JP2006328032A - Nucleic acid probe and method for fluorescence detection of multiple-stranded nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound capable of easily detecting a multiple-stranded nucleic acid in high sensitivity without using any exciting light cut filter and without causing photolysis by exciting light, and to provide a method for detecting such a multiple-stranded nucleic acid. <P>SOLUTION: The compound is represented by the general formula(I):A-L-B( wherein, A is a fluorescent semiconductor nanoparticle 1-20 nm in size; B is a multiple-stranded nucleic acid-binding fluorescent molecule; and L is a bivalent linkage group adsorbed or bound to the surface of A ). <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel compound and a multi-stranded nucleic acid fluorescence detection method, and more particularly to a novel compound that can be used as a nucleic acid probe and a multi-stranded nucleic acid fluorescence detection method using the same.

With the development of molecular biology in recent years, the presence or amount of the target nucleic acid is detected by hybridizing the nucleic acid extracted from the living body with a probe nucleic acid having a complementary sequence, and genetic Obtaining information or gene expression information of a pathological tissue has been performed. In order to efficiently obtain such gene information and the like, as a means for obtaining information on a large number of genes and gene expression at once, for example, a detection technique called a DNA chip or a DNA array (hereinafter collectively referred to as “DNA array”) ) Has been developed and attracts great attention.
The target of analysis using the DNA array is mainly genomic DNA and mRNA. The DNA array method requires detection of hybrid multi-stranded nucleic acid formed by complementary nucleic acids, but the amount of nucleic acid collected from the living body is very small, and what are the suitable chemical species for detection? It's hard to say.

  Therefore, at present, the method most widely used for detection is a method called a nucleic acid labeling method. The principle of labeling is to use a labeled nucleotide triphosphate as a raw material for replication when making a copy of the nucleic acid to be detected, and to label the nucleic acid polymer (oligomer) that is produced. . Known labeling methods include direct labeling methods such as RI (radioisotope) and fluorescent dyes, and indirect labeling methods such as biotin and digoxigenin. In the case of RI labeling, radiation detection is performed, in the case of fluorescent dye labeling, fluorescence detection is performed, and in the case of biotin and digoxigenin, detection is performed after appropriate processing so that it can be detected by fluorescence or chemiluminescence. The detection system is designed so that it can be detected with sensitivity.

  However, two main problems have been pointed out regarding detection of hybrids based on these labeling methods. One is to replicate the nucleic acid used for labeling, that is, in PCR (polymerase chain reaction) and RT (reverse transcription) reactions, the quantitative relative relationship of the original nucleic acid mixture is precisely It may not be reflected. The other is that, except for the RI labeling method, the duplicated gene nucleic acid becomes another chemical species that cannot be accurately replicated due to the binding of the labeling compound. Specific problems in this regard include reduced hybridization efficiency (quantitative decrease) and misrecognition (reduced information quality) based on reduced recognition of probe and sample nucleic acids. , Could be a fundamental problem with labeling.

In order to solve this, a method for detecting a hybrid double-stranded nucleic acid without labeling, that is, without labeling, has been proposed.
For example, Patent Document 1 describes a method in which a hybrid is formed by a nucleic acid probe immobilized on an electrode and a gene sample, and the formation of this hybrid is detected using an electrochemically active intercalator or the like. Yes.
However, this method requires the probe nucleic acid to be immobilized on the electrode surface for electrochemical detection. For this reason, in order to perform a large number of simultaneous detections, a large number of electrodes must be prepared on a plane, and a nucleic acid probe must be immobilized on each electrode. Further, even in the signal / background ratio, the discrimination between hybrid and non-hybrid (probe single strand) is not always sufficient, and it is difficult to perform quantitative detection.

On the other hand, as a means for detecting a hybrid double-stranded nucleic acid with high sensitivity and sensitivity, a method using a fluorescent dye whose fluorescence intensity is increased by interaction with a multi-stranded nucleic acid has been proposed (for example, Patent Document 2). Such fluorescent dyes have low fluorescence intensity when interacting with probe nucleic acid (single strand), and significantly increase fluorescence intensity due to interaction with multiple strands formed by hybridization. Double strands can be detected.
However, the types of fluorescent dyes that can be used are limited. For fluorescent dyes whose excitation wavelength and fluorescence wavelength are close to each other, an excitation light cut filter is attached to the fluorescence detection unit to reduce noise derived from excitation light. In addition to this, the excitation light cut filter also cuts the fluorescent signal, so sensitivity loss is an essential problem.

