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    • 专利摘要:
    METHOD FOR SEALING SUBSTANCES AND METHOD OF DETECTION OF TARGET MOLECULE. The present invention provides a method for sealing a substance as a technique for effectively sealing many substances, such as microspheres, nucleic acid, protein, virus, cells and lipid membrane complex, in an array, the present invention providing a method for sealing a substance, including: (i) a step of introducing a first solvent containing a substance on a substrate in which a plurality of containers capable of storing the substance are formed which are separated from each other by a side wall; and (ii) a step of introducing a second solvent having a specific gravity greater than that of the first solvent on the first solvent, with step (ii) being carried out after step (i).
    公开号:BR112017000270B1
    申请号:R112017000270-1
    申请日:2015-07-01
    公开日:2021-03-23
    发明作者:Hiroyuki Noji；Lisa YAMAUCHI
    申请人:Japan Science And Technology Agency；
    IPC主号:
    专利说明:

    [0001] [001] The present invention relates to a method for storing substances and detecting a target molecule. BACKGROUND OF THE INVENTION
    [0002] [002] A single molecule assay is known as a method for carrying out multiple assays looking at biomolecules, such as proteins and nucleic acids in such a way that biomolecules are individually identified. In order to perform the single molecule assay, some methods have been known.
    [0003] [003] Patent Literature 1 describes a microchamber for the detection of single molecule enzyme activity. This microchamber includes a container part in which a liquid droplet can be sealed and which has the capacity to store a liquid droplet of up to 1,000 fL (fentoliters). The container part is made of a recess provided in at least one of a first element and a second element that are connected to each other. According to Patent Literature 1, an enzymatic reaction is carried out on the liquid droplet. With such a configuration, the enzymatic reaction can be carried out with a high concentration of the reaction products, even if the number of molecules of the reaction products is quite small. Thus, it is possible to detect an activity of an enzyme molecule.
    [0004] [004] Different Patent Literature 1 describes a method for performing a single molecule enzyme assay employing a matrix where a liquid droplet is covered with oil, in an order of fentoliter, and accessible directly from the outside. This matrix includes a hydrophilic region pattern obtained from a hydrophilic surface on which a hydrophobic region with a height of 17 nm is provided.
    [0005] [005] Different Patent Literature 2 describes a method for detecting a protein by a single molecule enzyme-linked immunosorbent assay (ELISA). According to this method, a very small amount of protein is captured by tiny microspheres covered with specific antibodies to the protein, and the microsphere complexes and proteins are fluorescently labeled. Then, the microspheres including the complexes are introduced into a reaction chamber by centrifugal force. After that, the number of microspheres that capture the proteins is counted. In this way, proteins are quantitatively tested. REFERENCES OF QUOTES Patent Literature
    [0006] [006] Patent Literature 1 - Publication of Japanese Patent Application, Tokukai, No. 2004-309405 A Different Patent Literature
    [0007] [007] Different Patent Literature 1 - S. Sakakihara et al., Lab Chip, 2010, 10, 3355-3362
    [0008] [008] Different Patent Literature 2 - David M Rissin et al., Nature Biotechnology: doi: 10.1038 / nbt.1641 BRIEF SUMMARY OF THE INVENTION Technical problem
    [0009] [009] In order to detect, for example, low concentration disease markers for early detection of diseases, infectious diseases and the like, there is a demand for biosensitivity techniques developed to provide higher sensitivities. For example, in a case where a million cancer cells included in a tumor with a volume of 1 mm3 secrete marker proteins (100 molecules per cell) in 5 liters of blood, a concentration of the proteins in the blood is approximately 30 aM. A technique is needed to detect target molecules of such a low concentration.
    [0010] [0010] A possible method for detecting such target molecules may be to detect the target molecules by the aforementioned single molecule enzyme assay at a single molecule level sensitivity. Specifically, such a method is performed by (i) sealing the target molecule specifically in a liquid droplet of the order of phentoliter (very small liquid droplet), (ii) binding the target molecule to a substance such as an antibody labeled with an enzyme and ( iii) detecting an enzyme activity by labeling the antibody in the manner mentioned above. The sealing of the target molecule specifically in the very small liquid droplet can be accomplished by a method using, for example, a microsphere labeled with a substance such as another antibody for specific binding to the target molecule. In this method, after the microsphere is attached to the target molecule, the microsphere is sealed in the very small droplet of solution.
    [0011] [0011] Incidentally, in order to effectively detect target molecules that are contained in a solution only in a very small amount, for example, approximately 30 aM target molecules, as described above, it is necessary to prepare a large number of very liquid droplet matrices small, as many as approximately one million, and cause the matrices to capture the microspheres.
    [0012] [0012] However, according to the method published by Patent Different Literature 2, microspheres need to be introduced into the matrices by strong centrifugal force and, therefore, a lot of time and efforts are needed. In addition, the number of matrices used in the Different Patent Literature 2 method is approximately fifty thousand. Therefore, the Different Patent Literature 2 method is quite difficult to apply to the case requiring approximately one million matrices. Thus, with the Patent Different Literature 2 method, it is difficult to effectively store a large number of microspheres in the matrices. In fact, none of the publications in Patent 1 and Non-Patent Literature 1 reveal any method for solving such a problem.
