How to introduce liquid into a well

Abstract

To provide a technique of replacing liquid in the inside of a small section individually sealed.SOLUTION: The method includes introducing a second liquid on one surface of a fluid device including a substrate and a plurality of wells opening in one surface of the substrate and storing a first liquid containing a surface-active agent in the inside, the first sealing liquid being laminated on one surface and the opening parts of the wells being sealed by the first sealing liquid. The method introduces the second liquid into the wells in such a manner that the second liquid is thus introduced in the inside of the wells instead of the first sealing liquid.SELECTED DRAWING: Figure 4

Description

Technical Field

[0006]

[0001] The present invention relates to a method for introducing a liquid into a well. This application claims priority from Japanese Patent Application No. 2019-101305 filed in Japan on May 30, 2019, the content of which is incorporated herein by reference.

Background Art

[0002] By quantitatively detecting a target molecule in a biological sample, early detection of a disease and prediction of the effect of medication are carried out. Conventionally, protein quantification has been performed by enzyme-linked immunosorbent assay (ELISA) or the like, and nucleic acid quantification has been performed by real-time PCR method or the like.

[0003] In recent years, for the purpose of detecting a disease earlier, there has been an increasing need to detect a target molecule more accurately. As a technique for accurately detecting a target molecule, for example, Patent Document 1, Patent Document 2, Non-Patent Document 1, etc. describe techniques for performing an enzyme reaction in a large number of microcompartments. These techniques are called digital measurement.

[0004] In digital measurement, a sample solution is divided into an extremely large number of microcompartments. Then, the signal from each microcompartment is binarized, and only whether the target molecule is present or not is discriminated to measure the number of molecules of the target molecule. According to digital measurement, the detection sensitivity and quantification can be significantly improved as compared with conventional ELISA, real-time PCR method, etc.

[0005] Digital measurement technology is not limited to use in the above diagnostic applications, but is widely used in applications for analyzing a large number of target molecules separately for each molecule. For example, Non-Patent Document 2, etc. describe its use in functional analysis of transmembrane proteins.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

[0007] [Non-Patent Document 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. [Non-Patent Document 2] Rikiya Watanabe, et al., High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition., Scientific Reports volume 4, Article number: 7076, 2014. [Overview of the project] [Problems that the invention aims to solve]

[0008] In digital measurement techniques, target molecules mixed with reaction reagents are divided into numerous microcompartments using a sealing solution or lipid bilayer, and then the reaction is carried out individually within each microcompartment to detect the target molecules. Therefore, typically, only analytical results corresponding to the reaction reagents contained within each microcompartment can be obtained. Thus, the present invention aims to provide a technique for replacing the liquid inside individually sealed microcompartments. [Means for solving the problem]

[0009] The present invention includes the following embodiments. [1] A method for introducing a second liquid into a well, comprising: a substrate; a plurality of wells opening on one side of the substrate and containing a first liquid containing a surfactant, wherein a first sealing liquid is laminated on the one side of the fluid device and the openings of the plurality of wells are sealed by the first sealing liquid, wherein the second liquid is introduced into the wells, thereby replacing the first sealing liquid. The introduction method wherein the concentration of the surfactant is 0.001 v / v% or more and 1.0 v / v% or less relative to the total volume of the first liquid. . [2] The method according to [1], further comprising introducing a second sealing liquid into the fluid device after introducing the second liquid, such that the second sealing liquid is deposited on one side to seal the openings of the plurality of wells, and the second liquid, or a mixture of the first liquid and the second liquid, is sealed inside the wells. [3] The method according to [1] or [2], further comprising introducing the first liquid into the fluid device before introducing the second liquid, and introducing the first sealing liquid into the fluid device such that the first sealing liquid is laminated on one side to seal the openings of the plurality of wells and the first liquid is sealed inside the wells. [4] The method according to any one of [1] to [3], wherein the first liquid and the second liquid are miscible. [5] The method according to any one of [1] to [4], wherein the fluid device further comprises a cover member disposed opposite to the one surface, and the space between the cover member and the one surface forms a flow path. [6] The method according to [5], wherein the second liquid is introduced into the fluid device through the flow path. [7] The method according to any one of [1] to [6], wherein the first liquid and the second liquid are reaction reagents. [8] The method according to any one of [1] to [7], wherein, after the second liquid is introduced into the well, at least a portion of the components contained in the first liquid is retained inside the well. [9] The method according to [8], wherein at least a portion of the components contained in the first liquid is held inside the well by being held on a carrier.

[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.

[11] The method according to

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

Advantages of the Invention

[0010] According to the present invention, it is possible to provide a technique for replacing the liquid inside individually sealed microcompartments.

Brief Description of the Drawings

[0011] [Figure 1] It is a schematic cross-sectional view showing an example of a fluid device. [Figure 2] It is a schematic cross-sectional view showing an example of a fluid device. [Figure 3] It is a schematic cross-sectional view showing an example of a fluid device. [Figure 4] It is a schematic cross-sectional view showing an example of a fluid device. [Figure 5] It is a schematic cross-sectional view showing an example of a fluid device. [Figure 6] It is a schematic cross-sectional view showing an example of a fluid device. [Figure 7] It is a schematic cross-sectional view showing an example of a fluid device. [Figure 8] It is a schematic cross-sectional view showing an example of a fluid device. [Figure 9] It is a schematic cross-sectional view showing an example of a fluid device. [Figure 10] It is a schematic cross-sectional view showing an example of a fluid device. [Figure 11] It is a schematic cross-sectional view showing an example of a fluid device. [Figure 12] [[ID=第51]]It is a microscopic image showing the results of Example 1. ; [Figure 13] It is a microscopic image showing the results of Example 1. [Figure 14]This is a microscopic image showing the results of Example 2. [Figure 15] This is a microscopic image showing the results of Example 2. [Figure 16] This is a microscopic image showing the results of Comparative Example 1. [Figure 17] This is a microscope image showing the results of Comparative Example 2. [Figure 18] This is a microscopic image showing the results of Comparative Example 1. [Figure 19] This is a microscope image showing the results of Comparative Example 2. [Figure 20] This is a microscopic image showing the results of Example 3. [Figure 21] This is a microscopic image showing the results of Example 3. [Figure 22] This is a microscopic image showing the results of Example 3. [Figure 23] This is a microscopic image showing the results of Example 4. [Figure 24] This is a microscopic image showing the results of Example 4. [Figure 25] This is a microscopic image showing the results of Example 4. [Figure 26] This is a microscopic image showing the results of Comparative Example 3. [Figure 27] This is a microscopic image showing the results of Comparative Example 3. [Figure 28] This is a microscopic image showing the results of Comparative Example 3. [Figure 29] This is a microscopic image showing the results of Example 5. [Figure 30] This is a microscopic image showing the results of Example 5. [Figure 31] This is a microscopic image showing the results of Example 6. [Figure 32] This is a microscopic image showing the results of Example 6. [Figure 33] This is a microscopic image showing the results of Example 7. [Figure 34] This is a microscopic image showing the results of Example 7. [Figure 35] This is a microscopic image showing the results of Comparative Example 4. [Figure 36]This is a microscopic image showing the results of Comparative Example 4. [Figure 37] This is a microscopic image showing the results of Comparative Example 5. [Figure 38] This is a microscopic image showing the results of Comparative Example 5. [Figure 39] This is a microscopic image showing the results of Comparative Example 6. [Figure 40] This is a microscopic image showing the results of Comparative Example 6. [Modes for carrying out the invention]

[0012] The embodiments of the present invention will be described in detail below, with reference to the drawings as appropriate. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and redundant explanations are omitted. Note that the dimensional ratios in each figure are exaggerated for illustrative purposes and do not necessarily correspond to the actual dimensional ratios.

