WO2018221271A1 - 検出対象の分析用センサ作製用基材、検出対象の分析用センサ、及び検出対象の分析法 - Google Patents
検出対象の分析用センサ作製用基材、検出対象の分析用センサ、及び検出対象の分析法 Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Definitions
- the present invention relates to a technique for quickly detecting a detection target on a substrate. More specifically, the present invention relates to a substrate for preparing an analysis sensor to be detected and a manufacturing method thereof, an analysis sensor to be detected and a manufacturing method thereof, and an analysis method of the detection target.
- the exosome is one of the vesicles released from the cell, and is a lipid bilayer vesicle having a diameter of 20 to 150 nm.
- the exosome encapsulates proteins and nucleic acids such as miRNA and mRNA inside, and also has proteins on its surface. Since exosomes are characterized by such substances, it is thought that by analyzing the characteristics of exosomes, it is possible to guess what kind of cells are secreted. Exosome has been confirmed to exist in various body fluids and can be collected relatively easily.
- Exosomes secreted from cancer cells contain tumor-derived substances. Therefore, it is expected that cancer can be diagnosed by analyzing substances contained in exosomes in body fluids. Furthermore, since exosomes are actively secreted by cells, it is expected to exhibit some characteristics even at an early stage of cancer.
- Patent Document 1 discloses a method for detecting an exosome by capturing an exosome with a substrate surface-modified with an anchor substance called BAM (Biocompatible anchor for membrane) and using an antibody against an antigen molecule on the exosome.
- BAM Biocompatible anchor for membrane
- Patent Document 2 in a fluid device including an exosome purification unit, a biomolecule purification unit, and a biomolecule detection unit that are connected to each other through a channel, the exosome purification unit modified with BAM performs exosome capture and exosome disruption.
- a method for purifying and detecting biomolecules inside exosomes by a biomolecule purification unit and a biomolecule detection unit, respectively, is disclosed.
- Patent Document 3 an exosome is captured by a 96-well plate on which an antibody for capturing an exosome surface antigen is fixed, then a detection antibody for another surface antigen is allowed to act, and further an enzyme-labeled secondary antibody for the detection antibody A method for detecting exosomes by using the method is disclosed.
- Patent Document 4 in a device having a recess in which an antibody against a surface antigen of exosome is immobilized, after the exosome is captured in the recess, a bead to which an antibody against another surface antigen of exosome is bound is allowed to act, and then the beads are counted.
- a method for detecting exosomes is disclosed.
- Patent Document 5 two types of antibodies are used: an antibody against an exosome surface antigen and capable of binding to an excitation label, and an antibody against another surface antigen of the exosome and bound with a signal generating label.
- Patent Document 6 a transparent flat substrate having a metal film having a plurality of nano-openings with a predetermined pattern for generating a detection region that generates surface plasmon resonance during illumination, and an exosome marker provided on the metal film
- a method of detecting exosomes using surface plasmon resonance using a nanoplasmon sensor containing a specific capture agent is disclosed.
- Non-Patent Document 1 avidin bound with an antibody against CD63, which is an exosome surface antigen, is bound to a biotinylated self-assembled monolayer provided on a gold substrate, and exosomes bound via CD63 are surface-plasmon-bonded. A method of measuring using resonance is disclosed.
- Exosomes have been reported to play an important role as one of intercellular communication tools that are deeply involved in immune regulation, development of neurodegenerative diseases, organ-specific metastasis of cancer, and the like. Therefore, the rapid detection of exosomes secreted from cells and the specific capture of what is detected is a simple method for detecting intercellular communication tools. is important.
- the exosome detection method disclosed in Patent Document 1 inevitably has a time lag because it requires an additional step of binding the antibody after capturing the exosome, and rapid detection cannot be performed.
- the exosome detection method disclosed in Patent Document 2 requires a complicated system in which an internal molecule generated by exosome disruption is purified and then detected using a substance specific to the internal molecule. Lack. Since the exosome detection method disclosed in Patent Document 3 is to detect exosomes by sandwich ELISA, an additional step of binding antibodies after capturing exosomes is unavoidably caused, resulting in rapid lag. It cannot be detected.
- the exosome detection method disclosed in Patent Document 4 also inevitably has a time lag because an additional step of binding antibody beads after capturing the exosome is unavoidably impossible to perform rapid detection. Since the exosome detection method disclosed in Patent Document 5 uses a fluorescence resonance energy transfer method, it requires a complicated combination of antibodies, and thus lacks simplicity. Since the exosome detection method disclosed in Patent Document 6 and Non-Patent Document 1 uses surface plasmon resonance to detect the state of the metal surface, an object non-specifically adsorbed on the surface is inevitably detected, It lacks the specificity to detect only specifically recognized objects.
- an object of the present invention is to provide a simple measurement system that can quickly and highly accurately grasp an object to be detected.
- the inventor has formed a polymer film having a recess for receiving a detection target on the surface, and can arrange an antibody that ensures binding specificity for the detection target in the recess, and in the recess. It was found that the object of the present invention can be achieved by configuring the base material so that only the signal substance can be arranged. Based on this finding, the present invention has been completed through further studies.
- the present invention includes a base material for producing an analysis sensor to be detected and a manufacturing method thereof, an analysis sensor to be detected and a manufacturing method thereof, and an analysis method of the detection object. That is, this invention provides the invention of the aspect hung up below.
- Item 1 A base material, and a polymer film provided on the surface of the base material, The polymer film has a recess for receiving a detection target; A substrate for preparing an analytical sensor to be detected, having an antibody substance binding group and a signal substance binding group in the recess.
- Item 2. Item 2. The detection according to Item 1, wherein the polymer film is composed of a molecularly imprinted polymer using the detection target or a target having a size larger than the detection target as a template, and the concave portion corresponds to a part of the surface shape of the template.
- Target substrate for analytical sensor production Item 3. Item 3.
- Item 4. The substrate for preparing an analytical sensor to be detected according to any one of Items 1 to 3, wherein the signal substance binding group is a thiol group.
- Item 5. A substrate for preparing an analytical sensor to be detected according to any one of Items 1 to 4, An antibody substance specific for the detection target bound to the antibody substance-binding group; A signal substance bound to the signal substance binding group; A sensor for analysis to be detected.
- Item 6. Item 6. The detection target analysis sensor according to Item 5, wherein the detection target is a microparticle having a film structure.
- Item 7. Item 7.
- Item 8. Item 8. The detection target according to any one of Items 5 to 7, wherein the antibody substance specific for the detection target has a specific binding ability to a specific antigen expressed on the surface of the microparticle having the membrane structure. Sensor for analysis.
- Item 9. A step of bringing a sample containing the detection target into contact with the analysis sensor of the detection target according to any one of Items 5 to 8, and binding the detection target to the antibody substance; Detecting a change in signal derived from the signal substance; Analytical methods to be detected, including Item 10.
- a polymer is added to the surface of the substrate by adding a polymerizable monomer, synthesizing a molecular imprint polymer for a part of the surface of the template using the polymerizable monomer as a substrate and the polymerization initiating group as a polymerizable initiator.
- a polymerization step to form a film A removing step of cleaving the first reversible linking group and the second reversible linking group to convert them into an antibody substance binding group and a signal substance binding group, respectively, and removing the template;
- the artificial particle binds to the surface of the antibody substance-binding group on the surface so that the first reversible linking group can be formed and to the signal substance-binding group.
- the antibody substance-binding group is a chelate-binding group, and the group capable of forming the first reversible linking group by binding to the antibody substance-binding group is a histidine tag.
- the manufacturing method of the base material for analytical sensor preparation of the detection object in any one.
- Item 14 Item 10 to 13 wherein the signal substance binding group is a thiol group, and the reversible binding group capable of forming the second reversible linking group by binding to the signal substance binding group is a thiol group.
- the manufacturing method of the base material for analytical sensor preparation of the detection object in any one of these.
- FIG. 4 is a schematic diagram for explaining a template introduction step subsequent to FIG. 3 (when exosome is used as a template).
- FIG. 5 is a schematic diagram for explaining a surface modification step subsequent to FIG. 4 (when exosome is used as a template).
- FIG. 6 is a schematic diagram for explaining a polymerization step subsequent to FIG. 5 (when exosome is used as a template).
- FIG. 6 It is a schematic diagram explaining the removal process following FIG. 6 (when using exosome as a template). It is a schematic diagram explaining the casting_mold
- FIG. 10 is a schematic diagram for explaining a polymerization step subsequent to FIG. 9 (when artificial particles are used as a template). It is a schematic diagram explaining the removal process following FIG. 10 (when using an artificial particle for a casting_mold
- the graph showing the change in fluorescence intensity with respect to the exosome concentration logarithmically obtained in the exosome detection by fluorescence analysis using the analytical sensor of the present invention (produced by using exosome as a template) and the curve fitting result in Example 3. is there. It is the graph which shows the SPR signal change with respect to the exosome density
- Example 12 shows the results of detecting exosomes in tears using the analytical sensor of the present invention (produced using silica nanoparticles as a template) in Example 12.
- the base material for producing an analytical sensor to be detected of the present invention is a base material that becomes a material for producing the analytical sensor of the invention described later.
- This base material for sensor production is configured so that the user can easily customize a sensor capable of quickly detecting a detection target such as an exosome.
- the base material for preparing an analytical sensor of the present invention includes a base material and a polymer film provided on the surface of the base material; the polymer film has a recess for receiving the detection target; An antibody substance binding group and a signal substance binding group are provided in the recess.
- FIG. 1 an example of the base material for analytical sensor preparation of the detection target of this invention is shown typically.
- the analytical sensor production substrate 10 includes a substrate 20 and a polymer film 30.
- the polymer film 30 is provided on the surface of the substrate 20 and has a recess 31.
- the recess 31 is a hole formed in a size capable of receiving a detection target (a detection target 60 described later).
- the base material 10 for analytical sensor production has an antibody substance binding group 22 and a signal substance binding group 32 in the recess 31.
- the signal substance binding group 32 is provided in the vicinity of the antibody substance binding group 22.
- the material of the base material 20 may be a material selected from the group consisting of metal, glass, and resin, for example.
- the metal include gold, silver, copper, aluminum, tungsten, molybdenum and the like.
- Resins include poly (meth) acrylate, polystyrene, ABS (acrylonitrile-butadiene-styrene copolymer), polycarbonate, polyester, polyethylene, polypropylene, nylon, polyurethane, silicone resin, fluorine resin, methylpentene resin, phenol resin, melamine Resins, epoxy resins, vinyl chloride resins and the like can be mentioned.
- the base material 20 may be formed by combining a plurality of materials selected from the above materials.
- the base material 20 may be a glass or resin surface provided with a metal film.
- a shape of the base material 20 plate shape and particle shape are not ask
- Preferred examples include gold substrates, glass substrates, gold nanoparticles, glass beads and the like.
- the polymer film 30 is provided in a layered manner on the substrate 20 and has a plurality of recesses 31.
- the concave portion 31 is a portion that becomes a sensor field in the analysis sensor to be detected of the present invention.
- the concave portion 31 is not limited as long as it is formed so as to be capable of receiving a detection target.
- a molecular imprint polymer MIP; molecularly formed by using a molecular imprint polymerization method as described later). imprinted polymer).
- the recess 31 is formed by a mold (mold 40 described later) used in the molecular imprint polymerization method, and has a shape corresponding to a part of the surface shape of the mold.
- the template of the concave portion 31 may be the same as the detection target, or a target that is larger in size than the detection target. Also good.
- the size of the recess 31 that can accept a detection target means that an antibody substance (an antibody substance 52 described later) and a signal substance (a signal substance 53 described later) are combined to form an analysis sensor (an analysis sensor described later). 50), the recess 31 has a sufficiently large opening on the surface of the base material 20 so that at least a part of the detection target enters the recess 31 and approaches the antibody substance to be able to bind. That means.
- the opening diameter of the recess 31 is not particularly limited because it may vary depending on the detection target, but may be, for example, 1 nm to 10 ⁇ m.
- the thickness of the polymer film 30 is not particularly limited because it may vary depending on the detection target, but may be, for example, 1 nm to 1 ⁇ m.
- the polymer constituting the polymer film 30 may be, for example, a biocompatible polymer including a component derived from a biocompatible monomer.
- Biocompatibility refers to a property that does not induce adhesion of biological substances.
- the biocompatible monomer is preferably a hydrophilic monomer, more preferably a zwitterionic monomer.
- the zwitterionic monomer includes an anionic group derived from an acidic functional group (for example, a phosphate group, a sulfuric acid group, and a carboxyl group) and a basic functional group (for example, a primary amino group, a secondary amino group, a tertiary group). Both amino groups and cationic groups derived from quaternary ammonium groups, etc.) are contained in one molecule.
- an acidic functional group for example, a phosphate group, a sulfuric acid group, and a carboxyl group
- a basic functional group for example, a primary amino group, a secondary amino group, a tertiary group.
- Both amino groups and cationic groups derived from quaternary ammonium groups, etc. are contained in one molecule.
- phosphobetaine, sulfobetaine, carboxybetaine and the like can be mentioned.
- examples of the phosphobetaine include molecules having a phosphorylcholine group in the side chain, and preferably 2-methacryloyloxyethyl phosphorylcholine (MPC) and the like.
- examples of sulfobetaines include N, N-dimethyl-N- (3-sulfopropyl) -3′-methacryloylaminopropaneaminium inner salt (SPB), N, N-dimethyl-N- (4-sulfobutyl) -3 ′.
- SPB N-dimethyl-N- (4-sulfobutyl) -3 ′.
- SBB -Methacryloylaminopropaneaminium inner salt
- Carboxybetaines include N, N-dimethyl-N- (1-carboxymethyl) -2′-methacryloyloxyethanaminium inner salt (CMB), N, N-dimethyl-N- (2-carboxyethyl)- And 2'-methacryloyloxyethaneaminium inner salt (CEB).
- the ratio of the biocompatible monomer-derived component in the polymer film 30 may be, for example, 5 mol% or more and 100 mol% or less. It is preferable that the content of the biocompatible monomer-derived component is not less than the above lower limit in terms of suppressing nonspecific adsorption on the surface of the polymer film 30.
