Microstructures and molecular detection methods

Abstract

To provide a micro structure equipped with a mechanism that selectively detects marker molecules on which individual cells forming a cell population are expressed or biomolecules that the cells secrete, a method for manufacturing the structure, and concrete solving means for detecting / identifying a molecule being a detection target by using the micro structure.SOLUTION: Provided is a method for manufacturing a micro structure of a semi-spherical shell shape on an inner face of which a material surface capable of immobilizing a probe for detecting a biomolecule is arranged, and which is manufactured in a thin film of a desired thickness and diameter, and a method for detecting a target biomolecule by using the structure.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a microstructure having a structure such as a hemispherical shell or a semi-elliptical shell, the structure being composed of two or more layers of thin film bonding material whose inner and outer surfaces are made of different materials, and a substance detection method using the same. [Background technology]

[0002] Microstructures such as microparticles are widely used as materials for the development of materials with new physical properties, and as labels for visualizing target proteins and DNA in the field of life science. Although spherical microparticles are generally used because they are easy to fabricate, microparticles with complex shapes such as ellipses and polygons have a wide range of applications because they have anisotropy in optical properties, and the development of fabrication methods for them is being actively pursued.

[0003] JP 2011-101941 A ("Hollow microparticles and their manufacturing method") (Patent Document 1) discloses a method for manufacturing bowl-shaped hemispherical shelled microparticles, and WO 2013 / 069732 ("Magnetic nanoparticles") (Patent Document 2) discloses a method for manufacturing such hemispherical shelled microparticles using a magnetic material and applying them to cell purification technology.

[0004] Japanese Patent Laid-Open Publication No. 2011-101941 (Patent Document 1) discloses a method for producing hemispherical shell microparticles by forming a metal thin film by vacuum deposition or sputtering on polystyrene particles aligned on a flat substrate, and removing the polystyrene particles by chemical treatment, heating, etc. However, there is no disclosure of specific applications of the produced microparticles in the field of life sciences, particularly applications for detecting biomolecules such as proteins and DNA, which are important in medical diagnosis.

[0005] WO2013 / 069732 (Patent Document 2) discloses, as one application of the above-mentioned JP-A-2011-101941 (Patent Document 1), a method of producing hemispherical shell-shaped microparticles of the same size as cells (diameter about 10 μm) using a magnetic material such as nickel or iron, and size-selectively capturing cells in the inner recess of the microparticles to purify and recover them. A method of producing hemispherical shell-shaped microparticles by sandwiching an insulating layer between magnetic thin films to form a multi-layer structure and making the physical properties of the magnetic microparticles superparamagnetic is also disclosed. However, applications other than cell recovery, particularly a method of identifying the type and properties of cells by detecting biomolecules expressed on the surface of recovered cells, is not disclosed.

[0006] Among biomolecules, secretions secreted by cells have been attracting attention in recent years as "message substances," and the type and amount of secretions are important indicators that reflect the "individuality" of the cell. Cells interact with surrounding cells through secretions, creating an optimal environment for themselves or adapting to the surrounding environment. This has also been linked to many diseases. For example, in tumor tissue, cancer cells are thought to affect surrounding normal cells through secretions, creating a microenvironment that is favorable for their own growth. For these reasons, precise measurement of substances secreted by individual cells in a cell population at the single-cell level is important for elucidating disease mechanisms and discovering new therapeutic targets.

[0007] There have been several reports on techniques for measuring secretions at the single cell level. In Jun Arita, "Analysis of the Secretion from Single Anterior Pituitary Cells by Cell Immunoblot Assay", Endocrine Journal, Vol. 40, 1, 1-15 (1993) (Non-Patent Document 1), cells are cultured on a sheet on which an antibody that captures secretions is immobilized, and the secretions released from the cells and captured by the antibody are later stained and visualized to measure the secretion amount of each cell. In JP 2014-233208 A ("Apparatus and method for comprehensively analyzing cell secretions") (Patent Document 3), cells are captured in tiny chambers of several tens of microns each, and the secretions released from each cell are detected by accumulating and concentrating them in the chamber, thereby measuring the secretion amount of each cell. However, all of these methods disclose only a method for measuring the secretions of completely isolated individual cells in which adjacent cells are separated from each other, and no means for measuring the secretions of individual cells in a cell population that constructs a network and interacts with each other is disclosed.

[0008] On the other hand, in order to detect a specific biomolecule, it is useful to use a probe switch that does not produce any reaction when the biomolecule is not present, but produces a signal (e.g., emits fluorescence) only when the biomolecule is present. For this purpose, aptamer molecules are often used. Like antibodies, aptamers are a general term for peptides and nucleic acids that selectively bind to specific target molecules, and are useful as target molecule detection probes from many perspectives, such as ease of synthesis and modification. Ueno et al., "Molecular design for enhanced sensitivity of a FRET aptasensor built on the graphene oxide surface", Chem. Commun., 49, 10346-10348, (2013) (Non-Patent Document 2) shows a biomolecule detection method that uses an aptamer probe modified with a fluorescent dye and a graphene membrane. When a fluorescent aptamer is immobilized on a flat graphene surface, the aptamer is adsorbed on the graphene surface, so the distance between the fluorescent dye and the graphene surface becomes short, and fluorescence resonance energy transfer (FRET) occurs between the fluorescent dye and graphene, resulting in quenching. However, when a target molecule binds, the aptamer structure changes, and the fluorescent dye moves away from the graphene surface, resulting in fluorescence. In other words, a sensing technology has been shown that detects the presence of a target biomolecule as the generation of fluorescence. However, because graphene is ultra-flat at the atomic level, it is difficult to form a continuous, large-area thin film on a curved surface, and there have been no examples of film formation on a three-dimensional material such as a microstructure. In addition, Non-Patent Document 2 shows a method of mounting the sensing technology on a microchannel device to detect biomolecules in a solution, but does not show a method of detecting biomolecules expressed by individual cells or secretions released by them. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] JP 2011-101941 A [Patent Document 2] WO2013 / 069732 [Patent Document 3] JP 2014-233208 A [Non-patent literature]

