Method for detecting target molecule

Abstract

To provide a method for detecting a target molecule that can detect the target molecule with high accuracy.SOLUTION: A method for detecting a target molecule comprises the steps of: preparing a fluidic device comprising a substrate and a well array disposed on the substrate; feeding a liquid in which a structure containing a target molecule is dispersed to the well array in the fluidic device, and introducing the structure into wells of the well array; feeding a sealing liquid to the well array, forming a layer of the sealing liquid on a top layer of the liquid introduced into the wells, and sealing the liquid in the wells; extracting the target molecule from the structure; and detecting the target molecule.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for detecting a target molecule. This application claims priority based on Japanese Patent Application No. 2017-222128 filed in Japan on November 17, 2017, and incorporates its content herein.

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 performed. Conventionally, quantification of proteins has been performed by enzyme-linked immunosorbent assay (ELISA) or the like, and quantification of nucleic acids has been performed by real-time PCR method or the like.

[0003] In recent years, for the purpose of detecting a disease earlier, the need to detect a target molecule more accurately has been increasing. 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 minute solutions. Then, the signals from each minute solution are binarized, and only whether the target molecule is present or not is discriminated to measure the number of target molecules. According to digital measurement, the detection sensitivity and quantification can be remarkably improved as compared with conventional ELISA, real-time PCR method, etc.

[0005] In digital PCR, the mixture of PCR reaction reagent and nucleic acid is diluted so that there are zero or one template nucleic acid molecules in each microdroplet. In digital PCR, it is preferable for the volume of each microdroplet to be small in order to increase the sensitivity of nucleic acid amplification and to perform nucleic acid amplification on a large number of microdroplets simultaneously. For example, Patent Document 3 discloses a microarray-like reaction vessel formed so that the volume of each well is 6 nl (nanoliters). Patent Document 1 also discloses a method for introducing the sample into each well of a microarray in which many wells with a depth of 3 μm and a diameter of 5 μm are formed in a channel, by flowing the sample through the channel and then pushing out the excess reagent in the channel with oil. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent No. 6183471 [Patent Document 2] Japan Special Publication No. 2014-503831 [Patent Document 3] International Publication No. 2013-151135 [Non-patent literature]

[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. [Overview of the project] [Problems that the invention aims to solve]

[0008] In digital measurement, target molecules are sometimes extracted from a structure containing the target molecule (for example, a cell as a structure having nucleic acid as the target molecule, as described later), delivered and distributed, and then a signal is generated if the target molecule is present in a microdroplet to detect its presence. However, the target molecule may be lost or reduced during the extraction stage from the structure. Furthermore, in detection methods such as those described in Patent Document 1, in which a sample is introduced into each well of a microarray using a channel and oil, target molecules in the sample may remain in the channel and be washed away by the oil without being distributed to each well, resulting in a decrease in the number of target molecules. As a result, the detection accuracy of the target molecule may decrease.

[0009] This invention has been made in view of the circumstances described above, and aims to provide a technology that can accurately detect target molecules. [Means for solving the problem]

[0010] (1) A method for detecting a target molecule according to a first aspect of the present invention comprises: preparing a fluid device comprising a substrate and a well array disposed on the substrate; supplying a liquid in which a structure containing the target molecule is dispersed into the well array of the fluid device to introduce the structure into the wells of the well array; supplying a sealing liquid to the well array to form a layer of the sealing liquid on top of the liquid introduced into the wells to seal the liquid in the wells; extracting the target molecule from the structure; and detecting the target molecule. (2) The step of extracting the target molecule may be carried out within the well. (3) The step of extracting the target molecule may be performed after the step of sealing the liquid in the well. (4) The step of detecting the target molecule may be performed after the step of extracting the target molecule. (5) The step of detecting the target molecule may include a signal amplification reaction. (6) The step of detecting the structure may be further included. (7) The steps of detecting the target molecule and the steps of detecting the structure may be performed simultaneously. (8) In the step of detecting the target molecule, two or more types of the target molecules may be detected. (9) The structure may be at least any one of exosomes, cells, bacteria, viruses, fungi, and endoplasmic reticulum. (10) The target molecule may be a nucleic acid or a protein. (11) In the step of introducing, one or less of the structures may be introduced into one well in the well array. (12) The step of extracting the target molecule may be performed by any one of physical, chemical, and biological methods. (13) The method may further include a step of binding a capture having a specific binding substance to the structure to the structure. (14) The step of introducing the structure may be performed by introducing the capture into the well of the well array. (15) The specific binding substance may be an antibody. [Advantages of the Invention]

[0011] According to the present invention, it is possible to provide a technique capable of accurately detecting a target molecule. [Brief Description of the Drawings]

[0012] [Figure 1] It is a schematic cross-sectional view for explaining a method of feeding a reagent solution to a fluid device. [Figure 2] It is a schematic cross-sectional view for explaining a method of feeding oil to a fluid device. [Figure 3] It is a schematic cross-sectional view for explaining a method of detecting a signal from inside a fluid device. [Figure 4] (a) and (b) are schematic cross-sectional views for explaining a method according to a first embodiment of detecting a target molecule in a microcompartment. [Figure 5](a) and (b) are schematic cross-sectional views for explaining a method according to a second embodiment of detecting a target molecule within a minute compartment. [Figure 6] These are the results of fluorescence microscopic observation according to Example 1. [Figure 7] These are the results of fluorescence microscopic observation according to Example 2. [Figure 8] This is a graph showing the number of wells in which fluorescence was observed with respect to the phage concentration according to Example 2. [Figure 9] These are the results of fluorescence microscopic observation according to Example 3. [Figure 10] This is a graph showing the change in fluorescence intensity with respect to time when examining the detection of phage internal nucleic acid by a method of collapsing various phages according to Example 4. [Figure 11] This is an enlarged view of FIG. 10 and is a graph showing the change in fluorescence intensity with respect to time when examining the detection of phage internal nucleic acid by a method of collapsing various phages according to Example 4. [Figure 12] These are the results of fluorescence microscopic observation according to Example 5. [Figure 13] These are the results of fluorescence microscopic observation according to Example 6. [Figure 14] These are the results of fluorescence microscopic observation according to Example 6. [Figure 15] These are the results of fluorescence microscopic observation according to Example 6. [Embodiments for Carrying Out the Invention]

[0013] Hereinafter, embodiments of the present invention will be described in detail while referring to the drawings as appropriate. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and duplicate explanations are omitted. Note that the dimensional ratios in each figure are exaggerated for the purpose of explanation and do not necessarily match the actual dimensional ratios.

[0014] [Fluid Device] One embodiment of the present invention relates to a method for detecting target molecules using a fluid device. The fluid device according to this embodiment comprises a substrate and a well array disposed on the substrate. Furthermore, the fluid device according to this embodiment may include a flow path and a well array arranged within the flow path.

[0015] The shape, dimensions, and arrangement of the wells are not particularly limited, but it is preferable to use a well array consisting of a structure used in the method according to this embodiment and wells capable of holding a certain amount of reagent solution used in the detection step. The wells may be used without treatment, or depending on the purpose, they may be pre-treated by immobilizing extraction reagents, detection reagents such as antibodies, specific binding substances, etc. on the inner walls of the wells beforehand, or by covering the well openings with a lipid bilayer.

[0016] The flow path is a route for delivering the liquid in which the structure is dispersed and the sealing liquid. The shape, structure, and capacity of the flow path are not particularly limited, but when a flow path is formed in a fluid device, it is preferable to use a fluid device with a flow path such that when the liquid in which the structure is dispersed is delivered, the structure is introduced into each well of the well array, and when the sealing liquid is inserted, each well is individually sealed and a microdroplet is formed. In this embodiment, the flow path in the fluid device may be any internal space formed to be filled with and contain liquids such as reagent solutions and sealing solutions, as described later, and the flow path may be referred to as the internal space. For example, a fluid device may have a configuration that allows a well array to be filled by dropping a reagent solution (e.g., an aqueous solution) into it, and then a sealing solution (e.g., oil) to be gently dropped from above to seal the well array with the sealing solution. Alternatively, the internal space may be formed by filling the well array with reagent solution, sealing the well array with sealing solution, and then covering it with a lid material.

[0017] (An example of a fluid device) Figure 1 is a schematic cross-sectional view of a fluid device. As shown in Figure 1, the fluid device 100 comprises a base material 104 and a cover material 101.

[0018] The substrate 104 may be formed from a light-transmitting resin. The substrate 104 according to this embodiment may be substantially transparent.

