JP7190273B2 - Method for manufacturing substrate having modified layer - Google Patents

Description

The present invention relates to a substrate having a modified layer and a manufacturing method thereof.

Various substrates having modified layers on their surfaces have been developed. For example, Non-Patent Document 1 describes a stainless steel substrate having an array of fine through holes. The substrate described in Non-Patent Document 1 is surface-treated so that the inside of the through-hole is hydrophilic and the surface where the through-hole opens is hydrophobic. It is reported that the substrate described in Non-Patent Document 1 can easily and quantitatively introduce a liquid into each through-hole.

In addition, Non-Patent Document 2 describes a microchamber. The microchamber described in Non-Patent Document 2 is a well array formed on a glass substrate, and a polydimethylsiloxane (PDMS) thin film is laminated on the surface where the wells are opened. Since the microchamber described in Non-Patent Document 2 has a PDMS layer with high sealing properties, it is reported that each chamber can be sealed by covering with a glass substrate. In addition, it has been reported that since the PDMS layer is sufficiently thin, inhibition of enzymatic reactions by PDMS is suppressed, and cell-free protein synthesis can be efficiently performed in the microchamber.

Morrison T., et al., Nanoliter high throughput quantitative PCR, Nucleic Acids Research, 34 (18), e123, 2006. Okano T., Cell-free protein synthesis from a single copy of DNA in a glass microchamber, Lab Chip, 12, 2704-2711, 2012.

However, the substrate described in Non-Patent Document 1 has a complicated manufacturing process. The microchamber described in Non-Patent Document 2 is manufactured by a method of forming a well array by laminating a PDMS layer on a glass substrate, masking it with a photoresist, and etching it. Therefore, the microchamber described in Non-Patent Document 2 cannot be produced by stacking a modified layer on the surface of an already formed well array. An object of the present invention is to provide a technique for easily laminating a modified layer on the surface of a substrate having recesses.

The present invention includes the following aspects.
[1] A method for manufacturing a substrate having a modified layer, comprising: laminating a protective layer on a surface of a substrate having a concave portion where the concave portion opens and an inner surface of the concave portion; By removing, the surface where the recess opens is exposed, obtaining a substrate in which the protective layer is laminated on the inner surface of the recess, and a solvent that dissolves the protective layer on the surface where the recess opens is transparent, and the solvent is brought into contact with the substrate to remove the protective layer laminated on the inner surface of the recess, so that the recess is opened. obtaining a substrate in which the first modified layer is laminated on the surface facing the substrate.
[2] After obtaining a substrate in which the first modified layer is laminated on the surface where the recess opens, a second modified layer is formed on the first modified layer and the inner surface of the recess. By laminating and removing the second modified layer laminated on the first modified layer, the first modified layer is laminated on the surface where the recess opens, and the recess is formed. The manufacturing method according to [1], further comprising obtaining a substrate having the second modified layer laminated on the inner surface thereof.
[3] A method for manufacturing a substrate having a modified layer, comprising: laminating a first modified layer on a surface of a substrate having a concave portion where the concave portion opens and an inner surface of the concave portion; By laminating a protective layer on one modified layer and removing a part of the protective layer and the first modified layer, the surface where the recess opens is exposed, and the inner surface of the recess is Obtaining a substrate in which the first modified layer and the protective layer are laminated in this order, and contacting the substrate with a solvent that dissolves the protective layer to laminate the protective layer on the inner surface of the recess. and obtaining a substrate in which the opening surface of the recess is exposed and the first modified layer is laminated on the inner surface of the recess by removing.
[4] After obtaining a substrate in which the surface where the recess opens is exposed and the first modified layer is laminated on the inner surface of the recess, the first modified layer laminated on the inner surface of the recess The recess is opened by laminating a second modified layer on the surface where the quality layer and the recess are opened, and removing the second modified layer stacked on the inner surface of the recess. The manufacturing method according to [3], further comprising obtaining a substrate in which the second modified layer is laminated on the surface and the first modified layer is laminated on the inner surface of the recess.
[5] A method for manufacturing a substrate having a modified layer, comprising: laminating a first modified layer on a surface of a substrate having a concave portion where the concave portion opens and an inner surface of the concave portion; By laminating a protective layer on one modified layer and removing a part of the protective layer and the first modified layer, the surface where the recess opens is exposed, and the inner surface of the recess is Obtaining a substrate in which the first modified layer and the protective layer are laminated in this order, and laminating a second modified layer on the surface where the protective layer laminated in the recess and the recess opens. By removing the protective layer laminated on the recess and the second modified layer laminated on the protective layer, the second modified layer is laminated on the surface where the recess opens. and obtaining a substrate in which the first modified layer is laminated on the inner surface of the recess.
[6] The manufacturing method according to any one of [1] to [5], wherein the volume of each recess is 1 to 1×10 ⁹ fL.
[7] A substrate having recesses, wherein a first modified layer is laminated on a surface where the recesses open, and a volume per one of the recesses is 1 to 1×10 ⁹ fL; A substrate, wherein the first modified layer is an ultrathin film.
[8] The substrate according to [7], wherein a second modified layer is laminated on the inner surface of the recess.
[9] The substrate according to [8], wherein biomolecules are immobilized on the first modified layer or the second modified layer.
[10] The substrate according to any one of [7] to [9], which is for reaction of biomolecules.

ADVANTAGE OF THE INVENTION According to this invention, the technique which laminates|stacks a modified layer simply on the board|substrate which has a recessed part can be provided.

