WO2016190236A1 - Réactif sonde pour hybridation in situ - Google Patents

Réactif sonde pour hybridation in situ Download PDF

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WO2016190236A1
WO2016190236A1 PCT/JP2016/064996 JP2016064996W WO2016190236A1 WO 2016190236 A1 WO2016190236 A1 WO 2016190236A1 JP 2016064996 W JP2016064996 W JP 2016064996W WO 2016190236 A1 WO2016190236 A1 WO 2016190236A1
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nucleic acid
phosphor
acid molecule
binding
nanoparticles
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PCT/JP2016/064996
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Japanese (ja)
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武寿 磯田
満 関口
義一 栗原
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コニカミノルタ株式会社
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Priority to JP2017520679A priority Critical patent/JP6711352B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • 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
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase

Definitions

  • the present invention relates to a probe reagent for in situ hybridization, in particular, fluorescence in situ hybridization (FISH) using phosphor nanoparticles, and a staining method using the probe reagent.
  • FISH fluorescence in situ hybridization
  • Patent Document 1 is an invention of a method of indirectly detecting fluorescence while sensitizing a target nucleic acid molecule through two stages of nucleic acid amplification, and discloses the use of probes as a plurality of types of cocktails.
  • a sample such as a DNA solution
  • the target nucleic acid N1 is captured through the hinge nucleic acid N3 with respect to the nucleic acid group N2s.
  • the first proliferation multimer N4 which is still another nucleic acid having a plurality of branch portions, is bound to the target nucleic acid N1 via the nucleic acid N5 of the second hinge.
  • a plurality of oligomers N6 are bound and captured at a plurality of branches of the first multimer N4 for proliferation. Washing is performed in this state, and only the immobilized nucleic acid remains, and other free nucleic acid molecules and the like are removed.
  • the hinge nucleic acid N5 is prepared as a plurality of kinds of cocktails and bound to different sequence regions on N1. Further, the plurality of captured oligomers N6 are released, and as shown in FIG.
  • a third hinge N8 is attached to the nucleic acid group N7s immobilized on another second solid support S2.
  • a second growth multimer N10 having a branched portion is bound to the captured oligomer N6 via a fourth hinge N9.
  • the nucleic acid N9 of the hinge is prepared as a plurality of kinds of cocktails and bound to different sequence regions on N6.
  • a plurality of alkaline phosphatases P1 are bound to the branched portion of the combined multimer N10 for proliferation, and fluorescence is detected by supplying a fluorescent substrate.
  • fluorescence is indirectly detected while sensitizing the target nucleic acid molecule.
  • the nucleic acid N1 of FIG. 5 (A) is not bound, the nucleic acid N6 of FIG. 5 (B) is not detected, and thus sensitization is indirectly performed.
  • Patent Document 1 The prior art of Patent Document 1 is mainly assumed to detect with a probe (by performing PCR) in order to lyse cells and examine whether a predetermined gene is transcribed or translated.
  • a kit “QuantiGene” manufactured by Affitrix As a technique for using probes as a plurality of types of cocktails, a kit “QuantiGene” manufactured by Affitrix is known, which includes about 40 types of Z-type probes, and is used for fluorescent labeling of mRNA by the mechanism shown in FIG. Realize sensitization. It is presumed that a kit is produced by applying the technique of Patent Document 1.
  • the Z-type probe includes a nucleic acid portion D1 that hybridizes to mRNA, a nucleic acid portion D2 that hybridizes to a detection probe, and a linker portion L that connects them.
  • the nucleic acid part D1 is different for each Z-type probe, and when mRNA is fluorescently stained using the above kit, for example, as shown in FIG. Bind specifically.
  • the main nucleic acid molecule D3 binds to the nucleic acid portions D2 and D2 where the two Z-shaped probes are continuous (see FIG. 4B). Sensitization is realized by binding a large number of nucleic acid molecules D4 labeled with a fluorescent dye F as branches to the nucleic acid molecule D3 (see FIG. 4C).
  • the present invention has been made in view of the above-described problems, and provides a probe reagent for in situ hybridization that can increase the number of bright spot signals and realize more accurate measurement than before.
  • the purpose is to do.
  • the present inventors have used a plurality of nucleic acid molecules having different sequences as probes, and by combining the probes and phosphor nanoparticles in a predetermined manner, the number of bright spot signals is sufficient. As a result, the present invention was found.
  • the probe reagent for in situ hybridization reflecting one aspect of the present invention can bind to or bind to a plurality of nucleic acid molecules having different sequences and the nucleic acid molecule.
  • a probe reagent comprising one or more phosphor nanoparticles.
  • a probe reagent for in situ hybridization that can increase the number of bright spot signals closer to the true value than before and can realize more accurate measurement.
  • HER2 genes in healthy people, there are 2 (at most 4) HER2 genes in one nucleus, but in breast cancer patients, it is known that more than 100 HER2 genes are amplified in one nucleus. Yes.
  • the number of bright spot signals is underestimated by the conventional method, only a few HER2 genes are observed even in breast cancer patients, and accurate diagnosis cannot be made. Absent.
  • FIG. 1 (A) is a diagram showing a state where two types (or more) of DNA probes (nucleic acid molecules) contained in the probe reagent according to the present invention are prepared from a human genome by PCR.
  • a DNA probe obtained by PCR is labeled with a hapten by using a nucleic acid substrate (dUTP-hapten) labeled with a hapten (antigenic small molecule) as a nucleic acid substrate.
  • FIG. 1B shows two (or more) DNA probes labeled with the hapten, which are obtained by the PCR of (A).
  • FIG. 1C shows an anti-hapten antibody ((E)) labeled with biotin (indicated by “ ⁇ ”, first biomolecule) bound to the hapten portion of the DNA probe of (B).
  • FIG. 1 (D) shows the phosphor nanoparticle (F) labeled with streptavidin (second biomolecule) bound to the biotin moiety of the anti-hapten antibody of (C).
  • the probe reagent according to the present invention include (B) a DNA probe, (E) an anti-hapten antibody, and (F) a fluorescent nanoparticle bound thereto ((D)),
  • the probe reagent which has said (B), (E), and (F) separately is also included.
  • FIG. 2 is a diagram showing another aspect of the FISH according to the present invention.
  • a probe reagent containing two or more types of DNA probes bound to phosphor nanoparticles is used as a hybridization reaction system. After adding and binding, excitation light is irradiated to detect fluorescence.
  • FIG. 3 is a diagram showing an aspect of FISH according to the present invention.
  • Two or more DNA probes A and B, an anti-hapten antibody labeled with a first biomolecule, and phosphor nanoparticles for a specific region containing a gene to be stained in a tissue section eg, HER2 gene region
  • a tissue section eg, HER2 gene region
  • FIG. 4 is a diagram for explaining a conventional in situ hybridization (Affitrix kit “QuantiGene”).
  • FIG. 5 is a diagram for explaining another conventional hybridization method (Patent Document 1: US Pat. No. 7,615,351).
  • the probe reagent for in situ hybridization according to the present invention will be described below with reference to FIGS.
  • the probe reagent for in situ hybridization according to the present invention includes a plurality of nucleic acid molecules having different sequences and one or more phosphor nanoparticles that can be bound to or bound to the nucleic acid molecule.
  • the probe reagent may be (1) a probe reagent in which a plurality of complexes in which phosphor nanoparticles and nucleic acid molecules are bound at 1: 1 are formed, and the sequence of the nucleic acid molecules differs between the complexes. 2) It may be a complex in which phosphor nanoparticles and nucleic acid molecules are combined at 1: many, and a probe reagent having a different sequence between the nucleic acid molecules of the complex may be used.
  • a probe reagent in which a plurality of complexes in which a nucleic acid molecule and a nucleic acid molecule are combined at a ratio of 1 to 2 is formed, and the sequence of the nucleic acid molecule differs between the complexes may be used, or any one of (4) (1) to (3) Two or more types of probe reagents may be mixed.
  • nucleic acid molecules having different sequences for example, nucleic acid molecules (i ) With phosphor nanoparticles (I), nucleic acid molecules (ii) with phosphor nanoparticles (II), and nucleic acid molecules (iii) with phosphor nanoparticles (III). If the observed number of bright spots is the same for each of the phosphor nanoparticles (I) to (III), the number of genes matches the observed number of bright spots with high accuracy. Can be estimated. In addition, in the case of the fluorescent substance nanoparticle which emits the same fluorescence, it becomes the same as that of embodiment (multiple: 1).
  • nucleic acid molecules can be bound to a single gene at a plurality of locations (sequences), and the stability of the binding is improved.
  • the number can be estimated to match the observed number of bright spots with high accuracy.
  • the target gene is multiple copies (there are multiple target genes of the same sequence on the genome)
  • the bright spot of the probe reagent bound to each gene is Observed as separate bright spots. In the case of cluster luminescent spots, they are also observed as separate luminescent spots.
  • the probe reagent according to the present invention includes (1) a plurality of nucleic acid molecules in which a first binding portion is directly or indirectly bound to each nucleic acid molecule, or (2) each phosphor.
  • a first reagent containing a plurality of modified nucleic acid molecules and a second reagent containing phosphor nanoparticles modified as in (2) are packaged, and these are sequentially reacted with a tissue section of a specimen slide.
  • the reagent is preferably a reagent that forms a complex containing nucleic acid molecules and phosphor nanoparticles.
  • first binding portion and the second binding portion may be (a) a combination in which they can be directly and specifically bound, or (b) as illustrated in FIGS.
  • each of the intervening molecules contained in the third reagent used in combination with the first reagent and the second reagent can specifically bind to the third binding part and the fourth binding part (first binding part-second (3 coupling portions, 4th coupling portion-second coupling portion) combination.
  • the third reagent is also preferably packaged from the first reagent and the second reagent.
  • the nucleic acid molecule is a nucleic acid molecule having a part of a sequence (probe sequence) of a specific region (eg, region of HER2 gene) on a chromosome.
  • a plurality of nucleic acid molecules having different sequences are a plurality of nucleic acid molecules having a base sequence selected so that each nucleic acid molecule does not overlap from a specific region on a chromosome (eg, region of HER2 gene). Means.
  • nucleic acid molecule examples include naturally occurring nucleic acids such as DNA and RNA (mRNA, tRNA, miRNA, siRNA, non-coding RNA, etc.), PNA, and LNA (or BNA: Bridged Nucleic Acid (crosslinked structure type nucleic acid molecule)).
  • Artificial nucleic acids such as The nucleic acid molecule may be a natural nucleic acid, an artificial nucleic acid, or a nucleic acid molecule in which a natural nucleic acid and an artificial nucleic acid are linked.
