光に対して透明であることが好ましい。部材５９の材料が、このようなレーザー光に対して透明であることによって、後述するように、前記部材にレーザー照射して改質部を形成することができる。
　また、可視光領域（波長約０．３６μｍ～約０．８３μｍ）の光に対して透明であることが、より好ましい。可視光領域の光に対して透明であることによって、形成した脂質膜Ｍを、前記単一部材を透して肉眼で容易に観察することができる。
　なお、本発明における「透明」とは、前記部材に光を入射して、前記部材から透過光が得られる状態の全てをいう。
　図１３Ａ～図１３Ｄでは、単一の部材５９は透明なガラス基板である（以下、ガラス基板５９と呼ぶ）。
　以下では、部材５９がガラス基板である場合について説明するが、部材５９がその他の部材、例えばシリコン、石英、又はサファイアの場合であっても、同様に行うことができる。後述する工程Ａ２における加工性はシリコン、石英、ガラスがより好適である。 Further, 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 μm to 10 μm.
Specifically, it is preferably transparent to light in at least a part of the wavelength region of a general wavelength region (0.1 μm to 10 μm) 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 μm to about 0.83 μm). 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.
In the present invention, “transparent” refers to all states in which light is incident on the member and transmitted light is obtained from the member.
In FIGS. 13A to 13D, the single member 59 is a transparent glass substrate (hereinafter referred to as a glass substrate 59).
Hereinafter, 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.
　ガラス基板５９は、例えば石英で構成されるガラス基板、珪酸塩を主成分とするガラス、ホウ珪酸ガラスで構成されるガラス基板等を用いることができる。合成石英で構成されるガラス基板が、加工性が良いため好適である。また、ガラス基板５９の厚さは特に制限されない。 As 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. Further, the thickness of the glass substrate 59 is not particularly limited.
　レーザー光Ｌの照射方法としては、図１３Ａに示す方法が挙げられる。すなわち、ガラス基板５９の内部に集光して焦点を結ぶようにレーザー光Ｌを照射して、前記焦点を矢印方向に走査することによって、ガラスが改質された改質部５１を形成する。
　微小チャンバー５５が形成される領域に、前記焦点をガラス基板５９内部で走査することによって、所望の形状の改質部５１を形成することができる。 As the irradiation method of the laser beam L, the method shown in FIG. That is, 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.
By scanning the focal point inside the glass substrate 59 in a region where the micro chamber 55 is formed, the modified portion 51 having a desired shape can be formed.
　レーザー光Ｌを照射する際、照射強度をガラス基板５９の加工上限閾値に近い値（加工適正値に近い値）又は加工上限閾値未満（加工適正値未満）にすると共に、レーザー光Ｌの偏波方向（電場方向）を走査方向に対して垂直となるようにすることが好ましい。このレーザー照射方法を、以下ではレーザー照射方法Ｓと呼ぶ。 When irradiating the laser beam L, 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. Hereinafter, this laser irradiation method is referred to as a laser irradiation method S.
　レーザー照射方法Ｓを、図１４で説明する。レーザー光Ｌの伝播方向は矢印Ｚであり、前記レーザー光Ｌの偏波方向（電場方向）は矢印Ｙである。レーザー照射方法Ｓでは、レーザー光Ｌの照射領域を、前記レーザー光の伝播方向と、前記レーザー光の偏波方向に対して垂直な方向と、で構成される平面５９ａ内とする。これと共に、レーザー照射強度をガラス基板５９の加工上限閾値に近い値又は加工上限閾値未満とする。 The laser irradiation method S will be described with reference to FIG. The propagation direction of the laser light L is an arrow Z, and the polarization direction (electric field direction) of the laser light L is an arrow Y. In the laser irradiation method S, 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. At the same time, 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.
　このレーザー照射方法Ｓによって、ガラス基板５９内にナノオーダー（例えば１ｎｍ以上１μｍ未満）の口径を有する改質部５１を形成することができる。例えば、短径が２０ｎｍ程度、長径が０．２μｍ～５μｍ程度の略楕円形状の断面を有する改質部５１が得られる。この略楕円形状は、レーザーの伝播方向に沿った方向が長軸で、レーザーの電場方向に沿った方向が短軸となる。レーザー照射の具合によっては、前記断面は矩形に近い形状となることもある。 By this laser irradiation method S, the modified portion 51 having a nano-order (for example, 1 nm or more and less than 1 μm) diameter can be formed in the glass substrate 59. For example, 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 μm to 5 μm is obtained. In this substantially elliptical shape, the direction along the laser propagation direction is the major axis, and the direction along the laser electric field direction is the minor axis. Depending on the condition of laser irradiation, the cross section may be a shape close to a rectangle.
　レーザー照射強度をガラス基板５９の加工上限閾値（加工適正値）以上とした場合、得られる改質部５１は周期構造を伴って形成されることがある。すなわち、ピコ秒オーダー以下のパルスレーザーを加工上限閾値以上で集光照射させることで、集光部で電子プラズマ波と入射光の干渉が起こり、レーザーの偏波に対して垂直であり、偏波方向に沿って周期性をもつ周期構造が自己形成的に形成されることがある。 When the laser irradiation intensity is set to be equal to or higher than the processing upper limit threshold (processing appropriate value) of the glass substrate 59, the obtained modified portion 51 may be formed with a periodic structure. In other words, by condensing and irradiating a pulse laser of picosecond order or less above the processing upper threshold, interference between the electron plasma wave and incident light occurs at the condensing part, and it is perpendicular to the laser polarization. A periodic structure having periodicity along the direction may be formed in a self-forming manner.