[0013] [How to introduce liquid into a well] One embodiment of the present invention provides a method for introducing a second liquid into a well, comprising introducing a second liquid onto one surface of a fluid device, the fluid device comprising a substrate and a plurality of wells opening on one surface of the substrate and containing a first liquid containing a surfactant inside, wherein a first sealing liquid is laminated on the one surface and the openings of the plurality of wells are sealed by the first sealing liquid, so that the second liquid replaces the first sealing liquid and is introduced into the well.

[0014] According to the method of this embodiment, the liquid inside individually sealed microcompartments can be replaced. This makes it possible, for example, in digital measurement technology to obtain analytical results using a reaction reagent contained in a first liquid contained inside a microcompartment, then replace the liquid inside the microcompartment with a second liquid, and perform a different analysis using the reaction reagent contained in the second liquid.

[0015] (Fluid devices) First, a fluid device that can be used in the method of this embodiment will be described. Figure 1 is a schematic cross-sectional view showing an example of a fluid device. As shown in Figure 1, the fluid device 100 comprises a substrate 110 and a cover member 120 positioned opposite one surface 111 of the substrate 110. The cover member 120 has a protrusion 121. The tip of the protrusion 121 is in contact with the substrate 110. In the fluid device 100, a plurality of wells 141 are integrally molded on one surface 111 of the substrate 110, forming a well array 140. One surface 111 faces the cover member 120. The cover member 120 may be welded or bonded to the substrate 110.

[0016] The wells 141 open to the surface of the substrate 110. The shape, dimensions, and arrangement of the wells 141 are not particularly limited, but it is preferable that one target molecule is introduced into each well 141. The wells 141 are preferably small wells with a small volume. For example, the volume of one well 141 may be about 10 fL to 100 pL. In the fluid device 100, multiple wells 141 of the same shape and size constitute a well array 140. By "same shape and size," they should be identical in shape and volume to the extent required for digital measurement, and variations within the range of manufacturing tolerances are acceptable.

[0017] The diameter of the well 141 may be, for example, about 1 to 30 μm. The depth of the well 141 may also be, for example, about 1 to 30 μm. Furthermore, the arrangement of the wells 141 is not particularly limited; for example, they may be arranged in a triangular lattice, a square lattice, or randomly.

[0018] In the fluid device 100, the presence of the protrusion 121 creates a space between one surface 111 and the cover member 120. This 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, which will be 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.

[0019] The shape, structure, and capacity of the flow path 130 are not particularly limited, but the height of the flow path 130 (i.e., 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, 100 μm or less.

[0020] The protrusion 121 may be molded integrally with the cover member 120. The cover member 120 can be formed into a plate shape having the protrusion 121 by, for example, molding a fluid of thermoplastic resin using a mold. The cover member 120 may also have a reagent introduction port 122 and an discharge port 123 formed therein.

[0021] If the cover member 120 has a protrusion 121, the cover member 120 and the substrate 110 are stacked such that the protrusion 121 contacts the surface 111 of the substrate 110 where the well 141 opens. As a result, the space between the cover member 120 and the substrate 110 becomes a flow path 130. The cover member 120 and the substrate 110 may be welded together by laser welding or the like.

[0022] (Modified example 1 of a fluid device) The fluid device used in the method of this embodiment is not limited to the fluid device 100 described above. Figure 7 is a schematic cross-sectional view showing an example of a fluid device. As shown in Figure 7, the fluid device 200 comprises a substrate 110 and a wall member 210. In the fluid device 200, the well array 140 is integrally molded with the substrate 110 on one side 111 of the substrate 110. The well array 140 has a plurality of wells 141.

[0023] The fluid device 200 differs from the fluid device 100 described above mainly in that it does not have a cover member 120. Therefore, the fluid device 200 does not have a flow path.

[0024] (Variation 2 of the fluid device) In the fluid device 100 described above, the cover member 120 and the protrusion 121 are integrally molded. However, the cover member 120 and the protrusion 121 may be molded as separate parts.

[0025] Furthermore, in the fluid devices 100 and 200 described above, the well array 140 was integrally molded with the substrate 110 on one surface 111 of the substrate 110. However, the well array does not have to be integrally molded with the substrate 110. For example, the well array 140, which is molded separately from the fluid device, may be placed on the substrate 110 of the fluid device. Alternatively, a resin layer may be laminated on the surface of the substrate 110, and the well array may be formed in the resin layer by etching or the like.

[0026] (Materials for fluid devices) The substrate 110 is formed using, for example, a resin. The type of resin is not particularly limited, but it is preferable that the resin is resistant to the first liquid, the second liquid, and the sealing liquid. Furthermore, if the signal to be detected is fluorescence, it is preferable that the resin has low autofluorescence. Examples of resins include, but are not limited to, cycloolefin polymers, cycloolefin copolymers, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesins, and amorphous fluororesins.

[0027] Multiple wells 141 may be formed on one side 111 of the substrate 110 in the thickness direction. Methods for forming wells using resin include injection molding, thermal imprinting, and optical imprinting.

[0028] Alternatively, for example, a fluororesin may be laminated on the substrate 110, and the fluororesin may be processed by etching or the like to form a well array. As the fluororesin, for example, CYTOP® (Asahi Glass Co., Ltd.) can be used.

[0029] Furthermore, if the fluid device has a cover member 120, the material of the cover member 120 is preferably a resin with low autofluorescence, and may be a thermoplastic resin such as a cycloolefin polymer or cycloolefin copolymer.

[0030] Furthermore, the cover member 120 may be made of a material that does not transmit light of wavelengths detected when observing the signal by fluorescence, or of wavelengths near that wavelength, or it may be made of a material that does not transmit light at all. For example, the cover member 120 may be made of a thermoplastic resin to which carbon or metal particles have been added.

[0031] (First Embodiment) As described above, the method of this embodiment is a method for introducing a second liquid into a well, comprising a substrate and a plurality of wells opening on one side of the substrate and containing a first liquid containing a surfactant, wherein a first sealing liquid is laminated on the one side of the fluid device and the openings of the plurality of wells are sealed by the first sealing liquid, and as a result the second liquid replaces the first sealing liquid and is introduced into the well.

[0032] The method of the first embodiment will be described using the case where the fluid device 100 is used, with reference to Figures 1 to 6 as needed.

[0033] 《Introduction of the first liquid》 First, as shown in Figure 1, the first liquid L110 is introduced from the introduction port 122 of the fluid device 100 and delivered 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 includes serum, plasma, urine, and cell culture medium. The environmental sample includes, for example, river water and factory wastewater.

