HK1097046B - Method for detecting biomolecule, labeling dye used therefor, and labeling kit - Google Patents

Description

Biomolecule detection method, and labeling dye and labeling kit used in same

Technical Field

The present invention relates to a method for detecting biomolecules such as nucleic acids, proteins, peptides, and saccharides using a fluorescent dye, and a labeled dye and a labeled kit used for the detection method.

Background

Background genomic research aimed at specific gene analysis techniques, gene therapy, and tailored medicine (テ - ラ - メイド) is currently being actively carried out worldwide. As a gene analysis technique, for example, a DNA detection method using a DNA microarray is used. By this detection method, simultaneous analysis of expression, functionality, mutation, and the like of a plurality of genes can be easily and rapidly performed.

The detection method using a DNA microarray employs a DNA chip in which a large number of sequences of DNA or oligonucleotides (probe nucleic acids) are spotted on a substrate such as glass or silicon wafer. By hybridizing the probe nucleic acid immobilized on the substrate with the labeled RNA sample (target nucleic acid), the target nucleic acid having a base sequence complementary to the probe nucleic acid is selectively bound to the probe nucleic acid. The microarray is then dried and the fluorescence intensity of the labeled target nucleic acid is measured.

Fluorescent dyes are widely used for labeling, and are required to have high fluorescence intensity, emit light even in a dry state (solid state), have water solubility, and the like. As the fluorescent dye, for example, Cy3 or Cy5 can be used (see, for example, non-patent document 1).

Non-patent document 1: science283, 1, January, 1999, 83-87

Disclosure of Invention

However, although Cy3 or Cy5 has the advantage of high fluorescence intensity and the ability to emit light even in a solid state, it is very expensive, and therefore the detection method has to be expensive. Further, the incorporation rate of the fluorescent dye into the RNA sample is low, and the RNA sample cannot be sufficiently labeled, so that there is a problem that the detection sensitivity is insufficient. On the other hand, a fluorescent dye replacing Cy3 or Cy5 has not been found to be the current situation.

Accordingly, an object of the present invention is to solve the above problems and to provide a method for detecting a biomolecule at a lower cost and with high sensitivity.

The present inventors have searched for fluorescent dyes that replace Cy3 or Cy5, and have found that organic EL dyes (organic electroluminescent dyes) used for organic electroluminescent elements have high fluorescence intensity when used as labels for biomolecules, thereby completing the present invention.

That is, the method for detecting a biomolecule according to the present invention is characterized by reacting a biomolecule sample with an organic EL dye and measuring fluorescence of the biomolecule sample labeled with the organic EL dye. Here, the biomolecule of the present invention refers to a molecular species existing in a living body, and includes a molecular species for constructing a structure of a living body, a molecular species involved in energy generation and conversion, and a molecular species for controlling biological information. Specifically, the nucleic acid includes nucleic acids, proteins, saccharides, lipids, peptides, nucleotides, metabolic intermediates or metabolic enzymes, hormones, neurotransmitters, and the like.

Further, between the organic EL dye and the biomolecule, an amide bond, an imide bond, a urethane bond, an ester bond, or a guanidine bond can be formed. Before the reaction with the biomolecule, any 1 kind of reactive group selected from the group consisting of an isocyanate group, an isothiocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group and an active esterified carbonyl group can be introduced into the organic EL dye. In addition, the biomolecule sample may employ any 1 selected from the group consisting of nucleic acids, proteins, peptides and saccharides.

Further, the biomolecule detecting method of the present invention is characterized in that a biomolecule sample is labeled with a labeling dye containing a five-membered ring compound having a conjugated system containing 1 or more kinds of hetero atom, selenium atom or boron atom, and fluorescence of the labeled biomolecule sample is measured.

Further, a fused ring compound containing the above five-membered ring compound and a six-membered ring compound having a conjugated system may also be used. Further, the five-membered ring compound may be an azole derivative or an imidazole derivative. Before the reaction with the biomolecule, any 1 kind of functional group selected from the group consisting of an isocyanate group, an isothiocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group and an active esterified carbonyl group can be introduced into the organic EL dye.

The labeled dye of the present invention is used for detecting a biomolecule by fluorescence detection, and is characterized by containing an organic EL dye having a reactive group that binds to the biomolecule. The reactive group can adopt any 1 functional group selected from carboxylic acid group, isocyanate group, isothiocyanate group, epoxy group, halogenated alkyl group, triazine group, carbodiimide group and active esterified carbonyl group. In addition, the organic EL dye may employ a compound containing a five-membered ring compound having a conjugated system, the five-membered ring compound containing 1 or more of a hetero atom, a selenium atom or a boron atom. Further, a fused ring compound containing the five-membered ring compound and a six-membered ring compound having a conjugated system may also be used. Further, the five-membered ring compound may employ an azole derivative or an imidazole derivative.

The labeling kit for biomolecules of the present invention is characterized by containing an organic EL dye for labeling biomolecules. The biomolecule may be any 1 selected from nucleic acids, proteins, peptides and saccharides. The reactive group can adopt any 1 functional group selected from carboxylic acid group, isocyanate group, isothiocyanate group, epoxy group, halogenated alkyl group, triazine group, carbodiimide group and active esterified carbonyl group. In addition, the organic EL dye may employ a compound containing a five-membered ring compound having a conjugated system, the five-membered ring compound containing 1 or more of a hetero atom, a selenium atom or a boron atom. Further, a fused ring compound containing the five-membered ring compound and a six-membered ring compound having a conjugated system may also be used. Further, the five-membered ring compound may employ an azole derivative or an imidazole derivative.

In addition, another biomolecule detection method of the present invention is characterized in that a biomolecule sample is reacted with a probe labeled with an organic EL dye, and fluorescence of the biomolecule sample is measured. Here, the biomolecule sample is a nucleic acid, and the probe may be an oligonucleotide or PNA (peptide nucleic acid) complementary to the base sequence of the nucleic acid. In addition, when the oligonucleotide is a primer or a terminator, the nucleic acid can be amplified and fluorescence can be measured. In addition, before the amplification of the nucleic acid, the primer may be labeled with an organic EL dye. Furthermore, the oligonucleotide or PNA may be constituted by a molecular beacon.

Another method for detecting a biomolecule according to the present invention is characterized by comprising a step of separating a biomolecule sample into different sizes by electrophoresis, and labeling the biomolecule sample with an organic EL dye before or after the electrophoresis. Here, the biomolecule sample is a nucleic acid, and the base sequence of the nucleic acid can be determined from an electrophoretogram of the labeled nucleic acid. Furthermore, the biomolecule sample is a protein, and mass spectrometry of the separated protein can be performed based on an electrophoretogram of the labeled protein.

When the labeling kit of the present invention is used as a kit for a DNA microarray, for example, a probe nucleic acid is immobilized on the microarray using a nucleic acid as a biomolecule sample, and a target nucleic acid as a sample is reacted with an organic EL dye to label the sample, and the labeled target nucleic acid is spotted on the microarray to perform hybridization. The labeling kit of the present invention can also be used as a biological assay kit such as ELISA (enzyme-linked immunosorbent assay) or western blotting using avidin modified with the dye, which is bound between avidin (streptavidin) and biotin. In addition, the labeling kit of the present invention can also be used as a kit for protein microarrays.

Further, the staining method of the present invention is characterized in that a biomolecule in a tissue or cell sample is labeled with an organic EL dye. Here, the above-mentioned biomolecule may use nucleic acid or protein.

The staining dye of the present invention is used for staining a tissue or cell sample, and is characterized by containing an organic EL dye having a reactive group that binds to a biomolecule in the tissue or cell.

The present invention can obtain the following effects by using an organic EL dye as a labeling dye for a biomolecule.

