WO2018173390A1 - Microwell-sealing cover plate and microchip - Google Patents

Microwell-sealing cover plate and microchip Download PDF

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Publication number
WO2018173390A1
WO2018173390A1 PCT/JP2017/045315 JP2017045315W WO2018173390A1 WO 2018173390 A1 WO2018173390 A1 WO 2018173390A1 JP 2017045315 W JP2017045315 W JP 2017045315W WO 2018173390 A1 WO2018173390 A1 WO 2018173390A1
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Prior art keywords
microwell
convex portion
lid plate
sealing lid
height
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PCT/JP2017/045315
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French (fr)
Japanese (ja)
Inventor
増原　慎
友照 阿部
加藤　義明
マルクオレル ブルン
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ソニー株式会社
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Publication of WO2018173390A1 publication Critical patent/WO2018173390A1/en

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  • This technology relates to a microwell sealing lid plate and a microchip.
  • Patent Document 1 a microwell is formed by covering a cell solution on a dish with a film of ethylene vinyl alcohol (EVOH) having gas barrier properties attached to the surface of a microchip made of polydimethylsiloxane (PDMS).
  • EVOH ethylene vinyl alcohol
  • PDMS polydimethylsiloxane
  • Non-Patent Document 1 discloses a technique for measuring the oxygen consumption of cells in a microwell using an oxygen sensor film.
  • the main object of the present technology is to provide a microwell sealing lid plate capable of sealing the microwell.
  • the present technology includes a projecting portion for microwell sealing, and the projecting portion has E ⁇ 5 GPa or less, where Young's modulus is E, and a cross-sectional area in a direction perpendicular to the projecting portion.
  • E Young's modulus
  • a lid plate is provided.
  • the convex portion may include a tapered side surface.
  • the convex portion may have a tapered tip.
  • the convex portion may be formed of a resin having an oxygen permeability at 25 ° C.
  • the convex portion may be formed of a resin having an oxygen permeability at 25 ° C. of 0.1 mL ⁇ cm / m 2 ⁇ 24 h ⁇ atm or less.
  • the convex portion may be formed of an ethylene-vinyl alcohol copolymer resin or a polyvinylidene chloride resin.
  • the convex portion may be provided with a film of a metal, an inorganic compound, or a parylene resin.
  • the convex portion may include a parylene resin film on a metal or inorganic compound film.
  • An antibody or a fluorescent dye may be immobilized on the surface of the convex portion.
  • a reagent may be fixed or coated on the surface of the convex portion.
  • the convex portion may include an oxygen sensor.
  • the present technology includes a substrate on which a microwell is formed and a lid plate on which a convex portion is formed, and the convex portion has a Young's modulus at least at a tip portion corresponding to the microwell.
  • E E ⁇ 5 GPa or less
  • the average cross-sectional area obtained by averaging the cross-sectional area of the cross section in the direction orthogonal to the convex portion over the height direction is S (mm 2 )
  • the height is H
  • the convex section has a circular cross-sectional shape at the tip
  • the microwell has a cylindrical shape
  • the diameter of the tip of the convex is ⁇ 1 ( ⁇ m)
  • the diameter of the microwell is ⁇ 2 ( ⁇ m).
  • ⁇ 1 ⁇ ⁇ 2-10 ( ⁇ m) may be satisfied.
  • the height of the convex portion corresponding to the microwell having ⁇ 2 of 50 ⁇ m may be 150 ⁇ m or more.
  • the height of the convex portion corresponding to the microwell having ⁇ 2 of 40 ⁇ m may be 95 ⁇ m or more.
  • the height of the convex portion corresponding to the microwell having ⁇ 2 of 30 ⁇ m may be 55 ⁇ m or more.
  • microwell sealing lid plate capable of sealing a microwell.
  • the effect of this technique is not necessarily limited to the effect described here, The effect described in this specification may be sufficient.
  • FIG. 1 is a schematic diagram of a microchip 1 having a cover plate 10 and a substrate 20. As shown in FIG. 1, a microwell 21 is formed on the substrate 20. The microwell 21 can accommodate microparticles 30 such as cells. Further, the lid plate 10 includes a convex portion 11 to be inserted into the microwell 21. By inserting the convex portion 11 into the microwell 21, the microwell 21 is sealed, and liquid movement between the microwells 21 can be blocked.
  • FIG. 2 is a schematic diagram showing the process of sealing the microwell.
  • FIG. 2A shows a cover plate 10 and a microwell 21 having a convex portion 11.
  • FIG. 2B the microwell 21 is sealed by inserting the convex portion 11 of the cover plate 10 into the microwell 21. Thereafter, as shown in FIG. 2C, the sealing can be released by removing the convex portion 11 from the microwell 21.
  • a position shift of up to 5 ⁇ m may occur between the corresponding microwell and the convex portion due to shrinkage of the cover plate or substrate or manufacturing error.
  • a microchip has a large number of microwells, and in order to seal all the microwells by inserting convex portions into all the microwells, it is necessary to take measures to eliminate the above-mentioned misalignment. It is.
  • the convex portion provided in the microwell sealing lid plate of the present technology has a structure capable of sealing the microwell even when there is a maximum positional deviation of 5 ⁇ m between the microwell and the convex portion.
  • the convex portion has E ⁇ 5 GPa or less when the Young's modulus is E, and the average cross section of the cross section in the direction orthogonal to the convex portion is averaged over the height direction.
  • the area is S (mm 2 ) and the height is H (mm)
  • the convex portion is formed of a material having a Young's modulus E of 5 GPa or less, and thus has a deformable elasticity.
  • the rigidity of the convex portion is G
  • the shearing force applied in the lateral direction to the convex portion is F
  • the displacement in the lateral direction is ⁇ X
  • ⁇ X G ⁇ 1 ⁇ F ⁇ (H / S) is there.
  • the rigidity G is generally 30 to 40% with respect to the Young's modulus E, and is about 1.5 Gpa or less for plastic resin and about 0.001 Gpa for rubber material.
  • the required displacement ⁇ X is set to 5 ⁇ m
  • the realistic shearing force is assumed to be 10 g
  • the rigidity G is calculated as an assumed maximum value of 1.5 Gpa, H / S ⁇ 75 (mm ⁇ 1 ) If so, ⁇ X ⁇ 5 ⁇ m, and a maximum positional deviation of 5 ⁇ m can be corrected. Therefore, a convex portion satisfying E ⁇ 5 GPa and H / S ⁇ 75 (mm ⁇ 1 ) can be easily fitted into the microwell by elastic deformation even when there is a maximum positional deviation of 5 ⁇ m from the corresponding microwell. Clogging and microwells can be sealed.
  • FIG. 3 is a schematic diagram showing a state in which the convex portion 11 is inserted into the microwell 21.
  • the convex portions 11a and 11b are elastically deformed, respectively. However, it fits into the microwell 21a and the microwell 21b.
  • the material for forming the convex portion is not particularly limited as long as the Young's modulus is E ⁇ 5 GPa or less, but plastic resins such as acrylic and polystyrene, and silicone rubbers such as polydimethylsiloxane (PDMS) are suitable. From the viewpoint of manufacturing efficiency, it is preferable to form not only the convex portions but also the entire lid plate with a material of E ⁇ 5 GPa or less.
  • the shape of the convex portion is not particularly limited as long as the microwell can be sealed, and is cylindrical, elliptical, polygonal, frustoconical, inverted frustoconical, elliptical frustum, inverted elliptical frustum, polygonal frustum, A reverse polygon frustum shape, a taper shape, a reverse taper shape, etc. are mentioned.
  • the convex portion has a side surface that is tapered from the root side, which is the lid plate side, toward the tip portion, that is, a tapered side surface.
  • the tip of the convex portion is more tapered.
  • the shape of the convex portion that satisfies the above-mentioned H / S ⁇ 75 (mm ⁇ 1 ) is, for example, a cylindrical shape having a diameter of 50 ⁇ m and a height H of 150 ⁇ m or more, and a diameter of 40 ⁇ m. And a columnar shape having a height H of 95 ⁇ m or more and a columnar shape having a diameter of 30 ⁇ m and a height of 55 ⁇ m or more.
  • the height of the convex portion is preferably 150 ⁇ m or more for a microwell having a diameter of 50 ⁇ m, and the height of the convex portion is preferably 95 ⁇ m or more for a microwell having a diameter of 40 ⁇ m.
  • the height of the convex portion is preferably 55 ⁇ m or more for a microwell having a diameter of 30 ⁇ m.
  • the microchip of the present technology includes the substrate and the lid plate.
  • a material for forming the substrate a material known in the art can be used. Examples thereof include plastic resins such as acrylic and polystyrene, silicone rubbers such as PDMS, and glass.
  • the shape of the microwell formed on the substrate is not particularly limited as long as it can be sealed by the convex portion formed on the cover plate, and is cylindrical, elliptical, polygonal, frustoconical, inverted frustoconical, or elliptical cone.
  • a trapezoid, a reverse elliptical frustum, a polygonal frustum, a reverse polygon frustum, a taper shape, a reverse taper shape, etc. are mentioned.
  • At least the tip of the convex portion formed on the cover plate is at a position corresponding to the microwell formed on the substrate. That is, when the lid plate and the substrate are overlapped, it is preferable that the tip of the convex portion is located inside the corresponding microwell. With such a configuration, even if there is a positional shift between the corresponding convex portion and the microwell, the convex portion is fitted into the microwell while being elastically deformed, and the microwell is sealed. It is possible.
  • the convex portion when the convex portion is inserted into the microwell in a state where the convex portion and the microwell are coaxially located, it is preferable that a gap of 5 ⁇ m or more is provided between the tip portion of the convex portion and the inner side surface of the microwell. .
  • FIG. 4 is a schematic diagram showing the tip 111 and the microwell 21 of the protrusion 11.
  • the diameter of the tip portion 111 is ⁇ 1. ( ⁇ m), and when the diameter of the microwell 21 is ⁇ 2 ( ⁇ m), it is preferable that ⁇ 1 ⁇ ⁇ 2-10 ( ⁇ m).
  • FIG. 5 is a schematic diagram showing the convex portion 11 and the microwell 21.
  • the convex portion 11 shown in FIG. 5 has a cylindrical shape except for the tapered tip portion 111.
  • the microwell 21 has a cylindrical shape.
  • the height H of the convex portion 11 is 80 ⁇ m.
  • the height Ht of the tapered portion of the tip 111 is 30 ⁇ m.
  • the diameter ⁇ 1 of the tip 111 is 10 ⁇ m.
  • the diameter ⁇ 3 of the cylindrical portion of the convex portion 11 is 30 ⁇ m.