(a) to (e) are schematic diagrams for explaining the substrate manufacturing method according to the first embodiment. (a) and (b) are schematic diagrams showing a state in which a solvent S is brought into contact with a substrate having a modified layer laminated thereon. (a) to (c) are schematic diagrams for explaining a method for manufacturing a substrate according to a second embodiment. (a) to (e) are schematic diagrams for explaining a method for manufacturing a substrate according to the third embodiment. (a) to (c) are schematic diagrams for explaining a method for manufacturing a substrate according to a fourth embodiment. (a) to (c) are schematic diagrams for explaining a method for manufacturing a substrate according to a fifth embodiment. (a) to (c) are schematic diagrams for explaining the experimental steps of Experimental Example 2. FIG. (a) is a photograph showing the results of Experimental Example 2. FIG. (b) and (c) are fluorescence micrographs showing the results of Experimental Example 2. FIG.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as the case may be. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and overlapping descriptions are omitted. Note that the dimensional ratios in each drawing are exaggerated for the sake of explanation, and do not necessarily match the actual dimensional ratios.

[Method for manufacturing a substrate having a modified layer]
(First embodiment)
In one embodiment, the present invention provides a method for manufacturing a substrate having a modified layer, comprising a step of laminating a protective layer on a surface of a substrate having recesses where the recesses open and on the inner surface of the recesses (1a ), a step (1b) of obtaining a substrate in which a surface on which the recess is opened is exposed by removing a part of the protective layer, and the protective layer is laminated on the inner surface of the recess; A step (1c) of laminating a first modified layer, which is permeable to a solvent that dissolves the protective layer, on the opening surface, and bringing the solvent into contact with the substrate and laminating it on the inner surface of the recess. a step (1d) of obtaining a substrate in which the first modified layer is laminated on the surface where the recess is opened by removing the protective layer. According to the manufacturing method of this embodiment, the modified layer can be easily laminated on the substrate having the concave portion.

Materials for the substrate used in the manufacturing method of the present embodiment include glass; semiconductors such as silicon; metals such as chromium, stainless steel, gold, and magnesium alloys; and resins such as polycarbonate and polymethyl methacrylate. The substrate may be composed of one kind of material, or may be composed of a combination of a plurality of materials. Also, the surface of the substrate may be coated with a metal oxide. Examples of metal oxides include Al ₂ O ₃ , Ta ₂ O ₅ , Nb ₂ O ₅ , ZrO ₂ , TiO ₂ , HfO ₂ , Ni oxides, Cr oxides, Fe oxides, Mn oxides, W oxides, Cu oxide, Ag oxide, etc. are mentioned.

Substrates with recesses are manufactured by processing substrates by dry etching using reactive gas, ions, radicals, etc., wet etching using reactive liquids, cutting, embossing by pressing a mold against the substrate, etc. be able to.

A plurality of recesses are preferably present on the substrate, and preferably form an array in which the plurality of recesses are regularly arranged. Further, the recess may be formed only on one surface of the substrate, or may be formed on both surfaces.

The shape of the recess is not particularly limited, and may be, for example, a cylindrical shape, a polyhedral shape (eg, a rectangular parallelepiped, a hexagonal prism, an octagonal prism, etc.), a truncated cone, or a truncated pyramid. (Triangular truncated pyramid, quadrangular truncated pyramid, pentagonal truncated pyramid, hexagonal truncated pyramid, polygonal truncated pyramid with seven or more sides, etc.), may be an inverted truncated cone, and an inverted truncated pyramid (inverted truncated triangular pyramid, inverted truncated quadrangular pyramid, inverted truncated pentagonal pyramid, inverted truncated hexagonal pyramid, inverted truncated polygonal pyramid with seven or more angles, etc., or a combination of two or more of these shapes.

The volume per recess may be, for example, 1 to 1×10 ⁹ fL, for example, 1 to 1×10 ⁶ fL, or, for example, 1 to 1×10 ³ fL.

The density of the recesses on the substrate may be, for example, 1-2×10 ⁷ /cm ² , such as 1-2×10 ⁵ /cm ² , such as 1-2×10 ³ . /cm ² .

The shape of the substrate is not particularly limited, and examples thereof include rectangles, polygons (eg, triangles, pentagons, hexagons, polygons with heptagons or more), circles, ellipses, combinations thereof, and the like.

Also, the area of the substrate can be appropriately changed depending on the design of the recess, and may be, for example, 1 to 1000 cm ² , or may be, for example, 1 to 100 cm ² .

1(a) to 1(e) are schematic diagrams for explaining the method for manufacturing a substrate according to the first embodiment. The manufacturing method of the first embodiment will be described below with reference to FIGS.

(Step (1a))
FIG. 1(a) is a cross-sectional view of a substrate 110 having a recess 120. FIG. In this step, as shown in FIG. 1B, a protective layer 140 is laminated on the surface 130 of the substrate 110 having the concave portion 120 and the inner surface 121 of the concave portion 120 . In this specification, the term "surface on which the recess is opened" refers to a region other than the recess on the surface of the substrate on which the recess is formed.

The protective layer 140 is not particularly limited as long as it can be easily removed and does not change the shape of the recess 120. For example, AZ photoresist (Merck), SU-8 photoresist (MicroChem). a resist such as chrome; a metal film such as chromium; a paint such as ink.

Lamination of the protective layer 140 can be performed by coating, dipping, spin coating, vapor deposition, spraying, sputter deposition, chemical vapor deposition (CVD), plating, or the like, depending on the material of the protective layer used.

(Step (1b))
Subsequently, as shown in FIG. 1C, by removing a part of the protective layer 140, the surface 130 where the recess 120 opens is exposed, and the protective layer 140 is laminated on the inner surface 121 of the recess 120. get the substrate.

Part of the protective layer 140 can be removed by dry etching, wet etching, chemical mechanical polishing, or the like. Such a processing method is sometimes called etchback. As a result, the material of the substrate 110 is exposed on the surface 130 . Moreover, the recess 120 is filled with the protective layer 140 , and the protective layer 140 forms an exposed surface 141 at the opening of the recess 120 . In other words, the inner surface 121 of the recess 120 is in a state where the protective layer 140 is laminated.