[0019] The substrate 104 has a plurality of wells 105 (hereinafter, the plurality of wells may be referred to as a "well array"). The wells 105 of the substrate 104 open to the surface of the substrate 104. The shape, dimensions, and arrangement of the wells 105 are not particularly limited, but it is preferable to design them to have a shape and dimensions suitable for introducing one structure into each well 105. Typically, it is preferable for the wells 105 to be small wells with a small volume. For example, the volume of one well 105 may be between 1 aL and 100 fL. In the example shown in Figure 1, multiple wells 105 of the same shape and size are formed on the substrate 104, each capable of holding a fixed amount of reagent solution 107 (a liquid in which a structure containing a target molecule is dispersed) used in biochemical analysis performed using the fluid device 100. Furthermore, if particles are used in biochemical analysis performed using the fluid device 100, the substrate 104 may also have wells 105 of the same shape and size formed thereon, each having a shape and dimensions capable of holding one or more particles and a fixed amount of reagent solution 107 containing the particles. "Same shape and size" means that the wells have the same shape and volume to the extent required for digital measurement, and variations within the range of manufacturing tolerances are acceptable.

[0020] In the fluid device 100, the diameter of the well 105 may be, for example, about 3 μm, and the depth of the well 105 may be, for example, about 4.5 μm. Furthermore, the wells 105 may be formed on the substrate 104 in an aligned manner to form a triangular lattice or a square lattice.

[0021] The region of the substrate 104 that includes multiple wells 105 is the region where the reagent solution 107 to be analyzed in biochemical analysis is filled. In the fluid device 100, a flow path 106 (an internal space that serves as a flow path) is provided between the substrate 104 and the lid material 101 inside the region where the reagent solution 107 is filled (internal space). In this embodiment, the flow path 106 in the fluid device 100 may be any internal space formed to be filled with and filled with liquids such as the reagent solution 107 and the sealing solution 201 described later, and the flow path 106 may sometimes be referred to as the internal space. For example, the fluid device 100 may have a configuration that allows a plurality of wells 105 (well arrays) to be filled by dropping a reagent solution 107 (e.g., aqueous solution) into them, and then a sealing solution 201 (e.g., oil) to be gently dropped from above to seal the plurality of wells 105 with the sealing solution 201.

[0022] The lid material 101 may be welded or bonded to the base material 104. For example, it is preferable to select a resin with low autofluorescence for the lid material 101, and it may be formed from a thermoplastic resin such as a cycloolefin polymer or cycloolefin copolymer. Furthermore, the lid material 101 may be formed from a material that does not transmit light of wavelengths near the wavelength detected when observing the signal by fluorescence, or it may be formed from a material that does not transmit light at all. For example, it may be formed from a thermoplastic resin to which carbon or metal particles have been added.

[0023] The substrate 104 is formed using, for example, a resin. The type of resin is not particularly limited, but it is preferable to use a resin that is resistant to the reagent and the sealing solution used when forming the droplet. Furthermore, when observing the signal by fluorescence, it is preferable to choose a resin with low autofluorescence. Examples include cycloolefin polymers, cycloolefin copolymers, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesins, and amorphous fluororesins. Note that these materials shown as examples of substrate 104 are merely examples, and the material is not limited to these.

[0024] Multiple wells 105 may be formed on one side of the substrate 104 in the thickness direction. The resin may be formed by injection molding, thermal imprinting, optical imprinting, or other methods. Furthermore, when using fluororesin, for example, a layer of CYTOP® (Asahi Glass) may be placed on the substrate 104, with minute pores formed in CYTOP® serving as the wells 105.

[0025] The lid material 101 is molded to have a protrusion on the surface that faces the base material 104 during assembly. For example, a fluid of thermoplastic resin may be molded into a plate shape with protrusions using a mold. Furthermore, a liquid supply port 102 and a waste liquid port 103 may be formed in the lid material 101.

[0026] Once the lid material 101 and the base material 104 are formed as described above, the lid material 101 and the base material 104 are stacked on top of each other such that the protrusion of the lid material contacts the surface of the base material 104 on which the well 105 opens. This forms the flow path 106. Furthermore, with the lid material 101 and the base material 104 stacked as described above, they are welded together by laser welding or the like.

[0027] [Conventional detection methods] First, we will explain conventional methods for detecting target molecules using fluid devices, using schematic cross-sectional diagrams of typical fluid devices shown in Figures 1 to 3.

[0028] First, as shown in Figure 1, a reagent solution 107 diluted so that one target molecule is contained in each well 105 of the fluid device 100 is delivered from the liquid delivery port 102 of the lid material 101 to the flow path 106 between the substrate 104 and the lid material 101. The reagent solution 107 delivered to the flow path 106 between the substrate 104 and the lid material 101 during the liquid delivery step is contained inside the multiple wells 105. Next, as shown in Figure 2, a sealing liquid 201, such as an oily oil, is delivered from the liquid delivery port 102 of the lid material 101 to the flow path 106 between the substrate 104 and the lid material 101 to individually seal the multiple wells 105. In the sealing step, the sealing liquid 201 replaces the reagent solution 107 that was delivered to the flow path 106 between the substrate 104 and the lid material 101 during the liquid delivery step but is not contained in the wells 105. As a result, the sealing solution 201 individually seals multiple wells 105, and each well 105 becomes an independent reaction space (micro-compartment 202). Subsequently, as shown in Figure 3, a predetermined biochemical reaction (reaction step) is carried out in the wells 105, and then the signal amplified by the signal amplification reaction in the reaction step is observed.

[0029] In conventional detection methods, when detecting target molecules such as nucleic acids and proteins contained in structures such as cells and viruses, the target molecules are first extracted from the structure and then delivered as reagent solution 107 in the liquid delivery step. However, the target molecules may be lost or reduced during the extraction step from the structure and during the liquid delivery step. In addition, the target molecules may not be distributed into the well 105 but remain in the channel 106, be washed away by the sealing solution 201 in the sealing step, and the signal amplification reaction may not be observed in the reaction step, resulting in their presence not being detected.

[0030] Thus, with conventional detection methods, if the target molecules are not properly distributed into the wells during the liquid delivery and sealing processes, it may not be possible to accurately detect the target molecules contained in the structure.

[0031] [Methods for detecting target molecules] A method for detecting a target molecule according to one embodiment of the present invention comprises: preparing a fluid device comprising a substrate and a well array disposed on the substrate; supplying a liquid in which a structure containing the target molecule is dispersed into the well array of the fluid device to introduce the structure into the wells of the well array; supplying a sealing liquid to the well array to form a layer of the sealing liquid on top of the liquid introduced into the wells to seal the liquid in the wells; extracting the target molecule from the structure; and detecting the target molecule. Furthermore, in this embodiment, if the fluid device has a flow channel, the method for detecting target molecules according to this embodiment may include the steps of: preparing a fluid device comprising a flow channel and a well array disposed within the flow channel; sending a liquid in which a structure containing the target molecule is dispersed into the flow channel of the fluid device and introducing the structure into the wells of the well array; sending a sealing liquid into the flow channel to form a layer of sealing liquid on top of the liquid introduced into the wells and sealing the liquid in the wells; extracting the target molecule from the structure; and detecting the target molecule.

[0032] According to the detection method of this embodiment, the number of target molecules lost without being distributed into the wells is reduced, thus improving detection accuracy. Furthermore, since there is no need to extract target molecules from the structure in advance, target molecules can be detected simply. Each step will be described in detail below.

[0033] (The process of introducing the structure into the well) In this process, a liquid containing a structure with target molecules dispersed in it is delivered into a fluid device (the flow path of the fluid device), and the structure is introduced into the wells of a well array.

[0034] The structure is not particularly limited, and any structure can be used. Specific examples of structures include exosomes, cells, bacteria, viruses, fungi, and endoplasmic reticulum in biological samples. The biological sample is not particularly limited, and examples include serum, plasma, and urine.

[0035] The structure contains the target molecule. In this specification, "contains" the target molecule in the structure may mean that the structure encapsulates the target molecule, or that part or all of the target molecule is present on the surface of the structure. Specific examples of target molecules include nucleic acids such as DNA, RNA, miRNA, and mRNA; proteins such as structural proteins and enzymes; sugars; lipids; and the like.

[0036] The liquid used to disperse the structure can be a general liquid used in biochemical analysis performed using a fluid device, and is preferably an aqueous solution. It may also contain surfactants or the like to facilitate the sealing of the liquid within the wells. Furthermore, it may contain reagents necessary for the steps described later, such as the steps for extracting the target molecule or detecting the target molecule. For example, when using the Invasive Cleavage Assay (ICA) reaction to detect the target molecule, the liquid used to disperse the structure may contain ICA reaction reagents such as allergen probes, ICA oligonucleotides, flap endonuclease-1 (FEN-1), and fluorescent substrates.

[0037] "Introducing" a structure into a well refers to distributing the structure into each well of a well array. For example, the structure may be introduced into each well so that the entire structure containing the target molecule is contained within a microdroplet, or the structure may be placed at the top of each well, and only the target molecule may be introduced into the well by performing the extraction process described later while the structure is positioned at the top of each well. A specific example of the latter is to attach a virus as a structure to a lipid bilayer covering the opening at the top of the well, extract RNA using the viral infection mechanism, and introduce only the extracted RNA into the well. In other words, in this example, the introduction process and the extraction process are performed simultaneously.