  • the diameter ⁇ 2 of the microwell 21 is 20 ⁇ m.
  • the depth Dw of the microwell 21 is 35 ⁇ m.
  • the convex portion 11 enters 15 ⁇ m into the microwell 21 and seals the microwell 21.
  • the substrate size may be 5.0 mm ⁇ 5.0 mm
  • the number of wells may be 500 ⁇ 500
  • the microwell pitch may be 80 ⁇ m in the vertical and horizontal directions.
  • the size of the cover plate may be 4.5 mm ⁇ 4.5 mm, and the number of protrusions and the pitch may be matched with the number of wells and the pitch of the substrate.
  • a space is formed between the substrate and the lid plate in a state where the convex portion is inserted into the microwell. Referring to FIG. 3 again, a space 40 is formed between the cover plate 10 and the substrate 20.
  • the liquid when a liquid such as a culture solution is stored in the microwell 21 together with the microparticles to be analyzed, the liquid may overflow from the microwell 21 by inserting the convex portion 11 into the microwell 21. is there.
  • the space 40 With the space 40 with the microchip of the present technology, it is possible to prevent the liquid overflowing from the microwell 21 from escaping into the space 40 and remaining between the lid plate 10 and the substrate 20.
  • the microchip of the present technology allows the liquid overflowing from the microwell to escape by the space formed between the cover plate and the substrate, and improves the sealing performance of the microwell.
  • Second embodiment (configuration example including a convex portion formed of a highly airtight material)
  • gas transfer as well as liquid transfer between the microwells is possible. It is also necessary to shut off.
  • high air tightness to the convex portion for sealing the microwell.
  • a resin having an oxygen permeability at 25 ° C. of 0.5 mL ⁇ cm / m 2 ⁇ 24 h ⁇ atm or less is preferable. If the material has an oxygen permeability in such a range, a change in the amount of gas dissolved in the liquid in the microwell can be measured.
  • a resin having an oxygen permeability at 25 ° C. of 0.1 mL ⁇ cm / m 2 ⁇ 24 h ⁇ atm or less is preferred.
  • the oxygen permeability is also referred to as “oxygen permeability” and can be calculated based on JIS K 7126 (reference: Japan Plastics Industry Federation “Plastics” 51 (6), 119-127, 2000-06. ).
  • the convex portion is formed of an elastically deformable material having a Young's modulus E ⁇ 5 GPa or less.
  • the resin having E ⁇ 5 GPa or less and oxygen permeability at 25 ° C. of 0.1 mL ⁇ cm / m 2 ⁇ 24 h ⁇ atm or less is an ethylene-vinyl alcohol copolymer (EVOH) resin or polyvinylidene chloride (PVDC). Resins are preferred.
  • Third embodiment (configuration example including a coating film on a convex portion)
  • a film having a gas barrier property on the convex portion.
  • the material for forming the film is not particularly limited as long as it has a gas barrier property, but is preferably a metal such as aluminum or aluminum oxide, an inorganic compound such as silicon dioxide or silicon nitride, or a parylene resin (paraxylylene resin). These materials have high gas barrier properties, and can impart high airtightness that can measure changes in the amount of dissolved gas in the microwell to the protrusions.
  • FIG. 6 is a cross-sectional view showing the convex portion 11 including a coating film having gas barrier properties.
  • FIG. 6A shows an example in which the convex portion 11 includes a metal film (metal vapor deposition film) 51 such as aluminum oxide.
  • Parylene resin is suitable not only for gas barrier properties but also for high biocompatibility, and is therefore suitable for handling biological samples such as cells in a microwell.
  • FIG. 6B shows an example in which the convex portion 11 includes a parylene resin coating 52.
  • a product “Parylene C” manufactured by Specialty Coating System can be deposited at room temperature using a vacuum deposition apparatus.
  • the convex portion may be provided with a parylene resin film on a metal or inorganic compound film. Since the parylene resin also has chemical resistance, the biocompatibility and chemical resistance of the convex portion can be improved by laminating the parylene resin film.
  • FIG. 6C shows an example in which the convex portion 11 includes a parylene resin film 52 on a metal film 51.
  • the convex part may be provided with a film having gas barrier properties on the entire surface, but it is sufficient that at least the part in contact with the microwell when the microwell is inserted has the film.
  • the tip of the convex portion is tapered, only the tapered portion may have a coating.
  • the corresponding microwell when the convex portion has a high airtightness, the corresponding microwell preferably has a high airtightness. Thereby, the inflow and outflow of the gas in a microwell can be interrupted
  • a gas barrier film in the microwell As a means for imparting high airtightness to the microwell, it is conceivable to form a gas barrier film in the microwell. From the viewpoint of manufacturing cost, a substrate having a microwell is formed of a material having a high gas barrier property. It is preferable to do. Since the substrate material does not necessarily need to be elastically deformable, glass may be used.
  • Fourth embodiment (configuration example having antibody, fluorescent dye or reagent on the surface of the convex portion)
  • An antibody or a fluorescent dye may be immobilized on the surface of the convex part, and a reagent may be fixed or coated.
  • a reagent may be fixed or coated.
  • the substance present in the microwell can be detected, captured and recovered.
  • cell-derived substances can be detected and captured.
  • the secretion of the cell may be detected and captured by immersing the convex portion according to the present embodiment in a microwell containing a culture solution containing cells.
  • cells may be crushed in a microwell to elute target substances such as nucleic acids and proteins, and the protrusions according to the present embodiment may be immersed therein to detect and capture the substances in the cells.
  • FIG. 7 is a cross-sectional view schematically showing an example of detecting cell secretions.
  • the convex portion 11 includes a parylene resin coating 52, and the antibody 55 is immobilized on the parylene resin coating 52.
  • FIG. 7B shows a state in which the convex portion 11 is inserted into the microwell 21.
  • the antibody 55 is bound to the cell secretion 63 in the microwell 21.
  • FIG. 7C shows a state in which the convex portion 11 is pulled out from the microwell 21.
  • FIG. 7D shows a state in which the cell secretion 63 bound to the antibody 55 present on the surface of the convex portion 11 is reacted with the antibody 72 present on the surface of the fluorescent bead 71.
  • the microchip of this embodiment can be used to optically detect a target substance.
  • the convex portion may have an antibody, a fluorescent dye or a reagent on the entire surface, but the portion inserted into the microwell only needs to have at least the antibody, the fluorescent dye or the reagent.
  • the tip of the convex portion is tapered, only the tapered portion may have an antibody, a fluorescent dye, or a reagent.
  • the convex portion includes an antibody, a fluorescent dye, or a reagent. It is preferable to do. For example, after detecting a target substance by inserting a convex portion coated with a reagent into a microwell containing cells, the convex portion is pulled out and another reagent is applied with the cells remaining in the microwell. By inserting the projected portions, it is possible to continuously observe a plurality of chemical reactions targeting a specific cell.
  • the convex portion may include an oxygen sensor.
  • the amount of oxygen in the microwell can be measured. For example, by immersing a convex portion having an oxygen sensor in a microwell containing a culture solution containing cells, the oxygen consumption of the cells can be measured from the change in the dissolved oxygen amount in the microwell.
  • the configuration according to the second embodiment or the third embodiment is adopted to increase the airtightness of the microwell. Is preferred.
  • the oxygen sensor is not particularly limited as long as measurement in a microwell is possible, and a known oxygen sensor may be used.
  • a configuration will be described in which a film-like oxygen sensor formed using platinum octaethylformylphylline (PlatinumtaOctaethylprophyrin, PtOEP) is provided on a convex portion.
  • FIG. 8 is a cross-sectional view showing a convex portion provided with a film-like oxygen sensor.
  • FIG. 8A shows an example in which the convex portion 11 includes a parylene resin coating 52 on a metal coating 51.
  • FIG. 8B shows an example in which an oxygen sensor 53 containing PtOEP is provided on a parylene resin coating.
  • the oxygen sensor may be provided on the entire convex portion, but may be provided at least in a portion to be inserted into the microwell.
  • the tip of the convex portion is tapered, only the tapered portion may include an oxygen sensor.
  • the oxygen sensor After the oxygen sensor is inserted into the microwell, laser light having a wavelength of about 350 nm collected by the objective lens is irradiated onto the oxygen sensor from the bottom surface of the substrate of the microwell, and phosphorescence having a wavelength of about 615 nm is emitted.
  • laser light having a wavelength of about 350 nm collected by the objective lens is irradiated onto the oxygen sensor from the bottom surface of the substrate of the microwell, and phosphorescence having a wavelength of about 615 nm is emitted.
  • FIG. 8C shows an example in which a protective film 54 is provided on the oxygen sensor 53.
  • the protective film is preferably formed of a material having a high oxygen permeability and low autofluorescence so that measurement by the oxygen sensor is not hindered.
  • a material having a high oxygen permeability and low autofluorescence so that measurement by the oxygen sensor is not hindered.
  • polystyrene can be used.
  • the protective film may be provided as long as the oxygen sensor can be protected, and may not be provided on the entire convex portion.
  • the convex portion has an oxygen sensor.
  • the oxygen consumption is measured using the convex part having an oxygen sensor. It is possible to analyze specific cells continuously from various viewpoints.
  • a unique address may be assigned to each of the convex portion and the microwell.
  • a projection for microwell sealing is provided,
  • the convex portion is When Young's modulus is E, E ⁇ 5 GPa or less, H / S ⁇ 75, where S (mm 2 ) is the average cross-sectional area obtained by averaging the cross-sectional areas in the direction perpendicular to the convex portions over the height direction, and H (mm) is the height.
  • the convex portion includes a metal, inorganic compound, or parylene resin coating.
  • the convex portion includes a parylene resin coating on a metal or inorganic compound coating.
  • microwell sealing lid plate according to any one of [1] to [8], wherein an antibody or a fluorescent dye is immobilized on the surface of the convex portion.
  • an antibody or a fluorescent dye is immobilized on the surface of the convex portion.
  • Young's modulus is E, E ⁇ 5 GPa or less, H / S ⁇ 75, where S (mm 2 ) is the average cross-sectional area obtained by averaging the cross-sectional areas in the direction perpendicular to the convex portions over the height direction, and H (mm) is the height.
  • the distance between the tip of the convex portion and the inner surface of the microwell is 5 ⁇ m or more.
  • the microchip according to [12].
  • the cross-sectional shape of the tip of the convex portion is circular,
  • the microwell is cylindrical, [12] or [13], wherein ⁇ 1 ⁇ ⁇ 2-10 ( ⁇ m) when the diameter of the tip of the convex portion is ⁇ 1 ( ⁇ m) and the diameter of the microwell is ⁇ 2 ( ⁇ m).

