WO2008013260A1 - Spécimen biologique et procédé de préparation de celui-ci - Google Patents

Spécimen biologique et procédé de préparation de celui-ci Download PDF

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WO2008013260A1
WO2008013260A1 PCT/JP2007/064755 JP2007064755W WO2008013260A1 WO 2008013260 A1 WO2008013260 A1 WO 2008013260A1 JP 2007064755 W JP2007064755 W JP 2007064755W WO 2008013260 A1 WO2008013260 A1 WO 2008013260A1
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group
biological specimen
aromatic hydrocarbon
alkyl
atom
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PCT/JP2007/064755
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English (en)
Japanese (ja)
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Shinichiro Isobe
Keiichiro Nakamura
Takaaki Kanemaru
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Shinichiro Isobe
Keiichiro Nakamura
Takaaki Kanemaru
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Application filed by Shinichiro Isobe, Keiichiro Nakamura, Takaaki Kanemaru filed Critical Shinichiro Isobe
Priority to JP2008526829A priority Critical patent/JP5415760B2/ja
Publication of WO2008013260A1 publication Critical patent/WO2008013260A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples

Definitions

  • the present invention relates to a biological specimen that is composed of a tissue or a cell of a living body and is used for microscopic observation for pathological diagnosis, a manufacturing method thereof, and an observation method thereof.
  • a biological specimen for observation with an optical microscope is usually prepared by a procedure for collecting, fixing, dehydrating, replacing, embedding, slicing, and staining a tissue or cell from a specimen. Fixing is performed for the purpose of maintaining the structure and physical properties of tissues or cells as they are, and formaldehyde, dartal aldehyde, or the like is used as a fixing agent. In dehydration, alcohol or acetone is used as a dehydrating agent, and the water in the tissue is replaced with the dehydrating agent.
  • the tissue is replaced with a replacement agent having a high affinity for the dehydrating agent prior to embedding.
  • Embedding is performed by allowing an embedding agent such as paraffin to penetrate into the tissue and then curing the embedding agent.
  • the embedded tissue is sliced to about 2 to 10 m by a microtome, and the obtained thin section is fixed on a glass slide, and after removing the embedding agent as necessary, it is counterstained. Then, it is sealed with a cover glass using an encapsulant to make a biological specimen.
  • Pathological diagnosis is performed not only by morphological observation of a biological specimen, but also by an analysis method for visualizing a target substance by an immunohistochemical staining method or an in-situ hybridization method (for example, Patent Document 1).
  • the immunohistochemical staining method is a method in which a specific antigen on a tissue is detected by an antibody that specifically recognizes the antigen. The antibody that recognizes the specific antigen is reacted with the tissue, and the presence or absence of the reacted antibody is detected. In this method, the presence of an antigen is judged. An antibody that reacts with a tissue is labeled with a fluorescent dye, and the distribution of the antigen on the tissue is analyzed.
  • the in-situ hybridization method uses oligonucleotide probes and D that hybridize with the target gene. This is a method for determining whether a target gene is present in a tissue or cell using an NA probe. A fluorescent dye is also used for the probe.
  • Patent Document 1 Japanese Unexamined Patent Publication No. 2003-130866
  • multi-labeling using a plurality of fluorescent dyes having different fluorescence wavelengths facilitates the identification of cells and tissues by multi-color imaging, as well as information on interactions between cells or tissues.
  • Interest is growing because of this.
  • a plurality of excitation lights for example, laser lights
  • there are technical difficulties such as a focal point shift due to a difference in laser wavelengths. Therefore, with regard to the excitation light source, a plurality of excitation light sources are used to sequentially excite a plurality of fluorescent dyes, or even when a single excitation light source is used, a plurality of excitation filters are used to adjust a plurality of excitation wavelengths. Multiple fluorescent dyes are sequentially excited.
  • the present invention is a permanent specimen in which fluorescence from a fluorescent dye does not disappear even when stored for a long period of time, a biological specimen that can be produced at a lower cost, a method for producing the same, and a multi-label
  • An object of the present invention is to provide a simpler observation method of a biological specimen.
  • the biological specimen of the present invention is a biological specimen in which a tissue or cells labeled with a fluorescent dye is fixed on a support substrate, and the fluorescent dye is at least It has a coloring part composed of an organic EL dye, and the organic EL dye is a azole derivative or imidazole derivative having a conjugated system and containing one or more hetero atoms, selenium atoms, or boron atoms.
  • the organic EL dye is a azole derivative or imidazole derivative having a conjugated system and containing one or more hetero atoms, selenium atoms, or boron atoms.
  • the biological specimen of the present invention preferably includes one in which a tissue or a cell is embedded with an embedding agent and fixed on the supporting substrate.
  • one kind of embedding resin selected from the group consisting of epoxy resin, polyester resin and methacrylic resin can be used as the embedding agent.
  • the fluorescent dye includes the azole derivative, the following general formula (1), (2) or (3)
  • a compound represented by V, shear force, or one type can be used.
  • R, R, R, and R each independently represent a hydrogen atom, a halogen atom, or a substituent.
  • X represents an aromatic hydrocarbon group, a hydrocarbon group, a heterocyclic group or an aromatic group containing a hetero atom in the ring, and X represents a substituent having a substituent, and may be a nitrogen atom.
  • R ′ is an aliphatic hydrocarbon group or an aromatic hydrocarbon group comprising an alkyl group or an alkenyl group which may contain an aromatic ring
  • An— is a halogen atom. Shows fluoride ion, CF SO-, BF- or PF-.
  • an aryl group having a sulfonyl group can be used for R and R described above.
  • imidazole derivative a compound represented by the following general formula (4), (5), (6), (7) or (8) can also be used.
  • R, R, R, R, and R are each independently a hydrogen atom, a halogen atom, or a substituent.
  • An— represents a halide ion, CF 2 SO—, BF— or PF—.
  • R and R may be thiophene derivatives, furan derivatives, pyrrole derivatives, imidazo.
  • Derivatives oxazole derivatives, thiazole derivatives, pyrazole derivatives and pyr
  • gin derivatives One selected from the group consisting of gin derivatives can be used.
  • an aryl group having a sulfonyl group may be used for R and R described above.
  • R, R, and R each independently represent a hydrogen atom, a halogen atom, or a substituent.
  • Alkyl group alkenyl group, alkynyl group, alkoxy group, alkyl ester group, phosphate ester group, sulfate ester group, nitrile group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group or heterocyclic ring It may have a group! Represents an aromatic hydrocarbon group, an aliphatic hydrocarbon group or a heterocyclic group, and R, R and R may be the same or different.
  • the fluorescent dye used for the biological specimen of the present invention includes a carboxylic acid group, an isocyanate group, an isothiocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carpositimide group, and an active ester at the bonding portion. Any one kind of reactive group selected from the activated carbonyl group can be used.
  • the fluorescent dye used in the biological specimen of the present invention preferably has a part of a spacer that connects the coloring portion and the binding portion.
  • An aliphatic hydrocarbon group such as a hydrogen atom or an alkyl group or an alkenyl group which may contain an aromatic ring, or an aromatic hydrocarbon group, which optionally comprises a sulfonyl group, a hydroxyl group, a quaternary amine group and a carboxyl group
  • Ar represents an aryl group
  • p and q each independently represents an integer from
  • a peptide linker consisting of an amino acid or 2 to 20 amino acids as a part of the spacer is determined by the force S.
  • the force S is used.
  • amino acid was selected from the group force consisting of cysteic acid, 2-amino-3-sulfosulfanylpropanoic acid, 2-amino-3-sulfoxypropanoic acid, tyrosine, threonine, and serine 1 Seeds can be used.