    [0013] In view of this, one of the objectives of the present invention is to provide a technique for effectively storing a large number of substances, such as microspheres, nucleic acid, protein, viruses, cells and lipid membrane complex in a matrix. Solution of the problem
    [0014] [1] Método para armazenar uma substância, incluindo: (i) uma etapa de Introdução de um primeiro solvente contendo uma substância sobre um substrato no qual é formada uma pluralidade de recipientes capazes de armazenar a substância os quais são separados uns dos outros por uma parede lateral; e (ii) uma etapa de introdução de um segundo solvente com uma gravidade específica maior do que a do primeiro solvente sobre o primeiro solvente, sendo a etapa (ii) realizada após a etapa (i). [2] O método de acordo com [1], em que pelo menos um do primeiro solvente e do segundo solvente contém um agente tensoativo. [3] O método de acordo com [2], em que o agente tensoativo no primeiro solvente tem uma concentração de 0,01% a 1%. [4] O método de acordo com [2] ou [3], em que o agente tensoativo é TWEEN 20 ou Triton X-100. [5] Método de acordo com qualquer um de [1] a [4], em que o segundo solvente é pelo menos um selecionado do grupo consistindo em hidrocarboneto saturado, hidrocarboneto insaturado, hidrocarboneto aromático, óleo de silicone, polímero epóxido de hexafluorpropileno, polímero tendo uma estrutura de hidrofluoréter, perfluorpoliéter, polímero de clorotrifluoretileno e um polímero com uma estrutura de perfluorcarboneto, ou é uma mistura incluindo pelo menos um dos mesmos. [6] O método de acordo com qualquer um de [1] a [5], em que o primeiro solvente é pelo menos um selecionado do grupo que consiste em água, álcool hidrofílico, éter hidrofílico, cetona, solventes de nitrila, sulfóxido de dimetila e N,N-dimetilformamida, ou é uma mistura incluindo pelo menos um desses. [7] O método de acordo com [5] ou [6], em que a parede lateral tem uma superfície superior hidrofóbica incluindo uma resina de polímero fluorcarbonado e o segundo solvente é um polímero tendo uma estrutura perfluorcarbonada. [8] O método de acordo com [7], em que a resina de polímero fluorcarboneto é resina fluorcarbonada amorfa. [9] O método de acordo com qualquer um de [1] a [8], em que uma região incluindo os recipientes do substrato é aberta para o exterior. [10] Método de acordo com qualquer um de [1] a [9], em que a substância é selecionada dentre um ou mais de microesferas, ácido nucleico, proteína, vírus, células e complexo de membrana lipídica. [11] Um método para detectar uma molécula alvo, incluindo: (i) uma etapa de reação das microesferas capturando especificamente as moléculas alvo com as moléculas alvo; uma etapa de realização, por emprego das microesferas, de um método citado em qualquer um de [1] a [9], a etapa (ii) sendo realizada após a etapa (i); e (iii) uma etapa para determinar se ou não uma das microesferas que capturaram a molécula alvo está armazenada em cada um da pluralidade de recipientes, a etapa (iii) sendo realizada após a etapa (ii). [12] O método de acordo com [11], em que as microesferas são tais microesferas às quais as moléculas especificamente anexáveis às moléculas alvo estão ligadas. [13] Um método para armazenar uma substância em recipientes providos em um substrato por introdução de um solvente hidrofílico contendo a substância nos recipientes e revestimento do solvente hidrofílico contendo a substância introduzida nos recipientes com um solvente hidrofóbico, incluindo: (i) uma etapa de Introdução de um primeiro solvente contendo uma substância sobre um substrato no qual é formada uma pluralidade de recipientes capazes de armazenar a substância os quais são separados uns dos outros por uma parede lateral; e (ii) uma etapa de introdução de um solvente hidrofóbico com uma maior gravidade específica que a do solvente hidrofílico no solvente hidrofílico, a etapa (ii) sendo realizada após a etapa (i). [14] O método de acordo com [13], em que pelo menos um dentre o solvente hidrofílico e solvente hidrofóbico contém um surfactante. [15] O método de acordo com [14], em que o surfactante no solvente hidrofílico tem uma concentração de 0,01% a 1%. [16] O método de acordo com [14] ou [15], em que o surfactante é TWEEN 20 ou Triton X-100. [17] Método de acordo com qualquer um de [13] a [16], em que o solvente hidrofóbico é pelo menos um selecionado do grupo consistindo em hidrocarboneto saturado, hidrocarboneto insaturado, hidrocarboneto aromático, óleo de silicone, polímero epóxido de hexafluorpropileno, um polímero possuindo uma estrutura de hidrofluoréter, perfluorpoliéter, polímero de clorotrifluoretileno, e um polímero possuindo uma estrutura perfluorcarbonada, ou é uma mistura incluindo pelo menos um dos citados. [18] O método de acordo com qualquer um de [13] a [17], em que o solvente hidrofílico é pelo menos um selecionado do grupo que consiste em água, álcool hidrofílico, éter hidrofílico, cetona, solventes de nitrila, dimetil sulfóxido e N,N-dimetilformamida, ou é uma mistura incluindo pelo menos um dos mesmos. [19] O método de acordo com [17] ou [18], em que a parede lateral tem uma superfície superior hidrofóbica incluindo uma resina de polímero fluorcarbonado e o solvente hidrofóbico é um polímero tendo uma estrutura perfluorcarbonada. [20] O método de acordo com [19], em que a resina de polímero fluorcarbonado é resina fluorcarbonada amorfa. [21] O método de acordo com qualquer um de [13] a [20], em que uma região incluindo os recipientes do substrato é aberta para o exterior. [22] O método de acordo com qualquer um de [13] a [21], em que a substância é uma ou mais selecionada dentre microesferas, ácido nucleico, proteína, vírus, células e complexo de membrana lipídica. [23] Um método para detectar uma molécula alvo, incluindo: (i) uma etapa de reação das microesferas capturando especificamente as moléculas alvo com as moléculas alvo; (ii) uma etapa de realização, por emprego das microesferas, de um método citado em qualquer um de [13] a [21], a etapa (ii) sendo realizada após a etapa (i); e (iii) uma etapa para determinar se ou não uma das microesferas que capturaram a molécula alvo está armazenada em cada um da pluralidade de recipientes, a etapa (iii) sendo realizada após a etapa (ii). [24] O método de acordo com [21], em que as microesferas são tais microesferas às quais as moléculas especificamente anexáveis às moléculas alvo estão ligadas. [0014] In order to achieve the above objective, the present invention provides the following method for storing a substance and the like. [1] Method for storing a substance, including: (i) a step of introducing a first solvent containing a substance on a substrate in which a plurality of containers capable of storing the substance are formed which are separated from each other by a side wall; and (ii) a step of introducing a second solvent with a specific gravity greater than that of the first solvent on the first solvent, with step (ii) being carried out after step (i). [2] The method according to [1], in which at least one of the first solvent and the second solvent contains a surfactant. [3] The method according to [2], in which the surfactant in the first solvent has a concentration of 0.01% to 1%. [4] The method according to [2] or [3], where the surfactant is TWEEN 20 or Triton X-100. [5] Method according to any one of [1] to [4], wherein the second solvent is at least one selected from the group consisting of saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, silicone oil, hexafluorpropylene epoxy polymer, polymer having a hydrofluorether structure, perfluorpolyether, chlorotrifluorethylene polymer and a polymer with a perfluorocarbon structure, or is a mixture including at least one of them. [6] The method according to any one of [1] to [5], in which the first solvent is at least one selected from the group consisting of water, hydrophilic alcohol, hydrophilic ether, ketone, nitrile solvents, sulfur dioxide dimethyl and N, N-dimethylformamide, or is a mixture including at least one of these. [7] The method according to [5] or [6], wherein the side wall has a hydrophobic upper surface including a fluorocarbon polymer resin and the second solvent is a polymer having a perfluorocarbon structure. [8] The method according to [7], wherein the fluorocarbon polymer resin is amorphous fluorocarbon resin. [9] The method according to any one of [1] to [8], in which a region including the substrate containers is opened to the outside. [10] Method according to any one of [1] to [9], in which the substance is selected from one or more of microspheres, nucleic acid, protein, virus, cells and lipid membrane complex. [11] A method for detecting a target molecule, including: (i) a reaction step of the