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Abstract

The present invention provides a microwell-sealing cover plate that is capable of tightly sealing microwells. This microwell-sealing cover plate is provided with protrusions for sealing microwells, wherein each of the protrusions satisfies the condition: E≤5 GPa, where E represents Young's modulus, and further satisfies the condition: H/S≥75 (mm-1), where S (mm2) represents an average cross-sectional area obtained by averaging, along the height direction, the cross-sectional areas of cross-sections in the direction orthogonal to the protrusion, and H (mm) represents the height.

Description

マイクロウェル封止用蓋板及びマイクロチップMicrowell sealing lid plate and microchip
　本技術は、マイクロウェル封止用蓋板及びマイクロチップに関する。 This technology relates to a microwell sealing lid plate and a microchip.
　マイクロウェルを用いて細胞を解析する手法が種々提案されている。例えば、特許文献１には、ポリジメチルシロキサン（ＰＤＭＳ）製のマイクロチップ表面にガスバリア性を有するエチレンビニルアルコール（ＥＶＯＨ）のフィルムを付着した状態で、ディッシュ上の細胞液に覆い被せて、マイクロウェル内の細胞の酸素消費量を測定する技術が開示されている。また、非特許文献１には、酸素センサー膜を用いてマイクロウェル内での細胞の酸素消費量を測定する技術が開示されている。 Various techniques for analyzing cells using microwells have been proposed. For example, in Patent Document 1, a microwell is formed by covering a cell solution on a dish with a film of ethylene vinyl alcohol (EVOH) having gas barrier properties attached to the surface of a microchip made of polydimethylsiloxane (PDMS). A technique for measuring the oxygen consumption of cells in the cell is disclosed. Non-Patent Document 1 discloses a technique for measuring the oxygen consumption of cells in a microwell using an oxygen sensor film.
特開２０１２－１９６１９８号公報JP 2012-196198 A
　しかしながら、マイクロウェルを密封して隣接するマイクロウェル間の液体移動を遮断することで、他の細胞とのコミュニケーションを断然した状態で細胞解析を行う手法は未だ提案されていない。 However, there has not yet been proposed a method for performing cell analysis in a state in which communication with other cells is unsettled by sealing a microwell and blocking liquid movement between adjacent microwells.
　そこで、本技術は、マイクロウェルを密封することが可能なマイクロウェル封止用蓋板を提供することを主目的とする。 Therefore, the main object of the present technology is to provide a microwell sealing lid plate capable of sealing the microwell.
　すなわち、本技術は、マイクロウェル封止用の凸部を備え、前記凸部は、ヤング率をＥとした場合に、Ｅ≦５ＧＰａ以下であり、前記凸部と直交する方向の断面の断面積を高さ方向に亘って平均化した平均断面積をＳ（ｍｍ２）とし、高さをＨ（ｍｍ）とした場合に、Ｈ／Ｓ≧７５（ｍｍ－１）である、マイクロウェル封止用蓋板を提供する。
　前記凸部は、テーパー状の側面を備えてもよい。
　前記凸部は、先端部がテーパー状であってもよい。
　前記凸部は、２５℃における酸素透過率が０．５ｍＬ・ｃｍ／ｍ２・２４ｈ・ａｔｍ以下の樹脂で形成されてもよい。
　前記凸部は、２５℃における酸素透過率が０．１ｍＬ・ｃｍ／ｍ２・２４ｈ・ａｔｍ以下の樹脂で形成されてもよい。
　前記凸部は、エチレン－ビニルアルコール共重合体樹脂又はポリ塩化ビニリデン樹脂で形成されてもよい。
　前記凸部は、金属、無機化合物又はパリレン樹脂の被膜を備えてもよい。
　前記凸部は、金属又は無機化合物の被膜の上にパリレン樹脂の被膜を備えてもよい。
　前記凸部の表面に抗体又は蛍光色素が固定化されていてもよい。
　前記凸部の表面に試薬が固着又は塗布されていてもよい。
　前記凸部は、酸素センサーを備えてもよい。 That is, the present technology includes a projecting portion for microwell sealing, and the projecting portion has E ≦ 5 GPa or less, where Young's modulus is E, and a cross-sectional area in a direction perpendicular to the projecting portion. Is a microwell sealing where H / S ≧ 75 (mm −1 ), where S (mm 2 ) is the average cross-sectional area averaged over the height direction and H (mm) is the height A lid plate is provided.
The convex portion may include a tapered side surface.
The convex portion may have a tapered tip.
The convex portion may be formed of a resin having an oxygen permeability at 25 ° C. of 0.5 mL · cm / m 2 · 24 h · atm or less.
The convex portion may be formed of a resin having an oxygen permeability at 25 ° C. of 0.1 mL · cm / m 2 · 24 h · atm or less.
The convex portion may be formed of an ethylene-vinyl alcohol copolymer resin or a polyvinylidene chloride resin.
The convex portion may be provided with a film of a metal, an inorganic compound, or a parylene resin.
The convex portion may include a parylene resin film on a metal or inorganic compound film.
An antibody or a fluorescent dye may be immobilized on the surface of the convex portion.
A reagent may be fixed or coated on the surface of the convex portion.
The convex portion may include an oxygen sensor.
　また、本技術は、マイクロウェルが形成された基板と、凸部が形成された蓋板と、を備え、前記凸部は、少なくとも先端部が前記マイクロウェルに対応する位置にあり、ヤング率をＥとした場合に、Ｅ≦５ＧＰａ以下であり、前記凸部と直交する方向の断面の断面積を高さ方向に亘って平均化した平均断面積をＳ（ｍｍ２）とし、高さをＨ（ｍｍ）とした場合に、Ｈ／Ｓ≧７５（ｍｍ－１）である、マイクロチップを提供する。
　前記凸部と前記マイクロウェルとが同軸上に位置する状態で前記凸部を前記マイクロウェルに挿入した時に、前記凸部の先端部と前記マイクロウェルの内側面との間に５μｍ以上の空隙を有してもよい。
　前記凸部の先端部の横断面形状が円形であり、前記マイクロウェルが円柱形であり、前記凸部の先端部の直径をΦ１（μｍ）とし、前記マイクロウェルの直径をΦ２（μｍ）とした場合に、Φ１≦Φ２－１０（μｍ）であってもよい。
　前記Φ２が５０μｍの前記マイクロウェルに対応する前記凸部の高さが１５０μｍ以上であってもよい。
　前記Φ２が４０μｍの前記マイクロウェルに対応する前記凸部の高さが９５μｍ以上であってもよい。
　前記Φ２が３０μｍの前記マイクロウェルに対応する前記凸部の高さが５５μｍ以上であってもよい。 In addition, the present technology includes a substrate on which a microwell is formed and a lid plate on which a convex portion is formed, and the convex portion has a Young's modulus at least at a tip portion corresponding to the microwell. When E is E ≦ 5 GPa or less, the average cross-sectional area obtained by averaging the cross-sectional area of the cross section in the direction orthogonal to the convex portion over the height direction is S (mm 2 ), and the height is H Provided is a microchip where H / S ≧ 75 (mm −1 ) when (mm).
When the convex portion and the microwell are coaxially positioned and the convex portion is inserted into the microwell, a gap of 5 μm or more is formed between the tip portion of the convex portion and the inner side surface of the microwell. You may have.
The convex section has a circular cross-sectional shape at the tip, the microwell has a cylindrical shape, the diameter of the tip of the convex is Φ1 (μm), and the diameter of the microwell is Φ2 (μm). In this case, Φ1 ≦ Φ2-10 (μm) may be satisfied.
The height of the convex portion corresponding to the microwell having Φ2 of 50 μm may be 150 μm or more.
The height of the convex portion corresponding to the microwell having Φ2 of 40 μm may be 95 μm or more.
The height of the convex portion corresponding to the microwell having Φ2 of 30 μm may be 55 μm or more.
　本技術によれば、マイクロウェルを密封することが可能なマイクロウェル封止用蓋板を提供することができる。なお、本技術の効果は、ここに記載された効果に必ずしも限定されるものではなく、本明細書中に記載されたいずれかの効果であってもよい。 According to the present technology, it is possible to provide a microwell sealing lid plate capable of sealing a microwell. In addition, the effect of this technique is not necessarily limited to the effect described here, The effect described in this specification may be sufficient.
蓋板及び基板を有するマイクロチップを示す模式図である。It is a schematic diagram which shows the microchip which has a cover plate and a board | substrate. マイクロウェルを密封する過程を示す模式図である。It is a schematic diagram which shows the process of sealing a microwell. 凸部をマイクロウェル内へ挿入した状態を示す模式図である。It is a schematic diagram which shows the state which inserted the convex part in the microwell. 凸部の先端部とマイクロウェルを示す模式図である。It is a schematic diagram which shows the front-end | tip part of a convex part, and a microwell. 凸部とマイクロウェルを示す模式図である。It is a schematic diagram which shows a convex part and a microwell. ガスバリア性を有する被膜を備える凸部を示す断面図である。It is sectional drawing which shows a convex part provided with the film which has gas barrier property. 細胞分泌物を検出する例を模式的に示す断面図である。It is sectional drawing which shows typically the example which detects a cell secretion. 酸素センサーを備える凸部を示す断面図である。It is sectional drawing which shows a convex part provided with an oxygen sensor.
　以下、本技術を実施するための好適な形態について図面を参照しながら説明する。なお、以下に説明する実施形態は、本技術の代表的な実施形態を示したものであり、これにより本技術の範囲が狭く解釈されることはない。説明は以下の順序で行う。
　１．第１実施形態
　２．第２実施形態（高気密性材料で形成された凸部を備える構成例）
　３．第３実施形態（凸部に被膜を備える構成例）
　４．第４実施形態（凸部の表面に抗体、蛍光色素又は試薬を有する構成例）
　５．第５実施形態（凸部に酸素センサーを備える構成例） Hereinafter, preferred embodiments for carrying out the present technology will be described with reference to the drawings. In addition, embodiment described below shows typical embodiment of this technique, and, thereby, the range of this technique is not interpreted narrowly. The description will be made in the following order.