  • Biomarkers include diagnostic biomarkers, biomarkers that determine disease stages, disease prognostic biomarkers, and monitor biomarkers that are intended to see responses to therapeutic treatments.
  • HER2, TOP2A, HER3, EGFR, P53, MET, etc. are mentioned as genes related to cancer growth and the response rate of molecular target drugs.
  • the following are mentioned as a gene known as a cancer related gene.
  • Tyrosine kinase-related genes include ALK, FLT3, AXL, FLT4 (VEGFR3, DDR1, FMS (CSF1R), DDR2, EGFR (ERBB1), HER4 (ERBB4), EML4-ALK, IGF1R, EPHA1, INSR, EPHA2, IRR (INSRR) ), EPHA3, KIT, EPHA4, LTK, EPHA5, MER (MERTK), EPHA6, MET, EPHA7, MUSK, EPHA8, NPM1-ALK, EPHB1, PDGFR ⁇ (PDGFRA), EPHB2, PDGFR ⁇ (PDGFRB), EPHEP3, T RON (MST1R), FGFR1, ROS (ROS1), FGFR2, TIE2 (TEK), FGFR3, TRKA (NTRK1), FGFR4, TRKB (NT RK2), FLT1 (VEGFR1), TRKC (NTRK3) and breast cancer-related genes such as ATM, BRCA1, BRCA2, BRCA3, CC
  • Cancer-related genes include APC, MSH6, AXIN2, MYH, BMPR1A, p53, DCC, PMS2, KRAS2 (or Ki-ras), PTEN, MLH1, SM D4, MSH2, STK11, and MSH6 Lung cancer-related genes include ALK, PTEN, CCND1, RASSF1A, CDKN2A, RB1, EGFR, RET, EML4, ROS1, KRAS2, TP53, and MYC.
  • genes include Axin1, MALAT1, b-catenin, p16 INK4A, c-ERBB-2, p53, CTNNB1, RB1, Cyclin D1, SMAD2, EGFR, SMAD4, IGFR2, TCF1, and KRAS.
  • Related genes include Alpha, PRCC, ASPSCR1, PSF, CLTC, TFE3, p54nrb / NONO, and TFEB As thyroid cancer-related genes, AKAP10, NTRK1, AK P9, RET, BRAF, TFG, ELE1, TPM3, H4 / D10S170, TPR and the like.
  • Examples of ovarian cancer-related genes include AKT2, MDM2, BCL2, MYC, BRCA1, NCOA4, CDKN2A, p53, ERBB2, PIK3CA, GATA4, RB, HRAS, RET, KRAS, and RNASET2.
  • Examples of prostate cancer-related genes include AR, KLK3, BRCA2, MYC, CDKN1B, NKX3.1, EZH2, p53, GSTP1, and PTEN.
  • Examples of bone tumor-related genes include CDH11, COL12A1, CNBP, OMD, COL1A1, THRAP3, COL4A5, and USP6.
  • DNA probe As the base sequence used for the nucleic acid molecule (DNA probe), it is preferable to design a unique sequence region in a specific region on the chromosome to be detected. In addition, when detecting the copy number of a specific gene on a chromosome by FISH, it is necessary to design a probe sequence in consideration of a genomic sequence including an intron before splicing. As a method of obtaining a genome sequence containing a gene to be detected, search using the name of the organism, gene name, chromosome number, etc. as search words against the publicly available gene database DDBJ (DNA Data Bank of Japan) Or by searching for “Cancer cell lines BACS” as a search word.
  • DDBJ publicly available gene database
  • the number of the target chromosome in the public database “HD FISH” http: ///www.hdfish.eu./Find#probes.php) (eg HER2 is the 17th).
  • the number of the base sequence of a specific region of the chromosome (for example, the region on the chromosome related to HER2) is numbered and a unique sequence portion is extracted.
  • the sequence of the BAC clone library of “Cancer cell lines BACS” containing the sequence of the cancer (original) gene is suitable.
  • the number of copies when detecting a normal structural gene, the number of copies, such as indel, VNTR (Variable Number of Tandem Repeat), microsatellite etc. It is preferable not to include a base sequence portion unrelated to the target gene that is polymorphic.
  • the number of copies of a normal gene per cell (nucleus) is 1 to 2, so the number of copies estimated from the number of bright spots of the phosphor is 3 or more. Is abnormal in the chromosome to which the gene is amplified. Conversely, when the copy number is 0, it can be determined that an abnormality in the chromosome in which the gene is defective has occurred.
  • the base sequence of the polymorphic gene as described above is included in the base sequence of the nucleic acid molecule (DNA probe), the number of bright spots of the phosphor does not match the target copy number of the specific gene, as described above. This will hinder the detection of the correct number of copies.
  • nucleic acid molecules if the number of bases of the nucleic acid molecule on the chromosome is from several tens of bases to 1000 bases or less, submit the sequence data used for the nucleic acid molecule (DNA probe) and the nucleic acid synthesis contract service such as Funakoshi It is preferable to obtain a nucleic acid molecule by requesting.
  • the nucleic acid molecule has a large number of bases (for example, more than 1000 bases), it can be synthesized as described above, but it takes time, so the DNA base sequence is sequenced to form the nucleic acid molecule correctly. For example, it may be performed as follows on the premise that it is confirmed whether or not
  • a plurality of primer sets (a set of forward primers and reverse primers) are set so as to sandwich two or more regions of the probe sequence contained in the genomic DNA of the organism to be detected. ) was designed and synthesized, and a PCR method using pfu DNA polymerase or the like having a high replication accuracy with respect to genomic DNA (or a genomic library such as the BAC clone library described above) using a set of two or more primers. Do. When performing this PCR, it is preferable to perform PCR for each primer set from the viewpoint of PCR efficiency.
  • the PCR is performed using a nucleic acid substrate having a hapten. It is preferable to label the nucleic acid molecule with a hapten.
  • the PCR reaction solution is separated by electrophoresis, and a band corresponding to the length of the target nucleic acid molecule is cut out and eluted using a nucleic acid purification kit (kit such as MonoFas (registered trademark) DNA purification kit I).
  • kit such as MonoFas (registered trademark) DNA purification kit I.
  • a plasmid containing a probe sequence (BAC plasmid or the like) is transformed into E. coli (E.coli HST08 Premium Electro-Cells (Takara Bio) etc.), cultured (amplified), and collected. Nucleic acid extraction is carried out. Next, two or more regions are selected from the probe sequence so as not to overlap each other, and each nucleic acid molecule corresponding to the region is cut out with a predetermined restriction enzyme, followed by electrophoresis and nucleic acid purification. "Molecules" can be obtained.
  • the phosphor nanoparticles include mainly inorganic semiconductor nanoparticles containing a semiconductor component and phosphor integrated nanoparticles.
  • phosphor-integrated nanoparticles it is possible to increase the amount of fluorescence emitted per particle, that is, the brightness of a bright spot that marks a predetermined biomolecule, as compared with the phosphor itself.
  • semiconductor nanoparticles examples include II-VI group compounds, III-V group compounds, or quantum dots containing group IV elements as components ("II-VI group quantum dots", " Or III-V quantum dots ”or“ IV quantum dots ”). You may use individually or what mixed multiple types.
  • the quantum dots may be commercially available. Specific examples include, but are not limited to, CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge. It is also possible to use a quantum dot having the above quantum dot as a core and a shell provided thereon.
  • quantum dots having a shell when the core is CdSe and the shell is ZnS, it is expressed as CdSe / ZnS.
  • CdSe / ZnS, CdS / ZnS, InP / ZnS, InGaP / ZnS, Si / SiO 2 , Si / ZnS, Ge / GeO 2 , Ge / ZnS, and the like can be used, but are not limited thereto.
  • the quantum dots those subjected to surface treatment with an organic polymer or the like may be used as necessary. Examples thereof include CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like.
  • a liquid phase method can be employed as a method for producing semiconductor nanoparticles.
  • the liquid phase method include a precipitation method, a coprecipitation method, a sol-gel method, a uniform precipitation method, and a reduction method.
  • the reverse micelle method, the supercritical hydrothermal synthesis method, and the like are also excellent methods for producing semiconductor nanoparticles (for example, JP 2002-322468, JP 2005-239775, JP 10-310770 A). No., JP 2000-104058 A, etc.).
  • the semiconductor precursor according to the present invention is a compound containing an element used as the semiconductor material.
  • the semiconductor precursor includes SiCl 4 .
  • Other semiconductor precursors include InCl 3 , P (SiMe 3 ) 3 , ZnMe 2 , CdMe 2 , GeCl 4 , tributylphosphine selenium and the like.
  • the reaction temperature of the reaction precursor is not particularly limited as long as it is not lower than the boiling point of the semiconductor precursor and not higher than the boiling point of the solvent, but is preferably in the range of 70 to 110 ° C.
  • reducing agent As the reducing agent for reducing the semiconductor precursor, various conventionally known reducing agents can be selected and used according to the reaction conditions.
  • lithium aluminum hydride (LiAlH 4 ) is preferable because of its reducing power.
  • solvent Various known solvents can be used as the solvent for dispersing the semiconductor precursor. Alcohols such as ethyl alcohol, sec-butyl alcohol and t-butyl alcohol, and hydrocarbon solvents such as toluene, decane and hexane are used. It is preferable to use it. In the present invention, a hydrophobic solvent such as toluene is particularly preferable as the dispersion solvent.
  • surfactant various conventionally known surfactants can be used, and anionic, nonionic, cationic, and amphoteric surfactants are included. Of these, tetrabutylammonium chloride, bromide or hexafluorophosphate, tetraoctylammonium bromide (TOAB), or tributylhexadecylphosphonium bromide, which are quaternary ammonium salt systems, are preferred. Tetraoctyl ammonium bromide is particularly preferable.
  • the reaction by the liquid phase method varies greatly depending on the state of the compound containing the solvent in the liquid.
  • special care must be taken.
  • the size and state of the reverse micelle serving as a reaction field vary depending on the concentration and type of the surfactant, so that the conditions under which nanoparticles are formed are limited. Therefore, an appropriate combination of surfactant and solvent is required.
  • the phosphor-integrated nanoparticles are obtained by accumulating the following phosphors. By using such phosphor-integrated nanoparticles, it is possible to increase the amount of fluorescence emitted per particle, that is, the brightness of a bright spot marking a predetermined biomolecule, compared to the phosphor itself.