[0010] [Non-Patent Document 1] Jun Arita, "Analysis of the Secretion from Single Anterior Pituitary Cells by Cell Immunoblot Assay", Endocrine Journal, Vol. 40, 1, 1-15 (1993) [Non-Patent Document 2] Ueno et al., "Molecular design for enhanced sensitivity of a FRET aptasensor built on the graphene oxide surface", Chem. Commun., 49, 10346-10348, (2013) Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, it is desirable to provide a microstructure equipped with a mechanism for selectively detecting marker molecules expressed or biomolecules secreted by individual cells that form a cell population, a method for producing the same, and a concrete solution for detecting and identifying the target molecules using the microstructure. [Means for solving the problem]

[0012] In view of the above circumstances, the present invention provides a method for producing a hemispherical shell-shaped microstructure, the inner surface of which is provided with a material surface capable of immobilizing a probe for detecting a biomolecule, and which is produced from a thin film of a desired thickness and diameter, and a method for detecting a target biomolecule using the same. The present invention also provides a method for producing and controlling a microstructure, the outer surface of which is made of a magnetic material, and the orientation of which can be controlled by application of an external magnetic field, and a method for producing and controlling a microstructure, the inner surface of which is made of a SP 2 The present invention provides a method for producing a microstructure which is a thin-film structure having hybrid orbitals or a thin-film structure made of metal, and a method for detecting a target molecule by fluorescence by selective reaction between the target biomolecule and a protein, peptide, or nucleic acid molecule which emits fluorescence due to a change in molecular structure and is immobilized on the inner surface of the microstructure after capturing a biomolecule or cell.

[0013] More specifically, this disclosure includes the following [1] to

[21] . [1] A hemispherical shell-shaped hollow multi-layer microstructure for use in detecting a target molecule, comprising: a first thin film layer in the form of a substantially minute hemispherical shell made of a first material including a magnetic material; a second thin film layer that is made of a second material including a material capable of detachably fixing a probe labeled with a fluorescent dye and capable of causing fluorescence resonance energy transfer between the fluorescent dye and the second thin film layer, the second thin film layer being disposed on the inner surface of the micro-hemispherical shell; Equipped with having a size capable of capturing at least one target cell or a portion thereof in a hollow space defined by the second thin film layer; The probe is a molecule capable of specifically binding to the target molecule, and changes in structure upon binding to the target molecule, thereby causing a change in emission / quenching of the fluorescent dye. [2] The hemispherical shell-shaped hollow multilayer microstructure according to [1] above, wherein the first material comprises a magnetic material selected from the group consisting of nickel, iron, cobalt, gadolinium, ruthenium, iron oxide, chromium oxide, ferrite, and neodymium. [3] The second material is SP 2 Elements with hybrid orbitals, SP 2 Binding regions and SPs 3 3. The hollow multi-layer microstructure of the hemispherical shell according to the above [1] or [2], wherein the bonding region comprises a mixture of elements or metals. [4] The hemispherical shell-shaped hollow multilayer microstructure according to [3] above, wherein the second material comprises nanocarbon, nanographene, or gold. [5] The hemispherical shell-shaped hollow multilayer microstructure according to the above [4], wherein the second thin film layer has amino groups on its surface. [6] The hemispherical shell-shaped hollow multilayer microstructure according to any one of the above [1] to [5], wherein the probe is a protein, a peptide, or a nucleic acid molecule. [7] The hemispherical shell-shaped hollow multilayer microstructure according to [6] above, wherein the probe is a nucleic acid molecule having one end modified with a fluorescent dye and the other end modified with a pyrene molecule or a thiol group. [8] The hemispherical shell-shaped hollow multilayer microstructure according to any one of [1] to [7] above, wherein the target molecule comprises a protein, a peptide, a nucleic acid, a cell surface molecule, or a cell secretory vesicle. [9] The hemispherical shell-shaped hollow multilayer microstructure according to any one of the above [1] to [8], wherein the thickness of each of the thin film layers is in the range of 0.1 nm to 1 mm.

[10] An array of hemispherical shell-shaped hollow multi-layer microstructures comprising a plurality of the hemispherical shell-shaped hollow multi-layer microstructures according to any one of the above [1] to [9].

[11] The hemispherical shell-shaped hollow multilayer microstructure or an array thereof according to [1] to

[10] above, comprising the probe detachably fixed to the surface of the second thin film layer.

[12] The hemispherical shell-shaped hollow multilayer microstructure or an array thereof according to

[11] above, wherein the probe is detachably fixed to the surface of the second thin film layer via a spacer molecule, and / or the fluorescent dye is bound to the probe via a spacer molecule.

[13] A method for producing a hemispherical shell-shaped hollow multilayer microstructure or an array thereof for use in detecting a target molecule according to any one of [1] to

[12] above, comprising the steps of: a) providing template particles of a desired size arranged in a monolayer on a substrate, the template particles being made of a material removable by a predetermined removal process; b) coating the template particles disposed on the substrate in the monolayer with the second material; c) further coating the template particles coated with the second material with the first material; and d) removing the template microparticles by the predetermined removal process to obtain the hemispherical shell-shaped hollow multi-layer microstructure. A method for producing a hemispherical shell-shaped hollow multi-layered microstructure or an array thereof, comprising:

[14] A method for producing a hemispherical shell-shaped hollow multi-layered microstructure or an array thereof according to

[13] above, comprising at least one step of coating with another material between the steps b) and c).