  • a peptide linker is used as a part of the spacer, and at least one selected from the group consisting of a sulfonyl group, a hydroxyl group, a quaternary amine group and a carboxyl group is used as the peptide linker. Those having a charged group of can also be used.
  • the peptide linker includes cysteic acid, 2-amino-3-sulfosulfanylpropanoic acid.
  • azole derivative a compound represented by any one of the following general formulas (9), (10) and (11) can be used.
  • R is, and in (10), one of R and R is the general formula LM
  • M is an optionally substituted pyridinium group, secondary amino group, tertiary amino group, quaternary amino group, piperidinium group, piperazium group, imidazolium group, thiazolium group, oxazolium
  • a nitrogen cation-containing group which is a group, a quinolium group, a benzimidazolium group, a benzothiazolium group or a benzoxazolium group, L is a direct bond or-(CH) n- (n is ;!
  • alkyl group alkoxy group, alkyl ester group, phosphate ester group, sulfate ester group, nitrile group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group or heterocyclic group.
  • An aromatic hydrocarbon group an aliphatic hydrocarbon group or a heterocyclic group which may be substituted
  • X is an optionally substituted carbon atom, nitrogen atom, sulfur atom, oxygen atom, selenium atom or Represents a boron atom
  • R ′ may contain an aromatic ring, an aliphatic hydrocarbon group comprising an alkyl group or an aromatic hydrocarbon group, An— It represents fluoride ion, CF 2 SO—, BF— or PF—.
  • each of R and R above is independently selected from the group consisting of thiophene derivatives, furan derivatives, pyrrolinole derivatives, imidazole derivatives, oxazole derivatives, thiazole derivatives, pyrazole derivatives, pyridine derivatives, and quinoline derivatives. Use the selected one.
  • an aryl group having a sulfonyl group may be used for R and R described above.
  • imidazole derivative includes the following general formula (12), (13), (14), (15) or (1
  • Any one of 1 4 1 4 is represented by the general formula L-M, and M is a pyridinium which may have a substituent.
  • R, R and R in (16), and R and R are each independently a hydrogen atom, halo
  • alkyl group alkoxy group, alkyl ester group, phosphate ester group, sulfate ester group, nitrile group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group or heterocyclic group.
  • An— is a halide ion, CF SO—, B
  • each of R and R is independently selected from thiophene derivatives, furan derivatives,
  • One kind selected from the group consisting of a nore derivative, an imidazole derivative, an oxazole derivative, a thiazole derivative, a pyrazonole derivative, a pyridine derivative and a quinoline derivative is used.
  • an aryl group having a sulfonyl group may be used for R and R described above.
  • the biological specimen of the present invention can be prepared, for example, by the following method. That is, the method for preparing a biological specimen of the present invention is a method for preparing a biological specimen in which a tissue or cell labeled with a fluorescent dye is fixed on a support substrate, and wraps the tissue or cell collected from the specimen. Embedding with an embedding material to form an embedding body, slicing the embedding body into a section, fixing the section on a supporting substrate, and separating the section from a coloring portion composed of an organic EL dye, a living body
  • the organic EL color has a binding part for binding molecules, and a spacer part for connecting the coloring part and the binding part.
  • the element has a conjugated system and is labeled with a fluorescent dye which is an azole derivative or an imidazole derivative containing one or more hetero atoms, selenium atoms, or boron atoms.
  • the embedding agent can also be removed prior to labeling the section with a fluorescent dye.
  • ether alcohols or gnoresidyl ethers can be used as a dehydrating solvent.
  • the biological specimen of the present invention can be observed, for example, by the following method.
  • the fluorescent dye has a coloring portion composed of at least an organic EL dye, and the organic EL dye has a conjugated system and includes one or more heteroatoms, selenium atoms, or boron atoms.
  • a fluorescent dye that is an imidazole derivative is used to simultaneously excite the fluorescent dye by irradiating it with one type of excitation light, and the generated fluorescence is transmitted through only one type of filter that absorbs the excitation light, and then a biological specimen. It is characterized by observing multiple fluorescence from
  • the microscope observation used for the observation of the biological specimen of the present invention means observation performed using a microscope capable of fluorescence observation, specifically, a fluorescence microscope, a laser scanning microscope, a confocal laser. Observations using a microscope or the like are included.
  • the present invention can provide a permanent specimen in which the fluorescence from the fluorescent dye does not disappear even when stored for a long period of time. Since it can be observed any number of times during the storage period, a highly reliable pathological diagnosis can be performed. For example, a biological specimen using a conventional fluorescent dye fades in about one week, whereas the biological specimen of the present invention can be stored semipermanently as long as it can be stored refrigerated. In addition, since the biological specimen of the present invention uses a fluorescent dye containing an organic EL dye that is less expensive than Cy3 or Cy5 as the fluorescent dye, the biological specimen can be produced at a lower price.
  • the biological specimen of the present invention includes an embodiment in which a tissue or a cell is embedded with an embedding agent.
  • a resin is used as the embedding agent, the fluorescence of the fluorescent dye does not occur due to the autofluorescence of the resin. There was a problem that it was not clear or could not be confirmed.
  • the biological specimen of the present invention has the effect of clearly confirming the fluorescence of the fluorescent dye that is not disturbed by the autofluorescence of the embedded resin, so that more reliable pathological diagnosis is possible. It becomes.
  • a multiple-labeled biological specimen is irradiated with one type of excitation light and simultaneously excited, and only one type of filter that absorbs the generated fluorescence is absorbed.
  • a plurality of fluorescent lights can be observed simultaneously.
  • observation in a short time becomes possible, and it is not necessary to provide a plurality of filters for separating a plurality of excitation light sources and a plurality of fluorescences, so that the microscope apparatus used for observation should have a simpler configuration. This makes it possible to reduce the cost of the microscope apparatus.
  • FIG. 1 Fluorescent stereomicrographs of mouse livers in Example 1. The upper row shows an example of labeling using GFP, and the lower row shows an example of labeling with the fluorescent dye used in the present invention.
  • FIG. 2 is a fluorescence micrograph of mouse lymph nodes in Example 2.
  • FIG. 3 is a fluorescent stereomicrograph showing the effect of autofluorescence of the embedding resin in Example 3.
  • the upper row is an example using a conventional fluorescent dye, and the lower row is using the fluorescent dye used in the present invention. It is an example.
  • FIG. 4 is a fluorescence micrograph showing the results of Example 4.
  • FIG. 5 is a fluorescence micrograph showing the results of Example 5.
  • FIG. 6 is another fluorescence micrograph showing the results of Example 5.
  • the present invention is a biological specimen in which tissues or cells labeled with a fluorescent dye are fixed on a support substrate, wherein the fluorescent dye has a color-developing portion composed of at least an organic EL dye, and the organic EL dye Is a azole derivative or imidazole derivative having a conjugated system and containing one or more hetero atoms, selenium atoms, or polone atoms.
  • the tissue or cells targeted by the biological specimen of the present invention are collected from mammals including humans. And biological samples collected from experimental animals can be used.
  • tissue include tissues such as brain, lung, stomach, liver, kidney, bladder, spleen, small intestine and large intestine, skin tissue, nerve tissue, blood vessel tissue, muscle tissue and cartilage tissue.
  • cells include cells (eg, brain cells, hepatocytes, epithelial cells, endothelium cells, nerve cells, muscle cells, chondrocytes, etc.), blood cells, cells in body fluids. Use the power S to lift up.