(Step (1c))
Subsequently, as shown in FIG. 1(d), a first modified layer (indicated by "A" in FIG. 1) is laminated on the surface 130 where the recess 120 opens. The first modified layer may be laminated not only on the surface 130 but also on the exposed surface 141 of the protective layer 140 . Here, a protective layer 140 is laminated on the inner surface 121 of the recess 120 . Therefore, the first modified layer is not laminated on the inner surface 121 .

As used herein, a modified layer means a layer of material that changes the properties of the surface of the substrate. For example, a modification layer can be laminated on the surface of a hydrophilic substrate to change it to be hydrophobic. Alternatively, a modifying layer can be laminated on the surface of a hydrophobic substrate to make it hydrophilic. The modified layer may be made of one material, or may be made of a mixture of two or more materials. Moreover, the modified layer may be formed from one layer, or may be formed from a plurality of laminated layers.

(Step (1d))
Subsequently, the substrate laminated with the first modified layer is brought into contact with a solvent that dissolves the protective layer 140 to remove the protective layer 140 laminated on the inner surface 121 of the recess 120 . As a result, as shown in FIG. 1(e), the substrate 100 having the first modified layer laminated on the surface 130 where the recess 120 opens can be obtained. In the substrate 100, the first modified layer A is not laminated on the inner surface 121 of the recess 120, and the material of the substrate 110 is exposed.

According to the manufacturing method of the first embodiment, it is possible to simply laminate the modified layer only on the surface where the concave portion opens.

In the manufacturing method of the first embodiment, a layer having permeability to a solvent that dissolves the protective layer 140 is used as the first modified layer. From the viewpoint that the solvent that dissolves the protective layer 140 is permeable, the first modified layer is preferably an extremely thin film.

In this specification, an ultra-thin film means a film having a thickness of about 0.5 to 1000 nm. The thickness of the ultra-thin film is, for example, preferably 0.5 to 10 nm, more preferably 1 to 2 nm. It is particularly preferred that the ultrathin film is a self-assembled monolayer.

In FIG. 2A, a substrate on which a first modified layer (indicated by “A” in FIG. 2) is laminated is brought into contact with a solvent S that dissolves the protective layer 140, and the inner surface 121 of the concave portion 120 is exposed. 1 is a schematic diagram showing a state in which a protective layer 140 laminated on a layer is removed. FIG.

As shown in FIG. 2A, when the first modified layer is an ultra-thin film, the solvent S permeates the first modified layer, and the protective layer 140 laminated on the inner surface 121 of the recess 120 can be removed. Moreover, the first modified layer laminated on the exposed surface 141 of the protective layer 140 is also removed together with the protective layer 140 . On the other hand, the first modified layer laminated on the surface 130 where the recess 120 opens remains without being peeled off by the solvent S.

FIG. 2(b) is a schematic diagram illustrating the case where the solvent S does not have permeability to the first modified layer. As shown in FIG. 2B, when the solvent S cannot permeate the first modified layer, the protective layer 140 laminated on the inner surface 121 of the recess 120 cannot be removed.

Thus, in the manufacturing method of the first embodiment, it is preferable that the first modified layer is an extremely thin film. A material for forming the first modified layer can be appropriately selected according to the material of the substrate 110 .

Specific materials for forming the first modified layer include biomolecules, biomolecule-capturing molecules, and materials other than biomolecules or biomolecule-capturing molecules. Examples of biomolecules include nucleic acids, peptides, antibodies, antibody fragments, enzymes and the like. Examples of biomolecule-capturing molecules include nucleic acid aptamers, peptide aptamers, antibodies, antibody fragments, and the like. Materials other than biomolecules or biomolecule-capturing molecules include, for example, hexamethyldisilazane (HMDS), tetraethyl orthosilicate (TEOS), tetramethylsilane (TMS) and other methyl group-terminated organic silanes; Amino-terminated organosilanes such as aminopropyltriethoxysilane (APTES); Fluoroalkyl-terminated organosilanes such as perfluoroalkylsilane; Glycidyl-terminated silanes such as 3-glycidoxypropyltrimethoxysilane (GLYMO) Alkanethiols such as sulfopetine 3-undecanethiol; Alkylphosphonic acids such as octadecylphosphonic acid; Phosphate esters such as tricresyl phosphite; Fatty acids such as stearic acid; Thermoplastic resins such as acrylic resins; Thermosetting resins such as epoxy resins; Silane coupling agents to which the above biomolecules or biomolecule-capturing molecules are bound; Metals such as chromium, gold, and aluminum; Metal oxides such as aluminum oxide; paste materials containing glass or inorganic materials; molecular alignment films such as polyimide; metal chelate complexes such as nitrilotriacetic acid Ni complex (Ni-NTA) and carboxymethylaspartic acid Co complex (Co-CMA); chelating agents such as carboxymethylaspartic acid (CMA); silicone polymers such as PDMS;

Lamination of the first modified layer can be carried out by coating, dipping, spin coating, spraying, sputtering, chemical vapor deposition (CVD), plating, etc., depending on the material of the first modified layer used. can.

The first modified layer may be a layer composed of the biomolecule or the biomolecule-capturing molecule itself, or may be a layer composed of a material other than the biomolecule or the biomolecule-capturing molecule. In addition, biomolecules, biomolecule-capturing molecules, or materials other than biomolecules or biomolecule-capturing molecules may be further bound to the first modified layer.