[15] After step d) above, Transferring the hemispherical shell-shaped hollow multi-layer microstructure from the substrate surface to the adhesive surface using an adhesive; and / or Removably fixing the probe to the second thin film layer. A method for producing a hemispherical shell-shaped hollow multi-layer microstructure or an array thereof according to the above

[13] or

[14] , comprising:

[16] The method of claim 14, wherein the further material comprises a material containing an element or element alloy different from the first or second material.

[17] The manufacturing method described in

[15] above, wherein the adhesive is a soluble adhesive.

[18] The manufacturing method according to

[17] above, wherein the soluble adhesive is polydimethylsiloxane, and the manufacturing method includes a step of solubilizing the adhesive in a solvent.

[19] A method for detecting a target molecule using at least one hemispherical shell-shaped hollow multilayer microstructure or an array thereof according to the above

[11] or

[12] , or at least one hemispherical shell-shaped hollow multilayer microstructure or an array thereof manufactured by the manufacturing method according to the above

[15] ,

[17] , or

[18] , comprising: a) placing the hemispherical shell-shaped hollow multi-layer microstructure or an array thereof, including the probes removably fixed to the second thin film layer, in a solution containing or suspected of containing the target molecule; and b) measuring the fluorescent emission of the fluorescent dye of the probe, detection of the fluorescent emission infers binding of the target molecule to the probe and confirms the presence of the target molecule in the solution; A method comprising:

[20] In step a), Controlling the orientation of the hemispherical shell-shaped hollow multi-layered microstructure dispersed in the solution by applying an external magnetic field. The method according to

[19] above, comprising:

[21] The method according to

[19] or

[20] above, wherein the target molecule is a secretion product of a cell, and the method further comprises, between steps a) and b), a step of capturing the cell or a part thereof in the hollow space of the hemispherical shell-shaped hollow multilayer microstructure. Effect of the Invention

[0014] Prior to the present invention, there was no technology capable of measuring the secretion of each individual cell in a cell network. The present invention makes it possible to identify the type and amount of substances secreted by each cell forming a population. For example, in a biopsy performed when a disease is suspected, a portion of tissue is taken and the characteristics of each cell are examined, and the present invention can be a simple examination method for identifying diseased cells, etc., using secretions as an indicator. In other words, it is expected that this will lead to the development of a technology for distinguishing cancer cells from other cells by secretions. Furthermore, the present invention can be applied not only to disease examinations, but also to the detection of specific substances and microorganisms in the environment. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram of a microstructure having a two-layer structure according to one embodiment of the present invention. [Diagram 2] 1A to 1C are schematic diagrams illustrating a method for producing a microstructure according to one embodiment of the present invention. [Diagram 3] As one embodiment of the present invention, a method for detecting a target molecule using a fluorescent aptamer immobilized on the inner surface of a microstructure whose inner surface is a nanocarbon film is shown. Figure 3-1 shows an example of a method for detecting a target molecule using a DNA aptamer with a fluorescent dye attached. Figure 3-2 shows an example of a method for detecting a target molecule using a DNA aptamer with a fluorescent dye and a pyrene group attached. [Figure 4] FIG. 1 shows representative examples of fluorescence microscope images obtained when the target molecule vaspin is detected using a DNA aptamer having a fluorescent dye attached thereto, or a DNA aptamer having a fluorescent dye and a pyrene group attached thereto, immobilized on the inner surface of a microstructure as one embodiment of the present invention, and a graph comparing the change in fluorescence brightness based on the images. [Diagram 5] 5A and 5B are schematic diagrams of another example of a method for detecting a target molecule using a fluorescent aptamer immobilized on the inner surface of a microstructure as one embodiment of the present invention. Fig. 5-1 shows an example of a method for detecting a target molecule using a fluorescent aptamer immobilized on the inner surface of a microstructure whose inner surface is made of Au. Fig. 5-2 shows an example of a method for detecting a target molecule by adsorbing a fluorescent aptamer to a microstructure whose inner surface has nanographene immobilized thereon. [Figure 6] As an embodiment of the present invention, this is a schematic diagram of a specific example of a method for detecting a target molecule using a microstructure with a fluorescent aptamer immobilized on its inner surface. Figure 6-1 shows an example of a method for detecting a target molecule using microstructures arranged in an array on a substrate. Figure 6-2 shows an example of a method for detecting a target molecule using a microstructure attached to the tip of a micro cantilever. Figure 6-3 shows an example of a method for detecting a target molecule by attaching a magnetic microstructure dispersed in a solution to a cell. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] 1. Hemispherical shell-shaped hollow multi-layer microstructures for use in detecting target molecules

[0017] In one aspect, the present invention provides a hollow multi-layered microstructure in the shape of a hemispherical shell (hereinafter, simply referred to as a "microstructure") for use in detecting a target molecule. The microstructure of the present invention comprises a first thin film layer in the form of a substantially micro-hemispherical shell made of a first material including a magnetic material, and a second thin film layer arranged on the inner surface of the micro-hemispherical shell, made of a second material including a material capable of detachably fixing a probe labeled with a fluorescent dye and causing fluorescence resonance energy transfer (FRET) between the fluorescent dye and the first thin film layer. Typically, the microstructure of the present invention has a size capable of capturing at least one target cell or a part thereof in its hollow space, and the probe arranged on the second thin film layer is a molecule capable of specifically binding to a target molecule, changing its structure by binding to the target molecule, thereby causing a change in emission / quenching of the fluorescent dye.

