Method for removing sealing oil

Abstract

To provide a method for efficiently removing sealing oil after filling fine storage pores of a base material with a filling liquid and sealing the pores with the sealing oil, particularly a method that enables repeated reactions in the fine storage pores by efficiently removing the sealing oil while retaining the filling liquid.SOLUTION: In a method for removing sealing oil 120 from a base material 100, the base material 100 has a plurality of fine storage holes 100a in the surface of the base material 100, the fine storage holes 100a are filled with a first filling liquid 111, and the fine storage holes 100a storing the first filling liquid 111 are sealed by the sealing oil 120. The method includes: (a) replacing the sealing oil 120 with a first removal liquid 131 mutually soluble with the sealing oil 120; and (b) volatilizing and removing the first removal liquid 131.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for removing an oil for sealing a substrate having fine accommodation pores.

Background Art

[0002] The following reactions using the fine accommodation pores of a substrate are known.

[0003] For example, Patent Document 1 discloses a substrate having a plurality of first surfaces having hydrophilicity and a second surface surrounding each of the plurality of first surfaces and having lower hydrophilicity than the first surface, the method including: a step of preparing a substrate on which a molecule that specifically binds to a nucleic acid serving as a template is immobilized on the first surface; a step of supplying, onto the substrate, a mixed solution of a sample solution containing the nucleic acid serving as the template, a nucleic acid amplification substrate, and a reaction solution containing a nucleic acid synthase, the mixed solution being disposed on the first surface, and the nucleic acid serving as the template binding to the molecule that specifically binds to the nucleic acid serving as the template; a step of supplying a hydrophobic solvent onto the substrate to form a droplet in which the mixed solution disposed on the first surface is encapsulated; a step of performing an amplification reaction of the nucleic acid within the droplet; a step of removing the hydrophobic solvent from the substrate; a step of supplying a reaction solution containing a nucleic acid amplification substrate and a nucleic acid synthase onto the substrate; and a step of performing an amplification reaction of the nucleic acid. According to the nucleic acid amplification method of Patent Document 1, it is possible to form clusters of amplified nucleic acid fragments in a regular arrangement on the substrate without amplification bias, thereby enabling efficient and highly accurate nucleic acid amplification and subsequent nucleic acid analysis in a parallel-type sequencer.

[0004] Patent Document 2 discloses a method for encapsulating beads, which includes a bead introduction step of introducing a hydrophilic solvent containing the beads into a space between a lower layer portion formed by separating a plurality of accommodating portions each capable of accommodating only one bead from each other by side walls having a hydrophobic upper surface, and an upper layer portion facing the surface of the lower layer portion where the accommodating portions are formed, and a hydrophobic solvent introduction step of introducing a hydrophobic solvent into the space after the bead introduction step. According to the bead encapsulation method of Patent Document 2, since a large number of beads can be efficiently encapsulated into an array, it is said that it is possible to contribute to a technique capable of detecting a low-concentration target molecule with high sensitivity.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] In the reaction in the fine accommodation holes of a substrate, it is known that a filling liquid, for example, a liquid containing a substrate, is introduced into the fine accommodation holes, and then the upper part of the fine accommodation holes is sealed with a sealing oil to isolate the fine accommodation holes for reaction. In order to repeatedly perform the reaction in the fine accommodation holes, it is necessary to remove this sealing oil, wash the filling liquid filled in the fine accommodation holes, and introduce the next filling liquid into the fine accommodation holes.

[0007] When the filling liquid in the fine accommodation holes is aqueous, it is known to use a water-insoluble solvent, for example, silicone oil, as the sealing oil. When washing the substrate, if a part of the sealing oil remains in the fine accommodation holes of the substrate, there is a problem that the fine accommodation holes cannot be washed sufficiently or the next filling liquid cannot be introduced into the fine accommodation holes.

[0008] Therefore, the present invention aims to provide a method for efficiently removing the sealing oil when a filling liquid is filled into the fine accommodation holes of a base material and sealed with the sealing oil, and particularly a method for efficiently removing the sealing oil while maintaining the filling liquid and performing repeated reactions in the fine accommodation holes.

Means for Solving the Problems

[0009] The present invention achieves the above object by the following means.

[0010] <Aspect 1> A method for removing the sealing oil from a base material, wherein the base material has a plurality of fine accommodation holes on the surface of the base material, a first filling liquid is accommodated in the fine accommodation holes, and the fine accommodation holes accommodating the first filling liquid are sealed with a sealing oil, and the removal method includes (a) replacing the sealing oil with a first removal liquid that is compatible with the sealing oil, and (b) volatilizing and removing the first removal liquid, A method for removing the sealing oil, including the above. <Aspect 2> After step (b), (c) removing the first filling liquid with a second removal liquid that is compatible with the first filling liquid The method according to Aspect 1, further including the above. <Aspect 3> The method according to Aspect 1 or 2, wherein the first removal liquid is a liquid having a higher volatility value than the first filling liquid. <Aspect 4> The method according to any one of Aspects 1 to 3, wherein the second removal liquid is the same liquid as the solvent contained in the first filling liquid. <Aspect 5> A method for removing the sealing oil from a base material, wherein the base material has a plurality of fine accommodation holes on the surface of the base material, A first filling liquid is contained in the fine accommodation holes, and the fine accommodation holes containing the first filling liquid are sealed with a sealing oil, the removal method is (d) replacing the sealing oil with a third removal liquid that is compatible with the sealing oil, (e) replacing the third removal liquid with a fourth removal liquid that is compatible with the third removal liquid, and (f) volatilizing and removing the fourth removal liquid, A method for removing a sealing oil, comprising the above steps. <Aspect 6> After step (f), (g) removing the first filling liquid with a fifth removal liquid that is compatible with the first filling liquid The method according to aspect 5, further comprising the above step. <Aspect 7> The method according to aspect 5 or 6, wherein the fourth removal liquid is a liquid having a higher volatility value than the first filling liquid. <Aspect 8> The method according to any one of aspects 5 to 7, wherein the fifth removal liquid is the same liquid as the solvent contained in the first filling liquid. <Aspect 9> A method for removing a sealing oil from a substrate, wherein the substrate has a plurality of fine accommodation holes on the surface of the substrate, a first filling liquid is contained in the fine accommodation holes, and the fine accommodation holes containing the first filling liquid are sealed with a sealing oil, the removal method is (h) replacing the sealing oil with a sixth removal liquid that is compatible with the sealing oil, (i) replacing the sixth removal liquid with a seventh removal liquid that is compatible with the sixth removal liquid, (j) replacing the seventh removal liquid with an eighth removal liquid that is compatible with the seventh removal liquid, and (k) volatilizing and removing the eighth removal liquid, A method for removing sealing oil, including <Aspect 10> After step (k), (l) Removing the first filling liquid with a ninth removing liquid that is compatible with the first filling liquid The method according to aspect 9, further including <Aspect 11> The method according to aspect 9 or 10, wherein the eighth removing liquid is a liquid having a higher volatility value than the first filling liquid <Aspect 12> The method according to any one of aspects 9 to 11, wherein the ninth removing liquid is the same liquid as the solvent contained in the first filling liquid <Aspect 13> (p) Accommodating the first filling liquid in a plurality of fine accommodation holes on the surface of the base material, (q) Sealing the fine accommodation holes containing the first filling liquid with sealing oil, (r) Causing a reaction of the first filling liquid in the fine accommodation holes to generate a first reaction product, and (s) Removing the sealing oil by the method according to any one of aspects 1 to 12 A method for generating a reaction product in a fine accommodation hole, including <Aspect 14> After step (s), removing the first filling liquid with the removing liquid that is compatible with the first filling liquid, and (p’) Accommodating a second filling liquid in a plurality of fine accommodation holes on the surface of the base material, (q’) Sealing the fine accommodation holes containing the second filling liquid with sealing oil, And (r’) Causing a reaction of the second filling liquid in the fine accommodation holes to generate a second reaction product The method according to aspect 13, further including <Aspect 15> The method according to aspect 13 or 14, wherein the second filling liquid is the same as the first filling liquid <Aspect 16> The method according to aspect 13 or 14, wherein the second filling liquid is different from the first filling liquid.