  • the term “phosphor” refers to a general substance that emits light in a process from an excited state to a ground state by being excited by irradiation with external X-rays, ultraviolet rays, or visible rays. Therefore, the “phosphor” in the present invention is not limited to the transition mode when returning from the excited state to the ground state, but is a substance that emits narrowly defined fluorescence that is light emission accompanying deactivation from the excited singlet. It may be a substance that emits phosphorescence, which is light emission accompanying deactivation from a triplet. Further, the “phosphor” referred to in the present invention is not limited by the emission lifetime after the excitation light is blocked.
  • phosphorescent substance such as zinc sulfide or strontium aluminate.
  • phosphors can be broadly classified into organic phosphors (fluorescent dyes) and inorganic phosphors.
  • organic phosphors examples include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (registered trademark, manufactured by Invitrogen Corporation) dye molecules, BODIPY (registered trademark, manufactured by Invitrogen Corporation) dyes Molecule, cascade (registered trademark, Invitrogen) dye molecule, coumarin dye molecule, NBD (registered trademark) dye molecule, pyrene dye molecule, Texas Red (registered trademark) dye molecule, cyanine dye molecule, perylene dye Examples thereof include substances known as organic fluorescent dyes, such as dye molecules and oxazine dye molecules.
  • Inorganic phosphor In the case of producing phosphor-integrated nanoparticles by accumulating inorganic phosphors, examples of usable inorganic phosphors include the semiconductor nanoparticles described above.
  • the method for producing the phosphor-integrated nanoparticles in which the phosphor is integrated is not particularly limited, and can be produced by a known method. In general, a production method can be used in which phosphors are gathered together using a resin or silica as a base material (the phosphors are immobilized inside or on the surface of the base material).
  • the particle diameter of the phosphor-integrated nanoparticles is not limited as long as the average particle diameter is within a range in which fluorescence observation is possible, but from the viewpoint of preferably performing fluorescence observation, the average particle diameter of the phosphor-integrated nanoparticles is 40 nm to 300 nm. Preferably there is.
  • Examples of a method for producing phosphor-integrated nanoparticles using organic phosphors include a method of forming resin particles having a diameter of nanometer order by fixing a fluorescent dye, which is a phosphor, inside or on the surface of a matrix made of resin. Can do.
  • the method for preparing the phosphor-integrated nanoparticles is not particularly limited.
  • a (co) monomer for synthesizing a resin (thermoplastic resin or thermosetting resin) that forms the matrix of the phosphor-integrated nanoparticles While (co) polymerizing the phosphor, a method of adding the phosphor and incorporating the phosphor into the inside or the surface of the (co) polymer can be used.
  • thermoplastic resin for example, polystyrene, polyacrylonitrile, polyfuran, or a similar resin
  • thermosetting resin for example, polyxylene, polylactic acid, glycidyl methacrylate, polymelamine, polyurea, polybenzoguanamine, polyamide, phenol resin, polysaccharide or similar resin
  • Thermosetting resins, particularly melamine resins are preferred in that elution of the dye encapsulated in the dye resin can be suppressed by treatments such as dehydration, penetration, and encapsulation using an organic solvent such as xylene.
  • polystyrene nanoparticles encapsulating an organic fluorescent dye can be obtained by a copolymerization method using an organic dye having a polymerizable functional group described in US Pat. No. 4,326,008 (1982), or US Pat. No. 5,326,692 (1992). ), And the method of impregnating polystyrene nanoparticles with a fluorescent organic dye is described.
  • silica nanoparticles in which an organic phosphor is immobilized inside or on the surface of a matrix made of silica can also be produced.
  • the method for synthesizing FITC-encapsulated silica nanoparticles described in Langmuir Vol. 8, Vol. 2921 (1992) can be referred to.
  • Various fluorescent dye-containing silica nanoparticles can be synthesized by using a desired fluorescent dye instead of FITC.
  • Inorganic phosphor As a method for producing phosphor-integrated nanoparticles using an inorganic phosphor, there can be mentioned a method of forming silica nanoparticles in which the aforementioned semiconductor nanoparticles, which are phosphors, are fixed inside or on the surface of a matrix made of silica. This production method can be referred to the synthesis of CdTe-containing silica nanoparticles described in New Journal of Chemistry Vol. 33, p. 561 (2009).
  • silica nanoparticles are treated with a silane coupling agent to aminate the ends, and semiconductor nanoparticles as phosphors having carboxyl group ends are obtained.
  • the particles are accumulated by binding to the surface of silica beads by amide bonds to form phosphor-integrated nanoparticles.
  • a reverse micelle method and a mixture of organoalkoxysilane and alkoxide having an organic functional group with good adsorptivity to semiconductor nanoparticles at the molecular end as a glass precursor are used.
  • glass-like particles in which semiconductor nanoparticles are dispersed and fixed are formed to form phosphor-integrated nanoparticles.
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
  • a preferred example is an embodiment having a structure in which semiconductor nanoparticles are directly cross-linked by first activating the carboxyl group of the mercapto acid using EDC and then reacting with a diamine to form an amide bond.
  • Such an embodiment can be applied to core / shell semiconductor nanoparticles having a shell having high affinity with a mercapto group (thiol group) of mercapto acid, for example, a shell of ZnS or the like containing a sulfur atom.
  • mercaptoundecanoic acid as the mercapto acid and ethylenediamine or hexaethylenediamine as the diamine.
  • an aggregate in which an inorganic phosphor is immobilized inside or on the surface of a matrix made of resin can be made using the method of impregnating quantum nanoparticles into polystyrene nanoparticles described in Nature Biotechnology Vol. 19, p. 631 (2001).
  • a preferred example is a mode in which semiconductor nanoparticles are directly cross-linked using polyethylene glycol (PEG) having mercapto groups at both ends.
  • PEG polyethylene glycol
  • the average particle diameter of the phosphor-integrated nanoparticles is preferably 40 nm or more and 300 nm or less.
  • the average particle diameter of the base particles is less than 40 nm, the bright spots that should be observed in the fluorescence observation due to the fluorescent aggregate nanoparticles are not observed at all, or are difficult to observe.
  • the average particle diameter of the base particles exceeds 300 nm, the bright spots observed in the fluorescence observation become too large, and it is difficult to accurately distinguish non-specific bright spots from specific bright spots. It becomes difficult to count the bright spots.
  • the average particle diameter of the produced pigment particles can be measured by a method known in the art. For example, gas adsorption method, light scattering method, X-ray small angle scattering method (SAXS), transmission electron microscope (TEM) ) Or a method of measuring the average particle diameter by observing with a scanning electron microscope (SEM).
  • SAXS X-ray small angle scattering method
  • TEM transmission electron microscope
  • SEM scanning electron microscope
  • the nucleic acid molecule that is a DNA probe and the phosphor nanoparticle may be bonded via any known bond as exemplified in Table 1 below. It is preferable to bind the body nanoparticle by a bond other than the bond between the nucleic acid molecules. This is because nonspecific binding is likely to occur in binding between nucleic acid molecules. Examples of bonds other than the bonds between nucleic acid molecules include, as described later, a bond between a hapten and an anti-hapten antibody, a bond between a first biomolecule and a second biomolecule, and a bond between a first binding group and a second binding group. Examples include bonding.
  • the substances listed as the first bonding part and the substances listed as the second bonding part in Table 1 may be interchanged. That is, as the first binding part that the nucleic acid molecule has, the substances listed as the second binding part that the phosphor nanoparticles have in Table 1 may be used, and conversely, the second binding part that the phosphor nanoparticles have. The substances listed as the first binding part of the nucleic acid molecule in Table 1 may be used.
  • the first binding part possessed by the nucleic acid molecule and the second binding part possessed by the phosphor nanoparticles may be a combination in which they can be directly and specifically bound (direct method).
  • the first coupling portion and the second coupling portion are included in the same group of any one of A, B, and C in Table 1.
  • a group A hapten can be selected as the first binding portion
  • a group A anti-hapten antibody can be selected as the second binding portion.
  • the term “having” refers to an embodiment in which the first binding portion and the second binding portion are covalently bonded to the nucleic acid molecule and the phosphor nanoparticle by using a linker as described later, Both embodiments that are attached without such a linker are included.
  • the first binding part possessed by the nucleic acid molecule and the second binding part possessed by the phosphor nanoparticle are the third binding part and the second joint possessed by the “intervening molecule” used in combination with the nucleic acid molecule and the phosphor nanoparticle. It may be a combination that can specifically bind to each of the four binding portions (first binding portion-third binding portion, fourth binding portion-second binding portion) (indirect method).
  • An “intervening molecule” is a compound (conjugate) having a third binding part at one end (first end) and a fourth binding substance at the other end (second end).
  • the first coupling portion and the second coupling portion are included in different groups of A, B, and C in Table 1, and the third coupling portion is the same as the first coupling portion. It becomes a joint part mentioned as the 2nd joint part which belongs to a group, and the 4th compound becomes a joint part mentioned as the 1st joint part which belongs to the same group as the 2nd joint part.
  • a group A hapten is selected as the first binding portion of the nucleic acid molecule
  • a group A anti-hapten antibody is selected as the third binding portion of the intervening molecule.
  • Group B biotin can be selected as the binding site, and Group B streptavidin can be selected as the fourth substance of the phosphor nanoparticles.
  • Binding of a nucleic acid molecule and a phosphor nanoparticle “via” as exemplified in Table 1 includes both direct and indirect binding modes as described above.
  • the matters described for the case where the nucleic acid molecule has the first binding portion and the phosphor nanoparticle has the second binding portion are as follows: the nucleic acid molecule has the first binding portion and the intervening molecule is the third. The same applies to the case of having a binding part, and the case where the intervening molecule has a fourth binding part and the phosphor nanoparticle has the second binding part, or the same is applied as necessary or after appropriate modification. It is possible.
  • hapten also called an antigenic small molecule, means a substance that does not produce an antibody even when injected into an animal or the like (lack of immunogenicity), and only causes an antigen-antibody reaction with the antibody (has only reactiveness) To do.
  • a hapten is a substance that reacts with a specific antibody but does not induce the proliferation or differentiation of antibodies or lymphocytes, and has a low molecular weight with a weight average molecular weight (Mw) of less than 1000 that has only the above-mentioned reactivity. included.
  • haptens usable in the present invention digoxigenin (DIG), fluorescein isothiocyanate (FITC), 2,4-dinitrophenol (DNP), and the like can be preferably used. Any one of these haptens may be used alone, or two or more thereof may be used in combination.