[0018] FIG. 1 shows an example of a microstructure 6 of the present invention. In this example, a two-layered hemispherical shell-shaped microstructure 6 is shown, consisting of a magnetic metal thin film as the first thin film layer 1 and a nanocarbon thin film as the second thin film layer 2, but the number of layers is not limited to two, and multiple thin film layers of different elements or element alloys may be sandwiched as intermediate layers. The type of thin film element is, in the case of detecting target molecules by switching the fluorescence ON / OFF, a SP 2 Elements with hybrid orbitals, SP 2 Binding regions and SPs 3The second material may be a thin film structure containing elements or metals (i.e., the second material) mixed with the binding region, but is not limited to this category in other cases. The first material constituting the first thin film layer may typically be a metal such as nickel, iron, cobalt, gadolinium, and ruthenium, or a magnetic material (e.g., ferromagnetic material, superparamagnetic material), including but not limited to metal oxides (e.g., iron oxide, chromium oxide, etc.), ferrite, or alloys such as neodymium. Typical examples of the second material include nanocarbon, nanographene, and gold. When gold is used as the second material, an amino group may be added to the gold forming the second thin film layer to positively charge the surface of the second thin film layer, as shown in the specific example described below, thereby promoting the binding of, for example, a negatively charged DNA aptamer to the surface of the second thin film layer.

[0019] In the present invention, when referring to a "magnetic material," the term "magnetic material" is used in the usual sense used in the art. For the purpose of the present invention, it is desirable that at least the "magnetic material" used in the present invention has magnetism to the extent that the orientation of the microstructure can be controlled by an external magnetic field when the magnetic field is applied.

[0020] The thickness of the thin film can be freely selected within a range in which the structure of the microstructure 6 can be maintained, and the thickness of each layer is typically about 0.1 nm to 1 mm, more preferably about 1 nm to 10 μm, and most preferably about 1 nm to 1 μm, but is not limited to these ranges and can be appropriately determined depending on the purpose. The shape of the microstructure changes depending on the shape of the template used to produce the microstructure, and may be hemispherical, cylindrical, conical, elliptical, or prismatic, but is not limited to these ranges. It will be understood from the description of this specification that the template microparticles themselves for producing the hemispherical shell-shaped microstructure 6 do not necessarily have to be hemispherical, and may be spherical. In this specification, when the terms "almost hemispherical", "almost hemispherical shell", or "almost spherical" are used, unless otherwise specified, they include all of the shapes exemplified herein or their shell shapes, and also include those having shape distortions that are acceptable in actual manufacturing situations. The size (diameter) of the microstructure can also be changed according to the shape of the template at the time of preparation, and is in the range of about 1 nm to about 1 cm, preferably about 1 nm to about 500 μm, more preferably about 5 nm to about 100 μm, and most preferably about 10 nm to about 50 μm. The size (diameter) of the hollow part (concave side cavity part) of the hemispherical shell structure of the microstructure of the present invention can also be freely prepared according to the shape of the template, and is in the range of about 1 nm to about 1 cm, preferably about 1 nm to about 500 μm, more preferably about 5 nm to about 100 μm, and most preferably about 10 nm to about 50 μm. Typically, the size of this cavity part can be made to a size (diameter) that can accommodate at least a single cell or a part thereof.

[0021] In the context of the present invention, a "subject cell" is typically a cell obtained from a mammal, including a human (e.g., a human, a cow, a pig, a goat, a sheep, a monkey, a dog, a cat, a mouse, a rat, etc.), but may also include, without being limited to these, cells of birds, reptiles, amphibians, insects, microorganisms, plants, etc.

[0022] 2. Method for producing hollow, multi-layered microstructures in the shape of a hemispherical shell for use in detecting target molecules

[0023] In another aspect, the present invention provides a method for producing a microstructure of the present invention. This production method includes the steps of: a) providing template particles of a desired size arranged in a monolayer on a substrate; b) coating the template particles disposed on the substrate in the monolayer with a second material; c) further coating the template particles coated with the second material with a first material; and d) removing the template fine particles by a predetermined removal process to obtain a hollow multi-layered microstructure in the shape of a hemispherical shell.

[0024] Typically, the template microparticles are made of a material that can be removed by a predetermined removal process. Optionally, between the steps b) and c), at least one step of coating with another material may be included, and after the step d), the method may include a step of transferring the hemispherical shell-shaped hollow multilayer microstructure from the substrate surface to the adhesive surface using an adhesive, and / or a step of removably fixing a probe to the second thin film layer.

[0025] Note that typical requirements for the first material, second material, and probe are described with respect to the microstructures of the present invention in Section 1 above.

[0026] FIG. 2 shows a typical example of a method for producing a microstructure 6 of the present invention. First, a single layer of microparticles 4 serving as a template is arranged on a flat substrate 3. The material of the flat substrate 3 may be glass, silicon, plastic, etc., but is not limited to this range as long as it is a substrate with a surface flatness smaller than the size of the template microparticles 4, and any substrate can be used depending on the purpose. The template microparticles 4 may also be polystyrene particles, cellulose particles, glass particles, etc., but are not limited to this range as long as they are microparticles of the same size and shape as the microstructure to be produced. The size of the template microparticles 4 is in the range of about 1 nm to 1 cm, typically about 1 nm to 500 μm, more typically about 5 nm to 100 μm, and most typically about 10 nm to 50 μm, depending on the size of the microstructure to be produced. The flat substrate on which the template microparticles 4 are placed is placed in a sample chamber of a thin film forming device 5 capable of forming a thin film material inside the microstructure 6.

[0027] The thin film forming device 5 may be a sputtering device, a resistance heating type vacuum deposition device, a chemical vapor deposition device, etc., but is not limited to this range as long as it is an device capable of forming a thin film having a thickness within a range of approximately 0.1 nm to 1 mm, which is approximately the same as or smaller than the size of the mold microparticles.

[0028] In addition, when preparing nanocarbon thin films, SP 2 Bonds and SPs 3 We use an unbalanced magnetron sputtering device that can form carbon thin films with mixed bond regions. Nanocarbon thin films are SP 2 Bonds and SPs 3 A carbon thin film with mixed bonding regions, SP 2 In the binding region, molecules with ring structures such as pyrene, nanographene, and DNA can be bound through π-π bond interactions. 3 Because of the presence of the bonding region, it is possible to form a continuous film on a curved surface such as a hemispherical shell-shaped microstructure. 2 Bonds and SPs 3 The ratio of the bonded area to the total area can be freely adjusted by changing the sputtering conditions.