- the proportion of the biocompatible monomer-derived component in the polymer film 30 may be preferably 10 mol% or more and 80 mol% or less, more preferably 20 mol% or more and 60 mol% or less.
- the antibody substance-binding group 22 is a group that allows an antibody substance to be introduced into the analytical sensor preparation substrate 10 by directly or indirectly binding an antibody substance (an antibody substance 52 described later).
- the user can freely target the detection target, and can freely select an antibody substance specific to the target detection target and introduce it into the antibody substance binding group 22.
- one base material 20 one kind of antibody substance is introduced into one recess 31, and another kind of antibody substance is introduced into another recess 31, whereby one base material 20. Can be customized so as to enable analysis of a detection target using a plurality of types of antibody substances.
- an antibody substance that binds to an exosome is introduced into one recess 31 and an antibody substance that binds to a protein is introduced into the other recess 31 to thereby form one substrate 20.
- Both exosomes and proteins can be analyzed.
- the antibody substance binding group 22 is not provided in a portion other than the concave portion 31.
- the antibody substance-binding group 22 may be an irreversible binding group, a reversible binding group, a covalent binding group, or a non-covalent binding group. .
- the antibody substance binding group 22 is a reversible binding group, more preferably a non-covalent group.
- Such groups include chelate binding groups.
- the chelate-binding group can bind the antibody substance indirectly via a substance that binds to the Fc region of the antibody substance, such as protein G.
- the chelate-binding group is not particularly limited as long as it is a group that binds (coordinates) to a metal ion by having a ligand having a plurality of coordination sites (polydentate ligand).
- Examples include polycarboxylic acid-based chelating agent-derived groups, hydroxycarboxylic acid-based chelating agent-derived groups, deferoxamine-derived groups, deferacyclos-derived groups, deferiprone-derived groups, histidine tags, etc., preferably aminopolycarboxylic acid-based chelates
- agent-derived groups include agent-derived groups.
- aminopolycarboxylic acid examples include ethylenediaminetetraacetic acid (EDTA), ethylenediaminediacetic acid, hydroxyethylethylenediaminetriacetic acid (HEDTA), dihydroxyethylethylenediaminetetraacetic acid (DHEDDA), nitrilotriacetic acid (NTA), and hydroxyethyliminodiacetic acid.
- EDTA ethylenediaminetetraacetic acid
- HEDTA ethylenediaminediacetic acid
- HEDTA hydroxyethylethylenediaminetriacetic acid
- DHEDDA dihydroxyethylethylenediaminetetraacetic acid
- NTA nitrilotriacetic acid
- hydroxyethyliminodiacetic acid examples include hydroxyethyliminodiacetic acid.
- NTA N- (2-hydroxyethyl) iminodiacetic acid, ⁇ -alanine diacetic acid, cyclohexanediaminetetraacetic acid, iminodiacetic acid, N- (2-hydroxyethyl) iminodiacetic acid, diethylenetriaminepentaacetic acid (DTPA), N- ( 2-hydroxyethyl) ethylenediaminetriacetic acid, glycol etherdiaminetetraacetic acid, glutamic acid diacetic acid, aspartic acid diacetic acid, methylglycine diacetic acid, iminodisuccinic acid, serine diacetic acid, And droxyimino disuccinic acid, dihydroxyethyl glycine, aspartic acid, glutamic acid, and triethylenetetramine-N, N, N ′, N ′′, N ′ ′′, N ′ ′′-hexaacetic acid, etc.
- Nitrilotriacetic acid (NTA) is prefer
- the reversible binding group is a group that can form a reversible linking group by binding to other reversible binding groups (whether covalent bonding or non-covalent bonding). It means that the conversion (bonding) from the reversible linking group to the reversible linking group and the conversion (cleavage) from the reversible linking group to the reversible linking group are possible in both directions (the same applies hereinafter).
- the signal substance binding group 32 is a group that allows a signal substance to be introduced into the analysis sensor production substrate 10 by binding a signal substance (a signal substance 53 described later). The user can freely select the signal substance and introduce it into the signal substance binding group 32.
- a signal substance a signal substance 53 described later.
- the user can freely select the signal substance and introduce it into the signal substance binding group 32.
- different types of antibody substances are provided in one concave portion 31 and other concave portions 31 in one base material 20, it can be customized to provide different signal substances for each type of antibody substance.
- a plurality of signal substance binding groups 32 are usually provided for each recess 31.
- the signal substance binding group 32 has a sufficient amount so that a change in signal intensity can be detected during sensing in the recess 31 of the analysis sensor of the present invention (when a detection target is received in the recess 31). It only has to be prepared. Accordingly, the amount of the signal substance binding group 32 provided in one recess 31 is not particularly limited, but depends on the size of the recess 31 and the size of the target substance to be detected. For example, it may be about 100 to about 1000 per 31. In addition, on the surface of the base material 20, the signal substance binding group 32 is not provided in a portion other than the concave portion 31.
- the signal substance binding group 32 may be an irreversible binding group, a reversible binding group, a covalent binding group, or a non-covalent binding group. .
- the signal substance binding group 32 is a reversible binding group, more preferably a covalent bond group.
- Examples of such a group include a thiol group (the corresponding reversible linking group is a disulfide group), Aminooxy group or carbonyl group (corresponding reversible linking group is oxime group), boronic acid group and diol group (corresponding reversible linking group is cyclic diester group), amino group and carbonyl group (corresponding reversible linking group is Schiff base), aldehyde group or ketone group and alcohol (the corresponding reversible linking group is an acetal group).
- a thiol group the corresponding reversible linking group is a disulfide group
- Aminooxy group or carbonyl group corresponding reversible linking group is oxime group
- boronic acid group and diol group corresponding reversible linking group is cyclic diester group
- amino group and carbonyl group corresponding reversible linking group is Schiff base
- aldehyde group or ketone group and alcohol the corresponding reversible linking group
- the substrate for preparing an analytical sensor to be detected according to the present invention only needs to be configured so that at least one of the antibody substance and the signal substance can be customized by the user. Therefore, as another embodiment, an antibody substance specific for the detection target may already be bound to the antibody substance binding group. In this case, the user can freely select and introduce a signal substance.
- the signal substance may already be bound to the signal substance binding group.
- the user can freely target the detection target, freely select an antibody substance specific to the target detection target, and introduce the antibody substance.
- the analytical sensor of the present invention comprises the above-described base material for preparing an analytical sensor for detection target; an antibody substance specific to the detection target bound to the antibody substance binding group; and the signal substance binding group. And a bound signal substance.
- FIG. 2 schematically shows an example of an analysis sensor to be detected according to the present invention. As shown in FIG. 2, in the analysis sensor 50, the antibody substance 52 is bonded to the antibody substance binding group 22 of the above-described analysis sensor preparation substrate 10, and the signal substance 53 is bonded to the signal substance binding group 32. is doing.
- the detection target (detection target 60 described later) of the analytical sensor of the present invention is not particularly limited in principle as long as it has specific binding ability to the antibody substance 52.
- Specific examples include a low-molecular substance, a protein, and a microparticle having a membrane structure.
- the low molecular weight substance include arbitrary substances such as hormones, drugs, herbicides, agricultural chemicals, sugars, cholesterol, lipids, uric acid, environmental hormones, peptides, and the like.
- the protein include any protein such as HSA, IgG, fibrinogen, transferrin, AST, ALT, LDH, ALP, LAP, ⁇ -GTP, CRP, AFP, and PSA.
- microparticles having a membrane structure include extracellular microparticles, intracellular vesicles, organelles, and cells.
- the membrane structure include a lipid bilayer membrane structure.
- extracellular microparticles include exosomes, microvesicles, and apoptotic bodies.
- intracellular vesicles include lysosomes and endosomes.
- organelle include mitochondria.
- the cells include cancer cells such as circulating tumor cells (CTC) and other disease-related cells.
- the detection target 60 previously binds the other of the fluorescent dye pairs.
- the antibody substance 52 only needs to have a specific binding ability to the detection target.
- the antibody substance 52 includes an antibody and an antibody-like substance.
- An antibody refers to a protein having the complete basic structure of an immunoglobulin, and an antibody-like substance refers to an immunoglobulin fragment (antibody fragment).
- Examples of the antibody include immunoglobulin (Ig), chimeric antibody, and the like, and more specifically, IgG, IgA, IgM, IgE, IgD and the like.
- Examples of the chimeric antibody include a humanized antibody.
- the antibody may be derived from mammals such as mice, rabbits, cows, pigs, horses, sheep and goats, birds such as chickens, and animal species such as humans, and is not particularly limited.
- the antibody may be prepared, for example, from serum derived from the animal species by a conventionally known method, or a commercially available antibody may be used.
- the antibody may be, for example, either a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody.
- Examples of the antibody-like substance include Fab, Fab ′, F (ab ′) 2 , ScFv, and the like.
- the antibody substance 52 can be appropriately selected by those skilled in the art depending on the detection target. As long as the detection target has a specific antigen on its surface, the antibody substance 52 having a specific binding ability for a specific antigen can be used.
- the exosome is a membrane protein (exosome-specific antigen), for example, CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin V, Since it has LAMP1 or the like, an antibody against an exosome-specific antigen can be used as the antibody substance 52.
- the cancer cell is a cancer cell-specific antigen, for example, Caveolin-1, EpCAM, FasL, TRAIL, Galectine3, CD151, Tetraspanin 8, EGFR, HER2, RPN2, CD44, TGF- ⁇ , etc. Therefore, an antibody against a cancer cell-specific antigen can be used as the antibody substance 52.
- an antibody against a disease cell-specific antigen can be used as the antibody substance 52.
- the antibody substance 52 does not have a modifying group from the viewpoint of convenience and good affinity of the antibody substance with respect to the detection target and from the viewpoint of ease of production of the analytical sensor 50 or versatility. Can be used.
- the modifying group in this case refers to a modifying group provided for a purpose different from affinity, such as a signal substance.
- the modifying group does not include a group (for example, a histidine tag) that contributes to the binding with the antibody substance binding group 22.
- the signal substance 53 functions to read out binding information between the antibody substance 52 specific to the detection target and the antibody substance binding group 22.
- the signal substance 53 is not particularly limited as long as the signal intensity detected by the binding of the detection target to the recess 31 changes or the spectrum changes (for example, the peak shifts).
- fluorescent materials, radioactive element-containing materials, magnetic materials and the like can be mentioned.
- the signal substance is preferably a fluorescent substance.
- fluorescent substances include fluorescent dyes such as cyanine dyes and rhodamine dyes such as fluorescein dyes and indocyanine dyes; fluorescent proteins such as GFP; nanoparticles such as gold colloids and quantum dots.
- radioactive element-containing substances include sugars, amino acids, and nucleic acids labeled with radioactive isotopes such as 18 F.
- the magnetic substance include those having a magnetic material such as ferrichrome, those found in ferrite nanoparticles, nanomagnetic particles, and the like.
- the signal substance 53 can be configured as one of a fluorescent dye pair that causes fluorescence resonance energy transfer (FRET).
- the fluorescent dye pair that causes FRET is not particularly limited, and it is not limited which of the donor dye and the acceptor dye is selected as the signal substance 53.
- a donor dye can be selected as the signal substance 53.
- Specific examples of the donor dye / acceptor dye constituting the fluorescent dye pair that causes FRET include fluorescein isothiocyanate (FITC) / tetramethylrhodamine isothiocyanate (TRITC), Alexa Fluor647 / Cy5.5, HiLyte Fluor647 / Cy5.5.
- the manufacturing method of the base material for analytical sensor preparation of the detection target of this invention includes the following processes.
- a polymer is added to the surface of the substrate by adding a polymerizable monomer, synthesizing a molecular imprint polymer for a part of the surface of the template using the polymerizable monomer as a substrate and the polymerization initiating group as a polymerizable initiator.
- FIGS. 3 to 7 schematically show a method for producing a substrate for preparing an analytical sensor to be detected according to the present invention when exosome is used as a template.
- the method for producing the analytical sensor production substrate includes the following steps.
- a template introduction step for introducing a template 40 that specifically binds to the antibody substance 24 to the binding functional group 22a via the first reversible linking group 22b and the antibody substance 24 (FIG.
- a surface modification step of modifying the surface of the template 40 with the polymerizable functional group 33 via the second reversible linking group 32b (FIG. 5);
- a polymerizable monomer 35 By adding a polymerizable monomer 35, using the polymerizable functional group 33 and the polymerizable monomer 35 as a substrate, and using the polymerization initiating group 23a as a polymerizable initiator, a molecular imprint polymer for a part of the surface of the template 40 is synthesized.
- a polymerization step for forming the polymer film 30 on the surface of the substrate 20 (FIG.
- Molecular film formation process As shown in FIG. 3, in the molecular film formation step, a molecular film 21 having a binding functional group 22 a and a polymerization initiating group 23 a on the surface is formed on the base material 20.
- the binding functional group 22a is a group different from the polymerization initiating group 23a, and a group corresponding to the reagent used for extending the molecular chain to the template 40 in the template introduction step described later is appropriately determined by those skilled in the art. . In the illustrated embodiment, the case where the binding functional group 22a is an amino group is illustrated.
- the polymerization initiating group 23a is not particularly limited as long as it has a structure that can function as a polymerization initiator, and can be appropriately determined by those skilled in the art depending on the polymerization reaction used in the polymerization step described later.
- a group having a structure that generates a radical during the polymerization reaction specifically, a carbon-halogen bonding group derived from an organic halogen (—CX group; X represents a halogen atom).
- —CX group organic halogen
- the molecular film 21 can be formed by a conventionally known method as a monomolecular film by hybrid self-assembly using a molecule having a binding functional group 22a at its terminal and a molecule having a polymerization initiating group 23a at its terminal.
- the molecular film 21 can be formed as a hybrid self-assembled monolayer (mixed SAMs).
- a template 40 that specifically binds to the antibody substance 24 is introduced into the binding functional group 22a via the first reversible linking group 22b and the antibody substance 24. .