That is, the organic EL dye has a high quantum yield in a solid state (including solid and semi-solid) and has high fluorescence intensity. Further, the organic EL dye is cheaper than Cy3 and Cy5, and thus detection of biomolecules can be performed at a lower cost. Furthermore, since the organic EL dye reacts with the biomolecule substantially quantitatively and has a high incorporation efficiency, high detection sensitivity can be obtained. In addition, the degree of freedom in selecting the fluorescence wavelength is increased, and various fluorescence wavelengths such as orange, yellow, green, and blue can be used. Thus, 2 or more kinds of fluorescent dyes having a large Stokes shift (a large difference between an excitation wavelength and a fluorescence wavelength) can be used, so that a plurality of target nucleic acids contained in one sample can also be detected at the same time. Further, compared to Cy3 and Cy5, the organic EL dye needs to be stored in a frozen state, and is chemically stable and can be stored for a long period of time at room temperature, and therefore, the organic EL dye is easy to handle.

Drawings

FIG. 1A is an HPLC chromatogram of the labeled oligonucleotide of example 1 of the present invention.

FIG. 1B is a UV spectrum of a target of the labeled oligonucleotide in example 1 of the present invention.

FIG. 2 is a TOF MS spectrum of a labeled oligonucleotide in example 1 of the present invention.

FIG. 3 is a luminescence pattern of the labeled oligonucleotide in example 1 of the present invention. (a) The results of (b), (c) and (d) are 110fmol, 10fmol, 1fmol and 0.5fmol, respectively.

FIG. 4A is an HPLC chromatogram of the labeled oligonucleotide of example 2 of the present invention.

FIG. 4B is a UV spectrum of a target of the labeled oligonucleotide in example 2 of the present invention.

FIG. 5 is a luminescence pattern of the labeled oligonucleotide in example 2 of the present invention. (a) The results of (b), (c), (d) and (e) are 500fmol, 250fmol, 100fmol, 50fmol and 10fmol, respectively.

FIG. 6A is an HPLC chromatogram of a labeled peptide of example 3 of the present invention before purification.

FIG. 6B is an HPLC chromatogram of the labeled peptide of example 3 of the present invention after purification.

FIG. 7 is a TOF MS spectrum of a labeled peptide in example 3 of the present invention.

FIG. 8 is a luminescence pattern of the labeled peptide of example 3 of the present invention. (a) The results of (b), (c), (d) and (e) are 10fmol, 5fmol, 1fmol, 0.5fmol and 0.1fmol, respectively.

FIG. 9A is a TOF MS spectrum of a protein labeled in example 4 of the present invention before labeling.

FIG. 9B is a TOF MS spectrum of a protein labeled in example 4 of the present invention after labeling.

FIG. 10 is a luminescence pattern of the labeled protein in example 4 of the present invention.

FIG. 11 is a schematic diagram showing the mechanism of light emission when a molecular beacon is used as a probe in the detection method of the present invention.

FIG. 12 is a schematic diagram showing an example of a method for producing a Fab' fragment of an IgG antibody in the detection method of the present invention.

FIG. 13 is a schematic diagram showing an example of a method for introducing an organic EL dye into a Fab' fragment of an IgG antibody in the detection method of the present invention.

Description of the symbols

1 substrate

2 electrode

3 electrodes

4 Probe nucleic acid

4a organic EL dye

5 target nucleic acid

Detailed Description

The embodiments of the present invention will be described in detail below.

The organic EL dye used in the present invention is not particularly limited as long as it is a dye that is sandwiched between a pair of an anode and a cathode in a solid state and can emit light by energy generated when holes injected from the anode and electrons injected from the cathode are recombined. For example, the following may be employed: polycyclic aromatic compounds such as tetraphenylbutadiene and perylene, cyclopentadiene derivatives, distyrylpyrazine derivatives, acridone derivatives, quinacridone derivatives, stilbene derivatives, phenothiazine derivatives, pyrazinopyridine derivatives, oxazole derivatives, imidazole derivatives, carbazole derivatives, tetraphenylthiophene derivatives, and the like. And preferably a dye containing carboxylic acid groups or carboxylic acid groups that can be introduced into the molecule. This is because, as described below, a reactive group for binding to a biomolecule is easily introduced.

The organic EL dye preferably has a reactive group for binding to a biomolecule sample (hereinafter referred to as a target molecule), and the reactive group preferably has a functional group capable of reacting with an amino group, an imino group, a thiol group or a hydroxyl group of the target molecule. It is preferable to form an amide bond, an imide bond, a urethane bond, an ester bond or a guanidine bond between the organic EL dye and the biomolecule. The functional group can be, for example, an isothiocyanate group, an isocyanate group, an epoxy group, a halosulfonyl group, an acid chloride group, a haloalkyl group, a glyoxal group, an aldehyde group, a triazine group, a carbodiimide group, an activated esterified carbonyl group, or the like. It is preferable to use any 1 selected from the group consisting of an isothiocyanate group, an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group and an active esterified carbonyl group. More preferably, 1 kind of compound selected from the group consisting of an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group and an activated esterified carbonyl group is used. This is because it can form an amide bond with an amino group of a target molecule or can directly bind to an imino group within a biomolecule. Further preferred are triazinyl groups, carbodiimide groups and activated esterified carbonyl groups. When these organic EL dyes contain a carboxylic acid group, an amino group and an imino group present in a biomolecule may be directly modified in the presence of a carbodiimide derivative or a triazine derivative. Further, the organic EL dye having a triazinyl group which may have a substituent and a carbodiimide group which may have a substituent is capable of reacting directly with an imino group in guanine or thymine among DNA bases, so that a mismatch can be detected (a base which does not form a double strand is detected) without introducing a dye by a PCR (polymerase chain reaction) method, and it can be used as a reagent for analyzing an SNP (single nucleotide polymorphism).

For example, an N-hydroxysuccinimide ester or maleimide ester may be used as the active esterified carbonyl group. Since N-hydroxysuccinimide is used, the EL dye and the target molecule are bound by an amide bond via an N-hydroxysuccinimide ester by using DCC as a condensing agent as shown in formula I of scheme 1 below. In addition, as shown in scheme 1 of formula II, through the active esterification of carbonyl can also use three triazine derivatives. As the carbodiimide group, a carbodiimide reagent such as N, N' -Dicyclohexylcarbodiimide (DCC) or 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide can be used. The EL dye can be bound to the target molecule via carbodiimide in an amide bond (formula III). The EL dye having a carbodiimide group or triazine group introduced into a molecule in advance may be directly bonded to an amino group or an imino group in a biomolecule (formula IV).

Further, by changing the substituent of the organic EL dye, the excitation wavelength and the emission wavelength can be changed, so that simultaneous detection of a plurality of samples can be performed on all substrates by multicolor formation.

[ chemical formula 1]

Scheme 1

The reactive group can be bonded to an amino group residue modified at the end of the oligo-DNA when the target molecule is DNA, an amino group residue when the target molecule is protein, an amino group of the polypeptide when the target molecule is peptide, an amino group residue of, for example, a polylysine derivative, and an amino group in the backbone of a polysaccharide derivative when the target molecule is sugar.

The organic EL dye preferably used in the detection method of the present invention is a compound containing a five-membered ring compound having a conjugated system, and examples of the five-membered ring compound include five-membered ring compounds containing 1 or more kinds of hetero atoms, selenium atoms or boron atoms. More specifically, there are mentioned a monocyclic compound containing a five-membered ring compound having a conjugated system and a fused ring compound containing the five-membered ring compound and a six-membered ring compound having a conjugated system. This is because such an organic EL dye has a large quantum yield even in a solid state, and thus exhibits strong fluorescence. The five-membered ring compound is preferably an azole derivative or an imidazole derivative. Further, the azole derivative and the imidazole derivative preferably contain 1 or more quaternary ammonium groups. This is because the water solubility can be improved.

Specific examples of the fused ring compound are described below.

(Monozole derivative 1)

[ chemical formula 2]

Here, in the formula R₁、R₂、R₃、R₄、R₆、R₇Each independently represents an aromatic hydrocarbon group or a heterocyclic group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group or a sulfonyl group, or an aromatic group containing a hetero atom in the ring. R₁、R₂、R₃、R₄、R₆、R₇May be the same or different.

R' represents An aliphatic or aromatic hydrocarbon group such as An alkyl or alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-. Unless otherwise specified, the following general formula is the same as above.