  • the fluorescent dye used in the biological specimen of the present invention can be used for staining including immunohistochemical staining, and is used by binding to a specific biomolecule or by incorporation into a tissue or cell.
  • the biomolecule to which the fluorescent dye binds means the molecular species that exists in tissues and cells, and is used to build the structure of the living body, to participate in energy production and conversion, and to connect biological information. Power and things are included. Specific examples include nucleic acids, proteins, sugars, lipids, peptides, nucleotides, metabolic intermediates and metabolic enzyme systems, hormones, and neurotransmitters.
  • the organic EL dye constituting the fluorescent dye is sandwiched in a solid state between a pair of anode and cathode, and when holes injected from the anode and electrons injected from the cathode are recombined.
  • the dye is not particularly limited as long as it can emit light with the energy of
  • polycyclic aromatic compounds such as tetraphenylbutadiene and perylene, cyclopentagen derivatives, distyrylvirazine derivatives, attaridone derivatives, quinacdrine derivatives, stilbene derivatives, phenothiazine derivatives, birazinopyridine derivatives, azole derivatives, imidazole derivatives, force A rubazole derivative, a tetraphenylthiophene derivative, or the like can be used.
  • it is preferably a dye having a carboxylic acid group in the molecule or capable of introducing a carboxylic acid group. This is because, as described below, a reactive group for binding to a biomolecule can be easily introduced.
  • the fluorescent dye has a reactive group that binds to a biomolecule (hereinafter referred to as a target molecule) as a binding portion.
  • a target molecule a biomolecule
  • the reactive group is the amino group of the target molecule
  • a functional group capable of reacting with a mino group, a thiol group or a hydroxyl group is preferred.
  • the covalent bond between the organic EL dye and the target molecule it is preferable to form an amide bond, an imide bond, a urethane bond, an ester bond, or a guanidine bond.
  • the functional group includes, for example, an isothiocyanate group, an isocyanate group, an epoxy group, a halogenated sulfonyl group, a chlorinated acylol group, a halogenated alkyl group, a darioxal group, an aldehyde group, a triazine group, a strong positive imide group, and a maleimide group.
  • An active esterified carbonyl group or the like can be used. It is preferable to use one selected from an isothiocyanate group, an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carpositimide group, and an active esterified carbonyl group.
  • an isothiocyanate group it is preferable to use any one selected from an isothiocyanate group, an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carboimide group, and an active esterified carbonyl group.
  • an amide bond can be formed with the amino group of the target molecule, and it can be directly bonded to the imino group in the target molecule.
  • More preferred are a triazine group, a carpositimide group or an active esterified carbonyl group.
  • organic EL dyes When these organic EL dyes have a carboxylic acid group, it is also possible to directly modify the amino group and imino group present in the target molecule in the presence of a carpositimide derivative or triazine derivative.
  • organic EL dyes that have a triazine group that may have a substituent and a calpositimide group that may have a substituent react directly with the guanine and thymine imino groups in the DNA base. Can be used as a reagent for SNP (single nucleotide polymorphism) analysis. Is possible.
  • an anionic group such as a sulfonyl group or a carboxyl group can be used as a reactive group that forms an ionic bond with a target molecule.
  • anionic groups are ionically bonded to a cationic group of a biomolecule, such as an amino group.
  • the amino acid is modified at the end of the oligo DNA; when the protein is a protein, the amino acid residue; when the peptide is a peptide, the amino group of the polypeptide; for example, polylysine Reactive groups can be linked to the amino residues of the derivatives, and in the case of saccharides, the amino groups in the polysaccharide derivative backbone.
  • the fluorescent dye is used by being incorporated into a tissue or a cell that is not bound to a biomolecule, the fluorescent dye does not need to have the above reactive group, but improves the solubility in water. Therefore, it is preferable to have a hydrophilic group.
  • an alkyl ester group As this hydrophilic group, an alkyl ester group, a nitrile group, a hydroxyl group, an amino group, a quaternary amino group, a phosphoric acid group, a cyano group or a sulfonyl group can be used.
  • the fluorescent dye may have a part of a spacer that connects the coloring portion and the coupling portion.
  • a part of the spacer is a component that links the coloring portion and the reactive group, and includes a covalent bond or an atomic chain.
  • Those containing one or more functional groups selected from the group consisting of _CH ⁇ CH 2 —, —C ⁇ C 2 —, —Ar—, and —CO—Ar—NR— can be used. That is, a part of the spacer may be composed of only one kind of functional group selected from the above group, or may contain two or more kinds of functional groups. It can also be configured to contain two or more selected functional groups.
  • R ′ and R ′′ are each independently a hydrogen atom, an aliphatic hydrocarbon group such as an alkyl group or an alkenyl group that may contain an aromatic ring, or an aromatic hydrocarbon group, and if necessary, Sulfonyl group, hydroxyl group, quaternary amine Any force selected from the group consisting of a group and a carboxyl group can be used, which can be substituted with one kind of charged group.
  • Ar is an aryl group, preferably a phenylene group or a naphthylene group, and if necessary, those substituted with a sulfonyl group can be used.
  • ⁇ and q are each independently an integer of 0 to 20, preferably an integer of 0 to 10, more preferably an integer of 0 to 5, and p + q ⁇ l.
  • spacers include:-(CH) P -CONH- (CH) q-, _ (CH) p-COO- (CH-,
  • R is a hydrogen atom, an aliphatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring, or an aromatic hydrocarbon group, and if necessary, a sulfonyl group, a hydroxyl group, Grade 4 Any force selected from the group consisting of a amine group and a carboxyl group can be used, and the force replaced with one charged group can be used.
  • Ar is an aryl group, preferably a phenylene group or a naphthylene group, and if necessary, those substituted with a sulfonyl group can be used.
  • r is 0 force, an integer of 20, an integer of 0 to 10, more preferably an integer of 0 to 5.
  • this spacer are: -CONH- (CH) r-COO_, -CONH -CH (-R _OH)-COO_, -CONH-CH (-R _COOH)-COO_, _CONH- CH (R -SO H)-
  • COO_, -COO- (CH) r-COO- and the like can be mentioned.
  • a peptide linker composed of an amino acid or 2 to 20 amino acids can be used as a part of the spacer.
  • Natural or synthetic amino acids can be used as amino acids.
  • natural amino acids include glycine, alanine, norine, leucine, isoleucine, serine, homoserine, threonine, aspartic acid, gnoletamic acid, asparagine, gnoletamine, lysine, hydroxylysine, arginine, cysteine, cysteic acid, 2- Amino-3-sulfosulfanyl propanoic acid, 2-amino-3-sulfoxypropanoic acid, cystine, methionine, phenylalanine, tyrosine, tryptophan, histidine, proline, 4-hydroxyproline, etc.
  • Synthetic amino acids include D-forms of the above natural amino acids and modified amino acids having at least an amino group and a carboxyl group in the molecule.
  • the modified amino acid has the general formula: H-N (R)-(R-
  • each of the hydrocarbon group, aromatic group or heterocyclic group may be substituted with at least one of a halogen atom, an anolenoquinole group, an alkenyl group, an alkynyl group or an alkoxy group.
  • An amino acid is an amino acid having a sulfonyl group, cysteic acid, 2-amino-3-sulfosulfanylpropanoic acid, 2-amino- It is any one selected from the group consisting of 3-sulfoxypropanoic acid and tyrosine, threonine, serine and homoserine having a hydroxyl group. Improve the water solubility of fluorescent dyes The power that can be S. More preferred is cysteic acid, serine or homoserine.
