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    • 专利摘要:
    MICRO-SPHERE SEALING METHOD, METHOD FOR DETECTION OF TARGET MOLECULE, MATRIX, KIT AND DEVICE FOR TARGET MOLECULE DETECTION This invention provides a technique that makes it possible to detect target molecules of low concentration with high sensitivity. This invention includes (i) a step of introducing a hydrophilic solvent (42) containing microspheres (41), (41 ') into a space (30) between (a) a section of the lower layer (10) including a plurality of receptacles (13) each of which is capable of storing only one of the microspheres (41), (41 ') and which are separated from each other by a side wall (12) with a hydrophobic upper surface and (b) a section of the upper layer (20) facing a surface of the lower layer section (10) on which the surface of the plurality of receptacles is provided; and (ii) a step of introducing a hydrophobic solvent (43) into the space (30), step (ii) being carried out after step (i).
    公开号:BR112013022933B1
    申请号:R112013022933-0
    申请日:2012-03-07
    公开日:2020-07-07
    发明作者:Hiroyuki Noji；Ryota Lino；Suguru Araki
    申请人:Japan Science And Technology Agency；
    IPC主号:
    专利说明:

    Technical Field
    The present invention relates to a method for sealing microspheres (i.e., a method for sealing microspheres), a method for detecting a target molecule, a matrix, a kit and a device for detecting a target molecule. Principles of the Technique
    An individual molecule analysis has been known as a method to conduct various analyzes by observing biomolecules such as proteins and nucleic acids so that biomolecules are individually identified. To perform the analysis of individual molecules, there are some known methods.
    Patent Literature 1 describes a microchamber for detecting enzyme activity of the individual molecule. This microchamber includes part of a container in which a liquid droplet can be sealed and which has the capacity to store a liquid droplet of up to 1000 fL (fentoliters). Part of the container is made of a niche provided in at least one of a first member and a second member that are connected together. According to Patent Literature 1, an enzymatic reaction is conducted in 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 very small. In this way, it is possible to detect an activity of an enzyme molecule.
    Non-Patent Literature 1 describes a method for conducting an individual molecule analysis using a matrix in which a droplet of liquid is covered with oil, in an order of fentoliter, and accessible directly from the outside. This matrix includes a pattern of the hydrophilic region made of a hydrophilic surface on which a hydrophobic region with a height of 17 nm is provided.
    Non-Patent Literature 2 describes a method for detecting a protein by an Enzyme-Binding Absorbent Immunoassay (ELISA) of the individual molecule. According to this method, a very small amount of protein is captured by tiny spheres covered with specific protein antibodies and complexes of microspheres and proteins are marked by fluorescence. Then, microspheres including complexes are introduced into a reaction chamber by centrifugal force. Then, the number of microspheres that captured the proteins is counted. In this way, the proteins are quantitatively evaluated. Citation List
    [Patent Literature] [Patent Literature 1] Japanese Patent Application Publication, Tokukai, No. 2004-309405 A (Publication Date: November 4, 2004) [Non-Patent Literature] [Non-Patent Literature 1] S. Sakakihara et al., Lab Chip, 2010, 10, 3355-3362 [Non-Patent Literature 2] David M Rissin et al., Nature Biotechnology: doi: 10.1038 / nbt.1641 Summary of the Invention Technical problem
    To detect, for example, low concentration disease markers for the purpose of detecting diseases, infectious diseases and the equivalent, there is a demand for the development of biosensitive techniques that have higher sensitivities. For example, in a case where one 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 amM. A technique capable of detecting target molecules of this very low concentration is necessary.
    A possible method for detecting such target molecules may be that for detecting the target molecules by enzymatic analysis of the individual molecule mentioned above with a sensitivity in terms of a single molecule. Specifically, this method is conducted by (i) sealing the target molecule specifically into a liquid droplet in a fentoliter order (very small liquid droplet), (ii) binding the target molecule to a substance such as an enzyme-labeled antibody and (iii ) detecting an activity of the enzyme that labels the antibody in the aforementioned manner. The sealing of the target molecule specifically in a liquid droplet can be performed by a method that uses, for example, a microsphere labeled with a substance such as another antibody to specifically bind to the target molecule. In this method, after the microsphere binds to the target molecule, the microsphere is sealed in a very small solution droplet.
    Incidentally, in order to efficiently detect target molecules that are contained in a solution in only a very small amount, such as target molecules of approximately 30 µM as described above, it is necessary to prepare a large number of very small liquid droplet matrices, as much as about a million, and induce the matrices to capture the microspheres.
    However, according to the method described by Patent Literature 2, it is necessary that the microspheres are introduced into matrices by intense centrifugal force, therefore, a lot of time and efforts are required. In addition, the number of matrices used in the method of Non-Patent Literature 2 is approximately fifty thousand. Thus, the Non-Patent Literature 2 method is very difficult to be applied to the case that requires about one million matrices. Thus, with the Non-Patent Literature 2 method, it is difficult to efficiently join a large number of microspheres in the matrices. In fact, neither Patent 1 Literature nor Non-Patent Literature 1 describes any method for solving such a problem.
    In view of this, the present invention has an objective to provide a technique for efficient sealing of large numbers of microspheres in a matrix. Solution of the problem
    To achieve the above objective, a method of the present invention for sealing microspheres includes (i) a step of introducing a hydrophilic solvent containing microspheres into a space between (a) a section of the lower layer that includes a plurality of receptacles each of which is capable of storing only one of the microspheres and which are separated from each other by a side wall with a hydrophobic upper surface and (b) a section of the upper layer that faces a surface of the section of the lower layer on whose surface the plurality of receptacles is provided; and (ii) a step of introducing a hydrophobic solvent into space, with step (ii) performed after step (i).
    To achieve the above objective, a matrix of the present invention includes: a section of the bottom layer provided with a plurality of receptacles being separated from each other by a side wall that has a hydrophobic upper surface; and an upper layer section which faces, through a space, a surface of the lower layer section on which the plurality of receptacles is provided. Advantageous Effects of the Invention
    The use of the microsphere sealing method of the present invention makes it possible to join a large number of microspheres in a matrix, thereby contributing to a technique by which target molecules of low concentration are detectable with high sensitivity. Brief Description of Drawings
    (a) to (e) of Figure 1 are schematic illustrations of a method for sealing microspheres according to the present invention and show cross-sectional projections of a matrix 1.
    Figure 2 is a schematic illustration of an embodiment of a target molecule detection device according to the present invention.
    Figure 3 shows a fluorescence image of a matrix in which microspheres were sealed in an example of the present invention.
    Figure 4 is a graph showing fluorescence intensities observed when target molecules were detected by a conventional method. (a) to (f) of Figure 5 show microscopic images of matrices in which microspheres were sealed in another example of the present invention.
    Figure 6 shows a graph illustrating a relationship, seen in another example of the present invention, between (i) a streptavidin concentration and (ii) a ratio between the number of microspheres with streptavidin captured and the number of microspheres stored in the matrix.
    Figure 7 is an incidence to explain a method of preparing a standard hydrophilic-hydrophobic slide according to an example of the present invention.