(Monozole derivative 2)

[ chemical formula 3]

Here, in the formula R₈、R₉Each represents an aromatic hydrocarbon group or a hydrocarbon group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group, a sulfonyl group or the like, a heterocyclic group or an aromatic group containing a hetero atom in the ring. R₈、R₉May be the same or different. Unless otherwise specified, the following general formula is the same as above. N is an integer of 1 or more, preferably 1 to 5, and the same applies to the following general formula.

(diazole derivative 1)

[ chemical formula 4]

(diazole derivative 2)

[ chemical formula 5]

(diazole derivative 3)

[ chemical formula 6]

Here, in the formula R₁、R₂、R₃、R₄Each independently represents an aromatic hydrocarbon group or a heterocyclic group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group or a sulfonyl group, or an aromatic group containing a hetero atom in the ring. R₁、R₂、R₃、R₄、R₆、R₇May be the same or different. R₂、R₃It is preferable to use an aromatic hydrocarbon group which may have a substituent, and the substituent is preferably an alkyl group or an alkoxy group having 1 to 4 carbon atoms, or a bromine atom. Further, methyl is preferably used as the alkyl group, and methoxy is preferably used as the alkoxy group. Further, X is a nitrogen atom, a sulfur atom, an oxygen atom, a selenium atom or a boron atom which may have a substituent, and the same meanings as described above in the following general formula are also applicable unless otherwise specified.

(diazole derivative 4)

[ chemical formula 7]

(diazole derivative 5)

[ chemical formula 8]

Here, N → O represents a state where a nitrogen atom forms a coordinate bond with an oxygen atom.

(diazole derivative 6)

[ chemical formula 9]

(oxadiazole derivatives 7)

[ chemical formula 10]

(diazole derivative 8)

[ chemical formula 11-1]

[ chemical formula 11-2]

Here, in the formula R₁₀、R₁₁Each represents an aromatic hydrocarbon group or a hydrocarbon group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group or a sulfonyl group, a heterocyclic group or an aromatic group containing a hetero atom in the ring. R₁₀、R₁₁May be the same or different. Furthermore, R₁₂Is an alkenyl or alkanyl group which may have a substituent, and n is an integer of 1 to 3, preferablyIs selected as 1. Unless otherwise specified, the following general formula is the same as above.

(oxadiazole derivatives 9)

[ chemical formula 12-1]

[ chemical formula 12-2]

The oxadiazole derivative is not particularly limited as long as it is an oxadiazole derivative represented by the following general formula.

[ chemical formula 13]

After synthesis of the carboxylic acid derivative of the oxazolopyridine derivative, for example, by the reaction shown in the following scheme 2, a derivative derived to form an active ester body including an N-hydroxysuccinimide ester can be used using N, N' -Dicyclohexylcarbodiimide (DCC) as a condensing agent.

[ chemical formula 14]

Flow chart 2

(triazole derivative 1)

[ chemical formula 15]

(triazole derivative 2)

[ chemical formula 16]

The five-membered ring compound may also use the following derivatives containing a thienyl group.

(thiophene derivative 1)

[ chemical formula 17]

(thiophene derivative 2)

[ chemical formula 18]

(thiophene derivative 3)

Further, when a thiophene derivative is used, a non-condensed type compound such as a 2, 3, 4, 5-tetraphenyl thiophene derivative represented by the following general formula may also be used.

[ chemical formula 19]

Here, in the formula R₁₃、R₁₄、R₁₅Each independently represents a hydrogen atom, a linear, branched or cyclic alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group; ar (Ar)₁And Ar₂Represents a substituted or unsubstituted aryl group, and Ar₁And Ar₂May form a nitrogen-containing heterocyclic ring together with the nitrogen atom to which it is bonded. Furthermore, Y₁And Y₂Represents a hydrogen atom, a halogen atom, a linear, branched or cyclic alkyl group, a linear, branched or cyclic alkoxy group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, or a substituted or unsubstituted amino group.

(thiophene derivative 4)

Further, 2, 3, 4, 5-tetraphenylthiophene derivatives represented by the following general formula may be used.

[ chemical formula 20]

Here, in the formula Ar₁～Ar₆Each independently represents a substituted or unsubstituted aryl group, and Ar₁And Ar₂、Ar₃And Ar₄And Ar₅And Ar₆May form a nitrogen-containing heterocyclic ring together with the nitrogen atom to which it is bonded.

Further, imidazole and imidazole derivatives represented by the following general formula may be used as the five-membered ring compound. Here, the imidazole group constituting the imidazole derivative preferably contains a quaternary ammonium group. This is because the water solubility can be improved. When a pyridyl group is contained, the pyridyl group may contain a quaternary ammonium group to further improve water solubility. In the general formula below, R "represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group such as an alkyl group or an alkenyl group which may have an aromatic ring.

(imidazole derivative 1)

[ chemical formula 21]

(imidazole derivative 2)

[ chemical formula 22]

(imidazole derivative 3)

[ chemical formula 23]

(imidazole derivative 4)

[ chemical formula 24-1]

[ chemical formula 24-2]

Here, the imidazole skeleton may be in the central benzene ring R₈、R₉、R₁₀、R₁₁In combination with a plurality of units at arbitrary positions. In addition, R₁₂Is an optionally substituted alkenyl or alkanyl group, and n is an integer of 1 to 3, preferably 1.

(carbazole derivative)

Further, carbazole derivatives represented by the following general formula may also be used.

[ chemical formula 25]

Five-membered ring compounds having a conjugated system, i.e., monocyclic compounds containing 1 or more hetero atoms, selenium atoms or boron atoms, may also be used. There are no particular restrictions on the examples, and azole derivatives represented by the following general formula can be used.

[ chemical formula 26]

Here, in the formula R₁、R₄、R₅Each independently represents an aromatic hydrocarbon group or a heterocyclic group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group or a sulfonyl group, or an aromatic group containing a hetero atom in the ring. R₁、R₄、R₅May be the same or different.

The detection method of the present invention can be applied to all methods for detecting biomolecules as long as it is a detection method for measuring fluorescence of labeled biomolecules in a solid or semisolid state. By using an organic EL dye instead of a conventional fluorescent dye, a detection method having high sensitivity, chemical stability, excellent operability and low cost can be provided. The method usable in the present invention may be a method in which a biomolecule sample is directly reacted with an organic EL dye to label the biomolecule sample with the organic EL dye, or a method in which a biomolecule sample is reacted with a probe labeled with an organic EL dye to label the biomolecule sample with the organic EL dye, as described above. Further, a method of separating a biomolecule sample labeled with an organic EL dye by electrophoresis in size can also be employed.

For example, in the DNA microarray method in which nucleic acids are detected, the following procedure is performed.

(DNA microarray method)

The detection method comprises reacting a target nucleic acid to be detected with an organic EL dye to label the target nucleic acid with the organic EL dye, preparing a single-stranded probe nucleic acid having a base sequence complementary to the target nucleic acid, hybridizing the single-stranded target nucleic acid with the probe nucleic acid on a substrate, and measuring the fluorescence of the target nucleic acid. In the present detection method, when gene expression is to be investigated, the probe nucleic acid immobilized on the substrate may be prepared as follows: that is, cDNA is amplified by PCR using a cDNA library, a genomic library or the whole genome as a template. In addition, for the purpose of studying gene mutation or the like, various oligonucleotides corresponding to the mutation or the like can be synthesized based on a standard known sequence.

The method for immobilizing the probe nucleic acid on the substrate may be appropriately selected depending on the type of the nucleic acid and the type of the substrate. For example, a method of electrostatically bonding a DNA to a substrate surface-treated with a cation such as polylysine by using the charge of the DNA can be used. On the other hand, the target nucleic acid denatured into single strands is mixed with an organic EL dye and reacted to prepare a target nucleic acid labeled with an organic EL dye. The reaction is preferably carried out at room temperature to 60 ℃ for 2 to 48 hours.

