Method of introducing liquid into the hole

Abstract

The present invention relates to a method for introducing a second liquid into a hole, which includes the step of introducing the second liquid on one surface of a fluid device, the fluid device including a substrate and a plurality of holes that are open on one surface of the substrate and contain a first liquid containing a surfactant, a first sealing liquid is stacked on the one surface, the openings of the plurality of holes are sealed by the first sealing liquid, and the result of introducing the second liquid on one surface of the fluid device is that the second liquid replaces the first sealing liquid and is introduced into the interior of the hole.

Description

Technical Field

[0001] The present invention relates to a method for introducing a liquid into a well.

[0002] This application claims priority to Japanese Patent Application No. 2019-101305 filed in Japan on May 30, 2019, the contents of which are incorporated herein by reference. Background Art

[0003] Early detection of diseases or prediction of the effects of drug administration can be achieved by quantitatively detecting target molecules in biological samples. Conventionally, protein quantification has been performed by enzyme-linked immunosorbent assay (ELISA) and nucleic acid quantification by real-time PCR.

[0004] In recent years, for the purpose of earlier disease detection, the demand for more accurate detection of target molecules has increased. As a method for detecting target molecules with good accuracy, for example, Patent Document 1, Patent Document 2, and Non-Patent Document 1 describe technologies for performing enzyme reactions in multiple micro-partitions. These methods are called digital measurements.

[0005] In digital measurement, the sample solution is divided into a large number of micro-partitions. Then, the signal from each micro-partition is binarized to identify only the presence or absence of the target molecule, and then the number of target molecules is measured. Compared with existing ELISA or real-time PCR methods, digital measurement can significantly improve detection sensitivity and quantitativeness.

[0006] Digital measurement technology is not limited to the above-mentioned diagnostic applications, but is also widely used for analyzing multiple target molecules individually. For example, in Non-Patent Document 2, it is used for functional analysis of transmembrane proteins.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent No. 5551798

[0010] Patent Document 2: Japanese Patent Application No. 2014-503831

[0011] Non-patent literature

[0012] Non-patent literature 1: KiM SH, et al., Large-scale femtoliter droplet array for digital counting of single biomolecules., Lab on a Chip, 12(23), 4986-4991, 2012.

[0013] Non-patent document 2: Rikiya Watanabe, et al., High-throughput formation of lipidbilayer membrane arrays with an asymmetric lipid composition., Scientific Reports volume 4, Article number: 7076, 2014. Summary of the invention

[0014] Technical problem to be solved by the invention

[0015] In digital measurement technology, after the target molecule mixed with the reaction reagent is divided into a plurality of micro-partitions using a sealing liquid or a lipid bilayer membrane, the target molecule is detected by reacting separately inside each micro-partition. Therefore, usually only the analysis result corresponding to the reaction reagent contained inside the micro-partition can be obtained. Therefore, the purpose of the present invention is to provide a technology for replacing the liquid inside the individually sealed micro-partition.

[0016] Means for solving technical problems

[0017] The present invention includes the following aspects.

[0018] [1] A method for introducing a second liquid into a hole, comprising the step of introducing the second liquid onto one surface of a fluid device, the fluid device comprising a substrate and a plurality of holes opening onto one surface of the substrate and containing a first liquid including a surfactant, a first sealing liquid being stacked on the one surface, the openings of the plurality of holes being sealed by the first sealing liquid, and the result of introducing the second liquid onto one surface of the fluid device is that the second liquid replaces the first sealing liquid and is introduced into the interior of the hole.

[0019] [2] The method according to [1], wherein after introducing the second liquid, there is further included a step of introducing a second sealing liquid into the fluid device, so that the second sealing liquid is stacked on the one surface to seal the openings of the plurality of holes, and the second liquid, or a mixture of the first liquid and the second liquid, is sealed inside the holes.

[0020] [3] The method according to [1] or [2], further comprising: a step of introducing the first liquid into the fluid device before introducing the second liquid; and a step of introducing the first sealing liquid into the fluid device so that the first sealing liquid is stacked on the one surface to seal the openings of the plurality of holes and the first liquid is sealed inside the holes.

[0021] [4] The method according to any one of [1] to [3], wherein the first liquid and the second liquid are miscible.

[0022] [5] The method according to any one of [1] to [4], wherein the fluid device further includes a cover member disposed facing the one surface, and a space between the cover member and the one surface forms a flow path.

[0023] [6] The method according to [5], wherein the second liquid is introduced into the fluid device through the flow path.

[0024] [7] The method according to any one of [1] to [6], wherein the first liquid and the second liquid contain a reaction reagent.

[0025] [8] The method according to any one of [1] to [7], wherein after the second liquid is introduced into the interior of the pore, at least a portion of the components contained in the first liquid is retained in the interior of the pore.

[0026] [9] The method according to [8], wherein at least a portion of the components contained in the first liquid is retained inside the pore by being retained by a carrier.

[0027]

[10] The method according to any one of [1] to [9], wherein the affinity between the one surface and the first sealing liquid is equal to or lower than the affinity between the one surface and the second liquid.

[0028]

[11] The method according to

[10] , wherein the material of the one surface is cycloolefin polymer, the main components of the first liquid and the second liquid are water, and the first sealing liquid is fluorine-based oil.

[0029] Effects of the Invention

[0030] According to the present invention, a technique for replacing the liquid inside each of the sealed micro-partitions can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic cross-sectional view showing an example of a fluid device.

[0032] Figure 2 This is a schematic cross-sectional view showing an example of a fluid device.

[0033] Figure 3 This is a schematic cross-sectional view showing an example of a fluid device.

[0034] Figure 4 This is a schematic cross-sectional view showing an example of a fluid device.

[0035] Figure 5 This is a schematic cross-sectional view showing an example of a fluid device.

[0036] Figure 6 This is a schematic cross-sectional view showing an example of a fluid device.

[0037] Figure 7 This is a schematic cross-sectional view showing an example of a fluid device.

[0038] Figure 8 This is a schematic cross-sectional view showing an example of a fluid device.

[0039] Fig. 9 This is a schematic cross-sectional view showing an example of a fluid device.

[0040] Fig.10 This is a schematic cross-sectional view showing an example of a fluid device.

[0041] Fig.11 This is a schematic cross-sectional view showing an example of a fluid device.

[0042] Fig.12 This is a microscope image showing the results of Example 1.

[0043] Fig.13 This is a microscope image showing the results of Example 1.

[0044] Fig.14 This is a microscope image showing the results of Example 2.

[0045] Fig.15 This is a microscope image showing the results of Example 2.

[0046] Fig.16 This is a microscope image showing the result of Comparative Example 1.

[0047] Fig.17 This is a microscope image showing the results of Comparative Example 2.

[0048] Fig.18 This is a microscope image showing the result of Comparative Example 1.

[0049] Fig.19 This is a microscope image showing the results of Comparative Example 2.

[0050] Fig. 20 The microscope images show the results of Example 3.

[0051] Fig.21 The microscope images show the results of Example 3.

[0052] Fig. 22 The microscope images show the results of Example 3.

[0053] Fig.23 The microscope images show the results of Example 4.

[0054] Fig.24 The microscope images show the results of Example 4.

[0055] Fig.25 The microscope images show the results of Example 4.

[0056] Fig.26 This is a microscope image showing the results of Comparative Example 3.

[0057] Fig. 27 This is a microscope image showing the results of Comparative Example 3.

[0058] Fig.28 This is a microscope image showing the results of Comparative Example 3.

[0059] Fig.29 This is a microscope image showing the results of Example 5.

[0060] Fig.30 This is a microscope image showing the results of Example 5.

[0061] Fig.31 The microscope images show the results of Example 6.

[0062] Fig.32 The microscope images show the results of Example 6.

[0063] Fig.33 This is a microscope image showing the results of Example 7.

[0064] Fig.34 This is a microscope image showing the results of Example 7.

[0065] Fig.35 This is a microscope image showing the results of Comparative Example 4.

[0066] Fig.36 This is a microscope image showing the results of Comparative Example 4.