- the mold introduction step can include the following steps. Introducing an antibody substance-binding group 22c into the binding functional group 22a via the first reversible linking group 22b; A step of binding the antibody substance 24 to the antibody substance-binding group 22c; and a step of binding the template 40 to the antibody substance 24.
- the first reversible linking group 22b includes a cleavage from the first reversible linking group 22b to a reversible binding group (that is, the above-described antibody substance binding group 22), and a reversible binding group (that is, the above-described antibody).
- a reversible binding group that is, the above-described antibody substance binding group 22
- a reversible binding group that is, the above-described antibody
- an NTA group (a kind of antibody substance-binding group 22) that is an aminopolycarboxylic acid chelating agent-derived group is bound to the antibody substance-binding group 22
- the histidine tag which is a group 25 capable of forming the first reversible linking group 22b (the histidine tag in the figure is schematically shown, and does not show an accurate molecular structure.
- the imidazolyl group of a part of the tag is highlighted.
- the antibody substance-binding group 22c is not particularly limited as long as it is a group capable of binding to the antibody substance 24. However, the antibody substance-binding group 22c binds the antibody substance 24 by orientation (that is, binds in a direction in which the template 40 binding site of the antibody substance 24 faces outward). From the viewpoint, a group having a specific binding ability to the Fc region of the antibody substance 24 is preferable. In the illustrated embodiment, protein G is exemplified as such a group.
- Antibody substance 24 can be determined according to template 40. Specifically, an antibody substance having specific binding ability to the template 40 is selected as the antibody substance 24.
- the template 40 is determined by considering the detection target. Specifically, an object that is the same as the detection target (detection target 60 described later) or a larger size than the detection target is selected as a template. As a target having a size larger than the detection target, a recess 31 formed by synthesis of a polymer film 30 in a polymerization process described later can be opened on the surface of the substrate 20 with a size sufficient to accept the detection target. And if it is a thing of a magnitude
- the template 40 is bound via the antibody substance 24, it is preferable to use a template having an antigen to which the antibody substance 24 specifically binds on the surface.
- a template having an antigen to which the antibody substance 24 specifically binds on the surface For example, an antibody against a specific antigen (surface protein) on the exosome surface can be used as the antibody substance 24, and an exosome can be used as the template 40.
- a molecule having an NTA group which is an aminopolycarboxylic acid chelating agent-derived group, is bound to an amino group as the binding functional group 22a, and a Histag is present in the presence of nickel ions.
- an exosome surface protein as the antibody substance 24 is further introduced.
- An embodiment in which a specific antibody is bound and exosome as the template 40 is finally bound is illustrated.
- the surface modification step can include the following steps. Anchoring anchor material 41 having anchor group 43 and reversible binding group 42 to template 40; and converting reversible binding group 42 into second reversible linking group 32b and polymerizable functional group 33. The process of introducing.
- the anchor substance 41 is a substance including an anchor group 43 and a reversible binding group 42.
- the anchor group 43 is a group that can be anchored to the template 40.
- the template 40 is a membrane body having a lipid bilayer membrane, it is a hydrophobic chain group that can be anchored to the lipid bilayer membrane. It is preferable.
- the hydrophobic chain may be a single chain or a double chain, may be a saturated hydrocarbon or an unsaturated hydrocarbon, and may or may not have a substituent. Also good.
- a linear or branched alkyl group or alkenyl group having 6 to 24 carbon atoms is preferable, and more specifically, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, Dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, stearyl group (octadecyl group), nonadecyl group, icosyl group, heicosyl group, docosyl group, tricosyl group, tetracosyl group, myristolyl group, palmitoleyl group Oleyl group, linoleyl group, linoleyl group, ricinoleyl group, isostearyl group and the like.
- a myristol group a myristol group
- the anchor substance 41 may contain a hydrophilic chain group in addition to the anchor group 43 and the reversible binding group 42.
- the hydrophilic chain group may be a linking group located between the anchor group 43 and the reversible binding group 42.
- protein, oligopeptide, polypeptide, polyacrylamide, polyethylene glycol (PEG), dextran, etc. are mentioned, More preferably, PEG is mentioned.
- the reversible binding group 42 is a group that is converted into the second reversible linking group 32b by binding to another reversible binding group (specifically, the signal substance binding group 32 described above corresponds).
- a thiol group corresponding second reversible linking group 32b is a disulfide group
- an aminooxy group or a carbonyl group corresponding second reversible linking group 32b is an oxime group
- boronic acid group Diol group corresponding second reversible linking group 32b is a cyclic diester group
- amino group and carbonyl group corresponding second reversible linking group 32b is Schiff base
- aldehyde group or ketone group and alcohol corresponding Examples of the second reversible linking group 32b include an acetal group).
- a specific example of the preferred anchor material 41 is a material represented by the following formula (BAM-SH).
- n represents an integer of 0 to 182, for example.
- the polymerizable functional group 33 only needs to have a polymerizable unsaturated bond, and a typical example is a (meth) acryl group.
- the anchor substance 41 represented by the above formula is anchored to the lipid bilayer of the exosome as the template 40, and then the second reversible binding of the anchor substance 41 is performed.
- the thiol group as the second reversible linking group 32b is obtained by disulfide exchange of the molecule 34 containing the (meth) acryl group as the polymerizable functional group 33 and the disulfide bond with the thiol group as the functional group 42.
- An example in which the surface of the template 40 is modified with a polymerizable functional group 33 while being converted into a group is illustrated.
- the second reversible linking group 32b capable of generating the signal substance binding group 32 is introduced by a method called surface modification of the template 40, whereby the second reversible linking group 32b is introduced only on the surface of the template 40.
- the reversible linking group 32b can be delivered.
- a polymer synthesized by imprinting polymerization using a template is referred to as a molecular imprint polymer, and the molecular imprint polymer is not a molecule as a template (for example, a cell Polymers synthesized by imprinting polymerization using) are also included.
- the polymerizable monomer 35 is preferably a hydrophilic monomer, more preferably a zwitterionic monomer.
- the zwitterionic monomer includes an anionic group derived from an acidic functional group (for example, a phosphate group, a sulfuric acid group, and a carboxyl group) and a basic functional group (for example, a primary amino group, a secondary amino group, a tertiary group). Both amino groups and cationic groups derived from quaternary ammonium groups, etc.) are contained in one molecule.
- an acidic functional group for example, a phosphate group, a sulfuric acid group, and a carboxyl group
- a basic functional group for example, a primary amino group, a secondary amino group, a tertiary group.
- Both amino groups and cationic groups derived from quaternary ammonium groups, etc. are contained in one molecule.
- phosphobetaine, sulfobetaine, carboxybetaine and the like can be mentioned.
- examples of the phosphobetaine include molecules having a phosphorylcholine group in the side chain, and preferably 2-methacryloyloxyethyl phosphorylcholine (MPC) and the like.
- examples of sulfobetaines include N, N-dimethyl-N- (3-sulfopropyl) -3′-methacryloylaminopropaneaminium inner salt (SPB), N, N-dimethyl-N- (4-sulfobutyl) -3 ′.
- SPB N-dimethyl-N- (4-sulfobutyl) -3 ′.
- SBB -Methacryloylaminopropaneaminium inner salt
- Carboxybetaines include N, N-dimethyl-N- (1-carboxymethyl) -2′-methacryloyloxyethanaminium inner salt (CMB), N, N-dimethyl-N- (2-carboxyethyl)- And 2'-methacryloyloxyethaneaminium inner salt (CEB).
- the surface initiation control / living radical polymerization proceeds by constructing a polymerization reaction system in which the polymerizable functional group 33, the polymerizable monomer 35, the polymerization initiating group 23a and the template 40 coexist on the surface of the base material 20.
- the polymerization reaction system further preferably contains a transition metal complex formed from a transition metal or a transition metal compound and a ligand as a polymerization catalyst, and more preferably a reducing agent.
- the transition metal or transition metal compound include copper metal or copper compound.
- the copper compound include chloride, bromide, iodide, cyanide, oxide, hydroxide, acetate, sulfate, nitrate, Preferably, bromide is used.
- the ligand is preferably a polydentate amine, and specific examples include bidentate to hexadentate ligands. Among these, a bidentate ligand is preferable, and 2,2-bipyridyl, 4,4′-di- (5-nonyl) -2,2′-bipyridyl, N- (n-propyl) is more preferable. ) Pyridylmethanimine, N- (n-octyl) pyridylmethanimine and the like, more preferably 2,2-bipyridyl.
- the reducing agent include alcohols, aldehydes, phenols, organic acid compounds, and the like, and preferably organic acid compounds.
- the organic compound examples include citric acid, oxalic acid, ascorbic acid, ascorbate, ascorbate and the like, preferably ascorbic acid, ascorbate and ascorbate, and more preferably ascorbic acid. It is done. Specifically, when the polymer chain extends from the polymerization initiator group 23a, which is a radical generation source, using the polymerizable monomer 35 as a substrate, the thickness of the polymer film increases, and when the extended polymer chain reaches the surface of the template 40, By incorporating the polymerizable functional group 33 modified as a substrate, a polymer is synthesized so that the concave portion 31 having a shape along the surface shape of the template 40 is formed.
- the polymerization initiator group 23a which is a radical generation source
- the polymer film can be grown to a thickness corresponding to about 1 ⁇ 2, preferably about ⁇ of the upper and lower diameters of the mold 40 introduced into the substrate 20 (when the top of the drawing is the top). Thereby, the polymer film 30 is obtained.
- an aqueous solvent such as a buffer is preferably used from the viewpoint of suppressing the denaturation of the template 40 and the like.
- a method for producing a substrate for preparing an analytical sensor to be detected when artificial particles are used as a template is a molecular film forming step in which a molecular film having a binding functional group and a polymerization initiating group on the surface is formed on the substrate. And a template introduction step of introducing artificial particles as a template to the binding functional group via a first reversible linking group; and polymerizing the surface of the template via a second reversible linking group.
- a surface modification step of modifying with a functional functional group adding a polymerizable monomer, using the polymerizable monomer as a substrate, and using the polymerization initiating group as a polymerizable initiator, a molecular imprint polymer for a part of the surface of the template
- conversion to the base It can include, a removal step of removing the mold.
- FIG. 8 to FIG. 11 schematically show a method for producing a base material for producing an analytical sensor to be detected according to the present invention when artificial particles are used as a template.
- 8 shows a mold introduction process
- FIG. 9 shows a surface modification process
- FIG. 10 shows a polymerization process
- FIG. 11 shows a removal process.
- the molecular film formation step is the same as that in the case of using exosome as a template, and specifically, as described in “3-1-1. Molecular film formation step” and FIG.
- the binding functional group 22a is an amino group
- the first reversible linking group 22b is a nitrilotriacetic acid (NTA) group (a kind of antibody substance-binding group 22) and a histidine tag (antibody substance binding).
- NTA nitrilotriacetic acid
- the artificial particles used as the template 40 are not particularly limited as long as they can be used as a template, and include artificially produced inorganic particles and organic particles.
- the inorganic particles include metals, metal oxides, nitrides, fluorides, sulfides, borides, and complex compounds thereof, and hydroxyapatite.
- silicon dioxide (silica) is used.
- the organic particles include latex cured products, dextran, chitosan, polylactic acid, poly (meth) acrylic acid, polystyrene, and polyethyleneimine. Since the size of the recess 31 (see FIG.
- the size of the artificial particle depends on the size of the template 40, the size of the artificial particle can be determined as appropriate depending on the size of the detection target.
- artificial particles having a size comparable to or larger than the detection target may be used.
- the size of the artificial particles can be selected from those of 1 nm to 10 ⁇ m.
- the template 40 as an artificial particle includes a group 25 capable of forming the above-described first reversible linking group 22b by binding to the above-described antibody substance-binding group 22 on the surface, and the above-described signal substance-binding group. And a reversible linking group 42 capable of forming the second reversible linking group 32b described above.
- the reversible linking group 42 is as described above.
- the antibody substance-binding group 22 is bonded to the nitrilotriacetic acid (NTA) group and the antibody substance-binding group 22 to form the first reversible group.
- the group 25 capable of forming a general linking group 22b is a histidine tag, and the reversible linking group 42 is a thiol group.
- the reversible linking group 42 shown as a thiol group binds to the signal substance binding group 32 to form a second reversible linking group 32b.
- the amino group as the binding functional group 22a on the substrate 20 is bonded to the amino group.
- the artificial particle template 40 is introduced through the first reversible linking group 22b.
- the surface of the template 40 is modified with the polymerizable functional group 33 via the second reversible linking group 32b. More specifically, the reversible binding group 42 on the surface of the template 40 is converted into the second reversible linking group 32b and the polymerizable functional group 33 is introduced.
- the polymerizable functional group 33 is as described above in “3-1-3. Surface modification step”.
- disulfide exchange is performed on a molecule 34 including a (meth) acryl group as a polymerizable functional group 33 and a disulfide bond with a thiol group which is the second reversible binding group 42 on the surface of the template 40.
- the thiol group is converted into a disulfide group that is the second reversible linking group 32 b and the surface of the template 40 is modified with the polymerizable functional group 33.
- the second reversible linking group 32 b is used as the template 40 by using the template 40 having the thiol group as the second reversible binding group 42 on the surface in advance. It can be formed only on the surface.
- the analysis sensor 50 to be detected includes an antibody substance binding group 22 that binds to an antibody substance binding group 22 and a signal substance 53 to a signal substance binding group 32 in the analysis sensor preparation substrate 10. Can be combined.
- the antibody-like substance 52 is not particularly limited as long as it is specific to the detection target.
- the antibody-like substance 52 may be the same antibody-like substance as the antibody-like substance 24 used in the production of the analytical sensor production substrate 10, or the antibody An antibody-like substance different from the like-like substance 24 may be used.
- the base material 10 for sensor preparation for analysis has the signal substance binding group 32 only in the recess 31 that becomes the sensor field on the surface of the substrate 20, the signal substance 53 can be disposed only in the recess 31.