Advantages of the Invention

[0011] According to the method for removing the sealing oil of the present invention, when a filling liquid is filled into the fine accommodation holes of the base material and sealed with the sealing oil, the sealing oil can be efficiently removed. In particular, while maintaining the filling liquid, the sealing oil can be efficiently removed, and repeated reactions can be performed in the fine accommodation holes.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Embodiments for Carrying Out the Invention

[0013] Hereinafter, embodiments of the present invention will be described in detail. It should be noted that the present invention is not limited to the following embodiments, and can be variously modified and implemented within the scope of the gist of the present invention. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant descriptions are omitted.

[0014] In the present invention, "compatible" in the description of liquid B "compatible" with liquid A means that even if the target liquid B is mixed with liquid A at any ratio, no interface is formed. Also, "incompatible" in the description of liquid B "incompatible" with liquid A means that an interface is formed when the target liquid B is mixed with liquid A at any ratio.

[0015] In the present invention, a "non-volatile" liquid means a liquid having a volatility value of less than 1, and a "volatile" liquid means a liquid having a volatility value of 1 or more. Here, the "volatility value" is a relative value with water as 1, which is the amount of weight loss after leaving a certain amount of the solvent in a container without a lid such as a beaker or a reagent tube under normal temperature, normal pressure and constant humidity conditions for a certain period of time.

[0016] 《Method for Removing Sealing Oil: First Aspect》 The present invention is a method for removing sealing oil from a substrate, wherein the substrate has a plurality of fine accommodation holes on the surface of the substrate, a first filling liquid is accommodated in the fine accommodation holes, and The fine accommodation holes containing the first filling liquid are sealed with sealing oil, The removal method is, (a) replacing the sealing oil with a first removal liquid that is compatible with the sealing oil, and (b) volatilizing and removing the first removal liquid. It includes.

[0017] According to the method for removing the sealing oil of the present invention, when a filling liquid is filled in the fine accommodation holes of a substrate and sealed with the sealing oil, the sealing oil can be efficiently removed. In particular, while maintaining the filling liquid, the sealing oil can be efficiently removed, and repeated reactions can be performed in the fine accommodation holes.

[0018] The sealing oil is usually a liquid that is incompatible with the first filling liquid in order to seal the fine accommodation holes. Therefore, it is predicted that even if the first filling liquid or the solvent contained in the first filling liquid is circulated on the surface of the substrate having the fine accommodation holes, the removal of the sealing oil will be insufficient. Although not limited to theory, since the first removal liquid is compatible with the sealing oil, the first removal liquid is circulated to remove the sealing oil and replace it with the first removal liquid. For example, by circulating a gas, the first removal liquid is volatilized, thereby efficiently removing the sealing oil that seals the fine accommodation holes. Furthermore, since the sealing oil can be removed with high efficiency, repeated reactions can be performed in the fine accommodation holes.

[0019] FIG. 1 is a schematic diagram showing one embodiment of the method for removing the sealing oil of the present invention, but it is not limited to this case.

[0020] In a substrate 100 having fine accommodation holes 100a, fine accommodation holes 110a containing a first filling liquid 111 are sealed with a sealing oil 120 (FIG. 1A). For the substrate 100 having the fine accommodation holes 100a sealed with the sealing oil 120, a first removing liquid 131 is circulated to replace the sealing oil 120 with the first removing liquid 131 (FIG. 1B). Since the first removing liquid 131 is compatible with the sealing oil 120, the sealing oil 120 can be removed by circulating the first removing liquid 131 and replaced with the first removing liquid 131. Next, for example, by circulating a gas, the first removing liquid 131 is volatilized and removed (FIG. 1C). The sealing oil is replaced with the first removing liquid 131 that is compatible with the sealing oil, and the first removing liquid 131 is removed by volatilization, whereby the sealing oil 120 sealing the fine accommodation holes 100a can be efficiently removed. Further, since the sealing oil 120 is removed with high efficiency, another filling liquid (for example, a second filling liquid 112) can be introduced again and the fine accommodation holes 110a can be utilized.

[0021] The method for removing the sealing oil of the present invention may further include, after step (b), (c) removing the first filling liquid with a second removing liquid that is compatible with the first filling liquid, and may further include.

[0022] In FIG. 1 described above, for the substrate 100 having the fine accommodation holes 110a from which the first removing liquid 131 has been volatilized and removed (FIG. 1C), a second removing liquid 132 is circulated to fill the substrate 100 having the fine accommodation holes 100a with the second removing liquid 132, and the first filling liquid 111 can be removed from the fine accommodation holes 100a (FIG. 1D). Here, when the second removing liquid 132 is the same solvent as the first filling liquid 110, the first filling liquid 111 can be removed from the fine accommodation holes 100a more efficiently.

[0023] 〈Method of replacing with a removing liquid〉 The method of replacement with a removal liquid is not particularly limited. For example, the removal liquid can be replaced with the removal liquid from the sealing oil or the like by flowing the removal liquid on the surface of the substrate having the fine accommodation holes, but it is not limited to this case.

[0024] 〈Method of volatilizing the removal liquid〉 The method of volatilizing the removal liquid is not particularly limited. For example, gas flow, natural drying, heating, pressurization, negative pressure, etc. can be mentioned, but from the viewpoint of efficient volatilization, gas flow is preferable.

[0025] 〈Method of removing the first filling liquid〉 The method of removing the first filling liquid is not particularly limited. As a method of removing the first filling liquid, for example, the removal liquid is caused to flow on the surface of the substrate having the fine accommodation holes, the fine accommodation holes are filled with the removal liquid, and replaced with the removal liquid, whereby the first filling liquid can be removed.

[0026] 《Method of removing the sealing oil: Second aspect》 The present invention is a method for removing the sealing oil from a base material, wherein the base material has a plurality of fine accommodation holes on the surface of the base material, a first filling liquid is accommodated in the fine accommodation holes, and the fine accommodation holes accommodating the first filling liquid are sealed with a sealing oil, the removal method is (d) replacing the sealing oil with a third removal liquid that is compatible with the sealing oil, (e) replacing the third removal liquid with a fourth removal liquid that is compatible with the third removal liquid, and (f) volatilizing and removing the fourth removal liquid. including.

[0027] According to the method for removing the sealing oil of the present invention, when a filling liquid is filled into the fine accommodation holes of a base material and sealed with the sealing oil, the sealing oil can be efficiently removed. In particular, while maintaining the filling liquid, the sealing oil can be efficiently removed, and repeated reactions can be performed in the fine accommodation holes. Furthermore, according to the method for removing the sealing oil of the present invention, if the third removal liquid is compatible with the sealing oil, even when the third removal liquid is a non-volatile liquid, the sealing oil can be efficiently removed.

[0028] The sealing oil is usually a liquid that is not compatible with the first filling liquid in order to seal the fine accommodation holes. Therefore, it is predicted that even if the first filling liquid or the solvent contained in the first filling liquid is circulated on the surface of the base material having the fine accommodation holes, the removal of the sealing oil will be insufficient. Without being limited to theory, the sealing oil is replaced with a third removal liquid that is compatible with the sealing oil, the third removal liquid is replaced with a fourth removal liquid that is compatible with the third removal liquid, and the fourth removal liquid is volatilized and removed, whereby the sealing oil sealing the fine accommodation holes can be efficiently removed. According to the method of the present invention, if the third removal liquid is compatible with the sealing oil, even when the third removal liquid is a non-volatile liquid, the sealing oil can be efficiently removed by sequentially replacing the sealing oil with the third removal liquid and then the fourth removal liquid. Furthermore, since the sealing oil can be removed with high efficiency, repeated reactions can be performed in the fine accommodation holes.