For example, the above-described alkanethiol, alkylphosphonic acid, phosphate ester, or the like may be further bonded to the first modified layer made of the above-described metal or metal oxide film. Alternatively, the first modified layer made of the above-mentioned metal or the like may be modified to have an OH group terminal, and further the above-mentioned isocyanate or the like may be bound. Alternatively, the surface of the first modified layer described above is modified to have an amino group terminal, and further, a chelating agent such as the above-described fatty acid, nitrilotriacetic acid (NTA), carboxymethylaspartic acid (CMA), etc. may be bound. good. Nitrilotriacetic acid (NTA) may be further coordinated with nickel ions, and carboxymethylaspartic acid (CMA) may be further coordinated with cobalt ions. Alternatively, a biomolecule or a biomolecule-capturing molecule may be further bound to the first modified layer made of a material other than the biomolecule or the biomolecule-capturing molecule, or the first modified layer made of the biomolecule or the biomolecule-capturing molecule itself may be used. A biomolecule or a biomolecule-capturing molecule may be further bound to the modified layer.

Biomolecules or biomolecule-capturing molecules are the same as those described above, and include, for example, nucleic acids, peptides, proteins, and the like. Here, the nucleic acid and peptide may be nucleic acid aptamers and peptide aptamers, respectively. Examples of proteins include antibodies, antibody fragments, enzymes, and the like. Nucleic acids, peptides and proteins may each also be members of a mutant library. In the present specification, the terms "protein" and "peptide" are used without strict distinction, and the terms "peptide" and "protein" may be used depending on the number of amino acids.

Binding of biomolecules to the first modified layer uses, for example, chemical crosslinker binding; antigen-antibody reaction binding; protein capturing molecule binding; avidin-biotin binding; It can be done by

Protein-capturing molecules bound to the first modified layer include, for example, avidin; biotin; metal ions such as nickel and cobalt; maltose;

Binding of the protein-capturing molecule to the first modified layer can be carried out by using, for example, binding with a chemical crosslinker.

Also, the solvent that dissolves the protective layer 140 can be appropriately selected according to the material of the protective layer 140 to be used. -butyrolactone, propylene glycol monomethyl ether (PGME), propylene glycol monomethyl ether acetate (PGMEA), butyl carbitol (BDG), monoethanolamine (MEA), methanol, ethanol, denatured ethanol, 2-propanol (IPA), methyl lactate , ethyl lactate, ethyl acetate, butyl acetate, acetone, 2-butanone (MEK), 4-methyl-2-pentanone (MIBK), organic solvents such as acetone; sodium hydroxide solution, potassium hydroxide solution, tetramethylammonium hydroxy TMAH, alkaline solutions such as aqueous ammonia; An acid solution and the like can be mentioned. For example, when the protective layer 140 is a resist, an organic solvent or the like can be used depending on the material of the resist. Moreover, when the protective layer 140 is a metal film, an alkaline solution, an acid solution, or the like can be used. Moreover, when the protective layer 140 is a paint, water, an organic solvent, or the like can be used depending on the material of the paint.

(Second embodiment)
The manufacturing method of the second embodiment mainly differs from the manufacturing method of the first embodiment in that a second modified layer is further laminated on the substrate 100 manufactured by the manufacturing method of the first embodiment.

The manufacturing method of the second embodiment includes a step (2a) of laminating a second modified layer on the first modified layer and the inner surface 121 of the recess 120 after the step (1d) of the first embodiment. By removing the second modified layer laminated on the first modified layer, the first modified layer is laminated on the surface 130 where the recess 120 opens, and the second modified layer is formed on the inner surface 121 of the recess 120. and a step (2b) of obtaining a substrate laminated with the modified layer of (2b).

3(a) to 3(c) are schematic diagrams for explaining a method for manufacturing a substrate according to the second embodiment. The manufacturing method of the second embodiment will be described below with reference to FIGS.

(Step (2a))
FIG. 3(a) is a cross-sectional view of the substrate 100 obtained in step (1d) of the first embodiment, and corresponds to the substrate 100 shown in FIG. 1(e). As shown in FIG. 3A, in the substrate 100, the first modified layer (indicated by "A" in FIG. 3) is laminated on the surface 130 where the recess 120 opens. In the substrate 100, the first modified layer A is not laminated on the inner surface 121 of the recess 120, and the material of the substrate 110 is exposed.

In this step, as shown in FIG. 3B, the first modified layer A laminated on the surface 130 of the substrate 100 and the second modified layer ( 3, indicated by “B”) are laminated.

As a result, on the surface 130 , the first modified layer A and the second modified layer B are laminated in this order on the substrate 110 . Also, in the recess 120 , the second modified layer B is laminated on the substrate 110 .

The material of the second modified layer can be appropriately selected according to the material of the first modified layer, and after lamination on the first modified layer A, only the second modified layer B can be used.

The material for the second modified layer can be appropriately selected from materials similar to those exemplified for the first modified layer. Lamination of the second modified layer can be carried out by coating, dipping, spin coating, spraying, sputtering, chemical vapor deposition (CVD), plating, etc., depending on the material of the second modified layer used. can.

The second modified layer B, like the first modified layer A, may be a layer composed of biomolecules or biomolecule-capturing molecules themselves, or a layer composed of a material other than biomolecules or biomolecule-capturing molecules. may be Further, in the same manner as described above for the first modified layer in the manufacturing method of the first embodiment, the second modified layer B contains biomolecules or biomolecule-capturing molecules, or biomolecules or biomolecule-capturing molecules. Other materials may also be combined.

The binding of biomolecules to the second modified layer B can be performed in the same manner as the binding of biomolecules to the first modified layer A described above, for example, binding by a chemical crosslinker; binding; binding by a protein-capturing molecule; avidin-biotin binding; binding between a histidine tag and a nickel ion, or the like.