[0034] Biological and environmental samples may contain target molecules for detection. Conversely, biological and environmental samples may not contain target molecules. Examples of target molecules include DNA, RNA, proteins, viruses, cells, and specific compounds. Here, RNA includes miRNA and mRNA. Cells include bacteria, yeast, animal cells, plant cells, and insect cells.

[0035] The first liquid L110 may contain a reaction reagent for detecting the target molecule. Examples of reaction reagents include buffers, enzymes, substrates, antibodies, and antibody fragments. The enzyme is selected according to the nature of the biochemical reaction, for example, if the target molecule is a nucleic acid, to carry out a biochemical reaction such as an enzymatic reaction with a template nucleic acid related to the target molecule. The biochemical reaction with the template nucleic acid is, for example, a reaction in which signal amplification occurs under the condition that the template nucleic acid is present. The reaction reagent is selected according to the detection reaction to be adopted. Specific detection reactions include the Invasive Cleavage Assay (ICA) method, the Loop-Intermediated Isothermal Amplification (LAMP) method (trademark registered), the 5'→3' nuclease method (TaqMan® method), and the fluorescent probe method.

[0036] The first liquid L110 contains a surfactant. Examples of surfactants include Triton-X100 (also known as polyethylene glycol mono-4-octylphenyl ether (n=approximately 10)), sodium dodecyl sulfate, Nonidet P-40 (also known as octylphenoxypoly(ethyleneoxy)ethanol), and Tween20 (also known as polyoxyethylene sorbitan monolaurate).

[0037] The concentration of the surfactant is preferably 0.001 v / v% to 1.0 v / v%, more preferably 0.005 v / v% to 0.5 v / v%, and even more preferably 0.01 v / v% to 0.1 v / v% 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, substitution with the second liquid L410 is easily performed. When the concentration of the surfactant is 1.0 v / v% or less relative to the total volume of the first liquid L110, the effect on the reaction in the subsequent detection of the target molecule is minimal.

[0038] The first liquid L110, delivered through channel 130, is contained within well 141. As a result, the reaction reagent, surfactant, and, if present, the target molecule are introduced into well 141.

[0039] The number of target molecules introduced into a single well 141 is not particularly limited, but preferably, one or fewer target molecules are introduced into each well 141, i.e., zero or one target molecule. This allows for detection of target molecules on a single-molecule basis, i.e., enables digital measurement. Furthermore, it is not necessary for target molecules to be introduced into all wells 141 of the well array 140.

[0040] The means of introducing the target molecule into well 141 are not particularly limited, and an appropriate method can be selected depending on the chosen target molecule. For example, one method is to allow the target molecule to settle in the fluid device (specifically in the flow path) by its own gravity and distribute it into well 141. Alternatively, a carrier (i.e., a capture material) that captures the target molecule may be used, and the capture material may be attached to the target molecule that is difficult to settle by its own gravity before being delivered. Furthermore, the efficiency of introducing the target molecule into the well can be improved by pre-immobilizing the capture material in well 141 and capturing the delivered target molecule.

[0041] The step of binding the capture to the target molecule can be performed at any point. For example, this step may be performed by bringing the target molecule and the capture into contact in the sample tube before introducing the target molecule into well 141. Alternatively, the target molecule may be introduced into the well after the capture has been introduced into well 141, and the capture and target molecule may be brought into contact in the well.

[0042] The capture substance is a substance capable of capturing the target molecule. The capture substance may be, for example, a combination of a solid phase and a substance that specifically binds to the target molecule.

[0043] Examples of solid phases include particles, films, and substrates. Furthermore, the specific binding material to the target molecule may be one type or multiple types. For example, there may be three types, four types, or five or more types.

[0044] The particles are not particularly limited and include polymer particles, magnetic particles, and glass particles. Particles that have undergone surface treatment to avoid nonspecific adsorption are preferred. Furthermore, particles having functional groups such as carboxyl groups on their surface are preferred for immobilizing specific binding substances. More specifically, JSR's product name "Magnosphere LC300" can be used as particles.

[0045] Alternatively, for example, when using a virus as the target molecule, cells to which the virus can attach (i.e., cells possessing viral receptors) may be used as the capture material.

[0046] Examples of specific binding substances in the capture include antibodies, antibody fragments, and aptamers. Examples of antibody fragments include Fab, F(ab')2, Fab', single-chain antibodies (scFv), disulfide-stabilized antibodies (dsFv), dimerized V-region fragments (Diabody), and peptides containing CDRs. The antibody may be a monoclonal antibody or a polyclonal antibody. Commercially available antibodies may also be used.

[0047] Furthermore, if the target molecule contains a sugar chain, the specific binding substance may be a lectin. Also, if the target molecule contains a lipid membrane, the specific binding substance may be a substance that binds to the lipid membrane. Examples of substances that bind to lipid membranes include hydrocarbons such as hexanediol and membrane proteins such as transmembrane proteins. Examples of membrane proteins include α-hemolysin.

[0048] Methods for immobilizing the specific binding substance on a solid phase are not particularly limited and include methods by physical adsorption, chemical bonding, methods utilizing avidin-biotin bonding, and methods utilizing the bonding of protein G or protein A to an antibody. Methods by physical adsorption include immobilizing the specific binding substance on the particle surface through hydrophobic or electrostatic interactions. Methods by chemical bonding include using a crosslinking agent. For example, if the particle surface has hydroxyl groups, the specific binding substance can be immobilized on the particle surface by reacting the carboxyl groups of the specific binding substance with a crosslinking agent to activate esterification, and then reacting the hydroxyl groups with these ester groups. Furthermore, it is preferable to provide a spacer between the specific binding substance and the particle surface so as not to hinder the target molecule recognition ability of the specific binding substance.

[0049] When introducing target molecules into well 141 using a trapping agent, it is preferable to form a conjugate between the trapping agent and the target molecule under conditions where zero or one target molecule is trapped per trapping agent. Furthermore, it is preferable that each well 141 is configured to introduce zero or one trapping agent. This enables digital measurement.

[0050] 《Introduction of sealing fluid》 Next, as shown in Figures 2 and 3, the first sealing liquid L120 is introduced from the introduction port 122 and delivered to the flow path 130.

[0051] The first sealing liquid is a liquid that can individually seal the liquids introduced into multiple wells 141 so that they do not mix with each other, thereby forming droplets (microdroplets). The first sealing liquid is preferably an oily solution, and more preferably an oil. As the oil, a fluorinated oil, a silicone oil, a hydrocarbon oil, or a mixture thereof can be used. More specifically, a product such as "FC-40" manufactured by Sigma Corporation 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.

[0052] The first sealing liquid L120 delivered to the channel 130 displaces the first liquid L110 introduced into the channel 130 that is not contained in the wells 141. As a result, the first sealing liquid L120 individually seals each of the multiple wells 141, and the wells 141 become independent reaction spaces (micro-compartments 142).

[0053] When the flow path 130 is filled with the first sealing liquid L120, any excess first sealing liquid L120 is discharged from the discharge port 123. Figure 3 shows the state in which all the wells 141 of the well array 140 are sealed with the first sealing liquid L120, and sealed wells (micro-compartments) 142 are formed.