  • R, R, and R are hydrogen atoms, alkyl groups having 1 to 6 carbon atoms, alcohol groups, indole groups,
  • peptides may be homo or heteropeptides. Specific examples include Ala_Ser, Glu_Ala, Glu-Ala-Leu, Gly_Pro, Gly-Pro_Asn, Ile_Val, Ile-Val-Met, and the like.
  • a part of the peptide linker having at least one charged group selected from the group consisting of a sulfonyl group, a hydroxyl group, a quaternary amine group and a carboxyl group as necessary.
  • a peptide linker containing one or more amino acids having any one of these charged groups can be used. Thereby, the water solubility of the fluorescent dye can be improved.
  • a peptide linker comprising at least one amino acid prepared can be used.
  • the distance between the coloring part and the labeling part of the biomolecule is changed to suppress steric hindrance between the biomolecule and the fluorescent dye.
  • the structure of the fluorescent dye can be designed to suppress the steric hindrance in accordance with the three-dimensional structure of biomolecules such as proteins, peptides, and DNAs that have complex structures, so that the labeling rate can be improved. It becomes possible.
  • reaction shown in Scheme 1 When a reactive group is introduced into the fluorescent dye, for example, the reaction shown in Scheme 1 is used. Can do.
  • Reaction formula (I) shows an example in which an active esterified carbonyl group is used as a reactive group, and -coo- is used as a functional group of a part of the spacer bonded to the reactive group.
  • N-hydroxy-succinimide ester and maleimide ester can be used for the active esterified carbonyl group.
  • N-hydroxymonosuccinimide and DCC as the condensing agent, the organic EL dye and the target molecule are bonded by an amide bond via the N-hydroxy-succinimide ester.
  • Reaction formula (II) shows an example in which a triazine derivative is used for the active esterified carbonyl group and -COO- is used for the functional group of a part of the spacer bonded to the reactive group.
  • reaction formula ( ⁇ ⁇ ) shows an example in which a calpositimide group is used as a reactive group and —COO— is used as a functional group of a part of a spacer bonded to the reactive group.
  • a carpositimide reagent such as ⁇ , ⁇ ′-dicyclohexyl carpositimide (DCC) or 1-cyclohexyl-3_ (2-morpholinoethyl) carpositimide can be used.
  • the organic EL dye and the target molecule can be bound by an amide bond via a carpositimide body.
  • Reaction formula (IV) is an example in which a carposimide group or triazine group is introduced in advance to a part of the spacer, that is, an example in which a functional group of a part of the spacer bonded to the reactive group also serves as a reactive group. Is shown.
  • the fluorescent dye can be directly bonded to the amino group or imino group in the target molecule without separately introducing a reactive group into the fluorescent dye.
  • a preferred organic EL dye used for the fluorescent dye is a compound containing a 5-membered ring compound having a conjugated system, and the 5-membered ring compound is one or more heteroatoms, selenium atoms, or boron atoms.
  • the 5-membered ring compound is preferably an azole derivative or an imidazole derivative. Further, the azole derivative or imidazole derivative preferably has one or more quaternary ammonium groups. It is because water solubility can be improved. Below, the specific example of a condensed polycyclic compound is demonstrated.
  • R, R, R, R, and R are each independently a hydrogen atom, a halogen atom, a substituent,
  • Alkyl group alkenyl group, alkynyl group, alkoxy group, alkyl ester group, phosphate ester group, sulfate ester group, nitrile group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group, heterocyclic ring
  • An aromatic hydrocarbon group, a hydrocarbon group or a heterocyclic group which may have a group, and R, R, R, R and R may be the same or different.
  • the alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.
  • the alkenyl group is preferably a bur group, a allyl group, a crotyl group, a tiglyl group, or a prenyl group.
  • the alkynyl group is preferably an ethur group or an open pargyl group.
  • the alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group.
  • the alkyl ester group is a methyl ester group, an ethyl ester group, a propyl ester group, an isopropyl ester group, a butyl ester group or an isobutyl ester group. It is.
  • the aromatic hydrocarbon group includes a monocyclic ring or a polycyclic ring, and is preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenyl group.
  • the heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazolone group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group, an imidazole group or a thiophene group. It is a group.
  • the hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.
  • R ′ represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring.
  • alkyl group and the alkenyl group aromatic hydrocarbon group the same groups as described above can be used.
  • An— represents a halide ion such as Cl—, Br— or ⁇ , CF 2 SO—, BF— or PF—.
  • R and R each independently represent a hydrogen atom, a halogen atom, or a substituent.
  • Alkynole group alkenyl group, alkynyl group, alkoxy group, alkyl ester group, phosphate ester group, sulfate ester group, nitrile group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group, heterocyclic group
  • An aromatic hydrocarbon group, a hydrocarbon group or a heterocyclic group which may be present, and R and R may be the same or different.
  • the above alkyl group is
  • a linear or branched alkyl group having 1 to 6 carbon atoms is preferred.
  • the alkenyl group is preferably a bur group, a allyl group, a crotyl group, a tiglyl group, or a prenyl group.
  • the above alkynyl group is preferably an ethur group or a propargyl group.
  • the alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group.
  • the alkyl ester group is a methyl ester group, an ethyl ester group, a propyl ester group, an isopropyl ester group, a butyl ester group or an isobutyl ester group.
  • the aromatic hydrocarbon group includes a monocyclic ring or a polycyclic ring, and is preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenyl group.
  • the above-described bicyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group or an imidazole group.
  • the hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.
  • N is an integer of 1 or more, preferably;! To 5, and the same applies to the following general formulas.
  • R, R, R, and R are each independently a hydrogen atom, a halogen atom, or a substituent.
  • R, R, R, R, R, and R may be the same or different from each other.
  • R is an aromatic hydrocarbon group which may have a substituent, preferably a phenyl group.
  • alkyl group having 1 to 4 carbon atoms, an alkoxy group, or a bromine atom is preferable to use. Furthermore, it is preferable to use a methyl group for the alkyl group and a methoxy group for the alkoxy group.
  • X is a nitrogen atom, sulfur atom, oxygen atom, selenium atom or boron atom which may have a substituent, and the same applies to the following general formulas unless otherwise specified.
  • R and R are independently a hydrogen atom, a ha atom, or a substituent.
  • Alkyl group, alkenyl group, alkynyl group, alkoxy group, alkyl ester group, phosphate ester group, sulfate ester group, nitrile group, hydroxyl group, cyan group, sulfol group Represents an aromatic hydrocarbon group, a hydrocarbon group or a heterocyclic group which may have an aromatic hydrocarbon group or a heterocyclic group, and R 1 and R 2 may be the same or different.
  • the above alkyl group is
  • the alkenyl group is preferably a bur group, a allyl group, a crotyl group, a tiglyl group, or a prenyl group.
  • the above alkynyl group is preferably an ethur group or a propargyl group.
  • the alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group.
  • the alkyl ester group is a methyl ester group, an ethyl ester group, a propyl ester group, an isopropyl ester group, a butyl ester group or an isobutyl ester group.
  • the aromatic hydrocarbon group includes a monocyclic ring or a polycyclic ring, and is preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenyl group.
  • the above-described bicyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group or an imidazole group.
  • the hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. R is a substitution
  • n is an integer of 1 to 3, preferably 1. The same applies to the following general formulas unless otherwise specified.
  • thiophene derivative it is a non-condensed compound, and a 2,3,4,5-tetraphenylthiophene derivative represented by the following general formula can also be used.
  • R 1 and R 2 are each independently a hydrogen atom, linear, branched or ring
  • Y and Y are hydrogen atoms, halogen atoms, or linear, branched or cyclic carbons.
