Method for evaluating efficiency of cleaning base material

Abstract

To provide a method for evaluating the efficiency of cleaning a base material, the method detecting a filling liquid remaining in a fine storage hole after the filling liquid is cleaned by the cleaning liquid.SOLUTION: In the method for evaluating the efficiency of cleaning a base material, a base material 100 has a plurality of fine storage holes 100a in the surface of the base material 100, a first filling liquid 111 is stored in the fine storage holes 100a, and the first filling liquid 111 contains a first indicator. The method sequentially includes the steps of: (i) cleaning the base material 100 by a cleaning liquid 131 not including the first indicator; and (ii) detecting the first indicator in the base material 100 after the step (i).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for evaluating the cleaning efficiency of a substrate having fine accommodation pores.

Background Art

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

[0003] For example, Patent Document 1 discloses a substrate having a plurality of first surfaces having hydrophilicity and second surfaces surrounding each of the plurality of first surfaces and having lower hydrophilicity than the first surfaces, wherein a molecule that specifically binds to a nucleic acid serving as a template is fixed to the first surface, a step of preparing the substrate, a step of supplying a mixed solution of a sample solution containing the nucleic acid serving as the template, a reaction solution containing a nucleic acid amplification substrate and a nucleic acid synthase onto the substrate, 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 droplets enclosing the mixed solution disposed on the first surface, a step of performing an amplification reaction of the nucleic acid within the droplets, 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 in which a plurality of accommodating portions capable of accommodating only one bead each are formed and separated from each other by side walls having a hydrophobic upper surface, and an upper layer portion facing the surface on which the accommodating portions are formed in the lower layer portion, 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 in 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 order to perform repeated reactions in fine accommodation pores, it is necessary to wash the filling liquid filled in the fine accommodation pores, for example, a liquid containing a substrate, and then introduce the next filling liquid into the fine accommodation pores.

[0007] Therefore, an object of the present invention is to provide a method for detecting the presence or absence of a filling liquid in the fine accommodation pores or a sealing liquid that seals the filling liquid in the fine accommodation pores after washing the substrate with a washing liquid, and thereby evaluating the washing efficiency of the substrate.

Means for Solving the Problems

[0008] The present invention achieves the above object by the following means.

[0009] 〈Aspect 1〉 A method for evaluating the cleaning efficiency of a substrate, comprising: 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 first filling liquid contains a first indicator; the method for evaluating the cleaning efficiency includes: (i) cleaning the substrate with a cleaning liquid that does not contain the first indicator; and (ii) detecting the first indicator on the substrate after step (i). A method for evaluating the cleaning efficiency of a substrate, comprising the above steps. <Aspect 2> A method for evaluating the cleaning efficiency of a substrate, comprising: 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; the first filling liquid in the fine accommodation holes is sealed with a first sealing liquid, and the first sealing liquid contains a first indicator; the method for evaluating the cleaning efficiency includes: (i) cleaning the substrate with a cleaning liquid that does not contain the first indicator; and (ii) detecting the first indicator on the substrate after step (i). A method for evaluating the cleaning efficiency of a substrate, comprising the above steps. <Aspect 3> The method according to Aspect 1 or 2, wherein the first indicator contains fluorescent molecules. <Aspect 4> The method according to any one of Aspects 1 to 3, wherein when the first indicator is detected in step (ii), it is determined that the first filling liquid or the first sealing liquid remains. <Aspect 5> The method according to any one of Aspects 1 to 4, wherein the cleaning liquid contains a second indicator different from the first indicator. <Aspect 6> (p) Accommodating the above-described first filling liquid in a plurality of fine accommodation holes on the surface of the above-described base material; (r) Causing a reaction of the above-described first filling liquid in the above-described fine accommodation holes to generate a first reaction product; and (s) Cleaning the above-described base material by the method according to any one of Aspects 1 to 5; A method for generating a reaction product in a fine accommodation hole, comprising: <Aspect 7> After step (s), (p’) Accommodating a second filling liquid in a plurality of fine accommodation holes on the surface of the above-described base material; and (r’) Causing a reaction of the above-described second filling liquid in the above-described fine accommodation holes to generate a second reaction product; The method according to Aspect 6, further comprising: <Aspect 8> The method according to Aspect 6 or 7, wherein the above-described second filling liquid is the same as the above-described first filling liquid. <Aspect 9> The method according to Aspect 6 or 7, wherein the above-described second filling liquid is different from the above-described first filling liquid. [Advantages of the Invention]

[0010] According to the method of the present invention, after cleaning the base material with a cleaning liquid, it is possible to detect the presence or absence of a filling liquid in the fine accommodation holes or a sealing liquid that seals the filling liquid in the fine accommodation holes, and thereby evaluate the cleaning efficiency of the base material. [Brief Description of the Drawings]

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0012] 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 various modifications can be made 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.