[0054] Alternatively, by dissolving lipids in the first liquid L110, sending the first sealing liquid L120 into the flow path 130, and then sending the lipid-containing liquid again, a lipid bilayer can be formed at the opening of the well 141, and each of the multiple wells 141 can be individually sealed with the lipid bilayer to form a sealed well 142. Examples of lipids that form the lipid bilayer 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.

[0055] Detection of target molecules Here, the target molecule may be detected by reacting the reaction reagent contained in the first liquid L110. For example, a signal amplification reaction is carried out inside the sealed well 142. That is, the signal is amplified by the reaction step to an observable level so that a signal originating from the reaction reagent can be detected inside well 142. Examples of signals include fluorescence, color development, potential change, and pH change.

[0056] A signal amplification reaction is, for example, an enzymatic reaction. As an example, a signal amplification reaction is an isothermal reaction in which a fluid device 100 is maintained for a predetermined time at a constant temperature condition that yields the desired enzymatic activity, with a first liquid L110 containing an enzyme for signal amplification contained inside a well 142. Specifically, an ICA reaction can be used as the signal amplification reaction. In this case, the well 142 contains an ICA reaction reagent and a nucleic acid, which is the target molecule. As a result of the enzymatic reaction, if the target molecule is contained in well 142, a fluorescent substance is released from the quenching substance, emitting a predetermined fluorescence signal in response to excitation light. In Figure 3, the label 142R indicates a well containing the target molecule and emitting a signal.

[0057] 《Introduction of the second liquid》 Next, as shown in Figure 4, 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 well 141. Here, it is preferable 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 well 141, some of the first sealing liquid L120 may remain.

[0058] Conventionally, it has been believed that the liquid inside a well 142 sealed with a sealing liquid cannot be replaced. However, as will be described later in the examples, the inventors have shown that, by the method of this embodiment, a second liquid L410 can be introduced into the well 142 sealed with the first sealing liquid L120.

[0059] The second liquid L410 may or may not contain a surfactant. If the second liquid L410 contains a surfactant, the type and concentration of the surfactant may be the same as that contained in the first liquid L110.

[0060] The second liquid L410, like the first liquid L110, may also contain a reaction reagent for detecting the target molecule. The reaction reagent may be similar to those that can be contained in the first liquid L110, but it is preferable that it be a different reagent from that of the first liquid L110. This allows for a different reaction to be carried out than that using the reaction reagent contained in the first liquid L110.

[0061] The second liquid L410, delivered 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 on the individually sealed wells 142 are released, leaving well 141 unsealed. In addition, the second liquid L410 is introduced into well 141, which contains the first liquid L110.

[0062] In the method of the first embodiment, it is preferable that the affinity between one surface 111 and the sealing liquid L120 is about the same as the affinity between one surface 111 and the second liquid L410, or that the affinity between one surface 111 and the first sealing liquid L120 is lower than the affinity between one surface 111 and the second liquid L410. This makes it easier to release the seal of the well 142 when the second liquid L410 is introduced, as the second liquid L410 pushes away the first sealing liquid L120.

[0063] Examples of combinations in which the affinity between one surface 111 and the first sealing liquid L120 is similar to or lower than the affinity between one surface 111 and the second liquid L410 include a combination in which the material of one surface 111 is a cycloolefin polymer, the main components of the first liquid L110 and the second liquid L410 are water, and the first sealing liquid L120 is a fluorine-based oil.

[0064] In this specification, "the main component of the liquid is water" means that 50% or more by mass of the liquid is water, for example, 60% or more by mass, for example, 70% or more by mass, for example, 80% or more by mass, for example, 90% or more by mass, for example, 95% or more by mass, for example, 98% or more by mass. As the fluorine-based oil, for example, a product name "FC-40" manufactured by Sigma Corporation can be used.

[0065] Because the first liquid L110 contains a surfactant, the second liquid L410 is introduced into the unsealed well 141, and as a result, the inside of well 141 can be replaced with the second liquid L410. In other words, the first liquid L110 containing a surfactant is introduced into well 141, the first liquid L110 is sealed by the first sealing liquid L120, and then the second liquid L410 is introduced, and the inside of well 141 can be replaced with the second liquid L410.

[0066] After the second liquid L410 is introduced into the well 141, at least a portion of the components contained in the first liquid L110 may be retained inside the well 141. For example, a target molecule contained in the first liquid L110 may be bound to a capture, and the capture may be retained inside the well 141. That is, at least a portion of the components contained in the first liquid L110 may be retained inside the well 141 by being held by the capture (carrier). In this case, the second liquid L410 will be present inside the well 141 along with the capture and the target molecule.

[0067] Alternatively, for example, a target molecule contained in the first liquid L110 may be bound to the capture, and an antibody against the target molecule contained in the first liquid L110 may be bound to the target molecule, and the capture may be retained inside well 141. In this case, the second liquid L410 will be present inside well 141 along with the capture, the target molecule, and the antibody bound to the target molecule.

[0068] It is preferable that the first liquid L110 and the second liquid L410 are miscible. If the first liquid L110 and the second liquid L410 are miscible, the liquid inside the well 141 can be replaced efficiently. Here, miscible means that when the first liquid L110 and the second liquid L410 are mixed, a uniform solution or dispersion is formed without layer separation.

[0069] When the flow path 130 is filled with the second liquid L410, any excess second liquid L410 is discharged from the discharge port 123.

[0070] 《Introduction of sealing fluid》 After the step of introducing the second liquid L410, the step of introducing the second sealing liquid L120 into the fluid device 100 may be performed further. Here, the second sealing liquid may be the same as the first sealing liquid described above, or it may be different. Specifically, as shown in Figure 5, the second sealing liquid L120 may be introduced again from the introduction port 122 and sent to the flow path 130.

[0071] As a result, the second sealing liquid L120 is layered on one side 111, sealing the openings of the multiple wells 141, and the second liquid L410, or a mixture of the first liquid L110 and the second liquid L410, is sealed inside the wells 141, making each well 141 an independent reaction space (micro-compartment 142).

[0072] When the flow path 130 is filled with the second sealing liquid L120, any excess second sealing liquid L120 is discharged from the discharge port 123. Figure 6 shows the state in which all the wells 141 of the well array 140 are sealed with the second sealing liquid L120, and sealed wells (micro-compartments) 142 are formed.

[0073] Alternatively, by dissolving lipids in the second liquid L410, sending the second sealing liquid L120 into the flow path 130, and then sending the lipid-containing liquid again, a lipid bilayer can be formed at the opening of the well 141. Multiple wells 141 can then be individually sealed with this lipid bilayer, forming sealed wells 142. The lipids used to form the lipid bilayer are the same as those described above.

[0074] Detection of target molecules 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, the signal amplification reaction may be carried out inside the sealed well 142. In Figure 6, the label 142R indicates the well containing the target molecule that emitted a signal as a result of reacting with the reaction reagent.

[0075] (Second Embodiment) Next, with reference to Figures 7 to 11, the method of the second embodiment will be described using the fluid device 200 as an example. The method of the second embodiment differs from the method of the first embodiment in that the fluid device does not have a cover member. Furthermore, the first and second sealing liquids in the method of the second embodiment differ from the first and second sealing liquids in the method of the first embodiment in that they must satisfy the specific gravity conditions described later.