  • a first hapten eg, FITC
  • a second hapten eg, DIG
  • FITC fluorescein isothiocyanate
  • DNP 2,4-dinitrophenol
  • nucleic acid molecule labeled with hapten When a hapten is bound to a nucleic acid molecule, the following method can be used. Since the nucleic acid molecule itself is also an antigenic small molecule, it is not necessary to label the nucleic acid molecule with the hapten other than the nucleic acid when utilizing the binding between the nucleic acid molecule and the anti-hapten antibody.
  • a method for labeling a nucleic acid with a hapten for example, a PCR labeling method, a nick translation method, or a random prime method can be used.
  • a nucleic acid molecule is labeled with a hapten by performing PCR using a nucleic acid substrate (eg, dUTP-DIG, dUTP-FITC, etc.) labeled with a hapten (eg, DIG) using the nucleic acid molecule as a template.
  • a nucleic acid substrate eg, dUTP-DIG, dUTP-FITC, etc.
  • a hapten eg, DIG
  • a nucleic acid molecule can be labeled with a hapten by performing nick translation using a nucleic acid substrate (eg, dUTP-DIG or the like) labeled with a hapten.
  • a nucleic acid substrate eg, dUTP-DIG or the like
  • a double strand is formed between a primer having a different length and a nucleic acid molecule serving as a template, and a nucleic acid substrate labeled with a hapten (eg, dUTP ⁇
  • the nucleic acid molecule can be labeled with a hapten by allowing klenowEnzyme to act in the presence of DIG) or the like.
  • the labeled nucleic acid molecule can be purified by separating it from contaminants by polyacrylamide gel electrophoresis (PAGE) or the like, and cutting out and eluting the corresponding part.
  • PAGE polyacrylamide gel electrophoresis
  • a first functional group eg, amino group
  • the first functional group eg, amino group
  • the hapten are combined.
  • a nucleic acid molecule can be labeled with a hapten (eg, FITC) by a binding reaction with a second functional group (eg, isothiocyanate group) in the molecule of (eg; FITC).
  • the site of the nucleic acid molecule to be introduced is enzymatically phosphorylated, And the amino group can be introduced into the nucleic acid molecule by reacting at room temperature for about 12 to 18 hours.
  • a reagent for introducing an amino group for example, a solution of about 0.2 M carbodiimide dissolved in a MES buffer can be suitably used.
  • a DMF solution containing 1 to 10 times the number of moles of FITC is mixed with the phosphorylated nucleic acid molecule and reacted at a low temperature (eg, 4 ° C.) for about 10 to 20 hours, thereby allowing the FITC isothiocyanate to react.
  • the group can react with the amino group of the nucleic acid molecule.
  • the labeled nucleic acid molecule can be purified by separating it from contaminants by polyacrylamide gel electrophoresis (PAGE) or the like, and subjecting the corresponding portion to gel cutting and elution. Whether or not the nucleic acid molecule is labeled with FITC can be confirmed by examining the presence or absence of fluorescence having a FITC excitation wavelength of 500 nm and an emission wavelength of 520 nm for the purified nucleic acid molecule.
  • PAGE polyacrylamide gel electrophoresis
  • a nucleic acid molecule can be labeled with DNP, for example, with a DNP labeling kit “Label IT (registered trademark) DNP Labeling Kit (Takara Bio).
  • the labeled nucleic acid molecule can be purified by separating it from contaminants by polyacrylamide gel electrophoresis (PAGE) or the like, and cutting out and elution the corresponding part. Whether or not a nucleic acid molecule is labeled with DNP can be confirmed by examining the presence or absence of a DNP visible light wavelength of 570 to 590 nm for the purified nucleic acid molecule.
  • the antibody against hapten includes an antibody capable of specifically binding to the hapten.
  • anti-DIG antibodies, anti-FITC antibodies, and anti-DNP antibodies can be suitably used.
  • anti-DIG antibody commercially available antibodies such as “anti-digoxigenin monoclonal antibody” (manufactured by Roche Applied Science) can be used.
  • anti-FITC antibody “Anti-FITC antibody (ab19224)” (manufactured by Abcam) can be preferably used.
  • anti-DNP antibody “anti-DNP, monoclonal antibody (Clone: SPE7)” (manufactured by Japan Biotest Laboratories) can be preferably used. Any one of these anti-hapten antibodies may be used alone, or two or more thereof may be used in combination.
  • the anti-first hapten antibody and the anti-second hapten antibody are bound to the phosphor nanoparticles. Can be.
  • antibody is used to include any antibody fragment or derivative, and includes various antibodies such as Fab, Fab′2, CDR, humanized antibody, multifunctional antibody, and single chain antibody (ScFv). Used in
  • the anti-hapten antibody may be modified with biotin
  • the phosphor nanoparticles may be modified with streptavidin
  • the anti-hapten antibody and the phosphor nanoparticles may be bound via the binding of biotin and streptavidin. Good.
  • an anti-hapten antibody is reduced with a predetermined reducing reagent to introduce a thiol group (SH group), and a maleimide group, an aldehyde group, A functional group such as a bromoacetamide group (iodoacetamide group, bromoacetamide group) or the like may be introduced, and the thiol group and the functional group may be coupled to each other to bond the anti-hapten antibody and the phosphor nanoparticles.
  • a thiol group SH group
  • a maleimide group an aldehyde group
  • a functional group such as a bromoacetamide group (iodoacetamide group, bromoacetamide group) or the like may be introduced, and the thiol group and the functional group may be coupled to each other to bond the anti-hapten antibody and the phosphor nanoparticles.
  • a thiol group when introduced into an antibody, it can be introduced by forming a thiol group (SH group) from a disulfide bond (—S—S—) by treating the antibody with a reducing agent.
  • a reducing agent examples include 2-mercaptoethanol, 3-mercapto-1,2-propanediol, glutathione ( ⁇ -L-glutamyl-L-cysteinylglycine), and tris (2-carboxyethyl) phosphine hydrochloride.
  • cysteine 2-mercaptoethylamine and the like.
  • the reducing pH is, for example, pH 7.0 to 8.5 when 2-mercaptoethanol is used.
  • the conditions for the reduction reaction are 4 ° C. to 8 ° C. for 8 hours to 36 hours.
  • the molar concentration of the reducing agent is preferably 100,000,000,000 to 10,000,000,000,000 moles per mole of antibody.
  • the SH group in the antibody molecule is quantified by, for example, using an SH group quantification kit such as a known SH group quantification reagent (for example, 5,5′-Dithiobis (2-nitrobenzoic acid), same product code: D029, product name: DTNB). It can carry out by well-known methods, such as a method to use.
  • an SH group quantification kit such as a known SH group quantification reagent (for example, 5,5′-Dithiobis (2-nitrobenzoic acid), same product code: D029, product name: DTNB). It can carry out by well-known methods, such as a method to use.
  • the method for introducing the functional group onto the surface of the phosphor nanoparticle is not particularly limited.
  • the phosphor nanoparticle is a phosphor-integrated nanoparticle based on a resin particle
  • the phosphor-integrated nanoparticle is used.
  • a method of introducing a binding group into the phosphor-integrated nanoparticles by polymerizing the monomer having the functional group in the side chain at the main chain portion, or a linker having a binding group is added to the phosphor-integrated nanoparticle.
  • An example of the method is a method in which the particles are introduced by binding to the surface of the particles.
  • the functional group is coupled via a coupling agent (such as a silane coupling agent) that reacts with OH groups on the surface of the inorganic particles constituting the semiconductor nanoparticles. Group, etc.) can be introduced.
  • a coupling agent such as a silane coupling agent
  • a functional group As a method other than FT-IR, whether or not a functional group has been introduced can also be confirmed by the following method.
  • the functional group is a maleimide group
  • use the “AmpliteTM Fluorescent Maleimide Quantification Kit” manufactured by Cosmo Bio
  • to quantify the maleimide group on the surface of the phosphor nanoparticles before and after the introduction of the maleimide base. Can be confirmed.
  • the 2,4-dinitrophenylhydrazine (DNPH) method is used to remove the aldehyde group on the surface of the phosphor nanoparticles before and after the aldehyde group introduction treatment. It can be confirmed by quantifying.
  • the functional group is a haloacetamide group (iodoacetamide group or bromoacetamide group)
  • the halogen produced as a by-product by actually reacting with the SH group is quantified to the surface of the phosphor nanoparticles by a known method. It can be confirmed whether or not the haloacetamide group has been introduced.
  • the binding between the antibody introduced with the SH group and the phosphor nanoparticle introduced with the functional group is performed with 100,000 antibodies having an SH group after reduction with respect to 1 mol of the phosphor nanoparticle from the viewpoint of enhancing the reaction efficiency. It is preferable to use from mol to 100,000,000 mol.
  • the temperature and time of the binding reaction are preferably left at room temperature (1 to 40 ° C.) for 1 to 12 hours from the viewpoint of sufficiently performing the binding reaction.
  • the binding reaction can be stopped by adding about 30 to 50 nmol of a reducing agent such as mercaptoethanol to the reaction solution.
  • a hydrophilic polymer linker may be interposed between the anti-hapten antibody and the phosphor nanoparticles.
  • a functional group such as maleimide is introduced into one end of the linker, and a functional group such as an SH group is provided in the anti-hapten antibody.
  • One end of the linker and the phosphor nanoparticles can be bound by a binding reaction.
  • a functional group such as an NHS ester is introduced into the other end of the linker, which is different from the functional group used for binding one end of the linker.
  • a functional group that can be bonded to the functional group such as an amino group is introduced to the surface of the phosphor nanoparticle, and the other end of the linker is bonded to the phosphor nanoparticle by a bonding reaction between these functional groups. it can.
  • Such a linker can be obtained by purchasing a commercially available product. For example, if you want to use a linker that has a maleimide group at one end of the PEG and an NHS ester at the other end, you can purchase a PEG linker “SM (PEG) 12 ” manufactured by Thermo Fisher Scientific. It can be preferably used.
  • PEG linker “SM (PEG) 12 ” manufactured by Thermo Fisher Scientific. It can be preferably used.
  • an amino group is originally present on the particle surface.
  • an amino group may be introduced by introducing an amino group with an amino group introduction reagent such as 2-iminothiolane and adding a functional group.
  • an amino group introduction reagent such as 2-iminothiolane
  • the phosphor nanoparticle is a semiconductor nanoparticle, it is attached to the particle surface by a coupling agent for introducing an amino group that reacts with an OH group on the metal surface (described in International Publication 2012-165081 or the like).
  • An amino group can be introduced.
  • a linker is interposed between the nucleic acid molecule and the hapten
  • a nucleic acid substrate for hapten labeling eg, dUTP-hapten
  • a nucleic acid substrate eg, dUTP-linker
  • the hapten portion and the nucleic acid portion are bound via a linker.