[0013] 《Method for Evaluating Cleaning Efficiency of Substrate: First Aspect》 The present invention is a method for evaluating the cleaning efficiency of 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 first filling liquid contains a first indicator, and the method for evaluating the cleaning efficiency is (i) cleaning the substrate with a cleaning liquid that does not contain the first indicator, and (ii) detecting the first indicator on the substrate after step (i). including.

[0014] According to the first aspect of the method of the present invention, after cleaning the substrate with a cleaning liquid, the presence or absence of the filling liquid in the fine accommodation holes can be detected, and thereby the cleaning efficiency of the substrate can be evaluated.

[0015] Although not limited to theory, the first filling liquid contains a first indicator, for example, a fluorescent molecule, and in a state where the first filling liquid is contained in the fine accommodation pores, fluorescence derived from the first indicator can be detected from within the fine accommodation pores. Next, when the substrate is washed with a cleaning liquid that does not contain the first indicator, the first filling liquid in the fine accommodation pores is replaced with the cleaning liquid, the amount of the first filling liquid in the fine accommodation pores decreases, and accordingly, the amount of the first indicator in the fine accommodation pores also decreases, whereby the fluorescence intensity detected from the fine accommodation pores becomes smaller. When the washing progresses sufficiently, there is no first filling liquid in the fine accommodation pores, and accordingly, there is no first indicator in the fine accommodation pores, whereby no fluorescence is detected from the fine accommodation pores. By detecting the first indicator in this way, the filling liquid remaining in the fine accommodation pores can be detected.

[0016] FIG. 1 is a schematic diagram showing one aspect of the method of the present invention for evaluating the cleaning efficiency of a substrate, but is not limited to this case.

[0017] The first filling liquid 111 contains, for example, fluorescent molecules as the first indicator, and by detecting fluorescence (fluorescence observation), it is possible to detect the fluorescence derived from the first indicator contained in the first filling liquid 111. First, the first filling liquid 111 is accommodated in the fine accommodation holes 100a of the base material 100 (FIG. 1A). When detecting the fluorescence in this state, it is possible to detect the fluorescence derived from the first indicator in the fine accommodation holes 100a. Next, when a cleaning liquid 131 that does not contain the first indicator is circulated through the base material 100, the first filling liquid 111 in the fine accommodation holes 100a is replaced with the cleaning liquid 131 (FIGS. 1B and 1C). When detecting the fluorescence in this state, the first filling liquid 111 is replaced with the cleaning liquid 131, the first filling liquid 111 in the fine accommodation holes 100a decreases, and accordingly, the first indicator in the fine accommodation holes 100a also decreases, whereby the fluorescence intensity detected from the fine accommodation holes 100a becomes smaller. When the cleaning progresses sufficiently, the first filling liquid 111 no longer exists in the fine accommodation holes 100a (FIG. 1D). When detecting the fluorescence in this state, the first filling liquid 111 does not exist in the fine accommodation holes 100a, and accordingly, the first indicator also does not exist in the fine accommodation holes 100a, whereby fluorescence is no longer detected from the fine accommodation holes 100a. By detecting the first indicator in this way, it is possible to detect the first filling liquid 111 remaining in the fine accommodation holes 100a.

[0018] 《Method for Evaluating Cleaning Efficiency of Substrate: Second Aspect》 The present invention is a method for evaluating the cleaning efficiency of 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, the first filling liquid in the fine accommodation holes is sealed with a first sealing liquid, and the first sealing liquid contains a first indicator, and the method for evaluating the cleaning efficiency is (i) cleaning the substrate with a cleaning liquid that does not contain the first indicator, and (ii) After step (i), detecting the first indicator on the substrate, including.

[0019] According to a second aspect of the method of the present invention, after cleaning the substrate with a cleaning liquid, the presence or absence of a sealing liquid that seals the filling liquid in the fine accommodation holes can be detected, whereby the cleaning efficiency of the substrate can be evaluated.

[0020] Although not limited to theory, the first sealing liquid contains a first indicator, for example, a fluorescent molecule, and in a state where the first sealing liquid that seals the fine accommodation holes is present on the substrate surface, fluorescence derived from the first indicator can be detected. Then, when the substrate is cleaned with a cleaning liquid that does not contain the first indicator, the first sealing liquid is removed by the cleaning liquid, the first sealing liquid on the substrate surface decreases, and accordingly, the first indicator on the substrate surface also decreases, whereby the fluorescence intensity detected from the substrate surface becomes smaller. When the cleaning progresses sufficiently, the first sealing liquid does not exist on the substrate surface, and accordingly, the first indicator also does not exist on the substrate surface, whereby fluorescence is not detected from the substrate surface. By detecting the first indicator in this way, the sealing liquid remaining on the substrate surface can be detected.