In order to avoid such a problem, Patent Document 3 uses a fluorescence resonance energy transfer (FRET) between two kinds of fluorescent dyes to greatly increase fluorescence sensitization when intercalated into a nucleic acid duplex. A fluorescent dye having a large difference between an excitation wavelength and a fluorescence wavelength is disclosed. By using such a fluorescent dye having a large Stoke shift, the wavelength separation between the excitation light and the fluorescence signal is improved.
However, in general, a probe is excited by a strong laser. However, in a reagent in which two kinds of organic fluorescent dyes are linked, the photolysis of the probe often makes quantitative analysis difficult. In addition, there is a problem that a measuring instrument equipped with a laser light source is expensive and large, so that no one can use it easily.

JP-A-5-199898 Japanese Patent Laid-Open No. 8-211050 JP 2002-327130 A

  Therefore, the present invention provides a compound and a method for detecting a multi-stranded nucleic acid, which can detect a multi-stranded nucleic acid with high sensitivity and ease without using an excitation light cut filter and without causing photolysis by excitation light. That is.

The compound of the present invention is a compound represented by the following general formula (I).
A-L-B (I)
In the formula (I), A represents a fluorescent semiconductor nanoparticle having a particle diameter of 1 to 20 nm, B represents a multi-stranded nucleic acid-binding fluorescent molecule, and L represents a divalent linking group adsorbed or bound to the surface of A. To express.

Here, A in the general formula (I) is preferably a metal oxide, and Y, Eu, Tb, Tm, Ba, Ca, Mg, Al, Mn, Zn, Si, Sr, Ga, Yb, Cr, More preferred is an oxide of a metal selected from Ce, Pb and W.
Moreover, it is preferable that B of general formula (I) is a compound represented by the following general formula (II).

In formula (II), X represents an oxygen atom, a sulfur atom, C (R ³ ) (R ⁴ ), or N (R ³ ), wherein R ³ and R ⁴ represent an alkyl group having 1 to 3 carbon atoms. R ¹ and R ² each independently represents an alkyl group having 1 to 20 carbon atoms or a — (R ⁵ O) _k —R ⁶ group having or not having a nitrogen atom in a substituted or unsubstituted main chain. Here, R ⁵ represents an alkyl group having 2 to 3 carbon atoms, R ⁶ represents an alkyl group having 1 to 2 carbon atoms, and k represents an integer of 1 to 3, provided that R ¹ , R ² At least one of them has a group substituted with a functional group capable of being linked to the linking group L, and V and W are each independently a hydrogen atom, a halogen atom, an alkyl group having 1 to 5 carbon atoms, or a carbon number. An alkoxy group having 1 to 5 carbon atoms, an aryl group having 6 to 10 carbon atoms or a heteroaryl group having 1 to 5 carbon atoms; And, p and q is an integer from 0 4, n represents an integer from 0 3, M is necessary to neutralize the charge of the molecule and bonded with R ¹ or R ² M represents the number necessary for charge neutralization. )

Here, L in the general formula (I) is preferably a hydrolyzate of the compound represented by the general formula (III).
Q- (R ⁷ ) ₄ (III)
(In the formula, Q represents a Si or Ti atom, and R ⁷ represents an organic group. R ⁷ may be the same or different, but at least one of R ⁷ is a functional group for linking to B. Group.)
Moreover, it is preferable that L of general formula (I) is a hydrolyzate of the compound represented by general formula (IV).
E- (CH ₂ ) _r —Si (OR ⁸ ) ₃ (IV)
(In the formula (IV), E represents an amino group, a carboxyl group or a mercapto group, r represents an integer of 2 to 5, and R ⁸ represents an alkyl group having 3 or less carbon atoms.)
Here, L in the general formula (I) is more preferably a hydrolyzate of 3-aminopropyltrialkoxysilane.

  The nucleic acid probe of the present invention is a compound represented by the above general formula (I)

The multi-stranded nucleic acid detection method of the present invention is a multi-stranded nucleic acid fluorescence detection method for detecting multi-stranded nucleic acid using fluorescence, wherein the compound represented by the general formula (I) interacts with the multi-stranded nucleic acid. Photoexciting the fluorescent semiconductor nanoparticles of A in the general formula (I) to cause excitation energy transfer to the multi-stranded nucleic acid-binding fluorescent molecule of B in the general formula (I), the excitation Detecting fluorescence emitted from B as a result of energy transfer.
Here, the multi-stranded nucleic acid may be a multi-stranded nucleic acid in a solution, or a multi-stranded nucleic acid containing a single-stranded DNA or a single-stranded RNA immobilized on a solid support.

  Since the compound according to the present invention has a fluorescent semiconductor nanoparticle as the A moiety and a multi-stranded nucleic acid-binding fluorescent molecule as the B moiety, the multi-stranded nucleic acid binding whose photoexcitation energy is B part by photoexcitation of the fluorescent semiconductor nanoparticle. Fluorescence is released by moving to the fluorescent molecule. For this reason, the ability to express the fluorescence of the multi-stranded nucleic acid-binding fluorescent molecule changes depending on the presence or absence of the interaction with the multi-stranded nucleic acid, and also causes the transfer of photoexcitation energy from the fluorescent semiconductor nanoparticles in the A portion. Thus, a multi-stranded nucleic acid can be detected easily and with high sensitivity.