Then, the labeled target nucleic acid is spotted on the substrate, and hybridization is performed. Hybridization is preferably carried out at room temperature to 70 ℃ for 2 to 48 hours. By hybridization, a target nucleic acid having a base sequence complementary to the probe nucleic acid selectively binds to the probe nucleic acid. The substrate was then cleaned and dried at room temperature. Subsequently, the fluorescence intensity of the dried substrate surface was measured by a fluorescence laser scanning method. The level of gene expression can be monitored by fluorescence intensity. The above hybridization describes a method for immobilizing a probe nucleic acid on a substrate, and a method for spotting a probe nucleic acid on a substrate by immobilizing a target nucleic acid labeled with an organic EL dye on the substrate may be used.

In addition, PCR using primers and terminators can be performed in the following order also with nucleic acids as detection targets.

(PCR method)

In the detection method, a probe complementary to the base sequence of a target nucleic acid to be detected is labeled with an organic EL dye, and the target nucleic acid is reacted with the probe before or after amplification of the target nucleic acid, thereby measuring the fluorescence of the target nucleic acid. Specifically, the extension reaction of the target nucleic acid is carried out by an enzyme (DNA polymerase or RNA polymerase), and in this case, the nucleic acid sequence of 2 strands formed by the target nucleic acid and the oligonucleotide-containing primer is recognized by the enzyme, and the extension reaction is carried out from the recognized position, whereby only the target gene region is amplified. When the enzyme is synthesized, the synthesis reaction is carried out by taking nucleotide (dNTP and NTP) as raw materials. In this case, when nucleotides containing dyes are mixed in an arbitrary ratio among ordinary nucleotides (dNTPs, NTPs) as shown in, for example, chemical formula 27, nucleic acids into which dyes are introduced in the ratio can be synthesized. Further, nucleic acid into which an organic EL dye is introduced may be synthesized by PCR by introducing amino group-containing nucleotides at an arbitrary ratio and then binding to the organic EL dye.

[ chemical formula 27]

When the enzyme is synthesized, a synthesis reaction is carried out using a nucleotide as a starting material, but if a nucleotide in which OH at the 3' -end of the nucleotide is changed to H is used, the extension reaction of the nucleic acid does not proceed any more, and the reaction is terminated at that time. This nucleotide, namely dideoxynucleoside triphosphate (ddNTP), is called a terminator. When a terminator is incorporated into a common nucleotide to carry out a nucleic acid synthesis reaction, the terminator is incorporated with a certain probability, and nucleic acids of different lengths are synthesized by terminating the reaction. When the obtained nucleic acids are separated by size by gel electrophoresis, the DNAs are arranged in the order of length. When the terminator is labeled with different organic EL dyes depending on the type of each base, a tendency of dependence on each base is observed at the end point (3' -end) of the synthesis reaction, and the fluorescence information is read from the organic EL dye labeled on the terminator as a base point, whereby the base sequence information of the target nucleic acid can be obtained. Alternatively, a primer labeled with an organic EL dye in advance may be used instead of the terminator to hybridize with the target nucleic acid.

In addition, PNA (peptide nucleic acid) can also be used as a probe. PNA is a substance obtained by replacing a pentose phosphate skeleton, which is a basic skeleton structure of nucleic acid, with a polyamide skeleton composed of glycine as a unit, has a three-dimensional structure very similar to that of nucleic acid, and can bind to nucleic acid having a complementary base sequence very specifically and strongly. Therefore, the probe is effective as a probe for detecting a specific nucleic acid. Therefore, the method can be used not only in the original DNA analysis methods such as in situ hybridization, but also as a telomere research reagent by applying the method to a telomere PNA probe.

The detection can be performed as follows: for example, a PNA-DNA complex is prepared by contacting a double-stranded DNA with PNA labeled with an organic EL dye and having a base sequence complementary to all or part of the base sequence of the DNA to hybridize, heating the mixture to form a single-stranded DNA, slowly cooling the mixture to room temperature to prepare a PNA-DNA complex, and measuring the fluorescence of the PNA-DNA complex.

In the above examples, the method of amplifying a target nucleic acid by PCR and measuring the fluorescence of the product was described, and it was necessary to examine the amount of the amplified product by determining the fluorescence intensity after confirming the size of the product by electrophoresis. In this regard, the amount of the product can also be measured in real time using a probe designed to hybridize with the product of the PCR method by energy conversion using a fluorescent dye to generate fluorescence. Among them, for example, DNAs that label the donor and the acceptor can be used. Specific examples of the detection method include a molecular beacon method, a TaqMan-PCR method, and a cycling probe method, which are used to confirm the presence of a nucleic acid having a specific sequence.

For example, an example in which a molecular beacon is immobilized on a substrate and hybridized with a target gene will be described with reference to FIG. 11 for the light emission mechanism of the molecular beacon method. A DNA (probe) having a specific DNA sequence is labeled with an organic EL dye F at one end and a quencher Q at the other end. The quencher Q is immobilized on the substrate, and before the target gene is introduced, the quencher Q is close to the organic EL dye F, and the fluorescence of the fluorescent dye disappears. Here, when a target gene having a sequence complementary to the labeled DNA is introduced, the labeled DNA hybridizes with the target gene, thereby separating the distance between the organic EL dye F and the quencher Q, and fluorescence of the organic EL dye F can be observed. This allows observation of the hybridization of the DNA and determination of the amount of hybridization.

In addition, when a protein is used as a detection target, a staining dye is used for protein detection after electrophoresis. In general, a method of irradiating a gel after electrophoresis with ultraviolet light to emit light by staining proteins with a staining dye such as Coomassie Brilliant Blue (CBB) and permeating the gel with the dye. However, although the conventional method using a dye is simple, the sensitivity is as low as about 100ng, and thus it is not suitable for detecting a trace amount of protein. Further, since the dye is permeated through the gel, there is a problem that dyeing requires a long time.

In contrast, in the present invention, proteins are separated into different sizes by electrophoresis, and then the separated proteins are bound to an organic EL dye to label the proteins. The organic EL dye of the present invention has a reactive group, can react with a protein rapidly and quantitatively, has high sensitivity, and is very suitable for the detection of a trace amount of protein. And can also be identified by mass spectrometry of the size-separated proteins.

Here, any of the following proteins can be detected: simple proteins such as albumin, globulin, gluten, histone, protamine, and collagen, and binding proteins such as nucleoprotein, glycoprotein, lipoprotein, phosphoprotein, and metalloprotein. For example, phosphoproteins, glycoproteins, and total proteins can be stained in a protein sample separated by two-dimensional electrophoresis using 3 organic EL dyes corresponding to staining dyes for phosphoproteins, glycoproteins, and total proteins. Further, since proteins can be identified by Mass spectrometry such as TOF-Mass, they are useful for diagnosis and treatment of diseases such as cancer or viral infectious diseases that produce specific proteins. In addition, collagen is a protein constituting animal connective tissue, and has a unique fibrous structure. I.e. it consists of 3 polypeptide chains which are interlaced together to form a triple heavy chain. Collagen is generally a protein with extremely low immunogenicity, and is widely used in the fields of foods, cosmetics, pharmaceuticals, and the like. However, even when a fluorescent dye is introduced into the peptide chain of collagen, the stability of the conventional fluorescent dye is not sufficient, and a more stable fluorescent dye is required. Therefore, by using an organic EL dye as a fluorescent dye for labeling collagen, detection can be performed stably and with high sensitivity.

In addition, the protein can also be labeled by labeling an antibody that specifically binds to the protein with an organic EL dye. For example, as shown in FIG. 12, IgG antibody is treated with pepsin to obtain a protein called F (ab')₂A fragment of (a). This fragment is reduced with dithiothreitol or the like to obtain a fragment called Fab'. Fab' fragments have 1 or 2 sulfhydryl groups (-SH). The thiol group can be specifically reacted by reacting it with a maleimide group. That is, as shown in fig. 13, by reacting the maleimide group-introduced organic EL dye with the thiol group in the fragment, the antibody can be labeled with the organic EL dye. At this time, the physiological activity (antigen capturing ability) of the antibody is not lost.