[0021] FIG. 2 is a schematic diagram showing one aspect of the method of the present invention for evaluating the cleaning efficiency of a substrate, but is not limited to this case.

[0022] The first sealing liquid 121 contains a first indicator, for example, a fluorescent molecule, and by detecting fluorescence (fluorescence observation), it is possible to detect the fluorescence derived from the first indicator contained in the first sealing liquid 121. First, the substrate 100 has a plurality of fine accommodation holes 100a on the surface of the substrate 100, and the first filling liquid 111 is accommodated in the fine accommodation holes 100a. The fine accommodation holes 100a are sealed with the first sealing liquid 121, and the first filling liquid 111 is fractionated into the fine accommodation holes 100a respectively (FIG. 2A). When detecting the fluorescence in this state, it is possible to detect the fluorescence derived from the first indicator on the surface of the substrate 100. Next, when a cleaning liquid 131 that does not contain the first indicator is circulated through the substrate 100, a part of the first sealing liquid 121 is removed by the cleaning liquid 131 (FIGS. 2B and 2C). When detecting the fluorescence in this state, a part of the first sealing liquid 121 on the surface of the substrate 100 is removed by the cleaning liquid 131, and the liquid thickness of the first sealing liquid 121 on the surface of the substrate 100 becomes smaller, whereby the fluorescence intensity from the surface of the substrate 100 becomes smaller (FIG. 2B). Also, a part of the first sealing liquid 121 on the surface of the substrate 100 is removed by the cleaning liquid 131, and the fine accommodation holes 100a are partially sealed by the first sealing liquid 121 on the surface of the substrate 100, whereby fluorescence is observed from a part of the surface of the substrate 100 (FIG. 2C). When the cleaning progresses sufficiently, the first sealing liquid 121 is removed and no longer exists (FIG. 2D). When detecting the fluorescence in this state, the first sealing liquid 121 does not exist on the surface of the substrate 100, and accordingly, the first indicator on the surface of the substrate 100 also does not exist, whereby fluorescence is not detected from the surface of the substrate 100. By detecting the first indicator in this way, it is possible to detect the first sealing liquid 121 remaining on the surface of the substrate 100.

[0023] 〈Fine accommodation holes containing the first filling liquid〉 In the present invention, the substrate has a plurality of fine accommodation holes on the surface of the substrate, and the first filling liquid is accommodated in the fine accommodation holes. The first filling liquid accommodated in the fine accommodation holes is not particularly limited, and the first filling liquid may be sealed with the first sealing liquid.

[0024] For example, in FIG. 1A described above, the substrate 100 has a plurality of fine accommodation holes 100a on the surface of the substrate, and a first filling liquid is accommodated in the fine accommodation holes 100a. The first filling liquid 111 is respectively fractionated in the fine accommodation holes 100a. Similarly, for example, in FIG. 2A described above, 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 by the first sealing liquid 121, and the first filling liquid 111 is respectively fractionated in the fine accommodation holes 100a.

[0025] <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 may have a flow path through which a fluid flows, and may have an inlet (fluid inlet) and an outlet (fluid outlet).

[0026] 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.

[0027] (Fine accommodation hole) The shape of the fine accommodation hole may be entirely or partially circular or elliptical, or may be non-circular, for example, polygonal. The equivalent diameter of the fine accommodation hole 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 an outer peripheral length equal to the outer peripheral length of that surface.

[0028] 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 also 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.

[0029] The fine accommodation holes may be evenly distributed or may be unevenly distributed in some places. For example, the fine accommodation holes may not be present at the ends of the substrate.

[0030] The substrate having the fine accommodation holes is not particularly limited, but can be produced using general photolithography and soft lithography. Specifically, for example, a mold having fine accommodation holes, channels, etc. formed on a silicon wafer by photolithography can be created, and then a resin or the like is poured into this mold, the resin is cured, and a fine three-dimensional structure is transferred (soft lithography) to produce a substrate having fine accommodation holes, but it is not limited to this case.

[0031] 〈First filling liquid〉 The first filling liquid is not particularly limited, and examples thereof include water, aqueous solvents, hydrophilic alcohols, hydrophilic ethers, ketones, nitrile solvents, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), etc. Examples of the aqueous solvent include buffer solutions, specifically phosphate buffered saline. 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 kind may be used alone, or two or more kinds may be used in combination.

[0032] The first filling liquid may optionally contain other components. Other components that may be included in the first filling liquid are not particularly limited, and may include, for example, beads, reaction substrates, enzymes, proteins, nucleic acids, catalysts, etc.