  • An aryl group preferably a phenyl group, a tolyl group, a xylyl group or a naphthyl group, or a substituted or unsubstituted aralkyl group, preferably a benzyl group or a phenethyl group, or a substituted or unsubstituted amino group.
  • Ar to Ar are each independently a substituted or unsubstituted aryl group, preferably
  • imidazole may be used as the 5-membered ring compound, and an imidazole derivative represented by the following general formula may be used.
  • the imidazole group constituting the imidazole derivative preferably has a quaternary ammonium group. It is because water solubility can be improved.
  • the pyridino group may also have a quaternary ammonium group in order to further improve water solubility.
  • R ′′ may contain an aromatic ring V, an aliphatic hydrocarbon group such as an alkyl group or an alkenyl group! /, Represents an aromatic hydrocarbon group.
  • the imidazole skeleton is plural at any position of the central benzene ring R, R, R, R.
  • R is an olefin group which may have a substituent or
  • a paraffin group, and n is an integer of 1 to 3, preferably 1.
  • a 5-membered ring compound having a conjugated system and a monocyclic compound containing one or more hetero atoms, selenium atoms, or boron atoms can also be used.
  • an imidazole derivative represented by the following general formula can be used.
  • R, R, and R each independently represent a hydrogen atom, a halogen atom, or a substituent.
  • Alkyl group alkenyl group, alkynyl group, alkoxy group, alkyl ester group, phosphate ester group, sulfate ester group, nitrile group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group or heterocyclic ring
  • An aromatic hydrocarbon group, a hydrocarbon group or a heterocyclic group which may have a group, and R, R and R may be the same or different.
  • the kill group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.
  • the alkenyl group is preferably a bur group, a allyl group, a crotyl group, a tiglyl group, or a prenyl group.
  • the alkynyl group is preferably an ethuryl group or a propargyl group.
  • the alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group.
  • the alkyl ester group is a methyl ester group, an ethyl ester group, a propyl ester group, an isopropyl ester group, a butyl ester group or an isobutyl ester group.
  • the aromatic hydrocarbon group includes a monocyclic or polycyclic ring, preferably a biphenyl group, a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a biphenyl group or a phenyl group.
  • the heterocyclic group is preferably a pyrrole group or a furan group.
  • the hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.
  • the organic EL dye used for the fluorescent dye of the present invention is not particularly limited as long as it is a condensed polycyclic compound and a monocyclic compound as described above! /, But a diazole represented by the following general formula A derivative or an imidazole derivative can be preferably used.
  • diazole derivatives and imidazole derivatives diazoguchi pyridine derivatives or imidazolopyridine derivatives can be preferably used.
  • a particularly preferred fluorescent dye of the present invention contains the above-mentioned diazo-mouth pyridine derivative or imidazolo pyridine derivative in the coloring part, and can be represented by the following general formula.
  • _ (CHR ') pX- (CHR ") q- represents a part of the above-mentioned spacer.
  • Z represents the above-mentioned reactive group.
  • the above R and R are substituted.
  • an aromatic hydrocarbon group or a hydrocarbon group which may have a group A green fluorescent dye corresponding to Cy3 can be obtained, and examples of the aromatic hydrocarbon group include a phenyl group, a tolyl group, and a xylyl group.
  • R and R may be selected from the group consisting of a thiophene group, a furan group, a pyrrole group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, and a pyridine group, which may have a substituent. It is also possible to use a thiophene group, an imidazole group or a furan group. A red fluorescent dye corresponding to Cy5 can be obtained.
  • a fluorescent dye can be synthesized by various methods by combining a reactive group and a part of a spacer.
  • a carboxylic group which is an active ester as a reactive group
  • an active ester form of a diazo-mouth pyridine derivative or an imidazolopyridine derivative is synthesized in advance, and a spacer compound is added to the active ester form.
  • amino acids such as glycine, alanine, 4 aminobutanoic acid, cysteic acid, and serine
  • the introduced active ester can be obtained.
  • glycine when used as the spacer compound, a part of the spacer having —CONH— and — (CH 2) _ can be obtained.
  • ⁇ -alanine when used, it is possible to obtain a spacer part having -CONH- and-(CH)-.
  • 4-aminobutanoic acid when 4-aminobutanoic acid is used, a part of the spacer having —CONH— and — (CH 2) 2 — can be obtained.
  • cysteic acid When cysteic acid is used, -CONH- and -SO- A part of the spacer having the following can be obtained.
  • serine a part of the spacer having -CONH- and -0H can be obtained.
  • an azole derivative or imidazole derivative having a nitrogen cation-containing group can be used.
  • the azole derivative those represented by the following general formula (9), (10) or (1 1) can also be used.
  • R is represented by (9) and (11), and one of R and R is represented by the general formula L-M in (10).
  • the ability to use a linker is preferable.
  • the linker alleviates the steric hindrance between the coloring part and the biomolecule to be labeled, and facilitates the binding between the binding part and the labeling part of the biomolecule, so that a higher V labeling rate can be provided. Because it is possible.
  • R and R in (10) and R and R are each independently a hydrogen atom or a halogen atom.
  • the above alkyl group is, for example, a linear or branched alkyl group having 1 to 6 carbon atoms.
  • the above alkoxy group is, for example, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group.
  • the alkyl ester group is a linear or branched alkyl ester having 1 to 6 carbon atoms.
  • the above aromatic hydrocarbon group is an aryl group containing a monocyclic or polycyclic ring, specifically a phenyl group, a tolyl group, a xylyl group or a naphthyl group, more preferably a phenyl group.
  • the heterocyclic group is, for example, a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group, or a quinoline group, and more preferably a furan group, an imidazole group, or Thiophene group.
  • the aliphatic hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.
  • R independently, the thiophene derivative,
  • R is one selected from the group consisting of orchid derivatives, pyrrole derivatives, imidazole derivatives, oxazole derivatives, thiazole derivatives, pyrazole derivatives, pyridine derivatives and quinoline derivatives.
  • R force aryl having a sulfonyl group
  • X has a substituent! /, May! /, And represents a carbon atom, a nitrogen atom, a sulfur atom, an oxygen atom, a selenium atom or a boron atom.
  • R ' represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group composed of an alkyl group which may contain an aromatic ring.
  • aliphatic hydrocarbon group or aromatic hydrocarbon group those similar to the above can be used.
  • An- represents a halide ion such as Cl-, Br-, or ⁇ , CFSO-, BF-, or PF-.
  • a triazole derivative represented by the following general formula may be used. Even if a triazole derivative is used, the same effect as that of the diazole derivative can be obtained.
  • R and R in (18) and R and R are each independently a hydrogen atom or a halogen atom.
  • the above alkyl group is, for example, a linear or branched alkyl group having 1 to 6 carbon atoms.
  • the above alkoxy group is, for example, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group.
  • the alkyl ester group is a linear or branched alkyl ester having 1 to 6 carbon atoms.
  • the above aromatic hydrocarbon group is an aryl group containing a monocyclic or polycyclic ring, specifically a phenyl group, a tolyl group, a xylyl group or a naphthyl group, more preferably a phenyl group.
  • the heterocyclic group is, for example, a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group, or a quinoline group, and more preferably a furan group, an imidazole group, or Thiophene group.
  • the aliphatic hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.
  • the R and R forces are each independently a thiophene derivative,
  • An aryl group having a sulfonyl group is an aryl group having a sulfonyl group.
  • R ' represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group composed of an alkyl group which may contain an aromatic ring.
  • the aliphatic hydrocarbon group or aromatic hydrocarbon group those similar to the above can be used.
  • An- represents a halide ion such as Cl-, Br-, or ⁇ , CFSO-, BF-, or PF-.