[0029] A thin film of the inner surface material is formed on the template microparticle 4 on the flat substrate 3 by forming a thin film of the inner surface material with an arbitrary thickness on the sample placed in the sample chamber according to the operating procedure of the thin film forming device 5. Next, this sample is placed in the sample chamber of the thin film forming device 5, which is capable of forming a magnetic metal thin film on the outer surface of the microstructure, and a thin film is formed in the same manner as when forming the inner thin film. As a result, a two-layered thin film as shown in FIG. 1 is formed on the template microparticle 4. If the number of layers of the thin film is three or more, this procedure is repeated to increase the number of layers. After forming a multi-layered thin film on the template microparticle 4 according to the above procedure, the template microparticle 4 is removed to obtain the microstructure 6 as shown in FIG. 1.

[0030] Methods for removing the template microparticles 4 include high-temperature heating, organic solvent treatment, and active oxygen treatment. As an example, the template polystyrene particles can be removed by heat treatment at 500° C. for 1 hour, but the method is not limited to this range as long as the template microparticles 4 are removed by this method and the thin film layer is not removed. When producing a magnetic microstructure, it is recommended to remove the template microparticles in an atmosphere with an oxygen concentration of 15% or less in order to maintain the magnetic moment.

[0031] 3. Method for detecting target molecules using the microstructure of the present invention

[0032] In yet another aspect, the present invention provides a method for detecting a target molecule using at least one microstructure of the present invention or an array thereof, or at least one microstructure of the present invention or an array thereof manufactured by the manufacturing method of a microstructure of the present invention. This method typically includes: a) placing a hemispherical shell-shaped hollow multi-layer microstructure or an array thereof, comprising probes removably fixed to a second thin film layer, in a solution containing or suspected of containing a target molecule; and b) measuring the fluorescent emission of the fluorescent dye of said probe.

[0033] Typically, detection of said fluorescence emission infers binding between the target molecule and the probe and confirms the presence of the target molecule in solution.

[0034] Typical requirements for the second thin film layer and the probe are described in the description of the microstructure of the present invention in Section 1. In addition, the term "array" is used in the sense commonly used in the art, and the term "array of microstructures" in relation to the present invention means a group of microstructures in which two or more microstructures are arranged one-dimensionally or two-dimensionally (see, for example, Figures 2, 4, 6-1, 6-3, etc.).

[0035] 3 shows an example of a method for detecting a target molecule by selective reaction between a protein, peptide, or nucleic acid molecule as a probe and the target molecule. In this example, a DNA aptamer modified with a fluorescent dye is used as the probe, and a nanocarbon film is used as the second thin film layer, as a non-limiting example. In this specification, the term "aptamer" is used in the sense commonly used in the field, and refers to a peptide or nucleic acid molecule capable of binding to a specific molecule.

[0036] FIG. 3-1 shows a non-limiting example using a DNA aptamer 8 modified at one end with a fluorescent dye molecule 7 and a microstructure 6 in which the inner thin film (second thin film layer) 2 is a nanocarbon thin film. The length of the DNA aptamer 8 can vary depending on the purpose. Typically, it is about 80 to 100 nucleotides, but is not limited to this range, and non-limiting examples of the lower limit include 10, 20, 30, 40, 50, 60, 70, and 80 nucleotides, and non-limiting examples of the upper limit include 90, 100, 110, 120, 130, 140, and 150 nucleotides or more, and the optimal range can be appropriately selected from the combination of these lower and upper limits depending on the purpose.

[0037] In a non-limiting example of the present invention shown in FIG. 3-1, when the aptamer 8 is mixed with the microstructure 6, the DNA backbone of the aptamer 8 has an affinity with the surface of the nanocarbon thin film 2, so that the aptamer 8 is adsorbed to the surface of the nanocarbon thin film 2, and as a result, the fluorescent dye 7 approaches the surface of the nanocarbon thin film 2, causing fluorescence resonance energy transfer (FRET), and the fluorescent dye 7 no longer emits fluorescence. When a target molecule 9 for the aptamer 8 is bound thereto, the aptamer 8 dissociates from the nanocarbon thin film 2 due to the binding of the target molecule 9 to the aptamer DNA backbone, and at the same time, the fluorescent dye molecule 7 also dissociates from the surface of the nanocarbon thin film 2, eliminating FRET, and as a result, fluorescence is generated. That is, the presence of the target molecule 9 can be detected by the generation of fluorescence from inside the electrode microstructure 6 in which the target molecule 9 exists.

[0038] FIG. 3-2 shows a non-limiting example using a DNA aptamer 8 modified with a pyrene molecule 10 having a cyclic structure at one end and a fluorescent dye molecule 7 at the other end, and a microstructure 6 whose inner thin film 2 is a nanocarbon thin film. When the aptamer 8 is mixed with the microstructure 6, the SP of the nanocarbon thin film 2 2 The binding region and the pyrene molecule 10 are bound by π-π interaction, and as a result, the aptamer 8 can be immobilized on the inner surface of the microstructure 6. Moreover, since the DNA main chain of the aptamer 8 has an affinity with the surface of the nanocarbon thin film 2, the aptamer 8 is adsorbed to the surface of the nanocarbon thin film 2 (the molecule lies down), and as a result, the fluorescent dye 7 approaches the surface of the nanocarbon thin film 2, causing fluorescent FRET, and the fluorescent dye 7 no longer emits fluorescence. When a target molecule 9 for the aptamer 8 binds thereto, the structure of the aptamer 8 changes due to the binding of the target molecule 9 to the aptamer DNA main chain (the molecule rises), and the fluorescent dye 7 moves away from the surface of the nanocarbon thin film 2, eliminating FRET, and as a result, fluorescence is generated. In other words, the presence of the target molecule 9 can be detected by the fluorescence generated from the inner surface of the electrode microstructure 6.