    Figure 8 is an incidence illustrating (i) the trapping efficiency of a microsphere found in a case involving the use of a matrix with a structure for cell flow (Example 3) and (ii) the trapping efficiency of a microsphere in a case involving the use of a matrix without a structure for cell flow (Comparative Example 2). Description of Modalities
    The following describes an embodiment of the present invention in detail. [Microsphere Sealing Method]
    With reference to (a) to (e) of Figure 1, the following describes a method for sealing microspheres according to the present embodiment, (a) to (e) of Figure 1 are schematic illustrations of a method for sealing microspheres according to the present invention and show cross-sectional views of a matrix 1.
    The present embodiment deals with a case in which microspheres 41 and 41 'are sealed in a matrix 1 including a section of the lower layer 10 and a section of the upper layer 20. The section of the lower layer 10 includes a plurality of receptacles 13 each of which are capable of storing only one of the microspheres 41 and 41 'and are separated from each other by a side wall 12 with a hydrophobic upper surface. In addition, the upper layer section 20 faces a surface of the lower layer section 10 on which the surface of the receptacles 13 is provided.
    Preferably, the microspheres have an average particle diameter from 1 pm to 4 pm. In this way, the microspheres can be efficiently sealed in the matrix, and the matrix can achieve high density. Note that the term "average particle diameter" here refers to a value obtained as a result of measuring the microspheres by observation by electron microscope or dynamic light scattering.
    The present modality describes, but is not particularly limited to, a use case for microspheres that specifically capture target molecules. In the present embodiment, the microspheres to be sealed are a mixture of microspheres 41, which have not yet captured the target molecules, and microspheres 41 ', which have captured the target molecules.
    For example, it is possible to use, as the microspheres that specifically capture the target molecules, microspheres that are attached to a molecule to specifically capture the target molecule. The molecule for specific capture of the target molecule can be attached to a modifying group on a surface of the microsphere, that is, by means of a binding agent. For example, the present invention can be configured so that the molecule to specifically capture the target molecule is covalently linked to an amino group on a surface of a microsphere modified by the amino group by means of a cross-linking agent containing N-hydroxysuccinimide and / or the equivalent.
    The "target molecule" refers to a molecule that must be detected (chosen as a target). Specifically, the "target molecule" here refers to a molecule that must be a molecule. Examples of the target molecule include (i) biomolecules such as a protein, nucleic acid and sugar and (ii) viral particles.
    The molecule to specifically capture the target molecule (hereinafter, this molecule is also referred to as the "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 linked to hundreds of thousands or more of 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 approximate order of nM. However, with the aforementioned configuration, it is possible to induce 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 x 10 ° particles / mL , the concentration of the target capture molecules is approximately 1 nM).
    The method for sealing microspheres according to the present embodiment includes a step of introducing the microspheres, a de-aeration step and a step of introducing the hydrophobic solvent. Each of these steps will be described in detail below. (Microspheres introduction stage)
    The following describes the step of introducing microspheres with reference to (a) and (b) of Fig. 1.
    The step of introducing microspheres is a step of introducing a hydrophilic solvent 42 containing microspheres 41 and 41 'into a space 30 between the lower layer section 10 and the upper layer section 20. The hydrophilic solvent 42 can be introduced into the space 30 between the lower layer section 10 and the upper layer section 20 along a direction that is parallel to the surfaces of the lower layer section 10 and the upper section section 20, the surfaces of the lower section section 10 and of the top layer section 20 facing each other. For example, the hydrophilic solvent 42 can be introduced into space 30 through a through hole (not shown) provided in at least one of the upper layer section 20 and the lower layer section 10.
    It is preferably used as the hydrophilic solvent 42, for example, at least one selected from the group consisting of water, hydrophilic alcohol, hydrophilic ether, ketone, nitrile-based solvents, dimethylsulfoxide (DMSO) and N, N-dimethylformamide (DMF) or a mixture including at least one of them. Examples of hydrophilic alcohol include ethanol, methanol, propanol and glycerin. Examples of hydrophilic ether include tetrahydrofuran, polyethylene oxide and 1,4-dioxane. Examples of ketone include acetone and methyl ethyl ketone. Examples of nitrile-based solvents include acetonitrile.
    In addition to microspheres 41 and 41 ', the hydrophilic solvent 42 may also include, for example, a substance to specifically detect the target molecule captured by any of the microspheres 41'. Such a fluorescent that releases a fluorescent material when decomposed by a certain enzyme bound (i) to the target molecule captured by any of the microspheres 41 'or (ii) to a molecule specifically bound to the target molecule. Examples of the molecule specifically bound to the target molecule comprise a secondary antibody and a nucleic acid. Examples of the particular enzyme comprise β-galactosidase and peroxidase. Examples of the fluorescent substrate comprise fluorescein-di-Bgalactopyranoside (FDG) and red Amplex (Trademark). (Deaeration step)
    The following describes the de-aeration step with reference to (c) in Figure 1.
    The deaeration step is a step of deaerating the space 30 between the lower layer section 10 and the upper layer section 20, which is carried out after the introduction of the microspheres and before the introduction of the hydrophobic solvent. Deaeration is preferably carried out with the aid of, for example, a method that allows a break in the matrix 1 under reduced pressure. Specifically, the de-aeration is carried out with the aid, for example, of a method that allows the matrix 1 to paralyze in a vacuum desiccator of approximately 0.1 atm (10.1325 kPa) for about 30 seconds.
    The de-aeration step is not essential for the present invention. However, performing the de-aeration step removes air in receptacles 13, thus making it possible to efficiently introduce receptacles 13 with hydrophilic solvent 42 containing microspheres 41 and 41 '. This makes it possible to efficiently seal microspheres 41 and 41 'in receptacles 13. Therefore, it is preferable to perform the deaeration step. (Hydrophobic solvent introduction step) The following describes the hydrophobic solvent introduction step with reference to (d ) and (e) of Figure 1.
    The step of introducing the hydrophobic solvent is a step of introducing a hydrophobic solvent 43 into the space 30 between the section of the bottom layer 10 and the section of the top layer 20. The step of introducing the hydrophobic solvent is carried out after the step of introducing the microspheres and preferably performed after the deaeration step.
    The hydrophobic solvent 43 only needs to be a solvent that is difficult to mix with the hydrophilic solvent 42, which is used in the step of introducing the microspheres. It is preferably used as the hydrophobic solvent 43, for example, at least one selected from the group consisting of saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, silicone oil, perfluorocarbon, halogen-based solvents and hydrophobic ionic liquid or a mixture including at least least one of them. Examples of saturated hydrocarbon comprise alkane and cycloalkane. Examples of alkane comprise decane and hexadecane. Examples of unsaturated hydrocarbon comprise squalene. Examples of aromatic hydrocarbons include benzene and toluene. Examples of perfluorocarbon © include Fluorinert (Trademark) FC40 (available from SIGMA). Examples of the halogen-based solvents include chloroform, methylene chloride and chlorobenzene. Hydrophobic ionic liquid denotes ionic liquid that is not dissociated at least in water. Examples of such ionic liquids include 1-butyl-3-methylimidazolium hexafluorophosphate. The ionic liquid denotes a salt that is a liquid at room temperature.