[0076] 《Introduction of the first liquid》 First, as shown in Figure 7, the first liquid L110 is introduced into the fluid device 200. The first liquid L110 is the same as described above. As shown in Figure 7, the first liquid L110 is contained inside the well 141. As a result, the reaction reagent and, if present, the target molecule are introduced into the well 141. The number of target molecules introduced into one well 141 is not particularly limited, but it is preferable that one or fewer, i.e., zero or one target molecule, is introduced into one well 141.

[0077] 《Introduction of sealing fluid》 Next, as shown in Figure 8, the first sealing liquid L120 is introduced into the fluid device 200. The specific gravity of the first sealing liquid L120 is greater than that of the first liquid L110. Therefore, the first sealing liquid L120 sinks below the first liquid L110 and comes into contact with one surface 111. The sealing liquid L120 then individually seals each of the multiple wells 241 containing the first liquid L110, forming independent reaction spaces (micro-compartments 142).

[0078] Detection of target molecules Here, as shown in Figure 9, a predetermined reaction may be carried out in well 142 and the resulting signal may be observed. In Figure 9, well 142R is the well in which the target molecule is contained and a signal is detected, while well 142 is the well in which the target molecule is not contained and no signal is detected.

[0079] 《Introduction of the second liquid》 Next, as shown in Figure 10, a second liquid L410 is introduced into the fluid device 200. The specific gravity of the second liquid L410 is greater than that of the first sealing liquid L120. Therefore, the second liquid L410 sinks below the first sealing liquid L120. As a result, the seal on the individually sealed well 142 is released, leaving the well 141 unsealed. The second liquid L410 is also introduced into the well 141. In well 141, it is preferable 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 well 141, some of the first sealing liquid L120 may remain.

[0080] The second liquid L410 may contain a reaction reagent for detecting the target molecule. The reaction reagent may be similar to those that can be contained in the first liquid L110 described above, but it is preferable that it be a different reagent from that of the first liquid L110. This allows for a different reaction to be carried out than that using the reaction reagent contained in the first liquid L110.

[0081] In the second embodiment, as in the first embodiment, after the second liquid L410 is introduced into the well 141, at least a portion of the components contained in the first liquid L110 may be retained inside the well 141.

[0082] 《Introduction of sealing fluid》 After the step of introducing the second liquid L410, the step of introducing the second sealing liquid L620 into the fluid device 200 may be further performed. Specifically, as shown in Figure 11, the second sealing liquid L620 is introduced into the fluid device 200. The specific gravity of the second sealing liquid L620 is greater than that of the second liquid L410. Therefore, the second sealing liquid L620 sinks below the second liquid L410 and comes into contact with one surface 111. The second sealing liquid L620 then individually seals each of the multiple wells 241 containing the second liquid L410, or a mixture of the first liquid L110 and the second liquid L410, forming independent reaction spaces (micro-compartments 142).

[0083] Figure 11 shows the state in which all of the wells 141 of the well array 140 are sealed with the second sealing liquid L620, and sealed wells (micro-compartments) 142 are formed.

[0084] Detection of target molecules 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, the signal amplification reaction may be carried out inside the sealed well 142. In Figure 11, 142R indicates the well containing the target molecule and which emitted a signal as a result of reacting with the reaction reagent.

[0085] In the method of the second embodiment, it is preferable that the specific gravity of the first sealing liquid L120 is greater than that of the first liquid L110, the specific gravity of the second liquid L410 is greater than that of the first sealing liquid L120, and the specific gravity of the second sealing liquid L620 is greater than that of the second liquid L410. Furthermore, it is preferable that the main components of the first liquid L110 and the second liquid L410 are water. In this case, for example, the specific gravity can be adjusted by including sucrose or the like in the second liquid L410. In addition, the first sealing liquid L120 and the second sealing liquid L620 can be appropriately selected from those described above that satisfy the requirements such as specific gravity.

[0086] Up to this point, an embodiment has been described in which the liquid inside the individually sealed microcompartments is replaced once. However, according to one aspect of the present invention, the liquid inside the individually sealed microcompartments can be replaced two or more times. That is, by repeatedly carrying out one aspect of the present invention, it becomes possible to replace the liquid a desired number of times.

[0087] Specifically, in the example described above, the introduction of the first liquid L110, sealing with the first sealing liquid L120, and introduction of the second liquid L410 are performed in succession. However, these steps do not necessarily have to be performed in succession. For example, between sealing with 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 well 142 by the third sealing liquid. In this case, the third liquid may or may not contain a surfactant. Even if the third liquid does not contain a surfactant, the inside of the well 141 can be replaced from a surfactant-free liquid to the second liquid L410 by introducing the second liquid L410 into the fluid device 100.

[0088] The reason for this is that when the first liquid L110 is introduced, a surfactant is introduced into the well 141, and even if the inside of the well 141 is subsequently replaced with a surfactant-free liquid, the surfactant may remain in the well 141 or adhere to the inner wall of the well 141. Therefore, when the second liquid L410 is introduced, the surfactant remaining inside the well 141 can replace the inside of the well 141 with the second liquid L410.

[0089] Furthermore, a fourth liquid may be introduced between the sealing with the third sealing liquid and the introduction of the second liquid L410, and the fourth liquid may be sealed inside the well 141 by the fourth sealing liquid. The fourth liquid may or may not contain a surfactant.

[0090] According to one aspect of the present invention, when a sample contains multiple target molecules, for example when the sample is cells, the detection of target molecules can be simplified. One example is the following aspect.

[0091] The first liquid L110, containing cells and the first reaction reagent, is introduced into well 141. It is preferable to introduce these cells into well 141 using a capture agent. The capture agent used is one that allows cells bound to the capture agent to remain in well 142 even after the introduction of the second liquid L410. For example, this could involve using a capture agent with a sufficiently large mass, or using a capture agent immobilized in well 141.

[0092] Subsequently, well 142 is obtained using the first sealing solution, 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 well 141, replacing well 141 with the second liquid. Then, the second sealing solution is introduced into the channel 130 to obtain a sealed well 142. This well 142 contains cells bound to the captured material and the second liquid L410. Subsequently, the second target molecule is detected using the second reaction reagent.

[0093] In this way, when a sample contains multiple target substances, it is possible to easily detect multiple target substances without using multiple devices. [Examples]

[0094] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0095] (Manufacturing of fluid devices) First, a fluid device having the structure shown in Figure 1 was manufactured. The fluid device was manufactured by bonding a cycloolefin polymer substrate 110, which was formed by injection molding and had a well array 140, and a cycloolefin polymer cover member 120, which was colored by adding carbon black, with double-sided tape. The double-sided tape functioned as a protrusion 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) was 100 μm. The cover member 120 also had an inlet port 122 and an outlet port 123. The diameter of the wells 141 constituting the well array 140 was 5 μm, and the depth of the wells 141 was 3 μm. The volume Vd of each well 141 was 93 fL.

[0096] (First liquid and third liquid) The first and third liquids were solvents of water (AccuGENE Molecular Biology Grade Water (LONZA)) and contained 5 μg / ml of Redmond Red as a fluorescent reagent, 10 mM Tris-HCl (Nippon Gene, pH: 8.5) as a buffer, 6.25 mM MgCl2 (SIGMA-ALDRICH) as a salt, and 0.1 v / v% Tween 20 (SIGMA-ALDRICH) as a surfactant.