[0067] Fig.37 This is a microscope image showing the results of Comparative Example 5.

[0068] Fig.38 This is a microscope image showing the results of Comparative Example 5.

[0069] Fig.39 This is a microscope image showing the results of Comparative Example 6.

[0070] Fig.40This is a microscope image showing the results of Comparative Example 6. DETAILED DESCRIPTION

[0071] The following is based on the situation while referring to the attached Figure 1 The embodiments of the present invention are described in detail. In addition, the same or corresponding parts in the drawings are marked with the same or corresponding symbols, and repeated descriptions are omitted. In addition, the dimensions in each figure are exaggerated for the purpose of explanation and may not be consistent with the actual dimensions.

[0072] [Method of introducing liquid into the hole]

[0073] One embodiment of the present invention provides a method for introducing a second liquid into a hole, which includes the step of introducing the second liquid on one surface of a fluid device, the fluid device includes a substrate and a plurality of holes that are open on one surface of the substrate and contain a first liquid containing a surfactant, a first sealing liquid is stacked on the one surface, the openings of the plurality of holes are sealed by the first sealing liquid, and the result of introducing the second liquid on one surface of the fluid device is that the second liquid replaces the first sealing liquid and is introduced into the interior of the hole.

[0074] According to the method of this embodiment, the liquid inside the respectively sealed micro-partitions can be replaced. Thus, for example, in digital measurement technology, after obtaining the analysis result using the reaction reagent contained in the first liquid contained in the micro-partition, the liquid inside the micro-partition can be replaced with the second liquid, so that different analysis can be performed using the reaction reagent contained in the second liquid.

[0075] (Fluid Equipment)

[0076] First, a fluid device that can be used in the method of this embodiment will be described. Figure 1 FIG. 1 is a schematic cross-sectional view showing an example of a fluid device. Figure 1 As shown, the fluid device 100 includes a substrate 110 and a cover member 120 disposed facing one surface 111 of the substrate 110. The cover member 120 has a convex portion 121. The front end of the convex portion 121 contacts the substrate 110. In the fluid device 100, a plurality of holes 141 are integrally formed on one surface 111 of the substrate 110 to form a hole array 140. One surface 111 faces the cover member 120. The cover member 120 may also be welded or bonded to the substrate 110.

[0077] The hole 141 is opened on the surface of the substrate 110. The shape, size and arrangement of the hole 141 are not particularly limited, and it is preferred to introduce one target molecule into one hole 141. The hole 141 is preferably a tiny hole with a small volume. For example, the volume of one hole 141 can be about 10fL to 100pL. In the fluid device 100, a plurality of holes 141 of the same shape and size constitute the hole array 140. The same shape and size are sufficient as long as they are the same shape and the same capacity to the extent required for digital measurement, and the degree of manufacturing error is allowed to vary.

[0078] The diameter of the hole 141 may be, for example, about 1 to 30 μm. The depth of the hole 141 may be, for example, about 1 to 30 μm. In addition, the arrangement of the holes 141 is not particularly limited, and may be, for example, arranged in a triangular lattice, in a square lattice, or randomly arranged.

[0079] In the fluid device 100, a space is formed between the one surface 111 and the cover member 120 due to the presence of the convex portion 121. The space forms a flow path 130. The flow path 130 functions as a path for delivering the first liquid, the first sealing liquid, the second liquid, and the second sealing liquid described later. That is, the first liquid, the first sealing liquid, the second liquid, and the second sealing liquid are introduced into the fluid device 100 through the flow path 130.

[0080] The shape, structure and capacity of the flow channel 130 are not particularly limited. The height of the flow channel 130 (ie, the distance between one surface 111 of the substrate 110 and the surface of the cover member 120 facing the substrate 110 ) may be, for example, less than 100 μm.

[0081] The projection 121 may be formed integrally with the cover member 120. The cover member 120 may be formed into a plate having the projection 121 by, for example, molding a thermoplastic resin fluid using a molding die. The cover member 120 may also be formed with a reagent introduction port 122 and a discharge port 123.

[0082] When the cover member 120 has the convex portion 121, the cover member 120 and the substrate 110 are overlapped in such a manner that the convex portion 121 contacts the surface 111 where the hole 141 of the substrate 110 is opened. As a result, the space between the cover member 120 and the substrate 110 becomes the flow path 130. The cover member 120 and the substrate 110 can also be welded by laser welding or the like.

[0083] (Variation 1 of Fluid Device)

[0084] The fluid device used in the method of this embodiment is not limited to the above-mentioned fluid device 100 . Figure 7 FIG. 1 is a schematic cross-sectional view showing an example of a fluid device. Figure 7As shown, the fluid device 200 includes a substrate 110 and a wall member 210. In the fluid device 200, a hole array 140 is formed integrally with the substrate 110 on one surface 111 of the substrate 110. The hole array 140 has a plurality of holes 141.

[0085] The fluid device 200 is different from the above-described fluid device 100 mainly in that it does not have a cover member 120. Therefore, the fluid device 200 does not have a flow path.

[0086] (Variation 2 of Fluid Device)

[0087] In the above-mentioned fluid device 100, the cover member 120 and the convex portion 121 are integrally formed. However, the cover member 120 and the convex portion 121 may be separately formed.

[0088] In addition, in the above-mentioned fluid device 100 and fluid device 200, the hole array 140 is formed integrally with the substrate 110 on one surface 111 of the substrate 110. However, the hole array may not be formed integrally with the substrate 110. For example, the hole array 140 formed separately from the fluid device may be arranged on the substrate 110 of the fluid device. Alternatively, a resin layer may be stacked on the surface of the substrate 110, and the hole array may be formed on the resin layer by etching or the like.

[0089] (Material of fluid equipment)

[0090] The substrate 110 is formed of, for example, a resin. The type of resin is not particularly limited, and preferably a resin that is resistant to the first liquid, the second liquid, and the sealing liquid. In addition, when the detected signal is fluorescence, a resin with less autofluorescence is preferred. For example, as the resin, cycloolefin polymer, cycloolefin copolymer, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesin, and amorphous fluororesin can be cited, but are not limited to these.

[0091] A plurality of holes 141 may be formed on one surface 111 in the plate thickness direction of the substrate 110. Examples of a method for forming the holes using a resin include injection molding, thermal embossing, and photo embossing.

[0092] Alternatively, for example, a fluororesin may be stacked on the substrate 110 and the hole array may be formed by processing the fluororesin by etching or the like. As the fluororesin, for example, CYTOP (registered trademark) (Asahi Glass) or the like may be used.

[0093] When the fluid device includes the cover member 120 , the material of the cover member 120 is preferably a resin with little autofluorescence, for example, a thermoplastic resin such as a cycloolefin polymer or a cycloolefin copolymer.

[0094] In addition, the cover member 120 may be formed of a material that does not transmit light of the wavelength detected when the signal is observed by fluorescence or its adjacent wavelengths, or may be formed of a material that does not transmit light at all. For example, the cover member 120 may be formed of a thermoplastic resin to which carbon or metal particles are added.

[0095] (First Embodiment)

[0096] As described above, the method of this embodiment is a method for introducing a second liquid into a hole, which includes the step of introducing the second liquid on one surface of a fluid device, the fluid device includes a substrate and a plurality of holes that are open on one surface of the substrate and contain a first liquid containing a surfactant, a first sealing liquid is stacked on the one surface, the openings of the plurality of holes are sealed by the first sealing liquid, and the result of introducing the second liquid on one surface of the fluid device is that the second liquid replaces the first sealing liquid and is introduced into the interior of the hole.

[0097] Depending on the situation, refer to Figures 1 to 6 , The method of the first embodiment will be described by taking the case of using the fluid device 100 as an example.

[0098] 《Introduction of the First Liquid》

[0099] First, if Figure 1 As shown, the first liquid L110 is introduced from the inlet 122 of the fluid device 100 and sent to the flow path 130. The first liquid L110 includes, for example, a biological sample or an environmental sample. The biological sample is not particularly limited, and examples thereof include serum, plasma, urine, and cell culture fluid. In addition, examples of environmental samples include river water and factory wastewater.