[0038] The number of structures introduced into a single well during the introduction process is not particularly limited, but preferably, zero or one structure is introduced into each well. This allows for the detection of structures on an individual basis, i.e., enables digital measurement. Furthermore, it is not necessary to introduce structures into all wells of the well array.

[0039] The means of introducing the structure into the well are not particularly limited, and an appropriate method can be selected depending on the chosen structure. For example, one method is to allow the structure to settle in the fluid device (flow channel) by its own weight and then distribute it into the well. Alternatively, the introduction efficiency can be improved by using a substance that captures the structure (captured material), attaching the capture material to structures that are difficult to settle by their own weight and then sending them into the fluid, or by pre-fixing the capture material in the well and then capturing the structures as they are sent into the fluid.

[0040] (The sealing process) In this process, a sealing liquid is introduced into the internal space of the fluid device (e.g., the flow path), forming a layer of sealing liquid on top of the liquid introduced into the well, thereby sealing the liquid within the well. This process creates tiny droplets within the well. In this process, in a fluid device, after filling the well array with a reagent solution (e.g., aqueous solution), a sealing solution (e.g., oil) may be gently dropped from above to seal the well array with the sealing solution.

[0041] The sealing liquid is a liquid that can individually seal the liquids introduced into multiple wells to prevent them from mixing with each other, thereby forming droplets (microdroplets). Preferably, it is an oily solution, more preferably an oil. As the oil, a fluorine-based oil, a silicone-based oil, a hydrocarbon-based oil, or a mixture thereof can be used. More specifically, a product such as "FC-40" manufactured by Sigma Corporation can be used.

[0042] (The process of extracting target molecules from a structure) In this process, target molecules are extracted from the structure. In the extraction process, all target molecules may be extracted from the structure, or only a portion of the target molecules contained in the structure may be extracted.

[0043] There are no particular limitations on the method for extracting target molecules from a structure, and known methods can be used. Examples include physical methods such as heat, ultrasound, light, magnetism, and electromagnetic waves; chemical methods such as surfactants, antibiotics, osmotic pressure inducers, and necrosis / apoptosis inducers; and biological methods such as enzymes, viruses, and phages. More specifically, when using heat as an extraction method, target molecules can be extracted from the structure by heating the fluid device to 70°C or higher, preferably 75°C or higher, for example about 80°C, for at least 5 minutes, preferably at least 10 minutes, for example about 15 minutes or about 30 minutes. One specific example of a biological extraction method is to infect bacteria such as E. coli with phages such as f1 phage and induce lysis. Another specific example of a biological extraction method is to elute nucleic acids as target molecules from phages such as f1 phage as structures. More specifically, when a phage (structure) is brought into contact with bacteria, the phage first recognizes and binds to the bacterial surface membrane structure (protein, lipid bilayer). Subsequently, nucleic acids (target molecules) are injected into the bacterial cell from the phage. Then, the nucleic acids are replicated within the bacterial cell, and new phages are generated. After that, the action of the phages generated within the bacterial cell causes the bacterial cell membrane to break down (lysis), and the injected and replicated target molecule DNA is released. Alternatively, instead of lysis from within by the phage as described above, the bacterial cell may be broken down by an external force such as heat, and the phages inside may be extracted. Furthermore, in the above case, instead of using bacteria themselves, a lipid bilayer-like substance that mimics the membrane structure of bacteria may be used. By attaching phages to the lipid bilayer-like substance, the phages release nucleic acids contained within them, similar to when attached to bacterial cells. In such cases, the lysis step does not need to be performed.

[0044] The process of extracting target molecules from the structure may be performed within the well after the well has been sealed. This helps to suppress the loss of target molecules. As a result, target molecules can be detected with greater accuracy compared to conventional methods in which target molecules are extracted from the structure and then distributed to each well of the microarray using a channel.

[0045] (Steps to detect target molecules) In this step, the target molecule is detected. The method for detecting the target molecule can be any known detection method, depending on the characteristics of the target molecule to be detected. For example, it can be done by first performing a reaction (signal amplification reaction) to amplify the signal derived from the target molecule to a detectable level as needed, and then detecting the amplified signal using appropriate means. In this embodiment, the step of detecting the target molecule is performed by a method for detecting nucleic acids.

[0046] Examples of signals that can be used in the detection method according to this embodiment include fluorescence, chemiluminescence, color development, potential changes, pH changes, and the like.

[0047] The signal amplification reaction may be, for example, a biochemical reaction, or more specifically, an enzymatic reaction. As an example, the signal amplification reaction is an isothermal reaction in which a reagent solution containing an enzyme for signal amplification is contained in a well, and the fluid device is maintained at a constant temperature condition, for example 60°C or higher, preferably about 66°C, for a predetermined time, for example at least 10 minutes, preferably about 15 minutes, under conditions that yield the desired enzyme activity.

[0048] Examples of signal amplification reactions include, specifically, ICA reactions such as the Invader® method, the LAMP method (trademark registered trademark), the TaqMan® method, and the fluorescent probe method, when using a reaction to detect nucleic acids. Using an ICA reaction is particularly preferred. This is related to the principle of the ICA reaction, in which signal amplification proceeds through a cycle of two reactions: (1) complementary binding between nucleic acids and (2) recognition and cleavage of the triple-stranded structure by an enzyme. In such signal amplification reactions, the influence of reaction cycle inhibition by contaminants other than the target molecule is small. Therefore, even if various components other than the target molecule present in the structure are released into the microcompartment during the extraction process, the target molecule can be detected with high accuracy by using an ICA reaction. For example, when using an ICA reaction for signal amplification, the liquid for introducing the structure into the well (the liquid for dispersing the structure) contains the reaction reagents and template nucleic acid necessary for the ICA reaction. If the biochemical reaction in the reaction process is an ICA reaction, and the target molecule is present in the well, the fluorescent substance is released from the quenching substance by an isothermal enzymatic reaction, thereby emitting a predetermined fluorescence signal in response to the excitation light.

[0049] Alternatively, the target molecule can be detected by binding a substance that specifically binds to the target molecule (specific binding substance) to the target molecule and then detecting the bound specific binding substance. For example, if the target molecule is a protein, it can be detected using ELISA. More specifically, detection may be performed, for example, by a sandwich ELISA using the FRET principle. When using the sandwich method with the FRET principle, first, a first specific binding substance (e.g., an antibody) labeled with a first fluorescent substance (donor) and a second specific binding substance labeled with a second fluorescent substance (acceptor) having an absorption wavelength that overlaps with the fluorescence wavelength of the first fluorescent substance are prepared. Next, the target molecule (e.g., an antigen) is brought into contact with both the first and second specific binding substances to form a complex. Once the complex is formed, the distance between the donor and acceptor decreases, and the fluorescence wavelength of the acceptor can be detected by irradiation with the excitation wavelength of the donor. Alternatively, the specific binding substance may be labeled with a nucleic acid fragment, and the nucleic acid fragment may be detected by the ICA reaction.

[0050] As the specific binding substance, the same substances as the specific binding molecules for the structure described later can be used, such as antibodies, antibody fragments, aptamers, etc. To detect the specific binding substance bound to the target molecule, the specific binding substance may be directly or indirectly labeled with an enzyme such as horseradish peroxidase (HRP). When using two or more specific binding substances, each specific binding molecule can be labeled in a way that allows them to be distinguished from one another.

[0051] The method for observing the signal can be selected from known and appropriate methods depending on the type of signal to be observed. For example, when performing bright-field observation, white light is irradiated perpendicularly to the substrate on which the well array is provided. When observing a fluorescence signal, excitation light corresponding to the fluorescent substance is irradiated into the well from the bottom of the well, and the fluorescence emitted by the fluorescent substance is observed. An image of the entire or a part of the observed well array is captured and saved, and image processing is performed using a computer system.

[0052] (An example of a detection process) In this embodiment, a single target molecule can be detected using two or more specific binding substances. For example, a single protein can be detected using two monoclonal antibodies that recognize different epitopes. This allows for highly accurate detection of the target molecule even when specific binding substances misrecognize or when similar target molecules are mixed in the well.

[0053] The target molecule detection method according to this embodiment can detect two or more types of target molecules. In this case, each target molecule may be detected in any order. For example, when detecting two types of target molecules, the two target molecules may be detected simultaneously in a single detection step, or they may be detected separately in two independent detection steps. This makes it possible to detect multiple target molecules present in a structure. Furthermore, since the detection method according to this embodiment performs an extraction step after distributing the structure into each microdroplet, the detection data for each target molecule can be correlated and evaluated for each individual structure. Furthermore, when detecting two or more target molecules simultaneously, or when detecting them sequentially while each target molecule is present and detectable, it is necessary to design the reaction system so that the signals indicating the presence of each target molecule do not become confused. For example, when detecting using a fluorescence signal, by using different wavelength bands for the excitation light and fluorescence of the first target molecule and the second target molecule, fluorescence can be detected independently without interference from the presence of both molecules.