microspheres specifically capturing the target molecules with the target molecules; a step of carrying out, by using the microspheres, a method mentioned in any one of [1] to [9], step (ii) being carried out after step (i); and (iii) a step to determine whether or not one of the microspheres that captured the target molecule is stored in each of the plurality of containers, step (iii) being carried out after step (ii). [12] The method according to [11], in which the microspheres are such microspheres to which the molecules specifically attachable to the target molecules are attached. [13] A method for storing a substance in containers provided on a substrate by introducing a hydrophilic solvent containing the substance into the containers and coating the hydrophilic solvent containing the substance introduced into the containers with a hydrophobic solvent, including: (i) a step of Introduction of a first solvent containing a substance on a substrate in which a plurality of containers capable of storing the substance are formed which are separated from each other by a side wall; and (ii) a step of introducing a hydrophobic solvent with a greater specific gravity than that of the hydrophilic solvent in the hydrophilic solvent, step (ii) being carried out after step (i). [14] The method according to [13], wherein at least one of the hydrophilic solvent and hydrophobic solvent contains a surfactant. [15] The method according to [14], in which the surfactant in the hydrophilic solvent has a concentration of 0.01% to 1%. [16] The method according to [14] or [15], where the surfactant is TWEEN 20 or Triton X-100. [17] Method according to any one of [13] to [16], wherein the hydrophobic solvent is at least one selected from the group consisting of saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, silicone oil, hexafluorpropylene epoxy polymer, a polymer having a hydrofluorether structure, perfluorpolyether, chlorotrifluorethylene polymer, and a polymer having a perfluorocarbon structure, or is a mixture including at least one of those mentioned. [18] The method according to any one of [13] to [17], in which the hydrophilic solvent is at least one selected from the group consisting of water, hydrophilic alcohol, hydrophilic ether, ketone, nitrile solvents, dimethyl sulfoxide and N, N-dimethylformamide, or is a mixture including at least one of them. [19] The method according to [17] or [18], wherein the side wall has a hydrophobic upper surface including a fluorocarbon polymer resin and the hydrophobic solvent is a polymer having a perfluorocarbon structure. [20] The method according to [19], wherein the fluorocarbon polymer resin is amorphous fluorocarbon resin. [21] The method according to any of [13] to [20], in which a region including the substrate containers is opened to the outside. [22] The method according to any one of [13] to [21], in which the substance is one or more selected from microspheres, nucleic acid, protein, virus, cells and lipid membrane complex. [23] A method for detecting a target molecule, including: (i) a reaction step of the microspheres specifically capturing the target molecules with the target molecules; (ii) a step of carrying out, using microspheres, a method mentioned in any one of [13] to [21], step (ii) being carried out after step (i); and (iii) a step to determine whether or not one of the microspheres that captured the target molecule is stored in each of the plurality of containers, step (iii) being carried out after step (ii). [24] The method according to [21], in which the microspheres are such microspheres to which the molecules specifically attachable to the target molecules are attached.
    [0015] [0015] The use of the method for trapping a substance according to the present invention makes it possible to effectively store many substances, such as microspheres, nucleic acid, protein, viruses, cells and lipid membrane complexes, within a matrix, by which, contributing to a technique by which low concentration target molecules are detectable with high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS
    [0016] [0016] Figure 1 consists of a series of views that schematically illustrate the procedure of the method for storing a substance according to the present invention, which illustrates cross-sectional views of a matrix 1.
    [0017] [0017] Figure 2 is a view schematically illustrating an embodiment of a target molecule detection device according to the present invention. DETAILED DESCRIPTION OF THE INVENTION
    [0018] [0018] A preferred way of carrying out the present invention will be described below with reference to the drawings. The modalities described below are only intended to show an example of an exemplary embodiment of the present invention, however the scope of the present invention is not intended to be interpreted in a limiting sense.
    [0019] [0019] In the method for storing a substance according to the present invention, the sealed substance in the containers provided on a substrate can be microspheres, nucleic acid, protein, virus, cells, lipid membrane complex or the like; however, the following embodiment will be described using microspheres as an example. 1. Method for Storing Microspheres
    [0020] [0020] With reference to figure 1, the following describes a method for storing microspheres according to the present invention. Figure 1 is a series of views schematically illustrating the procedure of the method for storing microspheres according to the present invention, which illustrates cross-sectional views of a matrix 1.
    [0021] [0021] The present embodiment deals with a case that will be described, in which the microspheres 21 and 21 'are sealed in the matrix 1 including a substrate 10. The substrate 10 includes a plurality of containers 13, each of which is capable of storing only one of the microspheres 21 and 21 'and which are separated from each other by a side wall 12 which has a hydrophobic upper surface.
    [0022] [0022] In this document, "microsphere" is used as a synonym for "particle" and is a technical term commonly used in the art. The shape of the microsphere is not particularly limited; however, it is typically spherical. The material of the microsphere is also not particularly limited and can be glass, silica gel, polystyrene, polypropylene, membrane, magnetic material or the like. Specific examples of the material include cellulose, cellulose derivatives, acrylic resin, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, vinyl and acrylamide copolymers, polystyrenes cross-linked with divinylbenzene and the like, polyacrylamide, latex gel, polystyrene dextran, rubber, silicon, plastics, nitrocellulose, cellulose, natural sponge, silica gel, glass, metal plastics, cellulose, crosslinked dextran (Sephadex (Trademark)) and agarose gel (Sepharose (Trademark)). Microspheres can be porous. The microspheres preferably have an average particle diameter of 5 μm or less, for example, approximately 1 μm to 4 μm. With this, the microspheres can be sealed efficiently in the matrix, and the matrix can obtain high density. Note that the term "average particle diameter" mentioned in this document refers to a value obtained as a result of measuring the microspheres by means of observation under an electron microscope or dynamic light diffusion.
    [0023] [0023] The present modality describes, but is not particularly limited to, a case of using microspheres that specifically capture target molecules. In the present embodiment, the microspheres to be sealed are a mixture of the microspheres 21, which have not yet captured the target molecules, and the microspheres 21 ', which have captured the target molecules.
    [0024] [0024] For example, it is possible to use, as the microspheres that specifically capture the target molecules, microspheres being attached to a molecule to specifically capture the target molecule. The molecule to specifically capture the target molecule can be linked to a modifying group on a surface of the microsphere, for example, via a linker. For example, the present invention can be configured in such a way that the molecule to specifically capture the target molecule is covalently linked to an amino group on the surface of a microsphere modified by amino groups through a cross-linking agent having N-hydroxysuccinimide and / or similar.
    [0025] [0025] The "target molecule" refers to a molecule that must be detected (target molecule). Specifically, the "target molecule" refers, in this document, to a molecule that must be detected by causing the microsphere to capture the molecule. Examples of the target molecule include (i) biomolecules, such as a protein, nucleic acid and sugar, and (ii) the virus particles themselves.