    The step of introducing the hydrophobic solvent makes it possible to efficiently form, in the respective receptacles 13, droplets (liquid droplets) covered with the hydrophobic solvent 43. In addition, the step of introducing the hydrophobic solvent makes it possible to seal the microspheres 41 and 41 'in the droplets so that any of the microspheres 41 and 41' are stored in each of the droplets.
    According to the present modality, microspheres 41 and 41 'are introduced through the space 30 between the section of the lower layer 10 and the section of the upper layer 20, thus enabling highly efficient sealing of any of the microspheres in each of a large number of receptacles 13 that are provided over a large area (eg, an area of 1 cm2 or greater).
    The present method makes it possible to provide a droplet matrix for the large area including a large number of receptacles. For example, even with a matrix that includes a million or more receptacles, it is possible to efficiently seal microspheres 41 and 41 'in the receptacles so that any of the microspheres 41 and 41' are stored in each of the receptacles. Thus, with the present modality, it is possible to detect the target molecules with high sensitivity, thus making it possible to detect the target molecules in a very low concentration like approximately 0.1 aM. [Method for detecting the target molecule]
    Next, the method for detecting the target molecule according to the present modality is described.
    The method for detecting the target molecule according to the present modality includes a reaction step, a microsphere sealing step and a determination step.
    The present modality uses, like microspheres, microspheres that specifically capture the target molecules. For example, each of the microspheres can be the one that has been linked to a molecule to specifically capture the target molecule. Suitably used as the microspheres, the target molecule and the molecule to specifically capture the target molecule can be any of those exemplified in the descriptions for the microsphere sealing method of the present embodiment.
    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.
    The microsphere sealing step is a step of carrying out the aforementioned method for sealing the microspheres using the microspheres that reacted with the target molecules in the reaction step. That is, the step of sealing the microspheres is (i) a step that includes the step of introducing the microspheres and the step of introducing the hydrophobic solvent or (ii) a step that includes the step of introducing the microspheres, the deaeration step and the step of introducing the hydrophobic solvent. Note that the descriptions of the microspheres introduction step, the deaeration step and the hydrophobic solvent introduction step are omitted here, as these steps can be performed in the same way as described in the section "Microsphere sealing method" .
    The determination step is a step of determining, after the microsphere sealing step, on whether each of the receptacles 13 contains or not any of the microspheres 41 'with captured target molecules.
    Suitable examples of the method of determining whether or not each of the receptacles 13 contains any of the microspheres 41 'with the captured target molecules include known molecular identification reactions such as antigen-antibody reaction, streptavidin-biotin reaction and complementary acid binding nucleic. For example, this method can be a method of detecting a fluorescent material released from a fluorescent substrate when decomposed by a certain enzyme bound (i) to a target molecule or (ii) to a molecule specifically bound to the target molecule. The detection of the fluorescent material is carried out with the aid, for example, of a method of determining a fluorescent intensity of each receptacle using, for example, a fluorescent microscope or an image sensor.
    In the determination step, it is also preferable to determine whether each of the receptacles 13 contains any of the microspheres 41 or any of the microspheres 41 '. The determination of whether each of the receptacles 13 contains any of the microspheres 41 or any of the microspheres 41 'can be carried out by, for example, microscopic observation to determine the presence or absence of any of the microspheres 41 or any of the microspheres 41 'in each of the receptacles 13. Alternatively, the determination of the presence or absence of any of the microspheres 41 or any of the microspheres 41' in each of the receptacles 13 can be carried out by a method of light scattering detection of the microspheres or by a method of measuring an electrical potential with a field effect transistor (FET).
    After the determination step, based on (i) the number of receptacles 13 containing microspheres 41 or microspheres 41 'and (ii) the number of receptacles 13 containing microspheres 41' with the captured target molecules, it is possible to calculate a proportion the number of microspheres that 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.
    According to the present modality, it is possible to provide a drop matrix with a large area including a large number of receptacles; moreover, even with a matrix that includes a million or more receptacles, it is possible to efficiently seal microspheres 41 and 41 'in the receptacles. Thus, with the present modality, it is possible to detect the target molecules with high sensitivity, therefore allowing to detect the target molecules in a concentration as low as approximately 0.1 aM. [Matrix]
    Next, a configuration of the matrix 1 of the present embodiment is described with reference to (a) of Figure 1. The matrix 1 may be a matrix used in the method for sealing microspheres according to the present embodiment or it may be a matrix used in the method to detect the target molecule according to the present modality.
    The matrix 1 includes the lower layer section 10 and the upper layer section 20.
    The lower layer section 10 includes a blade-like member 11 and the side wall 12 with a hydrophobic upper surface. The bottom layer section 10 includes the plurality of receptacles 13 which are separated from each other by the side wall 12.
    Preferably, the blade-like member 11 has a hydrophilic surface. The term "hydrophilic surface" refers to a surface whose affinity for a hydrophilic solvent is greater than that with affinity for a hydrophobic solvent. The blade-like member 11 only needs to be made of a solid material. For example, the blade-like member 11 can be made of glass, silicone or a polymer resin.
    The side wall 12 is a structure that is provided on a surface of the blade-like member 11, preferably on the hydrophilic surface of the blade-like member 11 and is configured to separate the plurality of receptacles 13 from each other. The side wall 12 has a hydrophobic upper surface. The term "hydrophobic" here is used as a synonym for "lipophilic" and denotes a nature whose affinity for a hydrophobic solvent is greater than that with affinity for a hydrophilic solvent.
    Note that the side wall 12 needs to be configured so that its upper surface, that is, its surface facing the upper layer section 20, is hydrophobic. Whereas a side surface of the side wall 12, i.e., an inner wall of each of the receptacles 13, can be hydrophobic or hydrophilic.
    For example, the side wall 12 can be made of a hydrophilic structure and a hydrophobic layer that is formed on an upper surface of the hydrophilic structure. The hydrophilic structure can be made of, for example, glass, silicone or a polymer resin. The hydrophobic layer can be made of, for example, a water-repellent resin or a fluorocarbon polymer resin. Examples of the fluorocarbon polymer resin comprise amorphous fluorocarbon resin. The amorphous fluorocarbon resin is preferably used, because the amorphous fluorocarbon resin has high hydrophobic properties and low toxicity for a biological sample.
    Preferable examples of amorphous fluorocarbon resin comprise at least one item selected from CYTOP (Trademark), TEFLON (Trademark) AF2400 and TEFLON (Trademark) AF1600. Among them, CYTOP (Trademark) is more preferable, since it is easy to be microfabricated.
    Alternatively, the side wall 12 can be made of a hydrophobic material. For example, the side wall 12 can be made of a fluorocarbon polymer resin or a paraxylene polymer resin. Examples of the fluorocarbon polymer resin comprise a resin of amorphous fluorocarbon ©. It is preferably used as the amorphous fluorocarbon resin any of those exemplified above.
    The side wall 12 only needs to be configured in such a way that the plurality of receptacles 13 are provided on the blade-like member 11. For example, the side wall 12 can be a blade-like structure whose parts corresponding to the receptacles 13 are holes.