  According to the present invention, a multi-stranded nucleic acid can be detected with high sensitivity and ease without using an excitation light cut filter and without causing photolysis by excitation light.

The compound of the present invention is represented by the following general formula (I).
A-L-B (I)
(In the formula (I), A represents a fluorescent semiconductor nanoparticle having a particle diameter of 1 to 20 nm, B represents a multi-stranded nucleic acid-binding fluorescent molecule, and L represents a divalent linking group adsorbed or bound to the surface of A. Represents.)

  The compound represented by the above general formula (I) has fluorescent semiconductor nanoparticles having a particle diameter of 1 to 20 nm as the A portion via the linking group L, and a multi-stranded nucleic acid-binding fluorescent molecule as the B portion. Have. The multi-stranded nucleic acid-binding fluorescent molecule as the B portion of the present compound is a molecule whose fluorescence intensity is small when interacting with a single strand, but whose fluorescence intensity is increased by interaction with a multi-stranded nucleic acid. On the other hand, the fluorescent semiconductor nanoparticles as the A portion of the present compound are nanoparticles capable of photoexcitation energy transfer to the multi-stranded nucleic acid-binding fluorescent molecule of the B portion. For this reason, it is not necessary to directly excite the multi-stranded nucleic acid-binding fluorescent molecule, and it is only necessary to photo-excite the fluorescent semiconductor nanoparticles in the A portion, so that the multi-stranded nucleic acid can be detected easily and with high sensitivity.

  In addition, the present compound can exist in a substantially non-fluorescent state in an aqueous solution in the absence of the multi-stranded nucleic acid in an environment in which the solubility of the multi-stranded nucleic acid is maintained. b) In the presence of a multi-stranded nucleic acid, it has the property of substantially expressing fluorescence by interaction with the multi-stranded nucleic acid. The fluorescent properties of the present invention include the efficiency of excitation energy transfer from the A moiety, ie, the fluorescent semiconductor nanoparticles, to the B part, ie, the multi-stranded nucleic acid-binding fluorescent molecule, and the absorption / emission wavelength and fluorescence of the multi-stranded nucleic acid-binding fluorescent molecule. It is determined by multiplying the luminous efficiency.

In this specification, “interactable” means that the compound and the multi-stranded nucleic acid have an affinity, and perform some physicochemical interaction such as energy transfer by reversible binding. This term should not be construed as limiting in any way, but in a broad sense.
In addition, “in an environment where the solubility of the multi-stranded nucleic acid is maintained” means that when the present compound interacts with the multi-stranded nucleic acid, the conditions such as the pH and salt concentration of the solution are usually selected as appropriate. Is soluble. Conditions for using a conventional intercalator can be applied as they are to the solubilized form of such a multi-stranded nucleic acid, and those skilled in the art can easily select such an environment and / or conditions. it can.

In this specification, the multi-stranded nucleic acid refers to a state in which the nucleic acids are assembled by an interaction derived from a complementary sequence (the process of generating a multi-stranded nucleic acid by the interaction may be referred to as “hybridization”). . A multi-stranded nucleic acid is known to be in a double-stranded, triple-stranded, or quadruplex state, and the multi-stranded nucleic acid in the present specification includes these multiple strands.
In addition to DNA or RNA, many of these chemically modified substances are known as nucleic acids, and nucleic acid analogs having a polypeptide chain called PNA as the main chain are also known. All of these are included in the strand nucleic acid.
Nucleic acids that are more preferably used in the present invention are DNA, RNA, and chemical modifications thereof, and double strands are preferable among double strands, triple strands, or quadruple strands. In some cases, a multi-stranded nucleic acid generated by hybridization of a probe nucleic acid and a sample nucleic acid is appropriately referred to as a “hybrid multi-stranded nucleic acid” herein.

Such a nucleic acid is preferably a sequence in which at least one of the multi-strands is at least 20 bases or more in order to effectively detect the fluorescence, but the hybrid multi-strand nucleic acid that is effective as a target for fluorescence detection. Are known in the art.
Hereinafter, each component of the compound according to the present invention will be described.