1. First embodiment2. Second embodiment (configuration example including a convex portion formed of a highly airtight material)
3. Third embodiment (configuration example including a coating film on a convex portion)
4). Fourth embodiment (configuration example having antibody, fluorescent dye or reagent on the surface of the convex portion)
5). Fifth embodiment (configuration example including an oxygen sensor on the convex portion)
１．第１実施形態
　図１は、蓋板１０及び基板２０を有するマイクロチップ１の模式図である。図１に示すように、基板２０にはマイクロウェル２１が形成されている。マイクロウェル２１には、細胞などの微小粒子３０が収容されうる。また、蓋板１０は、マイクロウェル２１に挿入される凸部１１を備える。凸部１１をマイクロウェル２１に挿入することでマイクロウェル２１が密封され、マイクロウェル２１間の液体移動を遮断することができる。 1. First Embodiment FIG. 1 is a schematic diagram of a microchip 1 having a cover plate 10 and a substrate 20. As shown in FIG. 1, a microwell 21 is formed on the substrate 20. The microwell 21 can accommodate microparticles 30 such as cells. Further, the lid plate 10 includes a convex portion 11 to be inserted into the microwell 21. By inserting the convex portion 11 into the microwell 21, the microwell 21 is sealed, and liquid movement between the microwells 21 can be blocked.
　図２は、マイクロウェルを密封する過程を示す模式図である。図２Ａは、凸部１１を備える蓋板１０とマイクロウェル２１を示している。図２Ｂに示すように、蓋板１０の凸部１１をマイクロウェル２１内に挿入することでマイクロウェル２１を密封する。その後、図２Ｃに示すようにマイクロウェル２１から凸部１１を抜くことで密封を解除できる。 FIG. 2 is a schematic diagram showing the process of sealing the microwell. FIG. 2A shows a cover plate 10 and a microwell 21 having a convex portion 11. As shown in FIG. 2B, the microwell 21 is sealed by inserting the convex portion 11 of the cover plate 10 into the microwell 21. Thereafter, as shown in FIG. 2C, the sealing can be released by removing the convex portion 11 from the microwell 21.
　対応するマイクロウェルと凸部との間において、蓋板又は基板の収縮や製造誤差などによって最大５μｍの位置ずれが発生する可能性がある。通常、マイクロチップには多数のマイクロウェルが形成されており、多数のマイクロウェル全てに凸部を挿入して全てのマイクロウェルを密封するためには、上記位置ずれを解消するための対策が必要である。 There is a possibility that a position shift of up to 5 μm may occur between the corresponding microwell and the convex portion due to shrinkage of the cover plate or substrate or manufacturing error. Usually, a microchip has a large number of microwells, and in order to seal all the microwells by inserting convex portions into all the microwells, it is necessary to take measures to eliminate the above-mentioned misalignment. It is.
　そこで、本技術のマイクロウェル封止用蓋板が備える凸部は、マイクロウェルと凸部との間に最大５μｍの位置ずれが存在してもマイクロウェルを密封できる構造を備える。具体的には、上記凸部は、ヤング率をＥとした場合に、Ｅ≦５ＧＰａ以下であり、前記凸部と直交する方向の断面の断面積を高さ方向に亘って平均化した平均断面積をＳ（ｍｍ２）とし、高さをＨ（ｍｍ）とした場合に、Ｈ／Ｓ≧７５（ｍｍ－１）である。 Therefore, the convex portion provided in the microwell sealing lid plate of the present technology has a structure capable of sealing the microwell even when there is a maximum positional deviation of 5 μm between the microwell and the convex portion. Specifically, the convex portion has E ≦ 5 GPa or less when the Young's modulus is E, and the average cross section of the cross section in the direction orthogonal to the convex portion is averaged over the height direction. When the area is S (mm 2 ) and the height is H (mm), H / S ≧ 75 (mm −1 ).
　凸部は、ヤング率Ｅが５ＧＰａ以下の材料で形成されることで、変形可能な弾性を有している。ここで、凸部の剛性率をＧ、凸部に対して横方向に加わるせん断力をＦ、横方向への変位をΔＸと表すと、ΔＸ＝Ｇ－１×Ｆ×（Ｈ／Ｓ）である。剛性率Ｇは一般的にヤング率Ｅに対して３０～４０％であり、プラスチック樹脂で約１．５Ｇｐａ以下、ゴム材料で０．００１Ｇｐａ前後となる。この場合、必要な変位ΔＸを５μｍと設定して、現実的なせん断力を１０ｇと仮定し、剛性率Ｇを想定最大値の１．５Ｇｐａとして計算すると、Ｈ／Ｓ≧７５（ｍｍ－１）であれば、ΔＸ≧５μｍとなり、最大５μｍの位置ずれを補正することができる。したがって、Ｅ≦５ＧＰａ、Ｈ／Ｓ≧７５（ｍｍ－１）を満たす凸部は、対応するマイクロウェルとの間に最大５μｍの位置ずれがある場合でも、弾性変形によって容易にマイクロウェル内に嵌まり込み、マイクロウェルを密封することができる。 The convex portion is formed of a material having a Young's modulus E of 5 GPa or less, and thus has a deformable elasticity. Here, if the rigidity of the convex portion is G, the shearing force applied in the lateral direction to the convex portion is F, and the displacement in the lateral direction is ΔX, ΔX = G −1 × F × (H / S) is there. The rigidity G is generally 30 to 40% with respect to the Young's modulus E, and is about 1.5 Gpa or less for plastic resin and about 0.001 Gpa for rubber material. In this case, assuming that the required displacement ΔX is set to 5 μm, the realistic shearing force is assumed to be 10 g, and the rigidity G is calculated as an assumed maximum value of 1.5 Gpa, H / S ≧ 75 (mm −1 ) If so, ΔX ≧ 5 μm, and a maximum positional deviation of 5 μm can be corrected. Therefore, a convex portion satisfying E ≦ 5 GPa and H / S ≧ 75 (mm −1 ) can be easily fitted into the microwell by elastic deformation even when there is a maximum positional deviation of 5 μm from the corresponding microwell. Clogging and microwells can be sealed.
　図３は、凸部１１をマイクロウェル２１内へ挿入した状態を示す模式図である。図３に示す例では、凸部１１ａとマイクロウェル２１ａとの間、及び、凸部１１ｂとマイクロウェル２１ｂとの間に位置ずれが生じているため、凸部１１ａ及び１１ｂはそれぞれ、弾性変形しながらマイクロウェル２１ａ及びマイクロウェル２１ｂに嵌り込んでいる。 FIG. 3 is a schematic diagram showing a state in which the convex portion 11 is inserted into the microwell 21. In the example shown in FIG. 3, since the misalignment occurs between the convex portion 11a and the microwell 21a and between the convex portion 11b and the microwell 21b, the convex portions 11a and 11b are elastically deformed, respectively. However, it fits into the microwell 21a and the microwell 21b.
　凸部を形成する材料は、ヤング率Ｅ≦５ＧＰａ以下の材料であれば特に限定されないが、アクリル、ポリスチレンなどのプラスチック樹脂や、ポリジメチルシロキサン（ＰＤＭＳ）などのシリコーンゴム類が好適である。製造効率の観点からは、凸部のみならず蓋板全体をＥ≦５ＧＰａ以下の材料で形成することが好ましい。 The material for forming the convex portion is not particularly limited as long as the Young's modulus is E ≦ 5 GPa or less, but plastic resins such as acrylic and polystyrene, and silicone rubbers such as polydimethylsiloxane (PDMS) are suitable. From the viewpoint of manufacturing efficiency, it is preferable to form not only the convex portions but also the entire lid plate with a material of E ≦ 5 GPa or less.
　凸部の形状は、マイクロウェルを密封可能であれば特に限定されず、円柱形、楕円柱形、多角柱形、円錐台形、逆円錐台形、楕円錐台形、逆楕円錐台形、多角錐台形、逆多角錐台形、テーパー状、逆テーパー状などが挙げられる。マイクロウェル内への凸部の挿入をより容易にする観点からは、凸部は、蓋板側である根元側から先端部に向かって先細りとなった側面、すなわちテーパー状の側面を備えることが好ましく、凸部の先端部がテーパー状であることがより好ましい。 The shape of the convex portion is not particularly limited as long as the microwell can be sealed, and is cylindrical, elliptical, polygonal, frustoconical, inverted frustoconical, elliptical frustum, inverted elliptical frustum, polygonal frustum, A reverse polygon frustum shape, a taper shape, a reverse taper shape, etc. are mentioned. From the viewpoint of facilitating insertion of the convex portion into the microwell, the convex portion has a side surface that is tapered from the root side, which is the lid plate side, toward the tip portion, that is, a tapered side surface. Preferably, the tip of the convex portion is more tapered.
　凸部が円柱形である場合、上述したＨ／Ｓ≧７５（ｍｍ－１）を満たす凸部の形状としては、例えば、直径が５０μｍであり高さＨが１５０μｍ以上の円柱形、直径が４０μｍであり高さＨが９５μｍ以上の円柱形、直径が３０μｍであり高さが５５μｍ以上の円柱形などが挙げられる。したがって、マイクロウェルも円柱形である場合、直径５０μｍのマイクロウェルに対しては凸部の高さは１５０μｍ以上が好ましく、直径４０μｍのマイクロウェルに対しては凸部の高さは９５μｍ以上が好ましく、直径３０μｍのマイクロウェルに対しては凸部の高さは５５μｍ以上が好ましい。 When the convex portion is cylindrical, the shape of the convex portion that satisfies the above-mentioned H / S ≧ 75 (mm −1 ) is, for example, a cylindrical shape having a diameter of 50 μm and a height H of 150 μm or more, and a diameter of 40 μm. And a columnar shape having a height H of 95 μm or more and a columnar shape having a diameter of 30 μm and a height of 55 μm or more. Therefore, when the microwell is also cylindrical, the height of the convex portion is preferably 150 μm or more for a microwell having a diameter of 50 μm, and the height of the convex portion is preferably 95 μm or more for a microwell having a diameter of 40 μm. The height of the convex portion is preferably 55 μm or more for a microwell having a diameter of 30 μm.
　本技術のマイクロチップは、上記基板及び上記蓋板を備える。基板を形成する材料は、当技術分野で既知の材料を用いることができる。例えば、アクリル、ポリスチレンなどのプラスチック樹脂や、ＰＤＭＳなどのシリコーンゴム類、ガラスなどが挙げられる。 The microchip of the present technology includes the substrate and the lid plate. As a material for forming the substrate, a material known in the art can be used. Examples thereof include plastic resins such as acrylic and polystyrene, silicone rubbers such as PDMS, and glass.
　基板に形成されたマイクロウェルの形状は、蓋板に形成された凸部によって密封可能であれば特に限定されず、円柱形、楕円柱形、多角柱形、円錐台形、逆円錐台形、楕円錐台形、逆楕円錐台形、多角錐台形、逆多角錐台形、テーパー状、逆テーパー状などが挙げられる。 The shape of the microwell formed on the substrate is not particularly limited as long as it can be sealed by the convex portion formed on the cover plate, and is cylindrical, elliptical, polygonal, frustoconical, inverted frustoconical, or elliptical cone. A trapezoid, a reverse elliptical frustum, a polygonal frustum, a reverse polygon frustum, a taper shape, a reverse taper shape, etc. are mentioned.