[0054] The target molecule detection method according to this embodiment may further include a step of detecting the target molecule before performing the extraction step. This step is a step of detecting the target molecule contained in the structure before it is extracted from the structure. The target molecule detected in this step may be a target molecule present on the surface of the structure or a target molecule present inside the structure.

[0055] If the detection step is performed before the extraction step, the temperature of the detection step is preferably lower than the temperature at which the target component is extracted, more specifically in the range of room temperature to about 60°C.

[0056] Each of the steps detailed above may be performed sequentially in the order described above. Doing so (for example, by performing the step of detecting the target molecule after the step of extracting the target molecule) allows the target molecule extracted from the structure to be detected while it remains inside the well, thus enabling highly accurate detection of the target molecule. Alternatively, the order of each step may be changed, or two steps may be performed simultaneously. For example, the extraction step may be performed after the introduction step but before the sealing step, in which case the introduction step and the extraction step may be performed simultaneously. Also, if there are multiple detection steps as described above, some of the multiple detection steps may be performed before the extraction step.

[0057] [A detection method that further develops the process for detecting structures] A detection method according to one embodiment of the present invention may further include a step of detecting a structure in the target molecule detection method according to the above embodiment. The step of detecting a structure can be performed at any point in the detection method according to the above embodiment, for example, after the step of introducing the structure into a well and before the step of extracting the target molecule from the structure. Alternatively, the structure may be detected after the target substance has been extracted from the structure. In a method according to a specific embodiment of the present invention, the step of detecting a target molecule and the step of detecting a structure may be performed simultaneously. Furthermore, when performing simultaneous detection, the same considerations apply as when simultaneously detecting two or more target molecules, in order to ensure that the reaction system does not interfere with each other. Furthermore, when extracting target molecules within a well, extracting the target molecules within the well confirms that they were indeed extracted from the structure.

[0058] The detection of the structure may be performed directly, such as by bright-field observation of the structure, or indirectly, such as by detecting the target molecule contained in the structure using the same procedure as the detection steps described above. Examples of the latter include methods using fluorescent dyes that stain cell membranes or antibodies that recognize viral coat proteins.

[0059] The following describes more specific embodiments of the method for detecting target molecules according to the above embodiment of the present invention. (First Embodiment) A method for detecting a target molecule according to the first embodiment of the present invention will now be described. This embodiment comprises the steps of introducing a structure having a target molecule into a well, sealing the well, extracting the target molecule from the structure, and detecting the target molecule. Figures 4(a) and (b) are schematic cross-sectional views illustrating the method according to this embodiment for detecting a target molecule in a microcompartment.

[0060] First, a structure containing the target molecule is introduced into the well. This process is carried out, for example, by distributing the structure into the well 105 by sending the reagent solution 107 into the channel 106, as shown in Figure 1. Next, as shown in Figure 2, the sealing liquid 201 is delivered into the flow path 106 to individually seal the wells 105 and form micro-compartments 202. In Figure 4(a), a structure 12 containing the target molecule 11 is housed in a microcompartment 202 formed by a substrate 104 and a sealing solution 201. Subsequently, the target molecule is extracted from the structure within the sealed well. In Figure 4(b), the target molecule 11 is extracted from the structure 12' after the extraction process. Next, the target molecules extracted in the wells are detected using a signal amplification reaction in a reaction step, similar to conventional detection methods.

[0061] (Second Embodiment) A method for detecting a target molecule according to a second embodiment of the present invention will now be described. This embodiment further comprises the step of binding the captured substance to a structure in the method according to the first embodiment. Figures 5(a) and (b) are schematic cross-sectional views illustrating the method according to this embodiment for detecting a target molecule within a microcompartment.

[0062] In Figure 5(a), the structure 12 containing the target molecule 11, the specific binding substance 13, and the captured substance 14 are housed in a microcompartment 202 formed by the substrate 104 and the sealing liquid 201. A specific binding substance 13 is immobilized on the captured substance 14. Furthermore, the specific binding substance 13 recognizes and binds to the structure 12. In Figure 5(b), the target molecule 11 is extracted from the structure 12' after the extraction process.

[0063] The step of binding the captured material to the structure can be performed at any point in the detection method of this embodiment. For example, this step can be performed by bringing the structure and the captured material into contact in the sample tube before introducing the structure into the well. Alternatively, the binding may be performed by bringing the structure into contact with the captured material after introducing the captured material into the well.

[0064] A capture substance is a substance capable of capturing a structure. For example, it may be a substance that specifically binds to a target molecule (specific binding substance) immobilized on a solid phase, such as particles, but is not limited to this. The specific binding substance possessed by the capture substance may be one type, or two or more types, for example, three, four, five or more types. Alternatively, for example, when a virus is used as the structure, a cell to which the virus can attach (i.e., a cell possessing a viral receptor) may be used as the capture substance.

[0065] Examples of specific binding agents include antibodies, antibody fragments, and aptamers. Antibodies can be produced, for example, by immunizing animals such as mice with a target molecule or a fragment of a target molecule as an antigen. Alternatively, they can be produced, for example, by screening a phage library. 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. Antibodies may be monoclonal antibodies or polyclonal antibodies. Commercially available antibodies may also be used.

[0066] The particles are not particularly limited, and polymer particles, magnetic particles, glass particles, etc., can be used. It is preferable that the particles have been surface-treated to avoid nonspecific adsorption. Furthermore, it is preferable to use particles having functional groups such as carboxyl groups on their surface in order to immobilize specific binding substances. More specifically, products such as "Magnosphere LC300" manufactured by JSR Corporation can be used.

[0067] The method for immobilizing a specific binding substance on the particle surface is not particularly limited and includes methods by physical adsorption, chemical bonding, avidin-biotin bonding, and 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 and 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 a crosslinking agent with the carboxyl groups of the specific binding substance 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.

[0068] In one embodiment of the present invention, the step of introducing the structure may be performed by introducing the captured material into the wells of the well array. For example, a liquid in which the structure bound to the captured material to which the specific binding substance has been bound by the method described above is dispersed may be sent through a flow path and introduced into the wells.

[0069] Here, it is preferable to bring the captured material and the structure into contact under conditions where zero or one structure is captured by one captured material. Furthermore, it is preferable that zero or one captured material is introduced into each well. This enables digital measurement. In other words, in this embodiment, the detection of structures may be performed on a single-unit basis. In that case, the target molecule contained in the structure is detected on a single-unit basis.

[0070] Furthermore, in one embodiment of the present invention, the step of extracting a target molecule from a structure can be performed by a capture substance. In addition, in one embodiment of the present invention, the step of detecting a structure can be performed by detecting a specific binding substance possessed by the capture substance.

[0071] (Third embodiment) A method for detecting target molecules according to the third embodiment will be described. In this embodiment, the step of extracting target molecules from a structure is performed using a substance (extractant) that can act on the structure to extract the target molecules, as in the method according to the first embodiment. The extractant may be any substance used to perform the extraction steps described above, and examples include surfactants, antibiotics, osmotic pressure inducers, necrosis / apoptosis inducers, etc. used in chemical methods, or enzymes, viruses, phages, etc. used in biological methods. For example, when cells are used as the structure, a virus can be used as the extractant to infect the cells, lyse the cells, and extract nucleic acids as the target molecules.

[0072] The method according to this embodiment includes a step of bringing a structure into contact with an extractant. The structure and the extractant may be brought into contact in any order. For example, the structure and the extractant may be mixed before the step of introducing the structure into the well, and then the mixed liquid may be introduced into the well. In this case, the extraction conditions may be designed so that the target molecule is extracted from the structure in the well after the well is sealed, for example by adjusting the mixing ratio of the structure and the extractant. Alternatively, the extractant may be introduced into the well, and then the liquid in which the structure is dispersed may be introduced and brought into contact in the well.

[0073] After sealing the wells, the extractant acts within the wells, causing the structures to break down and the target molecules to be extracted. The extracted target molecules can then be detected using the detection method described above.

[0074] (Fourth Embodiment) A method for detecting target molecules according to the fourth embodiment will be described. In this embodiment, a first target molecule present on the surface of the structure and a second target molecule present inside the structure are detected, in addition to the method according to the first embodiment.

[0075] First, the structure is introduced into the well and sealed in the same manner as in the first embodiment. Next, the first target molecule is detected. The first target molecule can be detected by any of the methods described above, for example, by using a specific binding agent for the first target molecule, the specific binding agent may be bound to the first target molecule before introduction into the well or after introduction into the well.