[1] Fluorescent semiconductor nanoparticles (A)
A part in general formula (I) is a fluorescent semiconductor nanoparticle with a particle size of 1-20 nm.
The present fluorescent semiconductor nanoparticles are inorganic particles that fluoresce with a predetermined excitation light, and the average particle diameter thereof is 1 to 20 nm as an average particle diameter when expressed by the diameter in the projected area, preferably 1 -10 nm. If the number average particle diameter is 1 nm or more, the stability of the fluorescent semiconductor nanoparticles is good, and if it is 20 nm or less, light scattering is low and the dispersibility of the particles is good when detecting the target substance. Detection can be performed with high sensitivity. In addition, the particle size of the metal nanoparticles is a negative image obtained by placing a diluted fluorescent semiconductor nanoparticle on a Cu mesh with a carbon film and drying it, and photographing with a transmission electron microscope (TEM: 1200EX manufactured by JEOL, for example). It can be shown by an arithmetic average measured by a particle size measuring device (for example, KS-300 manufactured by Carl Zeiss).
The particle size distribution of the fluorescent semiconductor nanoparticles is preferably 0 to 50%, more preferably 0 to 20%, and still more preferably 0 to 10% in terms of variation coefficient. The coefficient of variation means a value (arithmetic standard deviation × 100 / number average particle size) obtained by dividing the arithmetic standard deviation by the number average particle size and expressing this as a percentage.

Preferred as the fluorescent semiconductor nanoparticles are metal oxide or metal sulfide fluorescent semiconductor nanoparticles. Examples of the metal constituting the metal oxide or metal sulfide include IIA group such as Ba and Ca, IIB group such as Zn, IIIA group such as Y, Eu, and Tb, IIIB group such as Ga and In, and Ti. Group IVA, IVB group such as Si and Pb, Group VIA such as W, Group VIIA such as Mn, and the like. Among these, an oxide of a metal selected from Y, Eu, Tb, Tm, Ba, Ca, Mg, Al, Mn, Zn, Si, Sr, Ga, Yb, Cr, Ce, Pb and W is preferable. . In addition, metal compounds such as ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, HgS, HgSe, HgTe, InP, InAs, GaN, GaP, GaAs, TiO ₂ , PbS, and PbSe can also be exemplified. Among these metal oxides, metal sulfides, and metal compounds, Y, Eu, Tb, Tm, Ba, and Ca that do not exhibit intense reactivity with living bodies and can emit fluorescence in a wide wavelength range as an energy donor. , Mg, Al, Mn, Zn, Si, Sr, Ga, Yb, Cr, Ce, Pb and W are more preferable, and Zn is particularly preferable. Fluorescent semiconductor nanoparticles can be manufactured stably, have little concern about toxicity, can be manufactured at low cost, have high monodispersity of particles, can produce strong luminescence, and have a wavelength range of emission spectrum. Zinc oxide (ZnO) is most preferable from the viewpoints of easy matching and excitation light wavelength in the visible or near-ultraviolet region.

  Furthermore, it is also preferable that the fluorescent semiconductor nanoparticles of these metal oxides or metal sulfides contain a small amount of metal ions different from the metal in the metal oxide or metal sulfide constituting the metal oxide or metal sulfide. Examples of the metal ion include metal ions such as Mn, Cu, Eu, Tb, Tm, Ce, Al, and Ag. Mn and Eu are preferable from the viewpoint of high light emission visibility and stable production. These metal ions are also preferably doped as a compound combining chloride ions and fluoride ions. The metal ion to be doped may be one kind of atom or a plurality of kinds of atoms. Therefore, examples of the fluorescent semiconductor nanoparticles containing such metal ions include ZnS: Mn and ZnO: Eu. The concentration of the metal ion varies depending on the metal constituting the fluorescent semiconductor nanoparticles and the type thereof, but is preferably in the range of 0.001 to 10 atomic%, more preferably in the range of 0.01 to 10 atomic%. preferable.

  The fluorescent semiconductor nanoparticles are preferably excited by light in the near ultraviolet region, more preferably from 300 nm to 400 nm, from the viewpoint of separation of excitation light and signal fluorescence, utilization of an inexpensive light source, and simple detection system construction. The near-ultraviolet light is used as excitation light, and it is preferable that the excitation light emits light in the visible range, more preferably visible light of 400 nm to 700 nm. By emitting visible light, energy can be transferred to the multi-stranded nucleic acid-binding fluorescent molecule in the B part of the compound, and another multi-stranded nucleic acid-binding fluorescent molecule in another visible region can be excited. Nucleic acid-binding fluorescent molecules can be colored.

[2] Linking group (L)
The L portion in the general formula (I) is a divalent linking group that is physically adsorbed or chemically bonded to the surface of the fluorescent semiconductor nanoparticles of the A portion. L has a structure for uniformly dispersing the fluorescent semiconductor nanoparticles (A) in water and a buffer solution and linking them with the multi-stranded nucleic acid-binding fluorescent molecule (B).

  Examples of the functional group for linking with B include an amino group (bonding group) and a carboxyl group (bonding group, water-soluble). In addition, L may have a structure formed by mixing two or more kinds of surface modifiers, and has a structure such as a sulfonic acid group (water-soluble) or polyalkylene glycol (water-soluble) in addition to the aforementioned functional group. Also good.