　形成された周期構造はエッチング耐性の弱い層となる。例えば石英の場合、酸素が欠乏した層と酸素が増えた層が周期的に配列され（図１５Ｂ）、酸素欠乏部のエッチング耐性が弱くなっており、エッチングを行うと周期的な凹部及び凸部が形成されうる。このような周期的な凹部及び凸部は、後述する微小チャンバー５５の形成においては不要である。 The formed periodic structure becomes a layer with weak etching resistance. For example, in the case of quartz, 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.
　一方、前述のレーザー照射方法Ｓのように、レーザー照射強度をガラス基板５９の加工上限閾値未満、且つガラス基板５９を改質してエッチング耐性を低下させることが可能なレーザー照射強度の下限値（加工下限閾値）以上とすれば、前記周期構造は形成されず、レーザー照射によって一つの酸素欠乏部（エッチング耐性の弱い層）が形成される（図１５Ａ）。これのエッチングを行うと、一つの微小チャンバー５５を形成することができる。 On the other hand, as in the laser irradiation method S described above, 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.
　前述のレーザー照射方法Ｓによれば、微小チャンバー５５の形状を楕円又は略楕円とすることができる。また、その短径をエッチングによってナノオーダーサイズで制御することが可能となる。微小チャンバー５５の形状が楕円又は略楕円形状である場合は、短径をナノオーダーサイズにすることによって、脂質膜をより容易に形成し易くなることがある。また、脂質膜を形成する前準備として、微細チャンバー５５に、あらかじめバッファ液等の液体を充填させる必要があるが、チャンバーが微細であるほど毛細管力が大きくなるため、微細チャンバー５５から、液体が外部（前記空間）に出てこなくなる弊害が発生する場合がある。しかしながら、微小チャンバー５５を楕円又は略楕円状に形成することによって、脂質膜を形成するのに十分な短径においても毛細管力を抑制させ、液体が空間などの外部に出てこなくなる弊害を抑制することができる。 According to the laser irradiation method S described above, the shape of the micro chamber 55 can be made elliptical or substantially elliptical. In addition, the minor axis can be controlled to a nano-order size by etching. When 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. In addition, as a preparation for forming the lipid membrane, it is necessary to preliminarily fill the microchamber 55 with a liquid such as a buffer solution. However, 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. However, by forming the micro chamber 55 in an elliptical shape or a substantially elliptical shape, 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.
　エッチング耐性が弱い層（石英又はガラスにおいては酸素欠乏部）がレーザー照射によって一つだけ形成される場合においても（本明細書では改質部５１と呼ぶ。）、前記酸素欠乏部は極めてエッチングの選択性（選択比）が高い層となる。このことは、本発明者らの鋭意検討によって見出された。 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.
　したがって、前記加工上限閾値は、前記周期構造が形成されうるレーザーパルスパワーの下限値（前記周期構造が形成されないレーザーパルスパワーの範囲における上限値）と定義される。
　また、前記「ガラス基板５９を改質してエッチング耐性を低下させうるレーザー照射強度の下限値（加工下限閾値）（閾値）」とは、エッチング処理により、ガラス基板５９に微小チャンバー５５を形成できる限界値である。この下限値よりも低いと、レーザー照射によってエッチング耐性の弱い層が形成出来ないため、微小チャンバー５５が形成できない。
　すなわち、本明細書及び特許請求の範囲において、「加工上限閾値（加工適正値）」とは、基材内に照射したレーザー光の焦点（集光域）において、基材とレーザー光との相互作用によって生じる電子プラズマ波と入射するレーザー光との干渉が起こり、前記干渉によって基材に縞状の改質部が自己形成的に形成されることが可能なレーザー照射強度の下限値を意味する。
　また、本明細書及び特許請求の範囲において、「加工下限閾値（閾値）」とは、基材内に照射したレーザー光の焦点（集光域）において、基材を改質した改質部を形成し、後段のエッチング処理によって選択的又は優先的にエッチングされうる程度に、前記改質部のエッチング耐性を低下させることが可能なレーザー照射強度の下限値である。この下限値よりも低いレーザー照射強度でレーザー照射した領域は、後段のエッチング処理において選択的又は優先的にエッチングされ難い。このため、エッチング後に微細孔となる改質部を形成するためには、加工下限閾値以上のレーザー照射強度に設定することが好ましい。 Therefore, 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).
Further, 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.
That is, in the present specification and claims, 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. .
In the present specification and claims, 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. It is 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.
　加工上限閾値及び加工下限閾値は、レーザー光の波長、レーザー照射対象である基材の材料（材質）及びレーザー照射条件によって概ね決定される。しかし、レーザー光の偏波方向と走査方向との相対的な向きが異なると、加工上限閾値及び加工下限閾値も多少異なる場合がある。例えば、偏波方向に対して走査方向が垂直の場合と、偏波方向に対して走査方向が平行の場合とでは、加工上限閾値及び加工下限閾値が異なる場合がある。したがって、使用するレーザー光の波長及び使用する基材において、レーザー光の偏波方向と走査方向との相対関係を変化させた場合の、それぞれの加工上限閾値及び加工下限閾値を、予め調べておくことが好ましい。 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. However, when the relative directions of the polarization direction of the laser beam and the scanning direction are different, the processing upper limit threshold and the processing lower limit threshold may be slightly different. For example, 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.