[0033] 〈The first sealing liquid〉 The first sealing liquid 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 preferred as the first sealing liquid. The first sealing liquid is not particularly limited, and examples include silicone oil, saturated hydrocarbons, unsaturated hydrocarbons, hydrophobic ionic liquids, etc. Examples of saturated hydrocarbons include alkanes, cycloalkanes, etc. Examples of alkanes include decane, hexadecane, etc. Examples of unsaturated hydrocarbons include squalene, etc. Examples of hydrophobic ionic liquids include 1-butyl-3-methylimidazolium hexafluorophosphate, etc. An ionic liquid is a salt that exists as a liquid at room temperature (23°C). The first sealing liquid is not particularly limited, and silicone oil is preferred. The first sealing liquid may be used alone as only one type, or two or more types may be combined and used.

[0034] 〈The first indicator〉 The first indicator is not particularly limited, and fluorescent molecules, chromogenic molecules, luminescent molecules, radioactive labels, etc. can be used. The first indicator is preferably a fluorescent molecule from the viewpoint of being able to detect with high sensitivity in a small amount. Also, the first indicator used in the first aspect is preferably a water-soluble fluorescent molecule, and the indicator used in the second aspect is preferably a water-insoluble fluorescent molecule.

[0035] The fluorescent molecule is not particularly limited, and water-soluble fluorescent molecules and water-insoluble fluorescent molecules can be used.

[0036] Examples of water-soluble fluorescent molecules include, for example, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), fluorescein (FAM), tetramethylrhodamine, ROX, DsRED, tdTomato, etc. Specific examples of water-soluble fluorescent molecules include, for example, ROX Reference Dye manufactured by Takara Bio Inc.; Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647 manufactured by Thermo Fisher Scientific; ATTO 488, ATTO 532, ATTO 600, ATTO 655 manufactured by ATTO-Tech; DY-547, DY-632, DY-633, DY-647 manufactured by emp Biotech, etc., but are not limited to these cases.

[0037] Examples of water-insoluble fluorescent molecules include, for example, dimethylaminocoumarin, etc. Specific examples of water-insoluble fluorescent molecules include, for example, Cy3, Cy5, Cy5.5 manufactured by ATT Bioquest; DY-505 manufactured by emp Biotech, etc., but are not limited to these cases.

[0038] The chromogenic molecule is not particularly limited, and spiropyran, azobenzene, diarylethene, fulgide, various dyes, various pigments, etc. can be used.

[0039] The luminescent molecule is not particularly limited, and rofin, skatole, luminol, lucigenin, luciferin, etc. can be used.

[0040] The radioactive label is not particularly limited, but 3 H, 14 C, 32 P, 35 S, 125Molecules containing isotopes such as I can be used. The structure of the molecules containing the above isotopes is not particularly limited, and a water-soluble or water-insoluble structure can be appropriately selected.

[0041] 〈Method for cleaning a substrate〉 The method for cleaning a substrate is not particularly limited. For example, the substrate can be cleaned by flowing a cleaning liquid on the surface of the substrate having fine accommodation pores, but it is not limited to this case.

[0042] 〈Method for detecting a first indicator〉 The method for detecting the first indicator is not particularly limited, and a known detection method suitable for the first indicator can be adopted. For example, when the first indicator contains a fluorescent molecule, fluorescence can be detected and observed by a fluorescence microscope, specifically, an inverted research microscope IX-71 (Olympus). Also, for example, when the first indicator contains a chromogenic molecule, it can be detected by bright-field observation of an optical microscope.

[0043] 〈Cleaning liquid〉 The cleaning liquid is not particularly limited, and examples include water, aqueous solvents, hydrophilic alcohols, hydrophilic ethers, ketones, 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. Also, the cleaning liquid is not particularly limited, and examples include silicone oil, saturated hydrocarbons, unsaturated hydrocarbons, hydrophobic ionic liquids, etc. The cleaning liquid is not particularly limited, and only one kind may be used alone, or two or more kinds may be used in combination.

[0044] When washing the first filling liquid, although not particularly limited, at least one selected from water, aqueous solvents, hydrophilic alcohols, hydrophilic ethers, ketones, nitrile solvents, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), etc. is preferable, and phosphate buffered saline is more preferable. When washing the first sealing liquid, although not particularly limited, hydrofluoroether, silicone oil, saturated hydrocarbon, unsaturated hydrocarbon, hydrophobic ionic liquid, etc. are preferable, and hydrofluoroether is more preferable.

[0045] The cleaning liquid is not particularly limited, but may contain a second indicator different from the first indicator. Regarding the second indicator, reference can be made to the description of "<the first indicator>" above. By detecting the second indicator contained in the cleaning liquid, the cleaning liquid remaining after cleaning can be detected.