[0097] (Second liquid) The second liquid was a solution containing water (AccuGENE Molecular Biology Grade Water (LONZA)) as the solvent, 1 μM fluorescein isothiocyanate (hereinafter referred to as FITC) as a fluorescent reagent, 10 mM Tris-HCl (Nippon Gene, pH: 8.5) as a buffer, 6.25 mM MgCl2 (SIGMA-ALDRICH) as a salt, and 0.1 mass% Tween 20 (SIGMA-ALDRICH) as a surfactant.

[0098] (Sealing liquid) Oil (Fluorinert FC-40, manufactured by SIGMA-ALDRICH) was used as the sealing fluid.

[0099] (Microscopic observation) The equipment used was as follows: Microscope: BZ-800 (manufactured by Keyence Corporation) Objective lens: CFI Plan Apochromatic Lambda 10X (Nikon) Filter 1: BZ-X filter Texas Red (manufactured by Keyence Corporation) Filter 2: BZ-X filter GFP (manufactured by Keyence Corporation)

[0100] [Example 1] (Introduction of the first liquid) 20 μL of the first liquid was injected through the introduction port 122 of the fluid device.

[0101] (Signal detection 1) Next, a fluorescence microscope was used to image the substrate 110 of the fluid device 100 from the side opposite to the side where the well array 140 is formed, with the focus set on the well 141. At this time, excitation light was irradiated from the side opposite to the side where the well array 140 is formed on the substrate 110. Using filter 1, a fluorescence image was taken with an exposure time of 1 / 4 second, and a bright-field image of the same field of view was also taken.

[0102] The image in Figure 12(a) is an observation image from signal detection 1. The upper part of the image is a bright-field image, and the lower part is a fluorescence image. As a result, fluorescence was observed across the entire field of view.

[0103] (Introduction of sealing liquid) 100 μL of sealing solution was injected through the introduction port 122 of the fluid device. As a result, well 141 was individually sealed, and a sealed well 142 was formed.

[0104] (Signal detection 2) Next, a fluorescence microscope was used to image the substrate 110 of the fluid device 100 from the side opposite to the side where the well array 140 is formed, with the focus set on the well 141. At this time, excitation light was irradiated from the side opposite to the side where the well array 140 is formed on the substrate 110. Using filter 1, a fluorescence image was captured with an exposure time of 5 seconds, and a bright-field image of the same field of view was also captured.

[0105] The image in Figure 12(b) is an observation image from signal detection 2. The upper part of the image is a bright-field image, and the lower part is a fluorescence image. As a result, fluorescence signals corresponding to the arrangement of wells 142 were observed. In other words, it was confirmed that the first liquid was contained individually in multiple wells 142.

[0106] (Introduction of the second liquid) A second liquid, 20 μL, was injected through the fluid device's introduction port 122.

[0107] (Signal detection 3) Next, a fluorescence microscope was used to image the wells 141 of the substrate 110 of the fluid device 100 from the side opposite to the side where the well array 140 is formed, with the focus on the wells 141. At this time, excitation light was irradiated from the side opposite to the side where the well array 140 of the substrate 110 is formed. A fluorescence image was taken using filter 1 with an exposure time of 5 seconds, and a fluorescence image was taken using filter 2 with an exposure time of 1 / 40 second, and a bright-field image of the same field of view was also taken.

[0108] The image in Figure 13(a) is an observation image from signal detection 3. The top row of the image is a bright-field image, the middle row is a fluorescence image of Redmond Red, and the bottom row is a fluorescence image of FITC. As a result, no fluorescence of Redmond Red was observed, and fluorescence of FITC was observed across the entire image.

[0109] (Introduction of sealing liquid) 100 μL of sealing solution was injected through the introduction port 122 of the fluid device. As a result, well 141 was individually resealed, and a sealed well 142 was formed.

[0110] (Signal detection 4) Next, a fluorescence microscope was used to image the wells 141 of the substrate 110 of the fluid device 100 from the side opposite to the side where the well array 140 is formed, with the focus on the wells 141. At this time, excitation light was irradiated from the side opposite to the side where the well array 140 of the substrate 110 is formed. A fluorescence image was taken using filter 1 with an exposure time of 5 seconds, and a fluorescence image was taken using filter 2 with an exposure time of 1.5 seconds, and a bright-field image of the same field of view was also taken.

[0111] The image in Figure 13(b) is the observation image of signal detection 4. The top row of the image is a bright-field image, the middle row is a fluorescence image of Redmond Red, and the bottom row is a fluorescence image of FITC. As a result, no fluorescence signal was observed in the Redmond Red fluorescence image, while a fluorescence signal corresponding to the arrangement of well 142 was observed in the FITC fluorescence image.

[0112] [Example 2] The experiment was conducted under the same conditions as in Example 1, except that the second liquid did not contain Tween 20.

[0113] The image in Figure 14(a) is an observation image from signal detection 1. The upper part of the image is a bright-field image, and the lower part is a fluorescence image. As a result, fluorescence was observed across the entire field of view.

[0114] The image in Figure 14(b) is an observation image from signal detection 2. The upper part of the image is a bright-field image, and the lower part is a fluorescence image. As a result, fluorescence signals corresponding to the arrangement of wells 142 were observed. In other words, it was confirmed that the first liquid was contained individually in multiple wells 142.

[0115] The image in Figure 15(a) is an observation image from signal detection 3. The top row of the image is a bright-field image, the middle row is a fluorescence image of Redmond Red, and the bottom row is a fluorescence image of FITC. As a result, no fluorescence of Redmond Red was observed, and fluorescence of FITC was observed across the entire image.

[0116] The image in Figure 15(b) is the observation image of signal detection 4. The top row of the image is a bright-field image, the middle row is a fluorescence image of Redmond Red, and the bottom row is a fluorescence image of FITC. As a result, no fluorescence signal was observed in the Redmond Red fluorescence image, while a fluorescence signal corresponding to the arrangement of well 142 was observed in the FITC fluorescence image.

[0117] [Comparative Example 1] The experiment was conducted under the same conditions as in Example 1, except that the first liquid did not contain Tween 20.

[0118] [Comparative Example 2] The experiment was conducted under the same conditions as in Example 1, except that the first and second liquids did not contain Tween 20.

[0119] The image in Figure 16(a) is an observation image of signal detection 1 in Comparative Example 1. The image in Figure 17(a) is an observation image of signal detection 1 in Comparative Example 2. The upper part of the image is a bright-field image, and the lower part is a fluorescence image. As a result, fluorescence was observed across the entire field of view.

[0120] The image in Figure 16(b) is an observation image of signal detection 2 in Comparative Example 1. The image in Figure 17(b) is an observation image of signal detection 2 in Comparative Example 2. The upper part of the image is a bright-field image, and the lower part is a fluorescence image. As a result, fluorescence signals corresponding to the arrangement of wells 142 were observed. In other words, it was confirmed that the first liquid was contained individually in multiple wells 142.