[0039] The biomolecules to be detected are assumed to be proteins, peptides, nucleic acids, cell surface molecules, cell secretory vesicles, etc., but are not limited thereto, and any molecule that can bind to an aptamer can be detected. In addition, although a DNA aptamer is used as an example in FIG. 3, different types of aptamers such as RNA aptamers, proteins, and peptides can be detected using exactly the same principle. Furthermore, it is easily understood by those skilled in the art that, not limited to aptamers, any molecule that can selectively bind to a target molecule and changes the emission and quenching of fluorescence due to a change in molecular structure can be detected using the same principle. In FIG. 3-2, an aptamer is immobilized on the surface of a nanocarbon thin film via pyrene molecules, but the number of carbon hexagonal structures is not limited to four as in pyrene molecules, and may be two, three, four or more as long as the aptamer can be immobilized by π-π bonds. The minimum number of six-membered carbon ring structures that can be immobilized is determined by the size of the aptamer molecule. For example, if the DNA aptamer has 80 bases, four (pyrene molecules) are sufficient for immobilization.

[0040] Regarding the difference between Figure 3-1 and Figure 3-2, in Figure 3-1, the aptamer 8 dissociates from the surface of the microstructure 6 due to the binding of the target molecule 9, so the number of aptamer 8 molecules adsorbed on the surface of the microstructure 6 decreases as the target molecule 9 binds. On the other hand, since the fluorescent dye 7 is sufficiently far from the surface of the microstructure 6, the change in fluorescence emission and quenching is more clear than in the case of Figure 3-2. Also, since the introduction of the pyrene molecule 10 into the aptamer 8 is not required, the molecule can be easily produced and is relatively inexpensive. On the other hand, in Figure 3-2, the distance that the fluorescent dye molecule 7 can move away from the surface of the microstructure 6 is limited compared to Figure 3-1. To address this issue, a spacer may be introduced between the fluorescent dye 7 and the DNA aptamer 8, or between the DNA aptamer 8 and the pyrene molecule 10. By doing so, the dissociation distance between the fluorescent dye molecule 7 and the surface of the microstructure 6 can be increased when the target molecule 9 binds and the structure of the aptamer 8 changes. As the spacer molecule, it is preferable to use a polymer such as polyethylene glycol, a polypeptide, a chain molecule such as DNA, RNA, etc. The spacer length may be in the range of about 0.1 nm to about 30 μm, typically about 0.1 nm to about 1 μm, but is not limited to these ranges as long as it is feasible with the polymer, polypeptide, nucleic acid molecule, etc.

[0041] FIG. 4 shows a non-limiting example of biomolecule detection using the fluorescent DNA aptamer described in FIG. 3, in which vaspin, a biomolecule, was detected. As shown in FIG. 3-1 and FIG. 3-2, a fluorescent DNA aptamer as a complex of a fluorescent dye 7 and an aptamer 8, and in the case of FIG. 3-2, a pyrene molecule 10, was further linked to the inner surface of a microstructure 6 having a diameter of 15 μm, an inner surface made of a nanocarbon thin film, and an outer surface made of nickel. The fluorescence brightness before and after the vaspin reaction was compared when a vaspin solution was added to the immobilized fluorescent DNA aptamer. The addition of vaspin, the target molecule 9, increased the fluorescence brightness in both FIG. 3-1 and FIG. 3-2 compared to before the reaction. Furthermore, when bovine serum albumin (BSA) was added as a control, no change in the fluorescence brightness was observed before and after the reaction. This result demonstrated that the presence or absence of a target molecule can be selectively detected as a change in the fluorescence brightness value using the fluorescent DNA aptamer.

[0042] FIG. 5 illustrates a non-limiting example of a method for detecting target biomolecules on the inner surface of a thin film other than the nanocarbon film illustrated in FIG.

[0043] FIG. 5-1 shows an example of biomolecule detection on a gold (Au) surface 14. When a DNA aptamer 13, which has a thiol group (S) attached to one end that bonds with the Au surface 14 and a fluorescent dye 12 attached to the other end, is mixed with a microstructure 6 whose inner surface is Au, the DNA aptamer 13 is immobilized on the Au surface 14 on the inner surface of the microstructure 6 via the thiol group (S). Since DNA bases have an affinity for the Au surface, the DNA aptamer 13 is adsorbed to the Au surface 14, causing FRET, and the fluorescence is quenched. When a target molecule 9 is bound thereto, the structure of the DNA aptamer 13 changes as in FIG. 3-2, causing fluorescence, and the presence of the target molecule 9 can be detected. Furthermore, the Au surface 14 can be positively charged by surface treatment such as attaching an amino group to the Au surface 14, so that the negatively charged DNA aptamer 13 is adsorbed to the Au surface 14 by electrostatic interaction, causing FRET. When attaching an amino group to the Au surface 14, an alkyl chain having an amino group at one end and a thiol group at the other end may be prepared and mixed in the same manner as when immobilizing the DNA aptamer 13 to the microstructure 6 whose inner surface is Au. The DNA aptamer 13 and the alkyl chain having the amino group and the thiol group are mixed in advance at any ratio, and then mixed with the microstructure 6 whose inner surface is Au and immobilized, so that the DNA aptamer 13 and the alkyl chain having the amino group added thereto can be immobilized on the Au surface 14 at any ratio. The alkyl chain length may be in the range of about 0.1 nm to about 20 nm, typically about 0.1 nm to about 5 nm, taking into consideration the efficiency of FRET generation.