[0076] Next, a second target molecule is extracted from the well, and the extracted second target molecule is detected using the detection method described above.

[0077] The first target molecule and the second target molecule may be detected sequentially as described above, or they may be detected simultaneously. [Examples]

[0078] The present invention will be described in more detail below based on examples. The present invention is not limited in any way to these examples.

[0079] In this embodiment, two resin components were used to manufacture an experimental fluid device: a COP base material formed by injection molding and a COP lid material (colored with carbon black, with fluid delivery and waste ports). The diameter of the wells (micro-compartments) formed in the base material was 5 μm, and the volume was sufficient to detect a signal by the Invader reaction within a few minutes. The base material was bonded to the lid material, forming an internal space (flow channel) of the fluid device with a height of 100 μm.

[0080] [Example 1] (Investigation of phage breakdown and nucleic acid extraction from phages) Using the f1 phage (reagent name: Escherichia coli phage f1, National Institute of Technology and Evaluation, model number: NBRC20010), a type of virus, as a structure (containing the base sequence of "SEQ ID NO: 1" as encapsulated DNA (f1 phage oligo)) (see Table 1), we investigated phage decay and detected target molecules using nucleic acids, specifically the f1 phage encapsulated DNA, as the target molecule.

[0081] (Preparation of nucleic acid detection reagents) To perform the Digital Invader reaction, a type of ICA reaction, an Invader reaction reagent (ICA reaction reagent) is prepared as a nucleic acid detection reagent. The Invader reaction reagent used in Example 1 was prepared using the reagents shown in Table 1 below, containing 0.5 μM Allele Probe 1 ((f1phage)Allele probe 1) (SEQ ID NO: 2), 1 μM Invader Oligo 1 (SEQ ID NO: 3) (ICA Oligo 1) (both from Fasmac), 4 μM FRET Cassette 1 (Alexa488-BHQ) (SEQ ID NO: 4) (Nippon Bioservices Co., Ltd.) (fluorescent substrate), 0.1 mg / mL FEN-1 (Flap endonuclease-1), 50 mM Tris-HCl (pH 8.5), and 20 mM MgCl2. The concentrations of each component in these Invader reaction reagents are the final concentrations in the Invader reaction reagent according to Example 1.

[0082] [Table 1]

[0083] <Investigation of phage decay and nucleic acid extraction reactions> We investigated whether surfactants could be used to break down phages. As a sample containing a surfactant, a solution (Solution A) was prepared by mixing the above-mentioned Invader reaction reagent and Bugbuster (Merck Millipore), a nucleic acid extraction reagent containing a surfactant, in a 1:1 ratio in a sample tube with the above-mentioned f1 phage (1%), and the mixture was stirred at room temperature for 15 minutes. As a control experiment, a solution (surfactant-free sample) (Solution B) was prepared by mixing the above Invader reaction reagent and the above f1 phage (1%) in a sample tube, and the mixture was stirred at room temperature for 15 minutes. Note that the concentrations of f1 phage in this example and the following examples are expressed as dilutions relative to the concentration of commercially available f1 phage suspension, which is set to 100%.

[0084] <Transportation of reaction mixture solution> 20 μL of solution A containing the surfactant was introduced into each well of the fluid device. Subsequently, 150 μL of FC-40 (Sigma) was introduced as a sealing solution to seal each well. As a result, one or fewer f1 phages were sealed in each well. Similarly, as a control experiment, 20 μL of the surfactant-free solution B described above was introduced into each well of the fluid device. Subsequently, 150 μL of FC-40 (Sigma) was introduced as a sealing solution to seal each well.

[0085] <Nucleic acid detection reaction> The above fluid device was placed on a hot plate and reacted at 66°C for 15 minutes. This resulted in the recognition of phage-encapsulated DNA by the allele probe and invader oligo, cleavage of the allele probe by FEN-1, binding of the released allele probe fragment to the FRET Cassette, cleavage of the FRET Cassette by FEN-1, and generation of a fluorescence signal from Alexa488.

[0086] <Fluorescence observation of wells> After heating at 66°C for 15 minutes, fluorescence images of the fluorescence signals obtained from the nucleic acid detection reaction in each well of the fluid device were captured using a 10x objective lens with a BZ-710 fluorescence microscope (KEYENCE). The exposure time was 3000 msec using a GFP fluorescence filter. The results of Example 1 are shown in Figure 6. As shown in Figure 6(a), when solution A, prepared using a mixed solution of Invader reaction reagent (ICA reagent) and Bugbuster and f1 phage, was applied to a fluid device, numerous fluorescent signals originating from the phage-encapsulated DNA, which was the target of detection, were observed in the wells of the fluid device. On the other hand, as shown in Figure 6(b), when solution B, prepared using the Invader reaction reagent (ICA reagent) and f1 phage without adding Bugbuster, was applied to the fluid device, almost no fluorescence signal originating from the phage-encapsulated DNA, which was the target of detection, was observed in the wells of the fluid device.

[0087] In this example, when Bugbuster was added, nucleic acids inside the phage (f1 phage oligo, f1 phage-encapsulated DNA) were detected, suggesting that nucleic acids were extracted from inside the phage. These results revealed that phages do not collapse with the Invader reaction reagent alone, but they do collapse when a reagent containing a surfactant such as Bugbuster is used. Furthermore, when a solution (Solution A) was prepared by mixing the above-mentioned Invader reaction reagent and Bugbuster, a nucleic acid extraction reagent containing a surfactant, in a 1:1 ratio with the above-mentioned f1 phage (1%), and after stirring in a sample tube at room temperature for 15 minutes, it is thought that the f1 phage had begun to disintegrate, albeit only partially, and the extraction of nucleic acids from within the f1 phage had begun.

[0088] [Example 2] (Investigation of phage decay, nucleic acid extraction, and nucleic acid detection within the wells of a fluid device) In this embodiment, instead of introducing the sample into the fluid device after pre-degrading the phage and extracting its internal nucleic acids, we investigated whether it is possible to degrade the phage inside the fluid device and detect the nucleic acids inside the phage inside the fluid device. In this example, as in Example 1, a type of virus called f1 phage (reagent name: Escherichia coli phage f1, National Institute of Technology and Evaluation, model number: NBRC20010) (containing the base sequence of "Sequence ID 1" as its encapsulated DNA) (see Table 1) was used as the structure. In this example, we investigated the decay of phages within the wells of a fluid device, and detected target molecules, specifically DNA encapsulated in f1 phage, within the wells of the fluid device.

[0089] (Preparation of nucleic acid detection reagents) The Invader reaction reagent in Example 2 uses the reagents shown in Table 1 and contains 0.5 μM AllerProbe 1 (SEQ ID NO: 2), 1 μM Invader Oligo 1 (SEQ ID NO: 3) (ICA Oligo 1) (both from Fasmac), 4 μM FRET Cassette 1 (Alexa488-BHQ) (SEQ ID NO: 4) (Nippon Bioservices Co., Ltd.) (fluorescent substrate), 0.1 mg / mL FEN-1, 50 mM Tris-HCl (pH 8.5), 20 mM MgCl2, and 0.05% Tween20. The concentrations of each component in these Invader reaction reagents are the final concentrations in the Invader reaction reagents of Example 2. A solution (Solution C) was prepared by mixing the above-mentioned Invader reaction reagent with Bugbuster (Merck Millipore), a nucleic acid extraction reagent containing a surfactant, in a 1:1 ratio.

[0090] <Transportation of mixed solution> A 20 μL solution, containing a suspension of the f1 phage (as a virus sample), and solution C (a mixture of the Invader reaction reagent and Bugbuster), was delivered to a fluid device and introduced into each well. Furthermore, a mixture of f1 phage suspension and solution C was delivered at four different f1 phage concentrations so that the final f1 phage concentrations in the fluid device were 0% (no f1 phage added), 0.1%, 0.5%, and 1.0%. As in Example 1 above, the f1 phage concentration is expressed as a dilution ratio when the concentration of commercially available f1 phage suspension is set to 100%. Furthermore, the concentrations of each component in solution C are adjusted so that the f1 phage does not disintegrate immediately after mixing with the f1 phage suspension, but disintegrates only after the nucleic acid extraction reaction described later is performed after the solution is introduced into the wells. Next, 150 μL of FC-40 (Sigma) was delivered as a sealing solution to seal each well.

[0091] <Nucleic acid extraction reaction> The above fluid device was placed on a hot plate and reacted at 66°C for 30 minutes. This broke down the capsid structure of the f1 phage within the sealed well, allowing for DNA extraction.

[0092] <Nucleic acid detection reaction> Upon heating of the above fluid device, each reaction of the Invader method proceeds as in Example 1, ultimately generating a fluorescence signal for Alexa488.