- one type of antibody-like substance 52 and one type of signal substance 53 may be introduced into all the concave parts 31, When one type of antibody-like substance is introduced, another type of antibody-like substance is introduced into the other recess 31, and a different type of signal substance 53 is introduced corresponding to the type of antibody-like substance. Good.
- FIG. 12 is a schematic diagram for explaining an example of the detection target analysis method of the present invention. As shown in FIG. 12, in the detection target analysis method of the present invention, the analysis sample liquid containing the detection target 60 is brought into contact with the surface of the base material 20 of the analysis sensor 50.
- the detection target 60 is not particularly limited in principle as long as it is a substance that specifically binds to the antibody substance 52, and includes, as described above, a low-molecular substance, a protein, and a microparticle having a membrane structure.
- low molecular weight substances include hormones, drugs, herbicides, agricultural chemicals, sugars, cholesterol, lipids, uric acid, environmental hormones, peptides, and other arbitrary substances.
- the protein include HSA, IgG, fibrinogen, transferrin, AST, ALT, LDH, ALP, LAP, --GTP, CRP, AFP, and PSA.
- microparticles having a membrane structure include extracellular microparticles, intracellular vesicles, organelles, and cells.
- the membrane structure include a lipid bilayer membrane structure.
- extracellular microparticles include exosomes, microvesicles, and apoptotic bodies.
- intracellular vesicles include lysosomes and endosomes.
- organelle include mitochondria.
- the cells include cancer cells such as circulating tumor cells (CTC) and other disease-related cells.
- the mode of the analysis sample liquid including the detection target 60 is not particularly limited, but it is preferable that the process for separating the detection target 60 has not been performed from the viewpoint of rapid analysis.
- Examples of the process for separating the detection target 60 include ultracentrifugation, ultrafiltration, continuous flow electrophoresis, filtration using a size filter, gel filtration chromatography, and the like.
- the analysis sample liquid containing the detection target 60 includes a sample obtained from the environment in which the detection target 60 exists (when the detection target 60 is a cell or extracellular particulate), or (the detection target 60 is an extracellular particulate). (If it is a product from a cell), it may be a sample obtained from the environment in which the detection target 60 can occur. Specifically, it may be a biological sample containing cells.
- the detection target 60 is an extracellular microparticle such as an exosome
- examples of cells that produce the detection target 60 include cancer cells, mast cells, dendritic cells, reticulocytes, epithelial cells, B cells, and nerve cells. .
- examples of the analysis sample liquid containing the detection target 60 include body fluids such as blood, milk, urine, saliva, lymph, cerebrospinal fluid, amniotic fluid, tears, sweat, rhinorrhea, and the like.
- Examples of the treatment liquid include pretreatment such as removal of unnecessary components from body fluids, and culture liquid obtained by culturing cells contained in these body fluids.
- body fluids such as urine, saliva, tears, sweat, and rhinorrhea are particularly preferred in terms of non-invasiveness and ease of collection.
- the detection target 60 is specifically captured by the antibody substance 52 in the recess 31.
- the exosome is used as a membrane protein (exosome-specific antigen), for example, CD63, CD9, CD81, CD37, CD53, CD82, CD13, CD11, CD86, ICAM-1, Rab5, Annexin Captured by specifically binding to the antibody substance 52 via V, LAMP1 or the like.
- the cancer cell is used as a cancer cell-specific antigen, for example, Caveolin-1, EpCAM, FasL, TRAIL, Galectine3, CD151, Tetraspanin 8, EGFR, HER2, RPN2, CD44, TGF- Captured by specifically binding to the antibody substance 52 via ⁇ or the like.
- a cancer cell-specific antigen for example, Caveolin-1, EpCAM, FasL, TRAIL, Galectine3, CD151, Tetraspanin 8, EGFR, HER2, RPN2, CD44, TGF- Captured by specifically binding to the antibody substance 52 via ⁇ or the like.
- the detection target 60 When the detection target 60 is specifically captured by the antibody substance 52 in the recess 31, the signal substance 53 is shielded by the detection target 60 at that moment, so that the signal intensity detected from the signal substance 53 is reduced.
- the detection target 60 is detected by the change in the signal intensity. Since the capture of the detection target 60 and the change in signal intensity occur almost simultaneously, there is no need to add a reagent for the detection of the detection target 60, and the detection can be performed quickly.
- the analysis sensor 50 is configured so that the signal substance 53 becomes one of the fluorescent dye pairs that cause fluorescence resonance energy transfer (FRET), and one of the fluorescent dye pairs is bonded to the detection target 60 in advance.
- FRET fluorescence resonance energy transfer
- the detection target 60 is specifically captured by the antibody substance 52 in the recess 31, the fluorescent dye in the signal substance 53 and the fluorescent dye in the detection target 60 are close to each other at that moment, so that fluorescence is emitted by FRET. Is done.
- the detection object 60 is detected by the fluorescence emission by this FRET. Since the capture of the detection target 60 and the fluorescence emission by FRET occur almost simultaneously, it is not necessary to add a reagent for the detection of the detection target 60, and the detection can be performed quickly.
- the fluorescent dye pair that causes FRET is not particularly limited, and it is not limited which of the donor dye and the acceptor dye is selected as the signal substance 53.
- a donor dye can be selected as the signal substance 53.
- Specific examples of the donor dye / acceptor dye constituting the fluorescent dye pair that causes FRET include fluorescein isothiocyanate (FITC) / tetramethylrhodamine isothiocyanate (TRITC), Alexa Fluor647 / Cy5.5, HiLyte Fluor647 / Cy5.5.
- the senor for analysis 50 of the present invention is not desired even if there is nonspecific adsorption on the surface of the substrate 20 outside the recess 31 because the signal substance 53 is not provided in addition to the recess 31 on the surface of the substrate 20. Unaffected by the background. Therefore, the detection target 60 can be detected with high sensitivity.
- Example 1 Substrate for preparing an analytical sensor to be detected using exosome as a template
- hybrid self-assembled monolayers mixed SAMs terminated with an amino group and a bromo group on a gold thin film-deposited glass substrate are prepared (molecular film forming step), and an NTA group is introduced at the end of the amino group.
- the antibody-binding protein ProteinG was bound by chelate binding, the Anti-CD9 antibody was immobilized, and the exosome was immobilized via the anti-CD9 antibody (template introduction step).
- the exosome membrane was subjected to methacrylic group modification using BAM (surface modification step), and a polymer thin film was synthesized by surface initiation control / living radical polymerization (polymerization step). Finally, the exosome was removed (removal step) to obtain a base material for preparing an analytical sensor to be detected.
- BAM surface modification step
- polymer thin film was synthesized by surface initiation control / living radical polymerization (polymerization step).
- the exosome was removed (removal step) to obtain a base material for preparing an analytical sensor to be detected.
- the substrate After washing the substrate with pure water, the substrate is immersed in an EtOH solution of 0.5 mM Amino-EG6-undecanthiol hydrochloride (Dojindo Laboratories) and 0.5 mM Bis [2- (2-bromoisobutyryloxy) undecyl] disulfide (Sigma-Aldrich). And allowed to stand at 25 ° C. for 24 hours.
- EtOH solution 0.5 mM Amino-EG6-undecanthiol hydrochloride (Dojindo Laboratories) and 0.5 mM Bis [2- (2-bromoisobutyryloxy) undecyl] disulfide (Sigma-Aldrich).
- Protein G and Anti-CD9 antibodies were immobilized because surface plasmon resonance analysis (measurement conditions were sample 1: 100 ⁇ M Protein G, sample 2: 0.3 ⁇ M Anti-CD9, flow rate: 30 ⁇ L / min. , Injection volume: 30 ⁇ L, running buffer: PBS (10 mM posphate, 140 mM NaCl, pH 7.4), temperature: 25 ° C., increase of SPR signal (response unit RU) ( ⁇ Ru is 4500 for Protein G) , Anti-CD9 was 5919).
- a surface plasmon resonance intermolecular interaction analyzer Biacore Q (GE Healthecare) was used (hereinafter, the same applies when SPR analysis is performed).
- exosome Exosomes from PC3 cell, HansaBioMed
- PBS 10 M posphate, 140 mM NaCl, pH7.4
- SPR measurement conditions were sample: exosome, concentration: 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000 ng / mL, flow rate: 10 ⁇ L / min, injection volume: 30 ⁇ L, running buffer: PBS (10 mM posphate, 140 mM NaCl, pH7.4), temperature : 25 ° C) was performed by confirming that the amount of exosome adsorbed was larger on the substrate on which the Anti-CD9 antibody was immobilized.
- 2- (2-Pyridyl) dithioethylmethacrylate was dissolved in 50 ⁇ L of DMSO and then diluted to 100 ⁇ M with PBS (10 M posphate, 140 mM NaCl, pH7.4) (DMSO is about 0.9 wt% of the whole)
- PBS PBS (pH 7.4) solution of 100 ⁇ M 2- (2-Pyridyl) dithioethylmethacrylate was prepared.
- the prepared solution substrate was immersed and reacted at 25 ° C. overnight.
- the substrate was immersed in 1 M EDTA-4Na aqueous solution for 15 minutes to remove Cu 2+ .
- the substrate was immersed in an aqueous 50 mM TCEP solution at 25 ° C. for 3 hours to reduce disulfide bonds.
- After washing the substrate with pure water, the substrate was immersed in 50 mM acetate buffer (pH 4.0) containing 0.5 wt% SDS and shaken at low speed for 1 hour to cut out Protein G, Anti-CD9 and exosome from the polymer thin film.
- Example 2 Sensor for analysis to be detected using exosome as a template
- the substrate for analysis sensor preparation (MIP substrate) obtained in Example 1 was added to 100 ⁇ L of 4 mM NiCl 2 aqueous solution, 100 ⁇ L of 100 ⁇ M Protein G PBS (10 mM posphate, 140 mM NaCl, pH 7.4) solution, and anti-CD9.
- 100 ⁇ L of 0.3 ⁇ M PBS (10 mM posphate, 140 mM NaCl, pH 7.4) solution of the antibody was dropped to fix the anti-CD9 antibody.
- 100 ⁇ L of a 500 ⁇ M POLARIC-MLI (pentamine drug) DMSO solution was dropped onto the substrate, left at 25 ° C. for 1 hour, and then washed. As a result, an analysis sensor to be detected was obtained.
- Example 3 Exosome detection by fluorescence analysis using an analysis sensor to be detected
- the analytical sensor obtained in Example 2 the analytical sensor obtained in Example 2, the exosome binding behavior was observed. Specifically, 10 ⁇ L of exosome solutions (estimated average molecular weight: 1.92 ⁇ 10 8 Da) having different concentrations were dropped onto the prepared substrate, covered with an 18 ⁇ 18 mm cover glass, and allowed to stand at room temperature for 10 minutes. The standing was performed under light shielding. The fluorescence intensity of the substrate after the reaction was measured.
- the fluorescence intensity was measured using a fluorescence microscope (inverted research microscope IX 73 (OLYMPUS)), and the measurement conditions were: 2.5, 5, 10, 50, 100, 250, 500, 1000, 2500, 5000 ng / mL, filter: BW, objective lens: x10, exposure time: 0.50 sec, light intensity: 100%, and measured values were average values of 5 points. Andor SOLIS was used as spectroscopic software.
- FIG. 13 shows the result of measuring the fluorescence intensity change ((Io-I) / Io) with respect to the exosome concentration, creating a graph, and curve fitting (regression analysis) to calculate the binding constant.
- Graph creation software DeltaGraph was used for graph creation and curve fitting, and the coupling constant was calculated by the following equation. As a result, the binding constant Ka was calculated to be 1.50 ⁇ 10 14 [M ⁇ 1 ].
- Example 2 [Reference example: exosome detection by SPR using an analytical sensor to be detected]
- the binding constant was determined by surface plasmon resonance (SPR) analysis.
- the SPR measurement conditions were: sample: Exosome, concentration: 25, 50, 100, 250, 500, 1000, 2500, 5000 ng / mL, flow rate: 10 ⁇ L / min, injection volume: 30 ⁇ L, running buffer: PBS (10 mM posphate, 140 mM NaCl, pH 7.4), temperature: 25 ° C.
- FIG. 14 shows the result of calculating the binding constant by performing curve fitting in the same manner as in Example 3 from the graph showing the SPR signal (response unit RU) with respect to the exosome concentration.
- the binding constant Ka was calculated to be 2.03 ⁇ 10 12 [M ⁇ 1 ].
- the reason why the binding constant larger than that in Example 3 was calculated was that Example 3 specifically detected a change in fluorescence only in the concave portion serving as the sensor field, and there was nonspecific adsorption outside the sensor field. This non-specific adsorption did not affect the detection result, whereas in this reference example, not only the sensor field but also non-specific adsorption other than the sensor field was detected on the principle of SPR analysis. Thus, according to the present invention, it is possible to detect only the specifically recognized object.
- Example 5 Reproducibility of creating sensor for analysis to be detected
- Three analysis sensors to be detected were prepared by the same method as in Example 2.
- the three detection sensors to be detected were reacted by dropping the exosome solutions having different concentrations by the same method as in Example 3, and the fluorescence intensity after the reaction was measured.
- the fluorescence intensity was measured for a total of nine points, three for each analytical sensor.
- FIG. 15 shows changes in fluorescence intensity at nine measurement points.
- the horizontal axis represents exosome concentration (ng / ml) and the vertical axis represents fluorescence intensity change ((II 0 ) / I 0 ).
- the measurement error between the sensors for analysis was small, indicating that the sensor could be created with good reproducibility.
- Example 6 Exosome detection by FRET using an analytical sensor to be detected.
- the horizontal axis indicates the exosome concentration
- the vertical axis indicates the amount of change in fluorescence intensity ( ⁇ I).
- fluorescence of rhodamine was observed at the excitation wavelength of fluorescein (that is, FRET was confirmed). FRET does not occur unless fluorescein and rhodamine are placed in close proximity to each other, indicating that exosomes have indeed bound to the fluorescein-labeled recess of the analytical sensor.
- Example 7 Exosome binding inhibition test by adding free anti-CD9 antibody
- an analytical sensor to be detected was produced in the same manner as in Example 2 except that Alexa-Fluor TM 647 was used instead of POLARIC-MLI.