[0100] Biological samples and environmental samples may contain target molecules to be detected. In addition, biological samples and environmental samples may not contain target molecules to be detected. Examples of target molecules include DNA, RNA, protein, virus, cell, and specific compound. Here, examples of RNA include miRNA and mRNA. In addition, examples of cells include bacteria, yeast, animal cells, plant cells, and insect cells.

[0101] The first liquid L110 may also contain a reaction reagent for detecting the target molecule. As reaction reagents, buffer substances, enzymes, substrates, antibodies and antibody fragments, etc. can be cited. When the target molecule is a nucleic acid, for example, the enzyme is selected according to the content of the biochemical reaction in order to carry out biochemical reactions such as enzyme reactions for template nucleic acids related to the target molecule. The biochemical reaction for the template nucleic acid is, for example, a reaction that causes signal amplification under the condition that the template nucleic acid exists. The reaction reagent is selected according to the detection reaction adopted. As specific detection reactions, the Invasive Cleavage Assay (Invasive Cleavage Analysis, ICA) method, the loop-mediated isothermal amplification (LAMP) method (registered trademark), the 5'→3' nuclease method (TaqMan (registered trademark) method) and the fluorescent probe method, etc. can be cited.

[0102] The first liquid L110 contains a surfactant. Examples of the surfactant include Triton-X100 (also known as polyethylene glycol mono-4-octylphenyl ether (n=about 10)), sodium lauryl sulfate, Nonidet P-40 (also known as octylphenoxy poly(ethyleneoxy)ethanol), and Tween 20 (also known as polyoxyethylene sorbitan monolaurate).

[0103] The concentration of the surfactant is preferably 0.001 v / v% or more and 1.0 v / v% or less, more preferably 0.005 v / v% or more and 0.5 v / v% or less, and further preferably 0.01 v / v% or more and 0.1 v / v% or less relative to the total volume of the first liquid L110. When the concentration of the surfactant is 0.001 v / v% or more relative to the total volume of the first liquid L110, it is easy to replace the second liquid L410. When the concentration of the surfactant is 1.0 v / v% or less relative to the total volume of the first liquid L110, it has little effect on the reaction in the subsequent detection of the target molecule.

[0104] The first liquid L110 sent to the flow channel 130 is accommodated in the well 141. As a result, the reaction reagent, the surfactant, and the target molecule, if present, are introduced into the well 141.

[0105] The number of target molecules introduced into one hole 141 is not particularly limited, and preferably one or less, that is, 0 or 1 target molecule is introduced into one hole 141. Thus, the target molecule can be detected in 1 unit, that is, digital measurement can be performed. In addition, it is not necessary to introduce the target molecule into all the holes 141 of the hole array 140.

[0106] The means for introducing the target molecule into the hole 141 is not particularly limited, and an appropriate means corresponding to the selected target molecule can be selected. For example, a method can be cited in which the target molecule is precipitated in the fluid device (specifically, in the flow path) by its own weight and distributed in the hole 141. Alternatively, a carrier (i.e., a capture object) for capturing the target molecule can be used to bind the capture object to the target molecule that is difficult to precipitate by its own weight to carry out liquid delivery. In addition, by fixing the capture object on the hole 141 in advance and capturing the target molecule to be delivered, the efficiency of introducing the target molecule into the hole can also be improved.

[0107] The process of binding the captured object to the target molecule can be performed at any time point. For example, the process can be performed by contacting the target molecule with the captured object in the sample tube before introducing the target molecule into the hole 141. Alternatively, the captured object can be introduced into the hole after the captured object is introduced into the hole 141, and the captured object and the target molecule are contacted in the hole.

[0108] The capture object is a substance that can capture the target molecule. For example, the capture object can be a combination of a solid phase and a specific binding substance for the target molecule.

[0109] As the solid phase, particles, films, substrates, etc. can be cited. In addition, the specific binding substances for the target molecule can be one or more. For example, it can be three, four, or five or more.

[0110] As particles, there is no particular limitation, and polymer particles, magnetic particles and glass particles can be cited. Particles are preferably particles that have been surface treated to avoid nonspecific adsorption. In addition, in order to immobilize specific binding substances, particles having functional groups such as carboxyl groups on the surface are preferred. More specifically, as an example, the trade name "Magnosphere LC300" manufactured by JSR Corporation can be used.

[0111] Alternatively, for example, when a virus is used as a target molecule, cells to which the virus can attach (ie, cells having a virus receptor) may be used as captured objects.

[0112] As specific binding substances in the captured object, antibodies, antibody fragments and aptamers can be cited. As antibody fragments, Fab, F(ab')2, Fab', single-chain antibody (scFv), disulfide bond stabilized antibody (dsFv), dimeric V region fragment (Diabody) and peptides containing CDR can be cited. The antibody can be a monoclonal antibody or a polyclonal antibody. In addition, it can also be a commercially available antibody.

[0113] In addition, when the target molecule contains a sugar chain, the specific binding substance may also be a lectin. In addition, when the target molecule contains a lipid membrane, the specific binding substance may also be a substance that has a binding property to the lipid membrane. As a substance that has a binding property to the lipid membrane, for example, hydrocarbons such as hexanediol and membrane proteins such as transmembrane proteins may be cited. As a membrane protein, for example, α-hemolysin may be cited.

[0114] There is no particular limitation on the method of immobilizing the specific binding substance on the solid phase, and methods using physical adsorption, chemical bonding, avidin-biotin bonding, and protein G or protein A bonding with antibodies can be cited. As a method using physical adsorption, a method using hydrophobic interaction or electrostatic interaction to immobilize the specific binding substance on the particle surface can be cited. As a method using chemical bonding, a method using a cross-linking agent can be cited. For example, when the surface of the particle has a hydroxyl group, after active esterification by reacting a cross-linking agent with the carboxyl group possessed by the specific binding substance, the hydroxyl group is reacted with the ester group, so that the specific binding substance can be immobilized on the particle surface. In addition, it is preferred to set a spacer between the specific binding substance and the particle surface so as not to hinder the recognition ability of the specific binding substance to the target molecule.

[0115] When the target molecule is introduced into the hole 141 using the capture object, it is preferred that a combination of the capture object and the target molecule is formed under the condition that one capture object captures 0 or 1 target molecule. In addition, it is preferred that 0 or 1 capture object is introduced into one hole 141. Thus, digital measurement can be performed.

[0116] 《Introduction of sealing liquid》

[0117] Then, if Figure 2 and Figure 3 As shown, the first sealing liquid L120 is introduced from the introduction port 122 and sent to the flow path 130 .

[0118] The first sealing liquid is a liquid that can form droplets (tiny droplets) by sealing the liquids introduced into the plurality of holes 141 in a manner that does not mix with each other. The first sealing liquid is preferably an oily solution, more preferably oil. As the oil, fluorine oil, silicone oil, hydrocarbon oil, or a mixture thereof can be used. More specifically, the trade name "FC-40" manufactured by Sigma can be used. FC-40 (CAS number: 86508-42-1) is a fluorinated aliphatic compound with a specific gravity of 1.85 g / mL at 25°C.

[0119] The first sealing liquid L120 sent to the flow path 130 washes away and replaces the first liquid L110 introduced into the flow path 130 that is not contained in the holes 141. Thus, the first sealing liquid L120 seals the plurality of holes 141 individually, and the holes 141 become independent reaction spaces (micropartitions 142).

[0120] When the flow path 130 is filled with the first sealing liquid L120 , excess first sealing liquid L120 is discharged from the discharge port 123 . Figure 3 This shows a state in which all the holes 141 of the hole array 140 are sealed by the first sealing liquid L120 to form micro-partitions (sealed holes) 142 .

[0121] In addition, lipids may be dissolved in the first liquid L110 in advance, and the liquid containing lipids may be sent again after the first sealing liquid L120 is sent to the flow path 130, so that a lipid bilayer membrane is formed in the opening of the hole 141, and the plurality of holes 141 are individually sealed by the lipid bilayer membrane to form micropartitions 142. Examples of lipids forming the lipid bilayer membrane include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), and mixtures thereof.