The protein-capturing molecules to be bound to the second modified layer B are the same as those exemplified for the first modified layer in the production method of the first embodiment. maltose; guanine nucleotide; glutathione; antigen;

The binding of the protein-capturing molecule to the second modified layer B is the same as the binding of the protein-capturing molecule to the first modified layer in the production method of the first embodiment. can be done by using

(Step (2b))
Subsequently, in this step, by removing the second modified layer B laminated on the first modified layer A, the first modified layer A is laminated on the surface 130 where the recess 120 opens, A substrate 200 in which the second modified layer B is laminated on the inner surface 121 of the recess 120 is obtained. In this step, the second modified layer B laminated on the first modified layer A is removed, but the second modified layer B laminated on the inner surface 121 of the recess 120 remains. The removal of the second modified layer B can be performed by a method such as removal using a solvent. The solvent can be appropriately selected according to the material of the second modified layer B, the material of the first modified layer A, etc., and is the same as that described above in the manufacturing method of the first embodiment.

In this step, the second modified layer B laminated on the first modified layer A is removed, but the second modified layer B laminated on the inner surface 121 of the recess 120 remains. The reasons for this include, for example, the following. That is, on the surface 130, the interaction between the material of the substrate 110 and the first modified layer A and the interaction between the modified layer A and the second modified layer B laminated on the modified layer A This is because the actions are different. Here, the interaction includes chemical bonding, electrostatic interaction, frictional resistance, adhesion, and the like. Due to the difference in these interactions, the adhesion between the material of the substrate 110 and the first modified layer A is lower than the adhesion between the first modified layer A and the second modified layer B. increases, and the second modified layer B stacked on the first modified layer A is removed. In addition, in the recess 120, a sufficiently large adhesive force acts due to the interaction between the material of the substrate 110 and the second modified layer B, so that the second modified layer B laminated on the inner surface 121 of the recess 120 is remain.

According to the manufacturing method of the second embodiment, different modified layers can be easily laminated on the inner surface of the recess and the surface where the recess opens.

(Third embodiment)
The manufacturing method of the third embodiment differs from the manufacturing method of the first embodiment mainly in the position where the modified layer is laminated. Specifically, in the manufacturing method of the first embodiment, the modified layer is laminated on the surface 130 where the recess 120 opens. In contrast, in the manufacturing method of the third embodiment, the modified layer is laminated on the inner surface 121 of the recess 120 .

The manufacturing method of the third embodiment includes a step (3a) of laminating a first modified layer on a surface of a substrate having a recess and an inner surface of the recess, and By the step (3b) of laminating a protective layer on the layer and removing a part of the protective layer and the first modified layer, the surface where the recess opens is exposed, and the inner surface of the recess is covered with the A step (3c) of obtaining a substrate in which the first modified layer and the protective layer are laminated in this order; and a step (3d) of obtaining a substrate in which the opening surface of the recess is exposed by removing the protective layer and the first modified layer is laminated on the inner surface of the recess.

4A to 4E are schematic diagrams for explaining a method for manufacturing a substrate according to the third embodiment. The manufacturing method of the third embodiment will be described below with reference to FIGS.

(Step (3a))
FIG. 4(a) is a cross-sectional view of the substrate 110 having the recess 120. FIG. In this step, as shown in FIG. 4B, a first modified layer (in FIG. 4, " A”) are laminated.

In the manufacturing method of the third embodiment, the substrate having recesses and the first modified layer are the same as in the manufacturing method of the first embodiment.

(Step (3b))
Subsequently, as shown in FIG. 4C, a protective layer 140 is laminated on the first modified layer. The protective layer 140 is the same as in the manufacturing method of the first embodiment.

(Step (3c))
Subsequently, as shown in FIG. 4D, by removing a part of the protective layer 140 and the first modified layer, the surface 130 where the recess 120 opens is exposed, and the inner surface 121 of the recess 120 is exposed. A substrate is obtained in which the first modified layer and the protective layer 140 are laminated in this order. Removal of the protective layer 140 can be performed in the same manner as in the manufacturing method of the first embodiment. As a result, the material of the substrate 110 is exposed on the surface 130 . Further, in the concave portion 120, the first modified layer and the protective layer 140 are laminated in this order on the substrate 110. As shown in FIG.

(Step (3d))
Subsequently, the substrate laminated with the first modified layer is brought into contact with a solvent that dissolves the protective layer 140 to remove the protective layer 140 laminated on the inner surface 121 of the recess 120 . The solvent that dissolves the protective layer 140 is the same as in the manufacturing method of the first embodiment. As a result, as shown in FIG. 4E, a substrate 300 is obtained in which the surface 130 where the recess 120 opens is exposed and the first modified layer is laminated on the inner surface 121 of the recess 120 . The material of the substrate 110 is exposed on the surface 130 of the substrate 300 . A first modified layer is laminated on the inner surface 121 of the recess 120 .

According to the manufacturing method of the third embodiment, the modified layer can be simply laminated only on the inner surface of the recess.

Thus, in the manufacturing method of the third embodiment, unlike the modified layer in the manufacturing method of the first embodiment, the solvent that dissolves the protective layer 140 does not need to permeate the modified layer. Therefore, the modified layer need not be an ultrathin film, but may be an ultrathin film.

(Fourth embodiment)
The manufacturing method of the fourth embodiment mainly differs from the manufacturing method of the third embodiment in that a second modified layer is further laminated on the substrate 300 manufactured by the manufacturing method of the third embodiment.

In the manufacturing method of the fourth embodiment, after the step (3d) of the third embodiment, a second The step (4a) of laminating the modified layer and removing the second modified layer laminated on the inner surface 121 of the recess 120 form the second modified layer on the surface 130 where the recess opens. , and a step (4b) of obtaining a substrate in which the first modified layer is laminated on the inner surface 121 of the concave portion 120 .

5(a) to 5(c) are schematic diagrams for explaining a method for manufacturing a substrate according to the fourth embodiment. The manufacturing method of the fourth embodiment will be described below with reference to FIGS.