In order to obtain such a linking group, it is preferably surface-modified with a surface modifier represented by the following general formula (III) or a decomposition product thereof.
Q- (R ⁷ ) ₄ (III)
(In the formula, Q represents a Si or Ti atom, and R ⁷ represents an organic group. R ⁷ may be the same or different, but at least one of R ⁷ is a functional group for linking to B. Group.)

Examples of R ⁷ excluding the functional group moiety include alkylene groups (eg, methylene group, ethylene group, trimethylene group, tetramethylene group, hexamethylene group, propylene group, ethylethylene group, cyclohexylene group, etc.). 1-10, preferably 1-8 chain or cyclic), alkenylene group (eg, vinylene group, propenylene group, 1-butenylene group, 2-butenylene group, 2-pentenylene group, 8-hexadecenylene group, 1 , 3-butanedienylene group, cyclohexenylene group or the like having 1 to 10 carbon atoms, preferably 1 to 8 chain or cyclic, or arylene groups (eg phenylene group, naphthylene group, etc.) having 6 to 10 carbon atoms. , Preferably 6 phenylene groups).

R ⁷ includes one or two or more heteroatoms (meaning any atom other than a carbon atom such as a nitrogen atom, an oxygen atom, or a sulfur atom) or a carbon atom adjacent to the heteroatom. One or more functional groups may be included as a partial structure. The hetero atom is preferably an oxygen atom or a sulfur atom, and most preferably an oxygen atom. The number of heteroatoms is not particularly limited, but is preferably 5 or less, more preferably 3 or less. Other functional groups include ester groups (including carboxylic acid esters, carbonic acid esters, sulfonic acid esters and sulfinic acid esters), amide groups (including carboxylic acid amides, urethanes, sulfonic acid amides and sulfinic acid amides), ethers Group, thioether group, disulfide group, amino group, imide group and the like. The other functional group may further have a substituent. Such other functional groups are preferably an ester group, an amide group, an ether group, a thioether group, a disulfide group or an amino group, and more preferably an alkenyl group, an ester group or an ether group.

The other organic group represented by R ⁷ includes any group, but preferably alkoxy such as methoxy group, ethoxy group, isopropoxy group, n-propoxy group, t-butoxy group and n-butoxy group. Group and phenoxy group. These alkoxy groups and phenoxy groups may further have a substituent, but those having a total carbon number of 8 or less are desirable. The surface modifier used in the present invention may be one in which an amino group, a carboxyl group or the like forms a salt with an acid or base.

  Among the surface modifiers used in the present invention, decomposition products of those represented by the general formula [I] are hydroxides obtained by hydrolyzing alkoxy groups, and low molecular weight oligomers generated by a dehydration condensation reaction between hydroxyl groups. (This may be any of a linear structure, a cyclic structure, a cross-linked structure, etc.), a dealcoholization condensation reaction product of a hydroxyl group and an unhydrolyzed alkoxy group, a sol and gel formed by further dehydration condensation reaction Say.

  The surface modifier usable in the present invention may cover the entire surface of the phosphor nanoparticles or may be bonded to a part thereof. In the present invention, the surface modifiers may be used alone or in combination.

Among such linking groups, a substituted alkylthiol substituted at the terminal of the alkyl group or a silane coupling agent of the following general formula (IV) having a functional group is from the viewpoint of production cost and dispersion stability in an aqueous medium. Further preferred.
E- (CH ₂ ) _r —Si (OR ⁸ ) ₃ (IV)
In formula (III), E represents an amino group, a carboxyl group, or a mercapto group, r represents 2 to 5, and R ⁸ represents an alkyl group having 3 or less carbon atoms.

  Examples of such silane coupling agents include N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropyltriethoxysilane, and 3-aminopropyltrimethoxy. Silane, aminophenyltrimethoxysilane, 3-aminopropyltriethoxysilane, bis (trimethoxysilylpropyl) amine, N- (3-aminopropyl) -benzamidetrimethoxysilane, 3-hydrazidepropyltrimethoxysilane, 3-maleimide Examples thereof include propyltrimethoxysilane, (p-carboxy) phenyltrimethoxysilane, 3-carboxypropyltrimethoxysilane, and 3- (2-pyridyl) -propyltrimethoxysilane.

  Among such linking groups, a structure obtained by mixing 3-aminopropyltrimethylsilane and 3-aminopropyltriethoxysilane with ZnO and performing surface modification while performing hydrolysis is dispersed in an aqueous medium. Most preferable from the viewpoint of stability.

[3] Multi-stranded nucleic acid-binding fluorescent molecule (B)
The B portion in the general formula (I) is a multi-stranded nucleic acid-binding fluorescent molecule, is substantially non-fluorescent in an aqueous solution in the absence of the multi-stranded nucleic acid, and when the multi-stranded nucleic acid is present, It corresponds to a dye compound that substantially exhibits fluorescence by interaction with a multi-stranded nucleic acid.