    [0026] [0026] The molecule to specifically capture a target molecule (hereinafter, such a molecule will also be referred to as a "target capture molecule") can be chosen according to the target molecule. Examples of the target capture molecule include a protein, an antibody and a nucleic acid. Preferably, a microsphere is limited to one hundred thousand or more target capture molecules. For example, in a case where the target capture molecule is an antibody, the target capture molecule has a dissociation constant in the order of nM or so. However, with the above-mentioned configuration, it is possible to cause the reaction between the microspheres and the target molecules with a sufficiently high concentration of target capture (for example, in a case where the concentration of the microspheres is 8 * 106 particles / mL , the concentration of the target capture molecules is approximately 1 nM).
    [0027] [0027] The method for storing microspheres in accordance with the present embodiment includes a step of introducing microspheres, a de-aeration step and a step of storing microspheres. Each of these steps will be described in detail below. Microspheres Introduction Stage
    [0028] [0028] The following describes the step of introducing microspheres with reference to figure 1A.
    [0029] The step of introducing microspheres is a step of introducing a first solvent 20 containing microspheres 21 and 21 'onto substrate 10. The method of introducing the first solvent 20 is not particularly limited; however, a method can be adopted which involves employing a region 14 including the containers 13 of the substrate 10 as an open well that is opened to the outside to introduce the first solvent 20 from the opening into the well.
    [0030] [0030] The first solvent 20 is preferably a hydrophilic solvent; preferably used as is, for example, at least one selected from the group consisting of water, hydrophilic alcohol, hydrophilic ether, ketone, nitrile solvents, dimethyl sulfoxide (DMSO) and N, N-dimethylformamide (DMF) or is a mixture including the at least one. Examples of hydrophilic alcohol include ethanol, methanol, propanol and glycerin. Examples of hydrophilic ether include tetrahydrofuran, polyethylene oxide and 1,4-dioxane. Examples of ketones include acetone and methyl ethyl ketone. Examples of nitrile solvents include acetonitrile.
    [0031] [0031] The first solvent 20 preferably contains a surfactant. In the microsphere storage step to be described below, a second solvent 30 with a specific gravity greater than that of the first solvent 20 is introduced into the first solvent 20 (see figure 1B), followed by a substitution based on difference in specific gravity between the first solvent 20 and the second solvent 30 to move the second solvent 30 to the lower layer of the first solvent 20 (see figure 1H). On this occasion, the surfactant can be added to the first solvent 20 and / or the second solvent 30 to promote the substitution between the first solvent 20 and the second solvent 30.
    [0032] [0032] The surfactant is not particularly limited; however, its examples include TWEEN 20 (CAS No. 9005-64-5, polyoxyethylene sorbitan monolaurate) and Triton X100 (CAS No. 9002-93-1, general name: polyethylene glycol mono-4-octylphenyl ether (n ≈ 10)). The concentration of the surfactant added to the first solvent 20 is not particularly limited; however, it is preferably 0.01 to 1%.
    [0033] [0033] In addition, it can be widely used as a surfactant, a nonionic surfactant, a zwitterionic surfactant, a surfactant derived from nature or the like.
    [0034] [0034] The anionic surfactant is classified, for example, in a carboxylic type, a sulfate type, a sulfonic type and a phosphate type. Specific examples include sodium dodecyl sulfate, sodium laurate, α-sulfo sodium fatty acid methyl ester, sodium dodecylbenzenesulfonate and sodium dodecylethoxylate sulfate; among these, sodium dodecylbenzenesulfonate is preferably used.
    [0035] [0035] The cationic surfactant is classified, for example, in a type of quaternary ammonium salt, a type of alkylamine and a type of heterocyclic amine. Specific examples include stearyltrimethylammonium chloride, distearyldimethylammonium chloride, didecyldimethylammonium chloride, cetyltripyridinium chloride and dodecyldimethylbenzylammonium chloride.
    [0036] [0036] Examples of the non-ionic surfactant include polyoxyethylene alkyl ethers, polyoxyethylene hydrogenated castor oils, polyoxyethylene fatty mono acid esters, polyoxyethylene sorbitan fatty acid esters, sucrose fatty acid esters, glycolic acid esters alkyl polyglycosides and N-methylalkyl glucamides. Among these, preferred nonionic surfactants are available under the names of Triton X (Triton X-100 and the like), Pluronic (Trademark) (Pluronic F-123, F-68 and the like), Tween (Tween 20, 40, 60 , 65, 80, 85 and similar), Brij (registered trademark) (Brij 35, 58, 98 and similar) and Span (Span 20, 40, 60, 80, 83 and 85) in addition to dodecyl ethoxylate, nonylphenol ethoxylate and lauroyl diethanol amide.
    [0037] [0037] Examples of the amphoteric surfactant include lauryl dimethyl aminoacetic acid betaine, dodecylaminomethyl dimethyl sulfopropyl betaine and 3- (tetradecyldimethylaminio) propane-1-sulfonate; however, its preferred examples include 3 - [(3-colamidopropyl) dimethylammonium] -1-propane sulfonate (CHAPS) and 3 - [(3-colamidopropyl) dimethylammonium] -2-hydroxy1-propane sulfonate (CHAPSO).
    [0038] [0038] The surfactant derived from nature is preferably, for example, lecithin or saponin. Among the compounds called lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid and phosphatidylglycerol are specifically preferred. Quilaja saponin is preferred as saponin.
    [0039] [0039] In addition to microspheres 21 and 21 ', the first solvent 20 may also include, for example, a substance to specifically detect the target molecule captured by any of the microspheres 21'. Such a substance can be, for example, a fluorescent substrate that releases a fluorescent material when decomposed by a certain enzyme linked to (i) the target molecule captured by any of the microspheres 21 'or (ii) a molecule specifically linked to the target molecule. Examples of the molecule specifically bound to the target molecule include a secondary antibody and a nucleic acid. Examples of the particular enzyme include β-galactosidase and peroxidase. Examples of the fluorescent substrate comprise fluorescein-di-β-galactopyranoside (FDG) and Amplex red (Trademark). Microsphere Storage Step
    [0040] [0040] The following describes the stage of storage of microspheres with reference to figures 1B to 1H.
    [0041] The step of storing the microspheres is a step of introducing the second solvent 30 with a specific gravity greater than that of the first solvent 20 in the first solvent 20. The method for introducing the second solvent 30 is not particularly limited; however, a method can be adopted which involves the use of region 14 including containers 13 of substrate 10 as an open well that is opened to the outside for introduction of the second solvent 30 from the opening in the well. On this occasion, the second solvent 30 is preferably introduced so that the layer of the second solvent 30 is laminated to the layer of the first solvent 20, as shown in figure 1B.