[0046] In the present invention, although not particularly limited, when the first indicator is detected in step (ii), it can be determined that the first filling liquid or the first sealing liquid remains.

[0047] In FIG. 1 described above, when the cleaning liquid 131 that does not contain the first indicator is circulated through the substrate, in the initial stage of cleaning, a part of the first filling liquid 111 in the fine accommodation hole 100a is replaced with the cleaning liquid 131 (FIGS. 1B and 1C). When the fluorescence in this state is detected, the first filling liquid 111 remains in the fine accommodation hole 100a, and accordingly, the first indicator also remains in the fine accommodation hole 100a, whereby fluorescence is detected from the fine accommodation hole 100a. When the cleaning progresses sufficiently, the first filling liquid 111 no longer exists in the fine accommodation hole 100a (FIG. 1D). When the fluorescence in this state is detected, the first filling liquid 111 does not exist in the fine accommodation hole 100a, and accordingly, the first indicator also does not exist in the fine accommodation hole 100a, whereby fluorescence is no longer detected from the fine accommodation hole 100a. Therefore, when the first indicator is detected, it can be determined that the first filling liquid remains.

[0048] Similarly, in FIG. 2 described above, when a cleaning liquid that does not contain the first indicator is circulated through the base material 100, in the initial stage of cleaning, a part of the first sealing liquid 121 is removed by the cleaning liquid 131 (FIGS. 2B and 2C). When the fluorescence in this state is detected, a part of the first sealing liquid 121 remains on the surface of the base material 100, and accordingly, the first indicator also remains on the surface of the base material, whereby fluorescence is detected from the surface of the base material. When the cleaning progresses sufficiently, the first sealing liquid 121 no longer exists on the surface of the base material (FIG. 2D). When the fluorescence in this state is detected, the first sealing liquid 121 on the surface of the base material 100 does not exist, and accordingly, the first indicator on the surface of the base material 100 also does not exist, whereby fluorescence is no longer detected from the surface of the base material 100. Therefore, when the first indicator is detected, it can be determined that the first sealing liquid remains.

[0049] 《Method for Generating Reaction Product in Fine Accommodation Pores》 Including the following steps, a reaction product can be generated in the fine accommodation pores: (p) Accommodating a first filling liquid in a plurality of fine accommodation pores on the surface of a base material, (r) Causing a reaction of the first filling liquid in the fine accommodation pores to generate a first reaction product, and (s) Cleaning the base material by the method of the present invention.

[0050] According to the method for generating a reaction product in the fine accommodation pores of the present invention, it is possible to detect the filling liquid remaining in the fine accommodation pores after cleaning with a cleaning liquid, particularly to confirm that no filling liquid remains, and to introduce the next filling liquid into the fine accommodation pores.

[0051] FIG. 3 shows one embodiment of the method for generating a reaction product in the fine accommodation pores of the present invention, but is a schematic view and is not limited to this case.

[0052] For a substrate 100 having fine accommodation holes 100a (FIG. 3A), a first filling liquid 111 containing a first indicator is circulated, and the fine accommodation holes 100a are filled with the first filling liquid 111 (FIG. 3B). A reaction of the first filling liquid 111 occurs in the fine accommodation holes 100a to generate a first reaction product 141 (FIG. 3C). Then, a cleaning liquid 131 is circulated to clean the fine accommodation holes 100a (FIGS. 3D and 3E). At this time, by detecting the first indicator, it is possible to detect the filling liquid remaining in the fine accommodation holes after cleaning with the cleaning liquid, and confirm the remaining of the filling liquid.

[0053] The method for generating a reaction product in the fine accommodation holes of the present invention, after step(s), (p’) accommodating a second filling liquid in a plurality of fine accommodation holes on the surface of the substrate, 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.

[0054] In FIG. 3 described above, for a substrate having fine accommodation holes in which no first filling liquid remains (FIG. 3E), a second filling liquid is circulated, and the fine accommodation holes are filled with the second filling liquid (FIG. 3F). A reaction of the second filling liquid 112 occurs in the fine accommodation holes 100a to generate a second reaction product 142 (FIG. 5G). For example, when using the first filling liquid containing the first indicator, the first indicator is detected, the first filling liquid remaining in the fine accommodation holes is detected, thereby confirming that the cleaning of the fine accommodation holes is completed, and the fine accommodation holes can be used for the next reaction.

[0055] Optionally, after generating the second reaction product, the substrate can be washed and the indicator detected again by the method of the present invention to evaluate the washing efficiency of the substrate. Therefore, according to the present invention, the generation of the reaction product in the fine accommodation pores of the substrate and the evaluation of the washing efficiency of the substrate can be repeated two or more times, three or more times, four or more times, or five or more times. The number of repetitions may be 1000 times or less, 100 times or less, 50 times or less, 30 times or less, or 10 times or less.