(Step (4a))
FIG. 5(a) is a cross-sectional view of the substrate 300 obtained in step (3d) of the third embodiment, and corresponds to the substrate 300 shown in FIG. 4(e). As shown in FIG. 5A, in the substrate 300, the material of the substrate 110 is exposed on the surface 130 where the recess 120 opens, and the inner surface 121 of the recess 120 is coated with the first modified layer (see FIG. 5). , indicated by “A”) are stacked.

In this step, as shown in FIG. 5B, a second modified layer ( , denoted by “B”).

As a result, on the surface 130 , the second modified layer B is laminated on the substrate 110 . In the recess 120, the first modified layer A and the second modified layer B are laminated in this order on the substrate 110. As shown in FIG.

(Step (4b))
Subsequently, in this step, by removing the second modified layer B laminated on the inner surface 121 of the recess 120, the second modified layer B is laminated on the surface 130, and the inner surface 121 of the recess 120 A substrate 400 on which the first modified layer A is laminated is obtained.

The removal of the second modified layer B can be performed by a method such as removal using a solvent. The solvent can be appropriately selected according to the material of the second modified layer B, the material of the first modified layer A, etc., and is the same as that described above in the manufacturing method of the first embodiment.

In this step, the second modified layer B laminated on the first modified layer A is removed, but the second modified layer B laminated on the surface 130 where the recess 120 opens remains. The reasons for this include, for example, the following. That is, in the concave portion 120, the interaction between the material of the substrate 110 and the first modified layer A and the interaction between the modified layer A and the second modified layer B laminated on the modified layer A This is because the actions are different. Here, the interaction includes chemical bonding, electrostatic interaction, frictional resistance, adhesion, and the like. Due to the difference in these interactions, the adhesion between the material of the substrate 110 and the first modified layer A is lower than the adhesion between the first modified layer A and the second modified layer B. increases, and the second modified layer B stacked on the first modified layer A is removed. Further, on the surface 130, a sufficiently large adhesive force acts due to the interaction between the material of the substrate 110 and the second modified layer B, so that the second modified layer B laminated on the surface 130 remains.

In the manufacturing method of the fourth embodiment, the material of the second modified layer B can be appropriately selected from materials similar to those exemplified for the first modified layer in the manufacturing method of the first embodiment. can. However, in the manufacturing method of the fourth embodiment, unlike the first modified layer A in the manufacturing method of the first embodiment, the solvent that dissolves the protective layer 140 does not need to pass through the first modified layer, Also, the solvent for removing the second modified layer does not need to pass through the first modified layer.

Therefore, each of the first modified layer and the second modified layer in the fourth embodiment may or may not be an ultrathin film. The film thicknesses of the first modified layer and the second modified layer in the fourth embodiment may be, for example, 0.5 to 100,000 nm, and may be, for example, 0.5 to 1,000 nm. , for example, from 0.5 to 10 nm.

Moreover, the film thickness of the first modified layer disposed on the inner surface of the recess is also limited by the volume of the recess. For example, if the first modified layer fills the interior of the recess, the function of the recess is lost.

This will be explained by taking as an example the case where the concave portion has a cubic shape with a side length of 1,000 nm. The volume of the cubic recess with a side length of 1,000 nm is 1 fL. In this case, unless the film thickness of the modified layer laminated on the inner surface of the recess is less than 500 nm, which is half the length of one side, the interior of the recess is filled with the modified layer.

On the other hand, for example, when the recess has a cubic shape with a side length of 10 μm, the volume of this recess is 1×10 ³ fL. In this case, for example, even a modified layer having a thickness of about 1,000 nm can be laminated without filling the interior of the recess.

Examples of materials for the modified layer that is not an ultrathin film include thermoplastic resins such as acrylic resins; thermosetting resins such as epoxy resins; biomolecules such as nucleic acids, peptides, antibodies, antibody fragments, enzymes; nucleic acid aptamers, peptide aptamers. , antibodies, antibody fragments and other biomolecule-capturing molecules; silane coupling agents to which the biomolecules or biomolecule-capturing molecules are bound; metals such as chromium, gold and aluminum; metal oxides such as aluminum oxide; Contained paste materials; silicone polymers such as PDMS, and the like.

According to the manufacturing method of the fourth embodiment, different modified layers can be easily laminated on the inner surface of the recess and the surface where the recess opens.

(Fifth embodiment)
The manufacturing method of the fifth embodiment differs from the manufacturing method of the fourth embodiment in part of the manufacturing process, although the final manufactured substrate is the same.

The manufacturing method of the fifth embodiment is a step of laminating a second modified layer on the protective layer 140 laminated in the recess 120 and the surface 130 where the recess 120 opens after the step (3c) of the third embodiment. (5a), by removing the protective layer 140 laminated on the recess 120 and the second modified layer laminated on the protective layer 140, the second modified layer is laminated on the surface 130 where the recess 120 opens. and a step (5b) of obtaining a substrate in which the first modified layer is laminated on the inner surface 121 of the recess.

FIGS. 6A to 6C are schematic diagrams for explaining the substrate manufacturing method according to the fifth embodiment. The manufacturing method of the fifth embodiment will be described below with reference to FIGS.

(Step (5a))
FIG. 6(a) is a cross-sectional view showing a state after step (3c) of the third embodiment is completed, and corresponds to FIG. 4(d). As shown in FIG. 6A, the material of the substrate 110 is exposed on the surface 130 of the substrate 110 where the recess 120 opens, and the inner surface 121 of the recess 120 is coated with the first modified layer (in FIG. 6, "A") and the protective layer 140 are laminated in this order.

In this step, as shown in FIG. 6B, a second modified layer (indicated by "B" in FIG. 6) is formed on the protective layer 140 laminated in the recess 120 and on the surface 130 where the recess 120 opens. ) are stacked.