In the detection method of the present invention, an aptamer which specifically binds to a specific biomolecule (particularly, a protein) can be used as a probe. Aptamers, which contain oligonucleotides, can acquire a steric structure having various characteristics depending on the base sequence, and thus can bind to all biomolecules containing proteins through such a steric structure. By utilizing this property, an aptamer labeled with an organic EL dye can be bound to a specific protein, and the structure of the protein is changed by the binding to a substance to be detected, and fluorescence is also changed in accordance with the change in the structure, whereby the substance to be detected can be indirectly detected. For example, a biosensor for cocaine detection using energy conversion using an aptamer labeled with a fluorescent dye has been proposed (j.am.chem.soc.2001, 123, 4928-. By using an organic EL dye instead of the above fluorescent dye, a highly sensitive, easily handled biosensor can be provided.

In addition, the detection method of the present invention can be used for detection of metal ions. All life phenomena generated in vivo, such as stability of DNA and proteins in vivo, maintenance of higher-order structures, functional manifestation, and activation of enzymes that govern all chemical reactions in the organism, are related to metal ions. Therefore, a metal ion sensor that can observe the activity of metal ions in a living body in real time is being advertised as an important thing represented by the medical field. Conventionally, a metal ion sensor that incorporates a fluorescent dye into a biomolecule has been known. For example, it is proposed to use at K⁺Metal ion sensor using nucleic acid having set K in the presence of ion⁺Ion to form a sequence of specific structures (J.AM. CHEM. SOC.2002, 124, 14286-14287). A fluorescent dye causing energy conversion is introduced into both ends of the nucleic acid. Energy conversion is generally not caused due to the distance between the dyes. But at K⁺In the presence of the ion, the nucleic acid forms a special shape, and as a result, the fluorescent dye approaches a distance causing energy conversion, and fluorescence can be observed. In addition, a zinc ion sensor in which a fluorescent dye is introduced into a peptide has been proposed (J.Am.chem.Soc.1996, 118, 3053-3054). By using the marker dye comprising the organic EL dye of the present invention in place of these conventional fluorescent dyes, a metal ion sensor having higher sensitivity and easier handling than conventional ones can be provided. All metal ions can be detected as long as they are present in the living body.

In addition, intracellular signal observation can be performed by the detection method of the present invention. Giant molecules ranging from ions to enzymes are involved in cellular responses to internal signals and environmental information. It is known that during signal transmission, specific protein kinases are activated, leading to phosphorylation of specific cellular proteins and thusBears the initial response of various cellular responses. Binding and hydrolysis of nucleotides play a significant role in their activity, and by using nucleotide derivatives, signal transduction changes can be rapidly observed. For example, protein kinase c (pkc) plays an important role in signal transmission in cell membranes. The Ca²⁺The serine/threonine-dependent protein kinase is activated on membrane-constituting lipids such as diacylglycerol or phosphatidylserine, and signals are transmitted by changing the membrane surface charge by phosphorylating serine or threonine present in ion channels or cytoskeletal proteins.

The observation of cell signaling can be performed by dynamic observation in living cells.

Here, the nucleotide derivative is supplied as a substrate or inhibitor of the enzyme, binds to a nucleotide-binding protein moiety of an organelle such as mitochondria and a desmoplastic fibrous tissue, and regulates the detection of the structure and mechanics of an isolated protein and the remodeling of a membrane-bound protease. Furthermore, it has recently become apparent that there are also compounds which influence signal transmission, such as inhibitors or activators of G-proteins. By introducing a labeled dye comprising the organic EL dye of the present invention into the nucleotide derivative, dynamic observation of intracellular signaling can be performed with high sensitivity and with ease of handling.

In addition, the detection method of the present invention can also be used for observation of the expression state of a gene utilizing RNA interference effect (RNAi). RNAi refers to a phenomenon in which, when RNA is introduced into a cell, expression of a gene having the same sequence as that of RNA is suppressed. RNAi involves introducing double-stranded RNA (dsRNA) into a cell, and decomposes mRNA of a target gene to suppress expression. In this process, firstly, long dsRNA (double-stranded RNA) is cleaved into 21-23 mer short siRNA by a cutting enzyme having ribonuclease activity. It is known that the generated siRNA is recombined by an intermediate complex (RNA-induced silencing complex (RISC)), and mRNA having a sequence complementary to the antisense strand of the siRNA recombined by the complex is cleaved. In this field, fluorescent dyes are also used for observing the expression state of genes. By using an organic EL dye as a labeled fluorescent dye, stable and highly sensitive detection can be performed.

The labeling kit of the present invention contains an organic EL dye or a derivative thereof for labeling a biomolecule, and may contain a reagent, an enzyme, a solvent, and the like for reacting the dye with the target biomolecule, if necessary. The target biomolecule is a nucleic acid, a protein, a peptide or a saccharide. The organic EL dye is preferably a derivative having a functional group capable of reacting with an amino group of a biomolecule, and the functional group is preferably 1 selected from the group consisting of an isocyanate group, an isothiocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group and an activated and esterified carbonyl group. More preferably, the organic EL dye derivative contains an active ester containing a triazinyl group, a carbodiimide compound and an active esterified carbonyl group.

The marker dyes of the present invention can also be used as a tissue or cell staining dye in the study of the expression level of a target nucleic acid or target protein in a tissue or cell sample. Staining of tissues or cells can be performed by binding the organic EL dye to the target nucleic acid or target protein through the reactive group, as described above.

That is, the dye of the present invention exhibits more excellent performance than conventional dyes in that it can emit fluorescence even in a dry state, can be stored after labeling, and the like, when an organic EL dye is used for staining eukaryotic cells. Furthermore, the dye can be used not only for eukaryotic cells but also for cytoskeleton. In addition, the labeling can be used for labeling mitochondria, Golgi apparatus, endoplasmic reticulum, lysosomes, lipid bilayer membranes, and the like. These labeled cells and the like can be observed under all conditions of wetting and drying, and therefore have a large versatility. For observation, a fluorescence microscope or the like can be used.

In a clinical stage, tissues collected from a human body are cut into thin films by an instrument such as a microtome, and then stained. This frequently uses Cy dyes and Alexa dyes. However, the stability of the original dye is very poor, and the sample must be prepared again at the time of the return visit. Furthermore, the prepared samples cannot be preserved as specimens. In contrast, the organic EL dye is a very stable dye compared to the conventional dyes, and therefore, the stained tissue can be stored as a specimen.

Example 1

The present invention will be described in more detail with reference to examples.

Synthesis example 1

The organic EL dye adopts 1, 2, 5-oxadiazole- [3, 4-c ] pyridine derivative.

The following shows a synthetic scheme of an active ester compound of 1, 2, 5-oxadiazolo- [3, 4-c ] pyridine (hereinafter abbreviated as EL-OSu).

[ chemical formula 28]

Flow chart 3

(1) Synthesis of diketone derivative (2)

37.5g (0.25mol) of 4-methoxyacetophenone and 0.15g of sodium nitrite were dissolved in 100mL of acetic acid in a 500mL three-necked flask. Dissolving 100mL of HNO in 100mL of acetic acid under the condition of water bath₃The substance (d) was dropped into a three-necked bottle for 2 hours. Then, the mixture was stirred at room temperature for 2 days. The reaction mixture was slowly added to 500mL of water to cause precipitation. The precipitate was filtered and dissolved in chloroform. The chloroform phase was washed with saturated sodium bicarbonate solution and then with 10% aqueous NaCl solution 2 times. With MgSO₄After dehydration, chloroform was distilled off under reduced pressure to obtain 34.5g (yield: 78%) of oxadiazole-N-oxide (2).

(2) Synthesis of diketone derivative (3)

In a 500mL three-necked flask, 17.7g (0.05mol) of oxadiazole-N-oxide (2) was dissolved in 400mL of acetonitrile. To which 12.0 was addedg of Zn, 7mL of AcOH, 20mL of Ac₂And O. Cooling in a water bath until the reaction temperature does not exceed 30 ℃. Stirring for 12 hours till the end of the reaction. The reaction mixture was filtered to remove insoluble components. Acetonitrile was distilled off under reduced pressure to obtain a residue. The residue was recrystallized from chloroform to give 10.2g (yield: 60%) of oxadiazole-N-oxide (3).