  • the nucleic acid substrate include “Fluorescein-12-dUTP” (manufactured by Roche Life Science).
  • hydrophilic polymer that can be used as a linker examples include polyethylene glycol, polypropylene glycol, Ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether- Maleic anhydride alternating copolymer, polyvinyl pyrrolidone, polyvinyl methyl ether, polyvinyl methyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxy For ethyl acrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyaspartamide, synthetic polyamino acids It can be used one or two or more hydrophilic polymers selected from the group formed Ri. Among the hydrophilic polymers selected from the group formed Ri. Among the hydrophilic polymers selected from the group formed Ri. Among the hydro
  • the length of the linker means the length of the portion derived from the hydrophilic polymer in a state where the linker is bound to the antibody or phosphor nanoparticle, as shown by the following chemical formula.
  • the length of the linker is preferably 30 angstroms or more and 1000 angstroms or less, and particularly preferably 15 angstroms or more and 65 angstroms or less.
  • One or more linkers may be bonded to one phosphor nanoparticle. When two or more linkers are bonded to a phosphor nanoparticle, one linker nanoparticle is bonded to one phosphor nanoparticle.
  • a plurality of linkers having different lengths may be bound. For example, when two or more kinds of second binding parts (for example, DIG and FITC) are bound to one phosphor nanoparticle, it is preferable that each is bound by a linker having a different length.
  • the binding between the nucleic acid molecule and the fluorescent nanoparticle may be based on the binding through the binding between the first biomolecule and the second biomolecule that can specifically bind to the first biomolecule.
  • the first biomolecule and the second biomolecule refer to biomolecules that specifically bind to each other, and the “specific binding” referred to here includes a covalent bond, an antigen-antibody reaction that occurs between an antigen and an antibody, and Hybridization occurring between nucleic acid molecules having complementary base sequences is not included.
  • the first biomolecule and the second biomolecule do not include a molecule contained in a reaction reagent that causes a covalent bond, an antibody itself, and a nucleic acid molecule itself.
  • the first biomolecule to be bound to the nucleic acid molecule is preferably a biomolecule having a weight average molecular weight (Mw) lower than that of the second biomolecule.
  • Mw weight average molecular weight
  • Examples of the first biomolecule include biotin, which is a low molecule.
  • Examples of the second biomolecule include streptavidin, avidin, and neutravidin, which are polymers. Of these, streptavidin can be suitably used.
  • a PCR labeling method As a method for binding the first biomolecule to the nucleic acid molecule, a PCR labeling method, a nick translation method, or a random prime method using a nucleic acid substrate having the first biomolecule can be used.
  • a nucleic acid molecule is used as a first biomolecule by performing PCR using a nucleic acid substrate (eg, dUTP-biotin) labeled with a first biomolecule (eg, biotin) using the nucleic acid molecule as a template.
  • a nucleic acid substrate eg, dUTP-biotin
  • a first biomolecule eg, biotin
  • a nucleic acid molecule can be labeled with a first biomolecule by performing nick translation using a nucleic acid substrate (eg, dUTP-biotin) labeled with the first biomolecule.
  • a nucleic acid substrate eg, dUTP-biotin
  • a nucleic acid substrate eg, dUTP-biotin
  • a primer having a different length is formed into a double-stranded nucleic acid molecule as a template, and a Klenow enzyme is labeled with the first biomolecule is used.
  • the nucleic acid molecule can be labeled with the first biomolecule by acting in the presence of.
  • the binding between the second biomolecule and the phosphor nanoparticle is not particularly limited.
  • the second biomolecule is a protein such as avidin
  • the second biomolecule is coupled with the antibody and the phosphor nanoparticle as described above.
  • Molecules can be bound to phosphor nanoparticles.
  • the second biomolecule may be bound to the phosphor nanoparticles by a method in which a linker is interposed between the antibody and the phosphor nanoparticles.
  • first and second linking groups When binding between a nucleic acid molecule and a phosphor nanoparticle does not use binding between a hapten and an anti-hapten antibody or binding between a first biomolecule and a second biomolecule, for example, a first binding group and a second binding group Can be introduced into a nucleic acid molecule and a phosphor nanoparticle, respectively, and the nucleic acid molecule and the phosphor nanoparticle can be bound via a bond between the first and second bonding groups.
  • a combination of known functional groups that can be bonded can be used.
  • the first binding group introduced into the nucleic acid molecule contained in the first reagent and the phosphor nanoparticle contained in the second reagent are introduced.
  • the second linking groups a bond by the Huisgen cycloaddition reaction of azidoalkyne, which is used in “click chemistry” in which the reaction proceeds rapidly at room temperature just by mixing, is utilized. It is desirable to do.
  • the first reagent and the second reagent are not packaged.
  • a covalent bond is formed under predetermined reaction conditions, for example, a maleimide group-thiol group reaction, in addition to the coupling by the Husgen cycloaddition reaction.
  • a maleimide group-thiol group reaction in addition to the coupling by the Husgen cycloaddition reaction.
  • the present invention is not limited to the above-described embodiment in which phosphor nanoparticles and nucleic acid molecules are directly bound to each other by using an intervening molecule or the like to bind between a hapten-anti-hapten antibody and between a first biomolecule and a second biomolecule.
  • the nucleic acid molecule and the phosphor nanoparticle may be bound with each other.
  • introduction of an azide group (azidation) into the phosphor-integrated nanoparticle can be performed using a known azido reagent (diazo group transfer reagent).
  • introduction of a carbon-carbon triple bond (alkyne modification) into a nucleic acid molecule can be performed by a nick translation method using “EdU (5-ethynyl-2′deoxyuridine)” (manufactured by Life Technology Co., Ltd.) or the like. it can.
  • a nucleic acid molecule having a maleimide group is obtained by performing nick translation on a nucleic acid molecule using a nucleic acid substrate having maleimide or the like, or by adding a maleimide group to a nucleic acid molecule with a reagent that maleates the 5 ′ end or 3 ′ end of the nucleic acid. Etc. can be introduced.
  • the phosphor nanoparticle when the phosphor nanoparticle is a phosphor-integrated nanoparticle based on a resin particle, the monomer having a thiol group is introduced by polymerization as described above. Can be introduced. In addition, about confirmation of bond molar ratio and functional group introduction
  • the specimen slide can be prepared, for example, by a method used for general histopathological diagnosis of tissues of subjects suspected of having cancer (human, dog, cat, etc.). First, a test subject's tissue is fixed using formalin or the like, dehydrated with alcohol, then subjected to xylene treatment, immersed in high temperature paraffin and embedded in paraffin to prepare a tissue sample. Subsequently, the tissue sample is cut into 3 to 4 ⁇ m sections and placed on a slide glass as a specimen slide.
  • model animals such as CDX mice and PDX mice have been used for examination of pathological tissues as subjects suspected of having the above-mentioned cancer, and are attracting attention as model animals used in drug discovery research.
  • specimen slides can be prepared and purchased by a method used for general histopathological diagnosis.
  • a CDX mouse is a model animal produced by planting cultured cells derived from tumor cells removed from a patient and growing in the mouse.
  • a PDX mouse is a tumor tissue or tumor removed from a patient.
  • the tissue section on the specimen slide is immersed in a container containing xylene or other deparaffinizing agent to remove paraffin (see FIGS. 2 and 3).
  • the temperature at this time is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, xylene may be exchanged during the immersion.
  • the section is immersed in a container containing ethanol to remove xylene.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. Further, if necessary, ethanol may be exchanged during the immersion.
  • the section is immersed in a container containing water to remove ethanol.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. Moreover, you may exchange water in the middle of immersion as needed.
  • pretreatment of specimen slide Before subjecting the probe to a hybridization reaction, perform pretreatment to enable the probe reagent to efficiently reach the nucleic acid on the tissue section, such as pretreatment (heat treatment, acid treatment), treatment by enzyme treatment, etc. It has been known. These processing conditions and combinations have different optimum conditions depending on the section type, thickness, slide adjustment conditions, and the like, and it is necessary to appropriately determine the procedure. Not all treatments need to be performed, for example there may be an option of not performing enzyme treatment.
  • pretreatment of a specimen slide for FISH is performed.
  • the pretreatment conditions are not particularly defined, but can be performed by the following procedure, for example.
  • the specimen slide is immersed in hydrochloric acid (about 0.2 mol / L) for a certain period of time. Then, it is immersed in water, and further washed by immersing it in a washing buffer (2 ⁇ SSC: standard sailline citrate).
  • a washing buffer (2 ⁇ SSC: standard sailline citrate).
  • a heated NaSCN solution for example, about 1 N
  • a pretreatment solution the two kinds of solutions as described above are used, and 0.01M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, 0.1M Tris-HCl buffer. It can also be performed under heating using a liquid or the like.
  • a heating device an autoclave, a microwave, a pressure cooker, a water bath, or the like can be used.
  • the temperature is not particularly limited, but the temperature can be 50 to 130 ° C. and the time can be 5 minutes to 30 minutes.
  • a hydrolase protease solution for a certain time. Subsequently, it is immersed in a washing buffer and washed, and this operation is repeated twice.
  • the specimen slide is dried by air drying or the like.
  • a known dehydration process using 70 to 100% ethanol may be performed instead of air drying.
  • the protease a proteinase suitable for protein hydrolysis, such as pepsin, proteinase K and the like is often used.
  • the efficiency of deproteinization can be determined by examining the combination of protease, the concentration of protease that maximizes the reaction with the target chromosome, and the degradation time, and then the morphological detail. The conditions are set so as not to be damaged. The optimum conditions vary depending on the tissue type and the fixing method. Further, additional fixation after protease treatment is useful.
  • the fixing process may be performed before and after the nuclear staining as necessary even in steps other than the pre-processing steps.
  • the sample may be fixed before and / or after hybridization.
  • [Dyeing process] [DNA denaturation treatment] After the above fixing treatment, for example, the following procedure is performed in order to denature the DNA present on the section (from double-stranded DNA to single-stranded DNA).
  • a denaturing solution formamide / SSC solution or the like
  • the specimen slide is taken out and immersed in several stages of ethanol (for example, 70% ethanol aqueous solution, 80% ethanol aqueous solution and 100% ethanol) with gradually increasing concentration in order to remove formamide. Thereafter, the specimen slide is dried by air drying or the like.