[0122] Detection of target molecules

[0123] Here, the target molecule can be detected by reacting the reaction reagent contained in the first liquid L110. For example, a signal amplification reaction is performed inside the micro-partition 142. That is, the signal is amplified to a level at which the signal can be observed by using a reaction process in such a way that the signal derived from the reaction reagent is detected inside the micro-partition 142. Examples of the signal include fluorescence, color development, potential change, and pH change.

[0124] The signal amplification reaction is, for example, an enzyme reaction. As an example, the signal amplification reaction is an isothermal reaction in which the first liquid L110 containing the enzyme for signal amplification is contained in the micropartition 142, and the fluid device 100 is maintained for a required time at a certain temperature condition that can obtain the required enzyme activity. As a specific example, an ICA reaction can be used as a signal amplification reaction. At this time, the interior of the micropartition 142 contains an ICA reaction reagent and a nucleic acid as a target molecule. As a result of the enzyme reaction, when the target molecule is contained in the micropartition 142, the fluorescent substance is released from the extinction substance, and a prescribed fluorescent signal is emitted corresponding to the excitation light. Figure 3 In FIG. 1 , reference numeral 142R indicates a well that contains a target molecule and emits a signal.

[0125] 《Introduction of the second liquid》

[0126] Then, if Figure 4 As shown, the second liquid L410 is introduced from the introduction port 122 and sent to the flow path 130. As a result, the second liquid L410 washes away the first sealing liquid L120 and is introduced into the inside of the hole 141. Here, the first sealing liquid L120 is preferably completely replaced and removed by the second liquid L410, but as long as the second liquid L410 is introduced into the inside of the hole 141, a part of the first sealing liquid L120 may remain.

[0127] In the past, it was believed that the liquid inside the micro-partition 142 sealed by the sealing liquid could no longer be replaced. However, in the embodiment, as described later, the inventors found that the second liquid L410 can be introduced into the micro-partition 142 sealed by the first sealing liquid L120 through the method of this embodiment.

[0128] The second liquid L410 may or may not contain a surfactant. When the second liquid L410 contains a surfactant, the type and concentration of the surfactant may be the same as the surfactant contained in the first liquid L110.

[0129] The second liquid L410 may also contain a reaction reagent for detecting the target molecule, similarly to the first liquid L110. As the reaction reagent, the same reagent as the reagent that can be contained in the first liquid L110 can be cited, but preferably a reagent different from the first liquid L110. Thus, a reaction different from the reaction reagent contained in the first liquid L110 can be performed.

[0130] The second liquid L410 sent to the flow path 130 washes away and replaces the first sealing liquid L120 introduced into the flow path 130. As a result, the seals of the respectively sealed micro-regions 142 are released, and the holes 141 are not sealed. In addition, the second liquid L410 is introduced into the inside of the hole 141 containing the first liquid L110.

[0131] In the method of the first embodiment, it is preferred that the affinity between the first surface 111 and the first sealing liquid L120 is equal to the affinity between the first surface 111 and the second liquid L410, or the affinity between the first surface 111 and the first sealing liquid L120 is lower than the affinity between the first surface 111 and the second liquid L410. Thus, when the second liquid L410 is introduced, the second liquid L410 easily washes away the first sealing liquid L120 and releases the seal of the micro-partition 142.

[0132] As an example in which the affinity between a surface 111 and the first sealing liquid L120 is equal to or lower than the affinity between a surface 111 and the second liquid L410, for example, a combination in which the material of the surface 111 is cycloolefin polymer, the main components of the first liquid L110 and the second liquid L410 are water, and the first sealing liquid L120 is fluorine-based oil can be cited.

[0133] In this specification, the main component of the liquid is water, which means that 50% by mass or more, for example, 60% by mass or more, for example, 70% by mass or more, for example, 80% by mass or more, for example, 90% by mass or more, for example, 95% by mass or more, for example, 98% by mass or more of the liquid is water. In addition, as fluorine-based oil, for example, the trade name "FC-40" made by Sigma Company can be used.

[0134] Since the first liquid L110 contains a surfactant, the second liquid L410 is introduced into the interior of the unsealed hole 141, and as a result, the interior of the hole 141 can be replaced with the second liquid L410. That is, the first liquid L110 containing a surfactant is introduced into the interior of the hole 141, and after the first liquid L110 is sealed with the first sealing liquid L120, the second liquid L410 is introduced to replace the interior of the hole 141 with the second liquid L410.

[0135] After the second liquid L410 is introduced into the inside of the hole 141, at least a part of the components contained in the first liquid L110 can also be retained inside the hole 141. For example, the target molecule contained in the first liquid L110 is bound to the captured object, and the captured object is retained inside the hole 141. That is, at least a part of the components contained in the first liquid L110 can be retained inside the hole 141 by being retained by the captured object (carrier). At this time, inside the hole 141, the second liquid L410 exists together with the captured object and the target molecule.

[0136] Alternatively, for example, when the target molecule contained in the first liquid L110 binds to the captured object, and then the antibody against the target molecule contained in the first liquid L110 binds to the target molecule, the captured object can also be retained inside the hole 141. At this time, inside the hole 141, the second liquid L410 exists together with the captured object, the target molecule, and the antibody bound to the target molecule.

[0137] It is preferred that the first liquid L110 and the second liquid L410 are miscible. When the first liquid L110 and the second liquid L410 are miscible, liquid replacement inside the hole 141 can be performed efficiently. Here, miscible means that when the first liquid L110 and the second liquid L410 are mixed, layer separation does not occur and a uniform solution or dispersion can be formed.

[0138] When the flow path 130 is filled with the second liquid L410 , excess second liquid L410 is discharged from the discharge port 123 .

[0139] 《Introduction of sealing liquid》

[0140] After the process of introducing the second liquid L410, a process of introducing a second sealing liquid into the fluid device 100 may be further performed. Here, the second sealing liquid may be the same as or different from the first sealing liquid. Figure 5 As shown, the first sealing liquid L120 can be introduced again from the introduction port 122 and sent to the flow path 130 .

[0141] As a result, the first sealing liquid L120 is stacked on a surface 111 to seal the openings of the plurality of holes 141, and the second liquid L410 or a mixture of the first liquid L110 and the second liquid L410 is sealed inside the hole 141, so that each hole 141 becomes an independent reaction space (micro partition 142).

[0142] When the flow path 130 is filled with the first sealing liquid L120 , excess first sealing liquid L120 is discharged from the discharge port 123 . Figure 6 The diagram shows a state in which all the holes 141 of the hole array 140 are sealed by the first sealing liquid L120 to form micro-partitions (sealed holes) 142 .

[0143] Alternatively, lipids may be dissolved in the second liquid L410 in advance, and after the first sealing liquid L120 is supplied to the flow path 130, a liquid containing lipids may be supplied again to form a lipid bilayer membrane at the opening of the hole 141, and the plurality of holes 141 may be individually sealed by the lipid bilayer membrane to form the micropartitions 142. Examples of the lipids forming the lipid bilayer membrane include the same lipids as described above.

[0144] Detection of target molecules

[0145] Here, the reaction reagent contained in the second liquid L410 may be reacted to detect the target molecule in the same manner as described above. For example, a signal amplification reaction may be performed inside the micropartition 142 . Figure 6 In the figure, reference numeral 142R indicates a well in which a target molecule is accommodated and a reaction reagent is reacted to generate a signal.

[0146] (Second Embodiment)

[0147] Next, refer to Figure 7 to Figure 11, while taking the case of using the fluid device 200 as an example, the method of the second embodiment is described. The method of the second embodiment is different from the method of the first embodiment in that the fluid device does not have a cover member. In addition, the first sealing liquid and the second sealing liquid in the method of the second embodiment are different from the first sealing liquid and the second sealing liquid in the method of the first embodiment in that they need to satisfy the specific gravity condition described later.