    [0042] [0042] The second solvent 30 can be a solvent with a higher specific gravity than that of the first solvent 20 used in the microsphere introduction step. The first solvent 20 and the second solvent 30 are preferentially and mutually amphiphilic to a degree capable of replacing layers; however, they are bound to be incompatible with each other. The fact that there is very little amphiphile between the first solvent 20 and the second solvent 30 does not produce the replacement of the layer between the first solvent 20 and the second solvent 30. Nor does the replacement of the layer between the first solvent 20 and the second solvent 30 occurs when the first solvent 20 and the second solvent 30 are compatible with each other. In order to avoid mixing the first solvent 20 and the second solvent 30, it is preferable to use a hydrophilic solvent as the first solvent 20 and a hydrophobic solvent as the second solvent 30. Like the first solvent 20, the second solvent 30 may contain a surfactant to promote the replacement of the layer.
    [0043] Such second solvent 30 is preferably a hydrophobic solvent; preferably used as is, for example, at least one selected from the group consisting of saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, silicone oil, hexafluorpropylene epoxy polymer, polymer having a hydrofluorether structure, perfluoropolyether, chlorotrifluorethylene polymer and a polymer having a perfluorocarbon structure, or is a mixture including at least one of those mentioned. Examples of saturated hydrocarbons include alkanes and cycloalkanes. Examples of saturated hydrocarbons include alkanes and cyanoalkanes. Examples of alkanes include decane and hexadecane. Examples of unsaturated hydrocarbons include squalene. Examples of aromatic hydrocarbons include benzene and toluene. Examples of epoxy hexafluorpropylene polymers include Krytox 143 (from DuPont Co., Ltd.) and Krytox GPL (from DuPont Co., Ltd.). Examples of the polymer with a hydroflower structure include Asahiklin AE3000 (from Asahi Glass Co., Ltd.) and Novec 7000 (from Sumitomo 3M Co., Ltd.). Examples of the polymer having a perfluorocarbon structure include Fluorinert FC-40 and Fluorinert FC-43 (from Sumitomo 3M Co., Ltd.).
    [0044] [0044] The second solvent 30 introduced and laminated to the first solvent 20 has a specific gravity greater than that of the first solvent 20 and thus moves down below the first solvent 20. Specifically, the substitution between the first solvent 20 and the second solvent 30 occurs and, thus, the state in which the upper layer is the second solvent 30 with the lower layer being the first solvent 20 (see figure 1B) becomes a state in which the upper layer is the first solvent 20 with the lower layer being the second solvent 30 (see figure 1H). Figures 1C to G show schematically how to replace layers. In this layer replacement, the microspheres 21 and 21 'that precipitate at the bottom of the region 14 are stored in the respective containers 13, in which case they are forced inside by the second solvent 30 which moves downstream below the first solvent 20 (see figures 1D to F). As a result, the microspheres can be sealed with high efficiency in each of a large number of containers 13 which are provided on the substrate 10. In the case of using a hydrophilic solvent as the first solvent 20 and a hydrophobic solvent as the second solvent 30 droplets (liquid droplets of the first solvent 20) covered by the second solvent 30 are efficiently formed in the respective containers 13.
    [0045] [0045] In this document, preferred examples of the first solvent 20 and the second solvent 30 used in the microsphere sealing method according to the present invention are found in "Table 1" and "Table 2".
    [0046] [0046] Table 1
    [0047] [0047] Table 2
    [0048] [0048] In the microsphere storage step, when magnetic microspheres such as microspheres 21 and 21 'are used, a magnetic means can be used to promote the movement of the microspheres into the containers 13. The first solvent 20 containing the microspheres 21 and 21 'is introduced into region 14 including containers 13 in the microsphere introduction step, followed by application of a magnetic field external to microspheres 21 and 21' before or after the introduction of the second solvent 30 in the first solvent 20 in the step. This can promote the movement of the microspheres 21 and 21 'into the containers 13, causing the force towards the containers 13 to act on the microspheres 21 and 21'. The application of the magnetic field can be carried out, for example, by making a magnet close to the opposite side of the side on which the containers 13 of the substrate 10 are provided (the side of the lower face of the matrix 1).
    [0049] [0049] The present modality allows to provide a large area matrix that includes a large number of containers. For example, even with a matrix including a million or more containers, it is possible to effectively store microsphere 21 or 21 'in the containers so that any of the microspheres 21 and 21' are stored in each of the containers. Thus, with the present modality, it is possible to detect the target molecules with high sensitivity, thus allowing to detect the target molecules with a concentration as low as approximately 10 aM. De-aeration stage
    [0050] [0050] Region 14 including containers 13 of substrate 10 can be obtained in an upper open space as described above; however, when region 14 is transformed into an enclosed space, a deaeration step can be performed from within region 14 between the microsphere introduction step and the microsphere storage step. Deaeration is preferably carried out, for example, by a method to allow the matrix 1 to still remain under reduced pressure. Specifically, de-aeration is carried out, for example, by a method that allows matrix 1 to remain stationary in a vacuum desiccator of approximately 0.1 atm for approximately 30 seconds.
    [0051] [0051] The de-aeration step is not essential for the present invention. However, carrying out the de-aeration step removes air in containers 13, thus making it possible to efficiently introduce microspheres 21 and 21 'into containers 13. 2. Method for Detecting the Target Molecule
    [0052] [0052] Next, the method for detecting the target molecule according to the present invention is described. The method for detecting a target molecule according to the present invention includes a reaction step, a microsphere sealing step and a determination step.
    [0053] [0053] The present modality uses, as microspheres, the microspheres that specifically capture the target molecules. For example, each of these microspheres may be the one that has been linked to a molecule to specifically capture the target molecule. Suitably used as microspheres, the target molecule and the molecule to specifically capture the target molecule can be any of those exemplified in the above descriptions for the method for storing microspheres.
    [0054] [0054] The reaction step is a reaction step of the microspheres with the target molecules. For example, the reaction between the microspheres and the target molecules can be carried out by mixing a solution containing the microspheres with a solution containing the target molecules.
    [0055] [0055] The microsphere sealing step is a step of carrying out the aforementioned method for storing microspheres using the microspheres that were reacted with the target molecules in the reaction step. Namely, the microsphere sealing step is a step that includes the microsphere introduction step and the microsphere storage step, or a step that includes the microsphere introduction step, the de-aeration step and the microsphere storage step . Note that the descriptions of the microsphere introduction step, the de-aeration step and the microsphere storage step are omitted in this document, as these steps can be performed in the same way as described in the section above "Method for store microspheres ".
    [0056] [0056] The determination step is a step to determine, after the microsphere sealing step, whether each of the containers 13 contains or not any of the microspheres 21 'having captured the target molecules.
    [0057] [0057] Suitable examples of the method for determining whether each of the containers 13 contains or not any of the microspheres 21 'having captured the target molecules cover known molecular recognition reactions, such as antigen-antibody reaction, streptavidin-biotin reaction or binding complementary nucleic acid. For example, this method can be a method of detecting a fluorescent material released from a fluorescent substrate, when decomposed by a particular enzyme linked to (i) a target molecule or (ii) a molecule specifically linked to the target molecule. The detection of the fluorescent material is carried out, for example, by a method to determine a fluorescence intensity of each container using, for example, a fluorescence microscope or an image sensor.