[0029] FIG. 2 is a schematic diagram showing one embodiment of the method for removing the sealing oil of the present invention, but it is not limited to this case.

[0030] In a substrate 100 having fine accommodation holes 110a, the fine accommodation holes 110a containing a first filling liquid 111 are sealed with a sealing oil 120 (FIG. 2A). With respect to the substrate 100 having the fine accommodation holes 100a sealed with this sealing oil 120, a third removal liquid 133 is passed therethrough to replace the sealing oil 120 with the third removal liquid 133 (FIG. 2B). Since the third removal liquid 133 is compatible with the sealing oil 120, the sealing oil 120 can be removed by passing it through and replaced with the third removal liquid 133. Next, a fourth removal liquid 134 is passed therethrough to replace the third removal liquid 133 with the fourth removal liquid 134 (FIG. 2C). Since the fourth removal liquid 134 is compatible with the third removal liquid 133, the third removal liquid 133 can be removed by passing it through and replaced with the fourth removal liquid 134. Next, for example, by passing a gas therethrough, the fourth removal liquid 134 is volatilized and removed (FIG. 2D). The sealing oil 120 is replaced with a third removal liquid 133 that is compatible with the sealing oil 120, the third removal liquid is replaced with a fourth removal liquid 134 that is compatible with the third removal liquid 133, and the fourth removal liquid 134 is volatilized and removed, whereby the sealing oil sealing the fine accommodation holes 100a can be efficiently removed. Furthermore, since the sealing oil is removed with high efficiency, another filling liquid (for example, a second filling liquid 112) can be introduced again and this fine accommodation hole can be utilized.

[0031] The method for removing the sealing oil of the present invention, after step (f), (g) removing the first filling liquid with a fifth removal liquid compatible with the first filling liquid, may further be included.

[0032] In the above-described FIG. 2, with respect to the substrate 100 (FIG. 2D) having the fine accommodation holes 100a from which the fourth removal liquid 134 has been volatilized and removed, the fifth removal liquid 135 is circulated, and the substrate 100 having the fine accommodation holes 110a is filled with the fifth removal liquid 135, and the first filling liquid 111 can be removed from the fine accommodation holes 100a (FIG. 2E). Here, when the fifth removal liquid 135 is the same solvent as the first filling liquid 111, the first filling liquid 111 can be removed from the fine accommodation holes 100a more efficiently.

[0033] Regarding the operation methods such as substitution and volatilization, reference can be made to the descriptions in the above "〈Method of Substituting with a Removal Liquid〉", the above "〈Method of Volatilizing a Removal Liquid〉", and the above "〈Method of Removing the First Filling Liquid〉".

[0034] 《Method for Removing Sealing Oil: Third Aspect》 The present invention is a method for removing sealing oil from a substrate, wherein the substrate has a plurality of fine accommodation holes on the surface of the substrate, a first filling liquid is accommodated in the fine accommodation holes, and the fine accommodation holes accommodating the first filling liquid are sealed with sealing oil, and the removal method is (h) substituting the sealing oil with a sixth removal liquid that is compatible with the sealing oil, (i) substituting the sixth removal liquid with a seventh removal liquid that is compatible with the sixth removal liquid, (j) substituting the seventh removal liquid with an eighth removal liquid that is compatible with the seventh removal liquid, and (k) volatilizing and removing the eighth removal liquid. It includes.

[0035] According to the method for removing the sealing oil of the present invention, when a filling liquid is filled into the fine accommodation holes of a base material and sealed with the sealing oil, the sealing oil can be efficiently removed. In particular, while maintaining the filling liquid, the sealing oil can be efficiently removed, and repeated reactions can be carried out in the fine accommodation holes. Furthermore, according to the method for removing the sealing oil of the present invention, if the sixth removal liquid is compatible with the sealing oil and the seventh removal liquid is compatible with the sixth removal liquid, even when the sixth and seventh removal liquids are non-volatile liquids, the sealing oil can be efficiently removed.

[0036] The sealing oil is usually a liquid that is not compatible with the first filling liquid in order to seal the fine accommodation holes. Therefore, it is predicted that even if the first filling liquid or the solvent contained in the first filling liquid is circulated on the surface of the base material having the fine accommodation holes, the removal of the sealing oil will be insufficient. Without being limited to theory, the sealing oil is replaced with a sixth removal liquid that is compatible with the sealing oil, the sixth removal liquid is replaced with a seventh removal liquid that is compatible with the sixth removal liquid, the seventh removal liquid is replaced with an eighth removal liquid that is compatible with the seventh removal liquid, and the eighth removal liquid is volatilized and removed, whereby the sealing oil sealing the fine accommodation holes can be efficiently removed. According to the method of the present invention, if the sixth removal liquid is compatible with the sealing oil, the seventh removal liquid is compatible with the sixth removal liquid, and the eighth removal liquid is compatible with the seventh removal liquid, even when the sixth and seventh removal liquids are non-volatile liquids, the sealing oil can be efficiently removed by sequentially replacing the sealing oil with the sixth removal liquid, the seventh removal liquid, and the eighth removal liquid. Furthermore, since the sealing oil can be removed with high efficiency, repeated reactions can be carried out in the fine accommodation holes.

[0037] FIG. 3 is a schematic diagram showing one embodiment of the method for removing the sealing oil of the present invention, but is not limited to this case.

[0038] In a substrate 100 having fine accommodation holes 110a, the fine accommodation holes 110a containing a first filling liquid 111 are sealed with a sealing oil 120 (FIG. 3A). With respect to the substrate 100 having the fine accommodation holes 100a sealed with this sealing oil 120, a sixth removing liquid 136 is circulated to replace the sealing oil 120 with the sixth removing liquid 136 (FIG. 3B). Since the sixth removing liquid 136 is compatible with the sealing oil 120, the sealing oil 120 can be removed by circulation and replaced with the sixth removing liquid 136. Next, a seventh removing liquid 137 is circulated to replace the sixth removing liquid 136 with the seventh removing liquid 137 (FIG. 3C). Since the seventh removing liquid 137 is compatible with the sixth removing liquid 136, the sixth removing liquid 136 can be removed by circulation and replaced with the seventh removing liquid 137. Next, an eighth removing liquid 138 is circulated to replace the seventh removing liquid 137 with the eighth removing liquid 138 (FIG. 3D). Since the eighth removing liquid 138 is compatible with the seventh removing liquid 137, the seventh removing liquid 137 can be removed by circulation and replaced with the eighth removing liquid 138. Next, for example, by circulating a gas, the eighth removing liquid 138 is volatilized and removed (FIG. 3E). The sealing oil is replaced with the sixth removing liquid 136 that is compatible with the sealing oil, the sixth removing liquid 136 is replaced with the seventh removing liquid 137 that is compatible with the sixth removing liquid 136, the seventh removing liquid 137 is replaced with the eighth removing liquid 138 that is compatible with the seventh removing liquid 137, and the eighth removing liquid 138 is removed by volatilization, whereby the sealing oil 120 sealing the fine accommodation holes 100a can be efficiently removed. Further, since the sealing oil is removed with high efficiency, another filling liquid (for example, a second filling liquid 112) can be introduced again and this fine accommodation hole can be used.

[0039] The method for removing the sealing oil of the present invention may further include, after step (k), (l) removing the first filling liquid with a ninth removing liquid that is compatible with the first filling liquid, and may further include.

[0040] In the above-described FIG. 4, with respect to the substrate 100 (FIG. 4E) having the fine accommodation holes 110a from which the eighth removal liquid 138 has been volatilized and removed, the ninth removal liquid 139 is caused to flow, the substrate 100 having the fine accommodation holes 110a is filled with the ninth removal liquid 139, and the first filling liquid 111 can be removed from the fine accommodation holes 100a (FIG. 4F). Here, when the ninth removal liquid 139 is the same solvent as the first filling liquid 110, the first filling liquid 111 can be more efficiently removed from the fine accommodation holes 100a.