[0148] 《Introduction of the First Liquid》

[0149] First, if Figure 7 As shown, the first liquid L110 is introduced into the interior of the fluid device 200. The first liquid L110 is the same as described above. Figure 7 As shown, the first liquid L110 is contained in the inside of the hole 141. As a result, the reaction reagent and the target molecule, if present, are introduced into the hole 141. The number of target molecules introduced into one hole 141 is not particularly limited, but it is preferred that one or less, that is, 0 or 1 target molecule, is introduced into one hole 141.

[0150] 《Introduction of sealing liquid》

[0151] Then, if Figure 8 As shown, the first sealing liquid L120 is introduced into the interior of the fluid device 200. The specific gravity of the first sealing liquid L120 is greater than the specific gravity of the first liquid L110. Therefore, the first sealing liquid L120 settles at a lower position than the first liquid L110 and contacts one surface 111. Furthermore, the first sealing liquid L120 individually seals the multiple holes 141 containing the first liquid L110 to form independent reaction spaces (micro-partitions 142).

[0152] Detection of target molecules

[0153] Here, if Fig. 9 As shown, a predetermined reaction can be performed in the micro-region 142 and the emitted signal can be observed. Fig. 9 In FIG. 1 , the well 142R is a well in which the target molecule is contained and a signal is detected, and the micropartition 142 is a well in which the target molecule is not contained and a signal is not detected.

[0154] 《Introduction of the second liquid》

[0155] Then, if Fig.10As shown, the second liquid L410 is introduced into the interior of the fluid device 200. The specific gravity of the second liquid L410 is greater than the specific gravity of the first sealing liquid L120. Therefore, the second liquid L410 settles at a lower position than the first sealing liquid L120. As a result, the seals of the respectively sealed micro-partitions 142 are released, resulting in unsealed holes 141. Furthermore, the second liquid L410 is introduced into the interior of the hole 141. In the hole 141, it is preferred that the first sealing liquid L120 is completely replaced and removed by the second liquid L410, but as long as the second liquid L410 is introduced into the interior of the hole 141, a portion of the first sealing liquid L120 may remain.

[0156] The second liquid L410 may also contain a reaction reagent for detecting the target molecule. As the reaction reagent, the same reagent as the reagent that can be contained in the first liquid L110 can be cited, but preferably a reagent different from the first liquid L110. Thus, a reaction different from the reaction reagent contained in the first liquid L110 can be implemented.

[0157] In the second embodiment, similarly to the first embodiment, after the second liquid L410 is introduced into the inside of the hole 141 , at least a part of the components included in the first liquid L110 may be retained inside the hole 141 .

[0158] 《Introduction of sealing liquid》

[0159] After the process of introducing the second liquid L410, a process of introducing the second sealing liquid L620 into the fluid device 200 may be further performed. Fig.11 As shown, the second sealing liquid L620 is introduced into the interior of the fluid device 200. The specific gravity of the second sealing liquid L620 is greater than the specific gravity of the second liquid L410. Therefore, the second sealing liquid L620 settles at a lower position than the second liquid L410 and contacts one surface 111. Furthermore, the second sealing liquid L620 individually seals the plurality of holes 141 containing the second liquid L410 or the mixture of the first liquid L110 and the second liquid L410 to form independent reaction spaces (micro-partitions 142).

[0160] Fig.11 This shows a state in which all the holes 141 of the hole array 140 are sealed by the second sealing liquid L620 to form micro-partitions (sealed holes) 142 .

[0161] Detection of target molecules

[0162] Here, the reaction reagent contained in the second liquid L410 can be reacted to detect the target molecule in the same manner as described above. For example, a signal amplification reaction can be performed inside the micropartition 142 . Fig.11 In the figure, 142R indicates a well in which a target molecule is accommodated and a reaction reagent is reacted to generate a signal.

[0163] In the method of the second embodiment, it is preferred that the specific gravity of the first sealing liquid L120 is greater than the specific gravity of the first liquid L110, the specific gravity of the second liquid L410 is greater than the specific gravity of the first sealing liquid L120, and the specific gravity of the second sealing liquid L620 is greater than the specific gravity of the second liquid L410. In addition, the main component of the first liquid L110 and the second liquid L410 is preferably water. In this case, for example, the specific gravity can be adjusted by adding sucrose or the like to the second liquid L410. In addition, as the first sealing liquid L120 and the second sealing liquid L620, liquids that meet the requirements such as specific gravity can be appropriately selected from the above.

[0164] So far, an embodiment of replacing the liquid inside the respectively sealed micro-partitions once has been described, but according to one aspect of the present invention, the liquid inside the respectively sealed micro-partitions may be replaced more than twice. That is, by repeatedly implementing one aspect of the present invention, the liquid may be replaced a desired number of times.

[0165] Specifically, in the above example, the introduction of the first liquid L110, the sealing using the first sealing liquid L120, and the introduction of the second liquid L410 are performed continuously. However, these processes do not have to be performed continuously. For example, between the sealing using the first sealing liquid L120 and the introduction of the second liquid L410, a third liquid may be introduced into the fluid device 100, and the third liquid may be sealed inside the micropartition 142 using the third sealing liquid. At this time, the third liquid may contain a surfactant or may not contain a surfactant. Even if the third liquid does not contain a surfactant, by introducing the second liquid L410 into the fluid device 100, the inside of the hole 141 may be replaced from a liquid that does not contain a surfactant to the second liquid L410.

[0166] The reason for this is that the surfactant is introduced into the inside of the hole 141 by the introduction of the first liquid L110, and then, even if the inside of the hole 141 is replaced with a liquid that does not contain a surfactant, the surfactant remains in the hole 141 or adheres to the inner wall of the hole 141. Therefore, when the second liquid L410 is introduced, the inside of the hole 141 can be replaced with the second liquid L410 by the action of the surfactant remaining in the hole 141.

[0167] Furthermore, the fourth liquid may be introduced between the sealing by the third sealing liquid and the introduction of the second liquid L410, and the fourth sealing liquid may seal the fourth liquid inside the hole 141. The fourth liquid may also contain a surfactant or may not contain a surfactant.

[0168] According to one aspect of the present invention, when a sample contains a plurality of target molecules, for example, when the sample is a cell, etc., detection of the target molecules can be simplified. As an example, the following aspect can be cited.

[0169] The first liquid L110 containing cells and the first reaction reagent is introduced into the hole 141. At this time, it is preferred to use a captured object to introduce the cells into the hole 141. As the captured object, a captured object is used in which the cells bound to the captured object after the second liquid L410 is introduced and also remain in the micro-partition 142. For example, a captured object with a sufficiently large mass or a captured object fixed in the hole 141 can be used.

[0170] Afterwards, the first sealing liquid is used to obtain the micropartition 142, and the first target molecule is detected using the first reaction reagent. Furthermore, the second liquid L410 containing the second reaction reagent is introduced into the hole 141, and the hole 141 is replaced with the second liquid. Afterwards, the second sealing liquid is introduced into the flow path 130 to obtain the micropartition 142. The micropartition 142 contains cells bound to the captured object and the second liquid L410. Afterwards, the second target molecule is detected using the second reaction reagent.

[0171] In this way, when a sample contains a plurality of target substances, the plurality of target substances can be detected simply without using a plurality of devices.

[0172] Example

[0173] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0174] (Manufacture of fluid equipment)

[0175] First, manufacture Figure 1 A fluid device of the structure shown. A cycloolefin polymer substrate 110 having a hole array 140 formed by injection molding and a cycloolefin polymer cover member 120 colored by adding carbon black are bonded together using double-sided tape to manufacture the fluid device. The double-sided tape functions as a convex portion 121, and the height of the flow path 130 (the distance between one surface 111 of the substrate 110 and the surface of the cover member 120 facing the substrate 110) is 100 μm. In addition, the cover member 120 has an inlet 122 and an outlet 123. In addition, the diameter of the hole 141 constituting the hole array 140 is 5 μm, and the depth of the hole 141 is 3 μm. The volume Vd of each hole 141 is 93 fL.