　前記偏波としては直線偏波に関して詳細に説明したが、多少の楕円偏波成分を持つレーザーパルスであっても同様な構造（改質部）が形成されることが容易に想像できる。 Although the above-described polarization has been described in detail with respect to linear polarization, it can be easily imagined that a similar structure (modified part) is formed even with a laser pulse having some elliptical polarization component.
　レーザー光Ｌの焦点を走査する方法は特に限定されないが、一度の連続走査によって形成できる改質部５１は偏波方向（矢印Ｙ方向）に対して垂直な１次元方向と、レーザー光Ｌの伝搬方向（矢印Ｚ方向）の２次元方向（平面５９ａ）内に限定される。この２次元方向内であれば任意の形状で改質部を形成することができる。 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.
　図１４においては、レーザー光Ｌの伝播方向は、ガラス基板５９の上面に対して垂直である場合を示したが、必ずしも垂直である必要はない。前記上面に対して所望の入射角で、レーザーＬを照射してもよい。 FIG. 14 shows the case where the propagation direction of the laser light L is perpendicular to the upper surface of the glass substrate 59, but it is not necessarily perpendicular. The laser L may be irradiated at a desired incident angle with respect to the upper surface.
　一般に、改質された部分のレーザーの透過率は、改質されていない部分のレーザーの透過率とは異なるため、改質された部分を透過させたレーザー光の焦点位置を制御することは通常困難である。したがって、レーザー照射する側の面から見て、奥に位置する領域について先に改質部を形成することが望ましい。 In general, 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.
　ガラス基板５９内に、３次元方向に任意形状を有する改質部を形成する場合には、レーザーの偏波方向（矢印Ｙ方向）を適宜変更することによって行うことができる。 In the case where a modified portion having an arbitrary shape in the three-dimensional direction is formed in the glass substrate 59, the modification can be performed by appropriately changing the polarization direction of the laser (arrow Y direction).
　また、図１４で示すように、レーザー光Ｌをレンズ５２を用いて集光して、前述の様に照射することによって改質部５１を形成してもよい。
　前記レンズとしては、例えば屈折式の対物レンズ若しくは屈折式のレンズを使用することができるが、他にも例えばフレネル、反射式、油浸若しくは水浸式の方法によって照射することも可能である。また、例えばシリンドリカルレンズを用いれば、一度にガラス基板５９の広範囲にレーザー照射することが可能になる。また、例えばコニカルレンズを用いればガラス基板５９の垂直方向に広範囲に一度にレーザー光Ｌを照射することができる。ただしシリンドリカルレンズを用いた場合には、レーザー光Ｌの偏波はレンズが曲率を持つ方向に対して水平である必要がある。 Further, as shown in FIG. 14, the modified portion 51 may be formed by condensing the laser beam L using a lens 52 and irradiating it as described above.
As the 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. Further, for example, if a cylindrical lens is used, it is possible to irradiate a laser on a wide area of the glass substrate 59 at a time. For example, if a conical lens is used, the laser beam L can be irradiated at once in a wide range in the vertical direction of the glass substrate 59. However, when a cylindrical lens is used, the polarization of the laser light L needs to be horizontal with respect to the direction in which the lens has a curvature.
　レーザー照射条件Ｓの具体例としては、以下の各種条件が挙げられる。例えばチタンサファイアレーザー（レーザー媒質としてサファイアにチタンをドープした結晶を使用したレーザー）又は１ｆｓ以上１０ピコ秒未満のパルス時間幅を有するパルスレーザーを用いることができる。照射するレーザー光は、例えば波長８００ｎｍ、繰返周波数２００ｋＨｚを使用し、レーザー走査速度１ｍｍ／秒としてレーザー光Ｌを集光照射する。これら波長、繰返周波数、走査速度の値は一例であり、本発明はこれに限定されず任意に変えることが可能である。なお、本明細書及び特許請求の範囲において、「ピコ秒オーダー以下のパルス時間幅」は、１フェムト秒以上１ナノ秒未満のパルス時間幅であることが好ましく、１フェムト秒以上１０ピコ秒未満のパルス時間幅であることがより好ましく、１フェムト秒以上３ピコ秒未満のパルス時間幅であることが更に好ましく、１フェムト秒以上２ピコ秒未満のパルス時間幅であることが特に好ましい。
　前記パルス時間幅がピコ秒オーダー以下、特に１０ピコ秒未満、であると、集光部における基材の電子温度とイオン温度とが非平衡状態となり加熱され、いわゆる非熱過程での加工が進行する。そして、熱拡散長が極限まで抑えられる。さらには多光子吸収に始まる非線形加工が支配的となるため、加工後に得られる形状はナノスケールからマイクロオーダースケールの微細孔とすることが可能である。
　一方、比較的大きなパルス時間幅を有するパルスレーザー、例えば１０ピコ秒以上のパルス時間幅を有するレーザー光を用いた場合では、集光部における基材の電子温度とイオン温度とが平衡状態となる熱的加工が支配的となる。熱的加工においては熱拡散長が大きくなり、ナノからマイクロオーダースケールの加工を行うことが困難になる場合がある。このように、パルス時間幅が約１～１０ピコ秒付近を境にして、全く異なる反応メカニズムとなる場合があるため、１０ピコ秒未満のパルス時間幅を有するパルスレーザーを用いることが好ましい。 Specific examples of the laser irradiation condition S include the following various conditions. For example, a titanium sapphire laser (laser using a crystal in which sapphire is doped with titanium as a laser medium) or a pulse laser having a pulse time width of 1 fs or more and less than 10 picoseconds can be used. As 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. In the present specification and claims, 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.