[0041] Regarding the operation methods such as substitution and volatilization, reference can be made to the descriptions in the above "〈Method of Substituting with a Removal Liquid〉", the above "〈Method of Volatilizing a Removal Liquid〉", and the above "〈Method of Removing the First Filling Liquid〉".

[0042] 《Method for Removing Sealing Oil: Each Component》 Hereinafter, each component of the method of the present invention for removing the sealing oil will be described.

[0043] 〈Sealing Oil Present in the Substrate Having Fine Accommodation Holes〉 In the present invention, the substrate has a plurality of fine accommodation holes on the surface of the substrate, the first filling liquid is accommodated in the fine accommodation holes, and the fine accommodation holes accommodating the first filling liquid are sealed with the sealing oil.

[0044] In the above-described FIG. 1A, the substrate 100 has a plurality of fine accommodation holes 100a on the surface of the substrate, and the first filling liquid 111 is accommodated in the fine accommodation holes 100a. The fine accommodation holes 100a are sealed with the sealing oil 120, and the first filling liquid 111 is fractionated in the fine accommodation holes 100a, respectively.

[0045] 〈Substrate〉 The substrate has a plurality of fine accommodation holes on its surface. The shape of the substrate is not particularly limited. It may be planar or tubular. The substrate is not particularly limited, but it may be provided with a flow path through which a fluid flows, and may be provided with an inlet (fluid inlet) and an outlet (fluid outlet).

[0046] The material of the substrate is not particularly limited, but resins, glass, metals, etc. can be used. As the resin, for example, polydimethylsiloxane (PDMS), silicone resin, polystyrene resin, polypropylene resin, epoxy resin, etc. can be used, but it is not limited to these cases.

[0047] (Fine accommodation holes) The shape of the fine accommodation holes may be all or part of them circular or elliptical, or non-circular, such as polygonal. The equivalent diameter of the fine accommodation holes is not particularly limited, but may be 0.001 mm or more, 0.005 mm or more, 0.01 mm or more, or 0.05 mm or more, and may be 1.0 mm or less, 0.5 mm or less, 0.3 mm or less, 0.2 mm or less, or 0.1 mm or less. Note that the equivalent diameter refers to the diameter of a perfect circle having the same outer perimeter length as the outer perimeter length of that surface.

[0048] The depth of the fine accommodation holes is not particularly limited, but may be 0.001 mm or more, 0.005 mm or more, 0.01 mm or more, or 0.05 mm or more, and may be 1.0 mm or less, 0.5 mm or less, 0.3 mm or less, 0.2 mm or less, or 0.1 mm or less.

[0049] The pitch of adjacent fine accommodation holes (the center-to-center distance of adjacent fine accommodation holes) is not particularly limited, but may be 0.01 mm or more, 0.02 mm or more, 0.05 mm or more, or 0.10 mm or more, and may be 5.00 mm or less, 3.00 mm or less, 1.00 mm or less, 0.50 mm or less, or 0.20 mm or less.

[0050] The fine accommodation pores may be evenly distributed or may be unevenly distributed in some places. The fine accommodation pores may not be present, for example, at the ends of the substrate.

[0051] The substrate having the fine accommodation pores is not particularly limited, but can be produced using general photolithography and soft lithography. Specifically, for example, a mold having fine accommodation pores, channels, etc. formed on a silicon wafer by photolithography can be created, and then a resin or the like can be poured into this mold, the resin can be cured, and a fine three-dimensional structure can be transferred (soft lithography) to produce a substrate having fine accommodation pores, but it is not limited to this case.

[0052] 〈First filling liquid〉 The first filling liquid may contain a solvent and optionally other components, etc.

[0053] The solvent of the first filling liquid is not particularly limited, and examples include water, aqueous solvents, hydrophilic alcohols, hydrophilic ethers, hydrophilic ketones, hydrophilic nitrile solvents, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), etc. Examples of the aqueous solvent include buffer solutions, specifically phosphate buffered saline, etc. Examples of the hydrophilic alcohol include methanol, ethanol, propanol, glycerin, etc. Examples of the hydrophilic ether include tetrahydrofuran, polyethylene oxide, 1,4-dioxane, etc. Examples of the ketone include acetone, etc. Examples of the nitrile solvent include acetonitrile, etc. The first filling liquid is not particularly limited, but water is preferred, and phosphate buffered saline is more preferred. The first filling liquid is not particularly limited, but only one type may be used alone, or two or more types may be used in combination.

[0054] Other components that may be contained in the first filling liquid are not particularly limited, and may include, for example, fluorescent dyes, chromogenic dyes, radioactive labels, beads, reaction substrates, enzymes, proteins, nucleic acids, catalysts, etc.

[0055] <Sealing oil> The sealing oil is not particularly limited as long as it is a solvent that is incompatible with the first filling liquid. When the first filling liquid is water or an aqueous solvent, a water-insoluble solvent is preferable as the sealing oil. The sealing oil is not particularly limited, and examples thereof include silicone oil, saturated hydrocarbons, unsaturated hydrocarbons, and hydrophobic ionic liquids. Examples of silicone oil include non-volatile oils such as KF-96-20CS (Shin-Etsu Chemical Co., Ltd.) and KF-96-50CS (Shin-Etsu Chemical Co., Ltd.). Examples of saturated hydrocarbons include alkanes and cycloalkanes. Examples of alkanes include decane and hexadecane. Examples of unsaturated hydrocarbons include squalene. Examples of hydrophobic ionic liquids include 1-butyl-3-methylimidazolium hexafluorophosphate. An ionic liquid is a salt that exists as a liquid at room temperature (23°C). The sealing oil is not particularly limited, but silicone oil is preferable. The sealing oil is not particularly limited, and only one type may be used alone, or two or more types may be used in combination.

[0056] The fine accommodation pores sealed with the sealing oil can be easily formed, for example, by flowing the first filling liquid through a substrate having the fine accommodation pores and then flowing the sealing oil through them.

[0057] FIG. 4 is a schematic diagram showing one embodiment of sealing the fine accommodation pores containing the first filling liquid with the sealing oil in the method for removing the sealing oil of the present invention, but the present invention is not limited to this case.

[0058] For a substrate 100 having fine accommodation holes 100a (Fig. 4A), a first filling liquid 111 is circulated to fill the fine accommodation holes 100a with the first filling liquid 111 (Fig. 4B). Next, a sealing oil 120 is circulated (Fig. 4C) to replace the upper part of the fine accommodation holes 100a from the first filling liquid 111 with the sealing oil 120, whereby the fine accommodation holes 100a containing the first filling liquid 111 can be sealed with the sealing oil 120 (Fig. 4D).

[0059] 〈First Removing Liquid〉 The first removing liquid is not particularly limited as long as it is compatible with the sealing oil. It is preferable that the first removing liquid is not compatible with the first filling liquid.

[0060] The first removing liquid is not particularly limited, but from the viewpoint of volatilizing the first filling liquid while maintaining it in the fine accommodation holes, a liquid having a higher volatility value than the first filling liquid is preferable, and a liquid having a higher volatility value than the sealing oil is also preferable.

[0061] The volatility value of the first removing liquid is not particularly limited, but when water is taken as 1, it may be 10 or more, 15 or more, or 20 or more, or may be 200 or less, 150 or less, 100 or less, or 50 or less. As described above, the "volatility value" is a relative value when the weight reduction amount after leaving a certain amount of the solvent in a lidless container such as a beaker or a reagent tube for a certain time under normal temperature, normal pressure and constant humidity conditions is compared with water taken as 1.