[0093] <Fluorescence observation of wells> After heating at 66°C for 30 minutes, fluorescence images of the fluorescence signals obtained from the nucleic acid detection reaction in each well of the fluid device were captured using a BZ-710 fluorescence microscope (KEYENCE) with a 10x objective lens. The exposure time was 3000 msec using a GFP fluorescence filter. The results of Example 2 are shown in Figures 7 and 8. As shown in Figures 7 and 8, in wells containing the target molecule, f1 phage-encapsulated DNA, a fluorescence signal originating from the target molecule could be observed. Furthermore, as shown in the fluorescence image in Figure 7 and the graph in Figure 8, the number of wells in which fluorescence was observed increased in a dependent manner with increasing f1 phage concentration.

[0094] This example suggests that, depending on the concentration of the f1 phage, the f1 phage was introduced into the well, and the nucleic acid inside the f1 phage could be detected. In other words, the above method suppresses DNA loss in f1 phages and allows for accurate detection of the target molecule, nucleic acid.

[0095] [Example 3] (Bead binding to phages, introduction of bead-bound phages into wells, and detection of nucleic acids within the virus (phage)) The f1 phage (reagent name: Escherichia coli phage f1, National Institute of Technology and Evaluation, model number: NBRC20010) (containing the base sequence of "SEQ ID NO: 1" as its encapsulated DNA) (see Table 1) was used as the target molecule for detecting intraviral nucleic acids. Magnetic beads containing antibodies were used as capture materials to introduce the structure into the wells as a specific binding agent.

[0096] <Preparation of nucleic acid detection reagents> The Invader reaction reagent in Example 3 uses the reagents shown in Table 1 and contains 0.5 μM Allele Probe 1 ((f1phage)Allele probe 1) (SEQ ID NO: 2), 1 μM Invader Oligo 1 (SEQ ID NO: 3) (ICA Oligo 1) (all from Fasmac), 4 μM FRET Cassette 1 (Alexa488-BHQ) (SEQ ID NO: 4) (Nippon Bioservices Co., Ltd.) (fluorescent substrate), 0.1 mg / mL FEN-1, 50 mM Tris-HCl (pH 8.5), 20 mM MgCl2, and 0.05% Tween 20. The concentrations of each component in these Invader reaction reagents are the final concentrations in the Invader reaction reagents of Example 3. A solution (Solution D) was prepared by mixing the above-mentioned Invader reaction reagent with Bugbuster (Merck Millipore), a nucleic acid extraction reagent containing a surfactant, in a 1:1 ratio.

[0097] <Preparation of antibody-immobilized magnetic beads> To immobilize phage antibodies onto magnetic beads, anti-f1 phage antibody (type "Anti-M-13 Phage Coat Protein", Funakoshi Co., Ltd.) is added to a solution of carboxyl-modified magnetic beads (Magnosphere, LC300, JSR Corporation) and mixed to 100 μL. The mixture is then reacted in a rotator for 30 minutes, and then the condensing agent EDC is added and the mixture is reacted for 3 hours to immobilize the antibody onto the carboxyl magnetic beads.

[0098] To remove unreacted antibodies and reagents after the reaction, antibody-immobilized carboxyl magnetic beads were magnetically collected using a magnetic stand. Subsequently, the beads were washed three times with PBS-T (PBS containing 0.1% Tween20) to prepare antibody-immobilized carboxyl magnetic beads. This resulted in the preparation of capture materials containing specific binding substances.

[0099] <Reaction of sample and antibody-immobilized beads> A complex was formed by mixing f1 phage (0% or 1%) as the structure with 100 μg / mL antibody-immobilized carboxyl magnetic beads as a capture substance containing a specific binding agent, totaling 100 μL. The mixture was reacted on a rotator at room temperature for 1 hour to form a complex.

[0100] After the reaction, the resulting magnetic beads were magnetically collected using a magnetic stand, and the supernatant was removed and washed by repeating the process of adding 0.1% Tween-containing PBS (PBS-T) three times. Finally, the supernatant was removed to obtain the complex. This complex is formed when an anti-f1 phage antibody immobilized on a bead recognizes and binds to the coat protein on the surface of the f1 phage. After washing the resulting complex, the reaction mixture was prepared by diluting it with PBS.

[0101] <Transportation of reaction mixture solution> 20 μL of a solution, which is a mixture of the above reaction mixture (0% and 1% of f1 phage recovered by beads) and a 1:1 solution D of the Invader reaction reagent and Bugbuster used in this example, is introduced into each well of the fluid device. Subsequently, 150 μL of FC-40 (Sigma) is introduced as a sealing solution to seal each well. As a result, if f1 phage recovered by beads is present, one or fewer f1 phage per well is sealed within the well.

[0102] <Well's observations> After the detection reaction, bright-field images of the fluid device were taken using a BZ-710 microscope with a 10x objective lens. In the well into which the beads were introduced, black was observed in the bright-field image, as shown on the right side of Figure 9.

[0103] <Nucleic acid extraction reaction> The above device was placed on a hot plate and reacted at 66°C for 15 minutes. This broke down the capsid structure of the f1 phage within the sealed well, allowing for DNA extraction.

[0104] <Nucleic acid detection reaction> After heating at 66°C for 15 minutes as described above, each reaction of the Invader reaction proceeds as in Example 1, and ultimately the fluorescent signal of Alexa488 is generated.

[0105] <Fluorescence observation of wells> After heating at 66°C for 15 minutes, fluorescence images of the fluorescence signals obtained from the nucleic acid detection reaction in each well of the fluid device were captured using a 10x objective lens with a BZ-710 fluorescence microscope (KEYENCE). The exposure time was 3000 msec using a GFP fluorescence filter. The results of Example 3 are shown in Figure 9. As shown on the right side of Figure 9 (bright-field image), the wells shown in black are the wells into which the beads were introduced. Furthermore, as shown in the 1% fluorescence image on the left side of Figure 9, fluorescence signals originating from the target molecule were observed in wells containing the target molecule, f1 phage-encapsulated DNA.

[0106] The above method suppressed DNA loss in f1 phages and enabled accurate detection of the target molecule, nucleic acid. Furthermore, by using magnetic beads containing antibodies as a specific binding agent, the structure could be efficiently introduced into the wells. Furthermore, the anti-f1 phage antibody recognizes the coat protein on the surface of the f1 phage. Therefore, in addition to detecting the target molecule contained within the f1 phage using the method described above, it can also be said that the target molecule on the surface of the f1 phage was detected by using the captured substance.

[0107] [Example 4] (Investigation of various phage decay methods) In this example, we investigated whether phage decay was possible using various methods. Using the f1 phage (reagent name: Escherichia coli phage f1, National Institute of Technology and Evaluation, model number: NBRC20010), a type of virus, as a structure (containing the base sequence of "SEQ ID NO: 1" as its encapsulated DNA) (see Table 1), we investigated phage decay using various methods, and detected target molecules using nucleic acids, specifically the f1 phage encapsulated DNA, as the target molecule.

[0108] (Preparation of nucleic acid detection reagents) To perform the Digital Invader reaction, a type of ICA reaction, an Invader reaction reagent (ICA reaction reagent) was prepared as a nucleic acid detection reagent. The invader reaction reagent in Example 4 was prepared using the reagents shown in Table 1, consisting of 0.5 μM Allele Probe 1 ((f1phage)Allele probe 1) (SEQ ID NO: 2), 1 μM Invader Oligo 1 (SEQ ID NO: 3) (ICA Oligo 1) (both from Fasmac), 2 μM FRET Cassette 1 (Alexa488-BHQ) (SEQ ID NO: 4) (Nippon Bioservices Co., Ltd.) (fluorescent substrate), and 0.1 mg / mL FEN-1 (Flap endonuclease-1). The solution was prepared to contain 50 mM MOPS (3-morpholinopropanesulfonic acid buffer) (pH 7.9) and 20 mM MgCl2. The concentrations of each component in these Invader reaction reagents are the final concentrations in the Invader reaction reagent according to Example 4.

[0109] <Investigation of phage breakdown using various methods, and investigation of nucleic acid extraction reactions> The following conditions were used to examine phage decay. • Sample 1: Phage (10%) was subjected to sonication (probe-type ultrasound) for 1 minute. Sample 2: Phage (10%) was mixed with Bugbuster (50%) and stirred at 37°C for 30 minutes. Sample 3: Phage (10%) was heated at 70°C for 30 minutes. Sample 4: Phage (10%) was mixed with Bugbuster (50%) and stirred at 70°C for 30 minutes.