When 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. And 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.
On the other hand, in the case of using 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.
　集光に用いるレンズ５２としては、例えばＮ．Ａ.＜０．７未満の対物レンズを用いることが好ましい。より微小な微小チャンバー５５を形成させるためにはパルス強度は、加工上限閾値に近い値、又は加工上限閾値未満且つ加工上限閾値に近い値に設定することが好ましい。
　具体的には、ガラス基板５９にレーザーを照射する場合には、例えば、パルス時間幅３００ｆｓ、繰返周波数２００ｋＨｚ、走査速度１ｍｍ／ｓ程度の条件に設定して、８０ｎＪ／ｐｕｌｓｅ程度以下のパルスエネルギーで、照射強度は５５０ｋＷ／ｃｍ２程度の照射強度で、１パルスあたりのレーザーフルエンスが２．７Ｊ／ｃｍ２程度で照射することが好ましい。一方、加工上限閾値以上の照射強度、或いはその加工上限閾値に相当する１パルスあたりのレーザーフルエンス（パワー）よりも大きくすると周期構造が形成され、エッチングによってそれらが繋がるため、ナノオーダーの口径を有する微小チャンバー５５を形成することが困難となり、ミクロンオーダーの口径になる、あるいは前記周期構造が形成されてしまうことがある。Ｎ．Ａ.≧０．７に設定しても加工は可能であるが、スポットサイズがより小さくなり、１パルスあたりのレーザーフルエンスが大きくなるため、より小さなパルス強度（パルスエネルギー）に設定したレーザー照射が求められる。なお、レーザーフルエンスは、単位面積あたりのエネルギー量を指し、Ｊ／ｃｍ２またはＷ／ｃｍ２で表される。 Examples of the lens 52 used for condensing include N. A. It is preferable to use an objective lens of <0.7. In order to form a finer micro chamber 55, 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.
Specifically, when irradiating the glass substrate 59 with a laser, for example, 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 . On the other hand, when 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 .
［工程Ａ２］
　つぎに、単一のガラス基板５９に、前記空間を構成する流路５７若しくは貫通孔を形成する。前記流路５７を形成する方法としては、次の方法が例示できる。 [Step A2]
Next, the flow path 57 or the through-hole which comprises the said space is formed in the single glass substrate 59. FIG. As a method for forming the flow path 57, the following method can be exemplified.
　まず、ガラス基板５９の上面に、例えばフォトリソグラフィなどによってレジスト５２をパターニングして形成する。つづいて、ドライエッチング、ウェットエッチング、又はサンドブラスト等の方法によって、ガラス基板５９の上面におけるレジスト５２が形成されていない領域を、所定の深さに達するまで浸食して除去する（図１３Ｂ）。不要となったレジスト５２を剥離すると、流路５７が形成されたガラス基板５９が得られる。 First, 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.
　工程Ａ２においては、形成する流路５７の側面に、工程Ａ１で形成した改質部５１の断面を露呈させることが好ましい。後段の工程Ａ３におけるエッチング処理によって、微小チャンバー５５を形成させることがより容易となる。 In 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.
　また、工程Ａ２において、流路５７として、ガラス基板５９の上面に凹部を形成する代わりに、貫通孔を形成してもよい。ガラス基板５９内の、流路となる領域を微小ドリル（レーザードリル）等によってガラス基板５９の表面から掘削して貫通孔を形成することによって、流路５７を形成してもよい。このドリルによる掘削方法は、種々のエッチング法と組合わせて使用しても良い。 Also, in step A2, instead of forming a recess on the upper surface of the glass substrate 59, 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.
［工程Ａ３］
　つぎに、単一のガラス基板５９から、工程Ａ１で形成した改質部５１をエッチングによって除去する（図１３Ｃ）。
　エッチング方法としては、ウェットエッチングが好ましい。流路５７（若しくは貫通孔）の側面に露呈する断面を有する改質部５１は、エッチング耐性が弱くなっているため、選択的又は優先的にエッチングすることができる。 [Step A3]
Next, the modified portion 51 formed in step A1 is removed from the single glass substrate 59 by etching (FIG. 13C).
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.
　このエッチングは、ガラス基板５９の改質されていない部分に比べて、改質部５１が非常に速くエッチングされる現象を利用している。エッチングの結果として改質部５１の形状に応じた微小チャンバー５５を形成することができる。
　前記エッチング液は特に限定されず、例えばフッ酸（ＨＦ）を主成分とする溶液、フッ酸に硝酸等を適量添加したフッ硝酸系の混酸等を用いることができる。また、部材５９の材料に応じて、他の薬液を用いることもできる。 This etching utilizes the phenomenon that the modified portion 51 is etched very quickly compared to the unmodified portion of the glass substrate 59. As a result of the etching, a micro chamber 55 corresponding to the shape of the modified portion 51 can be formed.
The etching solution is not particularly limited, and for example, a solution containing hydrofluoric acid (HF) as a main component, or a hydrofluoric acid-based mixed acid obtained by adding an appropriate amount of nitric acid or the like to hydrofluoric acid can be used. Also, other chemicals can be used depending on the material of the member 59.