[0062] When the sealing oil is, for example, non-volatile silicone oil KF-96-20CS (Shin-Etsu Chemical Co., Ltd.), the first removal liquid is not particularly limited, but hydrofluoroether, volatile silicone oil, etc. can be used. Examples of the hydrofluoroether include Novec7200 (manufactured by 3M). Examples of the volatile silicone oil include KF-96L-0.65CS (Shin-Etsu Chemical Co., Ltd.), KF-96L-1CS (Shin-Etsu Chemical Co., Ltd.), KF-96L-1.5CS (Shin-Etsu Chemical Co., Ltd.), KF-96L-2CS (Shin-Etsu Chemical Co., Ltd.), KF-86A-1CS (Shin-Etsu Chemical Co., Ltd.), KF-4422 (Shin-Etsu Chemical Co., Ltd.), KF-995 (Shin-Etsu Chemical Co., Ltd.), TMF1.5 (Shin-Etsu Chemical Co., Ltd.), octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, etc. The first removal liquid is not particularly limited, but Novec7200 is preferred. The first removal liquid is not particularly limited, but only one kind may be used alone, or two or more kinds may be used in combination.

[0063] 〈Second removal liquid〉 The second removal liquid is not particularly limited as long as it is compatible with the first filling liquid.

[0064] The second removal liquid is not particularly limited, but from the viewpoint of efficiently removing the first filling liquid present in the fine accommodation pores, the same liquid as the solvent contained in the first filling liquid is preferred. For example, when phosphate buffered saline is contained as the solvent of the first filling liquid, phosphate buffered saline is preferred as the second removal liquid.

[0065] Regarding the second removal liquid, reference can be made to the description of the above "〈First filling liquid〉".

[0066] 〈Third removal liquid〉 The third removal liquid is not particularly limited as long as it is compatible with the sealing oil. It is preferable that the third removal liquid is not compatible with the first filling liquid.

[0067] As the third removal liquid, for example, when the sealing oil is non-volatile silicone oil KF-96-20CS (Shin-Etsu Chemical Co., Ltd.), long-chain fatty acid esters, hydrocarbons, hydrophobic and non-volatile alcohols, hydrophobic and non-volatile ketones, hydrophobic and non-volatile nitrile solvents, etc. can be used. The long-chain fatty acid ester is not particularly limited, but an ester of a saturated or unsaturated straight-chain fatty acid having 8 to 22 carbon atoms and an alcohol can be used. Specifically, for example, isopropyl palmitate, isopropyl laurate, isopropyl myristate, isopropyl stearate, isopropyl oleate, etc. can be mentioned, but it is not limited to these cases. Examples of the hydrocarbon include decane, hexadecane, etc., but it is not limited to these cases. Examples of the hydrophobic and non-volatile alcohol include octanol, nonanol, etc., examples of the hydrophobic and non-volatile ketone include diisobutyl ketone, etc., and examples of the hydrophobic and non-volatile nitrile solvent include butanenitrile, etc., but it is not limited to these cases.

[0068] 〈Fourth Removal Liquid〉 The fourth removal liquid is not particularly limited as long as it is compatible with the third removal liquid. It is preferable that the fourth removal liquid is not compatible with the first filling liquid.

[0069] The fourth removal liquid is not particularly limited, but from the viewpoint of volatilizing the first filling liquid while maintaining it in the fine accommodation pores, a liquid having a higher volatility value than the first filling liquid is preferable, and a liquid having a higher volatility value than the sealing oil and / or the third removal liquid is also preferable. The volatility value of the fourth removal liquid is not particularly limited, but when water is set to 1, it may be 10 or more, 15 or more, or 20 or more, and may be 200 or less, 150 or less, 100 or less, or 50 or less. Regarding the volatility value, reference can be made to the description in the above-mentioned "〈First Removal Liquid〉".

[0070] As the fourth removal liquid, for example, when the sealing oil is non-volatile silicone oil KF-96-20CS (Shin-Etsu Chemical Co., Ltd.), hydrofluoroether, hydrophobic and volatile alcohol, ketone, nitrile-based solvent, etc. can be used. Examples of hydrofluoroether include AE-3000, etc., examples of hydrophobic and volatile alcohol include butanol, etc., examples of hydrophobic and volatile ketone include methyl ethyl ketone, etc., and examples of hydrophobic and volatile nitrile-based solvent include acrylonitrile, etc., but it is not limited to this case.

[0071] 〈The Fifth Removal Liquid〉 The fifth removal liquid is not particularly limited as long as it is compatible with the first filling liquid.

[0072] The fifth removal liquid is not particularly limited, but from the viewpoint of efficiently removing the first filling liquid present in the fine accommodation pores, the same liquid as the solvent contained in the first filling liquid is preferable. For example, when the first filling liquid is phosphate buffered saline, phosphate buffered saline is preferable as the fifth removal liquid.

[0073] Regarding the fifth removal liquid, the description in the above “〈The First Filling Liquid〉” can be referred to.

[0074] 〈The Sixth and Seventh Removal Liquids〉 The sixth removal liquid is not particularly limited as long as it is compatible with the sealing oil. The seventh removal liquid is not particularly limited as long as it is compatible with the sixth removal liquid. The sixth and seventh removal liquids are preferably not compatible with the first filling liquid.

[0075] The sixth removal liquid and the seventh removal liquid are not particularly limited. For example, when the sealing oil is non-volatile silicone oil KF-96-20CS (Shin-Etsu Chemical Co., Ltd.), long-chain fatty acid esters, hydrocarbons, etc. can be used. Regarding long-chain fatty acid esters and hydrocarbons, the description in the above “〈The Third Removal Liquid〉” can be referred to.

[0076] 〈The Eighth Removal Liquid〉 The eighth removal liquid is not particularly limited as long as it is compatible with the seventh removal liquid. It is preferable that the eighth removal liquid is not compatible with the first filling liquid.

[0077] The eighth removal liquid is not particularly limited. However, from the viewpoint of volatilizing the first filling liquid while maintaining it in the fine accommodation holes, a liquid having a higher volatility value than the first filling liquid is preferable, and a liquid having a higher volatility value than the sealing oil, the sixth removal liquid, and / or the seventh removal liquid is also preferable. The volatility value of the eighth removal liquid is not particularly limited, but when water is set to 1, it may be 10 or more, 15 or more, or 20 or more, and may also be 200 or less, 150 or less, 100 or less, or 50 or less. Regarding the volatility value, reference can be made to the description of "<the first removal liquid>" above.

[0078] The eighth removal liquid is not particularly limited. For example, when the sealing oil is non-volatile silicone oil KF-96-20CS (Shin-Etsu Chemical Co., Ltd.), hydrofluoroether or the like can be used. Regarding hydrofluoroether, reference can be made to the description of "<the fourth removal liquid>" above.

[0079] 〈The ninth removal liquid〉 The ninth removal liquid is not particularly limited as long as it is compatible with the first filling liquid.

[0080] The ninth removal liquid is not particularly limited. However, from the viewpoint of efficiently removing the first filling liquid present in the fine accommodation holes, the same liquid as the solvent contained in the first filling liquid is preferable. For example, when the first filling liquid is phosphate buffered saline, phosphate buffered saline is preferable as the ninth removal liquid.

[0081] Regarding the ninth removal liquid, reference can be made to the description of "<the first filling liquid>" above.

[0082] 《Method for generating reaction product in fine accommodation holes》 The following steps can be included to generate a reaction product in the fine accommodation holes: (p) Accommodating a first filling liquid in a plurality of fine accommodation holes on the surface of a substrate, (q) Sealing the fine accommodation holes containing the first filling liquid with a sealing oil, (r) Causing a reaction of the first filling liquid in the fine accommodation holes to generate a first reaction product, and (s) Removing the sealing oil by the method for removing the sealing oil of the present invention.

[0083] FIG. 5 is a schematic diagram showing one embodiment of the method for generating a reaction product in the fine accommodation holes of the present invention, but is not limited to this case.

[0084] For a substrate 100 having fine accommodation holes 100a (FIG. 5A), a first filling liquid 111 is circulated, and the first filling liquid 111 is filled into the fine accommodation holes 100a (FIG. 5B). Next, a sealing oil 120 is circulated, and the upper part of the fine accommodation holes 100a is replaced from the first filling liquid 111 with the sealing oil 120, thereby sealing the fine accommodation holes 100a containing the first filling liquid 111 with the sealing oil 120 (FIG. 5C). A reaction of the first filling liquid 111 occurs in the fine accommodation holes 100a to generate a first reaction product 141 (FIG. 5D). Next, the sealing oil is removed by the method for removing the sealing oil of the present invention, whereby the sealing of the fine accommodation holes 100a can be released (FIG. 5E).