(3) Synthesis of Oxadiazolopyridine ethyl ester (4)

15.6g (0.046mol) of oxadiazole-N-oxide (3) was dissolved in 300mL of butanol in a 500mL three-necked flask. 32.0g (0.23mol) of glycine ethyl ester hydrochloride was added thereto. Heating reflux was carried out for 24 hours. Butanol was distilled off under reduced pressure to obtain a residue. The residue was dissolved in 200mL of chloroform and then saturated NaHCO with 10% HCl₃And washing with 10% NaCl. With MgSO₄Drying and distilling off the solvent. The resulting residue was recrystallized from chloroform to give 13.0g (yield 70%) of oxadiazolyl-pyridylethyl ester (4).

(4) Hydrolysis of oxadiazolyl-pyridylethyl ester (4)

3.0g (0.007mol) of oxadiazolyl-pyridino-ethyl ester (4) were dissolved in 200mL of ethanol in a 500mL three-necked flask. 0.62g (0.01mol) of KOH was added thereto. After heating to reflux for 5 hours, the reaction mixture was added to 200mL of water. Concentrated hydrochloric acid was added dropwise to the aqueous solution to adjust the pH to 1, and then a precipitate was generated. The precipitate was filtered and dissolved in chloroform. The chloroform phase was treated with 10% NaHCO₃Washing with water solution and water. Chloroform was distilled off to obtain a residue. The residue was recrystallized from water-ethanol (1: 1). Oxadiazole pyridopyridine carboxylic acid (5) was obtained in 2.1g (yield 81%).

Synthesis of active ester (6)

1.0g (0.0026mol) of oxadiazolopyridine carboxylic acid (5) and 0.30g (0.0026mol) of N-hydroxysuccinimide are dissolved in 20mL of DMF in a 50mL three-necked flask. 0.54g (0.0026mol) of N, N' -dicyclohexylcarbodiimide was added dropwise thereto over 30 minutes. After dropping, the mixture was stirred at room temperature for 30 hours. DMF was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (chloroform) to give 0.76g (yield 62%) of oxadiazolyl pyridine active ester (6).

Synthesis example 2

As the organic EL dye, an imidazopyridine ethyl ester derivative is used. The following shows a synthetic scheme of an active ester compound of imidazopyridine ethyl ester (hereinafter abbreviated as im-EL-OSu).

[ chemical formula 29]

(1) Hydrolysis of Imidazopyridine Ethyl ester (1)

0.5g (1.5mol) of the ester body 1 was dissolved in 50mL of ethanol in a 500mL three-necked flask. 0.12g (2.1mol) of KOH was added thereto. After 5 hours of heating to reflux, the reaction mixture was added to 50mL of water. Concentrated hydrochloric acid was added dropwise to the aqueous solution to adjust the pH to 1, and then a precipitate was generated. The precipitate was filtered and dissolved in chloroform. The chloroform phase was treated with 10% NaHCO₃Washing with water solution and water. Chloroform was distilled off to obtain a residue. The residue was recrystallized from water to give 0.3g (yield: 63%) of carboxylic acid 2.

(2) Synthesis of active ester (3)

0.2g (0.6mmol) of carboxylic acid derivative 2 and 0.07g (0.6mmol) of N-hydroxysuccinimide are dissolved in 10mL of DMF in a 50mL three-necked flask. 0.12g (0.6mmol) of N, N' -dicyclohexylcarbodiimide was added dropwise thereto over 30 minutes. After dropping, the mixture was stirred at room temperature for 30 hours. DMF was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (chloroform) to give 0.14g (yield: 55%) of active ester 3.

Example 1

< dye labeling and detection of oligonucleotides (1) >

1. Dye labeling of oligonucleotides

Dye labeling of oligonucleotides was performed using the following scheme 5.

[ chemical formula 30]

(Experimental operation)

To contain H₂N-dT₂O (40mmol) of Na₂CO₃/NaHCO₃Mu.l of an anhydrous DMSO solution containing 5.0. mu. mol (2.4mg) of an active ester of an organic EL dye was added to 40. mu.l of the buffer (pH9.0), and the mixture was shaken at room temperature for 6 hours. After shaking, 0.1M TEAA (triethylamine acetate) buffer (pH7.0) was added to the solution to make a total volume of 1 ml. Fractions from the oligonucleotides were separated using a NAP-10 column (Parpharmacia Sephadex G-25). At this time, the NAP-10 column was used after previously being equilibrated with 15ml of 0.1M TEAA buffer. The mixed sample is filled into the column until the total amount reaches 1ml, and after 1ml of solution is eluted, 1.5ml of 0.1M TEAA buffer solution is injected. 1.5ml of the eluate was collected immediately thereafter. The resulting solution was lyophilized overnight, 20. mu.l of distilled water under reduced pressure was added, and analyzed by reverse phase HPLC. The solution injected into the HPLC was diluted to 40-fold for analysis.

(conditions for HPLC measurement)

A chromatographic column: lichrospher RP-18(Cica-MERCK) flow rate: 1ml/min

Detection wavelength: 260nm sample injection solvent used: ultrapure water

Eluent A: 0.1M TEAA buffer (pH7.0), 10% CH₃CN solution

Eluent B: 0.1M TEAA buffer (pH7.0), 40% CH₃CN solution

[ Table 1]

	0	30	35	40 (fen)
					A	100	0	0	100(％)
B	0	100	100	0(％)

Gradient conditions for HPLC determination

[0228]

The HPLC pattern of the labeled oligonucleotide and the UV pattern of the target are shown in FIG. 1A and FIG. 1B, respectively. As a result of HPLC, it was judged that a peak derived from the target product was obtained around RT 25min, and HPLC separation was performed. The identification of the resulting target was performed by MALDI TOF MASS. The results are shown in FIG. 2. The reaction rate was calculated from the peak area of the HPLC chromatogram, and as a result, about 90% was obtained, and the active ester (6) of the EL dye reacted with the oligo-DNA substantially quantitatively.

2. Detection of labeled oligonucleotides

Solutions of labeled oligonucleotides were prepared at different concentrations as shown in table 2 below. Then, 1nL of this solution was spotted (5X 5) on a glass substrate. And drying the glass substrate after spotting.

[ Table 2]

Concentration of solution (μ M)	Relative concentration of labeled oligonucleotide (fmol)
		110	110
11	11
		1	1
0.5	0.5

Then, the detection limit thereof was checked with a fluorescence scanner. The results are shown in FIG. 3. (a) The results of (b), (c) and (d) are 110fmol, 10fmol, 1fmol and 0.5fmol, respectively.

Here, the detector used BIO-RAD Molecular Imager FXPro. The laser wavelength was 488nm and the scan interval was 50 nm.

(results)

The excitation light used for detection at this time is 488nm laser, and the excitation wavelength of the fluorescent dye is 438 nm. Nevertheless, the detection limit of the relative concentration of labeled oligonucleotide is 0.5fmol (500amol), which allows high sensitivity detection. In addition, the reaction with DNA is basically quantitative, and the reaction time can be shortened from about 24 hours to about 6 hours. The EL dye was stable, and even when re-measurement was performed using an EL dye stored at room temperature for 15 days, the same results were obtained.

Example 2

< dye labeling and detection of oligonucleotides (2) >

1. Dye labeling of oligonucleotides

Dye labeling of oligonucleotides was performed using the following scheme 6. The labeling conditions were the same as in example 1, and the addition reaction of the imidazole derivative proceeded rapidly and substantially quantitatively.

[ chemical formula 31]

(conditions for HPLC measurement)

A chromatographic column: lichrospher RP-18(Cica-MERCK) flow rate: 1ml/min

Detection wavelength: 260nm samples were injected with all solvents: ultrapure water

Eluent A: 0.1M TEAA buffer (pH7.0), 10% CH₃CN solution

Eluent B: 0.1M TEAA buffer (pH7.0), 40% CH₃CN solution

The gradient conditions for the HPLC assay were the same as in example 1. The HPLC pattern of the labeled oligonucleotide and the UV pattern of the target are shown in FIGS. 4A and 4B, respectively. As a result of HPLC, it was judged that a peak derived from the target product was obtained around RT 25min, and HPLC separation was performed.