　前記エッチングの結果、ナノオーダーの口径を有する微小チャンバー５５を、ガラス基板５９内の所定位置に、流路５７の側面に開口するように、形成することができる。 As a result of the etching, 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.
　微小チャンバー５５の開口部は、例えば、短径が２０ｎｍ～２００ｎｍ程度、長径が０．２μｍ～５μｍ程度のサイズの略楕円形状の断面を有する開口部として、形成することができる。エッチング処理の条件を調整することによって、前記断面を矩形に近い形状にすることも可能である。 The opening of the micro chamber 55 can be formed, for example, as an opening having a substantially elliptical cross section with a minor axis of about 20 nm to 200 nm and a major axis of about 0.2 μm to 5 μm. It is also possible to make the cross section nearly rectangular by adjusting the etching conditions.
　前記ウェットエッチングの処理時間を調整することによって、改質部５１と微小チャンバー５５とのサイズ差を小さくしたり大きくしたりすることが可能である。
　前記処理時間を短くすることによって、前記短径を数ｎｍ～数十ｎｍにすることも理論的には可能である。これとは逆に、前記処理時間を長くすることによって、前記短径を１μｍ～２μｍ程度に、前記長径を５μｍ～１０μｍ程度とすることもできる。 By adjusting the processing time of the wet etching, the size difference between the modified portion 51 and the minute chamber 55 can be reduced or increased.
It is theoretically possible to make the minor axis several nanometers to several tens of nanometers by shortening the treatment time. On the contrary, by increasing the processing time, the minor axis can be set to about 1 μm to 2 μm and the major axis can be set to about 5 μm to 10 μm.
　ここで、形成した微小チャンバー１の開口部に適切なマスキングを施し、微小チャンバー１を更にエッチングすることによって、前述の図６，７に示したような、開口部Ｕに凸部Ｅを備えた微小チャンバー１を形成できる。具体的には、例えば微小チャンバー１の開口部Ｕにおいて、凸部Ｅを形成する領域に、ＣＶＤ法等を用いてエッチングマスクを成膜する。次にガラス基板５９を選択的にエッチング可能なエッチング液を微小チャンバー１の内部に導入し、微小チャンバー１の内奥（内部の奥）の内壁をエッチングすることによって、微小チャンバー１の内奥の内径を大きくすることができる。このとき、微小チャンバー１のエッチングマスクの近傍（エッチングマスクに近い位置）では、ガラス基板がエッチングされ難いため、当該領域が凸部Ｅとなる。このように、微小チャンバー１を再加工することで、内奥における内径より小さな開口径を有する開口部Ｕを備えた微小チャンバー１を形成することができる。 Here, 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. At this time, in the vicinity of the etching mask of the micro chamber 1 (position close to the etching mask), the glass substrate is difficult to be etched, so that the region becomes the convex portion E. Thus, 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.
　つぎに、形成された流路５７を覆うように、部材５６をガラス基板５９の上面に貼り合わせる（図１３Ｄ）。
　形成された流路５７は、そのままでも流路５７として使用しうるが、図１３Ｄに示すように流路５７の上面に蓋をすることによって、流路５７に圧力をかけて送液することができる。 Next, the member 56 is bonded to the upper surface of the glass substrate 59 so as to cover the formed flow path 57 (FIG. 13D).
The formed channel 57 can be used as the channel 57 as it is, but by covering the upper surface of the channel 57 as shown in FIG. it can.
　上記の方法によって、液体Ｐを流入させる空間として、流路５７の代わりにウェルを形成することも可能である。 It is also possible to form a well instead of the flow path 57 as a space into which the liquid P flows by the above method.
　部材５６とガラス基板５９の上面とを貼り合わせる方法は、部材５６の材料に応じて、公知の方法で行えばよい。 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.
　部材５６の材料としては特に制限されず、ＰＤＭＳ、ＰＭＭＡ等の樹脂基板や、ガラス基板を使用することができる。また、部材５６の材料は、観察装置の光線（例えば可視光線）に対して透明であっても不透明であっても良い。つまり、部材５６の材料は、観察装置の光線を透過させる材料であってもよいし、観察装置の光線を透過させない材料であってもよい。脂質膜の形成のみを目的とする場合は、必ずしも観察装置の光線に対して透明である必要はない。観察装置の光線に対して透明な部材であれば、上面からの光学的手法による観察が可能となるため好ましい。 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. When 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.
　工程Ａ２及び工程Ａ３におけるエッチングとしては、ウェットエッチングやドライエッチングが適用できる。ウェットエッチングは例えば１％以下のフッ酸を用いるのが最も好ましいが、その他の酸や塩基性を有するエッチャントを用いてもよい。 As the etching in the process A2 and the process A3, wet etching or dry etching can be applied. For wet etching, for example, 1% or less of hydrofluoric acid is most preferably used, but other acid or basic etchants may be used.
　前記ドライエッチングのうち、等方性エッチング法としては、例えばバレル型プラズマエッチング、平行平板型プラズマエッチング、ダウンフロー型ケミカルドライエッチング、などの各種ドライエッチング方式が挙げられる。 Among the dry etching methods, 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.