    A height (that is, a thickness in a vertical direction) of the side wall 12 measured from the surface of the blade-like member 11 only needs to be designed so that one of the microspheres 41 and 41 'contained in one of the receptacles 13 would not be discharged from there during the last described step of introducing the hydrophobic solvent. For example, the height of the sidewall 12 can be designed so that most, preferably the entire part of, one of the microspheres 41 and 41 'contained in one of the receptacles 13 is positioned lower than the upper surface of the sidewall 12.
    To efficiently store microspheres 41 and 41 'in receptacles 13, the height of side wall 12 is preferably equal to or greater than the average particle diameter of microspheres 41 and 41'. In addition, so that only one of the microspheres 41 and 41 'is stored in one of the receptacles 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 41 and 41'.
    Each of the plurality of receptacles 13 is a recess capable of storing only one of the microspheres 41 and 41 ', and the receptacles 13, in their plurality, are separated from each other by the side wall 12. Each of the receptacles 13 has a lower surface that it is a part of the surface of the blade-like member 11, and the bottom surface is hydrophilic.
    The receptacles 13 can be of any shape or size, as long as the shape or size allows each of the receptacles 13 to store only one of the microspheres 41 and 41 'in it. A region surrounded by the lower surface and the lateral surface of each of the receptacles 13 can be in the form of, for example, a circular cylinder or a rectangular column.
    The width "A" of each of the receptacles 13 in a horizontal direction (eg, in a case where a cross section of each receptacle 13 when viewed in the horizontal direction is shaped like a circle, the width "A" is a diameter of the circle; in a case where the cross section of each receptacle 13 when viewed in the horizontal direction is shaped like a square, the width "A" is a length on one side of the square) only needs to be greater than the average diameter particle size of microspheres 41 and 41 '. Preferably, the width "A" is 1.5 to 2 times greater than the average particle diameter of microsphere 41 and 41', for example. In the present embodiment, each of the receptacles 13 has a depth equal to the height of the side wall 12. In order to efficiently store the microspheres in the receptacles, the depth of each of the receptacles of the present invention is preferably equal to or greater than the average particle diameter of the microspheres. spheres is stored in one of the receptacles, the depth of each of the receptacles of the present invention is preferably equal to or less than 1.5 times the average particle diameter of the microspheres.
    According to the present embodiment, each of the receptacles 13 has a hydrophilic lower surface and the side wall 12 has a hydrophobic upper surface. This makes it possible to efficiently introduce the hydrophilic solvent 42 containing aspherical spheres 41 and 41 'into the receptacles 13 in the last described step of introducing the microspheres and prevent the hydrophobic solvent 43 from entering the receptacles 13 in the last described step of introducing the hydrophobic solvent. With this, the receptacles 13 that store the liquid droplets containing the microspheres 41 and 41 'can be hermetically sealed with the hydrophobic solvent in an efficient manner.
    The upper layer section 20 includes a blade-like member 21 and a hydrophobic layer 22. The hydrophobic layer 22 is provided on the surface of the blade-like member 21 whose surface faces the lower layer section 10. The member similar to blade 21 is made of, for example, glass, silicone or a polymer resin. The hydrophobic layer 22 is made of, for example, a water-repellent resin or a fluorocarbon polymer resin. Examples of the fluorocarbon polymer resin include amorphous fluorocarbon resin.
    The upper layer section 20 faces, through space 30, the surface of the lower layer section 10 on which the surface of the receptacles 13 is provided. That is, the space 30 exists between the side wall 12 and the hydrophobic layer 22. The space 30 serves as a flow path. In this way, matrix 1 is configured to have a cellular flow structure.
    The space 30 can be used as the flow path to allow a fluid to flow between the lower layer 10 section and the upper layer section 20 in a parallel direction with the surfaces of the lower layer section 10 and the upper layer section 20 , the surfaces of the lower layer section 10 and the upper layer section 20 facing each other.
    A distance between (i) the upper surface of the side wall 12 and (ii) the hydrophobic layer 22 of the blade-like member 21, that is, a width of the space 30 in the vertical direction, only needs to be greater than the average particle diameter. microspheres 41 and 41 'and is preferably from 10 pm to 150 pm.
    The bottom layer 10 section or the top layer 20 section can be provided with the through hole (not shown) through which fluid is introduced into space 30. For example, the bottom layer 10 section may have a region provided with the receptacles 13 and a region provided without receptacles 13. Furthermore, the section of the lower layer 10 may have the through hole in the region provided without receptacles 13; alternatively, the upper layer section 20 may have the through hole in a region facing the region of the lower layer section 10 provided without receptacles 13.
    According to the present embodiment, an upper side of the space 30 corresponds to the surface of the hydrophobic layer 22 and a lower side of the space 30 corresponds to the upper surface of the side wall 12 and the receptacles 13. Thus, with the exception of parts of the space 30 which correspond to the lower surfaces of the receptacles 13, the entire space 30 has a hydrophobic property. This configuration makes it possible to efficiently introduce the hydrophilic solvent 42 containing microspheres 41 and 41 'into receptacles 13 in the last described step of introducing the microspheres. In addition, this configuration prevents the hydrophobic solvent 43 from entering the receptacles 13 in the last described step of introducing the hydrophobic solvent. Thus, by introducing the hydrophobic solvent 43 into space 30, it is possible to efficiently form, in each of the receptacles 13, a droplet into which any of the microspheres 41 and 41 'is introduced.
    The matrix 1 of the present embodiment can be, for example, a matrix that includes one million or more receptacles. Even with the matrix having this large area, the use of the method for sealing microspheres of the present modality or the method for detecting the target molecule of the present modality makes it possible to efficiently seal the microspheres in the receptacles so that any of the microspheres are stored in each of the receptacles. Therefore, according to the present modality, it is possible to detect the target molecules with high sensitivity, therefore making it possible to provide a matrix that allows detection of target molecules in a concentration as low as 0.1 aM.
    In the following, a configuration of a kit of the present modality is described.
    The kit of the present embodiment includes at least matrix 1 and microspheres 41. Matrix 1 preferably having the configuration described above is used as matrix 1. Each of the receptacles 13 in the matrix 1 is configured to be able to store only one of the microspheres 41 included in this kit.
    Each of the microspheres 41 included in that kit can be the one that specifically captures the target molecule. For example, each of the microspheres 41 included in that kit can be the one that has been linked to a molecule for specific binding to the target molecule. It can be suitably used as the target molecule and the molecule to specifically bind to the target molecule any of those mentioned above.
    This kit can also include a substance to specifically detect the target molecule. It can preferably be used as the substance to specifically detect the target molecule any of those mentioned above. In addition, the kit may further include, for example, a water-soluble solvent and / or a hydrophobic solvent. [Target molecule detection device]
    In the following, a target molecule detection device 50 of the present embodiment is described with reference to Figure 2. Figure 2 is a schematic illustration of an embodiment of a target molecule detection device according to the present invention.
    The detection device for the target molecule 50 of the present embodiment includes matrix 1, an image sensor 51 and a light source 52. Preferably, matrix 1 can be used as the one with the configuration described above, and therefore explanations of the matrix 1 are omitted here.