　蓋板に形成された凸部は、少なくとも先端部が、基板に形成されたマイクロウェルに対応する位置にあることが好ましい。つまり、蓋板と基板とを重ね合わせる際に、凸部の先端部が対応するマイクロウェルの内側に位置することが好ましい。このような構成とすることで、対応する凸部とマイクロウェルとの間に位置ずれが生じていたとしても、凸部が弾性変形しながらマイクロウェル内に嵌まり込んで、マイクロウェルを密封することが可能である。 It is preferable that at least the tip of the convex portion formed on the cover plate is at a position corresponding to the microwell formed on the substrate. That is, when the lid plate and the substrate are overlapped, it is preferable that the tip of the convex portion is located inside the corresponding microwell. With such a configuration, even if there is a positional shift between the corresponding convex portion and the microwell, the convex portion is fitted into the microwell while being elastically deformed, and the microwell is sealed. It is possible.
　また、凸部とマイクロウェルとが同軸上に位置する状態で凸部をマイクロウェルに挿入した時に、凸部の先端部とマイクロウェルの内側面との間に５μｍ以上の空隙を有することが好ましい。このような構成とすることで、実際に蓋板と基板とを重ね合わせる際に、対応する１又は２以上の凸部とマイクロウェルとの間で軸が最大５μｍずれてしまっても、凸部が弾性変形しながらマイクロウェル内に嵌まり込んで、位置ずれを補正しながらマイクロウェルを密封することができる。 Further, when the convex portion is inserted into the microwell in a state where the convex portion and the microwell are coaxially located, it is preferable that a gap of 5 μm or more is provided between the tip portion of the convex portion and the inner side surface of the microwell. . With this configuration, even when the cover plate and the substrate are actually overlapped, even if the axis is shifted by a maximum of 5 μm between the corresponding one or more convex portions and the microwell, the convex portion It fits in the microwell while elastically deforming, and the microwell can be sealed while correcting the displacement.
　図４は、凸部１１の先端部１１１とマイクロウェル２１を示す模式図である。先端部１１１の横方向（凸部１１と直行する方向であり、蓋板表面と平行な方向）の断面形状が円形であり、マイクロウェル２１が円柱形である場合、先端部１１１の直径をΦ１（μｍ）とし、前記マイクロウェル２１の直径をΦ２（μｍ）とした時に、Φ１≦Φ２－１０（μｍ）であることが好ましい。このような構成とすることで、凸部１１とマイクロウェル２１との間に５μｍの位置ずれが存在しても、凸部１１の先端部１１１はマイクロウェル２１の内側に存在するため、凸部１１が弾性変形しながらマイクロウェル２１内に嵌まり込んで、マイクロウェル２１を密封することが可能である。 FIG. 4 is a schematic diagram showing the tip 111 and the microwell 21 of the protrusion 11. When the cross-sectional shape of the tip portion 111 in the lateral direction (the direction perpendicular to the convex portion 11 and parallel to the cover plate surface) is circular and the microwell 21 is cylindrical, the diameter of the tip portion 111 is Φ1. (Μm), and when the diameter of the microwell 21 is Φ2 (μm), it is preferable that Φ1 ≦ Φ2-10 (μm). With such a configuration, even if there is a positional deviation of 5 μm between the convex portion 11 and the microwell 21, the tip 111 of the convex portion 11 exists inside the microwell 21. 11 can fit into the microwell 21 while being elastically deformed, and the microwell 21 can be sealed.
　図５は、凸部１１とマイクロウェル２１を示す模式図である。次に、図５を参照しながら凸部１１及びマイクロウェル２１の実施形態の一例について更に説明する。図５に示す凸部１１は、テーパー状の先端部１１１以外は円柱形である。マイクロウェル２１は円柱形である。凸部１１の高さＨは８０μｍである。先端部１１１のテーパー状部分の高さＨｔは３０μｍである。先端部１１１の直径Φ１は１０μｍである。凸部１１の円柱形部分の直径Φ３は３０μｍである。マイクロウェル２１の直径Φ２は２０μｍである。マイクロウェル２１の深さＤｗは３５μｍである。図５に示す例の場合、凸部１１はマイクロウェル２１内に１５μｍ進入して、マイクロウェル２１を密封する。 FIG. 5 is a schematic diagram showing the convex portion 11 and the microwell 21. Next, an example of an embodiment of the convex portion 11 and the microwell 21 will be further described with reference to FIG. The convex portion 11 shown in FIG. 5 has a cylindrical shape except for the tapered tip portion 111. The microwell 21 has a cylindrical shape. The height H of the convex portion 11 is 80 μm. The height Ht of the tapered portion of the tip 111 is 30 μm. The diameter Φ1 of the tip 111 is 10 μm. The diameter Φ3 of the cylindrical portion of the convex portion 11 is 30 μm. The diameter Φ2 of the microwell 21 is 20 μm. The depth Dw of the microwell 21 is 35 μm. In the case of the example shown in FIG. 5, the convex portion 11 enters 15 μm into the microwell 21 and seals the microwell 21.
　図示はしないが、基板の一実施形態として、基板のサイズを５．０ｍｍ×５．０ｍｍ、ウェル数を５００×５００、マイクロウェルのピッチを縦横方向に各８０μｍとすることができる。また、蓋板の一実施形態として、蓋板のサイズを４．５ｍｍ×４．５ｍｍとし、凸部の数及びピッチを上記基板のウェル数及びピッチに一致させた形状とすることができる。 Although not shown, as an embodiment of the substrate, the substrate size may be 5.0 mm × 5.0 mm, the number of wells may be 500 × 500, and the microwell pitch may be 80 μm in the vertical and horizontal directions. Further, as an embodiment of the cover plate, the size of the cover plate may be 4.5 mm × 4.5 mm, and the number of protrusions and the pitch may be matched with the number of wells and the pitch of the substrate.
　また、本技術のマイクロチップは、蓋部に形成された凸部が所定の高さＨを有するため、凸部がマイクロウェルに挿入された状態において基板と蓋板との間に空間ができる。再び図３を参照して説明すると、蓋板１０と基板２０との間には空間４０が形成されている。 Further, in the microchip of the present technology, since the convex portion formed on the lid portion has a predetermined height H, a space is formed between the substrate and the lid plate in a state where the convex portion is inserted into the microwell. Referring to FIG. 3 again, a space 40 is formed between the cover plate 10 and the substrate 20.
　例えば、マイクロウェル２１内に、解析対象の微小粒子と共に培養液などの液体が収容されている場合、凸部１１をマイクロウェル２１に挿入することで液体がマイクロウェル２１の外へ溢れ出す場合がある。本技術のマイクロチップは空間４０を備えることで、マイクロウェル２１から溢れ出した液体を空間４０に逃がして、蓋板１０と基板２０との間に液体が残存することを防止できる。 For example, when a liquid such as a culture solution is stored in the microwell 21 together with the microparticles to be analyzed, the liquid may overflow from the microwell 21 by inserting the convex portion 11 into the microwell 21. is there. By providing the space 40 with the microchip of the present technology, it is possible to prevent the liquid overflowing from the microwell 21 from escaping into the space 40 and remaining between the lid plate 10 and the substrate 20.
　蓋板と基板との間に液体が残存すると、隣接するマイクロウェル間で液体が移動する可能性や、残存した液体を介して異なるマイクロウェルに収容された微小粒子同士が相互作用する可能性がある。本技術のマイクロチップは、蓋板と基板との間に形成された空間により、マイクロウェルから溢れ出た液体を逃がし、マイクロウェルの密封性を向上させることが可能である。 If liquid remains between the cover plate and the substrate, the liquid may move between adjacent microwells, and the microparticles contained in different microwells may interact with each other through the remaining liquid. is there. The microchip of the present technology allows the liquid overflowing from the microwell to escape by the space formed between the cover plate and the substrate, and improves the sealing performance of the microwell.
２．第２実施形態（高気密性材料で形成された凸部を備える構成例）
　マイクロウェル内の培養液中に存在する細胞の酸素消費量をモニタリングする場合など、マイクロウェル内における溶存気体量の正確な測定を実現するためには、マイクロウェル間における液体移動はもちろん、気体移動をも遮断する必要がある。この気体移動の遮断性を向上させるためには、マイクロウェル封止用の凸部に高気密性を付与することが好ましい。凸部に高気密性を付与する手段としては、気体の透過率が低い材料、即ちガスバリア性が高い材料により凸部を形成することが好ましい。 2. Second embodiment (configuration example including a convex portion formed of a highly airtight material)
In order to accurately measure the amount of dissolved gas in the microwell, such as when monitoring the oxygen consumption of the cells present in the culture medium in the microwell, gas transfer as well as liquid transfer between the microwells is possible. It is also necessary to shut off. In order to improve the barrier property of the gas movement, it is preferable to impart high air tightness to the convex portion for sealing the microwell. As a means for imparting high airtightness to the convex portion, it is preferable to form the convex portion with a material having a low gas permeability, that is, a material having a high gas barrier property.
　気体の透過率が低い材料としては、２５℃における酸素透過率が０．５ｍＬ・ｃｍ／ｍ２・２４ｈ・ａｔｍ以下の樹脂が好ましい。酸素透過率がこのような範囲の材料であれば、マイクロウェル内の液体に溶存する気体量の変化を測定しうる。単一細胞の酸素消費量を測定する場合など、より高度なガスバリア性が求められる場合には、２５℃における酸素透過率が０．１ｍＬ・ｃｍ／ｍ２・２４ｈ・ａｔｍ以下の樹脂が好ましい。酸素透過率は、「酸素透過度」とも称され、ＪＩＳ　Ｋ　７１２６に基づいて算出することができる（参考文献：日本プラスチック工業連盟誌「プラスチックス」51(6)，119-127，2000-06）。 As a material having a low gas permeability, a resin having an oxygen permeability at 25 ° C. of 0.5 mL · cm / m 2 · 24 h · atm or less is preferable. If the material has an oxygen permeability in such a range, a change in the amount of gas dissolved in the liquid in the microwell can be measured. When higher gas barrier properties are required, such as when measuring the oxygen consumption of a single cell, a resin having an oxygen permeability at 25 ° C. of 0.1 mL · cm / m 2 · 24 h · atm or less is preferred. The oxygen permeability is also referred to as “oxygen permeability” and can be calculated based on JIS K 7126 (reference: Japan Plastics Industry Federation “Plastics” 51 (6), 119-127, 2000-06. ).