　異方性ドライエッチング法としては、例えば反応性イオンエッチング(以下ＲＩＥ)を用いる方法として例えば平行平板型ＲＩＥ、マグネトロン型ＲＩＥ、ＩＣＰ型ＲＩＥ、ＮＬＤ型ＲＩＥなどの方法を適用することができ、ＲＩＥ以外にも例えば中性粒子ビームを用いたエッチングを適用することが可能である。異方性ドライエッチング法を用いる場合には、プロセス圧力を上げる等の手法によって、イオンの平均自由行程を短くし、等方性エッチングに近い加工も可能となる。 As the 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. When the anisotropic dry etching method is used, 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.
　使用するガスは例えばフロロカーボン系、ＳＦ系ガス、ＣＨＦ３、フッ素ガス、塩素ガス、など材料を化学的にエッチングすることができるガスが主で、それらに適宜その他ガス酸素、アルゴン、ヘリウムなどを混合し使用することが可能である。また、その他のドライエッチング方式による加工も可能である。 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.
　工程Ａ２において、より好適なエッチングは異方性エッチングであり、工程Ａ３において、より好適なエッチングは等方性エッチングである。 In step A2, more preferable etching is anisotropic etching, and in step A3, more preferable etching is isotropic etching.
＜脂質膜を形成するための基体の製造方法（第三態様）＞
　本発明の第三態様である製造方法の一例を、前述の第二実施形態の基体１０Ｂを例にとって説明する。この場合、前記製造方法は、図１６Ａ～図１６Ｅで示すように、単一の部材６９に、前記空間を構成する流路６７若しくは貫通孔を形成する工程Ｂ１（図１６Ｂ）と、ピコ秒オーダー以下のパルス時間幅を有するレーザーＬを、単一の部材６９の微小チャンバー６５を形成する領域に照射することによって、前記領域に改質部６１を形成する工程Ｂ２（図１６Ｃ）と、単一の部材６９から改質部６１をエッチングによって除去する工程Ｂ３（図１６Ｄ）と、を少なくとも有する。 <Method for Producing Substrate for Forming Lipid Membrane (Third Aspect)>
An example of the manufacturing method according to the third aspect of the present invention will be described by taking the substrate 10B of the second embodiment as an example. In this case, as shown in FIGS. 16A to 16E, the manufacturing method includes a step B1 (FIG. 16B) of forming a flow path 67 or a through hole constituting the space in a single member 69, and a picosecond order. Step B2 (FIG. 16C) for forming the modified portion 61 in the region by irradiating the region where the micro chamber 65 of the single member 69 is formed with a laser L having the following pulse time width, Step B3 (FIG. 16D) for removing the modified portion 61 from the member 69 by etching.
　部材６９の材料は、例えばシリコン、ガラス、石英、サファイアなどが挙げられる。これらの材料は、微小チャンバー６５の加工性に優れるので好ましい。なかでも、結晶方位による加工異方性の影響を受けにくい非結晶質である方が好ましい。
　更には、顕微鏡等の光学装置によって観察するには、ガラス、石英、サファイアを用いると、可視光線（波長０．３６μｍ～０．８３μｍ）に対して透明であるため、より好ましい。 Examples of the material of the member 69 include silicon, glass, quartz, and sapphire. These materials are preferable because they are excellent in workability of the micro chamber 65. 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 device such as a microscope, glass, quartz, or sapphire is more preferable because it is transparent to visible light (wavelength: 0.36 μm to 0.83 μm).
　また、部材６９の材料は、波長０．１μｍ～１０μｍを有する光のうち少なくとも一部の波長を有する光に対して透明であることが好ましい。
具体的には、加工用レーザー光として使用される一般的な波長領域（０．１μｍ～１０μｍ）の、少なくとも一部領域の光に対して透明であることが好ましい。このようなレーザー光に対して透明であることによって、後述するように、前記部材にレーザー照射して改質部を形成することができる。
　また、可視光領域（約０．３６μｍ～約０．８３μｍ）の光に対して透明であることが、より好ましい。可視光領域の光に対して透明であることによって、形成した脂質膜を、前記単一部材を透して肉眼で容易に観察することができる。
　なお、本発明における「透明」とは、前記部材に光を入射して、前記基材から透過光が得られる状態の全てをいう。
　図１６Ａ～図１６Ｅでは、単一の部材６９は透明なガラス基板である（以下、ガラス基板６９と呼ぶ）。 The material of the member 69 is preferably transparent to light having at least a part of light having a wavelength of 0.1 μm to 10 μm.
Specifically, it is preferably transparent to at least a part of light in a general wavelength region (0.1 μm to 10 μm) 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 μm to about 0.83 μm). 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.
In the present invention, “transparent” refers to all states in which light is incident on the member and transmitted light is obtained from the substrate.
In FIG. 16A to FIG. 16E, the single member 69 is a transparent glass substrate (hereinafter referred to as a glass substrate 69).
［工程Ｂ１］
　工程Ｂ１は、本発明の第二態様の製造方法における工程Ａ２と同様に行うことができる。すなわち、ガラス基板６９の上面に、フォトリソグラフィによってレジスト６２をパターニングして形成する（図１６Ａ）。つづいて、ドライエッチング、ウェットエッチング、又はサンドブラスト等の方法によって、ガラス基板６９の上面におけるレジスト６２が配されていない領域を、所定の深さに達するまで浸食して除去する（図１６Ｂ）。不要となったレジスト６２を剥離すると、流路６７が形成されたガラス基板５９が得られる。 [Step B1]
Process B1 can be performed similarly to process A2 in the manufacturing method of the 2nd aspect of this invention. That is, a resist 62 is patterned on the upper surface of the glass substrate 69 by photolithography (FIG. 16A). Subsequently, the region where the resist 62 is not disposed on the upper surface of the glass substrate 69 is eroded and removed until reaching a predetermined depth by a method such as dry etching, wet etching, or sand blasting (FIG. 16B). When the resist 62 that has become unnecessary is peeled off, the glass substrate 59 in which the flow path 67 is formed is obtained.