- a sample in which free antibodies do not coexist prepare 0.01, 0.05, 0.1, 0.5, 1, 5, 10 ng / ml exosome in PBS (10 mM phosphate, 140 mM NaCl, pH 7.4) solution
- a sample in which free antibody coexists 20 ⁇ g / ml exosome in PBS (10 ⁇ mM phosphate, 140 ⁇ mM NaCl, pH 7.4) solution in 100 ⁇ l 0.3 ⁇ M anti-CD9 antibody PBS (10 ⁇ mM phosphate, 140 ⁇ mM NaCl, Add pH 100) solution and incubate at 4 ° C for 1 hour, and dilute the resulting solution with PBS.
- Fluorescence intensity change was measured in the same manner as in Example 3 for each of the sample in which free antibody did not coexist and the sample in which free antibody coexisted. The result is shown in FIG. As shown in FIG. 17, it was found that exosome binding is inhibited when free antibodies coexist. It strongly suggests that exosome binding occurs via antibodies in the binding space. Therefore, it was found that the antibody and the fluorescent dye are provided in the recess of the analytical sensor, and the exosome binding information can be read out.
- Example 8 Substrate for preparing an analytical sensor to be detected using silica nanoparticles as a template.
- Template synthesis-Synthesis of silica nanoparticles with thiol group and histidine tag (His-tag) Disperse 200 ⁇ l of FITC-labeled silica nanoparticles (with -COOH 5 nmol on the surface per 200 ⁇ l, particle size 200 nm) in dichloromethane (DCM) (Silica nanoparticle dispersion).
- DCM dichloromethane
- His-tag terminal is a lysine residue and has a free ⁇ -amino group: 0.10 ⁇ mol, 40 eq
- 2-aminoethanethiol hydrochloride to surface-modified silica nanoparticles with 6 peptide bonds linked to histidine Salt 0.1 ⁇ mol, 40 eq
- silica nanoparticles SH / His-tagged silica nanoparticles
- thiol groups and His-tag were introduced were purified by centrifugation and filtration.
- SH / His-tagged silica nanoparticles are immobilized by dropping SH / His-tagged silica nanoparticles dispersed in 10 mM posphate, 140 mM NaCl, pH 7.4. Got.
- a substrate in which a dispersion of silica nanoparticles not subjected to His-tag modification was similarly dropped was also obtained.
- fluorescence intensity derived from the fluorescent molecule fluorescein (excitation 480 nm / fluorescence 510 nm) introduced into the silica nanoparticles before and 1 hour after the addition of the silica nanoparticles the silica nanoparticles were measured. Immobilization was confirmed.
- the substrate was treated with an acidic buffer (0.5 wt% SDS 50 mM acetic acid buffer (pH 4.0)), and then the fluorescence intensity on the substrate surface was measured again.
- an acidic buffer 0.5 wt% SDS 50 mM acetic acid buffer (pH 4.0)
- the fluorescence intensity increases after dropping the silica nanoparticles with His-tag modification onto the substrate. Particle immobilization was confirmed. Furthermore, in the case of His-tag-modified silica nanoparticles, the fluorescence intensity decreased to the same level as before the dropping after treatment with acidic buffer solution (0.5 wt% SDS 50 mM acetic acid buffer (pH 4.0)). It was found that the silica nanoparticles bonded via NTA were detached in the presence of acid and surfactant.
- a substrate for preparing an analytical sensor for detection using silica nanoparticles as a template A hybrid self-assembled monolayer (amino) and bromo group terminated on a gold thin film-deposited glass substrate as follows SAMs) were prepared (molecular film formation step), NTA groups were introduced to the amino group ends to form NTA-Ni complexes, and then silica nanoparticles were immobilized by chelate bonding (template introduction step). Thereafter, the silica nanoparticles were subjected to methacryl group modification (surface modification step), and a polymer thin film was synthesized by surface initiation control / living radical polymerization (polymerization step). Finally, the silica nanoparticles were removed (removal step) to obtain a substrate for preparing an analytical sensor to be detected.
- SAMs self-assembled monolayer
- NTA groups were introduced to the amino group ends to form NTA-Ni complexes
- silica nanoparticles were immobilized by chelate
- the above-mentioned EDTA-4Na treatment also removes NI-NTA nickel and the His-tag is likely to be free at this point.
- the substrate was immersed in 50 mM acetate buffer (pH 4.0) containing 0.5 wt% SDS to wash out silica nanoparticles bound via Ni-NTA and His-tag from the polymer thin film.
- Example 9 Sensor for analysis to be detected using silica nanoparticles as a template
- the MIP substrate was treated with a 4 mM NiCl 2 aqueous solution.
- 100 ⁇ M His-tag Protein G dissolved in PBS was added to the substrate to immobilize Protein G capable of binding the antibody via His-tag.
- 0.3 ⁇ M anti-CD9 antibody dissolved in PBS was added to the substrate, and the anti-CD9 antibody was immobilized via Protein G. Since Protein G binds to the Fc region of the antibody, the orientation of the immobilized antibody is uniform.
- thiol-reactive Alexa Fluor (R) 647 C 2 Maleimide was used as the fluorescent molecule, and the fluorescent molecule was selectively introduced into the recess of the analytical sensor.
- the fluorescence intensity before introduction was 113 ⁇ 0.6 (n-3), but after introduction, it was 151 ⁇ 2.1 (n-3), so the introduction of fluorescence was confirmed. As a result, an analysis sensor to be detected was obtained.
- Example 10 exosome detection by fluorescence analysis using an analysis sensor to be detected
- the analytical sensor (with antibody) obtained in Example 9 the binding behavior of exosomes was observed.
- the binding behavior of exosomes was also observed in the same manner for a substrate (without antibody) on which the anti-CD9 antibody of the analytical sensor was not immobilized.
- exosome fluorescence detection using exosome solution in PBS (10 mM posphate, 140 mM NaCl, pH 7.4) concentration of 0.01, 0.05, 0.1, 0.5, 1, 5, 10 ng / mL, respectively
- the fluorescence microscope measurement conditions are as follows. Filter: Cy5 Objective lens: ⁇ 5 Exposure time: 0.1 seconds
- Light source Mercury lamp
- Example 11 STED super-resolution image of sensor surface for analysis
- the obtained analytical sensor was subjected to a stimulated emission control (STED) microscope.
- STED stimulated emission control
- SP8-STED3X manufactured by Leica The obtained STED super-resolution image is shown in Fig. 19.
- dot-like fluorescence of about 200 nm size was observed, The size of the fluorescence was the same as that of the silica nanoparticles used for the template, and it was confirmed that it was a recess formed by the template effect. It was also shown that the reporter molecule was selectively introduced into the recess.
- Example 12 Measurement of exosomes in tears
- the test paper After collecting tears using Schirmer test paper, the test paper is immersed in 10 mM phosphate buffer (PBS) containing 500 ⁇ l of 140 mM NaCl at 4 ° C. for 6 hours, and the exosomes contained in the tears are removed from the tear sample. As recovered.
- the tear sample was subjected to fluorescence measurement using the analytical sensor of Example 9 to measure exosomes in the tear fluid.
- a tear sample was also measured using a substrate on which the antibody of the sensor was not fixed.
- FIG. 20 The results are shown in FIG. In FIG. 20, “PBS” is a result of providing PBS to the analysis sensor of Example 9, “Tear (with antibody)” is a result of providing a tear sample to the analysis sensor of Example 9, and “Tear (no antibody)” indicates the result of applying a tear sample to a substrate on which the antibody of the analytical sensor of Example 9 is not fixed.