[0110] Next, the above-mentioned invader reaction reagent and samples 1-4 were mixed in sample tubes to prepare solutions (samples 1A-4A) with a phage concentration of 1% and a volume of 10 μL. As a negative control, the invader reaction reagent (sample 5A) alone and a mixed solution of the invader reaction reagent and phage (sample 6A) were prepared and evaluated. Furthermore, as a positive control, a solution (Sample 7A) was prepared by adding phage-derived nucleic acid (phage oligo) (SEQ ID NO: 1), which had been previously extracted from phages, to the Invader reaction reagent to a concentration of 30 pM, and this was also evaluated. The sample solutions of these samples 1A to 7A were processed using a real-time PCR instrument, Light Cycler (manufactured by Roche Diagnostics, Inc.), at 66°C for 60 minutes. The results of Example 4 are shown in Figures 10 and 11. Figure 11 is a magnified view of Figure 10 up to approximately 1200 seconds. In the graphs in Figures 10 and 11, the horizontal axis represents processing time (seconds), and the vertical axis represents fluorescence intensity. The correspondence between the items in Figures 10 and 11 and the sample numbers is as follows. ·Sample 1A: ICA+phage+sonic ·Sample 2A: ICA+phage+bug ·Sample 3A: ICA+phage+heat Sample 4A: ICA + phage + bug + heat • Sample 5A: ICA • Sample 6A: ICA + phage ·Sample 7A: ICA+phage+phage oligo 30pM

[0111] In all samples—sonicated sample 1A, sample 2A with Bugbuster containing a surfactant added to the phage, sample 3A with heat treatment at 70°C, sample 4A with Bugbuster containing a surfactant added to the phage and heat treatment at 70°C for 30 minutes, and the positive control sample 7A—fluorescence intensity increased even at short treatment times of 300 seconds and 600 seconds. In sample 1A, where the phage was subjected to sonication, and in sample 4A, where Bugbuster containing a surfactant was added to the phage and then heat-treated at 70°C, a particularly short increase in fluorescence intensity was observed. As a negative control, sample 5A, which was subjected to the Light Cycler with only the Invader reaction reagent, showed almost no increase in fluorescence intensity. Furthermore, since the sample was processed at 66°C using a Light Cycler, an increase in fluorescence intensity was observed over time in sample 6A (ICA + phage), but the increase was more gradual compared to samples 1A to 4A. Compared to samples 5A and 6A, samples 1A to 4A, in which phages were disintegrated using various methods, showed an increase in fluorescence intensity over a short period of time. These results revealed that, in addition to treatment with surfactants, multiple methods such as sonication and heat treatment, as well as combinations of multiple methods, are effective in disrupting phages. This embodiment demonstrates that by combining the above-mentioned methods, phage lysis and nucleic acid extraction from within the phage can be performed in a short time.

[0112] [Example 5] (Detection of nucleic acids within viruses, detection of internal nucleic acids by lysis of E. coli) In this example, we investigated the detection of nucleic acids derived from phages using cell lysis with E. coli. The f1 phage (reagent name: Escherichia coli phage f1, National Institute of Technology and Evaluation, model number: NBRC20010) (containing the base sequence of "Sequence ID 1" as its encapsulated DNA) (see Table 1) is used as a structural agent to detect intraviral nucleic acids. Furthermore, a biological method is employed to extract the target molecule, intraviral nucleic acid, using Escherichia coli (Escherichia coli) (National Institute of Technology and Evaluation, model number: NBRC13965) as a lipid bilayer donor.

[0113] <Preparation of a mixture of sample and lipid bilayer donor> As a virus sample, a reconstituted aqueous solution of 1% final concentration f1 phage (NBRC20010) and a reconstituted aqueous solution of 1% final concentration Escherichia coli (NBRC13965) were mixed to a total volume of 100 μL. The f1 phage binds to a viral receptor on the surface of E. coli cells, forming a complex between the f1 phage and E. coli. Note that the final concentrations of f1 phage and E. coli are the final concentrations in the fluid device.

[0114] (Preparation of nucleic acid detection reagents) To perform the Digital Invader reaction, a type of ICA reaction, an Invader reaction reagent (ICA reaction reagent) was prepared as a nucleic acid detection reagent. The Invader reaction reagent in Example 5 was prepared using the reagents shown in Table 1, containing 0.5 μM Allele Probe 1 ((f1phage)Allele probe 1) (SEQ ID NO: 2), 1 μM Invader Oligo 1 (SEQ ID NO: 3) (ICA Oligo 1) (both from Fasmac), 4 μM FRET Cassette 1 (Alexa488-BHQ) (SEQ ID NO: 4) (Nippon Bioservices Co., Ltd.) (fluorescent substrate), 0.1 mg / mL FEN-1, 50 mM Tris-HCl (pH 8.5), 20 mM MgCl2, and 0.05% Tween 20. The concentrations of each component in these Invader reaction reagents are the final concentrations in the Invader reaction reagents within the fluid device according to Example 5.

[0115] <Transportation of mixed solution> A 20 μL solution, prepared by mixing the above-mentioned mixture (a mixture of f1 phage and E. coli) and the above-mentioned Invader reaction reagent in a 1:1 ratio, was delivered to a fluid device and introduced into each well. Subsequently, 150 μL of FC-40 (Sigma) was delivered as a sealing solution to seal each well. As a result, one or fewer f1 phage cells were sealed in each well. Furthermore, after sealing the wells, the fluid device was left undisturbed at room temperature for 60 minutes. As a negative control, the same procedure was performed on a solution using only f1 phage without E. coli, instead of the reaction mixture described above (a reaction mixture of f1 phage and E. coli). Specifically, 20 μL of a solution containing f1 phage and the above-mentioned invader reaction reagent in a 1:1 ratio was delivered to the fluid device and introduced into each well. Subsequently, 150 μL of FC-40 (Sigma) was delivered as a sealing solution to seal each well.

[0116] <Nucleic acid extraction reaction, nucleic acid detection reaction> By sealing the wells and allowing the reaction to proceed for 60 minutes, the f1 phage structure recognizes and binds to the surface membrane structure (protein and lipid bilayer) of E. coli, and the target molecule, DNA, is injected into the E. coli from the f1 phage. In other words, a complex between the f1 phage and E. coli is gradually formed during the 60-minute reaction. Furthermore, DNA is replicated within the E. coli, and phages are generated. The E. coli is then disintegrated by the f1 phage generated within the cell, releasing the injected and replicated DNA. Furthermore, the above fluid device was reacted on a hot plate at 66°C for 30 minutes. This allowed the released f1 phage DNA to be recognized, and each step of the invader reaction proceeded as in Example 1, ultimately resulting in the generation of an Alexa488 fluorescence signal.

[0117] <Fluorescence observation of wells> As described above, the fluid device was heated at 66°C for 30 minutes. Then, fluorescence images of the fluorescence signals obtained from the nucleic acid detection reaction in each well of the fluid device were captured using a BZ-710 fluorescence microscope (KEYENCE) with a 10x objective lens. The exposure time was 3000 msec using a GFP fluorescence filter. Figure 12 shows the results of fluorescence imaging according to Example 5. As shown on the left side of Figure 12, in wells containing both f1 phage and E. coli, a fluorescence signal originating from the target molecule could be observed. As shown in the fluorescence image in Figure 12, when f1 phage was used to infect E. coli and then introduced into the fluid device (left side of Figure 12), more DNA could be detected than when only f1 phage was introduced into the fluid device (right side of Figure 12). This result is thought to be because when the f1 phage infects E. coli, the f1 phage-derived DNA is amplified within the E. coli, and ultimately the E. coli lyses, releasing the target molecule, the f1 phage DNA, which leads to the observation of numerous fluorescence signals originating from the target molecule. Furthermore, as shown on the right side of Figure 12, when only the f1 phage was introduced into the fluid device without adding E. coli, a slight fluorescence signal was observed. In the case without E. coli, it is thought that a small amount of DNA was released into the well due to the effects of heating and Tween20, a surfactant contained in the buffer.

[0118] Using the method described above, DNA loss in f1 phages was suppressed, and the target molecule, nucleic acid, could be detected with high accuracy. Furthermore, by using E. coli with a lipid bilayer, the target molecule, DNA, could be extracted from the viral structure within the well. Furthermore, this study revealed that even with targets that have rigid membranes, such as phages, the DNA inside the phage can be easily detected by first transferring the DNA to another substance (for example, E. coli) and then subjecting it to heating or other methods.

[0119] [Example 6] (Detection of intracellular nucleic acids 1) Using human colon adenocarcinoma cells HT29 as a structure, the detection of a target molecule was carried out with a region containing a single nucleotide polymorphism (wild type) of the EGFR (epidermal growth factor receptor) gene, which is a nucleic acid, specifically DNA, as the target molecule. <PKH staining of HT29 cells> To confirm the introduction of HT29 cells into the fluid device, HT29 cells stained with PKH were prepared. The procedure for PKH staining of HT29 cells is as follows. The HT29 cultured cells were detached to prepare a suspension. Also, the suspension was centrifuged at 3500 rpm for 1 minute to collect the cells. Subsequently, a sufficient amount of Dilluent C reagent was added to the collected cells. The PKH solution was added to the cells to which the Dilluent C reagent was added, and the mixture was allowed to stand at room temperature for 10 minutes. Thereafter, the cell suspension to which the PKH solution was added was centrifuged at 3500 rpm for 1 minute to collect the PKH-stained cells, which were resuspended in the DMEM medium solution. The PKH-stained HT29 cells resuspended in the DMEM medium solution were mixed with the Invader reaction reagent described below and used for introduction into the fluid device. Note that the cells obtained by PKH staining of HT29 cells were prepared such that the number of cells in the fluid device to be introduced later was 5.16×10 5 cell / mL.