    [0058] [0058] In the determination step, it is also preferable to determine whether each of the containers 13 contains any of the microspheres 21 or any of the microspheres 21 '. The determination whether each of the containers 13 contains any of the microspheres 21 or any of the microspheres 21 'can be carried out, for example, by microscopic observation to determine the presence or absence of any of the microspheres 21 or any of the microspheres 21' in each of the containers 13. Alternatively, the determination of the presence or absence of any of the microspheres 21 or any of the microspheres 21 'in each of the containers 13 can be carried out by a method of detecting stray light from the microspheres or a method of measuring an electrical potential with a field effect transistor (FET).
    [0059] [0059] After the determination step, based on (i) the number of containers 13 containing microspheres 21 or microspheres 21 ', and (ii) the number of containers 13 containing microspheres 21' having captured the target molecules, it is possible to calculate a proportion of the number of microspheres having captured the target molecules in relation to the total number of microspheres. In this way, it is possible to quantify a concentration of the target molecules.
    [0060] [0060] In accordance with the present modality, it is possible to provide a large area matrix that includes a large number of containers; moreover, even with a set including one million or more containers, it is possible to efficiently store microspheres 21 or 21 'in each of the containers. Thus, with the present modality, it is possible to detect the target molecules with high sensitivity, thus allowing to detect the target molecules with a concentration as low as approximately 10 aM. 3. Matrix
    [0061] [0061] Next, the following describes a configuration of matrix 1 with reference to figure 1.
    [0062] [0062] In matrix 1, substrate 10 includes a plate-like element 11 and side wall 12 having a hydrophobic upper surface. The substrate 10 includes the plurality of containers 13 which are separated from each other by the side wall 12.
    [0063] [0063] Preferably, the plate-like element 11 has a hydrophilic surface. The term "hydrophilic surface" refers to a surface whose affinity for a hydrophilic solvent is greater than that for a hydrophobic solvent. The plate-like element 11 may only need to be made of a solid material. For example, the plate-like element 11 can be made of glass, silicon or a polymeric resin.
    [0064] [0064] The side wall 12 is provided on the surface, preferably on the hydrophilic surface of the plate-like element 11 and separates the plurality of containers 13. The side wall 12 has the upper surface hydrophobic. The term "hydrophobic" used in this document is used interchangeably with "lipophilic", and denotes a nature whose affinity with a hydrophobic solvent is greater than with a hydrophilic solvent.
    [0065] [0065] Note that the upper surface of the side wall 12 is preferably hydrophobic and its side surface, i.e., an inner wall of each of the containers 13, can be hydrophobic or hydrophilic.
    [0066] [0066] For example, the side wall 12 can be made of a hydrophilic structure and a hydrophobic layer that is formed on its upper surface. The hydrophilic structure can be made, for example, of glass, silicon or a polymeric resin. The hydrophobic layer can be made, for example, from a water-repellent resin or a fluorocarbon polymer resin. Examples of the fluorocarbon polymer resin include amorphous fluorocarbon resin. The amorphous fluorocarbon resin is preferably used because it has a high hydrophobic property and has a low toxicity for a biological sample.
    [0067] [0067] It is preferable to use, as the second solvent 30, a solvent with affinity for a hydrophobic layer that forms the upper surface of the side wall 12 to an extent that does not dissolve the hydrophobic layer. A lower affinity for the hydrophobic layer of the second solvent 30 may inhibit the replacement of the layer with the first solvent 20. Too much affinity for the hydrophobic layer of the second solvent 30 may result in the hydrophobic layer dissolving and not allowing the shape of the sidewall 12 is maintained. From this point of view, a polymer having a perfluorocarbon structure (Fluorinert FC-40, Fluorinert FC-43 or similar) is preferably used as the second solvent 30, when the upper surface of the sidewall 12 is formed by a polymeric resin of fluorocarbon.
    [0068] [0068] Preferred examples of amorphous fluorocarbon resin include at least one selected from CYTOP (registered trademark), TEFLON (registered trademark) AF2400 and TEFLON (registered trademark) AF1600. Among these, CYTOP (Trademark) is more preferred because it is easy to be microfabricated.
    [0069] [0069] Alternatively, the side wall 12 can be made of a hydrophobic material. For example, side wall 12 can be made from a polymeric fluorocarbon resin or a polymeric paraxylene resin. Examples of the fluorocarbon polymer resin include an amorphous fluorocarbon resin. Any of the above is preferably used as an amorphous fluorocarbon resin.
    [0070] [0070] The side wall 12 only needs to be configured in such a way that the various containers 13 are provided in the plate-shaped component 11. For example, the side wall 12 can be a plate-shaped structure, whose parts corresponding to the containers 13 are holes.
    [0071] [0071] A height of the sidewall 12 measured from the surface of the plate-shaped component 11 (that is, a thickness in a vertical direction) only needs to be designed so that one of the microspheres 21 and 21 ', once stored in containers 13, do not be discharged from containers 13 again during the microsphere storage step. For example, the height of the sidewall 12 can be designed so that most preferably the entire part of one of the microspheres 21 and 21 'stored in one of the containers 13 is positioned lower than the upper surface of the sidewall 12.
    [0072] In order to effectively store the microspheres 21 and 21 'in the containers 13, the height of the side wall 12 is preferably equal to or greater than the average particle diameter of the microspheres 21 and 21'. In addition, so that only one of the microspheres 21 and 21 'is stored in one of the containers 13, the height of the side wall 12 is preferably equal to or less than 1.5 times the average particle diameter of the microspheres 21 and 21'.
    [0073] [0073] Each of the plurality of containers 13 is a recess capable of storing only one of the microspheres 21 and 21 ', and the plurality of containers 13 are separated from each other by the side wall 12. Each of the containers 13 has a lower surface which is a part of the surface of the plate-like element 11, and the bottom surface is hydrophilic.
    [0074] [0074] Containers 13 can be of any shape or size, as long as the shape or size allows each of the containers 13 to store only one of the microspheres 21 and 21 'in it. The region surrounded by the lower surface and the lateral surface of each of the containers 13 can be shaped, for example, in a circular or rectangular cylindrical column.
    [0075] [0075] A width "w" of each of the containers 13 in a horizontal direction (for example, in the case where a cross section of each container 13 when viewed in the horizontal direction is formed in a circle, the width "w" is a diameter of the circle, in a case where the cross section of each container 13 when viewed in the horizontal direction is formed into a square, the width "w" is a length on one side of the square) only needs to be larger than the particle average Diameter of microspheres 21 and 21 '. Preferably, the width "w" is, for example, 1 to 2 times greater than the average particle diameter of microspheres 21 and 21 '. In the present embodiment, each of the containers 13 has a depth equal to the height of the side wall 12. To effectively store the microspheres in the containers, the depth of each of the containers of the present invention is preferably equal to or greater than the average particle diameter. of the microspheres. In addition, in order for only one of the microspheres to be stored in one of the containers, the depth of each of the containers of the present invention is preferably equal to or less than 1.5 times the average particle diameter of the microspheres.