  • Hybridization treatment Using a probe reagent containing a plurality of nucleic acid molecules (DNA probes) having different sequences, a known FISH (for example, “Agilent FISH General Purpose Reagents Protocol” and “Clinical FISH Protocol—Visual Chromosome / Gene Diagnosis Method (Separate Cell Engineering) -"Experimental protocol series” etc.), where the term “hybridization” refers to two DNAs or DNA and RNA complementary strands for the formation of double-stranded molecules. It means a binding process or a double-stranded molecule formed.
  • FISH for example, “Agilent FISH General Purpose Reagents Protocol” and “Clinical FISH Protocol—Visual Chromosome / Gene Diagnosis Method (Separate Cell Engineering) -"Experimental protocol series” etc.
  • hybridization As an example of the hybridization, as illustrated in FIG. 2, first, two types of complexes M, in which a plurality of nucleic acid molecules (DNA probes A and B) having different sequences and phosphor nanoparticles are bound, This is hybridization in which a probe reagent containing N is prepared and the complexes M and N are each bound to a target gene on a chromosome.
  • DNA probes A and B nucleic acid molecules having different sequences and phosphor nanoparticles
  • a probe reagent having a solution of DNA probes A and B and a dispersion of phosphor nanoparticles separately is prepared, and as shown in FIG.
  • B is hybridized to the target gene on the chromosome, and then an anti-hapten antibody is bound to the hapten bound to the hybridized DNA probe.
  • This is dynamic hybridization in which a second biomolecule that is bound to a phosphor nanoparticle is bound to one biomolecule.
  • nucleic acid molecules used for hybridization are not limited to two, but may be two or more, preferably 4 to 6.
  • the phosphor nanoparticles can be bound to a plurality of nucleic acid molecules (eg, DNA probes A and B) having different sequences that form complementary strands with the target gene on the chromosome, they can be separated from the above. It may be in the form of hybridization. For example, each bond exemplified in Table 1 (bond between hapten-anti-hapten antibody, bond between first biomolecule and second biomolecule, bond between first binding group and second binding group), or these bonds
  • the nucleic acid molecule and the phosphor nanoparticle may be dynamically bound using a combination of the above.
  • DAPI nuclear staining reagent
  • bisbenzimide derivatives such as Hoechst 33258 and Hoechst 33342, and other nuclear staining reagents may be used.
  • nuclear staining can be performed by the following procedure. First, the specimen slide subjected to the hybridization treatment is washed sequentially with deionized water and phosphate buffered saline (PBS). Subsequently, it is immersed in a DAPI staining reagent (2 ⁇ g / PBS) for a certain period of time.
  • PBS phosphate buffered saline
  • Specimen slides that have undergone FISH staining and nuclear staining are washed several times with PBS, air-dried or dehydrated, and then a mounting medium is dropped on the tissue section, covered with a cover glass, and dried. I do.
  • a known oil-based encapsulating agent such as Entellan (registered trademark) new
  • an aqueous encapsulating agent such as Aquatex (registered trademark)
  • the enclosed specimen slide prepared by the above processing becomes a preparation for performing pathological diagnosis and the like.
  • Bright field observation is performed as necessary in order to acquire distribution information of cell organs to be stained in cells or tissues.
  • a general method for bright field observation for example, it is preferable to perform observation with a microscope after performing the above-described staining, followed by hematoxylin / eosin staining (HE staining) or DAPI staining as described above.
  • HE staining for example, immunostained sections are stained with Mayer's hematoxylin solution for 5 minutes and stained with hematoxylin, and then the tissue sample is washed with running water at 45 ° C. for 3 minutes, and then 1% eosin solution. And eosin staining for 5 minutes.
  • eosin used for morphological observation staining can not only observe in a bright field, but also emits autofluorescence when irradiated with excitation light of a predetermined wavelength, so that an excitation light with an appropriate wavelength and output is applied to a stained tissue sample. Irradiation can be observed with a fluorescence microscope.
  • the other staining for example, in bright field observation when histological staining (DAB staining or the like) is performed using HER2 protein in breast cancer as an antigen to be detected, an optical microscope is used under irradiation with appropriate illumination light.
  • the HER2 protein positive staining image, the positive staining intensity, and the positive cell rate of cancer cells in the specimen tissue are observed using a 4 ⁇ objective lens.
  • the objective lens is switched to 10 times, it is confirmed whether the positive findings are localized in the cell membrane or the cytoplasm, and if necessary, further searching is performed with the objective lens 20 times.
  • Fluorescence observation Using a fluorescence microscope, the number of fluorescent bright spots or emission luminance is measured from a wide-field microscope image for the stained section. An excitation light source and a fluorescence detection optical filter corresponding to the absorption maximum wavelength and fluorescence wavelength of the fluorescent substance used are selected. The number of bright spots or emission luminance can be measured by using commercially available image analysis software, for example, all bright spot automatic measurement software G-Count manufactured by Zeonstrom Co., Ltd. Note that image analysis itself using a microscope is well known, and for example, a technique disclosed in Japanese Patent Laid-Open No. 9-197290 can be used.
  • the field of view of the microscopic image is preferably 3 mm 2 or more, more preferably 30 mm 2 or more, and further preferably 300 mm 2 or more.
  • the copy number of the target specific gene and the expressed protein are evaluated. Specifically, for example, a gene can be evaluated as normal if the copy number is 1 to 2, and abnormal (growth) if it is 3 or more.
  • the probe reagent for in situ hybridization according to the present invention comprises a plurality of nucleic acid molecules having different sequences and one or more phosphor nanoparticles that can be bound to or bound to the nucleic acid molecule. Is included.
  • DNA probes nucleic acid molecules
  • phosphor nanos are further bound to these bound DNA probes. Since each particle binds (is), the reaction probability compared with the case where FISH is performed with a probe reagent including one type of nucleic acid molecule (DNA probe) and a phosphor nanoparticle bonded thereto. And the fluorescence signal from the bright spot is strengthened. In addition, the fluorescent signal is further enhanced because phosphor nanoparticles are used instead of fluorescent dyes.
  • test accuracy for example, test accuracy such as cancer detection
  • Nucleic acid molecules are bound to a gene at a plurality of positions, and a plurality of phosphor nanoparticles are bound via the nucleic acid molecule. Since these bonded phosphor nanoparticles are physically close to each other, bright spots are gathered together and observed and counted as one bright spot (cluster bright spot) when observed with fluorescence. . Therefore, there is an advantage that a bright spot signal stronger than that when FISH is performed with a conventional probe reagent using only one kind of nucleic acid molecule is observed.
  • the first binding portion is directly or indirectly bound to each nucleic acid molecule or indirectly via the linker, and the second binding portion is directly connected to each phosphor nanoparticle. Or the one or more phosphor nanoparticles bound indirectly via the linker, and the first binding portion and the second binding portion are specific.
  • the nucleic acid molecule and the phosphor nanoparticle can be dynamically bound in the in situ hybridization process in addition to the effect of (2) above. That is, after the hybridization with the nucleic acid molecule, the phosphor nanoparticles can be added to the reaction system and the nucleic acid molecules hybridized to the target gene can be fluorescently stained. It can be stored in a state in which it is difficult to fade, and in this case, problems caused by the fading of the phosphor nanoparticles are less likely to occur, and the test accuracy for the target gene is increased.
  • the first binding portion is directly or indirectly bound to each nucleic acid molecule via the linker
  • the second nucleic acid molecule is second to each phosphor nanoparticle.
  • the plurality of nucleic acid molecules, intervening molecules, and phosphor nanoparticles can be combined with the target gene on the chromosome in this order by dividing the reagents and steps.
  • nucleic acid molecule can be bound to the target gene in a state where macromolecules such as intervening molecules and phosphor nanoparticles are not bound, the efficiency of hybridization is unlikely to decrease. Furthermore, since intervening molecules and phosphor nanoparticles can be stored under storage conditions suitable for each of them just before they are used for the above binding, the degradation of intervening molecules and the fading of phosphor nanoparticles are minimized. be able to.
  • the linker may be a plurality of linkers having different lengths bonded to one phosphor nanoparticle and / or one nucleic acid molecule.
  • a probe reagent including a nucleic acid molecule to which a plurality of linkers having different lengths are bound
  • a DNA probe hybridized to a target gene on a chromosome when FISH is performed using the probe reagent thus, a plurality of phosphor nanoparticles can be bonded at different distances. This avoids a situation in which phosphor nanoparticles cause steric hindrance and become difficult to bind, and more phosphor nanoparticles can be bound to one nucleic acid molecule to The strength can be further increased. In this case as well, bright spots obtained from a plurality of phosphor nanoparticles fixed (coupled) to one gene are gathered together and observed as one bright spot (cluster bright spot).
  • a probe reagent including a plurality of linkers having different lengths bonded to one phosphor nanoparticle
  • one phosphor nanoparticle can bind to a plurality of types of nucleic acid molecules
  • the binding is stronger than when the phosphor nanoparticles are bound to one nucleic acid molecule, and the target gene can be labeled more stably.
  • One phosphor nanoparticle has two or more types of second bonding portions, and each type of second bonding portion is related to each phosphor nanoparticle via a linker having a different length. Since one phosphor nanoparticle binds to a plurality of types of nucleic acid molecules via a plurality of linkers, and the plurality of types of nucleic acid molecules binds to one target gene, Binding becomes strong and the target gene can be labeled more stably. Furthermore, since the length of each linker is different, the phosphor nanoparticles are arranged within a specific position range from the target gene. As a result, it becomes easier to observe bright spots. In this case as well, bright spots obtained from a plurality of phosphor nanoparticles fixed (coupled) to one gene are gathered together and observed as one bright spot (cluster bright spot).
  • the first binding portion bound to the nucleic acid molecule is a hapten and the second binding portion bound to the phosphor nanoparticle is an anti-hapten antibody
  • a macromolecule such as an antibody or streptavidin is the first Compared with the case where it is used as a binding portion, hybridization with the nucleic acid molecule can be easily caused.
  • the anti-hapten antibody is stored in a state that is hardly inactivated until it is used, and the anti-hapten antibody (phosphor nanoparticle to which the anti-hapten antibody is bound) is added after hybridization. Fluorescent staining can be performed with increased reaction efficiency.
  • the effect (4) can be suitably obtained.
  • the hapten is one or more selected from the group consisting of digoxigenin (DIG), fluorescein isothiocyanate (FITC) and 2,4-dinitrophenol (DNP), and the anti-hapten antibody is If one or more selected from anti-DIG antibody, anti-FITC antibody and anti-DNP antibody are used, the amount of hapten labeled with the nucleic acid molecule is indicated to DIG, FITC or DNP respectively.
  • a hapten other than the FITC may be used as a hapten that emits fluorescence upon receiving excitation light.