- the degree of fluorescence quenching was the highest, so that exosomes in tear fluid were measured. It has been shown.
- reversible binding group (second reversible by binding to signal substance binding group)
- Anchor group 50 ...
- Analytical sensor 52 ...
- Antibody substance 53 specific to detection object 53 ...
- Signal substance 60 ... Detection object
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Abstract
Description
前記ポリマー膜が、検出対象を受け入れる凹部を有し、
前記凹部内に、抗体物質結合用基とシグナル物質結合用基とを有する、検出対象の分析用センサ作製用基材。
項2. 前記ポリマー膜が、前記検出対象又は前記検出対象よりサイズが大きい対象を鋳型とする分子インプリントポリマーで構成され、前記凹部が前記鋳型の表面形状の一部に対応する、項1に記載の検出対象の分析用センサ作製用基材。
項3. 前記抗体物質結合用基がキレート結合性基である、項1又は2に記載の検出対象の分析用センサ作製用基材。
項4. 前記シグナル物質結合用基がチオール基である、項1~3のいずれかに記載の検出対象の分析用センサ作製用基材。
項5. 項1から4のいずれかに記載の検出対象の分析用センサ作製用基材と、
前記抗体物質結合用基に結合した、前記検出対象に特異的な抗体物質と、
前記シグナル物質結合用基に結合したシグナル物質と、
を含む、検出対象の分析用センサ。
項6. 前記検出対象が膜構造を有する微小粒体である、項5に記載の検出対象の分析用センサ。
項7. 前記膜構造を有する微小粒体がエクソソームである、項6に記載の検出対象の分析用センサ。
項8. 前記検出対象に特異的な抗体物質が、前記膜構造を有する微小粒体の表面に発現している特異的抗原に対する特異的結合能を有する、項5から7のいずれかに記載の検出対象の分析用センサ。
項9. 項5から8のいずれかに記載の検出対象の分析用センサに、検出対象を含む試料を接触させ、前記抗体物質に前記検出対象を結合する工程と、
前記シグナル物質に由来するシグナルの変化を検出する工程と、
を含む、検出対象の分析法。
項10. 基材に、結合性官能基と重合開始性基とを表面に有する分子膜を形成する分子膜形成工程と、
前記結合性官能基に対し、第1の可逆的連結基を介して、人工粒子を鋳型として導入する鋳型導入工程と、
前記鋳型の表面を、第2の可逆的連結基を介して重合性官能基で修飾する表面修飾工程と、
重合性モノマーを加え、前記重合性モノマーを基質とし、前記重合開始性基を重合性開始剤として、前記鋳型の前記表面の一部に対する分子インプリントポリマーを合成することで前記基材表面にポリマー膜を形成する重合工程と、
前記第1の可逆的連結基及び第2の可逆的連結基を開裂させてそれぞれ抗体物質結合用基及びシグナル物質結合用基へ変換するとともに前記鋳型を除去する除去工程と、
を含む、検出対象の分析用センサ作製用基材の製造方法。
項11. 前記人工粒子が、表面に、前記抗体物質結合用基と結合することで前記第1の可逆的連結基の形成が可能な基と、前記シグナル物質結合用基と結合することで前記第2の可逆的連結基の形成が可能な可逆的結合基とを有する、項10に記載の検出対象の分析用センサ作製用基材の製造方法。
項12. 前記人工粒子がシリカ粒子である、項10又は11に記載の検出対象の分析用センサ作製用基材の製造方法。
項13. 前記抗体物質結合用基がキレート結合性基であり、前記抗体物質結合用基と結合することで前記第1の可逆的連結基の形成が可能な基がヒスチジンタグである、項10から12のいずれかに記載の検出対象の分析用センサ作製用基材の製造方法。
項14. 前記シグナル物質結合用基がチオール基であり、前記シグナル物質結合用基と結合することで前記第2の可逆的連結基の形成が可能な可逆的結合基がチオール基である、項10から13のいずれかに記載の検出対象の分析用センサ作製用基材の製造方法。
項15. 項10から14のいずれかに記載の検出対象の分析用センサ作製用基材の製造方法を行う工程と、
前記抗体物質結合用基に、検出対象に特異的な抗体物質を結合する工程と、
前記シグナル物質結合用基にシグナル物質を結合する工程と、
を含む、検出対象の分析用センサの製造方法。
本発明の検出対象の分析用センサ作製用基材は、後述の発明の分析用センサを作製するための材料となる基材である。この分析用センサ作製用基材は、エクソソームなどの検出対象を迅速に検出可能なセンサへユーザが簡便にカスタマイズできるように構成されている。
基材20の材料は、例えば、金属、ガラス、及び樹脂からなる群から選択される材料であってよい。金属としては、金、銀、銅、アルミニウム、タングステン、モリブデン等が挙げられる。樹脂としては、ポリ(メタ)アクリレート、ポリスチレン、ABS(アクリロニトリル-ブタジエン-スチレン共重合体)、ポリカーボネート、ポリエステル、ポリエチレン、ポリプロピレン、ナイロン、ポリウレタン、シリコーン樹脂、フッ素樹脂、メチルペンテン樹脂、フェノール樹脂、メラミン樹脂、エポキシ樹脂、塩化ビニル樹脂等が挙げられる。
ポリマー膜30は、基材20に層状に設けられており、複数の凹部31を有する。凹部31は、本発明の検出対象の分析用センサにおけるセンサ場となる部分である。凹部31は、検出対象を受け入れ可能に形成されていれば限定されるものではないが、たとえば、後述のように分子インプリント重合法を用いることによって形成された、分子インプリントポリマー(MIP;molecularly imprinted polymer)であってよい。この場合、凹部31は、分子インプリント重合法において用いられる鋳型(後述の鋳型40)によって型取られたものであり、当該鋳型の表面形状の一部に対応する形状を有する。凹部31は、検出対象を受け入れ可能な大きさで形成されていればよいため、凹部31の鋳型は、検出対象と同じものであってもよいし、検出対象よりもサイズが大きい対象であってもよい。なお、凹部31が検出対象を受け入れ可能な大きさであるとは、抗体物質(後述の抗体物質52)及びシグナル物質(後述のシグナル物質53)が結合して分析用センサ(後述の分析用センサ50)となった場合に、検出対象の少なくとも一部が凹部31内に進入し抗体物質にアプローチして結合可能となる程度に凹部31が基材20表面に十分な大きさで開口していることをいう。凹部31の開口径は、検出対象により異なり得るため特に限定されないが、たとえば1nm~10μmであってよい。ポリマー膜30の厚みも検出対象により異なり得るため特に限定されないが、たとえば1nm~1μmであってよい。
スルホベタインとしては、N,N-ジメチル-N-(3-スルホプロピル)-3’-メタクリロイルアミノプロパンアミニウムインナーソルト(SPB)、N,N-ジメチル-N-(4-スルホブチル)-3’-メタクリロイルアミノプロパンアミニウムインナーソルト(SBB)などが挙げられる。
カルボキシベタインとしては、N,N-ジメチル-N-(1-カルボキシメチル)-2’-メタクリロイロキシエタンアミニウムインナーソルト(CMB)、N,N-ジメチル-N-(2-カルボキシエチル)-2’-メタクリロイロキシエタンアミニウムインナーソルト(CEB)などが挙げられる。
抗体物質結合用基22は、抗体物質(後述の抗体物質52)を直接的又は間接的に結合させることで分析用センサ作製用基材10に抗体物質を導入可能とする基である。ユーザは、検出対象を自由にターゲティングすることができ、ターゲットとする検出対象に特異的な抗体物質を自由に選択し、抗体物質結合用基22へ導入することができる。また、1個の基材20において、一の凹部31に対し一の種類の抗体物質を導入し、他の凹部31に対し他の種類の抗体物質を導入することによって、1個の基材20において複数種の抗体物質を用いた検出対象の分析が可能となるようにカスタマイズすることもできる。このようなカスタマイズの具体例としては、一の凹部31に対しエキソソームに結合する抗体物質を導入し、他の凹部31に対しタンパク質に結合する抗体物質を導入することによって、1個の基材20において、エキソソームとタンパク質との両方の分析が可能となる。
シグナル物質結合用基32は、シグナル物質(後述のシグナル物質53)を結合させることで分析用センサ作製用基材10にシグナル物質を導入可能とする基である。ユーザは、シグナル物質を自由に選択し、シグナル物質結合用基32へ導入することができる。また、1個の基材20において、一の凹部31と他の凹部31とで異なる種類の抗体物質を設ける場合は、抗体物質の種類ごとに異なるシグナル物質を設けるようにカスタマイズすることもできる。
本発明の検出対象の分析用センサ作製用基材は、抗体物質及びシグナル物質の少なくともいずれかがユーザによってカスタマイズされるように構成されていればよい。したがって、他の態様として、抗体物質結合用基の方に、検出対象に特異的な抗体物質がすでに結合していてもよい。この場合、ユーザはシグナル物質を自由に選択して導入することができる。
本発明の分析用センサは、上述の検出対象の分析用センサ作製用基材と;前記抗体物質結合用基に結合した、前記検出対象に特異的な抗体物質と;前記シグナル物質結合用基に結合したシグナル物質と;を含む。図2に、本発明の検出対象の分析用センサの一例を模式的に示す。図2に示すように、分析用センサ50は、上述の分析用センサ作製用基材10の抗体物質結合用基22に抗体物質52が結合し、シグナル物質結合用基32にシグナル物質53が結合している。
本発明の分析用センサの検出対象(後述の検出対象60)は、抗体物質52への特異性結合能を有するものであれば原理上特に限定されるものではない。具体例としては、低分子物質、タンパク質、及び膜構造を有する微小粒体が挙げられる。低分子物質としては、ホルモン・薬剤・除草剤・農薬・糖・コレステロール・脂質・尿酸・環境ホルモン・ペプチド等の任意の物質が挙げられる。タンパク質としては、HSA, IgG,フィブリノーゲン,トランスフェリン、AST, ALT, LDH, ALP, LAP,γ-GTP, CRP, AFP, PSA等の任意のタンパク質が挙げられる。膜構造を有する微小粒体としては、細胞外微粒子、細胞内小胞、オルガネラ及び細胞が挙げられる。膜構造としては、脂質二重膜構造が挙げられる。細胞外微粒子としては、エクソソーム、マイクロベシクル、アポトーシス小体等が挙げられる。細胞内小胞としては、リソソーム、エンドソーム等が挙げられる。オルガネラとしてはミトコンドリア等が挙げられる。細胞としては、循環腫瘍細胞(CTC)等の癌細胞、その他の疾患関連細胞等が挙げられる。
抗体物質52は、検出対象への特異的結合能を有していればよい。抗体物質52とは、抗体と抗体様物質とを含む意である。抗体とは、免疫グロブリンの完全な基本構造を有するタンパク質をいい、抗体様物質とは、免疫グロブリンの断片(抗体フラグメント)をいう。抗体としては、例えば、免疫グロブリン(Ig)、キメラ抗体等が挙げられ、より具体的には、IgG、IgA、IgM、IgE、IgD等が挙げられる。前記キメラ抗体は、例えば、ヒト化抗体等が挙げられる。前記抗体は、例えば、マウス、ウサギ、ウシ、ブタ、ウマ、ヒツジ、ヤギ等の哺乳動物、ニワトリ等の鳥類、ヒト等の動物種由来のものでもよく、特に制限されない。前記抗体は、例えば、前記動物種由来の血清から、従来公知の方法により調製してもよいし、市販の抗体を使用してもよい。前記抗体は、例えば、ポリクローナル抗体およびモノクローナル抗体のいずれでもよく、モノクローナル抗体が好ましい。抗体様物質としては、例えば、Fab、Fab’、F(ab’)2、ScFv等が挙げられる。
シグナル物質53は、検出対象に特異的な抗体物質52と抗体物質結合用基22との結合情報を読み出すものとして機能する。シグナル物質53としては、凹部31への検出対象の結合により検知されるシグナル強度が変化したり、スペクトルが変化(例えばピークがシフト)したりするものであれば特に限定されない。たとえば、蛍光物質、放射性元素含有物質、磁性物質等が挙げられる。検出容易性等の観点から、シグナル物質としては蛍光物質であることが好ましい。蛍光物質としては、フルオレセイン系色素、インドシアニン色素などのシアニン系色素、ローダミン系色素などの蛍光色素;GFPなどの蛍光タンパク質;金コロイド、量子ドットなどのナノ粒子などが挙げられる。放射性元素含有物質としては、18Fなどの放射性同位体でラベルした、糖、アミノ酸、核酸などが挙げられる。磁性物質としては、フェリクロームなどの磁性体を有するもの、フェライトナノ粒子、ナノ磁性粒子などにみられるものが挙げられる。
本発明の検出対象の分析用センサ作製用基材の製造方法は、以下の工程を含む。
基材に、結合性官能基と重合開始性基とを表面に有する分子膜を形成する分子膜形成工程;
前記結合性官能基に対し、第1の可逆的連結基を介して、鋳型を導入する鋳型導入工程;
重合性モノマーを加え、前記重合性モノマーを基質とし、前記重合開始性基を重合性開始剤として、前記鋳型の前記表面の一部に対する分子インプリントポリマーを合成することで前記基材表面にポリマー膜を形成する重合工程;及び
前記鋳型を除去する除去工程。
鋳型には、エキソソームを用いることができる。図3~図7に、鋳型としてエキソソームを用いる場合の本発明の検出対象の分析用センサ作製用基材を製造する方法を模式的に示す。この場合における分析用センサ作製用基材を製造する方法は、以下の工程を含む。
基材20に、結合性官能基22aと重合開始性基23aとを表面に有する分子膜21を形成する分子膜形成工程(図3);
結合性官能基22aに対し、第1の可逆的連結基22b及び抗体物質24を介して、抗体物質24に特異的に結合する鋳型40を導入する鋳型導入工程(図4);
鋳型40の表面を、第2の可逆的連結基32bを介して重合性官能基33で修飾する表面修飾工程(図5);
重合性モノマー35を加え、重合性官能基33及び重合性モノマー35を基質とし、重合開始性基23aを重合性開始剤として、鋳型40の表面の一部に対する分子インプリントポリマーを合成することで基材20表面にポリマー膜30を形成する重合工程(図6);及び
第1の可逆的連結基22b及び第2の可逆的連結基32bを開裂させて、それぞれ抗体物質結合用基22及びシグナル物質結合用基32へ変換するとともに、抗体物質24及び鋳型40を除去する除去工程(図7)。
以下、各工程について図を参照しながら詳述する。
図3に示すように、分子膜形成工程では、基材20に、結合性官能基22aと重合開始性基23aとを表面に有する分子膜21を形成する。
図4に示すように、鋳型導入工程では、結合性官能基22aに対し、第1の可逆的連結基22b及び抗体物質24を介して、抗体物質24に特異的に結合する鋳型40を導入する。
結合性官能基22aに、第1の可逆的連結基22bを介して抗体物質結合性基22cを導入する工程;
抗体物質結合性基22cに抗体物質24を結合する工程;及び
抗体物質24に、鋳型40を結合する工程。
図5に示すように、表面修飾工程では、鋳型40の表面を、第2の可逆的連結基32bを介して重合性官能基33で修飾する。
アンカー基43と可逆的結合性基42とを有するアンカー物質41を鋳型40にアンカリングさせる工程;及び
可逆的結合性基42を第2の可逆的連結基32bに変換するとともに重合性官能基33を導入する工程。
図6に示すように、重合工程では、重合性モノマー35を加え、重合性官能基33及び重合性モノマー35を基質とし、重合開始性基23aを重合性開始剤として、鋳型40の表面の一部に対する分子インプリントポリマーを合成する。これによって、基材20表面に、凹部31を有するポリマー膜30を形成する。なお、本明細書においては、鋳型を用いたインプリンティング重合によって合成されるポリマーを、便宜上、分子インプリントポリマーと記載するものとし、分子インプリントポリマーには、鋳型として分子ではないもの(たとえば細胞)を用いたインプリンティング重合によって合成されるポリマーも含まれるものとする。
スルホベタインとしては、N,N-ジメチル-N-(3-スルホプロピル)-3’-メタクリロイルアミノプロパンアミニウムインナーソルト(SPB)、N,N-ジメチル-N-(4-スルホブチル)-3’-メタクリロイルアミノプロパンアミニウムインナーソルト(SBB)などが挙げられる。
カルボキシベタインとしては、N,N-ジメチル-N-(1-カルボキシメチル)-2’-メタクリロイロキシエタンアミニウムインナーソルト(CMB)、N,N-ジメチル-N-(2-カルボキシエチル)-2’-メタクリロイロキシエタンアミニウムインナーソルト(CEB)などが挙げられる。