    [0076] [0076] Techniques, such as photolithography, engraving and substrate lamination, to prepare matrix 1 are the same as techniques for preparing general purpose microchips and matrices.
    [0077] [0077] According to the present embodiment, each of the containers 13 has a hydrophilic lower surface, and the side wall 12 has a hydrophobic upper surface. Thus, the first solvent 20 containing microspheres 21 and 21 'can be introduced effectively into containers 13 when the first hydrophilic solvent 20 is used in the microsphere introduction step. In addition, since the second hydrophobic solvent 30 used in the microsphere storage step can be prevented from entering containers 13, the first hydrophilic solvent 20 in containers 13 can be coated and hermetically sealed with the second hydrophobic solvent 30 to form droplets (liquid drops).
    [0078] [0078] The matrix 1 of the present embodiment can be, for example, a matrix including one million or more containers. Even with the matrix having such a large area, the use of the method for storing microspheres of the present modality or the method for detecting a target molecule of the present modality makes it possible to effectively store the microspheres in the containers, so that any of the microspheres is stored in each of the containers. Thus, according to the present modality, it is possible to detect the target molecules with high sensitivity, thus providing a matrix that allows the detection of target molecules of a concentration as low as approximately 10 aM. 4. Kit
    [0079] [0079] The matrix 1 and the microspheres 21 can be composed in the form of a kit. Each of the containers 13 in the arrays 1 is configured to be able to store only one of the microspheres 21 included in this kit.
    [0080] [0080] Each of the microspheres 21 included in this kit may be the one that specifically captures the target molecule. For example, each of the microspheres 21 included in this kit can be the one that has been linked to a molecule to specifically bind to the target molecule. Any of the molecules cited above can be used appropriately as the target molecule and the molecule for specific binding to the target molecule.
    [0081] [0081] This kit can also include a substance to specifically detect the target molecule. The substance to specifically detect the target molecule preferably employed can be any one mentioned above. In addition, the kit may further include, for example, the first solvent and the second solvent. 5. Target Molecule Detection Device
    [0082] [0082] In the following, a target molecule detection device 50 of the present invention is described with reference to figure 2. Figure 2 is a view schematically illustrating an embodiment of a target molecule detection device according to the present invention.
    [0083] [0083] The target molecule detection device 50 according to the present embodiment includes matrix 1, an image sensor 51 and a light source 52. In the present document, matrix 1 is shown as a multi-well plate. Each well of the multi-well plate corresponds to region 14 in figure 1. The composition of matrix 1 is as described above and thus the explanations of matrix 1 are omitted in that part.
    [0084] [0084] The image sensor 51 is a sensor to detect the light emitted by each of the containers 13 when the microspheres that capture the target molecules are stored in the containers 13. For example, the image sensor 51 can be a sensor to detect fluorescence emitted by a fluorescent substrate when decomposed by a certain enzyme linked to (i) the target molecule or (ii) a molecule specifically bound to the target molecule. A CMOS image sensor can be used properly as an image sensor 51.
    [0085] [0085] Light source 52 is a light source for emitting light to matrix 1. In Figure 2, light source 52 is provided above matrix 1. However, the present invention is not particularly limited to this. Alternatively, the light source 52 may be the one that emits light to the side of the matrix 1, for example.
    [0086] [0086] Between matrix 1 and image sensor 51, for example, an interference filter and / or a light guide matrix can be provided. In addition, between the light source 52 and the matrix 1, an excitation filter can be provided, for example.
    [0087] [0087] According to the present modality, the matrix 1 and the image sensor 51 are directly connected to each other. This makes it possible to easily determine, without using another device, such as a microscope, whether or not any of the microspheres that have captured the target molecules are stored in each of the containers 13. This allows for quick and easy detection whether or not any of the microspheres that have captured the target molecules are stored in each of the containers 13 and provide the target molecule detection device at an affordable price. 6. Example of Application of the Method for Sealing Substance According to the Present Invention
    [0088] [0088] In the method for storing a substance according to the present invention, the sealed substance in the containers provided on the substrate can be constituted by microspheres, nucleic acid, protein, virus, cells, lipid membrane complex, or the like. In the above embodiment, the method has been described using microspheres as an example.
    [0089] [0089] In accordance with the present invention, nucleic acid includes DNA and RNA. Protein and viruses include polymers (oligomers) and complexes of them with other substances. The cells include, in particular, bacterial cells, and the lipid membrane complex includes, in particular, liposome and exosome, and other cellular organelles, such as mitochondria.
    [0090] [0090] When the sealing object is nucleic acid, protein, virus, cells, lipid membrane complex, or the like, the method for storing a substance according to the present invention can be used in applications such as ELISA-PCR. INDUSTRIAL APPLICABILITY
    [0091] [0091] The present invention is suitably applicable to a method for detecting low concentration target molecules, a matrix for this, a device and the like. LIST OF REFERENCE SYMBOLS 1: Matrix 10: Substrate 11: Element similar to the plate 12: Sidewall 13: Container 14: Region 20: First solvent 21, 21 ': microspheres 30: Second solvent
    权利要求:
    Claims (7)
    [0001]
    Method for storing a substance selected from spheres, nucleic acids, proteins, viruses, cells and lipid membrane complexes in a plurality of containers provided on a substrate and separated from each other by a side wall, in which a region including the containers of the substrate is opened to the outside forming an open pit and in which the side wall has a hydrophobic upper surface, said method characterized by comprising the steps of: (i) introducing, through the region of the open well in the substrate, a hydrophilic solvent containing the substance; and (ii) introducing, through the region of the open well in the hydrophilic solvent, a hydrophobic solvent with a specific gravity greater than that of the hydrophilic solvent, so that the hydrophobic solvent is immersed in the hydrophilic solvent, step (ii) being carried out after step (i), wherein after step (ii), the hydrophobic solvent moves down below the hydrophilic solvent and the hydrophilic solvent forms a layer over the hydrophobic solvent, and at least one of the hydrophilic solvent and the hydrophobic solvent contains a surfactant.
    [0002]
    Method according to claim 1, characterized in that the surfactant in the hydrophilic solvent has a concentration of 0.01% to 1%.
    [0003]
    Method according to claim 1 or 2, characterized in that the hydrophobic solvent is at least one solvent selected from the group consisting of saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, silicone oil, hexafluorpropylene epoxy polymer, a polymer having a hydrofluorether structure , perfluorpolyether, chlorotrifluorethylene and a polymer having a perfluorocarbon structure, or is a mixture including at least one of said solvents.