The principle of discriminating ability of the multi-stranded nucleic acid by the dye compound represented by B is that it interacts with the multi-stranded nucleic acid and returns to the original dye state (recovery of the dye-coupled system) by the interaction energy received from the multi-stranded nucleic acid. This is based on the fact that the equilibrium shifts to emit fluorescence, and that in the absence of multi-stranded nucleic acid, a substantially non-fluorescent state is maintained. Even if a single-stranded nucleic acid is present in the reaction system, the interaction energy between the dye compound and the single-stranded nucleic acid is small, so that a substantially non-fluorescent state is maintained. Thus, the non-fluorescent dye changes to a fluorescent dye by the interaction with the multi-stranded nucleic acid.
The dye compound in this compound emits light by receiving energy obtained by exciting the semiconductor nanoparticles A linked to B after interaction with the multi-stranded nucleic acid.

  A known intercalator having such a function can be applied to the dye compound as the B portion, but from the viewpoint of discrimination between single-stranded nucleic acid and multi-stranded nucleic acid and ease of absorption / fluorescence wavelength control. A dye compound having a cyanine dye structure represented by the following general formula (II) is preferable.

In the formula, X represents an oxygen atom, a sulfur atom, C (R ³ ) (R ⁴ ), or N (R ³ ). Here, R ³ and R ⁴ represent an alkyl group having 1 to 3 carbon atoms. X is preferably an oxygen atom and a sulfur atom from the viewpoint of high molecular planarity, high intercalation ability to a multi-stranded nucleic acid, and strong fluorescence intensity resulting from the interaction with the multi-stranded nucleic acid, Sulfur atoms are most preferred.

R ¹ and R ² each independently represents an alkyl group having 1 to 20 carbon atoms or a — (R ⁵ O) _k —R ⁶ group having or not having a nitrogen atom in a substituted or unsubstituted main chain. Here, R ⁵ represents an alkyl group having 2 to 3 carbon atoms, R ⁶ represents an alkyl group having 1 to 2 carbon atoms, and k represents an integer of 1 to 3.

When the alkyl group or — (R ⁵ O) _k —R ⁶ group has a substituent, the substituent is a halogen atom, or an alkyl group, alkenyl group, alkynyl group, aryl group, amino group having 1 to 20 carbon atoms. , Hydroxyl group, thiol group, alkoxy group, aryloxy group, alkylthio group, arylthio group, acylamino group, sulfonylamino group, heterocyclic group, cyano group, sulfonyl group, sulfinyl group, nitro group, sulfo group, carboxyl group, alkoxycarbonyl Group, aryloxycarbonyl group, carbamoyl group, sulfamoyl group, for example, chlorine, bromine, iodine, methyl group, ethyl group, n-propyl group, isopropyl group, t-butyl group, n-octyl group, Eicosyl group, 2-ethylhexyl group, cyclohexyl group, cyclopentyl group Vinyl group, allyl group, ethynyl group, propargyl group, trimethylsilylethynyl group, phenylethynyl group, p-sulfophenylethynyl group, p-sulfonylaminophenylethynyl group, phenyl group, p-tolyl group, naphthyl group, methoxy group, ethoxy group Group, isopropoxy group, t-butoxy group, phenoxy group, 2-methylphenoxy group, 4-t-butylphenoxy group, 3-nitrophenoxy group, methylthio group, ethylthio group, phenylthio group, formylamino group, acetylamino group , Methylsulfonylamino group, 2-furyl group, 2-thienyl group, 2-pyridyl group, 4-pyridyl group, 2-pyrimidinyl group, 2-benzothiazolyl group, methoxycarbonyl group, ethoxycarbonyl group, phenoxycarbonyl group, N- Methylcarbamoyl group, N-methyl This includes rusulfamoyl groups. Preferred are a hydroxyl group, an amino group, a thiol group, a sulfo group, and a carboxyl group.

At least one of R ¹ and R ² has a functional group capable of being linked to L on the particle surface. The functional group may be any group that can be linked to L, but from the viewpoint of reactivity with L, a thiol group, an amino group, and a carboxyl group are preferable, and a carboxyl group is most preferable.

  V and W are each independently a hydrogen atom, a halogen atom, an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an aryl group having 6 to 10 carbon atoms, or a heteroaryl group having 1 to 5 carbon atoms. For example, chlorine, bromine, iodine, methyl group, ethyl group, n-propyl group, isopropyl group, t-butyl group, methoxy group, ethoxy group, isopropoxy group, phenyl group, p-tolyl group, naphthyl group, Examples include 2-furyl group, 2-thienyl group, 2-pyridyl group, 4-pyridyl group, 2-pyrimidinyl group, 2-benzothiazolyl group, and the like. V and W are preferably a hydrogen atom, a halogen atom, an aryl group, or an alkoxy group, and most preferably a hydrogen atom or a halogen atom, from the viewpoint of water solubility and intercalation ability. p and q represent 0-4. Preferably it is 0 to 2, most preferably 0 or 1. n represents 0 to 3, preferably 0 and 1, and most preferably 0.