[0176] (First liquid and third liquid)

[0177] The first liquid and the third liquid are liquids using water (AccuGENE Molecular Biology Grade Water (manufactured by LONZA)) as a solvent, containing 5 μg / ml Redmond Red as a fluorescent reagent, 10 mM Tris-HCl (manufactured by Nippon Gene, pH: 8.5) as a buffer substance, 6.25 mM MgCl2 (manufactured by SIGMA-ALDRICH) as a salt, and 0.1 v / v% Tween 20 (manufactured by SIGMA-ALDRICH) as a surfactant.

[0178] (Second Liquid)

[0179] The second liquid is a liquid using water (AccuGENE Molecular Biology Grade Water (manufactured by LONZA)) as a solvent, containing 1 μM fluorescein isothiocyanate (hereinafter referred to as FITC) as a fluorescent reagent, 10 mM Tris-HCl (manufactured by Nippon Gene, pH: 8.5) as a buffer substance, 6.25 mM MgCl2 (manufactured by SIGMA-ALDRICH) as a salt, and 0.1 mass% Tween 20 (manufactured by SIGMA-ALDRICH) as a surfactant.

[0180] (Sealing fluid)

[0181] Oil (Fluorinert FC-40, manufactured by SIGMA-ALDRICH) was used as the sealing liquid.

[0182] (Microscope observation)

[0183] The apparatus used was as follows.

[0184] Microscope: BZ-800 (manufactured by Keyence)

[0185] Objective lens: CFIPlan-Apochromat Lambda10X (made by Nikon)

[0186] Filter 1: BZ-X filter TexasRed (manufactured by Keyence)

[0187] Filter 2: BZ-X filter GFP (manufactured by Keyence)

[0188] [Example 1]

[0189] (Introduction of First Liquid)

[0190] 20 μL of the first liquid was injected from the introduction port 122 of the fluid device.

[0191] (Signal detection 1)

[0192] Next, an image was taken using a fluorescence microscope from the opposite side of the substrate 110 of the fluidic device 100 to the side on which the hole array 140 was formed, with the hole 141 in focus. At this time, the excitation light was irradiated from the opposite side of the substrate 110 to the side on which the hole array 140 was formed. A fluorescence image was taken with an exposure time of 1 / 4 second using filter 1, and a bright field image of the same field of view was also taken.

[0193] Fig.12 The image of (a) is an observed image of signal detection 1. The upper part of the image is a bright field image, and the lower part is a fluorescent image. As a result, fluorescence was observed in the entire surface of the field of view.

[0194] (Introduction of sealing liquid)

[0195] 100 μL of the sealing liquid is injected from the inlet 122 of the fluid device. As a result, the holes 141 are sealed respectively to form micro-partitions 142 .

[0196] (Signal detection 2)

[0197] Next, an image was taken using a fluorescence microscope from the opposite side of the substrate 110 of the fluidic device 100 to the side on which the hole array 140 was formed, with the hole 141 in focus. At this time, the excitation light was irradiated from the opposite side of the substrate 110 to the side on which the hole array 140 was formed. A fluorescence image was taken with an exposure time of 5 seconds using filter 1, and a bright field image of the same field of view was also taken.

[0198] Fig.12 The image (b) is an observation image of signal detection 2. The upper part of the image is a bright field image, and the lower part is a fluorescent image. As a result, fluorescent signals corresponding to the configuration of the micro-partitions 142 are observed. That is, it is confirmed that the first liquid is contained in a plurality of micro-partitions 142, respectively.

[0199] (Introduction of Second Liquid)

[0200] 20 μL of the second liquid was injected from the introduction port 122 of the fluid device.

[0201] (Signal detection 3)

[0202] Next, an image was taken using a fluorescence microscope from the surface opposite to the surface on which the hole array 140 was formed of the substrate 110 of the fluidic device 100, with the hole 141 in focus. At this time, the excitation light was irradiated from the surface opposite to the surface on which the hole array 140 was formed of the substrate 110. The fluorescence image was taken with an exposure time of 5 seconds using filter 1, and with an exposure time of 1 / 40 seconds using filter 2, and a bright field image of the same field of view was also taken.

[0203] Fig.13 The image of (a) is an observation image of signal detection 3. The upper part of the image is a bright field image, the middle part is a fluorescence image of Redmond Red, and the lower part is a fluorescence image of FITC. As a result, no fluorescence of Redmond Red was observed, and fluorescence of FITC was observed on the entire surface.

[0204] (Introduction of sealing liquid)

[0205] 100 μL of the sealing liquid is injected from the introduction port 122 of the fluid device. As a result, the holes 141 are resealed respectively to form micro-partitions 142 .

[0206] (Signal detection 4)

[0207] Next, an image was taken using a fluorescence microscope from the surface opposite to the surface on which the hole array 140 was formed of the substrate 110 of the fluidic device 100, with the hole 141 in focus. At this time, the excitation light was irradiated from the surface opposite to the surface on which the hole array 140 was formed of the substrate 110. The fluorescence image was taken with an exposure time of 5 seconds using filter 1, and with an exposure time of 1.5 seconds using filter 2, and a bright field image of the same field of view was also taken.

[0208] Fig.13 The image (b) is an observation image of signal detection 4. The upper part of the image is a bright field image, the middle part is a fluorescent image of Redmond Red, and the lower part is a fluorescent image of FITC. As a result, no fluorescent signal was observed in the fluorescent image of Redmond Red, and a fluorescent signal corresponding to the configuration of the micro-partition 142 was observed in the fluorescent image of FITC.

[0209] [Example 2]

[0210] The experiment was conducted under the same conditions as Example 1, except that the second liquid did not contain Tween 20.

[0211] Fig.14 The image of (a) is an observed image of signal detection 1. The upper part of the image is a bright field image, and the lower part is a fluorescent image. As a result, fluorescence was observed in the entire field of view.

[0212] Fig.14 The image (b) is an observation image of signal detection 2. The upper part of the image is a bright field image, and the lower part is a fluorescent image. As a result, fluorescent signals corresponding to the configuration of the micro-partitions 142 are observed. That is, it is confirmed that the first liquid is contained in a plurality of micro-partitions 142, respectively.

[0213] Fig.15 The image of (a) is an observation image of signal detection 3. The upper part of the image is a bright field image, the middle part is a fluorescence image of Redmond Red, and the lower part is a fluorescence image of FITC. As a result, no fluorescence of Redmond Red was observed, and fluorescence of FITC was observed on the entire surface.

[0214] Fig.15 The image (b) is an observation image of signal detection 4. The upper part of the image is a bright field image, the middle part is a fluorescent image of Redmond Red, and the lower part is a fluorescent image of FITC. As a result, no fluorescent signal was observed in the fluorescent image of Redmond Red, and a fluorescent signal corresponding to the configuration of the micro-partition 142 was observed in the fluorescent image of FITC.

[0215] [Comparative Example 1]

[0216] The experiment was conducted under the same conditions as Example 1, except that the first liquid did not contain Tween 20.

[0217] [Comparative Example 2]

[0218] The experiment was conducted under the same conditions as in Example 1, except that the first liquid and the second liquid did not contain Tween 20.

[0219] Fig.16 The image of (a) is an observed image of signal detection 1 of Comparative Example 1. Fig.17 The image of (a) is an observed image of signal detection 1 of Comparative Example 2. The upper part of the image is a bright field image, and the lower part is a fluorescent image. As a result, fluorescence was observed over the entire surface of the field of view.

[0220] Fig.16 The image of (b) is an observed image of the signal detection 2 of Comparative Example 1. Fig.17 The image (b) is an observed image of the signal detection 2 of the comparative example 2. The upper part of the image is a bright field image, and the lower part is a fluorescent image. As a result, a fluorescent signal corresponding to the configuration of the micro-partitions 142 was observed. That is, it was confirmed that the first liquid was contained in a plurality of micro-partitions 142, respectively.