As a result, on the surface 130 , the second modified layer B is laminated on the substrate 110 . Further, in the concave portion 120, the first modified layer A, the protective layer 140, and the second modified layer B are laminated in this order on the substrate 110. As shown in FIG.

(Step (5b))
Subsequently, in this step, by removing the protective layer 140 laminated on the recess 120 and the second modified layer B laminated on the protective layer 140, the surface 130 where the recess 120 opens is subjected to the second modification. A substrate 400 is obtained in which the layer B is laminated and the first modified layer A is laminated on the inner surface 121 of the recess.

In the manufacturing method of the fifth embodiment, as the second modified layer B, a layer that is permeable to the solvent that dissolves the protective layer 140 is used. From the viewpoint that the solvent that dissolves the protective layer 140 is permeable, the second modified layer B is preferably an extremely thin film. The ultra-thin film is the same as that described above in the manufacturing method of the first embodiment.

The protective layer 140 and the second modified layer B can be removed by a method such as removal using a solvent that dissolves the protective layer 140 . The solvent can be appropriately selected according to the material of the protective layer 140, the material of the first modified layer A, the material of the second modified layer B, and the like. is similar to

In this step, the second modified layer B laminated on the protective layer 140 is removed, but the second modified layer B laminated on the surface 130 where the recess 120 opens remains. The reasons for this include, for example, the following. That is, in the recess 120, a sufficiently large adhesive force acts due to the interaction between the material of the substrate 110 and the first modified layer A, so that the first modified layer laminated on the inner surface 121 of the recess 120 A remains, and the protective layer 140 and the second modified layer B laminated on the protective layer 140 are removed. Here, the interaction includes chemical bonding, electrostatic interaction, frictional resistance, adhesion, and the like. Further, on the surface 130, a sufficiently large adhesive force acts due to the interaction between the material of the substrate 110 and the second modified layer B, so that the second modified layer B laminated on the surface 130 remains.

In the manufacturing method of the fifth embodiment, the material of the second modified layer B can be appropriately selected from materials similar to those exemplified for the first modified layer in the manufacturing method of the first embodiment. can.

[substrate]
In one embodiment, the present invention is a substrate having recesses, wherein a first modified layer is laminated on a surface where the recesses open, and the volume per one of the recesses is 1 to 1×10. ⁹ fL, and wherein the first modified layer is an ultra-thin film. The ultra-thin film is the same as that described above in the manufacturing method of the first embodiment.

The substrate of the present embodiment can be said to be a substrate having recesses, the surface on which the recesses open is modified, and the volume of each recess is 1 to 1×10 ⁹ fL. can also In this specification, the term "the surface of the substrate is modified" means that the surface of the substrate is endowed with properties different from those of the material of the substrate. Further, the modified portion of the surface where the recess opens can be said to be the first modified layer. Also, the first modified layer is an extremely thin film.

In the substrate of this embodiment, the volume per recess may be, for example, 1 to 1×10 ⁶ fL, or may be, for example, 1 to 1×10 ³ fL.

The substrate of this embodiment can be manufactured by the manufacturing method of the first embodiment described above. In the present embodiment, it is preferable that a plurality of recesses be present on the substrate, and preferably form an array in which the plurality of recesses are regularly arranged. Further, the recess may be formed only on one surface of the substrate, or may be formed on both surfaces.

The size, shape, and number of recesses, and the size, shape, and material of the substrate are the same as those described above in the manufacturing method of the first embodiment. Further, the material of the substrate may be exposed on the inner surface of the recess, or the second modified layer may be laminated on the inner surface of the recess.

Here, it can also be said that the second modified layer is laminated on the inner surface of the recessed portion to modify the inner surface of the recessed portion. Also, the modified portion of the inner surface of the recess can be said to be the second modified layer.

As the first modified layer and the second modified layer, the same layers as those described above can be used. The first modified layer and the second modified layer may be layers composed of biomolecules or biomolecule-capturing molecules themselves, or may be layers composed of materials other than biomolecules or biomolecule-capturing molecules. . Further, in the same manner as described above for the first modified layer in the manufacturing method of the first embodiment, the first modified layer or the second modified layer contains biomolecules, biomolecule-capturing molecules, or biomolecules. Materials other than molecules or biomolecule capture molecules may be further bound.

The substrate of this embodiment may have biomolecules immobilized on the first modified layer or the second modified layer. The substrate of this embodiment can be suitably used for biomolecular reactions. Reactions of biomolecules are not particularly limited, and examples thereof include enzyme reactions, antigen-antibody reactions, gene transcription, and mRNA translation. The substrate of the present embodiment may be used for measurement and analysis of biomolecules after reaction.

In the substrate of this embodiment, for example, the second modified layer laminated on the inner surface of the recess may contain an enzyme. In this case, an enzymatic reaction can be carried out in the recess by introducing a substrate for the enzyme into the recess. Enzyme activity can then be measured by detecting a signal according to the enzymatic reaction.

Here, the enzyme may be each enzyme mutant that constitutes an enzyme mutant library. For example, recesses on a substrate form an array, enzyme mutants having different amino acid sequences are immobilized in each recess, and an enzymatic reaction is performed, whereby enzymes can be screened on a large scale.

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental example 1]
(Production of substrate having modified layer)
A microreactor array chip was prepared in which the inner surface of the recess was hydrophilic and the opening surface of the recess was modified to be hydrophobic.

First, by dry etching, a quartz substrate having a width of 30 mm and a length of 30 mm was provided on one side thereof with one million cylindrical concave portions (microreactors) having a diameter of 4 μm and a height of 4 μm arranged at a pitch of 10 μm. .

Subsequently, a photoresist was laminated on the surface of the substrate where the recess was opened and the inner surface of the recess. Subsequently, soft baking was performed to form a protective layer.