　また、工程Ｂ１において、流路６７として、ガラス基板６９の上面に凹部を形成する代わりに、基板内部に流路を形成してもよい。ガラス基板６９内の、流路６７となる領域をドリル（レーザードリル）やピコ秒オーダー以下の時間幅を有するレーザー改質とその選択的なエッチング等によってガラス基板６９の表面から掘削して貫通孔を形成することによって、流路６７を形成してもよい。このドリルによる掘削方法は、種々のエッチング法と組合わせて使用しても良い。 Further, in step B1, instead of forming a recess on the upper surface of the glass substrate 69, a channel may be formed inside the substrate as the channel 67. A region to be a flow path 67 in the glass substrate 69 is drilled from the surface of the glass substrate 69 by drilling (laser drill) or laser modification having a time width of the picosecond order or less and selective etching thereof. The flow path 67 may be formed by forming. This drilling method may be used in combination with various etching methods.
［工程Ｂ２］
　つぎに、ピコ秒オーダー以下のパルス時間幅を有するレーザーＬを、単一のガラス基板６９の微小チャンバー６５となる領域に照射することによって、前記領域に改質部６１を形成する。
　具体的には、本発明の第二態様の製造方法における工程Ａ１と同様に行うことができる。このとき、流路６７の側面に露呈する部位にレーザー光Ｌを集光照射して改質部６１を形成する場合は、液浸露光によってレーザー光Ｌを照射することが、より望ましい（図１６Ｃ）。前記側面に露呈する部位に形成される改質部６１の形状（微小チャンバー６５の端部の形状）の精度を高めることができる。 [Step B2]
Next, the modified part 61 is formed in the said area | region by irradiating the area | region used as the micro chamber 65 of the single glass substrate 69 with the laser L which has a pulse time width below a picosecond order.
Specifically, it can carry out similarly to process A1 in the manufacturing method of the 2nd aspect of this invention. At this time, when the modified portion 61 is formed by condensing and irradiating the laser beam L to the portion exposed to the side surface of the flow path 67, it is more preferable to irradiate the laser beam L by liquid immersion exposure (FIG. 16C). ). The accuracy of the shape of the modified portion 61 (the shape of the end portion of the micro chamber 65) formed in the portion exposed on the side surface can be increased.
［工程Ｂ３］
　つづいて、単一のガラス基板６９から、工程Ｂ２で形成した改質部６１をエッチングによって除去する（図１６Ｄ）。
　エッチング方法としては、ウェットエッチングが好ましい。流路６７（若しくは貫通孔）の側面に露呈する断面を有する改質部６１は、エッチング耐性が弱くなっているため、選択的又は優先的にエッチングすることができる。
　具体的には、本発明の第二態様の製造方法における工程Ａ３と同様に行うことができる。 [Step B3]
Subsequently, the modified portion 61 formed in step B2 is removed from the single glass substrate 69 by etching (FIG. 16D).
As an etching method, wet etching is preferable. The modified portion 61 having a cross section exposed on the side surface of the flow path 67 (or the through hole) has low etching resistance, and can be selectively or preferentially etched.
Specifically, it can carry out similarly to process A3 in the manufacturing method of the 2nd aspect of this invention.
　前記エッチングの結果、ナノオーダーの口径を有する微小チャンバー６５を、ガラス基板６９内の所定位置に、流路６７の側面に開口するように、形成することができる。 As a result of the etching, 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.
　つぎに、形成された流路６７を覆うように、部材６６をガラス基板６９の上面に貼り合わせる（図１６Ｅ）。
　形成された流路６７は、そのままでも流路６７として使用しうるが、図１６Ｅに示すように流路６７の上面に蓋をすることによって、流路６７に圧力をかけて送液することができる。 Next, the member 66 is bonded to the upper surface of the glass substrate 69 so as to cover the formed flow path 67 (FIG. 16E).
The formed flow path 67 can be used as the flow path 67 as it is. However, by covering the upper surface of the flow path 67 as shown in FIG. it can.
　上記の方法によって、液体Ｐを流入させる空間として、流路６７の代わりにウェルを形成することも可能である。 It is also possible to form a well instead of the flow path 67 as a space into which the liquid P flows by the above method.
　部材６６とガラス基板６９の上面とを貼り合わせる方法は、部材６６の材料に応じて、公知の方法で行えばよい。 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.
　部材６６の材料としては特に制限されず、ＰＤＭＳ、ＰＭＭＡ等の樹脂基板や、ガラス基板を使用することができる。また、部材６６の材料は、観察装置の光線（例えば可視光線）に対して透明であっても不透明であっても良い。つまり、部材５６の材料は、観察装置の光線を透過させる材料であってもよいし、観察装置の光線を透過させない材料であってもよい。脂質膜の形成のみを目的とする場合は、必ずしも観察装置の光線に対して透明である必要はない。観察装置の光線に対して透明な部材であれば、上面からの光学的手法による観察が可能となるため好ましい。 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. When 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.