  • a detection system using Cy3 as a hapten and using an anti-Cy3 antibody can be exemplified.
  • the probe reagent was prepared as follows. [Preparation of DNA probe] (Amplification of nucleic acid molecule derived from HER2 gene from genome by PCR method and FITC labeling) HER2 gene using a set (1) of the following forward primer (FP1: 5′-cgatgtgactgtctcctcc-3 ′) and reverse primer (RP1: 5′-atcctactccatcccaagcc-3 ′) encoding a partial DNA sequence of the HER2 gene A nucleic acid molecule (DNA probe i) having a base number of 210 bp was derived.
  • FP1 forward primer
  • RP1 reverse primer
  • RP1 5′-atcctactccatcccaagcc-3 ′
  • PCR reaction product was subjected to 1.5% agarose electrophoresis, and a single band due to the PCR amplification product was confirmed only at a position corresponding to the 210 bp portion.
  • the entire remaining PCR reaction product was purified using “QIAquick PCR Purification Kit” (manufactured by QIAGEN) to obtain a DNA probe i derived from the HER2 gene labeled with 210 bp of FITC.
  • the 210 bp band is cut out from the electrophoresed gel, and the cut gel is “Wizard® PCR”
  • a solution containing DNA probe i derived from HER2 gene with 210 bp base labeled with FITC was obtained by purification using Preps DNA Purification System (manufactured by Promega).
  • Biotin-labeled anti-FITC antibody “Anti-FITC antibody with biotin” (Vector: cord BA-0601) was purchased as a biotin-labeled anti-FITC antibody.
  • FISH FISH was performed as follows using the probe reagent A prepared as described above, and the number of copies of the HER2 gene was measured.
  • FISH includes deparaffinization, specimen slide pretreatment, enzyme treatment, specimen immobilization, denaturation and hybridization, specimen slide washing, blocking, and biotin-labeled anti-FITC antibody binding to a DNA probe. Then, SA-labeled Qdot was bound to the anti-FITC antibody and DAPI staining was performed in this order.
  • Deparaffinization was performed by treating a specimen slide of a HER2-positive stained control specimen (“HER2-FISH Control Slide Code PS-09006” manufactured by Pathology Laboratories) in the order of (1) to (4) below. .
  • HER2-FISH Control Slide Code PS-09006 manufactured by Pathology Laboratories
  • (1) Immerse in Hemo-De for 10 minutes at room temperature.
  • (2) Immerse the specimen slide in new Hemo-De for 10 minutes at room temperature. Repeat the same operation three times.
  • the specimen slide is immersed in 100% ethanol at room temperature for 5 minutes, washed twice, and dehydrated.
  • the specimen slide is air-dried or dried on a slide warmer at 45 to 50 ° C.
  • the specimen slide was pretreated in the following order (1) to (6) to remove proteins from the cell membrane and the nuclear membrane.
  • (1) Treat the specimen slide with 0.2 mol / L HCl at room temperature for 20 minutes.
  • (2) Immerse the specimen slide in purified water for 3 minutes.
  • (3) The specimen slide is immersed in a washing buffer solution (2 ⁇ SSC: standard saline citrate) for 3 minutes.
  • (4) The specimen slide is immersed in a pretreatment solution (1N NaSCN) at 80 ° C. for 30 minutes.
  • (6) The specimen slide is immersed in a washing buffer solution (2 ⁇ SSC) for 5 minutes, and this immersion operation is repeated twice.
  • the sample treatment that had been pretreated was subjected to enzyme treatment by performing the following treatments (1) to (4) in this order.
  • (1) Take out the pretreated specimen slide, attach the lower end of the slide glass to a paper towel, and remove excess washing buffer.
  • (2) The specimen slide is immersed in a protease solution heated to 37 ° C. for 10 to 60 minutes. This immersion treatment is performed with 25 mg protease (2500-3000 Units / mg) [pepsin] / 1M NaCl [pH 2.0] in 50 mL at 37 ° C. for 60 minutes) in order to decompose cell membrane and nuclear membrane proteins, particularly collagen. Processing is desirable.
  • (3) Immerse the specimen slide in the washing buffer for 5 minutes. This operation is repeated twice.
  • (4) The specimen slide is air-dried or dried on a slide warmer at 45 to 50 ° C. for 2 to 5 minutes.
  • specimen fixation As the specimen fixing process, the following processes (1) to (3) were performed on the specimen slide that had been pretreated.
  • the specimen slide is immersed in 10% neutral buffered formalin (“4% paraformaldehyde / phosphate buffer solution” manufactured by Wako Pure Chemical Industries, Ltd., product number 163-20145) for 10 minutes at room temperature.
  • 10% neutral buffered formalin (“4% paraformaldehyde / phosphate buffer solution” manufactured by Wako Pure Chemical Industries, Ltd., product number 163-20145) for 10 minutes at room temperature.
  • the specimen slide is air-dried or dried on a slide warmer at 45 to 50 ° C. for 2 to 5 minutes.
  • the specimen slide is placed in a post-hybridization washing buffer kept at 63 ° C. and immersed for 10 minutes for washing. (5) Immerse for 3 minutes at room temperature using a Tris wash buffer (HER2 FISH PharmDx “Dako”) and wash twice. (6) Remove the specimen slide from the co-plinger and air-dry it under light shielding (closed drawer or closed cabinet shelf). [Specimen fixation] As the sample fixing process, the following processes (1) to (3) were performed on the sample slide subjected to hybridization. (1) The specimen slide is immersed in 10% neutral buffered formalin (“4% paraformaldehyde / phosphate buffer solution” manufactured by Wako Pure Chemical Industries, Ltd., product number 163-20145) for 10 minutes at room temperature. (2) Immerse the specimen slide in the washing buffer for 5 minutes. Repeat the same operation twice. (3) The specimen slide is air-dried or dried on a slide warmer at 45 to 50 ° C. for 2 to 5 minutes.
  • Blocking was performed by immersing in In Situ Hybridization Blocking Solution (Vector, MB1220) at room temperature for 30 minutes.
  • DAPI staining was performed as follows. First, 10 ⁇ L of DAPI counterstain was added to the hybridization area of the specimen slide. Next, after hybridization treatment, DAPI staining (2 ⁇ g / mL PBS) is performed at 25 ° C. for 10 minutes in order to count the number of cells. The cell nucleus is stained, covered with a cover glass, and the specimen slide is covered until signal measurement. Stored protected from light. For DAPI (2- (4-amidinophenyl) -1H-indole-6-carboamidine dihydrochloride), Molecular Probes (D1306) was used. [DAPI staining]
  • the specimen slide subjected to FISH as described above was observed as follows.
  • Fluorescence microscope observation In the fluorescence microscope observation, the section subjected to FISH as described above is subjected to fluorescence microscope observation (600 times) using a fluorescence microscope Zeiss imager (camera: MRm monochrome / with cooling function, objective lens ⁇ 60 oil immersion). , Fluorescence measurement, fluorescence image (fluorescence still image) and number of bright spots were measured.
  • 100 continuous fluorescent still images with a resolution of 200 to 400 milliseconds are acquired using a band pass filter of 690 nm to 730 nm, and a fluorescent moving image (200 to 400 ms / second) is acquired. frame ⁇ 100), and the number of bright spots was measured through the entire timeline of the fluorescent moving image.
  • [Comparative Example 2] [Preparation of DNA probe] (Amplification of nucleic acid molecule derived from HER2 gene from genome by PCR method and FITC labeling) PCR was performed using the primer set (1) in the same manner as in Comparative Example 1 to prepare a 210-bp nucleic acid molecule (DNA probe i) derived from the FITC-labeled HER2 gene.
  • Biotin-labeled anti-FITC antibody As a biotin-labeled anti-FITC antibody, the “biotin-attached anti-FITC antibody” of Comparative Example 1 (Vector: cord BA-0601) was used.
  • the mixture was centrifuged at 20000 G for 15 minutes in a centrifuge (Microcooled Centrifuge 3740 manufactured by Kubota), and after removing the supernatant, ultrapure water was added and ultrasonically irradiated to redisperse. Centrifugation, supernatant removal, and washing by redispersion in ultrapure water were repeated 5 times.
  • the obtained melamine particles were positively charged because the melamine resin itself contains many amino groups in the skeleton.
  • PBS phosphoric acid buffer physiological saline
  • EDTA ethylenediaminetetraacetic acid
  • concentration of the obtained phosphor-integrated nanoparticles (PID) was adjusted to 3 nM.
  • NHS-PEG 12 -maleimide succinimidyl-[(N-maleimidopropioamide) -dodecaethylene glycol]
  • Ester (manufactured by Thermo Fisher Scientific) and reacted at 20 ° C. for 1 hour to obtain a mixed solution containing phosphor-integrated nanoparticles (PID) having a fluorescent dye with a maleimide at the end. .
  • the mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM EDTA was added to disperse the precipitate, and the mixture was centrifuged again.
  • the above washing according to the same procedure was performed three times.
  • streptavidin manufactured by Wako Pure Chemical Industries, Ltd.
  • streptavidin and N-succinimidyl S-acetylthioacetate are used to add a thiol group to streptavidin and perform gel filtration.
  • SATA N-succinimidyl S-acetylthioacetate
  • the streptavidin capable of binding to the phosphor-integrated nanoparticles (PID) was prepared separately.
  • a probe reagent B having the above-described FITC-labeled DNA probe solution, biotin-labeled anti-FITC antibody solution, and SA-labeled phosphor-integrated nanoparticle dispersion was separately prepared.
  • the SA-labeled phosphor-aggregated nanoparticles are added to the reaction system and the reaction is performed by using 100 ⁇ L of a dispersion of SA-labeled phosphor-aggregated nanoparticles (particle size: 280 nm, concentration: 0.05 nM) as a specimen slide.
  • the reaction was carried out by dropping the solution above and causing a binding reaction at room temperature for 60 minutes. Subsequent washing was performed by performing the operation of immersing the specimen slide in PBS for 5 minutes three times.
  • Example 1 [Adjustment of DNA probe] [Amplification of nucleic acid molecules derived from multiple HER2 genes from genome by PCR method and FITC labeling] Nucleic acid molecules (DNA probes iv) having the number of bases derived from the HER2 gene were prepared using the primer sets 1 to 5 shown in Table 2 below that encode a partial DNA sequence of the HER2 gene. In each PCR for preparing DNA probes i to v, “dUTP-12-FITC” (manufactured by Roche Applied Science) is used as a nucleic acid substrate for PCR. Labeled with FITC.