Then, the detection limit was checked by a fluorescence scanner in the same manner as in example 1. The results are shown in FIG. 5. Here, (a), (b), (c), (d), (e) respectively represent light-emitting patterns of 500fmol, 250fmol, 100fmol, 50fmol, 10 fmol.

(results)

The detection limit of the relative concentration of the labeled oligonucleotide was 10fmol, and detection was possible with high sensitivity. In addition, the reaction of the oligonucleotide with the EL dye is substantially quantitative.

Example 3

(labeling and detection of peptides)

1.Ac-Lys(EL)-Lys-Lys-Lys(Acr)-Lys-Lys-Lys(Acr)-Lys-Lys-NH₂Synthesis of (1) Ac-Lys (Mtt) - (Lys (Boc))₂-Lys(Acr)-(Lys(Boc))₂-Lys(Acr)-(Lys(Boc))₂Synthesis of the resin

(Experimental operation)

The reactor was charged with 0.15g (0.61mmol/g) of Fmoc-NH-SAL resin, 0.26g of Fmoc-Lys (Acr) -OH was charged into each of barrels 3 and 6, 0.18g of Fmoc-Lys (Boc) -OH was charged into each of barrels 1, 2, 4, 5, 7 and 8, and 0.23g of Fmoc-Lys (Mtt) -OH was charged into each of barrel 9. Then, the peptide was synthesized using a model 431A peptide synthesizer from Applied Biosystems. The method is carried out by a standard Fmoc method, and acetylation is carried out at the N-terminal. The peptide resin was obtained as a yellow solid with a yield of 0.30 g.

(2)Ac-Lys(Mtt)-(Lys(Boc))₂-Lys(Acr)-(Lys(Boc))₂-Lys(Acr)-(Lys(Boc))₂Deprotection of the Mtt group of the resin, modification and cleavage of the EL from the resin, and deprotection of the side chain

(Experimental operation)

i) Deprotection of Mtt group

0.30g of the synthesized peptide resin was added to the spiral tube 1, and after an excess amount of Dichloromethane (DCM) was added thereto to swell it for 30 minutes, the excess DCM was removed with nitrogen gas. DCM was then added: TFA: TIPS (triisopropylsilane) ═ 94: 1: 5 was stirred for 2 minutes, and the solvent was removed with nitrogen. This operation was repeated 5 times, followed by suction filtration, washing with DCM, triethylamine and DCM, and drying under reduced pressure.

ii) modification of methoxy-type organic EL dyes

To the peptide resin dried under reduced pressure, 6ml of NMP was added, stirred for 30 minutes to swell, and 0.15ml of triethylamine was added and stirred. Then, 0.2g of active ester (6) was added thereto, and the mixture was stirred at room temperature for 24 hours. Then, the mixture was filtered with suction, washed with NMP and DCM, and dried under reduced pressure.

iii) cleavage from resin and deprotection of side chains

To the peptide resin dried under reduced pressure were added 0.08ml of m-cresol, 0.48ml of thioanisole and 3.44ml of TFA3, and the mixture was stirred at room temperature for 1.5 hours. Then, the mixture was filtered off with suction and washed with TFA. After TFA was distilled off under reduced pressure, 15ml of ether was added under ice-bath conditions. After sonication, the mixture was allowed to stand for a short time and the supernatant was separated and removed. Then 15ml ethyl acetate is added under the ice bath condition, and the mixture is placed for a short time after ultrasonic treatment. Then, the mixture was filtered with suction, washed with ether, and dried under reduced pressure.

A yellow-orange solid was obtained in a yield of 0.29 g. Fig. 6A and 6B show HPLC profiles of the product before and after purification, respectively. TOF-Mass measurement was performed on a sample of a peak near r.t. ═ 12.5min, and the molecular weight of a complex with an EL dye and a Peptide (EL-Peptide) was observed: 2055.30 was found to have a peak of 2057.33, confirming the formation of the target product. (Matrix: alpha-CHCA; FIG. 7)

2. Detection of peptides

The labeled peptide spotted on the glass substrate was detected by the same method as in example 1. The detector used BIO-RAD Molecular Imager FXPro. The laser wavelength was 488nm and the scan interval was 50 nm.

(results)

FIG. 8 is a luminescence pattern of a labeled peptide. (a) The results of (b), (c), (d) and (e) are 10fmol, 5fmol, 1fmol, 0.5fmol and 0.1fmol, respectively. The detection limit of the relative concentration of the labeled peptide was 0.1fmol (100amol), and detection was possible with high sensitivity. In addition, the reaction of the peptide with the EL dye was substantially quantitative.

Example 4

< dye labeling and detection of protein >

1. Dye labeling of proteins

The amino group of the lysine residue of BSA is reacted with an active ester of an organic EL dye to form an amide bond, thereby labeling BSA. Specifically, 40. mu.l of a DMSO solution containing 3.6mg (8.6. mu. mol) of organic EL dye active ester (EL-OSu) was added to 58. mu.l of a carbonic acid buffer (pH9.0) containing 4.0mg (58nmol) of BSA (bovine serum albumin), and the mixture was shaken at 37 ℃ for 24 hours. 0.1M TEAA buffer (pH7.0) was added to the reaction solution to make a total volume of 1 ml. The fractions from BSA were separated using a NAP-10 column (Parpharmacia Sephadex G-25), and the separated solution was lyophilized overnight.

BSA labeled with organic EL dye was identified by MALDI TOF MS. As shown in fig. 9, it was found that about 5 organic EL dyes were bonded together, as compared with the starting material (fig. 9A), the molecular weight of the labeled BSA (fig. 9B) was increased by about 2200.

2. Detection of proteins

(results)

As shown in fig. 10, the produced BSA fluoresced in a solid state, and it was found that proteins could be labeled with the organic EL dye active ester.

Claims (18)

1. A method for detecting a biomolecule, which comprises reacting a biomolecule sample with an organic EL dye containing a fused ring compound including an azole derivative or an imidazole derivative, measuring the fluorescence of the biomolecule sample labeled with the organic EL dye,

the azole derivative is a compound represented by any 1 of the following general formulae (1), (2) or (3),

in the formula, R₁、R₂、R₃、R₄Each independently represents An aromatic hydrocarbon group or a heterocyclic group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group or a sulfonyl group, or An aromatic group containing a hetero atom in the ring, X represents a nitrogen atom or a sulfur atom or An oxygen atom or a selenium atom which may have a substituent, R' represents An aliphatic hydrocarbon group or An aromatic hydrocarbon group such as An alkyl group or An alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-；

The imidazole derivative is a compound represented by any 1 of the following general formulae (4), (5), (6), (7) or (8),

in the formula, R₁、R₂、R₃、R₄、R₅Each represents an aromatic hydrocarbon group or hydrocarbon group which may have a substituent such as a hydrogen atom, halogen atom, hydroxyl group, cyano group or sulfonyl group, a heterocyclic group or an aromatic group containing a hetero atom in the ring, and R₁、R₂、R₃、R₄、R₅R 'and R' may be the same or different and each represents An aliphatic or aromatic hydrocarbon group such as An alkyl or alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-。

2. The detection method according to claim 1, wherein an amide bond, an imide bond, a urethane bond, an ester bond or a guanidine bond is formed between the organic EL dye and the biomolecule.

3. The detection method according to claim 2, wherein any 1 kind of reactive group selected from the group consisting of an isocyanate group, an isothiocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group and an active esterified carbonyl group is introduced into the organic EL dye before the reaction with the biomolecule.

4. The detection method according to claim 1, wherein the biomolecule sample is any 1 selected from the group consisting of nucleic acids, proteins, peptides and saccharides.