[0221] Fig.18 The image of (a) is an observed image of signal detection 3 of Comparative Example 1. Fig.19Image (a) is an observed image of signal detection 3 of Comparative Example 2. The upper part of the image is a bright field image, the middle part is a fluorescent image of Redmond Red, and the lower part is a fluorescent image of FITC. As a result, a fluorescent signal of Redmond Red corresponding to the configuration of the micro-partitions 142 was observed. That is, it was confirmed that the first liquid was contained in a plurality of micro-partitions 142, respectively. Furthermore, a fluorescent signal of FITC was observed on the entire surface.

[0222] Fig.18 The image of (b) is an observed image of the signal detection 4 of Comparative Example 1. Fig.19 The image (b) is an observed image of the signal detection 4 of Comparative Example 2. The upper part of the image is a bright field image, the middle part is a fluorescent image of Redmond Red, and the lower part is a fluorescent image of FITC. As a result, a fluorescent signal of Redmond Red corresponding to the configuration of the micro-partitions 142 was observed. That is, it was confirmed that the first liquid was contained in a plurality of micro-partitions 142, respectively. No fluorescent signal of FITC was observed.

[0223] From the results of Examples 1 and 2 and Comparative Examples 1 and 2, it can be seen that when the first liquid contains a surfactant, the first liquid can be replaced by the second liquid even if it is separately contained by the sealing liquid. At this time, it can be seen that regardless of whether the second liquid contains a surfactant, the second liquid can be used to replace the inside of the hole 141. Furthermore, it can be seen that even if the second liquid contains a surfactant, when the first liquid does not contain a surfactant, the inside of the hole 141 cannot be replaced by the second liquid.

[0224] [Example 3]

[0225] The process from (introduction of the first liquid) to (signal detection 4) was performed in the same order as in Example 1. Thereafter, the following operation was performed.

[0226] (Introduction of the Third Liquid)

[0227] 20 μL of the third liquid was injected from the introduction port 122 of the fluid device.

[0228] (Signal detection 5)

[0229] The fluorescence microscope was used to capture the image from the opposite side of the substrate 110 of the fluidic device 100 to the side on which the hole array 140 was formed, with the hole 141 in focus. At this time, the excitation light was irradiated from the opposite side of the substrate 110 to the side on which the hole array 140 was formed. The fluorescence image was captured with an exposure time of 1 / 4 second using filter 1, and with an exposure time of 1.5 seconds using filter 2, and a bright field image of the same field of view was also captured.

[0230] (Introduction of sealing liquid)

[0231] 100 μL of the sealing liquid is injected from the inlet 122 of the fluid device. As a result, the holes 141 are resealed respectively to form micro-partitions 142 .

[0232] (Signal detection 6)

[0233] Next, an image was taken using a fluorescence microscope from the surface opposite to the surface on which the hole array 140 was formed of the substrate 110 of the fluidic device 100, with the hole 141 in focus. At this time, the excitation light was irradiated from the surface opposite to the surface on which the hole array 140 was formed of the substrate 110. The fluorescence image was taken with an exposure time of 5 seconds using filter 1, and with an exposure time of 1.5 seconds using filter 2, and a bright field image of the same field of view was also taken.

[0234] Fig. 20 The image of (a) is the observation image of signal detection 1. Fig. 20 The image of (b) is an observation image of signal detection 2. The upper part of the image is a bright field image, and the lower part is a fluorescence image.

[0235] Fig.21 The image of (a) is the observation image of signal detection 3. Fig.21 The image of (b) is an observation image of signal detection 4. The upper part of the image is a bright field image, the middle part is a fluorescent image of Redmond Red, and the lower part is a fluorescent image of FITC.

[0236] Depend on Fig. 20 and Fig.21 The results show that the same results as in Example 1 were obtained.

[0237] Fig. 22 The image of (a) is an observation image of the signal detection 5. Fig. 22 The image of (b) is an observation image of signal detection 4. The upper part of the image is a bright field image, the middle part is a fluorescent image of Redmond Red, and the lower part is a fluorescent image of FITC.

[0238] like Fig. 22 As shown in the image (a), the fluorescence signal of Redmond Red was observed on the entire surface. However, the fluorescence signal of FITC was not observed. Fig. 22 As shown in the image of (b), a fluorescence signal of Redmond Red was observed corresponding to the arrangement of the micro-partitions 142. That is, it was confirmed that the third liquid was contained in each of the plurality of micro-partitions 142. However, a fluorescence signal of FITC was not observed.

[0239] [Example 4]

[0240] The experiment was conducted under the same conditions as Example 3, except that the second liquid did not contain Tween 20.

[0241] Fig.23 The image of (a) is the observation image of signal detection 1. Fig.23 The image of (b) is an observation image of signal detection 2. The upper part of the image is a bright field image, and the lower part is a fluorescence image.

[0242] Fig.24 The image of (a) is the observation image of signal detection 3. Fig.24 The image of (b) is an observation image of signal detection 4. The upper part of the image is a bright field image, the middle part is a fluorescent image of Redmond Red, and the lower part is a fluorescent image of FITC.

[0243] Depend on Fig.23 and Fig.24 The results show that the same results as in Example 2 were obtained.

[0244] Fig.25 The image of (a) is an observation image of the signal detection 5. Fig.25 The image of (b) is an observation image of signal detection 4. The upper part of the image is a bright field image, the middle part is a fluorescent image of Redmond Red, and the lower part is a fluorescent image of FITC.

[0245] like Fig.25 As shown in the image (a), the fluorescence signal of Redmond Red was observed on the entire surface. However, the fluorescence signal of FITC was not observed. Fig.25 As shown in the image of (b), a fluorescence signal of Redmond Red was observed corresponding to the arrangement of the micro-partitions 142. That is, it was confirmed that the third liquid was contained in each of the plurality of micro-partitions 142. However, a fluorescence signal of FITC was not observed.

[0246] From this result, it can be seen that even if the introduction of the liquid containing the surfactant (the first liquid in this embodiment), the sealing using the sealing liquid, and the introduction of the final replacement liquid (the third liquid in this embodiment) are not performed continuously, the final replacement liquid can be contained in the hole. In other words, it can be seen that even if the liquid contained immediately before the final replacement liquid (the third liquid in this embodiment) (the second liquid in this embodiment) does not contain a surfactant, as long as the liquid containing the surfactant (the first liquid in this embodiment) is contained in the hole 141 before the final replacement liquid, the final replacement liquid can be contained in the hole 141.

[0247] The reason for this is that the surfactant is introduced into the inside of the hole 141 by the introduction of the first liquid, and then even if the inside of the hole 141 is replaced with the second liquid that does not contain the surfactant, the surfactant remains in the hole 141 or adheres to the inner wall of the hole 141. Therefore, it is considered that when the third liquid is introduced, the inside of the hole 141 is replaced with the third liquid by the action of the surfactant remaining in the inside of the hole 141.

[0248] [Comparative Example 3]

[0249] The experiment was conducted under the same conditions as Example 3, except that the first liquid did not contain Tween 20.

[0250] Fig.26 The image of (a) is the observation image of signal detection 1. Fig.26 The image (b) is the observation image of signal detection 2. The upper part of the image is the bright field image, and the lower part is the fluorescence image. Fig.26 As can be seen from the results, the same results as those in Comparative Example 1 were obtained.

[0251] Fig. 27 The image of (a) is the observation image of signal detection 3. Fig. 27 The image (b) is an observation image of signal detection 4. The upper part of the image is a bright field image, the middle part is a Redmond Red fluorescence image, and the lower part is a FITC fluorescence image. Fig. 27 As can be seen from the image of (a), the same result as that of Comparative Example 1 was obtained. Fig. 27 In the image (b), in the Redmond Red fluorescence image, there is a black hollow portion corresponding to the configuration of the micro-partition 142. The black hollow portion is consistent with the portion where the fluorescence signal is observed in the FITC fluorescence image, so it can be considered that only a part of the micro-partition 142 is replaced by the second liquid. As can be seen, when the first liquid does not contain a surfactant, the second liquid cannot reliably replace the inside of the micro-partition 142.