[0121] The image in Figure 18(a) is an observation image of signal detection 3 in Comparative Example 1. The image in Figure 19(a) is an observation image of signal detection 3 in Comparative Example 2. The top row of the image is a bright-field image, the middle row is a fluorescence image of Redmond Red, and the bottom row is a fluorescence image of FITC. As a result, a fluorescence signal of Redmond Red corresponding to the arrangement of wells 142 was observed. In other words, it was confirmed that the first liquid was contained individually in multiple wells 142. Furthermore, a fluorescence signal of FITC was observed across the entire surface.

[0122] The image in Figure 18(b) is an observation image of signal detection 4 in Comparative Example 1. The image in Figure 19(b) is an observation image of signal detection 4 in Comparative Example 2. The top row of the image is a bright-field image, the middle row is a Redmond Red fluorescence image, and the bottom row is a FITC fluorescence image. As a result, a Redmond Red fluorescence signal corresponding to the arrangement of wells 142 was observed. In other words, it was confirmed that the first liquid was contained individually in multiple wells 142. No FITC fluorescence signal was observed.

[0123] The results from Examples 1-2 and Comparative Examples 1-2 revealed that when the first liquid contains a surfactant, the contents of well 141 can be replaced by the second liquid even if the first liquid is individually contained by a sealing liquid. It was found that the contents of well 141 can be replaced by the second liquid regardless of whether the second liquid contains a surfactant or not. Furthermore, it was found that even if the second liquid contains a surfactant, if the first liquid does not contain a surfactant, the contents of well 141 cannot be replaced by the second liquid.

[0124] [Example 3] The same procedure as in Example 1 was followed from (introduction of the first liquid) to (detection of the signal 4). After that, the following operations were performed.

[0125] (Introduction of the third liquid) A third liquid was injected in 20 μL through the introduction port 122 of the fluid device.

[0126] (Signal detection 5) Next, a fluorescence microscope was used to image the wells 141 of the substrate 110 of the fluid device 100 from the side opposite to the side where the well array 140 is formed, with the focus on the wells 141. At this time, excitation light was irradiated from the side opposite to the side where the well array 140 of the substrate 110 is formed. A fluorescence image was taken using filter 1 with an exposure time of 1 / 4 second, and a fluorescence image was taken using filter 2 with an exposure time of 1.5 seconds. Bright-field images of the same field of view were also taken.

[0127] (Introduction of sealing liquid) 100 μL of sealing solution was injected through the introduction port 122 of the fluid device. As a result, well 141 was individually resealed, and a sealed well 142 was formed.

[0128] (Signal detection 6) Next, a fluorescence microscope was used to image the wells 141 of the substrate 110 of the fluid device 100 from the side opposite to the side where the well array 140 is formed, with the focus on the wells 141. At this time, excitation light was irradiated from the side opposite to the side where the well array 140 of the substrate 110 is formed. A fluorescence image was taken using filter 1 with an exposure time of 5 seconds, and a fluorescence image was taken using filter 2 with an exposure time of 1.5 seconds, and a bright-field image of the same field of view was also taken.

[0129] The image in Figure 20(a) is an observation image from signal detection 1. The image in Figure 20(b) is an observation image from signal detection 2. The upper part of the image is a bright-field image, and the lower part is a fluorescence image.

[0130] The image in Figure 21(a) is an observation image from signal detection 3. The image in Figure 21(b) is an observation image from signal detection 4. The top row of the image is a bright-field image, the middle row is a Redmond Red fluorescence image, and the bottom row is a FITC fluorescence image.

[0131] The results in Figures 20 and 21 show that the same results as in Example 1 were obtained.

[0132] The image in Figure 22(a) is an observation image of signal detection 5. The image in Figure 22(b) is an observation image of signal detection 4. The top row of the image is a bright-field image, the middle row is a Redmond Red fluorescence image, and the bottom row is a FITC fluorescence image.

[0133] As shown in Figure 22(a), the fluorescence signal of Redmond Red was observed across the entire surface. On the other hand, the fluorescence signal of FITC was not observed. As shown in Figure 22(b), the fluorescence signal of Redmond Red corresponding to the arrangement of well 142 was observed. In other words, it was confirmed that the third liquid was contained individually in multiple wells 142. On the other hand, the fluorescence signal of FITC was not observed.

[0134] [Example 4] The experiment was conducted under the same conditions as in Example 3, except that the second liquid did not contain Tween 20.

[0135] The image in Figure 23(a) is an observation image from signal detection 1. The image in Figure 23(b) is an observation image from signal detection 2. The upper part of the image is a bright-field image, and the lower part is a fluorescence image.

[0136] The image in Figure 24(a) is an observation image from signal detection 3. The image in Figure 24(b) is an observation image from signal detection 4. The top row of the image is a bright-field image, the middle row is a Redmond Red fluorescence image, and the bottom row is a FITC fluorescence image.

[0137] The results shown in Figures 23 and 24 indicate that the same results as in Example 2 were obtained.

[0138] The image in Figure 25(a) is an observation image of signal detection 5. The image in Figure 25(b) is an observation image of signal detection 4. The top row of the image is a bright-field image, the middle row is a Redmond Red fluorescence image, and the bottom row is a FITC fluorescence image.

[0139] As shown in Figure 25(a), a Redmond Red fluorescence signal was observed across the entire surface. On the other hand, no FITC fluorescence signal was observed. As shown in Figure 25(b), a Redmond Red fluorescence signal corresponding to the arrangement of well 142 was observed. This confirmed that the third liquid was contained individually in multiple wells 142. On the other hand, no FITC fluorescence signal was observed.

[0140] These results show that the liquid to be ultimately replaced (the third liquid in this embodiment) can be contained in the well even if the introduction of the surfactant-containing liquid (the first liquid in this embodiment), the sealing with the sealing liquid, and the introduction of the liquid to be ultimately replaced (the third liquid in this embodiment) do not have to be performed sequentially. In other words, even if the liquid contained immediately before the liquid to be ultimately replaced (the third liquid in this embodiment) (the second liquid in this embodiment) does not contain an surfactant, if the surfactant-containing liquid (the first liquid in this embodiment) is contained in well 141 before the liquid to be ultimately replaced, the liquid to be ultimately replaced can be contained in well 141.

[0141] One possible reason for this is that the introduction of the first liquid introduced a surfactant into the well 141, and even after replacing the inside of the well 141 with the second liquid which does not contain a surfactant, some surfactant remained in the well 141 or adhered to the inner wall of the well 141. Therefore, it is thought that when the third liquid was introduced, the surfactant remaining inside the well 141 acted to replace the inside of the well 141 with the third liquid. [Comparative Example 3] The experiment was conducted under the same conditions as in Example 3, except that the first liquid did not contain Tween 20.

[0142] The image in Figure 26(a) is the observation image of signal detection 1. The image in Figure 26(b) is the 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. From the results in Figure 26, it can be seen that the same results as in Comparative Example 1 were obtained.

[0143] The image in Figure 27(a) is an observation image of signal detection 3. The image in Figure 27(b) is an observation image of signal detection 4. The top row of the image is a bright-field image, the middle row is a fluorescence image of Redmond Red, and the bottom row is a fluorescence image of FITC. From the image in Figure 27(a), it can be seen that the same results as in Comparative Example 1 were obtained. In the image in Figure 27(b), there is a black area in the fluorescence image of Redmond Red corresponding to the arrangement of well 142. Since this black area coincides with the area where a fluorescence signal was observed in the fluorescence image of FITC, it can be said that only a part of well 142 was replaced by the second liquid. Thus, it was found that if the first liquid does not contain a surfactant, well 142 cannot be reliably replaced by the second liquid.