5. A labeled dye for detecting a biomolecule by a fluorescence measurement, characterized by comprising an organic EL dye having a reactive group to bind to the biomolecule and containing a fused ring compound including an azole derivative or an imidazole derivative,

the azole derivative is a compound represented by any 1 of the following general formulae (1), (2) or (3),

in the formula, R₁、R₂、R₃、R₄Each independently represents An aromatic hydrocarbon group or a heterocyclic group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group or a sulfonyl group, or An aromatic group containing a hetero atom in the ring, X represents a nitrogen atom or a sulfur atom or An oxygen atom or a selenium atom which may have a substituent, R' represents An aliphatic hydrocarbon group or An aromatic hydrocarbon group such as An alkyl group or An alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-；

The imidazole derivative is a compound represented by any 1 of the following general formulae (4), (5), (6), (7) or (8),

in the formula, R₁、R₂、R₃、R₄、R₅Each represents an aromatic hydrocarbon group or hydrocarbon group which may have a substituent such as a hydrogen atom, halogen atom, hydroxyl group, cyano group or sulfonyl group, a heterocyclic group or an aromatic group containing a hetero atom in the ring, and R₁、R₂、R₃、R₄、R₅R 'and R' may be the same or different and each represents An aliphatic or aromatic hydrocarbon group such as An alkyl or alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-。

6. The marking dye according to claim 5, wherein the reactive group is selected from any 1 of a carboxylic acid group, an isocyanate group, an isothiocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group and a reactive esterified carbonyl group.

7. A labeling kit for biomolecules, comprising an organic EL dye for labeling biomolecules, wherein the organic EL dye contains a fused ring compound comprising an azole derivative or an imidazole derivative,

the azole derivative is a compound represented by any 1 of the following general formulae (1), (2) or (3),

in the formula, R₁、R₂、R₃、R₄Each independently represents An aromatic hydrocarbon group or a heterocyclic group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group or a sulfonyl group, or An aromatic group containing a hetero atom in the ring, X represents a nitrogen atom or a sulfur atom or An oxygen atom or a selenium atom which may have a substituent, R' represents An aliphatic hydrocarbon group or An aromatic hydrocarbon group such as An alkyl group or An alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-；

The imidazole derivative is a compound represented by any 1 of the following general formulae (4), (5), (6), (7) or (8),

in the formula, R₁、R₂、R₃、R₄、R₅Each represents an aromatic hydrocarbon group or hydrocarbon group which may have a substituent such as a hydrogen atom, halogen atom, hydroxyl group, cyano group or sulfonyl group, a heterocyclic group or an aromatic group containing a hetero atom in the ring, and R₁、R₂、R₃、R₄、R₅R' may be the same or different and represents An aliphatic or aromatic hydrocarbon group such as An alkyl or alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-。

8. The labeling kit according to claim 7, wherein the organic EL dye contains any 1 reactive group selected from the group consisting of a carboxylic acid group, an isocyanate group, an isothiocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group and an active esterified carbonyl group.

9. A method for detecting a biomolecule, characterized by reacting a biomolecule sample with a probe labeled with an organic EL dye containing a fused ring compound including an azole derivative or an imidazole derivative, measuring fluorescence of the biomolecule sample,

the azole derivative is a compound represented by any 1 of the following general formulae (1), (2) or (3),

in the formula, R₁、R₂、R₃、R₄Each independently represents An aromatic hydrocarbon group or a heterocyclic group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group or a sulfonyl group, or An aromatic group containing a hetero atom in the ring, X represents a nitrogen atom or a sulfur atom or An oxygen atom or a selenium atom which may have a substituent, R' represents An aliphatic hydrocarbon group or An aromatic hydrocarbon group such as An alkyl group or An alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-；

The imidazole derivative is a compound represented by any 1 of the following general formulae (4), (5), (6), (7) or (8),

in the formula, R₁、R₂、R₃、R₄、R₅Each represents an aromatic hydrocarbon group or hydrocarbon group which may have a substituent such as a hydrogen atom, halogen atom, hydroxyl group, cyano group or sulfonyl group, a heterocyclic group or an aromatic group containing a hetero atom in the ring, and R₁、R₂、R₃、R₄、R₅R 'and R' may be the same or different and each represents An aliphatic or aromatic hydrocarbon group such as An alkyl or alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-。

10. The detection method according to claim 9, wherein the biomolecule sample is a nucleic acid, and the probe is an oligonucleotide or PNA complementary to the base sequence of the nucleic acid.

11. The detection method according to claim 10, wherein the oligonucleotide is a primer or a terminator and the nucleic acid is amplified to perform fluorescence detection.

12. The detection method according to claim 11, wherein the primer is labeled with an organic EL dye before the nucleic acid amplification.

13. The detection method of claim 10, wherein the oligonucleotide or PNA is a molecular beacon.

14. A method for detecting a biomolecule, comprising the step of separating a biomolecule sample by size by electrophoresis, wherein the biomolecule sample is labeled with an organic EL dye containing a fused ring compound including an azole derivative or an imidazole derivative before or after the electrophoresis,

the azole derivative is a compound represented by any 1 of the following general formulae (1), (2) or (3),

in the formula, R₁、R₂、R₃、R₄Each independently represents An aromatic hydrocarbon group or a heterocyclic group which may have a substituent such as a hydrogen atom, a halogen atom, a hydroxyl group, a cyano group or a sulfonyl group, or An aromatic group containing a hetero atom in the ring, X represents a nitrogen atom or a sulfur atom or An oxygen atom or a selenium atom which may have a substituent, R' represents An aliphatic hydrocarbon group or An aromatic hydrocarbon group such as An alkyl group or An alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-；

The imidazole derivative is a compound represented by any 1 of the following general formulae (4), (5), (6), (7) or (8),

in the formula, R₁、R₂、R₃、R₄、R₅Each represents an aromatic hydrocarbon group or hydrocarbon group which may have a substituent such as a hydrogen atom, halogen atom, hydroxyl group, cyano group or sulfonyl group, a heterocyclic group or an aromatic group containing a hetero atom in the ring, and R₁、R₂、R₃、R₄、R₅R 'and R' may be the same or different and each represents An aliphatic or aromatic hydrocarbon group such as An alkyl or alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-。

15. The detection method according to claim 14, wherein the biomolecule sample is a nucleic acid, and the base sequence of the nucleic acid is determined on the basis of an electrophoretogram.

16. The detection method according to claim 14, wherein the biomolecule sample is a protein, and the protein separated according to the electrophoretogram is subjected to mass analysis.

17. A method for staining a tissue or a cell, characterized in that a biomolecule in a tissue or a cell sample is labeled with an organic EL dye containing a fused ring compound including an azole derivative or an imidazole derivative,

the azole derivative is a compound represented by any 1 of the following general formulae (1), (2) or (3),

in the formula, R₁、R₂、R₃、R₄Each independently represents a hydrogen atom or a halogen atomAn aromatic hydrocarbon group or heterocyclic group which may have a substituent such as a hydroxyl group, cyano group or sulfonyl group, or An aromatic group which may have a heteroatom in the ring, X represents a nitrogen atom or a sulfur atom or An oxygen atom or a selenium atom which may have a substituent, R' represents An aliphatic hydrocarbon group or aromatic hydrocarbon group such as An alkyl group or alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-；

The imidazole derivative is a compound represented by any 1 of the following general formulae (4), (5), (6), (7) or (8),

in the formula, R₁、R₂、R₃、R₄、R₅Each represents an aromatic hydrocarbon group or hydrocarbon group which may have a substituent such as a hydrogen atom, halogen atom, hydroxyl group, cyano group or sulfonyl group, a heterocyclic group or an aromatic group containing a hetero atom in the ring, and R₁、R₂、R₃、R₄、R₅R 'and R' may be the same or different and each represents An aliphatic or aromatic hydrocarbon group such as An alkyl or alkenyl group which may have An aromatic ring, An^-Represents Cl^-、Br^-、I^-Halide ion, CF₃SO₃ ^-、BF₄ ^-、PF₆ ^-。

18. The staining method of claim 17, wherein said biomolecule is a nucleic acid or a protein.