  • an imidazole derivative having a nitrogen cation-containing group can be represented by the following general formula.
  • M is a pyridinium group, a secondary amino group, a tertiary amino group, a quaternary amino group, a piperidinium group, a piperazium group, an imidazolium group.
  • L is a direct bond or- (CH) n- (n is an integer from!
  • R and R are each independently a hydrogen atom, a halogen atom, an alkyl group as a substituent,
  • Alkoxy group, alkyl ester group, phosphate ester group, sulfate ester group, nitrile group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group or heterocyclic group may be present, aromatic hydrocarbon A group, an aliphatic hydrocarbon group or a heterocyclic group;
  • the above alkyl group is, for example, a linear or branched alkyl group having 1 to 6 carbon atoms.
  • the above alkoxy group is, for example, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group.
  • the alkyl ester group is a linear or branched alkyl ester having 1 to 6 carbon atoms.
  • the above aromatic hydrocarbon group is an aryl group containing a monocyclic or polycyclic ring, specifically a phenyl group, a tolyl group, a xylyl group or a naphthyl group, more preferably a phenyl group.
  • the heterocyclic group is, for example, a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group, or a quinoline group, and more preferably a furan group or an imidazole group.
  • a thiophene group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.
  • R and R forces are independently derived from thiophene derivatives, furan derivatives, and pyrrole derivatives.
  • Imidazole derivative oxazole derivative, thiazole derivative, pyrazole derivative, pyridine derivative and quinoline derivative.
  • preferred is an aryl group having R, R force sulfonyl group.
  • the aliphatic hydrocarbon group or the aromatic carbon group As the hydrogen group, the same ones as described above can be used.
  • An- represents a halide ion such as Cl-, Br-, or ⁇ , CFSO-, BF-, or PF-.
  • R ′ and R ′′ are each independently an aliphatic hydrocarbon group or an aromatic hydrocarbon group comprising a hydrogen atom or an alkyl group which may contain an aromatic ring, and if necessary, a sulfonyl group, a hydroxyl group, 4 Selected from the group consisting of a primary amino group and a carboxyl group! /, Which is substituted by one charged group,
  • Ar is an aryl group, preferably a phenyl group or A naphthylene group substituted with a sulfonyl group as necessary can be used
  • p and q are each independently an integer of 0 to 20, preferably an integer of 0 to 10, more preferably 0 to 5 P + q ⁇ l.
  • linker examples include:-(CH) p-CONH- (CH) q-,-(CH) p-COO- (CH) q-,
  • R3 may contain a hydrogen atom or an aromatic ring V, an aliphatic hydrocarbon group composed of an alkyl group! /, Is an aromatic hydrocarbon group, and if necessary, a sulfonyl group, a hydroxyl group, 2 Any force selected from the group consisting of a tertiary amino group, a tertiary amino group, a quaternary amino group, and a force loxyl group can be used.
  • Ar is an arylene group, preferably a phenylene group or a naphthylene group, and those substituted with a sulfonyl group as necessary can be used.
  • r is 0 force, an integer of 20, preferably 0 to 10, more preferably 0 force is also an integer of 5.
  • this spacer are: coNH— (CH) r—COO—, -CONH-CH (-R—OH) —
  • a peptide linker can also be used as the linker.
  • peptide linkers -C (-R) -CONH-C (-R)-, -C (-R) _CONH_C (-R) _CONH_C (-R)-, _C (- R) -CONH-C (-R) -CONH-C (-R) _CONH_C (-R) _
  • These peptides may be homo or heteropeptides. Specific examples include Ala_Ser, Glu_Ala, Glu-Ala_Leu, Gly_Pro, Gly-Pro-Asn, Ile-VaU Ile-Val-Met, and the like.
  • the fluorescent dye having a nitrogen cation-containing group of the present invention can improve the labeling rate for biomolecules due to its high water solubility, and can detect biomolecules with high sensitivity. As a result, the amount of the labeling dye used can be greatly reduced, so that the cost for detecting the target molecule can be greatly reduced.
  • a bond can be provided in the nitrogen cation-containing group.
  • a reactive group that binds to a biomolecule to form a covalent bond or an ionic bond can be used for the binding portion.
  • the reactive group includes an amino group, an imino group, and a thiol group of the biomolecule A functional group capable of reacting with a carboxyl group or a hydroxyl group is preferred.
  • the functional group includes, for example, an isothiocyanate group, an isocyanate group, an epoxy group, a sulfonyl halide group, a chloride group, a halogenated alkyl group, a darioxal group, an aldehyde group, a triazine group, a carpositimide group, a maleimide group, and an active group.
  • An esterified carbonyl group or the like can be used.
  • any force selected from an isothiocyanate group, an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group, and an active esterified carbonyl group is used.
  • an isothiocyanate group an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carpositimide group, and an active esterified carbonyl group. More preferred are a triazine group, a carpositimide group, or an active esterified carbonyl group.
  • a functional group of the nitrogen cation-containing group that reacts with these reactive groups for example, a carboxylate group can be used.
  • an active esterified carbonyl group contains N-hydro A xy succinimide ester or a maleimide ester can be used.
  • a carpositimide reagent such as ⁇ , ⁇ ⁇ ⁇ '-dicyclohexyl carpositimide (DCC) and 1-cyclohexyl-3_ (2-morpholinoethyl) carpositimide can be used.
  • the force S is used to bind a fluorescent dye and a biomolecule through an amide bond via a carpositimide body.
  • an anionic group or a cationic group can be used as the reactive group that forms an ionic bond.
  • an anionic group for example, a sulfonyl group or a carboxyl group can be used. These anion 1 ⁇ biogroups ionically bond with cation 1 ⁇ biogroups of biomolecules, for example, amino groups.
  • a nitrogen cation-containing group such as a quaternary ammonium group or a pyridinium group can be used. These cationic groups ionically bond with anionic groups such as carboxyl groups of biomolecules.
  • the nitrogen cation-containing group bonded to the coloring portion can also serve as a cationic group as a reactive group.
  • the combination of the linker and the nitrogen cation-containing group is not particularly limited, but-(CH) ⁇ - ( ⁇ is an integer of 1 to 4)
  • a nitrogen cation-containing group is preferably a pyridinium group, a piperidinium group, a piperadium group or a quaternary amino group.
  • the preparation method of the biological specimen of the present invention is not particularly limited, and any preparation method conventionally used for microscopic observation can be used. For example, an embedding method using a thin section of a specimen And freezing method. In addition, a method for fixing the specimen piece itself on a supporting substrate for the purpose of observing the substance is also included.
  • paraffin As the embedding agent used for the embedding method, paraffin, celloidin, carbowax, gelatin, albumin, agarose, epoxy resin, polyester resin, polyamide resin, (meth) talyl resin, and the like can be used.
  • a glass support substrate, a resin support substrate, a semiconductor support substrate, and a metal support substrate can be used as the support substrate used for the biological specimen.
  • a glass support substrate A glass slide can be used, and a transparent or semi-transparent resin can be used for the resin support substrate.
  • the resin support substrate A support made of polyethylene terephthalate, polycarbonate, polymethylmethalate, etc.
  • the semiconductor support substrate may include a silicon wafer
  • the metal support substrate may include a grid mesh made of copper, gold, nickel, molybdenum, or the like.
  • a force slide glass that can use a glass support substrate or a resin support substrate is preferable.
  • any of a resin support substrate, a semiconductor support substrate, and a metal support substrate can be used.
  • paraffin is used as the embedding agent, for example, it is possible to prepare a biological specimen by the following method. In other words, the tissue or cells are collected from the specimen, fixed, dehydrated, replaced, embedded, sliced, and stained.