　第１実施形態において説明したように、凸部は、ヤング率Ｅ≦５ＧＰａ以下の弾性変形可能な材料で形成される。Ｅ≦５ＧＰａ以下であり、２５℃における酸素透過率が０．１ｍＬ・ｃｍ／ｍ２・２４ｈ・ａｔｍ以下の樹脂としては、エチレン－ビニルアルコール共重合体（ＥＶＯＨ）樹脂又はポリ塩化ビニリデン（ＰＶＤＣ）樹脂が好ましい。 As described in the first embodiment, the convex portion is formed of an elastically deformable material having a Young's modulus E ≦ 5 GPa or less. The resin having E ≦ 5 GPa or less and oxygen permeability at 25 ° C. of 0.1 mL · cm / m 2 · 24 h · atm or less is an ethylene-vinyl alcohol copolymer (EVOH) resin or polyvinylidene chloride (PVDC). Resins are preferred.
３．第３実施形態（凸部に被膜を備える構成例）
　凸部に高気密性を付与する他の手段としては、ガスバリア性を有する被膜を凸部に形成することが好ましい。これにより、ＰＤＭＳなどの気体透過率の高い材料で凸部を形成した場合であっても、凸部に高気密性を付与することが可能である。 3. Third embodiment (configuration example including a coating film on a convex portion)
As another means for imparting high airtightness to the convex portion, it is preferable to form a film having a gas barrier property on the convex portion. Thereby, even if it is a case where a convex part is formed with a material with high gas permeability, such as PDMS, it is possible to give a high airtightness to a convex part.
　上記被膜を形成する材料は、ガスバリア性を有する材料であれば特に限定されないが、アルミニウム、酸化アルミニウムなどの金属、二酸化ケイ素、窒化ケイ素などの無機化合物、又はパリレン樹脂（パラキシリレン樹脂）が好ましい。これらの材料は、ガスバリア性が高く、マイクロウェル内における溶存気体量の変化を測定しうる高気密性を凸部に付与することができる。 The material for forming the film is not particularly limited as long as it has a gas barrier property, but is preferably a metal such as aluminum or aluminum oxide, an inorganic compound such as silicon dioxide or silicon nitride, or a parylene resin (paraxylylene resin). These materials have high gas barrier properties, and can impart high airtightness that can measure changes in the amount of dissolved gas in the microwell to the protrusions.
　図６は、ガスバリア性を有する被膜を備える凸部１１を示す断面図である。図６Ａは、凸部１１が酸化アルミニウムなどの金属の被膜（金属蒸着膜）５１を備える例を示している。 FIG. 6 is a cross-sectional view showing the convex portion 11 including a coating film having gas barrier properties. FIG. 6A shows an example in which the convex portion 11 includes a metal film (metal vapor deposition film) 51 such as aluminum oxide.
　パリレン樹脂は、ガスバリア性だけではなく生体親和性も高いことから、マイクロウェル内において細胞などの生体試料を扱う場合に好適である。図６Ｂは、凸部１１がパリレン樹脂の被膜５２を備える例を示している。例えば、Specialty Coating System社製の商品「パリレンＣ」は、真空蒸着装置を用いて、室温で蒸着可能である。 Parylene resin is suitable not only for gas barrier properties but also for high biocompatibility, and is therefore suitable for handling biological samples such as cells in a microwell. FIG. 6B shows an example in which the convex portion 11 includes a parylene resin coating 52. For example, a product “Parylene C” manufactured by Specialty Coating System can be deposited at room temperature using a vacuum deposition apparatus.
　凸部は、金属又は無機化合物の被膜の上に、パリレン樹脂の被膜を備えていてもよい。パリレン樹脂は耐薬品性も有しているため、パリレン樹脂の被膜を積層することで、凸部の生体適合性と耐薬品性を向上できる。図６Ｃは、凸部１１が金属の被膜５１の上に、パリレン樹脂の被膜５２を備える例を示している。 The convex portion may be provided with a parylene resin film on a metal or inorganic compound film. Since the parylene resin also has chemical resistance, the biocompatibility and chemical resistance of the convex portion can be improved by laminating the parylene resin film. FIG. 6C shows an example in which the convex portion 11 includes a parylene resin film 52 on a metal film 51.
　凸部は、表面全体にガスバリア性を有する被膜を備えていてもよいが、マイクロウェル嵌入時にマイクロウェルと接する部分が少なくとも被膜を備えていればよい。例えば、凸部の先端部がテーパー状である場合、テーパー状の部分のみが被膜を備える構成としてもよい。 The convex part may be provided with a film having gas barrier properties on the entire surface, but it is sufficient that at least the part in contact with the microwell when the microwell is inserted has the film. For example, when the tip of the convex portion is tapered, only the tapered portion may have a coating.
　上記第２実施形態及び第３実施形態のように、凸部が高気密性を有する構成の場合、対応するマイクロウェルも高気密性を有することが好ましい。これにより、マイクロウェルにおける気体の流出入をより効果的に遮断することができる。マイクロウェルに高気密性を付与する手段としては、マイクロウェル内にガスバリア性の被膜を形成することが考えられるが、製造コストの観点からは、マイクロウェルを備える基板をガスバリア性の高い材料で形成することが好ましい。基板の材料は、必ずしも弾性変形可能である必要はないことから、ガラス類を用いてもよい。 As in the second embodiment and the third embodiment, when the convex portion has a high airtightness, the corresponding microwell preferably has a high airtightness. Thereby, the inflow and outflow of the gas in a microwell can be interrupted | blocked more effectively. As a means for imparting high airtightness to the microwell, it is conceivable to form a gas barrier film in the microwell. From the viewpoint of manufacturing cost, a substrate having a microwell is formed of a material having a high gas barrier property. It is preferable to do. Since the substrate material does not necessarily need to be elastically deformable, glass may be used.
４．第４実施形態（凸部の表面に抗体、蛍光色素又は試薬を有する構成例）
　上記凸部の表面には、抗体又は蛍光色素が固定化されてもよく、試薬が固着又は塗布されてもよい。これにより、マイクロウェル内に存在する物質の検出や、捕捉、回収が可能となる。マイクロウェル内に細胞が収容されている場合には、細胞由来の物質を検出や捕捉の対象とすることができる。例えば、細胞を含有する培養液を収容したマイクロウェルに本実施形態に係る凸部を浸漬することで、細胞の分泌物を検出、捕捉してもよい。また、細胞をマイクロウェル内で破砕して核酸やタンパク質などの対象物質を溶出させ、そこに本実施形態に係る凸部を浸漬することで、細胞内の物質を検出、捕捉してもよい。 4). Fourth embodiment (configuration example having antibody, fluorescent dye or reagent on the surface of the convex portion)
An antibody or a fluorescent dye may be immobilized on the surface of the convex part, and a reagent may be fixed or coated. As a result, the substance present in the microwell can be detected, captured and recovered. When cells are accommodated in the microwells, cell-derived substances can be detected and captured. For example, the secretion of the cell may be detected and captured by immersing the convex portion according to the present embodiment in a microwell containing a culture solution containing cells. In addition, cells may be crushed in a microwell to elute target substances such as nucleic acids and proteins, and the protrusions according to the present embodiment may be immersed therein to detect and capture the substances in the cells.
　図７は、細胞分泌物を検出する例を模式的に示す断面図である。図７Ａに示す例では、凸部１１はパリレン樹脂の被膜５２を備え、パリレン樹脂の被膜５２上には抗体５５が固定化されている。マイクロウェル２１の内部には、培養液６１、細胞６２、細胞分泌物６３が存在する。図７Ｂは、凸部１１をマイクロウェル２１に挿入した状態を示している。抗体５５はマイクロウェル２１内の細胞分泌物６３と結合している。図７Ｃは、凸部１１をマイクロウェル２１から引き抜いた状態を示している。図７Ｄは、凸部１１の表面上に存在する抗体５５に結合した細胞分泌物６３と、蛍光ビーズ７１の表面上に存在する抗体７２とを反応させた状態を示している。このようにして、対象とする物質を光学検出するために本実施形態のマイクロチップを用いることができる。 FIG. 7 is a cross-sectional view schematically showing an example of detecting cell secretions. In the example shown in FIG. 7A, the convex portion 11 includes a parylene resin coating 52, and the antibody 55 is immobilized on the parylene resin coating 52. Inside the microwell 21, there are a culture solution 61, cells 62, and a cell secretion 63. FIG. 7B shows a state in which the convex portion 11 is inserted into the microwell 21. The antibody 55 is bound to the cell secretion 63 in the microwell 21. FIG. 7C shows a state in which the convex portion 11 is pulled out from the microwell 21. FIG. 7D shows a state in which the cell secretion 63 bound to the antibody 55 present on the surface of the convex portion 11 is reacted with the antibody 72 present on the surface of the fluorescent bead 71. In this manner, the microchip of this embodiment can be used to optically detect a target substance.
　凸部は、表面全体に抗体、蛍光色素又は試薬を有していてもよいが、マイクロウェル内に挿入する部分が少なくとも抗体、蛍光色素又は試薬を有していればよい。例えば、凸部の先端がテーパー状である場合、テーパー状の部分のみが抗体、蛍光色素又は試薬を有する構成としてもよい。 The convex portion may have an antibody, a fluorescent dye or a reagent on the entire surface, but the portion inserted into the microwell only needs to have at least the antibody, the fluorescent dye or the reagent. For example, when the tip of the convex portion is tapered, only the tapered portion may have an antibody, a fluorescent dye, or a reagent.
　目的物質を検出、捕捉する手段として、マイクロウェルの内部に抗体、蛍光色素又は試薬を配置する構成を採用することもできるが、上述のように凸部が抗体、蛍光色素又は試薬を備える構成とすることが好ましい。例えば、細胞が収容されたマイクロウェルに試薬が塗布された凸部を挿入して対象物質を検出した後、当該凸部を引き抜き、マイクロウェル内に細胞を残留させた状態で別の試薬が塗布された凸部を挿入することで、特定の細胞を対象とした複数の化学反応を連続的に観察することが可能である。 As a means for detecting and capturing a target substance, a configuration in which an antibody, a fluorescent dye, or a reagent is arranged inside a microwell can be adopted, but as described above, the convex portion includes an antibody, a fluorescent dye, or a reagent. It is preferable to do. For example, after detecting a target substance by inserting a convex portion coated with a reagent into a microwell containing cells, the convex portion is pulled out and another reagent is applied with the cells remaining in the microwell. By inserting the projected portions, it is possible to continuously observe a plurality of chemical reactions targeting a specific cell.