[0085] The method for generating a reaction product in the fine accommodation holes of the present invention, after step (s), removes the first filling liquid with the removing liquid compatible with the first filling liquid, and (p’) Accommodating a second filling liquid in a plurality of fine accommodation holes on the surface of a substrate, (q’) Sealing the fine accommodation holes containing the second filling liquid with a sealing oil, and (r’) Causing a reaction of the second filling liquid in the fine accommodation holes to generate a second reaction product may further be included.

[0086] In the above-described FIG. 5, with respect to the substrate 100 having the fine accommodation holes 100a filled with the first reaction product 141 and with the seals released, the second filling liquid 112 is caused to flow through, and the fine accommodation holes 100a are filled with the second filling liquid 112 (FIG. 5F). Next, the sealing oil 120 is caused to flow through, and the upper part of the fine accommodation holes 100a is replaced from the second filling liquid 112 with the sealing oil 120, thereby sealing the fine accommodation holes 100a containing the second filling liquid 112 with the sealing oil 120 (FIG. 5G). A reaction of the second filling liquid 112 is caused to occur within the fine accommodation holes 100a to generate a second reaction product 142 (FIG. 5H).

[0087] 〈Second Filling Liquid〉 The second filling liquid is not particularly limited, and may be the same as the first filling liquid or may be different from the second filling liquid.

[0088] When the first filling liquid and the second filling liquid are the same, a stepwise reaction can be performed on the reaction substrate accommodated in the fine accommodation holes, or the reaction can be repeatedly performed by exchanging the reaction substrate accommodated in the fine accommodation holes. When the first filling liquid and the second filling liquid are different, a stepwise reaction can be performed on the reaction substrate accommodated in the fine accommodation holes.

Example

[0089] The present invention will be described in more detail with reference to the examples shown below, but the scope of the present invention is not limited to these examples.

[0090] 《Production Example 1》 〈Production of Substrate Having Fine Accommodation Holes〉 1. Formation of Mold for Substrate Having Fine Accommodation Holes After dropping photoresist SU-83005 (Microchem) onto a 4-inch bare silicon wafer (Filtech), a photoresist thin film was formed using a spin coater (MIKASA). Note that diluent Cyclopentanone (Tokyo Ohka Kogyo) was added to SU-83005 according to the target film thickness. A flow path pattern with fine accommodation holes was formed on the formed photoresist thin film using a mask aligner (USHIO Electric) and a chromium mask with an arbitrary pattern formed thereon. Subsequently, the flow path pattern was developed using SU-8 Developer (Microchem) to fabricate a mold of the flow path pattern with fine accommodation holes. Here, in order to suppress adsorption to SU-8, vapor deposition surface treatment with Trichloro(1H,1H,2H,2H-perfluoro-octyl)silane (Thermo Fisher Scientific) was performed.

[0091] 2. Fabrication of a polymer substrate with fine accommodation holes A mixture of an uncured siloxane monomer and a polymerization initiator (weight ratio 10:1) prepared using SYLGARD SILICONE ELASTOMER KIT (Toray Dow Corning) was poured into the mold of the flow path pattern with fine accommodation holes and heated at 80°C for 2 hours to fabricate a polymer (polydimethylsiloxane, PDMS) substrate onto which the shape of the flow path was transferred. The cured PDMS was carefully peeled from the mold, shaped into an arbitrary size using a cutter, and then inlets and outlets with a diameter of 1.0 mm were formed in the flow path using a puncher. The peeled PDMS cover glass (Matsunami Glass) was surface-treated with an oxygen plasma generator (Maywa Focus) and then bonded to the patterned surface of the PDMS substrate. The fabricated substrate was stored in a desiccator.

[0092] The flow path of the substrate with fine accommodation holes had a length of 20 mm × width of 0.5 mm × height of 0.08 mm. The fine accommodation holes had a diameter of 0.08 mm × depth of 0.09 mm and were arranged in an array of 662 in 4 rows with a pitch of 0.12 mm.

[0093] Example 1: Method for Removing Sealing Oil: First Aspect 〈Sealing of Fine Storage Holes with Sealing Oil〉 ROX Reference Dye (Takara Bio Inc.) having a fluorescent dye was diluted 50-fold with phosphate buffered saline (PBS) to prepare a ROX solution as the first filling liquid. The prepared ROX solution was introduced from the inlet of a substrate having fine storage holes, and left standing for 3 minutes to fill the ROX solution into the fine storage holes. The substrate having the fine storage holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus Corporation). The ROX solution as the first filling liquid was present in the fine storage holes and in the region outside the fine storage holes (Fig. 6A). Next, silicone oil KF96-20CS (Shin-Etsu Chemical Co., Ltd.) as the sealing oil was introduced from the inlet, and the substrate having the fine storage holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus Corporation). Since fluorescence derived from the ROX solution as the first filling liquid was no longer observed in the region outside the fine storage holes, and fluorescence derived from the ROX solution as the first filling liquid was observed in the fine storage holes, it was confirmed that the fine storage holes were sealed with the sealing oil and the ROX solution as the first filling liquid filled in each fine storage hole was fractionated (Fig. 6B).

[0094] 〈Replacement of Sealing Oil with First Removing Liquid〉 Novec 7200 (3M Company) as the first removing liquid was introduced from the inlet to a substrate having fine storage holes sealed with the sealing oil, and the substrate having the fine storage holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus Corporation) (Fig. 6C). The silicone oil KF96-20CS as the sealing oil was removed from the flow path with Novec 7200 as the first removing liquid that is compatible with the silicone oil KF96-20CS as the sealing oil.

[0095] 〈Removal of First Removing Liquid〉 Air was introduced from the inlet into the substrate having fine accommodation pores replaced with the first removal liquid, and the substrate having fine accommodation pores in this state was observed for fluorescence using an inverted research microscope IX-71 (Olympus Corporation) (Fig. 6D). Since Novec 7200 as the first removal liquid has a higher volatility value than the ROX solution as the first filling liquid and the sealing oil, the introduction of air made it possible to remove Novec 7200 while maintaining the filling liquid.

[0096] 〈Removal of the First Filling Liquid with the Second Removal Liquid〉 PBS, which is the same liquid as the solvent contained in the first filling liquid, was used as the second removal liquid. PBS as the second removal liquid was introduced from the inlet, and the substrate having fine accommodation pores in this state was observed for fluorescence using an inverted research microscope IX-71 (Olympus Corporation) (Fig. 6E). Since the fluorescence derived from the ROX solution as the first filling liquid filled in the fine accommodation pores was no longer observed, it was confirmed that the sealing oil was removed and the ROX solution as the first filling liquid filled in each fine accommodation pore was removed by the introduction of PBS as the second removal liquid.

[0097] 《Comparative Example 1》 Using PBS that has no compatibility with the sealing oil, in the same manner as in Example 1, PBS was introduced from the inlet into the fine accommodation pores (Fig. 7B) sealed with silicone oil KF96-20CS (Shin-Etsu Chemical Co., Ltd.) as the sealing oil, and the substrate having fine accommodation pores in this state was observed for fluorescence using an inverted research microscope IX-71 (Olympus Corporation) (Fig. 7C). The ratio of the fine accommodation pores in which the fluorescence derived from the ROX solution as the first filling liquid filled in the fine accommodation pores was no longer observed, that is, the removal rate of the first filling liquid, was 36%, and it was confirmed that this was significantly inferior to the removal rate of 100% of the first filling liquid in Example 1. (Table 1).