[0144] The image in Figure 28(a) is an observation image of signal detection 5. The image in Figure 28(b) is an observation image of signal detection 4. The top row of the image is a bright-field image, the middle row is a Redmond Red fluorescence image, and the bottom row is a FITC fluorescence image.

[0145] As shown in Figure 28(a), the fluorescence signal of Redmond Red was observed across the entire surface. On the other hand, the fluorescence signal of FITC was not observed. As shown in Figure 28(b), the fluorescence signal of Redmond Red corresponding to the arrangement of well 142 was observed. In other words, it was confirmed that the third liquid was contained individually in multiple wells 142. On the other hand, the fluorescence signal of FITC was not observed. In other words, it was found that if the second liquid contains a surfactant, the second liquid contained in well 142 is replaced by the third liquid.

[0146] [Example 5] 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.

[0147] [Example 6] The experiment was conducted under the same conditions as in Example 1, except that the material of the fluid device cover member 120 was changed to polypropylene (manufactured by AS ONE Corporation, part number: PPN-051001).

[0148] [Example 7] The experiment was conducted under the same conditions as in Example 1, except that the material of the fluid device cover member 120 was changed to silicon (manufactured by Togawa Rubber Co., Ltd., part number: K-125(50)).

[0149] [Comparative Example 4] The experiment was conducted under the same conditions as in Example 5, except that the first and second liquids did not contain surfactants.

[0150] [Comparative Example 5] The experiment was conducted under the same conditions as in Example 6, except that the first and second liquids did not contain surfactants.

[0151] [Comparative Example 6] The experiment was conducted under the same conditions as in Example 7, except that the first and second liquids did not contain surfactants.

[0152] The image in Figure 29(a) is an observation image of signal detection 1 in Example 5. The image in Figure 29(b) is an observation image of signal detection 2 in Example 5. The image in Figure 31(a) is an observation image of signal detection 1 in Example 6. The image in Figure 31(b) is an observation image of signal detection 2 in Example 6. The image in Figure 33(a) is an observation image of signal detection 1 in Example 7. The image in Figure 33(b) is an observation image of signal detection 2 in Example 7. The upper row of the image is a bright-field image, and the lower row is a fluorescence image.

[0153] The image in Figure 30(a) is an observation image of signal detection 3 in Example 5. The image in Figure 30(b) is an observation image of signal detection 4 in Example 5. The image in Figure 32(a) is an observation image of signal detection 3 in Example 6. The image in Figure 32(b) is an observation image of signal detection 4 in Example 6. The image in Figure 34(a) is an observation image of signal detection 3 in Example 7. The image in Figure 34(b) is an observation image of signal detection 4 in Example 7. The top row of the image is a bright-field image, the middle row is a Redmond Red fluorescence image, and the bottom row is a FITC fluorescence image.

[0154] The results shown in Figures 29 to 34 indicate that Examples 5 to 7 yielded the same results as Example 1. In other words, it became clear that even if the material of the fluid device cover member 120 is glass, polypropylene, or silicon, if the first liquid contains a surfactant, the sealing liquid can replace the wells 142 in which the first liquid is individually contained with the second liquid.

[0155] The image in Figure 35(a) is an observation image of signal detection 1 of Comparative Example 4. The image in Figure 35(b) is an observation image of signal detection 2 of Comparative Example 4. The image in Figure 37(a) is an observation image of signal detection 1 of Comparative Example 5. The image in Figure 37(b) is an observation image of signal detection 2 of Comparative Example 5. The image in Figure 39(a) is an observation image of signal detection 1 of Comparative Example 6. The image in Figure 39(b) is an observation image of signal detection 2 of Comparative Example 6. The upper row of the image is a bright-field image, and the lower row is a fluorescence image.

[0156] The image in Figure 36(a) is an observation image of signal detection 3 in Comparative Example 4. The image in Figure 36(b) is an observation image of signal detection 4 in Comparative Example 4. The image in Figure 38(a) is an observation image of signal detection 3 in Comparative Example 5. The image in Figure 38(b) is an observation image of signal detection 4 in Comparative Example 5. The image in Figure 40(a) is an observation image of signal detection 3 in Comparative Example 6. The image in Figure 40(b) is an observation image of signal detection 4 in Comparative Example 6. The top row of the images is a bright-field image, the middle row is a Redmond Red fluorescence image, and the bottom row is a FITC fluorescence image.

[0157] The results in Figures 35 to 40 show that Comparative Examples 4 to 6 yielded the same results as Comparative Example 2. In other words, it became clear that even if the material of the fluid device cover member 120 is glass, polypropylene, or silicon, if the first liquid does not contain a surfactant, the sealing liquid cannot replace the wells 142 in which the first liquid is individually contained with the second liquid. [Industrial applicability]

[0158] According to the present invention, a technique for replacing the liquid inside individually sealed micro-compartments can be provided. [Explanation of symbols]

[0159] 100, 200... Fluid device, 110... Substrate, 111... One side, 120... Cover member, 121... Protrusion, 122... Inlet port, 123... Outlet port, 130... Flow channel, 140... Well array, 141... Well, 142... Sealed well (micro-compartment), 210... Wall member, L110... First liquid, L120, L620... Sealing liquid, L410... Second liquid.

Claims

1. The fluid device comprises a substrate and a plurality of wells opening on one side of the substrate and containing a first liquid containing a surfactant, wherein a first sealing liquid is laminated on the one side of the substrate and the openings of the plurality of wells are sealed by the first sealing liquid, and the second liquid is introduced onto the one side of the fluid device. As a result, the second liquid is introduced into the well by replacing the first sealing liquid, and the second liquid is introduced into the well. An introduction method wherein the concentration of the surfactant is 0.001 v / v% or more and 1.0 v / v% or less with respect to the total volume of the first liquid.

2. The method further includes introducing a second sealing liquid into the fluid device after introducing the second liquid, The method according to claim 1, wherein, as a result, the second sealing liquid is laminated on one surface to seal the openings of the plurality of wells, and the second liquid, or a mixture of the first liquid and the second liquid, is sealed inside the wells.

3. The method according to claim 1 or 2, further comprising introducing the first liquid into the fluid device before introducing the second liquid, and introducing the first sealing liquid into the fluid device such that the first sealing liquid is laminated on one side to seal the openings of the plurality of wells and the first liquid is sealed inside the wells.

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

5. The fluid device further comprises a cover member positioned opposite to the one side, The method according to any one of claims 1 to 4, wherein the space between the cover member and the one surface forms a flow path.

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

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

8. The method according to any one of claims 1 to 7, wherein, after the second liquid is introduced into the well, at least a portion of the components contained in the first liquid is retained inside the well.

9. The method according to claim 8, wherein at least a portion of the components contained in the first liquid is held inside the well by being held on a carrier.

10. The method according to any one of claims 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.

11. The method according to claim 10, wherein the material of the one surface is a cycloolefin polymer, the main components of the first liquid and the second liquid are water, and the first sealing liquid is a fluorine-based oil.

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