図7に示すように、除去工程では、第1の可逆的連結基22b及び第2の可逆的連結基32bを開裂させて、それぞれ抗体物質結合用基22及びシグナル物質結合用基32へ変換するとともに、抗体物質24及び鋳型40を除去する。上述の表面修飾工程で第2の可逆的連結基32bが鋳型40の表面のみにデリバリされているため、鋳型40が除去された跡であるポリマー膜30の凹部31には、その内部に抗体物質結合用基22が残るとともに、凹部31内にのみ、第2の可逆的連結基32bから生じたシグナル物質結合用基32が配置される。これによって、分析用センサ作製用基材10が得られる。
鋳型には、上述のエクソソームの他に、人工粒子を用いることができる。人工粒子を用いる場合は、上述の鋳型にエクソソームを用いる場合に準じて検出対象の分析用センサ作製用基材の製造が可能であるが、鋳型を基板に固定するための抗体を用いる必要がないため安価に製造可能であり、工業的生産に好適である。また、鋳型に用いる人工粒子自体が工業生産品であり粒径制御されていることから、分析用センサ作製用基材に形成する凹部のサイズの制御及び均質化も容易であるため、得られる分析用センサ作製用基材からは、より分析特性に優れた分析用センサを作製することができる。
分子膜形成工程は、鋳型としてエクソソームを用いる場合と同様であり、具体的には、上記の「3-1-1.分子膜形成工程」及び図3に記載の通りである。
図8に示すように、鋳型導入工程では、結合性官能基22aに対し、第1の可逆的連結基22bを介して、人工粒子を鋳型40として導入する。結合性官能基22及び第1の可逆的連結基22bについては、それぞれ、上述の「3-1-1.分子膜形成工程」及び「3-1-2.鋳型導入工程」に記載の通りである。図示された態様では、結合性官能基22aはアミノ基であり、第1の可逆的連結基22bはニトリロ三酢酸(NTA)基(抗体物質結合用基22の一種)とヒスチジンタグ(抗体物質結合用基22と結合して第1の可逆的連結基22bを形成可能な基25の一種)との結合により形成される基である。
図9に示すように、表面修飾工程では、鋳型40の表面を、第2の可逆的連結基32bを介して重合性官能基33で修飾する。より具体的には、鋳型40表面の可逆的結合性基42を第2の可逆的連結基32bに変換するとともに重合性官能基33を導入される。重合性官能基33としては、上述の「3-1-3.表面修飾工程」で述べたとおりである。図示された態様では、鋳型40表面における第2の可逆的結合性基42であるチオール基に、重合性官能基33としての(メタ)アクリル基とジスルフィド結合とを含む分子34をジスルフィド交換することにより、チオール基を第2の可逆的連結基32bであるジスルフィド基に変換するとともに鋳型40の表面を重合性官能基33で修飾する態様を例示している。人工粒子を鋳型40として用いる場合においては、予め表面に第2の可逆的結合性基42であるチオール基を有している鋳型40を用いることで、第2の可逆的連結基32bを鋳型40の表面のみに形成することができる。
図10に示すように、重合工程では、重合性モノマー35を加え、重合性官能基33及び重合性モノマー35を基質とし、重合開始性基23aを重合性開始剤として、鋳型40の表面の一部に対する分子インプリントポリマーを合成する。これによって、基材20表面に、凹部31を有するポリマー膜30を形成する。重合性モノマー35及びポリマー膜30については、上述の「3-1-4.重合工程」で記載した通りである。
図11に示すように、除去工程では、第1の可逆的連結基22b及び第2の可逆的連結基32bを開裂させて、それぞれ抗体物質結合用基22及びシグナル物質結合用基32へ変換するとともに、鋳型40を除去する。上述の表面修飾工程で第2の可逆的連結基32bが鋳型40の表面のみに形成されるため、鋳型40が除去された跡であるポリマー膜30の凹部31には、その内部に抗体物質結合用基22が残るとともに、凹部31内にのみ、第2の可逆的連結基32bから生じたシグナル物質結合用基32が配置される。これによって、分析用センサ作製用基材10が得られる。
検出対象の分析用センサ50は、分析用センサ作製用基材10において、抗体物質結合用基22に、検出対象に特異的な抗体物質52を結合し、シグナル物質結合用基32にシグナル物質53を結合すればよい。
図12に、本発明の検出対象の分析法の一例を説明する模式図を示す。図12に示すように、本発明の検出対象の分析法では、分析用センサ50の基材20表面上に、検出対象60を含む分析試料液を接触させる。
本実施例では、金薄膜蒸着ガラス基板上にアミノ基及びブロモ基が末端となる混成自己組織化単分子膜(mixed SAMs)を作製し(分子膜形成工程)、アミノ基末端へNTA基を導入し、NTA-Ni錯体を形成した後、キレート結合により抗体結合タンパク質ProteinGを結合させ、Anti-CD9抗体を固定化し、さらに抗CD9抗体を介してエクソソームを固定化した(鋳型導入工程)。その後、BAMを用いてエクソソーム膜にメタクリル基修飾を施し(表面修飾工程)、表面開始制御/リビングラジカル重合によりポリマー薄膜を合成した(重合工程)。最後にエクソソームを除去し(除去工程)、検出対象の分析用センサ作製用基材を得た。
濃硫酸及び30w/v%過酸化水素水を3:1(体積基準)で混合し、ピラニア溶液を作製した。金薄膜蒸着ガラス基板(SPR測定に供する場合はGE Healthcare社のSIA Kit Auを用い、蛍光測定に供する場合はジャスコエンジニアリング社の金蒸着ミラーを用いた。)をピラニア溶液に室温で15分間浸漬させ有機残滓を除去した。基板を純水で洗浄後、0.5 mM Amino-EG6-undecanthiol hydrochloride(同仁化学研究所)、0.5mM Bis [2-(2-bromoisobutyryloxy) undecyl] disulfide(Sigma-Aldrich)のEtOH溶液に基板を浸漬させ、25℃で24時間静置した。
20 mM Isothiocyanobenzyl-NTA(同仁化学研究所)DMSO溶液80 μLを基板に滴下し、25℃で2時間静置した。基板をDMSO及び純水で洗浄後、4 mM NiCl2水溶液100 μLを基板に滴下し、室温で15分間静置した。基板を純水で洗浄後、100 μM Recombinant His-tagged Protein G(Bio Vision)PBS(10 mM posphate, 140 mM NaCl, pH7.4)溶液100 μLを基板に滴下して、25℃で30分間静置した。基板をPBSで洗浄後、0.3 μM Anti-CD9(Human)mAb(MBLライフサイエンス)PBS(10 mM posphate, 140 mM NaCl, pH7.4)溶液100 μLを基板に滴下して、25℃で30分間静置した。これによって、Protein G及びAnti-CD9抗体を固定化した。なお、Protein G及びAnti-CD9抗体が固定化されたことは、表面プラズモン共鳴分析(測定条件は、sample 1:100 μM Protein G、sample 2:0.3 μM Anti-CD9、flow rate:30 μL/min、injection volume:30 μL、running buffer:PBS (10 mM posphate,140 mM NaCl, pH7.4)、temperature:25℃)を用い、SPRシグナル(応答ユニットRU)の増大(ΔRuは、Protein Gで4500、Anti-CD9で5919であった。)により確認した。表面プラズモン共鳴分析には、表面プラズモン共鳴分子間相互作用解析装置Biacore Q(GE Healthecare)を用いた(以下、SPR分析を行う場合において同様)。
3-1.アンカー物質BAM-SH(図5中アンカー物質41に相当)の合成
BAM(Biocompatible Anchor for cell Membrane(Mw=2000);SUNBRIGHT OE-020CS、油化産業株式会社)10.3 mg (5.65 μmol)及び2-Aminoethanethiol hydrochloride(東京化成工業)31.9 mg (280 μmol, 49.7 eq)を、PB(10 mM posphate, pH7.0) 50 μLに溶解させて25°Cで4時間反応させ、アンカー物質BAM-SHを得た。ここでpH7.0の緩衝液を用いたのは、BAMのNHS基の加水分解速度を下げてアミノ基との反応性を上げるためである。
2,2-Dipyridyl disulfide (1.545 g, 7.01 mmol)をmethanol (15 mL)に溶解させ、acetic acid 180 μL を加えて攪拌した。methanolに溶解させた2-Mercaptoethanol (600 μL, 8.45 mmol)を室温で少量ずつ加え、一晩反応させた。シリカゲルクロマトグラフィー(EtOAc:hexane = 50:50(体積基準))によりRf=0.33のフラクションを回収して減圧留去することで,うすい黄色で透明な油状の精製物(2-hydroxyethyl 2-pyridyl disulfide)を得た。
下記表に示したレシピ(Recipe of SI-ATRP for synthesis of Exosome-MIP)に従い、表面開始原子移動ラジカル重合(SI-ATRP;Surface-initiated atom transfer radical polymerization)によってポリマー薄膜を合成した。
1 M EDTA-4Na 水溶液に基板を15分間浸してCu2+を取り除いた。50 mM TCEP水溶液に基板を25℃で3時間浸漬させ、ジスルフィド結合を還元した。純水で基板を洗浄後、0.5wt% SDSを含む50 mM酢酸バッファー(pH 4.0)に基板を浸して低速で1時間振とうし、ポリマー薄膜からProtein G、Anti-CD9及びエクソソームを切り出した。なお、エクソソームが実際に切り出されたことは、SPR(測定条件は、sample1:50 mM TCEP水溶液 x3、sample2:酢酸buffer(10 mM, pH4.0), 0.5wt% SDS solutions x2、flow rate:30 μL/min、injection volume:30 μL、running buffer:PBS (10 mM posphate,140 mM NaCl, pH7.4)、temperature:25℃)を用い、SPRシグナル(応答ユニットRu)の減少により確認した。これによって、検出対象の分析用センサ作製用基材(MIP基板)が得られた。
実施例1で得られた分析用センサ作製用基材(MIP基板)に、4 mM NiCl2水溶液100μL、100μM Protein G PBS(10 mM posphate,140 mM NaCl, pH7.4)溶液100μL、及び抗CD9抗体の0.3μM PBS(10 mM posphate, 140 mM NaCl, pH7.4)溶液100μLを滴下し、抗CD9抗体を固定した。さらに、500 μM POLARIC-MLI(五稜化薬)DMSO溶液100μLを基板に滴下して25℃で1時間静置し、その後洗浄した。これによって、検出対象の分析用センサが得られた。
実施例2で得られた分析用センサを用い、エクソソームの結合挙動を観察した。具体的には、作製した基板に濃度が異なるエクソソーム(推定平均分子量:1.92×108Da)溶液を10μLずつ滴下し、18x18mmのカバーガラスをかぶせ、室温で10分間静置した。静置は遮光下で行った。反応後の基板について蛍光強度の測定を行った。蛍光強度の測定には、蛍光顕微鏡(倒立型リサーチ顕微鏡IX 73(OLYMPUS))を用い、測定条件は、濃度:2.5, 5, 10, 50, 100, 250, 500, 1000, 2500, 5000 ng/mL、フィルター:BW、対物レンズ:x10、露光時間:0.50 sec、光量:100%とし、測定値は5点の平均値とした。分光学ソフトウェアとしては、Andor SOLISを用いた。エクソソーム濃度に対する蛍光強度変化((Io-I)/Io)を測定してグラフを作成し、それをカーブフィッティング(回帰分析)して結合定数を算出した結果を図13に示す。グラフの作成とカーブフィッティングにはグラフ作製ソフトDeltaGraphを用い、結合定数は下記式で算出式した。その結果、結合定数Kaは1.50×1014[M-1]と算出された。
実施例2で得られた分析用センサを用い、表面プラズモン共鳴(SPR)分析により結合定数を求めた。SPR測定条件は、sample:Exosome、concentration:25, 50, 100, 250, 500, 1000, 2500, 5000 ng/mL、flow rate:10 μL/min、injection volume:30 μL、running buffer:PBS (10 mM posphate, 140 mM NaCl, pH7.4)、temperature:25℃とした。エクソソーム濃度に対する、SPRシグナル(応答ユニットRU)を示すグラフから、実施例3と同様にしてカーブフィッティングを行い、結合定数を算出した結果を図14に示す。結果、結合定数Kaは2.03×1012[M-1]と算出された。上述の実施例3よりも大きい結合定数が算出されたのは、上記実施例3がセンサ場となる凹部のみにおける蛍光変化を特異的に検出しており、センサ場以外で非特異吸着があっても当該非特異吸着が検出結果に影響しなかったことに対し、本参考例ではSPR分析の原理上、センサ場だけでなくセンサ場以外における非特異吸着をも検出したことによる。このように、本発明によると特異的に認識された対象のみを検出することができる。
実施例2と同じ方法によって検出対象の分析用センサを3枚作成した。作成した3枚の検出対象の分析用センサに対し、実施例3と同じ方法によって、濃度が異なるエキソソーム溶液を滴下して反応させ、反応後の蛍光強度の測定を行った。蛍光強度の測定は、分析用センサそれぞれにおいて3点ずつ、合計9点について行った。9点の測定点における蛍光強度変化を図15に示す。図15中、横軸はエクソソーム濃度(ng/ml)、縦軸は蛍光強度変化((I-I0)/I0)を示す。図15に示されるように、分析用センサ間の測定誤差は小さく、センサを再現性よく作成できたことが示された。
1.エクソソームのローダミン標識
(1)チオール化BAM(BAM-SH)の合成
BAM (SUNBRIGHT OE-020CS, Mw=2000) 10 mg (5.6 μmol)及び2-aminoethanethiol hydrochloride 32 mg (280μmol, 50 eq)をPBS(pH 7.0) 50μLに溶解させ、25℃で一晩反応させた。
(2)チオール化BAMによるエクソソーム表面のチオール化
得られたチオール化BAM溶液を10 mM PBS (pH 7.4)で100倍希釈した溶液400μLとエクソソーム溶液2μg/100 μLとを混合し、4℃で3時間反応させた。
(3)チオール反応性ローダミンによるエクソソームのローダミン標識
得られた反応液について限外ろ過(Amicon Ultra 0.5 ; 100 kDa)を三回行い、回収液27μLに1 mMのチオール反応性ローダミンマレイミド溶液(10 mM PBS pH 7.4)を450μL加え、暗所、4℃で3時間反応させた。
(4)透析によるローダミン標識エクソソームの精製
孔径10kDaの透析膜を用いて4℃、24時間透析を行い、ローダミン標識エクソソームを精製した。
500μM POLARIC-MLI(五稜化薬)DMSO溶液100μLを滴下して25℃で1時間静置した代わりに100μMのチオール反応性フルオレセインマレイミド溶液を滴下して室温で3時間静置したことを除き、実施例2と同様にして検出対象の分析用センサを作製した。
3.フルオレセイン標識センサを用いたFRETによるローダミン標識エクソソームの測定
0, 5, 10, 50, 100, 500 ng/mLのエクソソーム溶液をセンサ上に滴下し、3分反応させ、純水及びPBSで十分に洗浄した。洗浄後、PBSを滴下し、カバーガラスを装着して、以下の測定条件で蛍光強度の測定を行った。
測定条件:フィルタ
励起:Fluiorescein用フィルタ
蛍光:Rhodamine用フィルタ
対物レンズ:40倍
露光時間: 0.1 sec;
本実施例では、エクソソームが分析用センサの凹部に抗体を介して結合することを確かめるため、エクソソームの滴下時にフリー(遊離)の抗体を共存させることによるエクソソームの結合挙動の変化を調べた。
1.鋳型の合成-チオール基およびヒスチジンタグ(His-tag)を導入したシリカナノ粒子の合成