[0044] FIG. 5-2 shows a non-limiting example of a method for detecting a target molecule 9 by immobilizing a nanographene 20, which is a nano-sized graphene film, on an amino group on the inner surface of a microstructure 6. In this case, the fluorescent aptamer (12, 13) is not firmly immobilized on the nanographene 20 by a covalent bond or the like, but the aptamer 13 is adsorbed on the surface of the nanographene 20. When the target molecule 9 binds, the structure of the aptamer 13 changes as in FIG. 3-1, and it dissociates from the surface of the nanographene 20, generating fluorescence. In this example, the aptamer 13 dissociates, but as shown in FIG. 4, the target molecule 9 can be detected with high sensitivity. The method for introducing an amino group on the inner surface of the microstructure 6 can be realized by using a material having a hydroxyl group, such as silicon dioxide, as the material on the inner surface of the microstructure, and reacting a silane coupling agent, such as 3-aminopropyltriethoxysilane, therewith, or by immobilizing the above-mentioned alkyl chain having an amino group on the Au surface. In the case of Figure 5-2, when an amino group is introduced using an alkyl chain having an amino group, there is no need to consider the efficiency of FRET generation, so the alkyl chain length may be in the range of about 0.1 nm to about 30 µm, typically in the range of about 0.1 nm to about 1 µm, and further, the material of the chain portion connecting the amino group and the thiol group may be a polymer, a polypeptide, a nucleic acid molecule, etc. in addition to the alkyl chain.

[0045] As a non-limiting example of detection of a specific biomolecule, Fig. 6 shows a method for detecting a secretion from a cell using a microstructure with a fluorescent aptamer immobilized thereon. In this example, fluorescence is generated only from a hemispherical shell-shaped microstructure 6 that captures a target cell 16 in a mixture of a general cell 15 that does not secrete a target molecule and a target cell 16 that secretes the target molecule. Three embodiments are shown in Fig. 6.

[0046] Fig. 6-1 shows a procedure for arranging microstructures 6 in an array on a substrate 3 and capturing and detecting cells on each microstructure 6. Arranging the microstructures 6 in an array can be achieved by attaching an adhesive material 17 onto the microstructures 6 on the substrate 3 prepared by the procedure in Fig. 2 and then peeling it off, thereby transferring the microstructures 6 onto the adhesive material 17.

[0047] Figure 6-2 shows an embodiment in which a microstructure 6 is attached to the tip of a microcantilever and the microstructure 6 is placed over a cell adhered to a substrate 3 for detection. As the microcantilever, a cantilever 18 of an atomic force microscope or the like may be used.

[0048] FIG. 6-3 shows an embodiment in which magnetic microstructures 6 are dispersed in a solution, and then a magnetic field is applied to attach the microstructures 6 to cells adhering to a substrate to detect secretions 19. In this case, microstructures 6 of approximately the same size as the protuberances (diameter approximately 5 μm) of cells adhering to the substrate 3 are fabricated, and after dispersing the microstructures 6 in the solution, a magnet is applied to the back side of the substrate 3 to which the cells are adhered, thereby accumulating the microstructures 6 toward the cells, thereby attaching the microstructures 6 to the cells. Since the microstructures 6 do not cover the entire cell surface, secretions 19 can be exchanged from other cells on the uncovered cell surface, and this is characterized by the fact that the response of cells to stimuli from the surrounding environment can be measured.

[0049] Needless to say, probe molecules such as fluorescent aptamers for detecting target molecules 19 are immobilized in advance on the inner surface of the minute structure 6 used in Figs. 6-1 to 6-3.

[0050] When dispersing the microstructure 6 produced in FIG. 2 in a solution, it is effective to drop a solution onto the substrate 3 and apply ultrasonic waves from the back side of the substrate 3 to peel off the microstructure 6 from the substrate 3, thereby dispersing the microstructure 6 in the solution. However, there is a problem that some of the microstructures 6 are destroyed by ultrasonic waves. To solve this problem, there is a method in which a solubilizable adhesive 17 is attached onto the microstructure 6 on the substrate 3 produced in FIG. 2 and then peeled off, and the microstructure 6 is transferred onto the surface of the adhesive 17 as shown in FIG. 6-1, and then the adhesive 17 is put into a tube and solubilized to dissolve the adhesive 17, thereby obtaining a microstructure 6 dispersed in the solution. For example, when polydimethylsiloxane (PDMS) is attached to the substrate 3 in FIG. 2 and peeled off, the microstructure 6 is transferred onto the PDMS surface, and when this PDMS is put into a tube and isopropanol is added to the tube, the PDMS surface is solubilized, and the microstructure 6 is dispersed in the isopropanol. By recovering the adhesive and replacing it with any solution, it is possible to obtain the microstructure 6 dispersed in the solution without being damaged. The "solubility" of the adhesive has been described above as being soluble in organic solvents, but is not limited thereto, and for example, the adhesive may be water-soluble.

[0051] The present invention has been described above using specific examples, but the technical scope of the present invention is not limited to these specific examples. Various variations are possible within the technical scope of the invention described in the appended claims and its equivalents, and these variations are also included in the technical scope of the present invention. [Industrial Applicability]

[0052] The microstructures of the present invention, the biomolecules fixed thereto (including those modified with fluorescent dyes, etc.), and combinations thereof, as well as their manufacturing methods, control methods, and usage methods, are applicable to a variety of fields, but are particularly useful in applications such as environmental testing chips for detecting substances and microorganisms in the environment, cell diagnosis for detecting specific cells from a large number of cells, and blood liquid biopsy for detecting specific cells from blood. [Explanation of symbols]

[0053] 1 First thin film layer, 2 Second thin film layer, 3 Flat substrate, 4 Template microparticle, 5 Thin film formation device, 6 Microstructure, 7 Fluorescent dye, 8 DNA aptamer, 9 Target molecule, 10 Pyrene molecule, 11 Thiol group, 12 Fluorescent dye, 13 DNA aptamer, 14 Au thin film, 15 Ordinary cell that does not secrete target molecule, 16 Target cell that secretes target molecule, 17 Adhesive material, 18 Atomic force microscope cantilever, 19 Secretion, 20 Nanographene.