[0056] (Second filling liquid) The second filling liquid is not particularly limited, and may be the same as the first filling liquid or different from the second filling liquid.

[0057] 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 pores, or the reaction substrate accommodated in the fine accommodation pores can be exchanged and the reaction can be repeated. 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 pores.

Example

[0058] The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

[0059] 《Production Example 1》 〈Substrate having fine accommodation pores〉 1. Formation of a mold for a substrate having fine accommodation pores After dropping photoresist SU-83005 (Microchem) onto a 4-inch bare silicon wafer (Filtec), a photoresist thin film was formed using a spin coater (MIKASA). Note that a 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) and a chromium mask with an arbitrary pattern. Next, 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.

[0060] 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 a mold of a 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. After surface treatment of the peeled PDMS cover glass (Matsunami Glass) with an oxygen plasma generator (Maywa Focus), the patterned surface of the PDMS substrate and the cover glass were bonded together. The fabricated substrate with fine accommodation holes was stored in a desiccator.

[0061] The flow path of the substrate with fine accommodation holes was 20 mm in length × 0.5 mm in width × 0.08 mm in height. The fine accommodation holes were 0.08 mm in diameter × 0.09 mm in depth and were arranged in an array of 662 in 4 rows with a pitch of 0.12 mm.

[0062] 《Example 1: Evaluation of the cleaning efficiency of the substrate》 〈Sealing of the fine accommodation holes with the sealing oil〉 ROX Reference Dye (Takara Bio Inc.) containing a fluorescent molecule as the first indicator was diluted 50-fold with phosphate-buffered saline (PBS) to prepare a ROX solution as the first filling liquid containing the first indicator. The prepared ROX solution was introduced from the inlet of the substrate having fine accommodation holes and allowed to stand for 3 minutes to fill the ROX solution into the fine accommodation holes. Next, silicone oil KF96-20CS (Shin-Etsu Chemical Co., Ltd.) was introduced from the inlet.

[0063] 〈Removal of silicone oil KF96-20CS〉 Novec7200 (3M) was introduced from the inlet to the substrate having fine accommodation holes sealed with the sealing oil. Novec7200, which is compatible with silicone oil KF96-20CS as the sealing oil, was used to remove silicone oil KF96-20CS as the sealing oil from the flow path.

[0064] 〈Removal of Novec7200〉 Air was introduced from the inlet to the substrate having fine accommodation holes replaced with the first removal liquid. Since Novec7200 as the first removal liquid is more volatile than the ROX solution as the first filling liquid, Novec7200 could be removed while maintaining the filling liquid by introducing air.

[0065] 〈Evaluation of the cleaning efficiency of the substrate〉 PBS, which is the same solvent as the solvent contained in the first filling liquid, was used as the cleaning liquid. The substrate having fine accommodation holes was fixed on the stage of an inverted research microscope IX-71 (Olympus), and PBS as the cleaning liquid was introduced at a flow rate of 2 μL / sec. The temporal change in fluorescence derived from the ROX solution as the first filling liquid when the cleaning liquid was introduced and the substrate having fine accommodation holes was being cleaned was acquired as a fluorescence imaging video by a digital CMOS camera (Hamamatsu Photonics) (Figure 4).

[0066] Figure 4A shows an image before introducing PBS. Figure 4B is an image when 1 second has elapsed after introducing PBS, Figure 4C is an image when 2 seconds have elapsed after introducing PBS, and Figure 4D is an image when 3 seconds have elapsed after introducing PBS.

[0067] In the image at 2 seconds (Figure 4C), fluorescence derived from the first indicator of the ROX solution as the first filling liquid filled in the fine accommodation holes was detected. Therefore, it was determined that the ROX solution as the first filling liquid remained in each fine accommodation hole. On the other hand, in the image at 3 seconds (Figure 4D), fluorescence derived from the first indicator of the ROX solution as the first filling liquid filled in the fine accommodation holes was not detected. Therefore, it was determined that the washing of the ROX solution as the first filling liquid filled in each fine accommodation hole was completed.

[0068] <<Example 2; Evaluation of the cleaning efficiency of the substrate and generation of reaction products (when the second filling liquid is the same as the first filling liquid)>> <Sealing of fine accommodation holes with sealing oil> A PCR reaction solution (Table 1) containing a β-actin gene (SEQ ID NO: 1) artificially synthesized by Euromedex, 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 artificially synthesized by Thermo Fisher Scientific was prepared as the first indicator. The PCR reaction solution as the first filling liquid was introduced from the inlet and allowed to stand for 3 minutes to fill the fine accommodation holes with the PCR reaction solution. The substrate having the fine accommodation holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus). (Figure 5A). 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). (Figure 5B).