[0120] <Preparation of nucleic acid detection reagent> To perform the Digital Invader reaction, a type of ICA reaction, an Invader reaction reagent was prepared as a nucleic acid detection reagent. The Invader reaction reagent in this example uses the reagents shown in Table 1 and contains 0.5 μM AllerProbe 2 (SEQ ID NO: 5), 1 μM Invader Oligo 2 (SEQ ID NO: 6) (ICA Oligo 2) (both from Fasmac), 4 μM FRET Cassette 1 (Alexa488-BHQ) (Nippon Bioservices Co., Ltd.) (fluorescent substrate) (SEQ ID NO: 4), 0.1 mg / mL FEN-1, 50 mM Tris-HCl (pH 8.5), 20 mM MgCl2, and 0.05% Tween20. The concentrations of each component in these Invader reaction reagents are the final concentrations in the Invader reaction reagent in the fluid device according to Example 6.

[0121] <Transportation of reaction mixture solution> A solution containing PKH-stained human colon adenocarcinoma cells (HT29) and the above-mentioned Invader reaction reagent was mixed in a fluid device (cell count: 5.16 × 10⁶). 5 20 μL of (cell / mL) was delivered and introduced into each well. Subsequently, 150 μL of FC-40 (Sigma) was delivered as a sealing solution to seal each well (Figures 14 and 15). As a result, one or fewer HT29 cells were sealed in each well. As a control experiment, a solution of human colon adenocarcinoma cells HT29 stained with PKH under the same conditions except that the Invader reaction reagent was not added (cell count: 5.16 × 10⁶). 5 We also investigated the case where HT29 cells were introduced into wells by delivering a fluid (cells / mL) into a fluid device (Figure 13).

[0122] <Nucleic acid extraction reaction> The above fluid device was placed on a hot plate and reacted at 66°C for 15 minutes. This disrupted the cell membrane structure of HT29 cells within the sealed wells, allowing for the extraction of genomic DNA.

[0123] <Nucleic acid detection reaction> The fluid device described above, after the extraction reaction, was placed on a hot plate and reacted at 66°C for 15 minutes. This resulted in the recognition of the EGFR gene region by the allele probe and invader oligo, cleavage of the allele probe by FEN-1, binding of the released allele probe fragment to the FRET Cassette, cleavage of the FRET Cassette by FEN-1, and generation of a fluorescence signal from Alexa488. As a control experiment, we also investigated the case where the above-mentioned fluid device was not heated to 66°C for 15 minutes (Figure 14).

[0124] <Fluorescence observation and bright-field observation of wells> After heating at 66°C for 15 minutes, fluorescence images of the fluorescence signals obtained from the nucleic acid detection reaction in each well of the fluid device were captured using a BZ-710 fluorescence microscope (KEYENCE) with a 10x objective lens. The exposure times were 3000 msec using a GFP fluorescence filter for Alexa488 fluorescence observation, and 2000 msec using a Texas Red fluorescence filter for PKH fluorescence observation. Furthermore, after sealing the wells, bright-field images were also taken using a BZ-710 microscope with a 10x objective lens. The results of Example 6 are shown in Figures 13-15.

[0125] Figure 13 shows a fluorescence image of PKH-stained HT29 cells in an example where the Invader reaction reagent was not added. As shown in Figure 13, PKH staining revealed that cells had been introduced into the wells. Figure 14 shows a fluorescence image of a fluid device before heating (without heating at 66°C for 15 minutes) after introducing a solution of PKH-stained human colon adenocarcinoma cells (HT29) mixed with the above-mentioned Invader reaction reagent into the fluid device. Only fluorescence originating from PKH was detected. Figure 15 shows a fluorescence image obtained after introducing a solution of PKH-stained human colon adenocarcinoma cells (HT29) mixed with the above-mentioned Invader reaction reagent into a fluid device and heating the fluid device. Both fluorescence from PKH and fluorescence from Alexa488 were detected.

[0126] As shown in Figure 13, PKH staining of HT29 cells confirmed that they were indeed present in the wells of the fluid device. Furthermore, the fluorescence of Alexa488 can be detected when DNA present inside HT29 cells is released into the solution. Therefore, as shown in Figure 15, the detection of Alexa488 revealed that the cell membrane of the HT29 cells was damaged. In this example, when the fluorescence images of PKH and Alexa488 were overlaid (overlay images in Figures 13 to 15), only PKH could be detected in the case where the invader reaction reagent containing the surfactant was not mixed (Figure 13), and in the case where the invader reaction reagent containing the surfactant was mixed but before heating (Figure 14). However, as shown in Figure 15, after heating, fluorescence from Alexa488 could be detected in the same well in which PKH was detected. Therefore, in the example shown in Figure 15, where a surfactant was added and heating was performed, it is thought that the Invader reaction proceeded in the wells containing HT29 cells, allowing for the detection of DNA inside the HT29 cells. These results demonstrate that, according to this embodiment, HT29 cells can be destroyed by heating, allowing for the detection of nucleic acids within them, and nucleic acids can be extracted from the cells present in the wells. Thus, in wells containing the EGFR gene region with the target molecule (single nucleotide polymorphism), we were able to observe a fluorescence signal originating from the target molecule.

[0127] The above method demonstrated that it is possible to suppress the loss of genomic DNA in HT29 cells and accurately detect target molecules, such as nucleic acids.

[0128] Although embodiments of the present invention have been described above, the configurations and combinations thereof in the embodiments are merely examples, and additions, omissions, substitutions, and other modifications are possible without departing from the spirit of the present invention. Furthermore, the present invention is not limited by the embodiments. [Industrial applicability]

[0129] According to the present invention, compared to conventional methods in which target molecules are extracted from a structure and then distributed to each well of a microarray using a channel, it is possible to provide a technology that can detect target molecules with high accuracy. [Explanation of symbols]

[0130] 100... Fluid devices 101…Lid material 102... Fluid delivery port 103...Waste liquid port 104...Base material 105... Well 106…flow channel 107…Reagent solution 108… Reagent solution filled in wells 201...Sealing liquid 202...miniature section 301... Tiny droplets that emit a signal 11…Target molecule 12...Structure 12'...Structure after extraction 13...Specific binding substance 14...Captured object

Claims

1. A method for detecting a target molecule, comprising: A step of preparing a fluidic device including a substrate and a well array disposed on the substrate, delivering a liquid having a structure containing a target molecule dispersed therein to the well array in the fluidic device, and introducing the structure into a well of the well array; a step of delivering a sealing liquid to the well array to form a layer of the sealing liquid on top of the liquid introduced into the wells, thereby sealing the liquid in the wells; extracting the target molecule from the structure; detecting the target molecule; Equipped with A method for detecting a target molecule, comprising: A method for detecting a target molecule, comprising detecting two or more types of the target molecule in the step of detecting the target molecule.

2. The method described in claim 1, wherein the step of extracting the target molecule is performed within the well.

3. A method according to claim 1 or 2, wherein the step of extracting the target molecule is performed after the step of sealing the liquid in the well.

4. The method described in claim 3, wherein the step of detecting the target molecule is performed after the step of extracting the target molecule.

5. A method according to any one of claims 1 to 4, wherein the step of detecting the target molecule comprises a signal amplification reaction.

6. The method of claim 1, further comprising a step of detecting the structure.

7. The method described in claim 6, wherein the step of detecting the target molecule and the step of detecting the structure are performed simultaneously.

8. A method according to any one of claims 1 to 7, wherein the structure is at least one of an exosome, a cell, a bacterium, a virus, a fungus, and an endoplasmic reticulum.

9. The method of claim 1, wherein the target molecule is a nucleic acid or a protein.

10. A method according to any one of claims 1 to 9, wherein in the introducing step, not more than one of the structures is introduced into one well in the well array.

11. A method described in any one of claims 1 to 10, wherein the step of extracting the target molecule is carried out by any one of physical, chemical, or biological techniques.

12. The method according to any one of claims 1 to 11, further comprising a step of binding a capture substance having a specific binding substance for the structure to the structure.

13. The method according to claim 12, wherein the step of introducing the structure is carried out by introducing the capture object into the well of the well array.

14. The method described in claim 12 or 13, wherein the specific binding substance is an antibody.

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