  • Biotin-labeled anti-FITC antibody As a biotin-labeled anti-FITC antibody, the “biotin-attached anti-FITC antibody” of Comparative Example 1 (Vector: cord BA-0601) was used.
  • Example 2 [Amplification of nucleic acid molecules derived from multiple HER2 genes from genome by PCR method and FITC labeling] Nucleic acid molecules derived from the HER2 gene (DNA probes iv) were prepared using the above primer sets 1 to 5 encoding a partial DNA sequence of the HER2 gene. In each PCR, the DNA probe was labeled with FITC using “dUTP-12-FITC” (Roche Applied Science) as a nucleic acid substrate for PCR.
  • Biotin-labeled anti-FITC antibody As a biotin-labeled anti-FITC antibody, the “biotin-attached anti-FITC antibody” of Comparative Example 1 (Vector: cord BA-0601) was used.
  • Example 2 [Streptavidin (SA) -labeled PID]
  • a probe reagent D having the above-mentioned mixed solution of FITC-labeled DNA probes iv, biotin-labeled anti-FITC antibody solution, and SA-labeled PID dispersion was prepared.
  • FISH and observation were performed using the probe reagent D instead of the probe reagent B used in Comparative Example 2.
  • the total amount ( ⁇ g) of DNA probes i to v used was about 5 times that of Comparative Example 1 (about 250 ng).
  • Comparative Example 3 In Comparative Example 3, unlike Comparative Example 1, a linker longer than Comparative Example 1 is interposed between the DNA probe and Qdot in Comparative Example 1, and the DNA probe, PEG linker, and Qdot are combined to form a single body. This is an example in which the sample is used as a probe reagent. Probe reagent preparation, FISH, observation, etc. were performed as follows.
  • probe reagent [Amplification of nucleic acid molecule derived from HER2 gene from genome by PCR method and thiol labeling]
  • “Thiol-11-dUTP” prepared as follows was used instead of dUTP-FITC (used at the same concentration as dUTP-FITC in Comparative Example 1).
  • PCR was performed to prepare a thiol-labeled HER2 gene-derived DNA probe i ′.
  • a gel filtration column was prepared by mixing 1 ⁇ L of Lumiprobe “Amino-11-dUTP” with N-succimidyl S acetylthioacetic acid (SATA) and incubating at 5 ° C. for 1 hour as a thiol group addition treatment. A Thiol-11-dUTP solution was obtained.
  • PEG linker “EZ-Link maleimide-PEG11 Biotin” (code21911, manufactured by Thermo Fisher Scientific) having maleimide group at one end and biotin at the other end was mixed with the DNA probe i ′ (5 ⁇ g) for 30 minutes at room temperature.
  • the PEG linker was bound to the DNA probe i ′ via the binding reaction between the thiol group of the DNA probe i ′ and the maleimide group of the PEG linker.
  • the length of the PEG linker (the length of the portion derived from the linker) was 55.5 angstroms.
  • the probe reagent E was used in the FISH of Comparative Example 1, that is, the FITC-labeled probe reagent i, the biotin-labeled anti-FITC antibody, and the SA-labeled Qdot performed in Comparative Example 1 were divided in this order.
  • Comparative Example 1 except that FISH and observation were performed using a DNA probe i ′ conjugated with a PEG linker and Qdot (probe reagent E) as an alternative to the adding operation (use of probe reagent A) FISH, observation, etc. were performed in the same manner as above.
  • the total amount ( ⁇ g) of DNA probes used in Comparative Example 3 was the same as that of Comparative Example 1.
  • Comparative Example 4 In Comparative Example 4, unlike Comparative Example 2, a linker longer than Comparative Example 2 is interposed between the DNA probe and PID in Comparative Example 2, and the DNA probe, PEG linker, and PID are combined to form a single body. This is an example in which the sample is used as a probe reagent. Probe reagent preparation, FISH, observation, etc. were performed as follows.
  • probe reagent [Amplification of nucleic acid molecule derived from HER2 gene from genome by PCR method and thiol labeling]
  • “Thiol-11-dUTP” prepared in Comparative Example 3 was used in place of dUTP-FITC (used at the same concentration as dUTP-FITC in Comparative Example 2).
  • PCR was performed to prepare a thiol-labeled HER2 gene-derived DNA probe i ′.
  • PEG linker “EZ-Link maleimide-PEG11 Biotin” (code21911, manufactured by Thermo Fisher Scientific) having maleimide group at one end and biotin at the other end was mixed with the DNA probe i ′ (5 ⁇ g) for 30 minutes at room temperature. By reacting, the PEG linker was bound to the DNA probe i ′ via a binding reaction between the thiol group of the DNA probe i ′ and the maleimide group of the PEG linker. The length of the PEG linker (the length of the portion derived from the linker) was 55.5 angstroms.
  • the probe reagent F was used in the FISH of Comparative Example 2, that is, the FITC-labeled probe reagent i, the biotin-labeled anti-FITC antibody, and the SA-labeled PID performed in Comparative Example 2 were added in this order.
  • FISH and observation were performed using a DNA probe i ′ conjugated with a PEG linker and PID (probe reagent F) instead of the operation (use of probe reagent B) FISH and observation were performed.
  • the total amount ( ⁇ g) of DNA probe i ′ used in Comparative Example 4 was almost the same as that in Comparative Example 2.
  • Example 3 In Example 3, unlike Example 1, a linker longer than that in Example 1 is interposed between the DNA probe and Qdot in Example 1, and the DNA probe, PEG linker, and Qdot are combined to form a single body. This is an example in which the sample is used as a probe reagent. Probe reagent preparation, FISH, observation, etc. were performed as follows.
  • probe reagent Amplification of nucleic acid molecules derived from multiple HER2 genes from genome by PCR method and thiol labeling
  • the PCR method performed in Example 1 was the same except that Thiol-11-dUTP prepared in Comparative Example 3 was used instead of dUTP-FITC (it was used at the same concentration as dUTP-FITC in Comparative Example 2).
  • dUTP-FITC dUTP-FITC
  • the probe reagent G was used in the FISH of Example 1, that is, the FITC-labeled probe reagent i, the biotin-labeled anti-FITC antibody, and the SA-labeled Qdot performed in Example 1 were added in this order.
  • FISH and observation were performed using a DNA probe i ′ to v ′ in which a PEG linker and a PID were bound (mixture thereof) (probe reagent G). Except for the above, FISH and observation were performed in the same manner as in Example 1.
  • the total amount ( ⁇ g) of DNA probes i ′ to v ′ used in Example 3 was about 5 times (about 250 ng) that of Example 1.
  • Example 4 In Example 4, unlike Example 2, a linker longer than that in Example 2 is interposed between the DNA probe and PID in Example 2, and the DNA probe, PEG linker, and PID are combined to form a single body. This is an example in which the sample is used as a probe reagent. Probe reagent preparation, FISH, observation, etc. were performed as follows.
  • probe reagent [Amplification and thiol labeling of nucleic acid molecules derived from multiple HER2 genes from genome by PCR method] In the same manner as in Example 3, DNA probes i ′ to v ′ derived from thiol-labeled HER2 gene were prepared.
  • the probe reagent H was used in the FISH of Example 2, that is, the FITC-labeled probe reagent i, biotin-labeled anti-FITC antibody, and SA-labeled PID, which were performed in Example 2, were added in this order.
  • FISH and observation were carried out using DNA probes i ′ to v ′ each having a PEG linker and PID bound thereto (mixture thereof) (probe reagent H). Except for the above, FISH and observation were performed in the same manner as in Example 2.
  • the total amount ( ⁇ g) of DNA probes i ′ to v ′ used in Example 4 was about 5 times (about 250 ng) that of Example 2.
  • the probe reagent according to the present invention has been described above based on the embodiments and examples. However, the present invention is not limited to these embodiments and examples, and the gist of the present invention described in the scope of claims. Design changes are allowed without departing from the above.

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Abstract

L'invention concerne un réactif sonde pour FISH, dans lequel le nombre de signaux de tache de lumière est augmenté par rapport à l'état de la technique, ce qui permet d'obtenir des mesures plus précises. Ce réactif sonde pour hybridation in situ contient une pluralité de molécules d'acide nucléique ayant différentes séquences, et une ou deux nanoparticules de phosphore qui sont liées ou susceptibles de se lier à ladite molécule d'acide nucléique.
PCT/JP2016/064996 2015-05-28 2016-05-20 Réactif sonde pour hybridation in situ WO2016190236A1 (fr)

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JPWO2020138444A1 (ja) * 2018-12-28 2021-11-04 株式会社クラレ 水溶性フィルムおよび包装体
CN114229827A (zh) * 2021-10-25 2022-03-25 广东石油化工学院 一种碳纳米点、基于碳纳米点制备的双模探针及其应用
EP3912995A4 (fr) * 2019-01-18 2022-06-15 Konica Minolta, Inc. Nanoparticules intégrées au phosphore pour la détection d'une substance cible

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WO2015159776A1 (fr) * 2014-04-16 2015-10-22 コニカミノルタ株式会社 Nanoparticules d'agregat de phosphore, reactif colorant utilisant celles-ci, trousse et procede d'immunocoloration fluorescente

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WO2008063378A2 (fr) * 2006-11-01 2008-05-29 Ventana Medical Systems, Inc. Haptènes, conjugués de haptène, compositions de haptène, procédé de fabrication et utilisation
WO2014139979A1 (fr) * 2013-03-12 2014-09-18 Ventana Medical Systems, Inc. Hybridation in situ de points quantiques
WO2015141856A1 (fr) * 2014-03-20 2015-09-24 コニカミノルタ株式会社 Réactif sonde, et réactif sonde utilisant la technique fish
WO2015159776A1 (fr) * 2014-04-16 2015-10-22 コニカミノルタ株式会社 Nanoparticules d'agregat de phosphore, reactif colorant utilisant celles-ci, trousse et procede d'immunocoloration fluorescente

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Publication number Priority date Publication date Assignee Title
JPWO2020138444A1 (ja) * 2018-12-28 2021-11-04 株式会社クラレ 水溶性フィルムおよび包装体
JP7240422B2 (ja) 2018-12-28 2023-03-15 株式会社クラレ 水溶性フィルムおよび包装体
EP3912995A4 (fr) * 2019-01-18 2022-06-15 Konica Minolta, Inc. Nanoparticules intégrées au phosphore pour la détection d'une substance cible
CN114229827A (zh) * 2021-10-25 2022-03-25 广东石油化工学院 一种碳纳米点、基于碳纳米点制备的双模探针及其应用

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