[0252] Fig.28 The image of (a) is an observation image of the signal detection 5. Fig.28 The image of (b) is an observation image of signal detection 4. The upper part of the image is a bright field image, the middle part is a fluorescent image of Redmond Red, and the lower part is a fluorescent image of FITC.

[0253] like Fig.28 As shown in the image (a), the fluorescence signal of Redmond Red was observed on the entire surface. However, the fluorescence signal of FITC was not observed. Fig.28As shown in the image (b), the fluorescence signal of Redmond Red corresponding to the configuration of the micro-partitions 142 was observed. That is, it was confirmed that the third liquid was contained in a plurality of micro-partitions 142. However, the fluorescence signal of FITC was not observed. That is, when the second liquid contains a surfactant, the second liquid contained in the micro-partitions 142 is replaced by the third liquid.

[0254] [Example 5]

[0255] The experiment was conducted under the same conditions as in Example 1 except that the material of the cover member 120 of the fluid device was changed to glass.

[0256] [Example 6]

[0257] The experiment was conducted under the same conditions as in Example 1 except that the material of the cover member 120 of the fluid device was changed to polypropylene (manufactured by AS ONE, model number: PPN-051001).

[0258] [Example 7]

[0259] The experiment was conducted under the same conditions as in Example 1 except that the material of the cover member 120 of the fluid device was changed to silicon (manufactured by Togawa Rubber Co., Ltd., model: K-125(50)).

[0260] [Comparative Example 4]

[0261] The experiment was conducted under the same conditions as in Example 5, except that the first liquid and the second liquid did not contain a surfactant.

[0262] [Comparative Example 5]

[0263] The experiment was conducted under the same conditions as in Example 6, except that the first liquid and the second liquid did not contain a surfactant.

[0264] [Comparative Example 6]

[0265] The experiment was conducted under the same conditions as in Example 7, except that the first liquid and the second liquid did not contain a surfactant.

[0266] Fig.29 The image of (a) is an observed image of signal detection 1 of Example 5. Fig.29 The image (b) is the observation image of signal detection 2 of Example 5. Fig.31 The image of (a) is an observed image of the signal detection 1 of Example 6. Fig.31 The image of (b) is the observation image of signal detection 2 of Example 6. Fig.33 The image of (a) is an observed image of signal detection 1 of Example 7. Fig.33The image of (b) is an observed image of signal detection 2 of Example 7. The upper part of the image is a bright field image, and the lower part is a fluorescent image.

[0267] Fig.30 The image of (a) is an observation image of signal detection 3 of Example 5. Fig.30 The image (b) is the observation image of the signal detection 4 of Example 5. Fig.32 The image of (a) is an observation image of signal detection 3 of Example 6. Fig.32 The image of (b) is the observation image of the signal detection 4 of Example 6. Fig.34 The image of (a) is an observed image of signal detection 3 of Example 7. Fig.34 The image of (b) is an observation image of the signal detection 4 of Example 7. The upper part of the image is a bright field image, the middle part is a fluorescent image of Redmond Red, and the lower part is a fluorescent image of FITC.

[0268] Depend on Figure 29 to Figure 34 It can be seen from the results that Examples 5 to 7 obtained the same results as Example 1. That is, even if the material of the cover member 120 of the fluid device is glass, polypropylene, or silicon, when the first liquid contains a surfactant, the micro-partitions 142 that respectively contain the first liquid through the sealing liquid can be replaced by the second liquid.

[0269] Fig.35 The image of (a) is an observed image of the signal detection 1 of Comparative Example 4. Fig.35 The image of (b) is an observed image of signal detection 2 of Comparative Example 4. Fig.37 The image of (a) is an observed image of the signal detection 1 of Comparative Example 5. Fig.37 The image of (b) is an observed image of signal detection 2 of Comparative Example 5. Fig.39 The image of (a) is an observed image of the signal detection 1 of Comparative Example 6. Fig.39 The image of (b) is an observed image of signal detection 2 of Comparative Example 6. The upper part of the image is a bright field image, and the lower part is a fluorescent image.

[0270] Fig.36 The image of (a) is an observed image of signal detection 3 of Comparative Example 4. Fig.36 The image of (b) is an observed image of the signal detection 4 of the comparative example 4. Fig.38 The image of (a) is an observed image of signal detection 3 of Comparative Example 5. Fig.38 The image of (b) is an observed image of the signal detection 4 of Comparative Example 5. Fig.40 The image of (a) is an observed image of signal detection 3 of Comparative Example 6. Fig.40The image of (b) is an observed image of the signal detection 4 of Comparative Example 6. The upper part of the image is a bright field image, the middle part is a fluorescent image of Redmond Red, and the lower part is a fluorescent image of FITC.

[0271] Depend on Figures 35 to 40 As can be seen from the results, Comparative Examples 4 to 6 obtained the same results as Comparative Example 2. That is, even if the material of the cover member 120 of the fluid device is glass, polypropylene, or silicon, when the first liquid does not contain a surfactant, the micro-partitions 142 that respectively contain the first liquid through the sealing liquid cannot be replaced by the second liquid.

[0272] Industrial Applicability

[0273] According to the present invention, a technique for replacing the liquid inside each of the sealed micro-partitions can be provided.

[0274] Explanation of symbols

[0275] 100, 200 fluid device, 110 substrate, 111 a surface, 120 cover member, 121 protrusion, 122 inlet, 123 outlet, 130 flow path, 140 hole array, 141 hole, 142 micro partition (sealed hole), 210 wall member, L110 first liquid, L120 first sealing liquid, L620 second sealing liquid, L410 second liquid.

Claims

1. A method for introducing a second liquid into a hole, comprising the step of introducing the second liquid on a surface of a fluid device, The fluid device includes a substrate and a plurality of holes that are open on one surface of the substrate and contain a first liquid containing a surfactant, a first sealing liquid is stacked on the one surface, and openings of the plurality of holes are sealed by the first sealing liquid. As a result of introducing the second liquid onto one surface of the fluid device, the second liquid washes away the first sealing liquid, thereby releasing the seal of the plurality of holes and leaving the holes unsealed. The second liquid replaces the first sealing liquid and is introduced into the holes.

2. The method according to claim 1, wherein: The specific gravity of the second liquid is greater than the specific gravity of the first sealing liquid.

3. The method according to claim 1 or 2, wherein: After the second liquid is introduced, the method further comprises the step of introducing a second sealing liquid into the fluid device. As a result, the second sealing liquid is stacked on the one surface to seal the openings of the plurality of holes, and the second liquid or a mixture of the first liquid and the second liquid is sealed in the holes.

4. The method according to claim 3, wherein: The specific gravity of the second sealing liquid is greater than the specific gravity of the second liquid.

5. The method according to claim 1 or 2, wherein: Further including: the step of introducing the first liquid into the fluidic device before introducing the second liquid; and The first sealing liquid is laminated on the one surface to seal the openings of the plurality of holes, and the first sealing liquid is introduced into the fluid device so that the first liquid is sealed inside the holes.

6. The method according to claim 1 or 2, wherein: The first liquid and the second liquid are miscible.

7. The method according to claim 1 or 2, wherein: The fluid device further includes a cover member disposed facing the one surface, A space between the cover member and the one surface forms a flow path.

8. The method according to claim 7, wherein: The second liquid is introduced into the fluid device through the flow path.

9. The method according to claim 1 or 2, wherein: The first liquid and the second liquid contain a reaction reagent.

10. The method according to claim 1 or 2, wherein: After the second liquid is introduced into the inside of the hole, at least a part of the components contained in the first liquid is retained in the inside of the hole.

11. The method according to claim 10, wherein: At least a part of the components contained in the first liquid is held in the pores by being held by the carrier.

12. The method according to claim 1 or 2, wherein: The affinity between the one surface and the first sealing liquid is equal to or lower than the affinity between the one surface and the second liquid.

13. The method according to claim 12, wherein: The material of the one surface is cycloolefin polymer, the main components of the first liquid and the second liquid are water, and the first sealing liquid is fluorine-based oil.

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