    Image sensor 51 is a sensor for detecting light emitted by each of the receptacles 13 when the microspheres that captured the target molecules are stored in receptacles 13. For example, the image sensor 51 can be a sensor for detecting fluorescence emitted by a fluorescent substrate when decomposed by a specific enzyme bound (i) to the target molecule or (ii) to a molecule specifically bound to the target molecule. It can be used properly as the image sensor 51, for example, a CMOS image sensor.
    Light source 52 is a moon source for emitting light to matrix 1. In Figure 2, light source 52 is provided over matrix 1. However, the present invention is not particularly limited to this. Alternatively, the light source 52 can be that which emits light to the side face of the matrix 1, for example.
    Between matrix 1 and image sensor 51, an interference filter and / or a light guide matrix can be provided, for example. In addition, between the light source 52 and the matrix 1, an excitation filter can be provided, for example.
    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 captured the target molecules are stored in each of the receptacles 13. This makes it possible to quickly and easily determine whether or not the targets are stored in each one of the receptacles 13 and provide the target molecule detection device at an affordable price.
    The present application comprises the following inventions.
    A method of sealing the microspheres of the present invention includes: (i) a step of introducing a hydrophilic solvent containing microspheres into a space between (a) a section of the lower layer including a plurality of receptacles each of which is capable of storing only one of the microspheres and which are separated from each other by a side wall with a hydrophobic upper surface and (b) a section of the upper wall which faces a surface of the section of the lower layer on whose surface the plurality of receptacles is provided; and (ii) a step of introducing a hydrophobic solvent into space, with step (ii) performed after step (i).
    Preferably, the method for sealing microspheres of the present invention further includes (iii) a step of deaerating the space, with step (iii) being performed after step (i) and before step (ii).
    Preferably, according to the microsphere sealing method of the present invention, the hydrophilic solvent is at least one selected from the group consisting of water, hydrophilic alcohol, hydrophilic ether, ketone, nitrile solvents, dimethylsulfoxide and N, N-dimethylformamide or is a mixture that includes the one referred to as at least one.
    Preferably, according to the method for hydrophobic, it is at least one selected from the group consisting of saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, silicone oil, perfluorocarbon, halogen-based solvents and hydrophobic ionic liquid or is a mixture that includes that referred to as at least one.
    To achieve the foregoing objective, a method for detecting a target molecule of the present invention includes: (i) a reaction step of the microspheres that specifically capture target molecules with the target molecules; (ii) a step of carrying out, using the microspheres, any of the aforementioned methods for sealing microspheres, with step (ii) performed after step (i); and (iii) a step of determining whether any of the microspheres that captured the target molecules is or is not stored in each of the plurality of receptacles, with step (iii) being carried out after step (ii).
    Preferably, according to the method for detecting a target molecule of the present invention, the microspheres are those microspheres to which molecules specifically agglutinate with the target molecules are attached.
    A matrix of the present invention includes: a section of the lower layer provided with a plurality of receptacles separated from each other by a side wall having a hydrophobic upper surface; and a section of the upper layer that faces, by means of a space, with a surface of the section of the lower layer on whose surface the plurality of receptacles is provided.
    Preferably, according to the matrix of the present invention, each of the plurality of receptacles has a hydrophilic bottom surface.
    Preferably, according to the matrix of the present invention, the upper layer section has a hydrophobic surface that faces the lower layer section.
    Preferably, according to the matrix of the present invention, with respect to the upper layer section and the lower layer section, at least one of them has a through hole through which a fluid is introduced into the space.
    To achieve the foregoing objective, a kit of the present invention includes: any of the matrices mentioned above; each of the plurality of receptacles capable of storing any of the microspheres.
    To achieve the foregoing objective, the target molecule detection device of the present invention includes: any of the matrices mentioned above; and an image sensor for detecting light that is emitted from each of the plurality of receptacles in a case where microspheres that have captured target molecules are stored in the plurality of receptacles.
    The present invention is not limited to the description of the above modalities, but it can be changed by specialized persons within the scope of the claims. The modalities of the present invention are described in more detail with the aid of the Examples below. Needless to say, the present invention is not limited to such Examples. As the invention is so described, it will be evident that it may undergo variations in several ways.
    The following describes materials and methods that were used in the Examples. (Materials)
    In the Examples, streptavidin (acquired from SIGMA) labeled with figalactosidase (hereinafter, also referred to simply as "streptavidin") was used as the target molecule. In addition, in the condition of the microspheres, biotinylated microspheres prepared by biotinylation of microspheres linked to an amino group with an average particle diameter of 3 pm (material: polystyrene; micromer-NH2-3pm; purchased from Micromod) were used. (Preparation of biotinylated microspheres)
    By the following method, amino groups of microspheres linked to amino group were subjected to reaction with NHSPE04-Biotin, so that the microspheres linked to amino group were biotinylated.
    First, 750 pL of buffer A (100 mM of phosphoric acid buffer, pH 8.0) was added to 250 pL of microspheres linked to amino group.
    Then, the resultant was subjected to centrifugation at 10,000 rpm at 4 ° C for 10 minutes, so that the microspheres linked to the amino group were collected and collected. Then, the microspheres bound to the amino group were suspended in 500 µl of buffer A (suspension A). After that, 50 µL of NHS-PEO4-Biotin (2 pg / 50 µL of DMSO) was added to suspension A, and then the microspheres bound to the amino group and NHS-PEO4-Biotin were subjected to reaction under gentle agitation at 25 ° C for at least 3 hours (the tube was subjected to an extreme to extreme rotation for mixing).
    Then, biotinylated microspheres thus obtained were washed. The mixture was subjected to centrifugation at 10,000 rpm at 4 ° C for 10 minutes, so that the biotinylated microspheres were collected and collected. Then, an aqueous phase was removed by a Pipetman pipette. To the precipitate resulting from the biotinylated microspheres, 1 ml of buffer A was added so that the precipitate from the biotinylated microspheres was suspended. This process was repeated six times, and then unreacted NHS-PEO4-Biotin was removed. Then, the resultant was suspended in 500 µl of buffer A (suspension B). Suspension B was preserved at 4 ° C.
    Subsequently, the concentration of the biotinylated microspheres in suspension B was measured. The number of biotinylated microspheres in a given volume was counted using a hemacytometer, and the concentration of biotinylated microspheres (approximately 3.0 x 108 microspheres / mL) was found. In order for the number of biotinylated microspheres to be easily counted, counting was performed after suspension B was diluted by buffer A approximately 5 times, for example.
    By the aforementioned method, biotinylated microspheres were obtained. (Streptavidin capture)
    Then, using the following method, streptavidin was captured using biotinylated microspheres.
    First, the biotinylated microspheres were diluted (8 x 106 microspheres / 500 pL). Next, B-galactosidase-labeled streptavidin was diluted by buffer B (100 mM phosphoric acid buffer, pH 8.0, containing 0.1% TWEEN20 [detergent]) so that a streptavidin concentration became greater than a concentration target (total amount: 500 pL).
    Then, 500 pL of the biotinylated microspheres and 500 pL of streptavidin so diluted were mixed together in a tube (total amount: 1 mL). The tube was gently shaken vertically, and the reaction between biotinylated microspheres and streptavidin was carried out at 25 ° C for 30 minutes.