FITC標識シリカナノ粒子200μl(200μlあたり表面に-COOH 5 nmolを有するもの、粒子径200nm)をジクロロメタン(DCM)に分散させた(シリカナノ粒子分散液)。エチル(ジメチルアミノプロピル) カルボジイミド(EDC: 50 nmol, 10 eq)、N-ヒドロキシコハク酸イミド(NHS: 50 nmol, 10 eq)、N,N-ジイソプロピルエチルアミン(DIEA: 50 nmol, 10 eq) をdry DCMに溶解させ、シリカナノ粒子分散液と混合した。一晩反応させることでシリカナノ粒子表面をNHSで修飾した。表面修飾したシリカナノ粒子に対して、ヒスチジンが6個ペプチド結合で連結されたHis-tag (末端はリジン残基でありフリーのεアミノ基を有する:0.10μmol, 40eq)及び2-アミノエタンチオール塩酸塩(0.1μmol, 40eq)を加えて、室温で反応させた。反応終了後、遠心分離およびろ過によりチオール基およびHis-tagを導入したシリカナノ粒子(SH・His-tag化シリカナノ粒子)を精製した。
実施例1の項目1で得られたmixed SAMsに、20 mM Isothiocyanobenzyl-NTA(同仁化学研究所)DMSO溶液80μLを基板に滴下し、25℃で2時間静置した。基板をDMSO及び純水で洗浄後、4 mM NiCl2水溶液100μLを基板に滴下し、室温で15分間静置した。基板を純水で洗浄後、10 mM posphate,140 mM NaCl, pH7.4に分散したSH・His-tag化シリカナノ粒子を滴下することにより、SH・His-tag化シリカナノ粒子が固定化された基板を得た。また、比較用に、His-tag修飾を行わなかったシリカナノ粒子の分散液も同様に滴下した基板も得た。これらの基板に対し、シリカナノ粒子の滴下前と滴下1時間後における、シリカナノ粒子に導入されている蛍光分子フルオレセイン(励起480 nm/ 蛍光 510 nm)由来の蛍光強度を測定することにより、シリカナノ粒子の固定化を確認した。また、結合したシリカナノ粒子が脱離するかどうか確かめるため、酸性緩衝液(0.5 wt% SDS 50 mM 酢酸buffer (pH4.0))で処理し、その後、再び基板表面の蛍光強度を測定した。
以下のようにして、金薄膜蒸着ガラス基板上にアミノ基及びブロモ基が末端となる混成自己組織化単分子膜(mixed SAMs)を作製し(分子膜形成工程)、アミノ基末端へNTA基を導入し、NTA-Ni錯体を形成した後、キレート結合によりシリカナノ粒子を固定化した(鋳型導入工程)。その後、シリカナノ粒子にメタクリル基修飾を施し(表面修飾工程)、表面開始制御/リビングラジカル重合によりポリマー薄膜を合成した(重合工程)。最後にシリカナノ粒子を除去し(除去工程)、検出対象の分析用センサ作製用基材を得た。
実施例1の項目1と同様に金薄膜蒸着ガラス基板の有機残滓の除去及び洗浄を行い、0.5 mM amino-EG6-undecanthiol hydrochloride及び0.5mM 2-(2-bromoisobutyryloxy) undecyl thiolのエタノール溶液に基板を浸漬させ、25℃で24時間静置することにより、ブロモ基およびアミノ基をもつ混合自己組織化単分子膜を形成させた。
5 mM isothiocyanobenzyl-nitrilotriacetic acid (ITC-NTA)のDMSO溶液80 μLを基板に滴下し、25℃で2時間静置してアミノ基をNTAで修飾した。基板をDMSO及び純水で洗浄後、4 mM NiCl2水溶液100 μLを基板に滴下し、室温で15分間静置することで、Ni-NTA錯体を形成した。その後、SH・His-tag化シリカナノ粒子(固形分濃度5.1 mg/ml)含有水溶液100 μlを基板に滴下し、25°Cで1時間静置した。
100 μM 2-(2-Pyridyl)dithioethyl methacrylateの PBS (pH 7.4)溶液に基板を浸して一晩静置させることで、ジスルフィド交換反応によってシリカナノ粒子表面のSH基にジスルフィドを介してメタクリロイル基の導入を行った。
重合時間を3時間としたことを除いて、実施例1の項目4と同様にして、基板上にポリマー薄膜を合成した。これによって、基板上に、表1のモノマーと共にシリカナノ粒子のメタクリロイル基も共重合されたポリマー薄膜を得た。重合終了後、1 M エチレンジアミン四酢酸-4Na 水溶液に基板を15分間浸し、ATRPに用いたCu2+を取り除いた。
50 mM トリス(2-カルボキシエチル)フォスフィン・HCl(TCEP)水溶液に25℃で3時間浸漬させ、ポリマーとシリカナノ粒子とを結合させているジスルフィド結合を還元・切断した。ポリマー側にはフリーのSH基が残るが、このSH基は、SH・His-tag化シリカナノ粒子由来であることから、ポリマー薄膜のうち鋳型に対応した凹部以外の部分には存在せず、鋳型に対応した凹部内のみに存在する。前述のEDTA-4Na処理で、NI-NTAのニッケルも除去されてHis-tagがこの時点でフリーとなる可能性が高いが、念のため以下の操作も行った。純水で洗浄後、0.5 wt% SDSを含む50 mM酢酸バッファー (pH 4.0)に基板を浸して、Ni-NTA及びHis-tagを介して結合していたシリカナノ粒子をポリマー薄膜から洗い出した。
実施例8で得られた分析用センサ作製用基材(MIP基板)に再度Ni-NTAを形成させるため、4 mM NiCl2水溶液でMIP基板を処理した。その後、PBSに溶解した100 μM His-tag Protein Gを基板に添加することで、His-tagを介して抗体を結合可能なProtein Gを固定化した。最後に、PBSに溶解した0.3 μM 抗CD9抗体を基板に添加し、抗CD9抗体をProtein Gを介して固定化した。Protein Gは、抗体のFc領域に結合することから、固定化された抗体の配向は一様となる。
実施例9で得られた分析用センサ(抗体あり)を用い、エクソソームの結合挙動を観察した。また、比較用として、当該分析用センサの抗CD9抗体が固定されていない基板(抗体なし)についても同様にエクソソームの結合挙動を観察した。試料としては、エクソソームのPBS (10 mM posphate,140 mM NaCl, pH7.4)溶液(濃度はそれぞれ、0.01, 0.05, 0.1, 0.5, 1, 5, 10 ng/mL)を用い、エクソソームの蛍光検出は顕微鏡下で行った。蛍光顕微鏡測定条件は以下の通りである。
フィルタ:Cy5
対物レンズ:×5
露光時間:0.1秒
光源:水銀ランプ
蛍光分子として(下記式に示すP=O-Rhodolを用いたことを除いて、実施例9と同様にして分析用センサを作製した。得られた分析用センサを、誘導放出制御(STED)顕微鏡(Leica社製SP8-STED3X)を用いて観察した。得られたSTED超解像イメージ像を図19に示す。図19に示すように、約200 nmサイズのドット状の蛍光が観察され、当該蛍光のサイズは、鋳型に用いたシリカナノ粒子と同等であり、鋳型効果により形成された凹部であることが確認できた。また、当該凹部以外にバックグラウンド蛍光がほとんど観察されなかったことから、蛍光レポーター分子が当該凹部に選択的に導入されたことも示された。
シルマー試験紙を用いて涙液を採取した後、試験紙を、500 μlの140 mMNaClを含む10 mM リン酸バッファー(PBS)に4℃,6時間浸漬し、涙に含まれるエクソソームを涙液試料として回収した。涙液試料を、実施例9の分析用センサを用いた蛍光測定に供することで、涙液中のエクソソームを測定した。比較用に、コントロールとして、実施例9の検出対象の分析用センサを用いたPBSの測定、及び、分析用センサの抗体への涙液エクソソームの結合性を確認するため、実施例9の分析用センサの抗体が固定されていない基板を用いた涙液試料の測定も行った。
20…基材
21…分子膜
22…抗体物質結合用基(可逆的結合性基)
22a…結合性官能基
22b…第1の可逆的連結基
22c…抗体物質結合性基
23a…重合開始性基
24…抗体物質
25…抗体物質結合用基と結合することで第1の可逆的連結基の形成が可能な基
30…ポリマー膜
31…凹部
32…シグナル物質結合用基(可逆的結合性基)
32b…第2の可逆的連結基
33…重合性官能基
35…重合性モノマー
40…鋳型
41…アンカー物質
42…可逆的結合性基(シグナル物質結合用基と結合することで第2の可逆的連結基の形成が可能な可逆的結合基)
43…アンカー基
50…分析用センサ
52…検出対象に特異的な抗体物質
53…シグナル物質
60…検出対象
Claims (15)
- 基材と、前記基材の表面上に設けられたポリマー膜と、を含み、
前記ポリマー膜が、検出対象を受け入れる凹部を有し、
前記凹部内に、抗体物質結合用基とシグナル物質結合用基とを有する、検出対象の分析用センサ作製用基材。 - 前記ポリマー膜が、前記検出対象又は前記検出対象よりサイズが大きい対象を鋳型とする分子インプリントポリマーで構成され、前記凹部が前記鋳型の表面形状の一部に対応する、請求項1に記載の検出対象の分析用センサ作製用基材。
- 前記抗体物質結合用基がキレート結合性基である、請求項1又は2に記載の検出対象の分析用センサ作製用基材。
- 前記シグナル物質結合用基がチオール基である、請求項1~3のいずれか1項に記載の検出対象の分析用センサ作製用基材。
- 請求項1から4のいずれか1項に記載の検出対象の分析用センサ作製用基材と、
前記抗体物質結合用基に結合した、前記検出対象に特異的な抗体物質と、
前記シグナル物質結合用基に結合したシグナル物質と、
を含む、検出対象の分析用センサ。 - 前記検出対象が膜構造を有する微小粒体である、請求項5に記載の検出対象の分析用センサ。
- 前記膜構造を有する微小粒体がエクソソームである、請求項6に記載の検出対象の分析用センサ。
- 前記検出対象に特異的な抗体物質が、前記膜構造を有する微小粒体の表面に発現している特異的抗原に対する特異的結合能を有する、請求項5から7のいずれか1項に記載の検出対象の分析用センサ。
- 請求項5から8のいずれか1項に記載の検出対象の分析用センサに、検出対象を含む試料を接触させ、前記抗体物質に前記検出対象を結合する工程と、
前記シグナル物質に由来するシグナルの変化を検出する工程と、
を含む、検出対象の分析法。 - 基材に、結合性官能基と重合開始性基とを表面に有する分子膜を形成する分子膜形成工程と、
前記結合性官能基に対し、第1の可逆的連結基を介して、人工粒子を鋳型として導入する鋳型導入工程と、
前記鋳型の表面を、第2の可逆的連結基を介して重合性官能基で修飾する表面修飾工程と、
重合性モノマーを加え、前記重合性モノマーを基質とし、前記重合開始性基を重合性開始剤として、前記鋳型の前記表面の一部に対する分子インプリントポリマーを合成することで前記基材表面にポリマー膜を形成する重合工程と、
前記第1の可逆的連結基及び第2の可逆的連結基を開裂させてそれぞれ抗体物質結合用基及びシグナル物質結合用基へ変換するとともに前記鋳型を除去する除去工程と、
を含む、検出対象の分析用センサ作製用基材の製造方法。 - 前記人工粒子が、表面に、前記抗体物質結合用基と結合することで前記第1の可逆的連結基の形成が可能な基と、前記シグナル物質結合用基と結合することで前記第2の可逆的連結基の形成が可能な可逆的結合基とを有する、請求項10に記載の検出対象の分析用センサ作製用基材の製造方法。
- 前記人工粒子がシリカ粒子である、請求項10又は11に記載の検出対象の分析用センサ作製用基材の製造方法。
- 前記抗体物質結合用基がキレート結合性基であり、前記抗体物質結合用基と結合することで前記第1の可逆的連結基の形成が可能な基がヒスチジンタグである、請求項10から12のいずれか1項に記載の検出対象の分析用センサ作製用基材の製造方法。
- 前記シグナル物質結合用基がチオール基であり、前記シグナル物質結合用基と結合することで前記第2の可逆的連結基の形成が可能な可逆的結合基がチオール基である、請求項10から13のいずれか1項に記載の検出対象の分析用センサ作製用基材の製造方法。
- 請求項10から14のいずれか1項に記載の検出対象の分析用センサ作製用基材の製造方法を行う工程と、
前記抗体物質結合用基に、検出対象に特異的な抗体物質を結合する工程と、
前記シグナル物質結合用基にシグナル物質を結合する工程と、
を含む、検出対象の分析用センサの製造方法。
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Families Citing this family (1)
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013094307A1 (ja) | 2011-12-22 | 2013-06-27 | Ochiya Takahiro | エクソソームの分析方法、エクソソーム分析用試薬およびエクソソーム分析装置 |
JP2014219384A (ja) | 2013-04-09 | 2014-11-20 | 株式会社Jvcケンウッド | 試料分析用デバイス及びエクソソームの捕捉方法 |
WO2015029979A1 (ja) | 2013-08-30 | 2015-03-05 | 国立大学法人東京大学 | エキソソームの分析方法、エキソソーム分析チップ、及びエキソソーム分析装置 |
WO2015045666A1 (ja) | 2013-09-25 | 2015-04-02 | 国立大学法人東京大学 | 流体デバイス、エキソソームの分析方法、生体分子分析方法及び生体分子検出方法 |
US20160334398A1 (en) | 2013-12-02 | 2016-11-17 | The General Hospital Corporation | Nano-plasmonic sensor for exosome detection |
JP2016197041A (ja) * | 2015-04-03 | 2016-11-24 | 国立大学法人神戸大学 | 分子インプリンティング膜、その製造方法、鋳型化合物、およびステロイドホルモン化合物の検出方法 |
JP2017019992A (ja) * | 2015-07-10 | 2017-01-26 | 国立大学法人神戸大学 | 分子インプリントポリマーの製造方法、分子インプリントポリマー、および標的タンパク質の検出方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4804361B2 (ja) * | 2003-12-08 | 2011-11-02 | ザ リサーチ ファウンデイション オブ ステイト ユニバーシティー オブ ニューヨーク | センサー利用のための、部位選択的にタグ付および鋳造された分子インプリントポリマー |
-
2018
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Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013094307A1 (ja) | 2011-12-22 | 2013-06-27 | Ochiya Takahiro | エクソソームの分析方法、エクソソーム分析用試薬およびエクソソーム分析装置 |
JP2014219384A (ja) | 2013-04-09 | 2014-11-20 | 株式会社Jvcケンウッド | 試料分析用デバイス及びエクソソームの捕捉方法 |
WO2015029979A1 (ja) | 2013-08-30 | 2015-03-05 | 国立大学法人東京大学 | エキソソームの分析方法、エキソソーム分析チップ、及びエキソソーム分析装置 |
WO2015045666A1 (ja) | 2013-09-25 | 2015-04-02 | 国立大学法人東京大学 | 流体デバイス、エキソソームの分析方法、生体分子分析方法及び生体分子検出方法 |
US20160334398A1 (en) | 2013-12-02 | 2016-11-17 | The General Hospital Corporation | Nano-plasmonic sensor for exosome detection |
JP2016197041A (ja) * | 2015-04-03 | 2016-11-24 | 国立大学法人神戸大学 | 分子インプリンティング膜、その製造方法、鋳型化合物、およびステロイドホルモン化合物の検出方法 |
JP2017019992A (ja) * | 2015-07-10 | 2017-01-26 | 国立大学法人神戸大学 | 分子インプリントポリマーの製造方法、分子インプリントポリマー、および標的タンパク質の検出方法 |
Non-Patent Citations (2)
Title |
---|
ANAL. CHEM., vol. 86, 2014, pages 5929 - 5936 |
See also references of EP3647786A4 |
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US20200110084A1 (en) | 2020-04-09 |
EP3647786B1 (en) | 2023-03-08 |
EP3647786A1 (en) | 2020-05-06 |
JP6573351B2 (ja) | 2019-09-11 |
JPWO2018221271A1 (ja) | 2019-06-27 |
EP3647786A4 (en) | 2021-03-10 |
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