[0098]

Table 1

[0099] Example 2: Reintroduction and resealing of the filling liquid into the fine accommodation holes <Introduction of the second filling liquid, sealing with the sealing oil for the second filling liquid> In the same manner as in Example 1, for the substrate having the fine accommodation holes from which the first filling liquid was removed, an ROX solution as the second filling liquid was reintroduced from the inlet, and the substrate having the fine accommodation holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus Corporation) (Fig. 6F). Next, silicone oil KF96-20CS (Shin-Etsu Chemical Co., Ltd.) as the sealing oil was introduced from the inlet, and the substrate having the fine accommodation holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus Corporation) (Fig. 6G). Since no fluorescence derived from the ROX solution as the second filling liquid was observed in the region outside the fine accommodation holes, it was confirmed that the fine accommodation holes were resealed with the sealing oil again and the ROX solution as the second filling liquid filled in each fine accommodation hole was fractionated. That is, it was confirmed that the filling liquid in the fine accommodation holes sealed with the sealing oil could be repeatedly exchanged.

[0100] Example 3: Method for removing the sealing oil: Second aspect <Replacement of the sealing oil with the third removing liquid> In the same manner as in Example 1, an ROX solution as the first filling liquid was sealed in the fine accommodation holes with the sealing oil (Figs. 8A and 8B). Next, Isopropyl palmitate (Merck & Co., Inc.) as the third removing liquid was introduced from the inlet to the substrate having the fine accommodation holes, and the substrate having the fine accommodation holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus Corporation) (Fig. 8C). The silicone oil KF96-20CS was removed from the flow path with Isopropyl palmitate as the third removing liquid that is compatible with the silicone oil KF96-20CS as the sealing oil.

[0101] <Replacement of the third removing liquid with the fourth removing liquid> Next, AE3000 (manufactured by AGC Inc.) as the fourth removal liquid was introduced from the inlet, and the substrate having the fine accommodation holes in this state was observed by fluorescence using an inverted research microscope IX-71 (manufactured by Olympus Corporation) (Fig. 8D). AE3000 as the fourth removal liquid removed Isopropyl palmitate from the flow path because it is compatible with Isopropyl palmitate as the third removal liquid. Since AE3000 as the fourth removal liquid is not compatible with silicone oil KF96-20CS as the sealing oil but is compatible with Isopropyl palmitate as the third removal liquid, Isopropyl palmitate could be removed from the flow path.

[0102] 〈Removal of the Fourth Removal Liquid〉 Next, air was introduced from the inlet, and the substrate having the fine accommodation holes in this state was observed by fluorescence using an inverted research microscope IX-71 (manufactured by Olympus Corporation) (Fig. 8E). Since AE3000 as the fourth removal liquid has a higher volatility value compared to the ROX solution as the first filling liquid and the sealing oil, AE3000 could be removed while maintaining the ROX solution by introducing air.

[0103] 〈Removal of the First Filling Liquid with the Fifth Removal Liquid〉 PBS, which is the same liquid as the solvent contained in the first filling liquid, was used as the fifth removal liquid. PBS as the fifth removal liquid was introduced from the inlet, and the substrate having the fine accommodation holes in this state was observed by fluorescence using an inverted research microscope IX-71 (manufactured by Olympus Corporation) (Fig. 8F). Since the fluorescence derived from the ROX solution as the first filling liquid filled in the fine accommodation holes was no longer observed, it was confirmed that even without using a removal liquid that is compatible with the sealing oil and has a higher volatility value compared to the filling liquid and the sealing oil, by sequentially replacing it with the third and fourth removal liquids, the silicone oil KF96-20CS as the sealing oil was removed, and the ROX solution as the first filling liquid filled in each fine accommodation hole was removed by the introduction of PBS as the fifth removal liquid.

[0104] <Re-introduction of the Second Filling Liquid and Re-sealing with Sealing Oil> The ROX solution as the second filling liquid was re-introduced from the inlet, and the substrate having the fine accommodation holes in this state was observed for fluorescence by an inverted research microscope IX-71 (Olympus Corporation) (Fig. 8G). Next, silicone oil KF96-20CS (Shin-Etsu Chemical Co., Ltd.) as the sealing oil was introduced from the inlet, and the substrate having the fine accommodation holes in this state was observed for fluorescence by an inverted research microscope IX-71 (Olympus Corporation) (Fig. 8H). Since fluorescence derived from the ROX solution as the first filling liquid was no longer observed in the region outside the fine accommodation holes and fluorescence derived from the ROX solution as the first filling liquid was observed inside the fine accommodation holes, it was confirmed again that the fine accommodation holes were sealed with the sealing oil and the ROX solution filled in each fine accommodation hole was fractionated. That is, it was confirmed that the filling liquid sealed in the fine accommodation holes with the sealing oil could be repeatedly exchanged.

[0105] <<Example 4>> Method for Generating Reaction Product in Fine Accommodation Hole: First Aspect <Sealing of Fine Accommodation Holes with Sealing Oil> A PCR reaction solution (Table 2) containing a β-actin gene (SEQ ID NO: 1) artificially synthesized by Eurofins, a forward primer (SEQ ID NO: 2) and a reverse primer (SEQ ID NO: 3) for amplifying the β-actin gene, and a TaqMan probe (SEQ ID NO: 4) that emits fluorescence during amplification of the β-actin gene and is artificially synthesized by Thermo Fisher Scientific was prepared. The PCR reaction solution as the first filling liquid was introduced from the inlet and allowed to stand for 3 minutes to fill the PCR reaction solution into the fine accommodation holes, and the substrate having the fine accommodation holes in this state was observed for fluorescence by an inverted research microscope IX-71 (Olympus Corporation) (Fig. 9A). Next, silicone oil KF96-20CS (Shin-Etsu Chemical Co., Ltd.) as the sealing oil was introduced from the inlet, and the substrate having the fine accommodation holes in this state was observed for fluorescence by an inverted research microscope IX-71 (Olympus Corporation) (Fig. 9B).

[0106] [Table 2]

[0107]

Table 3

[0108] 〈PCR Reaction in Fine Containment Holes〉 Mineral oil (Nacalai Tesque) was dropped onto the heat block of the Flat Type Thermal Cycler ProFlex PCR System - Dual Flat Sample Block (Thermo Fisher Scientific), and the substrate having the fine containment holes was placed thereon. The mineral oil was used to enhance the adhesion between the heat block and the substrate having the fine containment holes. Then, after performing the reaction shown in Table 4, the substrate having the fine containment holes in this state was observed for fluorescence with an inverted research microscope IX - 71 (Olympus). Since fluorescence emitted by the TaqMan probe was observed in the region inside the fine containment holes, it was confirmed that the β - actin gene as the first reaction product was amplified inside the fine containment holes (Figure 9C).

[0109]

Table 4

[0110] 〈Replacement from Sealing Oil to First Removing Liquid〉 In the same manner as in Example 1, replacement was performed from the silicone oil KF96 - 20CS, which is a sealing oil, to Novec7200, which is the first removing liquid.

[0111] 〈Removal of First Removing Liquid〉 In the same manner as in Example 1, the first removing liquid, Novec7200, was removed (Figure 9D).

[0112] 〈Removal of First Filling Liquid with Second Removing Liquid〉 In the same manner as in Example 1, the PCR reaction solution, which was the first filling solution, was removed with PBS, which was the second removal solution (Fig. 9E).

[0113] 〈Re-introduction of the second filling solution and re-sealing with sealing oil〉 The same PCR reaction solution as the first filling solution was introduced from the inlet as the second filling solution, and the PCR reaction solution was filled into the fine accommodation holes by allowing it to stand for 3 minutes. The substrate having the fine accommodation holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus Corporation) (Fig. 9F). Next, silicone oil KF96-20CS (Shin-Etsu Chemical Co., Ltd.), which was the sealing oil, was introduced from the inlet, and the substrate having the fine accommodation holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus Corporation) (Fig. 9G).