    The resultant was then centrifuged at 10,000 rpm at 4 ° C for 10 minutes, and the microspheres after the reaction (a mixture of [i] streptavidin complexes and the biotinylated microspheres and [ii] biotinylated microspheres that did not react) were collected and collected. Then, an aqueous phase was removed by a Pipetman pipette. To the resulting microsphere precipitate, 1 ml of buffer A was added and suspended. This process was repeated four times for washing, and the unreacted target molecules were removed.
    Then, to the precipitate of the microspheres after washing, 15 µl of buffer C (100 mM phosphoric acid buffer, pH 7.5, 1 mM MgC12) was added to the suspension. A final concentration of the microspheres was approximately 6.5 x 10 6 microspheres / 15 µl buffer C). (Matrix production)
    By the following method, a matrix containing the same cellular flow structure as that of matrix 1 shown in (a) to (e) of Figure 1 was produced. In the descriptions that follow, members with the same functions as those of matrix 1 are supplied with the same reference signals.
    First, a glass with a hydrophilic-hydrophobic pattern (lower layer section 10) and an upper side glass (upper layer section 20) (height: 24 mm x width: 26 mm x depth: 5 mm, SÍO2, with a through hole whose diameter is 1 mm) were produced. (Preparation of glass with hydrophilic-hydrophobic pattern)
    With reference to Figure 7, a specific method for preparing glass with a hydrophilic-hydrophobic pattern is described below. Figure 7 is a view for explaining a method of preparing glass with a hydrophilic-hydrophobic pattern according to an example of the present invention.
    According to the present modality, photolithography and dry engraving were performed so that a hydrophilic-hydrophobic pattern was formed on the glass. To form the hydrophilic-hydrophobic pattern, three steps including one step of applying CYTOP (Marca
    Registered), a photolithography step and an engraving and coating removal step were performed.
    In the application stage of CYTOP (Registered Trademark), CYTOP (Registered Trademark) CTL-809 (product name; available from ASAHI GLASS) was first applied to 24 mm (height) x 32 mm (width) glass (product name : NEO MICRO COVER GLASS Thickness No. 1; available from MATSUNAMI) (blade-like member 11), so that a layer of hydrophobic resin 61 was formed.
    Then, in the photolithography stage, a positive photocoat 62 (product name: AZ-4903; available from AZ Eletronic Materials USA) was applied to the hydrophobic resin layer 61. Then, using a 63 mask with a desired pattern, the resultant was exposed to UV emitted from above, so that an alkaline development process was carried out. As a result of the development process, photocoat 62 was dissolved only in the UV irradiated parts, so that fractions of the hydrophobic resin layer 61 whose parts face the UV irradiated 62 parts of the photocoat were exposed.
    After that, in the engraving and coating removal step, the glass was etched by O2 plasma by means of a partially dissolved photolayer 62 ', and thus fractions of the resin layer 61 were removed. As a result, a hydrophobic side wall 12 was obtained. Finally, photocoat 62 'was dissolved by an organic solvent. Thus, the hydrophilic-hydrophobic pattern was obtained.
    Additional detailed procedures for the above process are described below. The reference numbers (1) to (23) in Figure 7 correspond to (1) to (23) below, respectively.
    Application step of CYTOP (Trademark) (ie preparation of a CYTOP [Trademark] layer with a film thickness of approximately 3.3 pun at 3.5 pm by the following procedures)
    (1) First, the glass (blade-like member 11) was washed and CYTOP (Trademark) CTL-809 was applied to the glass.
    (2) Then, the glass was immersed in 10 N KOH for one night.
    (3) The coating glass that was immersed in KOH was washed with deionized water ten or more times.
    (4) The glass was dried with a hot blade at 180 ° C.
    (5) The glass thus dried was cooled to room temperature.
    (6) Approximately 70 pL of CYTOP (Trademark) CTL-809 was poured onto the glass.
    (7) Rotation coating was carried out according to the following program A: [Program A]
    Inclination: 5 seconds 500 rpm: 10 seconds Inclination: 5 seconds 2000 rpm: 30 seconds Inclination: 5 seconds End
    (8) The glass was dried on a hot slide at 180 ° C for one hour.
    By repeating procedures (6) to (8) four times, a layer of hydrophobic resin 61 with a depth of 3.3 pm to 3.5 pm was obtained. Photolithography step
    Then, photolithography was performed.
    (9) On the resin layer 61 prepared by the application stage of CYTOP (Trademark), a positive photocoat 62 (AZ-4903) was poured in such an amount that the positive photocoat 62 spread over the glass so that obtained a diameter of approximately 8 mm.
    (10) Rotation coating was carried out according to the following program B: [Program B]
    Slope: 5 seconds 500 rpm: 10 seconds Slope: 5 seconds 4000 rpm: 60 seconds Slope: 5 seconds End
    (11) The photocoat that remained on the edge of the glass was removed by a piece of gauze moistened with 100% EtOH.
    (12) The glass was dried at 55 ° C for 3 minutes.
    (13) The glass was dried at 110 ° C for 5 minutes.
    (14) A mask 63 was washed with acetone, and then mask 63 was fixed in a mask aligner (available from SAN-EI ELECTORIC).
    (15) The glass to which the photoforestry was applied was fixed on a sample table of the mask aligner, and the sample table was raised, so that the glass and the photo mask 63 were brought into contact with each other.
    (16) The glass then placed in contact with photomask 63 was irradiated with UV for 35 seconds (power: 256).
    (17) The glass was immersed in AZ Developer (available from AZ Electronic Materials USA) for 5 minutes or more for development.
    (18) The glass was rinsed with MilliQ (distilled water) for approximately 10 minutes.
    <Engraving and coating removal step>
    Subsequently, engraving and coating removal were carried out.
    (19) The glass was subjected to etching with O2 plasma using RIE-10NR (available from Samco) under certain process conditions (O2: 50 sccm, pressure: 10 Pa, power: 50 W, time: 30 min. ).
    (20) The glass that was subjected to the engraving was immersed in acetone and then the glass was subjected to ultrasound for 15 minutes.
    (21) The acetone was replaced by a new one, and then the glass was subjected to ultrasound for 15 minutes.
    (22) The glass was subjected to ultrasound in EtOH for 15 minutes.
    (23) The glass was washed with MilliQ (distilled water).
    In the method described above, a large number of wells (receptacles 13) were formed on the glass. A region surrounded by the lower surface and the lateral surface of each of the alveoli was formed in a circular cylinder. A cross section of each well in the horizontal direction was formed in a circle with a diameter of 5 pm. The height of the side wall, by which the alveoli were positioned, was approximately 3.3 pm to 3.5 pm. In addition, a distance "B", through which two adjacent wells were separated from each other, was 5 pm.
    (Preparation of the upper face glass)
    In the following method, an upper face glass was prepared. To prepare the top face glass, the glass used was 5 mm thick and had a 1 mm diameter through hole. One side of this glass was covered with approximately 70 pL of CYTOP (Trademark) CTL-809 (product name; available from ASAHI GLASS). Then, coating with rotation was carried out according to the following program C: [Program C] Slope: 5 seconds 500 rpm: 10 seconds Slope: 5 seconds 2,000 rpm: 30 seconds Slope: 5 secondsEnd
    After that, the glass was dried on a hot slide at 180 ° C for one hour.