    [0004]
    Method according to any one of claims 1 to 3, characterized in that the hydrophilic solvent is at least one solvent selected from the group consisting of water, hydrophilic alcohol, hydrophilic ether, ketone, nitrile solvents, dimethyl sulfoxide and N, N-dimethylformamide or it is a mixture including at least one of said solvents.
    [0005]
    Method according to claim 3 or 4, characterized in that the side wall has a hydrophobic upper surface comprising a fluorocarbon polymer resin and the hydrophobic solvent is a polymer having a perfluorocarbon structure.
    [0006]
    Method for detecting a target molecule characterized by comprising: (i) reacting microspheres that specifically capture the target molecules with the target molecules; (ii) carrying out a method, as defined in any one of claims 1 to 5, in which the microspheres of step (i) are used as the substance and in which step (ii) is carried out after step (i); and (iii) determining whether or not one of the microspheres that captured the target molecule is stored in each of the plurality of containers, in which step (iii) is carried out after step (ii).
    [0007]
    Method according to claim 6, characterized in that the microspheres are microspheres to which molecules that specifically bind to the target molecules are attached.
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    同族专利:
    公开号 | 公开日
    EP3168624A1|2017-05-17|
    RU2691694C2|2019-06-17|
    CN106662599B|2020-04-28|
    KR20170027716A|2017-03-10|
    WO2016006208A1|2016-01-14|
    SG10201811714UA|2019-01-30|
    BR112017000270A2|2018-06-26|
    US20170176430A1|2017-06-22|
    JPWO2016006208A1|2017-04-27|
    SG11201700133SA|2017-03-30|
    ES2831360T3|2021-06-08|
    EP3168624A4|2018-01-10|
    AU2015286488B2|2020-07-16|
    AU2015286488A1|2017-02-16|
    CA2953666A1|2016-01-14|
    JP6611714B2|2019-11-27|
    US10451619B2|2019-10-22|
    EP3168624B1|2020-08-26|
    CN106662599A|2017-05-10|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US7214540B2|1999-04-06|2007-05-08|Uab Research Foundation|Method for screening crystallization conditions in solution crystal growth|
    US20020039728A1|2000-02-10|2002-04-04|Robert Kain|Alternative substrates and formats for bead-based array of arrays|
    US7682565B2|2002-12-20|2010-03-23|Biotrove, Inc.|Assay apparatus and method using microfluidic arrays|
    JP3727026B2|2003-04-10|2005-12-14|博行 野地|Micro chamber used for detecting single-molecule enzyme activity and method for preparing droplets of 1000 fL or less|
    EP2662136A3|2003-08-27|2013-12-25|President and Fellows of Harvard College|Method for handling and mixing droplets|
    JP4547301B2|2005-05-13|2010-09-22|株式会社日立ハイテクノロジーズ|Liquid transport device and analysis system|
    CN101237934B|2005-05-21|2012-12-19|先进液体逻辑公司|Mitigation of biomolecular adsorption with hydrophilic polymer additives|
    JP2007017253A|2005-07-07|2007-01-25|Olympus Corp|Container and particle|
    CA2680061C|2006-04-18|2015-10-13|Duke University|Droplet-based biochemistry|
    JP4866208B2|2006-10-31|2012-02-01|株式会社島津製作所|Micro reactor|
    EP2837692A1|2007-03-22|2015-02-18|Advanced Liquid Logic, Inc.|Enzymatic assays for a droplet actuator|
    JP5009125B2|2007-10-26|2012-08-22|日立マクセル株式会社|Microdevice using functional particles and processing method using the same|
    JP2009162549A|2007-12-28|2009-07-23|Nec Corp|Electrophoresis chip|
    US20110065086A1|2008-02-21|2011-03-17|Otc Biotechnologies, Llc|Methods of producing homogeneous plastic-adherent aptamer-magnetic bead-fluorophore and other sandwich assays|
    CA2716950C|2008-02-29|2017-07-04|Northwestern University|Barriers for facilitating biological reactions|
    US20100022414A1|2008-07-18|2010-01-28|Raindance Technologies, Inc.|Droplet Libraries|
    JP5030897B2|2008-08-28|2012-09-19|メタウォーター株式会社|Microorganism measurement method|
    WO2011079176A2|2009-12-23|2011-06-30|Raindance Technologies, Inc.|Microfluidic systems and methods for reducing the exchange of molecules between droplets|
    US8236574B2|2010-03-01|2012-08-07|Quanterix Corporation|Ultra-sensitive detection of molecules or particles using beads or other capture objects|
    CA2791655C|2010-03-01|2014-11-04|Quanterix Corporation|Methods and systems for extending dynamic range in assays for the detection of molecules or particles|
    CN101947124B|2010-06-25|2012-07-04|博奥生物有限公司|Integrated microfluidic chip device and using method thereof|
    US9562897B2|2010-09-30|2017-02-07|Raindance Technologies, Inc.|Sandwich assays in droplets|
    JP5337324B2|2011-03-08|2013-11-06|独立行政法人科学技術振興機構|Bead encapsulation method, target molecule detection method, array, kit, and target molecule detection apparatus|
    WO2014034781A1|2012-08-31|2014-03-06|国立大学法人東京大学|Detector and detection method|WO2017043530A1|2015-09-08|2017-03-16|凸版印刷株式会社|Method for detecting biological substance|
    WO2018043733A1|2016-09-05|2018-03-08|国立研究開発法人科学技術振興機構|Method and kit for detecting pathogenic microorganism|
    US11008603B2|2016-09-05|2021-05-18|Japan Science And Technology Agency|Method and kit for detecting pathogenic microorganism|
    US10324041B2|2016-12-21|2019-06-18|Abbott Japan Co., Ltd.|Optical imaging system using lateral illumination for digital assays|
    US11047854B2|2017-02-06|2021-06-29|Abbott Japan Llc|Methods for reducing noise in signal-generating digital assays|
    SG11201908564RA|2017-03-29|2019-10-30|Univ Cornell|Devices, processes, and systems for determination of nucleic acid sequence, expression, copy number, or methylation changes using combined nuclease, ligase, polymerase, and sequencing reactions|
    US11231370B2|2017-03-29|2022-01-25|Japan Science And Technology Agency|Microscopic body enclosing method, microscopic body detection method, and microscopic body detection device|
    CN110476067A|2017-03-29|2019-11-19|国立研究开发法人科学技术振兴机构|Small substance detecting method and minim quality detection device|
    CN110520529A|2017-04-05|2019-11-29|株式会社日立高新技术|Nucleic acid amplification method and foranalysis of nucleic acids device|
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    法律状态:
    2018-10-30| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2020-02-04| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-09-01| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|
    2021-02-17| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-03-23| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/07/2015, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    JP2014140700|2014-07-08|
    JP2014-140700|2014-07-08|
    PCT/JP2015/003310|WO2016006208A1|2014-07-08|2015-07-01|Substance sealing method and target molecule detecting method|
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