[0069]

Table 1

[0070]

Table 2

[0071] 〈PCR Reaction in Fine Containment Holes〉 Mineral oil (Nacalai Tesque) was dropped onto the heat block of the Flat-Top Thermal Cycler ProFlex PCR System - Dual Flat Sample Block (Thermo Fisher Scientific), and the substrate having the above-described fine containment holes was placed thereon. The mineral oil was used to enhance the adhesion between the heat block and the substrate having the above-described fine containment holes. Subsequently, after performing the reaction shown in Table 3, the substrate having the fine containment holes in this state was subjected to fluorescence observation using 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 in the fine containment holes (Fig. 5C).

[0072]

Table 3

[0073] 〈Replacement from Sealing Oil to First Removing Liquid〉 In the same manner as in Example 1, replacement was performed from silicone oil KF96 - 20CS, which is a sealing oil, to Novec 7200, which is the first removing liquid.

[0074] 〈Removal of First Removing Liquid〉 In the same manner as in Example 1, the first removing liquid, Novec 7200, was removed (Fig. 5D).

[0075] 〈Removal of First Filling Liquid with Cleaning 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 cleaning solution. Since the fluorescence emitted by the TaqMan probe, which was the first indicator, in the region inside the fine accommodation holes was no longer detected, it was determined that the cleaning as the first filling solution filled in each fine accommodation hole was completed (Fig. 5E).

[0076] 〈Re-introduction of the second filling solution, re-sealing with sealing oil〉 A PCR reaction solution (Table 1) having the same composition as the first filling solution as the second filling solution was introduced from the inlet and allowed to stand for 3 minutes to fill the PCR reaction solution into the fine accommodation holes. The substrate having the fine accommodation holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus Corporation) (Fig. 5F). 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. 5G).

[0077] 〈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 3, the substrate having the fine accommodation holes in this state was observed for fluorescence with an inverted research microscope IX-71 (Olympus Corporation). Since the fluorescence emitted by the TaqMan probe was observed in the region inside the fine accommodation holes, it was confirmed that the β-actin gene as the second reaction product was amplified in the fine accommodation holes (Fig. 5H). That is, it was shown that repeated reactions can be performed in the fine accommodation holes in the aspect where the first filling solution and the second filling solution are the same.

[0078] <Replacement of the sealing oil with the first removal liquid> In the same manner as in Example 1, replacement was carried out from silicone oil KF96-20CS, which is a sealing oil, to Novec 7200, which is the first removal liquid.

[0079] <Removal of the first removal liquid> In the same manner as in Example 1, removal of Novec 7200, which is the first removal liquid, was carried out.

[0080] <Removal of the second filling liquid with the cleaning liquid> In the same manner as in Example 1, removal of the PCR reaction solution, which is the second filling liquid, was carried out with PBS, which is the cleaning liquid. Since fluorescence emitted by the TaqMan probe, which is the first indicator, in the region inside the fine accommodation holes was no longer detected, it was judged that cleaning as the second filling liquid filled in each fine accommodation hole was completed (Fig. 5I).

[0081] <<Example 3; Evaluation of the cleaning efficiency of the substrate and generation of reaction products (when the second filling liquid is different from the first filling liquid)>> <Sealing of the fine accommodation holes with the sealing oil> In the same manner as in Example 2, filling into the fine accommodation holes of a PCR reaction solution (Table 3) containing, as the first indicator, a TaqMan probe (SEQ ID NO: 4) that emits fluorescence during β-actin gene amplification, which is the first filling liquid (Fig. 6A), and sealing of the fine accommodation holes with silicone oil KF96-20CS, which is the sealing oil, were carried out (Fig. 6B).

[0082] <PCR reaction in the fine accommodation holes> In the same manner as in Example 2, a PCR reaction was carried out in the fine accommodation holes, and it was confirmed that the β-actin gene as the first reaction product was amplified in the fine accommodation holes (Fig. 6C).

[0083] <Replacement of the sealing oil with the first removal liquid> In the same manner as in Example 2, replacement was carried out from silicone oil KF96-20CS, which is the sealing oil, to Novec 7200, which is the first removal liquid.

[0084] <Removal of the first removal liquid> In the same manner as in Example 2, the removal of Novec 7200, which is the first removal liquid, was performed (Fig. 6D).