    In the method described above, an upper face glass having a side provided with a hydrophobic layer of a thickness of 1 pm was prepared.
    (Connection of glass with hydrophilic-hydrophobic pattern to glass on the upper side)
    Then, high vacuum lubricant (available from Dow CORNING TORAY) was applied to a portion of Parafilm backing paper (available from Peckiney Plastic Packaging), and then the portion of Parafilm backing paper was attached to a portion of the glass with hydrophilic-hydrophobic pattern, the part remaining on one side on which the hydrophilic-hydrophobic pattern was formed and the part without the hydrophilic-hydrophobic pattern. The upper face glass was attached to the side of the glass with hydrophilic-hydrophobic pattern on whose side the hydrophilic-hydrophobic pattern was formed, so that the side coated with coating agent on the upper face glass faced the hydrophilic pattern glass ilico-hydrophobic.
    Consequently, a space was made between the hydrophilic-hydrophobic pattern glass and the upper face glass. The width of this space in the vertical direction, that is, a distance between (i) the upper surface of the lateral wall of the hydrophilic-hydrophobic pattern glass and (ii) the glass of the upper face, was approximately 150 pm.
    (Sealing of microspheres in droplets)
    Subsequently, in the following method, the microspheres which reacted with streptavidin were sealed in droplets.
    First, 50 mM fluorescein-di-phalalpyrananoside (FDG) (available from Marker Gene Technology) / DMSO were diluted in FDG buffer (100 mM KPi buffer [H = 7.5], 1 mM MgCl2, 4 pL / mL of 2mercaptoethanol), so that 4 mM FDG solution was prepared. Then, 15 µl of the microspheres (6.5 x 106 microspheres / 15 µl of buffer C) and 15 µl of FDG solution were mixed together, and a microsphere solution was prepared.
    Then, 30 µL of the microsphere solution was loaded into the flow path by a yellow end through the through hole in the upper face glass [see (a) and (b) of Figure 1].
    Then, to remove air from the alveoli, deaeration was performed for one minute [see (c) of Figure 1]. Deaeration was performed in such a way that the matrix was allowed to pause in a vacuum desiccator of approximately 0.1 atm (10.1325 kPa) for about 30 seconds. Then, the matrix was left to stand for approximately 5 minutes, so that the microspheres were precipitated at the bottom of the wells.
    Thereafter, 200 pL to 1,000 pL of Fluorinert (Trademark) FC40 (available from SIGMA) were loaded into the flow path through the through hole in the upper face glass (see (d) and (e) in Figure 1).
    As a result, an aqueous phase was confined only to the alveoli, and droplets were formed. Thus, the microspheres were sealed in the droplets. [Example 1]
    By the method described above, biotinylated microspheres and 1 fM streptavidin were reacted with each other, and then the resultant was introduced into a matrix together with FDG, so that the microspheres were sealed in droplets, respectively. The matrix used in Example 1 was a 1.0 cm x 1.0 cm matrix comprising a total of 1097600 receptacles, specifically, including a matrix of 20 x 20 (horizontally and vertically) of submatrices each (i) with a size 512 pm x 512 pm and (ii) including 2744 receptacles. This matrix was observed with a fluorescence microscope (1X71 [available from OLYMPUS]).
    Figure 3 shows a fluorescence image of the matrix in which the microspheres were sealed in an example of the present invention. What is shown in Figure 3 is a submatrix. As shown in Figure 3, some bright spots were seen in the fluorescence image (138 bright spots in a field). These bright spots indicate positions of the receptacles in which biotinylated microspheres containing captured streptavidin have been sealed. This shows that the use of the method of the present invention makes it possible to adequately detect 1 fM of streptavidin. [Comparative Example 1]
    In a comparative example of the present invention, target molecules were detected by a conventional method of determining volume.
    12.5 fM streptavidin, which is a concentration 12.5 times higher than the (1 fM) of Example 1, were mixed with FDG, and the result was measured by fluorescence with the aid of a fluorescence spectrophotometer. In addition, a control experiment was performed in the same way by using 6.3 pM of streptavidin, which is a concentration 500 times higher than that of this comparative example.
    Figure 4 shows results from the comparative example and the control experiment. Figure 4 is a graph showing fluorescence intensities observed when target molecules were detected by the conventional method. In Figure 4, a "a" line in the graph shows a case result involving the use of 12.5 fM streptavidin, while a "b" line in the graph shows a case result involving the use of 6.3 pM streptavidin.
    As shown in Figure 4, in the case where the conventional method was used, it was impossible to detect 12.5 fM of streptavidin. This shows that the concentration of 12.5 fM is less than a detection limit of the conventional method. [Example 2]
    Then, biotinylated microspheres were subjected to reaction with streptavidin of five different concentrations (1 fM, 100 aM, 10 aM, 1 aM and 0.1 aM) and were then introduced into matrices together with FDG, so that the microspheres were sealed in droplets, respectively. Each of the matrices used in Example 2 had the same configuration as those used in Example 1. Each of these matrices was observed in a bright field image and in a fluorescence image by a microscope.
    (Detection results)
    (a) to (f) of Figure 5 show microscopic images of the matrices in which the microspheres were sealed in another example of the present invention. Note that each of (a), (c) and (e) in Figure 5 shows a bright field image of a respective submatrix, while each of (b), (d) and (f) in Figure 5 shows a fluorescence image of one of the respective submatrices shown in (a), (c) and (e) of Figure 5. In addition, (a) and (b) of Figure 5 show the results obtained in the case involving the use 1 fM streptavidin; (c) and (d) of Figure 5 show the results obtained in the case involving the use of 100 µM streptavidin; and (e) and (f) of Figure 5 show the results obtained in the case involving the use of 10 µM streptavidin.
    In the case involving the use of 1 fM of streptavidin, a total number of microspheres sealed in a submatrix was 1735; among these microspheres, the number of microspheres with streptavidin captured was 138. In the case involving the use of 100 µM streptavidin, a total number of microspheres sealed in a submatrix was 2008; among these microspheres, the number of microspheres with streptavidin captured was 6. In the case involving the use of 10 µM streptavidin, a total number of microspheres sealed in a submatrix was 1360; among these microspheres, the number of microspheres with streptavidin captured was 1.
    (Comparison between theoretical and experimental value)
    In addition, a theoretical value and an experimental value of a proportion (%) of the number of microspheres (active microspheres) containing streptavidin captured in relation to the total number of microspheres were calculated for each of the streptavidin concentrations.
    A proportion (%) of the number of streptavidin molecules in relation to the total number of microspheres used in the streptavidin reaction was calculated as the theoretical value. Whereas a proportion (%) of the number of microspheres containing streptavidin captured in relation to the total number of microspheres stored in the matrix was calculated as the experimental value.
    Figure 6 shows a graph illustrating a relationship, seen in said other example of the present invention, between (i) a streptavidin concentration and (ii) a ratio of the number of streptavidin-containing microspheres captured to the number of microspheres stored in the matrix. In Figure 6, a line "a" in the graph shows the theoretical values, a circular dashed line shows the experimental value and a circle represented with a dashed line shows means of the experimental values (N = 2 to 3). "b" in the graph is a line by which averages of the experimental values were approximated.