  • osmotic fixative thin and small sag example, a thickness of lmm, and shredded to a size lmm about 3.
  • Fixing is performed to preserve tissues and cells in a state close to that of a living body, and is performed using a reducing agent such as formaldehyde or dartal aldehyde.
  • a reducing agent such as formaldehyde or dartal aldehyde.
  • dehydration is performed using ethanol or acetone as a dehydrating agent.
  • the inside of the tissue is replaced with a replacement agent having affinity between paraffin and dehydrating agent, for example, xylene.
  • paraffin is permeated into the tissue and hardened to prepare a paraffin-embedded body.
  • the paraffin-embedded body is sliced with a microtome into slices of about 2 to 10 m thickness. Place the section on a slide glass, immerse it in warm water, crease the section and fix it on the support substrate.
  • deparaffinization is performed to remove the section force paraffin.
  • the section is labeled with a fluorescent dye. Further, counterstaining is performed using a matoxylin solution or the like. After that, dehydration with a dehydrating agent, penetration with xylene, etc.
  • a cover glass which is an encapsulating member together with an encapsulating agent in a state in which a thin section made of a tissue or cell labeled with a fluorescent dye is fixed on a support substrate made of a slide glass
  • a biological specimen encapsulated by can be produced. This biological specimen is stored frozen after microscopic observation.
  • a resin is used as the embedding agent, it can be carried out in the same manner as in the above production method except that a resin is used instead of paraffin.
  • a biological specimen When performing stereoscopic observation using a laser scanning microscope or a confocal laser microscope, it is possible to use a biological specimen in a state where the embedded resin is not removed.
  • a biological specimen in a state where a thin section obtained by embedding a tissue or cell labeled with a fluorescent dye with an embedding agent is fixed on a support substrate made of a slide glass, A biological specimen sealed with a cover glass that is an encapsulating member can be used together with the encapsulating agent.
  • ether alcohols or darcidyl ethers are preferably used as the dehydrating agent.
  • the power of using acetone, methanol, ethanol, etc. When these solvents are used, the fluorescent dye dissolves, making observation difficult, and there is a problem that the section is distorted or shrinks when dried.
  • ether alcohols or darcidyl ethers it is possible to prevent the fluorescent dye from dissolving and to prevent the section from shrinking during drying.
  • Ether alcohols include butoxypropanol, butoxyethanol, propoxypropanol, propoxyethanol, ethoxypropanol, ethoxyethanol, methoxypropanol, methoxyethanol, dipropylene glycolenobutenoreethenole, dipropylene Glycolol Monopropinoreateolate, Dipropylene Glycololone Monochineoleetenore, Dipropylene Glycolnole Monomethinoleatenore, Diethylene Glyconole Monobutinoleatenore, Diethylene Glyconole Monopropinoreateenore, Diethylene Glycol Monool Mentioning ethyl ether, diethylene glycol monomethyl ether, etc.
  • Glucidyl ethers include butyl daricidyl ether, pheninoreglycidinoreatenore, pendinoreglycidinoreatenore, glycidinoreacetate, glycidylpropionate, glycidyl 2-ethyl hexanoate, glycidyl neodeca Butyl daricidyl ether, glycidyl acetate, and glycidino repropionate are preferred! / ⁇ .
  • the freezing method can be produced, for example, by the following method. That is, if necessary, the tissue is frozen using an OCT compound, which is a water-soluble mounting agent, and sliced with a cryostat to obtain a section. Next, it is melted and adhered to the slide glass and dried. Then, it is labeled with a fluorescent dye. Further, after counterstaining, dehydration, penetration with xylene, etc. are performed, a water-insoluble encapsulant is dropped, a cover glass is placed on the tube, and the sample is observed under a microscope.
  • OCT compound which is a water-soluble mounting agent
  • the biological specimen of the present invention can be multi-labeled using a fluorescent dye. It is not necessary to use multiple excitation light sources to observe fluorescence, and a single excitation wavelength is adjusted using a filter.
  • the excitation wavelength includes not only a single wavelength but also an excitation wavelength having a predetermined width.
  • a plurality of fluorescence can be observed without providing a plurality of filters on the detection side.
  • the biological specimen of the present invention has a new effect not found in conventional biological specimens.
  • the organic EL dye has a coloring portion composed of at least an organic EL dye, and the organic EL dye has a conjugated system and includes one or more hetero atoms, selenium atoms, or boron atoms. Even if the self-fluorescence of the embedding resin exists, it is possible to clearly check the fluorescence of the fluorescent dye without being disturbed by the auto-fluorescence.
  • Synthesis Example 1 shows an example of a fluorescent dye having no spacer part
  • Synthesis Examples 2 to 6 show examples of a fluorescent dye into which a part of the spacer is introduced.
  • the following is an example of the reaction of an active ester of oxadiazo- [3,4-c] pyridine with a partial spacer (Schemes 2 and 3).
  • Oxadiazolopyridine active ester (6) was reacted with alanine in DMF to introduce part of the spacer Carboxylic acid compound (7) was synthesized. Thereafter, the carboxylic acid form (7) was reacted with N-hydroxysuccinimide in dioxane to synthesize an oxazizo-mouth pyridine active ester form (8) into which a part of the spacer was introduced. Examples of reactions are shown below.
  • An imidazolopyridine ethyl ester derivative was used as the organic EL dye.
  • the organic EL dye As the organic EL dye, the oxadiazo-mouth pyridine derivative used in Synthesis Example 1 was used, and cystine acid was used as a part of the spacer.
  • the oxadiazo mouth pyridine active ester (6) was reacted with cysteic acid to synthesize a carboxylic acid (9) into which a spacer was partially introduced.
  • the rubonic acid form (9) was reacted with N-hydroxysuccinimide in dioxane to synthesize an oxazizo-oral pyridine active ester form (10) into which a spacer was partially introduced. Examples of reactions are shown below.
  • the organic EL dye As the organic EL dye, the oxaziazo pyridine derivative used in Synthesis Example 1 was used, and serine was used as a part of the spacer.
  • the oxazazo mouth pyridine active ester (6) was reacted with serine to synthesize a carboxylic acid (11) into which a spacer was partially introduced.
  • the carboxylic acid form (11 ) was reacted with N-hydroxysuccinimide in dioxane to synthesize oxadiazolopyridine active ester (12) into which a part of the spacer was introduced.
  • a reaction example is shown below.
  • the organic EL dye As the organic EL dye, the oxadiazo oral pyridine derivative used in Synthesis Example 1 was used, and alanylserine (Ala-Ser) was used as a peptide linker for a part of the spacer.
  • the oxadiazolopyridine active ester (6) was reacted with valanylserine to synthesize a carboxylic acid (13) into which a spacer was partially introduced. Thereafter, the carboxylic acid form (13) was reacted with N-hydroxysuccinimide in dioxane to synthesize an oxazizo-oral pyridine active ester form (14) into which a spacer was partially introduced.
  • a reaction example is shown below.
  • Oxaziazo pyridine ester (4) is reduced in the presence of NaBH to
  • Methyl oxymethyl (15) was obtained and reacted with thionyl chloride to obtain thiadiazolopyridine chloromethyl (16), which was reacted with 3-ethyl ester pyridine active ester (17) to form nitrogen cation Body (18) was synthesized.
  • the liver extracted from the mouse was sprinkled with the powder of the active ester compound (6) synthesized in Synthesis Example 1 as a fluorescent dye and fixed on a slide glass to obtain a biological specimen.