Claims

1. A hollow, multi-layered microstructure in the form of a hemispherical shell for use in detecting a target molecule, comprising: a first thin film layer in the form of a substantially microscopic hemispherical shell composed of a first material including a magnetic material; a second thin film layer that is made of a second material including a material capable of detachably fixing a probe labeled with a fluorescent dye and capable of causing fluorescence resonance energy transfer between the fluorescent dye and the second thin film layer, the second thin film layer being disposed on the inner surface of the micro-hemispherical shell; Equipped with a size capable of trapping at least one target cell or a portion thereof in a hollow space defined by said second thin film layer; the probe is a molecule capable of specifically binding to the target molecule, and is capable of changing its structure upon binding to the target molecule, thereby causing a change in emission / quenching of the fluorescent dye; A hemispherical shell-shaped hollow multi-layer microstructure further comprising the probe removably fixed to the surface of the second thin film layer.

2. 2. The hemispherical shell shaped hollow multi-layer microstructure of claim 1, wherein the first material comprises a magnetic material selected from the group consisting of nickel, iron, cobalt, gadolinium, ruthenium, iron oxide, chromium oxide, ferrite, and neodymium.

3. 3. The hollow multi-layer microstructure of claim 1, wherein the second material comprises an element having an SP2 hybrid orbital, an element having a mixture of SP2 and SP3 bonding regions, or a metal.

4. The hemispherical shell-shaped hollow multi-layer microstructure of claim 3 , wherein the second material comprises nanocarbon, nanographene, or gold.

5. 5. The hemispherical shell-shaped hollow multi-layer microstructure according to claim 4, wherein the second thin film layer has an amino group on the surface thereof.

6. The hemispherical shell-shaped hollow multilayer microstructure according to any one of claims 1 to 5, wherein the probe is a protein, a peptide, or a nucleic acid molecule.

7. 7. The hemispherical shell-shaped hollow multilayer microstructure according to claim 6, wherein the probe is a nucleic acid molecule modified at one end with a fluorescent dye and at the other end with a pyrene molecule or a thiol group.

8. The hemispherical shell-shaped hollow multi-layered microstructure according to any one of claims 1 to 7, wherein the target molecule comprises a protein, a peptide, a nucleic acid, a cell surface molecule, or a cell secretory vesicle.

9. 9. The hemispherical shell-shaped hollow multi-layer microstructure according to claim 1, wherein the thickness of each of said thin film layers is in the range of 0.1 nm to 1 mm.

10. 10. An array of hemispherical shell-shaped hollow multi-layer microstructures comprising a plurality of the hemispherical shell-shaped hollow multi-layer microstructures according to any one of claims 1 to 9.

11. The hemispherical shell-shaped hollow multilayer microstructure or an array thereof according to any one of claims 1 to 10, wherein the probe is detachably fixed to the surface of the second thin film layer via a spacer molecule, and / or the fluorescent dye is bound to the probe via a spacer molecule.

12. A method for producing a hemispherical shell-shaped hollow multi-layered microstructure or an array thereof for use in detecting a target molecule according to any one of claims 1 to 11, comprising the steps of: a) providing template particles of a desired size arranged in a monolayer on a substrate, the template particles being made of a material that is removable by a predetermined removal process; b) coating the template particles disposed on the substrate in the monolayer with the second material; c) further coating the template particles coated with the second material with the first material; and d) removing the template particles by the predetermined removal process to obtain the hemispherical shell-shaped hollow multi-layered microstructure. A method for producing a hemispherical shell-shaped hollow multi-layered microstructure or an array thereof, comprising:

13. The method for producing a hemispherical shell-shaped hollow multi-layered microstructure or an array thereof according to claim 12, further comprising at least one step of coating with another material between said steps b) and c).

14. After step d), Transferring the hemispherical shell-shaped hollow multi-layer microstructure from the substrate surface to the adhesive surface using an adhesive; and / or Removably fixing the probe to the second thin film layer. A method for producing a hemispherical shell-shaped hollow multi-layered microstructure or an array thereof according to claim 12 or 13, comprising:

15. The method of claim 13 , wherein the further material comprises a material that includes a different element or elemental alloy than the first or second material.

16. The method according to claim 14, wherein the adhesive is a soluble adhesive.

17. The method of claim 16, wherein the soluble adhesive is polydimethylsiloxane, and further comprising the step of solubilizing the adhesive in a solvent.

18. A method for detecting a target molecule using at least one hemispherical shell-shaped hollow multilayer microstructure or an array thereof according to any one of claims 1 to 11, or at least one hemispherical shell-shaped hollow multilayer microstructure or an array thereof manufactured by the manufacturing method according to claim 14, 16, or 17, comprising: a) placing the hemispherical shell-shaped hollow multi-layer microstructure or an array thereof, including the probes removably fixed to the second thin film layer, in a solution containing or suspected of containing the target molecule; and b) measuring the fluorescence emission of the fluorescent dye of the probe, detection of the fluorescence emission infers binding of the target molecule to the probe and confirms the presence of the target molecule in the solution; A method comprising:

19. In step a), Controlling the orientation of the hemispherical shell-shaped hollow multi-layered microstructure dispersed in the solution by applying an external magnetic field.

20. The method of claim 18, comprising:

20. The method according to claim 18 or 19, wherein the target molecule is a secretion product of a cell, and the method further comprises, between steps a) and b), a step of capturing the cell or a part thereof in the hollow space of the hemispherical shell-shaped hollow multilayer microstructure.

Images