    As shown in Figure 6, the theoretical values and the experimental values are almost the same in relation to each other. This shows that the method of the present example has high quantitative precision and is able to determine exactly a concentration of target molecules. These results show that the method of the present example makes it possible to adequately detect even target molecules of 0.1 µM or more. [Example 3]
    By the method described above, a solution of microspheres (6.5 x 106 microspheres / 30 pL) was introduced (loaded) into the matrix with cellular flow structure prepared in Example 1, so that the microspheres were sealed (trapped) in droplets. Then, a trapping efficiency (that is, a proportion [%] of the number of trapped microspheres in relation to the number of loaded microspheres) during this process was calculated.
    A calculation result is shown in Figure 8. Figure 8 is an illustration (i) of a microsphere trapping efficiency found in a case involving the use of the matrix with the cellular flow structure (Example 3) and (ii) a microsphere trapping efficiency found in a case involving the use of a matrix without the cell flow structure (Comparative Example 2). [Comparative Example 2]
    In this comparative example, the matrix that lacks the cellular flow structure (that is, the matrix made only of glass with the hydrophilic-hydrophobic pattern described above) was used for sealing microspheres (microspheres connected to amino of <5 = 3 pm; micromer-NH2-3 pm; available from Micromod).
    By the method described below, microspheres were sealed to the glass with a hydrophilic-hydrophobic pattern using a microsphere solution that was diluted to the same concentration (2.2 x 108 microspheres / mL = 6.5 x 106 microspheres / 30 pL) as was used in the case involving the use of the matrix without flow cell structure (Example 3).
    (1) The microspheres were diluted to a concentration of 2.2 x 108 microspheres / ml with a buffer (100 mM KPi buffer [H = 7.5], 1 mM MgC12, 2 µl 2-mercaptoethanol).
    (2) The glass with hydrophilic-hydrophobic pattern (prepared by the method mentioned above) was fixed to the bottom of a Petri dish (35 mm petri dish, available from Becton Dickinson). An Araldite AR-R30 adhesive (available NICHIBAN).
    (3) A top surface of the glass with a hydrophilic-hydrophobic pattern was covered with 500 pL of the microsphere solution.
    (4) The resultant was subjected to deaeration and then incubated for 5 minutes.
    (5) 2 mL of FC40 (Fluorinert [Trademark] FC40, available from SIGMA) were loaded onto the glass with a hydrophilic-hydrophobic pattern, so that the microspheres were sealed in it.
    (6) To prevent evaporation of the droplets, water was introduced, so that an oily phase was covered with water.
    Subsequently, the number of microspheres confined to the droplets was counted, and then an entrapment efficiency (a proportion [%] of the number of microspheres trapped in relation to the number of microspheres loaded) was calculated.
    Calculation results are shown in Figure 8. As shown in Figure 8, the trapping efficiency found in the case involving the use of the matrix without cellular flow structure was 25 or more times greater than that found in the case involving the use of the matrix without structure flow cell. The reason for this is considered as follows: In the case involving the use of the matrix without a cellular structure of flow, a distance in which the microspheres could disperse in a vertical direction was increased, which made it difficult to approach the microspheres of the substrate.
    In addition, in the case involving the use of the matrix without the cell flow structure, the entire surface of the matrix substrate needs to be covered with the microsphere solution. This requires a large amount of microsphere solution (that is, the microsphere solution whose amount is approximately 16 times greater than that used in the case involving the use of the matrix without the cell flow structure). Thus, the use of the matrix with cellular flow structure makes it possible to perform sealing of microspheres with a small amount of microsphere solution.
    The present invention is suitably applicable to a method for detecting target molecules of low concentration, a respective matrix, a respective device and the equivalent. List of Reference Signs
    1 Matrix 10 Section of the lower layer 20 Section of the upper layer 12 Side wall 13 Receptacle 30 Space 41, 41 ’Microspheres 42 Hydrophilic solvent 43 Hydrophobic solvent
    权利要求:
    Claims (4)
    [0001]
    1. Method for sealing microspheres, characterized by comprising: (i) a step of introducing a hydrophilic solvent containing microspheres having a particle size from 1 pm to 4 pm, in a space within a flow cell structure, between (a) a section of the lower layer including a plurality of receptacles each of which is capable of storing only one of the microspheres and which are separated from each other by a side wall with a hydrophobic upper surface and (b) a section of the upper layer which faces a surface of the section of the lower layer on which the surface of the plurality of receptacles is provided; and (ii) a step of introducing a hydrophobic solvent into space, to displace the hydrophilic solvent, step (ii) be carried out after step (i) to form, in the plurality of receptacles, droplets of the hydrophilic solvent within the plurality of receptacles covered with the hydrophobic solvent, where a single granule is trapped within a drop of the hydrophilic solvent within each of the plurality of receptacles, and where the plurality of receptacles are 1.5 to 2 times wider in diameter average of the beads particles, the plurality of receptacles each have a depth equal to or less than 1.5 times the average diameter of the beads particles, and an efficiency of retention of the beads is twenty-five times or more superior in relation to the absence flow cell structure, where the structure does not have a section of the upper layer.
    [0002]
    2. Method, according to claim 1, characterized by further comprising: (111) a step of de-aerating the space, with step (iii) being performed after step (i) and before step (ii).
    [0003]
    3. Method according to claims 1, characterized by the fact that: the hydrophilic solvent is at least one selected from the group consisting of water, hydrophilic alcohol, hydrophilic ether, ketone, nitrile-based solvents, dimethylsulfoxide and N, N -dimethylformamide, or is a mixture including that referred to as at least one.
    [0004]
    4. Method according to claims 1, characterized by the fact that: the hydrophobic solvent is at least one selected from the group consisting of saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, silicone oil, perfluorocarbon, halogen-based solvents and hydrophobic ionic liquid, or is a mixture including that referred to as at least one.
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    同族专利:
    公开号 | 公开日
    WO2012121310A1|2012-09-13|
    CN103415774A|2013-11-27|
    AU2012226920A1|2013-10-10|
    JP5337324B2|2013-11-06|
    CA2829185A1|2012-09-13|
    US10809257B2|2020-10-20|
    US20170115284A1|2017-04-27|
    JPWO2012121310A1|2014-07-17|
    EP2685266A4|2014-01-15|
    RU2013144617A|2015-04-20|
    EP2685266A1|2014-01-15|
    US20160223531A1|2016-08-04|
    BR112013022933A2|2016-12-06|
    AU2012226920B2|2015-02-19|
    US20190154682A1|2019-05-23|
    US9329174B2|2016-05-03|
    US20130345088A1|2013-12-26|
    CN103415774B|2014-09-24|
    US10267793B2|2019-04-23|
    RU2548619C1|2015-04-20|
    CA2829185C|2017-11-14|
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    2018-12-18| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2019-09-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|
    2020-05-19| B09A| Decision: intention to grant|
    2020-07-07| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/03/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    JP2011050629|2011-03-08|
    JP2011-050629|2011-03-08|
    PCT/JP2012/055884|WO2012121310A1|2011-03-08|2012-03-07|Bead sealing method, method for detecting target molecule, array, kit, and target molecule detection device|
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