５．第５実施形態（凸部に酸素センサーを備える構成例）
　上記凸部は、酸素センサーを備えてもよい。これにより、マイクロウェル内における酸素量を測定できる。例えば、細胞を含有する培養液を収容したマイクロウェルに酸素センサーを備える凸部を浸漬することで、マイクロウェル内における溶存酸素量の変化から細胞の酸素消費量を測定できる。細胞の酸素消費量を測定する場合、特に単一細胞の酸素消費量を測定する場合、微量な酸素量の変化を検出しなければならない。酸素量測定の正確性を担保するためには気体の流出入を遮断する必要があることから、上記第２実施形態又は第３実施形態に係る構成を採用してマイクロウェルの気密性を高めることが好ましい。 5). Fifth embodiment (configuration example including an oxygen sensor on the convex portion)
The convex portion may include an oxygen sensor. Thereby, the amount of oxygen in the microwell can be measured. For example, by immersing a convex portion having an oxygen sensor in a microwell containing a culture solution containing cells, the oxygen consumption of the cells can be measured from the change in the dissolved oxygen amount in the microwell. When measuring the oxygen consumption of a cell, especially when measuring the oxygen consumption of a single cell, a change in a trace amount of oxygen must be detected. In order to ensure the accuracy of oxygen amount measurement, it is necessary to block the inflow and outflow of gas. Therefore, the configuration according to the second embodiment or the third embodiment is adopted to increase the airtightness of the microwell. Is preferred.
　酸素センサーは、マイクロウェル内での測定が可能であれば特に限定されず、公知の酸素センサーを用いればよい。一例として、白金オクタエチルホルフィリン（Platinum Octaethylprophyrin，ＰｔＯＥＰ）を用いて形成した膜状の酸素センサーを凸部に備える構成を説明する。 The oxygen sensor is not particularly limited as long as measurement in a microwell is possible, and a known oxygen sensor may be used. As an example, a configuration will be described in which a film-like oxygen sensor formed using platinum octaethylformylphylline (PlatinumtaOctaethylprophyrin, PtOEP) is provided on a convex portion.
　図８は、膜状の酸素センサーを備える凸部を示す断面図である。図８Ａでは、凸部１１が金属の被膜５１の上に、パリレン樹脂の被膜５２を備える例を示している。図８Ｂは、パリレン樹脂の被膜の上に、ＰｔＯＥＰを含有する酸素センサー５３を備える例を示している。 FIG. 8 is a cross-sectional view showing a convex portion provided with a film-like oxygen sensor. FIG. 8A shows an example in which the convex portion 11 includes a parylene resin coating 52 on a metal coating 51. FIG. 8B shows an example in which an oxygen sensor 53 containing PtOEP is provided on a parylene resin coating.
　酸素センサーは、凸部全体に設けてもよいが、マイクロウェル内に挿入する部分に少なくとも設ければよい。例えば、凸部の先端部がテーパー状である場合、テーパー状の部分のみが酸素センサーを備える構成としてもよい。 The oxygen sensor may be provided on the entire convex portion, but may be provided at least in a portion to be inserted into the microwell. For example, when the tip of the convex portion is tapered, only the tapered portion may include an oxygen sensor.
　酸素センサーをマイクロウェルに挿入した後、対物レンズによって集光した波長３５０ｎｍ付近のレーザー光をマイクロウェルの基板底面から酸素センサー上へ照射すると、波長６１５ｎｍ付近の燐光を発する。この燐光が減衰する数十μｓｅｃオーダーの時定数の変化を測定することで、マイクロウェル内の酸素消費状態をモニターすることができる。 After the oxygen sensor is inserted into the microwell, laser light having a wavelength of about 350 nm collected by the objective lens is irradiated onto the oxygen sensor from the bottom surface of the substrate of the microwell, and phosphorescence having a wavelength of about 615 nm is emitted. By measuring the change in the time constant on the order of several tens of microseconds at which this phosphorescence decays, the oxygen consumption state in the microwell can be monitored.
　酸素センサーをマイクロウェル内の培養液に浸漬すると、酸素センサーが溶解する場合があることから、酸素センサーの上に保護膜を備えることが好ましい。図８Ｃは、酸素センサー５３の上に保護膜５４を備える例を示している。 Since the oxygen sensor may be dissolved when the oxygen sensor is immersed in the culture solution in the microwell, it is preferable to provide a protective film on the oxygen sensor. FIG. 8C shows an example in which a protective film 54 is provided on the oxygen sensor 53.
　保護膜は、酸素センサーによる測定に支障が生じないよう、酸素透過率が高く、自家蛍光が低い材料で形成されることが好ましく、例えば、ポリスチレンを用いることができる。保護膜は、酸素センサーを保護しうる範囲で設ければよく、凸部全体に設けなくてもよい。 The protective film is preferably formed of a material having a high oxygen permeability and low autofluorescence so that measurement by the oxygen sensor is not hindered. For example, polystyrene can be used. The protective film may be provided as long as the oxygen sensor can be protected, and may not be provided on the entire convex portion.
　酸素量測定の手段として、マイクロウェルの内部に酸素センサーを配置する構成を採用することもできるが、凸部が酸素センサーを備える構成とすることが好ましい。例えば、マイクロウェル内に細胞を収容させた状態で、抗体、蛍光色素又は試薬を有する凸部を用いて目的物質の検出を行った後に、酸素センサーを備える凸部を用いて酸素消費量を測定するなど、特定の細胞を様々な観点から連続的に分析することが可能である。 As a means for measuring the amount of oxygen, a configuration in which an oxygen sensor is arranged inside the microwell can be adopted, but it is preferable that the convex portion has an oxygen sensor. For example, in a state where cells are housed in a microwell, after detecting a target substance using a convex part having an antibody, a fluorescent dye or a reagent, the oxygen consumption is measured using the convex part having an oxygen sensor. It is possible to analyze specific cells continuously from various viewpoints.
　上述した第４実施形態及び第５実施形態において、凸部とマイクロウェルのそれぞれに固有のアドレスを割り当ててもよい。凸部とマイクロウェルとの対応関係をアドレスにより把握することで、１つのマイクロウェルに対して複数の凸部を用いて複数の分析を行った場合でも、同一のマイクロウェルに対する異なる分析結果同士を容易に紐付けることが可能である。 In the fourth and fifth embodiments described above, a unique address may be assigned to each of the convex portion and the microwell. By grasping the correspondence between the convex part and the microwell by the address, even when performing a plurality of analyzes using a plurality of convex parts for one microwell, different analysis results for the same microwell can be obtained. It can be easily tied.
　なお、本技術は以下のような構成も採ることができる。
〔１〕マイクロウェル封止用の凸部を備え、
　前記凸部は、
　ヤング率をＥとした場合に、Ｅ≦５ＧＰａ以下であり、
　前記凸部と直交する方向の断面の断面積を高さ方向に亘って平均化した平均断面積をＳ（ｍｍ２）とし、高さをＨ（ｍｍ）とした場合に、Ｈ／Ｓ≧７５（ｍｍ－１）である、マイクロウェル封止用蓋板。
〔２〕前記凸部は、テーパー状の側面を備える、〔１〕に記載のマイクロウェル封止用蓋板。
〔３〕前記凸部は、先端部がテーパー状である、〔１〕又は〔２〕に記載のマイクロウェル封止用蓋板。
〔４〕前記凸部は、２５℃における酸素透過率が０．５ｍＬ・ｃｍ／ｍ２・２４ｈ・ａｔｍ以下の樹脂で形成されている、〔１〕から〔３〕のいずれかに記載のマイクロウェル封止用蓋板。
〔５〕前記凸部は、２５℃における酸素透過率が０．１ｍＬ・ｃｍ／ｍ２・２４ｈ・ａｔｍ以下の樹脂で形成されている、〔１〕から〔４〕のいずれかに記載のマイクロウェル封止用蓋板。
〔６〕前記凸部は、エチレン－ビニルアルコール共重合体樹脂又はポリ塩化ビニリデン樹脂で形成されている、〔１〕から〔５〕のいずれかに記載のマイクロウェル封止用蓋板。
〔７〕前記凸部は、金属、無機化合物又はパリレン樹脂の被膜を備える、〔１〕から〔６〕のいずれかに記載のマイクロウェル封止用蓋板。
〔８〕前記凸部は、金属又は無機化合物の被膜の上にパリレン樹脂の被膜を備える、〔１〕から〔７〕のいずれかに記載のマイクロウェル封止用蓋板。
〔９〕前記凸部の表面に抗体又は蛍光色素が固定化されている、〔１〕から〔８〕のいずれかに記載のマイクロウェル封止用蓋板。
〔１０〕前記凸部の表面に試薬が固着又は塗布されている、〔１〕から〔９〕のいずれかに記載のマイクロウェル封止用蓋板。
〔１１〕前記凸部は、酸素センサーを備える、〔１〕から〔１０〕のいずれかに記載のマイクロウェル封止用蓋板。
〔１２〕マイクロウェルが形成された基板と、凸部が形成された蓋板と、を備え、
　前記凸部は、
　少なくとも先端部が前記マイクロウェルに対応する位置にあり、
　ヤング率をＥとした場合に、Ｅ≦５ＧＰａ以下であり、
　前記凸部と直交する方向の断面の断面積を高さ方向に亘って平均化した平均断面積をＳ（ｍｍ２）とし、高さをＨ（ｍｍ）とした場合に、Ｈ／Ｓ≧７５（ｍｍ－１）である、マイクロチップ。
〔１３〕前記凸部と前記マイクロウェルとが同軸上に位置する状態で前記凸部を前記マイクロウェルに挿入した時に、前記凸部の先端部と前記マイクロウェルの内側面との間に５μｍ以上の空隙を有する、〔１２〕に記載のマイクロチップ。
〔１４〕前記凸部の先端部の横断面形状が円形であり、
　前記マイクロウェルが円柱形であり、
　前記凸部の先端部の直径をΦ１（μｍ）とし、前記マイクロウェルの直径をΦ２（μｍ）とした場合に、Φ１≦Φ２－１０（μｍ）である、〔１２〕又は〔１３〕に記載のマイクロチップ。
〔１５〕前記Φ２が５０μｍの前記マイクロウェルに対応する前記凸部の高さが１５０μｍ以上である、〔１４〕に記載のマイクロチップ。
〔１６〕前記Φ２が４０μｍの前記マイクロウェルに対応する前記凸部の高さが９５μｍ以上である、〔１４〕又は〔１５〕に記載のマイクロチップ。
〔１７〕前記Φ２が３０μｍの前記マイクロウェルに対応する前記凸部の高さが５５μｍ以上である、〔１４〕から〔１６〕のいずれかに記載のマイクロチップ。 In addition, this technique can also take the following structures.