M represents a counter ion that is necessary for neutralizing the charge of the molecule and may be bonded to R ¹ or R ^2, and m represents a number necessary for charge neutralization. M may be a cation or an anion. Examples of the cation include alkali metal ions such as sodium ion, potassium ion and lithium ion, and organic ions such as tetraalkylammonium ion and pyridinium ion. The anion may be either an inorganic anion or an organic anion, such as a halogen anion (for example, fluorine ion, chlorine ion, bromine ion, iodine ion), a substituted aryl sulfonate ion (for example, p-toluenesulfone). Acid ion, p-chlorobenzenesulfonate ion, etc.), aryl disulfonate ion (eg, 1,3-benzenedisulfonate ion, 1,5-naphthalenedisulfonate ion, etc.), alkyl sulfate ion (eg, methyl sulfate ion, etc.) ), Sulfate ions, thiocyanate ions, perchlorate ions, tetrafluoroborate ions, picrate ions, acetate ions, trifluoromethanesulfonate ions, and the like. M may be a hydrogen ion. The counter ion is preferably an ammonium ion, an alkali metal ion, a halogen anion, or a substituted aryl sulfonate ion, and more preferably an alkali metal ion, a halogen anion, or a substituted aryl sulfonate ion.

  As a synthesis of the dye compound corresponding to the partial structure B of the general formula (I), a general synthesis method is known and can be easily synthesized by those skilled in the art. For example, FMHarmer “Heterocyclic Compounds – Cyanine Dyes and Related Compounds”, John Wiley and Sons Sons, Inc.-New York, London, 1964; DMSturmer "Heterocyclic Compounds-Special Topics in Heterocyclic Chemistry" , Chapter 14, Section 14, 482-515, John Wiley and Sons (New York, London, 1977), and the like.

[4] Production method of this compound [4-1] Production method of fluorescent semiconductor nanoparticles and dispersion thereof The fluorescent semiconductor nanoparticles can be produced by a known synthesis method. For example, liquid phase synthesis methods such as homogeneous precipitation method (coprecipitation method), reverse micelle method (microemulsion method), hot soap method, sol-gel method, solvothermal method, molten urea method, metal complex method, CVD method, Special synthesis methods such as sputtering method, laser ablation method, Joule quench method, gas phase synthesis method such as vapor deposition method, spray pyrolysis method, supercritical method, etc. can be applied. These synthesis methods can also be used in combination. In the liquid phase synthesis method, microwave irradiation, ultrasonic irradiation or the like may be used together, or a micro reaction space such as a microreactor may be used.
In the present invention, the fluorescent semiconductor nanoparticles need to be colloidally dispersed, and it is desirable not to perform firing for use in ordinary phosphor production. Moreover, in order to suppress the crystal growth and aggregation of particles, it is preferable to react in the presence of an appropriate surface modifier or to react using micro or nano space.

  Fluorescent semiconductor nanoparticles composed of metal oxides include sol-gel methods that hydrolyze organometallic compounds such as metal alkoxides and acetylacetonates, and water added by adding alkali to an aqueous solution of the metal salt. Hydroxide precipitation method in which it is precipitated as an oxide, followed by dehydration and annealing, ultrasonic decomposition method using the above precursor solution of the metal, ultrasonic irradiation, solvothermal method in which decomposition reaction is performed under high temperature and high pressure It can be obtained by a liquid phase synthesis method such as spray pyrolysis sprayed at high temperature. Further, it can also be obtained by a vapor phase synthesis method such as a thermal CVD method or a plasma CVD method using an organometallic compound, a sputtering method or a laser ablation method using a target of the metal or the metal oxide.

  Fluorescent semiconductor nanoparticles composed of metal sulfides contain highly decomposable metal compounds such as diethyldithiocarbamate compounds of metals such as trialkylphosphine oxides, trialkylphosphines, and ω-aminoalkanes. Hot soap method for crystal growth in a boiling organic solvent, coprecipitation method for crystal growth by adding a sulfide solution such as sodium sulfide or ammonium sulfide to a solution of the metal salt, and the above aqueous raw material containing a surfactant as an alkane It can be obtained by a liquid phase synthesis method such as a reverse micelle method in which a reverse micelle is present in a non-polar organic solvent such as an organic hydrocarbon, an ether, or an aromatic hydrocarbon, and crystal is grown in the reverse micelle. It can also be obtained by the same gas phase synthesis method as in the case of the metal oxide fluorescent semiconductor nanoparticles.