[0114] 〈PCR reaction in the fine accommodation holes〉 Mineral oil (Nacalai Tesque Inc.) was dropped onto the heat block of a flat thermal cycler ProFlex PCR System-Dual Flat Sample Block (Thermo Fisher Scientific), and the substrate having the fine accommodation holes was placed thereon. The mineral oil was used to enhance the adhesion between the heat block and the substrate having the fine accommodation holes. Next, after performing the reaction shown in Table 4, the substrate having the fine accommodation holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus Corporation). Since fluorescence emitted by the TaqMan probe was observed in the region inside the fine accommodation holes, it was confirmed that the β-actin gene, which was the second reaction product, was amplified in the fine accommodation holes (Fig. 9H). That is, it was shown that repeated reactions can be performed in the fine accommodation holes in a mode where the first filling solution and the second filling solution are the same.

[0115] 《Example 5》Method for generating reaction products in fine accommodation holes: Second mode 〈Sealing of fine accommodation holes with sealing oil〉 In the same manner as in Example 4, the PCR reaction solution (Table 2), which is the first filling liquid, was filled into the fine accommodation holes (Fig. 10A), and the fine accommodation holes were sealed with silicone oil KF96-20CS, which is the oil for sealing (Fig. 10B).

[0116] 〈PCR Reaction in Fine Accommodation Holes〉 In the same manner as in Example 4, a PCR reaction was carried out in the fine accommodation holes, and it was confirmed that the β-actin gene, which is the first reaction product, was amplified in the fine accommodation holes (Fig. 10C).

[0117] 〈Replacement from Oil for Sealing to First Removing Liquid〉 In the same manner as in Example 4, replacement was carried out from silicone oil KF96-20CS, which is the oil for sealing, to Novec7200, which is the first removing liquid.

[0118] 〈Removal of First Removing Liquid〉 In the same manner as in Example 4, the first removing liquid, Novec7200, was removed (Fig. 10D).

[0119] 〈Removal of First Filling Liquid with Second Removing Liquid〉 In the same manner as in Example 4, the PCR reaction solution, which is the first filling liquid, was removed with PBS, which is the second removing liquid (Fig. 10E).

[0120] 〈Re-introduction of Second Filling Liquid, Re-sealing with Oil for Sealing〉 A CFPS (Cell-Free Protein Synthesis) reaction solution (Table 5) containing the fluorescent protein EGFP gene (SEQ ID NO: 5) synthesized artificially by Eurofins was introduced from the inlet as the second filling solution and allowed to stand for 3 minutes to fill the CFPS reaction solution into the fine accommodation pores. The substrate having the fine accommodation pores in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus). (Fig. 10F). Next, silicone oil KF96-20CS (Shin-Etsu Chemical Co., Ltd.) as the sealing oil was introduced from the inlet, and the substrate having the fine accommodation pores in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus). (Fig. 10G).

[0121]

Table 5

[0122]

Table 6

[0123] 〈CFPS reaction in fine accommodation pores〉 Mineral oil (Nacalai Tesque) was dropped onto the heat block of a flat thermal cycler ProFlex PCR System-Dual Flat Sample Block (Thermo Fisher Scientific), and the substrate having the above-mentioned fine accommodation pores was placed thereon. The mineral oil was used to enhance the adhesion between the heat block and the substrate having the above-mentioned fine accommodation pores. Next, after incubating at 30°C for 1 hour, the substrate having the fine accommodation pores in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus). Since fluorescence emitted by the fluorescent protein EGFP was observed in the region inside the fine accommodation pores, it was confirmed that the fluorescent protein EGFP as the second reaction product was being synthesized in the fine accommodation pores. (Fig. 10H). That is, it was shown that repeated reactions can be performed in the fine accommodation pores in a mode where the first filling solution and the second filling solution are different.

[0124] Although the preferred embodiments of the method for removing the sealing oil of the present invention and the method for generating the reaction product in the fine accommodation holes have been described, those skilled in the art understand that changes can be made without departing from the scope of the claims.

Explanation of Signs

[0125] 100 Substrate 100a Fine accommodation hole 111 First filling liquid 112 Second filling liquid 120 Sealing oil 131 First removing liquid 132 Second removing liquid 133 Third removing liquid 134 Fourth removing liquid 135 Fifth removing liquid 136 Sixth removing liquid 137 Seventh removing liquid 138 Eighth removing liquid 139 Ninth removing liquid 141 First reaction product 142 Second reaction product

Claims

1. 1. A method for removing sealing oil from a substrate, comprising: The substrate has a plurality of fine containing holes on a surface of the substrate, A first filling liquid is contained in the fine containing hole, and the fine containing hole containing the first filling liquid is sealed with a sealing oil, The removal method comprises: (a) replacing the sealing oil with a first removal solution that is compatible with the sealing oil; and (b) removing the first removal solution by volatilization; A method for removing sealing oil, comprising:

2. After step (b), (c) removing the first fill liquid with a second removal liquid that is compatible with the first fill liquid; The method of claim 1 further comprising:

3. The method of claim 1 , wherein the first removal liquid is a liquid having a higher volatility value than the first fill liquid.

4. The method of claim 2 , wherein the second removal liquid is the same liquid as a solvent contained in the first filling liquid.

5. 1. A method for removing sealing oil from a substrate, comprising: The substrate has a plurality of fine containing holes on a surface of the substrate, A first filling liquid is contained in the fine containing hole, and the fine containing hole containing the first filling liquid is sealed with a sealing oil, The removal method comprises: (d) replacing the sealing oil with a third removal liquid that is compatible with the sealing oil; (e) replacing the third removal liquid with a fourth removal liquid that is compatible with the third removal liquid; and (f) removing the fourth removal solution by volatilization; A method for removing sealing oil, comprising:

6. After step (f), (g) removing the first fill liquid with a fifth removal liquid that is compatible with the first fill liquid; The method of claim 5 further comprising:

7. The method of claim 5 , wherein the fourth removal liquid is a liquid having a higher volatility value than the first fill liquid.

8. The method of claim 6 , wherein the fifth removal liquid is the same liquid as a solvent contained in the first filling liquid.

9. 1. A method for removing sealing oil from a substrate, comprising: The substrate has a plurality of fine containing holes on a surface of the substrate, A first filling liquid is contained in the fine containing hole, and the fine containing hole containing the first filling liquid is sealed with a sealing oil, The removal method comprises: (h) replacing the sealing oil with a sixth removal solution that is compatible with the sealing oil; (i) replacing the sixth removal liquid with a seventh removal liquid that is compatible with the sixth removal liquid; (j) replacing the seventh removal liquid with an eighth removal liquid that is compatible with the seventh removal liquid; and (k) removing the eighth remover by volatilization; A method for removing sealing oil, comprising:

10. After step (k), (l) removing the first fill liquid with a ninth removal liquid that is compatible with the first fill liquid; The method of claim 9 further comprising:

11. The method of claim 9 , wherein the eighth removal liquid is a liquid having a higher volatility value than the first fill liquid.

12. The method of claim 10 , wherein the ninth removal liquid is the same liquid as a solvent contained in the first filling liquid.

13. (p) containing the first filling liquid in a plurality of fine containing holes in the surface of the substrate; (q) sealing the fine containing hole containing the first filling liquid with a sealing oil; (r) causing a reaction of the first filling liquid in the fine containing hole to generate a first reaction product; and (s) removing the sealing oil by the method according to any one of claims 1 to 12; A method for producing a reaction product in a fine containing hole, comprising:

14. After step (s), removing the first filling liquid with a removal liquid that is compatible with the first filling liquid; and (p') filling a second filling liquid into a plurality of fine holes on the surface of the substrate; (q') sealing the fine containing hole containing the second filling liquid with a sealing oil; and (r') causing a reaction of the second filling liquid in the fine containing holes to generate a second reaction product. The method of claim 13 further comprising:

15. The method of claim 14 , wherein the second filling liquid is the same as the first filling liquid.

16. The method of claim 14, wherein the second fill liquid is different from the first fill liquid.

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