[0085] <Removal of the first filling liquid with the cleaning liquid> In the same manner as in Example 2, the removal of the PCR reaction solution, which is the first filling liquid, was performed with PBS, which is the cleaning liquid. Since the fluorescence emitted by the TaqMan probe, which is the first indicator, in the region inside the fine accommodation holes was no longer detected, it was determined that the cleaning of the first filling liquid filled in each fine accommodation hole was completed (Fig. 6E).

[0086] <Re-introduction of the second filling liquid, re-sealing with the sealing oil> A CFPS (Cell-Free Protein Synthesis) reaction solution (Table 4) containing a fluorescent protein EGFP gene (SEQ ID NO: 5) synthesized artificially by Eurofins as the second filling liquid was introduced from the inlet, and the CFPS 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. 6F). Next, silicone oil KF96-20CS (Shin-Etsu Chemical Co., Ltd.), which is 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).

[0087] [Table 4]

[0088] [Table 5]

[0089] <CFPS reaction in the fine accommodation holes> Mineral oil (Nacalai Tesque) was dropped onto the heat block of the Flat Thermal Cycler ProFlex PCR System - Dual Flat Sample Block (Thermo Fisher Scientific), and the substrate having the fine accommodation holes described above was placed thereon. The mineral oil was used to enhance the adhesion between the heat block and the substrate having the fine accommodation holes. Then, after incubating at 30°C for 1 hour, the substrate having the fine accommodation holes in this state was observed for fluorescence using an inverted research microscope IX-71 (Olympus). Since fluorescence emitted by the fluorescent protein EGFP was observed in the region inside the fine accommodation holes, it was confirmed that the fluorescent protein EGFP was protein-synthesized as the second reaction product inside the fine accommodation holes (Fig. 6H). That is, it was shown that repeated reactions can be performed inside the fine accommodation holes in a mode where the first filling liquid and the second filling liquid are different.

[0090] 〈Replacement from Sealing Oil to First Removing Liquid〉 In the same manner as in Example 2, replacement was performed from silicone oil KF96-20CS, which is a sealing oil, to Novec7200, which is the first removing liquid.

[0091] 〈Removal of First Removing Liquid〉 In the same manner as in Example 2, the first removing liquid Novec7200 was removed.

[0092] 〈Removal of Second Filling Liquid with Cleaning Liquid〉 In the same manner as in Example 2, the CFPS reaction solution, which is the second filling liquid, was removed with PBS, which is a cleaning liquid. Since fluorescence emitted by EGFP, which is an indicator contained in the second filling liquid and was in the region inside the fine accommodation holes, was no longer detected, it was determined that the cleaning of the second filling liquid filled in each fine accommodation hole was completed (Fig. 6I).

[0093] Although the preferred embodiments of the method of the present invention for evaluating the cleaning efficiency of a substrate and the method for producing a reaction product in a fine accommodation hole have been described, those skilled in the art will understand that changes can be made without departing from the scope of the claims.

Explanation of Signs

[0094] 100 Substrate 100a Fine accommodation hole 111 First filling liquid 112 Second filling liquid 121 First sealing liquid 122 Sealing oil 131 Cleaning liquid 141 First reaction product 142 Second reaction product

Claims

1. 1. A method for evaluating the cleaning efficiency of 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 first filling liquid contains a first indicator; The method for evaluating cleaning efficiency comprises: (i) washing the substrate with a washing solution that does not contain the first indicator; and (ii) after step (i), detecting the first indicator at the substrate; A method for evaluating the cleaning efficiency of a substrate, comprising:

2. 1. A method for evaluating the cleaning efficiency of 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, The first filling liquid in the fine containing hole is sealed with a first sealing liquid, and the first sealing liquid contains a first indicator, The method for evaluating cleaning efficiency comprises: (i) washing the substrate with a washing solution that does not contain the first indicator; and (ii) after step (i), detecting the first indicator at the substrate; A method for evaluating the cleaning efficiency of a substrate, comprising:

3. The method of claim 1 or 2, wherein the first indicator comprises a fluorescent molecule.

4. The method according to claim 1 or 2, wherein it is determined that the first filling liquid or the first sealing liquid remains when a first indicator is detected in step (ii).

5. The method of claim 1 or 2, wherein the cleaning solution contains a second indicator different from the first indicator.

6. (p) containing the first filling liquid in a plurality of fine containing holes in the surface of the substrate; (r) causing a reaction of the first filling liquid in the fine containing hole to generate a first reaction product; and (s) cleaning the substrate with the method of claim 1 or 2; A method for producing a reaction product in a fine containing hole, comprising:

7. After step (s), (p') containing a second filling liquid in a plurality of fine containing holes on the surface of the substrate; 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 6 further comprising:

8. The method of claim 7 , wherein the second fill liquid is the same as the first fill liquid.

9. The method of claim 7 , wherein the second fill liquid is different from the first fill liquid.

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