Subsequently, dry etching using oxygen plasma was performed to partially remove the protective layer. As a result, a substrate was obtained in which the surfaces where the recesses were opened were exposed and the protective layer was laminated on the inner surfaces of the recesses.

Subsequently, a modified layer was formed on the surface on which the recess was opened. Specifically, organic silane was laminated first. Lamination of organic silane was performed by spin coating. Subsequently, by baking, a hydrophobic ultra-thin film was formed on the surface where the concave portions were opened. This ultra-thin film is the modified layer.

Subsequently, the substrate was immersed in a solvent to remove the protective layer laminated on the inner surface of the recess and the excess modified layer. As a result, a microreactor array chip having a modified layer was obtained in which a hydrophobic ultrathin film was laminated on the surface where the recess was opened and the inner surface of the recess was made of quartz.

[Experimental example 2]
(Liquid Encapsulation in Substrate)
A fluorescein solution was enclosed in the microreactor array of the microreactor array chip manufactured in Experimental Example 1. FIGS. 7A to 7C are schematic diagrams for explaining the experimental steps.

First, as shown in FIG. 7(a), a fluorescein solution F was dropped on the surface of the microreactor array chip produced in Experimental Example 1. Subsequently, as shown in FIG. 7(b), while pushing out the fluorescein solution on one side of the blade BL, the oil M was spread on the other side to seal the fluorescein solution in the microreactor array. FIG. 7(c) is a schematic diagram showing a state in which a fluorescein solution is enclosed in a microreactor array.

Subsequently, the microreactor array chip was observed with a fluorescence microscope to observe the encapsulation state of the fluorescein solution.

FIG. 8(a) is a photograph showing a state in which a fluorescein solution was dropped on the surface of a microreactor array chip. As shown in FIG. 8(a), the fluorescein solution was water-repellent, so it was confirmed that the surface of the microreactor array chip on which the recesses were opened was modified to be hydrophobic.

FIGS. 8(b) and (c) are fluorescence microscope photographs of the fluorescence of fluorescein. As a result, fluorescence of fluorescein was confirmed inside the microreactor array sealed with oil. This result indicates that the microreactor array contains liquid inside and that the inner surface of the microreactor array is hydrophilic.

From the above results, it was confirmed that by simply laminating the modified layer on the substrate on which the recesses were already formed, the inner surface of the recesses could be modified to be hydrophilic and the surface where the recesses open could be modified to be hydrophobic. .

ADVANTAGE OF THE INVENTION According to this invention, the technique which laminates|stacks a modified layer simply on the board|substrate which has a recessed part can be provided.

DESCRIPTION OF SYMBOLS 100, 200, 300... Substrate having a modified layer 110... Substrate 120... Recess 121... Inner surface of recess 130... Surface where recess opens 140... Protective layer 141... Exposed surface of protective layer, A , B... modified layer, S... solvent, F... fluorescein solution, BL... blade, M... oil.

Claims (6)

A method for manufacturing a substrate having a modified layer,
Laminating a protective layer on a surface of a substrate having a recess, where the recess opens, and on an inner surface of the recess;
Obtaining a substrate in which a surface where the recess is opened is exposed by removing a part of the protective layer, and the protective layer is laminated on the inner surface of the recess;
Laminating a first modified layer, which is permeable to a solvent that dissolves the protective layer, on the surface where the recess opens;
Obtaining a substrate having the first modified layer laminated on the surface where the recess opens by removing the protective layer laminated on the inner surface of the recess by bringing the solvent into contact with the substrate. When,
A manufacturing method, including: After obtaining the substrate in which the first modified layer is laminated on the surface where the recess opens,
Laminating a second modified layer on the first modified layer and the inner surface of the recess;
By removing the second modified layer laminated on the first modified layer, the first modified layer is laminated on the surface where the recess opens, and the inner surface of the recess is covered with the second modified layer. Obtaining a substrate on which the modified layer of 2 is laminated;
The manufacturing method of claim 1, further comprising: A method for manufacturing a substrate having a modified layer,
Laminating a first modified layer on a surface of a substrate having recesses on which the recesses open and an inner surface of the recesses;
Laminating a protective layer on the first modified layer;
By removing a part of the protective layer and the first modified layer, the surface where the recess opens is exposed, and the first modified layer and the protective layer are formed on the inner surface of the recess in this order. obtaining a laminated substrate;
A solvent that dissolves the protective layer is brought into contact with the substrate to remove the protective layer laminated on the inner surface of the recess. Obtaining a substrate on which one modified layer is laminated;
A manufacturing method, including: After obtaining a substrate in which the surface where the recess opens is exposed and the first modified layer is laminated on the inner surface of the recess,
Laminating a second modified layer on the first modified layer laminated on the inner surface of the recess and on the surface where the recess opens;
By removing the second modified layer laminated on the inner surface of the recess, the second modified layer is laminated on the surface where the recess opens, and the first modified layer is formed on the inner surface of the recess. Obtaining a substrate on which a modified layer is laminated;
4. The manufacturing method of claim 3, further comprising: A method for manufacturing a substrate having a modified layer,
Laminating a first modified layer on a surface of a substrate having recesses on which the recesses open and an inner surface of the recesses;
Laminating a protective layer on the first modified layer;
By removing a part of the protective layer and the first modified layer, the surface where the recess opens is exposed, and the first modified layer and the protective layer are formed on the inner surface of the recess in this order. obtaining a laminated substrate;
Laminating a second modified layer on the protective layer laminated in the recess and on the surface where the recess opens;
By removing the protective layer laminated on the recess and the second modified layer laminated on the protective layer, the second modified layer is laminated on the surface where the recess opens, and the recess is obtaining a substrate having the first modified layer laminated on the inner surface of the
A manufacturing method, including: The manufacturing method according to any one of claims 1 to 5, wherein the volume of each recess is 1 to ¹ x 109 fL.

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