  • GFP-labeled liver was used. That is, stem cells were extracted from the bone marrow of the Okabe mouse whose whole body shines green, and transplanted to mice whose own bone marrow cells did not function due to RI irradiation. In transplanted mice, GFP-positive bone marrow cells differentiated throughout the body, and cells with GFP expressed in each organ.
  • the prepared biological specimen was irradiated with ultraviolet excitation light and observed under a fluorescent stereomicroscope (manufactured by Nikon). Furthermore, this biological specimen was stored refrigerated for 2 weeks and then observed again under a fluorescent stereomicroscope.
  • Fig. 1 shows the observation images immediately after specimen preparation and after 2 weeks. In the case of a liver expressing GFP, the ability to confirm fluorescence after 2 weeks had passed. On the other hand, the biological specimen of the present invention was able to be observed without any fading of fluorescence.
  • mice were injected intramuscularly with 0.5 mL of a DMSO solution in which the active ester (6) was dissolved as a fluorescent dye, and allowed to stand for 3 days, and then 0.9 mL of the DMSO solution was injected from the blood vessel before perfusion fixation. Thereafter, the excised lymph nodes were fixed with Dartal + paraform perfusion, dehydrated with ethoxypropanol, dried under reduced pressure, embedded in technobit, and sliced with a microtome to obtain a section. The section was fixed on a slide glass and then encapsulated with a cover glass using an encapsulant to obtain a biological specimen. No shrinkage of the sections was observed after dehydration and drying.
  • the prepared biological specimen was irradiated with ultraviolet excitation light and observed under a fluorescence microscope (manufactured by Olympus). Furthermore, this biological specimen was refrigerated for one week and then observed again under a fluorescent stereomicroscope.
  • Figure 1 shows the observation image immediately after preparation of the specimen. In the part indicated by the arrow in the figure, high-intensity orange fluorescence due to the fluorescent dye was observed. It is thought that macrophages incorporating fluorescent dyes were collected. Even after refrigerated storage for 1 week, orange fluorescent light with high brightness was observed due to the fluorescent dye. For comparison, the force of preparing a section using acetone as a dehydrating agent. The section contracted by dehydration and drying.
  • the powder of the active ester (6) synthesized in Synthesis Example 1 as a fluorescent dye was placed on a slide glass, and the fluorescent dye was embedded in Ebon to prepare a sample. For comparison, a sample using Texas Red and epoxy resin was also prepared.
  • the sample was irradiated with visible light in a predetermined wavelength range and observed with a fluorescent stereomicroscope.
  • the upper part of Fig. 3 shows an example using (conventional fluorescent dye), and the lower part shows an example using an active ester (6). .
  • the orange color tone of the fluorescent dye that is not inhibited by Ebon's autofluorescence I was able to confirm.
  • DMSO solutions were prepared using an active ester (6) (orange) and two derivatives thereof (green and yellow) as fluorescent dyes. With these three fluorescent dyes, fluorescence was observed under the conditions of excitation wavelength / filter wavelength power of 515-560 nm / 580 nm, 490-577 nm / 528_633 nm, and 340_380 nm / 425 nm.
  • green has an excitation wavelength of 383 nm and a fluorescence wavelength of 520 ⁇ m
  • yellow has an excitation wavelength of 415 nm and a fluorescence wavelength of 535 nm
  • orange has an excitation wavelength of 460 nm and a fluorescence wavelength of 594 nm.
  • Figure 4 is a fluorescence micrograph showing the results.
  • the upper row is orange, the middle row is dark, and the lower row is yellow.
  • streptavidin 1.0 mg was dissolved in 0.2 M sodium bicarbonate buffer (pH 8.3) 400 ⁇ 1 to prepare a streptavidin solution.
  • a DMSO solution 2001 of a nitrogen cation (18) was added and stirred at room temperature for 1 hour. Thereafter, the reaction phase was filtered through Sephadex series G-25 to obtain labeled streptavidin.
  • a rat spinal cord tissue piece was reflux fixed with 4% paraformaldehyde / 0.1 M Phosphate Buffer (pH 7.2 to 7.4), and then fixed by immersion for 3 hours. Washed with 0.1 M Phosphate Buffer containing 20% sucrose to prepare 10 m frozen sections. 0.1 M Phosphate Buffer (pH 7.2-7.4) After washing with PBSTB, it was blocked with PBSTBF (0.8% fish gelatin, 1% bovine serum albumin, 0.2% Triton X-100 containing 0.1 M PBS).
  • an anti-glial fibrillary acidic protein (GFAP) mouse monoclonal antibody was loaded, reacted at 4 ° C for 14 hours, and washed with 0.1 M Phosphate Buffer.
  • a biotin-labeled anti-mouse antibody / PBSTBF was placed and reacted at room temperature for 90 minutes. Then, it was washed with 0.1 M Phosphate Buffer.
  • nitrogen cation (18) -labeled streptavidin (1: 200 to 800) / PBSTBF was placed and reacted at room temperature for 90 minutes. Thereafter, it was washed with 0.1 M Phosphate Buffer (pH 7.2 to 7.4). Then, it enclosed with glycerin: PBS (3: 1), and observed using the fluorescence microscope.

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Abstract

La présente invention concerne un spécimen biologique qui peut être préparé à un coût limité et dans lequel la fluorescence d'un colorant fluorescent ne disparaît pas, même après un stockage de longue durée. La présente invention décrit également un procédé de préparation d'un tel spécimen biologique et un procédé d'observation d'un tel spécimen biologique. Plus précisément, la présente invention concerne un spécimen biologique dans lequel un tissu ou une cellule marqué avec un colorant fluorescent est fixé sur une base de support. Le colorant fluorescent possède une partie colorante composée d'au moins un colorant organique EL, le colorant organique EL étant composé d'un dérivé d'azole ou d'imidazole qui possède un système conjugué, tout en contenant un ou plusieurs hétéroatomes, un atome de sélénium ou un atome de bore.
PCT/JP2007/064755 2006-07-28 2007-07-27 Spécimen biologique et procédé de préparation de celui-ci WO2008013260A1 (fr)

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JP2008286694A (ja) * 2007-05-18 2008-11-27 Shinichiro Isobe 生体標本の作製方法
CN106706643A (zh) * 2017-02-23 2017-05-24 马青山 一种肝癌对比切片检测装置及检测方法
CN109416730A (zh) * 2016-06-28 2019-03-01 康泰克斯特视觉公司 用于检测数字病理图像中的病理异常的方法和系统以及用于注释组织载玻片的方法
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JP2008286694A (ja) * 2007-05-18 2008-11-27 Shinichiro Isobe 生体標本の作製方法
CN109416730A (zh) * 2016-06-28 2019-03-01 康泰克斯特视觉公司 用于检测数字病理图像中的病理异常的方法和系统以及用于注释组织载玻片的方法
JP2019525151A (ja) * 2016-06-28 2019-09-05 コンテクストビジョン、アクチボラグＣｏｎｔｅｘｔｖｉｓｉｏｎ Ａｂ デジタル病理画像における病理学的異常を検出するための方法及びシステム並びに組織スライドに注釈を付けるための方法
CN106706643A (zh) * 2017-02-23 2017-05-24 马青山 一种肝癌对比切片检测装置及检测方法
CN106706643B (zh) * 2017-02-23 2019-07-02 马青山 一种肝癌对比切片检测方法
JP2019136023A (ja) * 2018-02-09 2019-08-22 株式会社シバサキ 細菌検出装置及び細菌検出方法
JP7260127B2 (ja) 2018-02-09 2023-04-18 株式会社シバサキ 細菌検出方法及び細菌検出装置

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