[1] A projection for microwell sealing is provided,
The convex portion is
When Young's modulus is E, E ≦ 5 GPa or less,
H / S ≧ 75, where S (mm 2 ) is the average cross-sectional area obtained by averaging the cross-sectional areas in the direction perpendicular to the convex portions over the height direction, and H (mm) is the height. (Mm −1 ) Microwell sealing lid plate.
[2] The microwell sealing lid plate according to [1], wherein the convex portion includes a tapered side surface.
[3] The microwell sealing lid plate according to [1] or [2], wherein the convex portion has a tapered tip.
[4] The microprojection according to any one of [1] to [3], wherein the convex portion is formed of a resin having an oxygen permeability at 25 ° C. of 0.5 mL · cm / m 2 · 24 h · atm or less. Well sealing lid plate.
[5] The microprojection according to any one of [1] to [4], wherein the convex portion is formed of a resin having an oxygen permeability at 25 ° C. of 0.1 mL · cm / m 2 · 24 h · atm or less. Well sealing lid plate.
[6] The microwell sealing lid plate according to any one of [1] to [5], wherein the convex portion is formed of an ethylene-vinyl alcohol copolymer resin or a polyvinylidene chloride resin.
[7] The microwell sealing lid plate according to any one of [1] to [6], wherein the convex portion includes a metal, inorganic compound, or parylene resin coating.
[8] The microwell sealing lid plate according to any one of [1] to [7], wherein the convex portion includes a parylene resin coating on a metal or inorganic compound coating.
[9] The microwell sealing lid plate according to any one of [1] to [8], wherein an antibody or a fluorescent dye is immobilized on the surface of the convex portion.
[10] The microwell sealing lid plate according to any one of [1] to [9], wherein a reagent is fixed or coated on the surface of the convex portion.
[11] The microwell sealing lid plate according to any one of [1] to [10], wherein the convex portion includes an oxygen sensor.
[12] A substrate on which a microwell is formed, and a lid plate on which a convex portion is formed,
The convex portion is
At least the tip is in a position corresponding to the microwell;
When Young's modulus is E, E ≦ 5 GPa or less,
H / S ≧ 75, where S (mm 2 ) is the average cross-sectional area obtained by averaging the cross-sectional areas in the direction perpendicular to the convex portions over the height direction, and H (mm) is the height. A microchip that is (mm −1 ).
[13] When the convex portion is inserted into the microwell in a state where the convex portion and the microwell are coaxially positioned, the distance between the tip of the convex portion and the inner surface of the microwell is 5 μm or more. [12] The microchip according to [12].
[14] The cross-sectional shape of the tip of the convex portion is circular,
The microwell is cylindrical,
[12] or [13], wherein Φ1 ≦ Φ2-10 (μm) when the diameter of the tip of the convex portion is Φ1 (μm) and the diameter of the microwell is Φ2 (μm). Microchip.
[15] The microchip according to [14], wherein the height of the convex portion corresponding to the microwell having Φ2 of 50 μm is 150 μm or more.
[16] The microchip according to [14] or [15], wherein the height of the convex portion corresponding to the microwell having Φ2 of 40 μm is 95 μm or more.
[17] The microchip according to any one of [14] to [16], wherein the height of the convex portion corresponding to the microwell having Φ2 of 30 μm is 55 μm or more.
１　マイクロチップ
１０　蓋板
１１　凸部
　１１１　先端部
２０　基板
２１　マイクロウェル
３０　微小粒子
４０　空間
５１　金属の被膜
５２　パリレン樹脂の被膜
５３　酸素センサー
５４　保護膜 DESCRIPTION OF SYMBOLS 1 Microchip 10 Cover plate 11 Convex part 111 Tip part 20 Substrate 21 Microwell 30 Microparticle 40 Space 51 Metal film 52 Parylene resin film 53 Oxygen sensor 54 Protective film

Claims (17)

  1. 　マイクロウェル封止用の凸部を備え、
    　前記凸部は、
    　ヤング率をＥとした場合に、Ｅ≦５ＧＰａ以下であり、
    　前記凸部と直交する方向の断面の断面積を高さ方向に亘って平均化した平均断面積をＳ（ｍｍ２）とし、高さをＨ（ｍｍ）とした場合に、Ｈ／Ｓ≧７５（ｍｍ－１）である、マイクロウェル封止用蓋板。     Providing projections for microwell sealing,
    The convex portion is
    When Young's modulus is E, E ≦ 5 GPa or less,
    H / S ≧ 75, where S (mm 2 ) is the average cross-sectional area obtained by averaging the cross-sectional areas in the direction perpendicular to the convex portions over the height direction, and H (mm) is the height. (Mm −1 ) Microwell sealing lid plate.
  2. 　前記凸部は、テーパー状の側面を備える、請求項１に記載のマイクロウェル封止用蓋板。 2. The microwell sealing lid plate according to claim 1, wherein the convex portion has a tapered side surface.
  3. 　前記凸部は、先端部がテーパー状である、請求項１に記載のマイクロウェル封止用蓋板。 2. The microwell sealing lid plate according to claim 1, wherein the convex portion has a tapered tip end portion.
  4. 　前記凸部は、２５℃における酸素透過率が０．５ｍＬ・ｃｍ／ｍ２・２４ｈ・ａｔｍ以下の樹脂で形成されている、請求項１に記載のマイクロウェル封止用蓋板。 2. The microwell sealing lid plate according to claim 1, wherein the convex portion is formed of a resin having an oxygen permeability at 25 ° C. of 0.5 mL · cm / m 2 · 24 h · atm or less.
  5. 　前記凸部は、２５℃における酸素透過率が０．１ｍＬ・ｃｍ／ｍ２・２４ｈ・ａｔｍ以下の樹脂で形成されている、請求項１に記載のマイクロウェル封止用蓋板。 2. The microwell sealing lid plate according to claim 1, wherein the convex portion is formed of a resin having an oxygen permeability at 25 ° C. of 0.1 mL · cm / m 2 · 24 h · atm or less.
  6. 　前記凸部は、エチレン－ビニルアルコール共重合体樹脂又はポリ塩化ビニリデン樹脂で形成されている、請求項１に記載のマイクロウェル封止用蓋板。 2. The microwell sealing lid plate according to claim 1, wherein the convex portion is formed of an ethylene-vinyl alcohol copolymer resin or a polyvinylidene chloride resin.
  7. 　前記凸部は、金属、無機化合物又はパリレン樹脂の被膜を備える、請求項１に記載のマイクロウェル封止用蓋板。 2. The microwell sealing lid plate according to claim 1, wherein the convex portion is provided with a coating of a metal, an inorganic compound, or a parylene resin.
  8. 　前記凸部は、金属又は無機化合物の被膜の上にパリレン樹脂の被膜を備える、請求項１に記載のマイクロウェル封止用蓋板。 2. The microwell sealing lid plate according to claim 1, wherein the convex portion includes a parylene resin coating on a metal or inorganic compound coating.
  9. 　前記凸部の表面に抗体又は蛍光色素が固定化されている、請求項１に記載のマイクロウェル封止用蓋板。 The microwell sealing lid plate according to claim 1, wherein an antibody or a fluorescent dye is immobilized on the surface of the convex portion.
  10. 　前記凸部の表面に試薬が固着又は塗布されている、請求項１に記載のマイクロウェル封止用蓋板。 The microwell sealing lid plate according to claim 1, wherein a reagent is fixed or coated on the surface of the convex portion.
  11. 　前記凸部は、酸素センサーを備える、請求項１に記載のマイクロウェル封止用蓋板。 2. The microwell sealing lid plate according to claim 1, wherein the convex portion includes an oxygen sensor.
  12. 　マイクロウェルが形成された基板と、凸部が形成された蓋板と、を備え、
    　前記凸部は、
    　少なくとも先端部が前記マイクロウェルに対応する位置にあり、
    　ヤング率をＥとした場合に、Ｅ≦５ＧＰａ以下であり、
    　前記凸部と直交する方向の断面の断面積を高さ方向に亘って平均化した平均断面積をＳ（ｍｍ２）とし、高さをＨ（ｍｍ）とした場合に、Ｈ／Ｓ≧７５（ｍｍ－１）である、マイクロチップ。     A substrate on which a microwell is formed, and a cover plate on which a convex portion is formed,
    The convex portion is
    At least the tip is in a position corresponding to the microwell;
    When Young's modulus is E, E ≦ 5 GPa or less,
    H / S ≧ 75, where S (mm 2 ) is the average cross-sectional area obtained by averaging the cross-sectional areas in the direction perpendicular to the convex portions over the height direction, and H (mm) is the height. A microchip that is (mm −1 ).
  13. 　前記凸部と前記マイクロウェルとが同軸上に位置する状態で前記凸部を前記マイクロウェルに挿入した時に、前記凸部の先端部と前記マイクロウェルの内側面との間に５μｍ以上の空隙を有する、請求項１２に記載のマイクロチップ。 When the convex portion and the microwell are coaxially positioned and the convex portion is inserted into the microwell, a gap of 5 μm or more is formed between the tip portion of the convex portion and the inner side surface of the microwell. The microchip according to claim 12.
  14. 　前記凸部の先端部の横断面形状が円形であり、
    　前記マイクロウェルが円柱形であり、
    　前記凸部の先端部の直径をΦ１（μｍ）とし、前記マイクロウェルの直径をΦ２（μｍ）とした場合に、Φ１≦Φ２－１０（μｍ）である、請求項１２に記載のマイクロチップ。     The cross-sectional shape of the tip of the convex part is circular,
    The microwell is cylindrical,
    13. The microchip according to claim 12, wherein Φ1 ≦ Φ2-10 (μm), where Φ1 (μm) is the diameter of the tip of the convex portion and Φ2 (μm) is the diameter of the microwell.
  15. 　前記Φ２が５０μｍの前記マイクロウェルに対応する前記凸部の高さが１５０μｍ以上である、請求項１４に記載のマイクロチップ。 15. The microchip according to claim 14, wherein the height of the convex portion corresponding to the microwell having Φ2 of 50 μm is 150 μm or more.
  16. 　前記Φ２が４０μｍの前記マイクロウェルに対応する前記凸部の高さが９５μｍ以上である、請求項１４に記載のマイクロチップ。 The microchip according to claim 14, wherein the height of the convex portion corresponding to the microwell having Φ2 of 40 μm is 95 μm or more.
  17. 　前記Φ２が３０μｍの前記マイクロウェルに対応する前記凸部の高さが５５μｍ以上である、請求項１４に記載のマイクロチップ。 The microchip according to claim 14, wherein the height of the convex portion corresponding to the microwell having Φ2 of 30 μm is 55 μm or more.
PCT/JP2017/045315 2017-03-21 2017-12-18 Microwell-sealing cover plate and microchip WO2018173390A1 (en)

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