　本発明の脂質膜を形成するための基体、及び前記基体の製造方法は、微小な脂質膜を基体に形成して、種々の観察、分析、及び測定を行うためのマイクロ流体デバイス等の使用及び製造に広く利用することができる。 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.
１…微小チャンバー、１ａ…微小チャンバーの端部（開口部）、２…ウェル、２ａ…ウェルの側面、２ｂ…ウェルの下面、Ｕ…開口部、Ｐ…液体、３…流路、４…基材、６…基板、１０Ａ，１０Ｂ，２０Ａ，３０Ａ,３０Ｂ…基体、１１…微小チャンバー、１４…基材、２１…微小チャンバー、２２…流路、２４…基材、２９…第二流路、５１…改質部、５２…レジスト、５５…微小チャンバー、５６…部材、５９…ガラス基板、６１…改質部、６２…レジスト、６５…微小チャンバー、６６…部材、６７…流路、６９…ガラス基板。 DESCRIPTION OF SYMBOLS 1 ... Micro chamber, 1a ... End part (opening part) of micro chamber, 2 ... Well, 2a ... Side surface of well, 2b ... Bottom surface of well, U ... Opening part, P ... Liquid, 3 ... Channel, 4 ... Base 6 ... Substrate, 10A, 10B, 20A, 30A, 30B ... Substrate, 11 ... Micro chamber, 14 ... Base material, 21 ... Micro chamber, 22 ... Channel, 24 ... Base material, 29 ... Second channel, DESCRIPTION OF SYMBOLS 51 ... Modified | denatured part, 52 ... Resist, 55 ... Micro chamber, 56 ... Member, 59 ... Glass substrate, 61 ... Modified | denatured part, 62 ... Resist, 65 ... Micro chamber, 66 ... Member, 67 ... Flow path, 69 ... Glass substrate.

Claims (6)

  1. 　脂質膜を形成するための基体であって、　
    　内壁面を有する基材と、
    　前記基材に内在され、脂質を含む液体が流入される空間と、
    　前記基材に内在され、前記空間を構成する前記基材の前記内壁面に設けられた開口部を有する微小チャンバーと、を含み、
    　前記基材のうち、少なくとも前記開口部を構成する部位は、単一の部材で構成されていることを特徴とする、脂質膜を形成するための基体。     A substrate for forming a lipid membrane,
    A base material having an inner wall surface;
    A space that is contained in the base material and into which a liquid containing lipid flows, and
    A micro-chamber having an opening provided in the inner wall surface of the base material, which is included in the base material and constitutes the space,
    The base | substrate for forming a lipid film characterized by the site | part which comprises the said opening part among the said base materials being comprised with the single member.
  2. 請求項１に記載の基体であって、
    　前記単一の部材が、波長０．１μｍ～１０μｍを有する光のうち少なくとも一部の波長を有する光を透過させることを特徴とする基体。     The substrate according to claim 1,
    The substrate characterized in that the single member transmits light having at least a part of light having a wavelength of 0.1 μm to 10 μm.
  3. 請求項１又は２に記載の基体であって、
    　前記単一の部材がシリコン、ガラス、石英、又はサファイアであることを特徴とする基体。     The substrate according to claim 1 or 2,
    A substrate characterized in that the single member is silicon, glass, quartz, or sapphire.
  4. 請求項１～３のいずれか一項に記載の基体であって、
    　前記微小チャンバーは、前記基体にレーザーを照射して改質された部分がエッチング処理によって除去されて形成されていることを特徴とする基体。     A substrate according to any one of claims 1 to 3,
    The micro chamber is formed by removing a portion modified by irradiating the base with a laser by an etching process.
  5. 　基体の製造方法であって、
    　ピコ秒オーダー以下のパルス時間幅を有するレーザーを、前記単一の部材の前記微小チャンバーを形成する領域に照射することによって、前記領域に改質部を形成し、
    　前記単一の部材に、前記空間を形成し、
    　前記単一の部材から前記改質部をエッチングによって除去することによって、請求項１～４のいずれか一項に記載の、脂質膜を形成するための基体を形成する、ことを特徴とする前記基体の製造方法。     A method for manufacturing a substrate, comprising:
    By irradiating a laser having a pulse time width of picosecond order or less to a region where the micro chamber of the single member is formed, a modified portion is formed in the region,
    Forming the space in the single member;
    The substrate for forming a lipid membrane according to any one of claims 1 to 4 is formed by removing the modified portion from the single member by etching. A method for manufacturing a substrate.
  6. 　基体の製造方法であって、
    　前記単一の部材に、前記空間を形成し、
    　ピコ秒オーダー以下のパルス時間幅を有するレーザーを、前記単一の部材の前記微小チャンバーを形成する領域に照射することによって、前記領域に改質部を形成し、
    　前記単一の部材から前記改質部をエッチングによって除去することによって、請求項１～４のいずれか一項に記載の、脂質膜を形成するための基体を形成する、ことを特徴とする前記基体の製造方法。     A method for manufacturing a substrate, comprising:
    Forming the space in the single member;
    By irradiating a laser having a pulse time width of picosecond order or less to a region where the micro chamber of the single member is formed, a modified portion is formed in the region,
    The substrate for forming a lipid membrane according to any one of claims 1 to 4 is formed by removing the modified portion from the single member by etching. A method for manufacturing a substrate.
PCT/JP2012/066574 2011-06-28 2012-06-28 Substrate for forming lipid membrane and method for producing substrate WO2013002339A1 (en)

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