  The above-described surface modifier can be added at the time of synthesis of the fluorescent semiconductor nanoparticles, but is preferably added after the synthesis, and at least a part thereof is hydrolyzed to bind to the fluorescent semiconductor nanoparticles, At least a part of the surface of the nanoparticles is coated (surface modification). The fluorescent semiconductor nanoparticles are washed and purified by a conventional method such as centrifugation or filtration, and then a solvent containing a surface modifier used in the present invention (preferably methanol, ethanol, isopropyl alcohol, 2-ethoxyethanol, etc.). It may be dispersed and coated in a hydrophilic organic solvent.

  The addition amount of the surface modifier varies depending on the particle size of the phosphor, the concentration of the particles, the type (size, structure) of the surface modifier, etc., but is preferably 0.001 with respect to the metal oxide or metal sulfide. -10 times mol, More preferably, it is 0.01-2 times mol.

  As described above, a known surface modifier may be used in combination with the surface modifier represented by the general formula (III) or (IV). The addition amount of the known surface modifier is not particularly limited, but is preferably 0.01 to 100 times mol, more preferably 0.05 to 10 times mol.

  In the dispersion liquid of the fluorescent semiconductor nanoparticles to which the surface modifier is bound, the concentration of the nanoparticles is not particularly limited because it varies depending on the fluorescence intensity, but is preferably 0.01 mM to 1000 mM, more preferably 0.1 mM to 100 mM. . As the dispersion medium, in addition to the above alcohols, hydrophilic organic solvents such as DMF, DMSO, and THF, and water are preferable.

  It should be noted that the surface of the fluorescent semiconductor nanoparticles is coated with a surface modifier because a certain interval between particles is observed when observed with a high-resolution TEM such as FE-TEM, and chemical analysis. Can be confirmed.

[4-2] Linkage with a multi-stranded nucleic acid-binding fluorescent molecule The fluorescent semiconductor nanoparticles coated with the surface modifier corresponding to the general formula (III) or (IV) are functionalized at the terminal of the surface modifier. The group is further linked to a multi-stranded nucleic acid-binding fluorescent molecule of the B portion of the compound by amidation reaction or the like using the group as a reactive group.
The amidation reaction is performed by condensation of a carboxyl group or a derivative group thereof (ester, acid anhydride, acid halide, etc.) and an amino group. When an acid anhydride or acid halide is used, it is desirable that a base coexists. When an ester such as methyl ester or ethyl ester of carboxylic acid is used, it is desirable to perform heating or decompression in order to remove the generated alcohol. When directly amidating a carboxyl group, amidation reagents such as DCC, Morpho-CDI, and WSC, condensation additives such as HBT, N-hydroxyphthalimide, p-nitrophenyl trifluoroacetate, 2,4,5- A substance that promotes an amidation reaction such as an active ester such as trichlorophenol may be allowed to coexist or may be reacted in advance. Further, during the amidation reaction, it is desirable to protect either the amino group or the carboxyl group of the affinity molecule to be bound by amidation with an appropriate protecting group according to a conventional method, and to deprotect after the reaction.

Fluorescent semiconductor nanoparticles to which affinity molecules are bound by an amidation reaction are washed and purified by a conventional method such as gel filtration, and then washed with water or a hydrophilic solvent (preferably methanol, ethanol, isopropanol, 2-ethoxyethanol, etc.). Use in a distributed manner. The concentration of the nanoparticle phosphor in the dispersion is not particularly limited because it varies depending on the fluorescence intensity, but is preferably 10 ⁻¹ M to 10 ⁻¹⁵ M, more preferably 10 ⁻² M to ^{10 −10} M.

  Specific examples of the compound suitably used in the method of the present invention are listed below, but the method of the present invention is not limited to the case of using these compounds.

In the compound of the present invention, energy transfer occurs to the dye compound in the B portion by photoexcitation of the fluorescent semiconductor nanoparticles in the A portion. Here, when the present compound is bound to the multi-stranded nucleic acid, the compound of the present invention intercalates into the multi-stranded nucleic acid via the B-strand-binding fluorescent molecule, so that the dye compound is substantially colored. And fluorescence is emitted. Therefore, the compound of the present invention can be preferably used as a fluorescent light-emitting nucleic acid probe.
In particular, since the fluorescent semiconductor nanoparticles in the A portion of the present compound are excited by near-ultraviolet light and can be detected in the visible region by the multi-chain-binding fluorescent molecule in the B portion, such as an ultraviolet illuminator. It is possible to detect a multi-stranded nucleic acid in the visible range that is excited by an inexpensive light source and has no influence of excitation light.

  When the nucleic acid probe of the present invention is used for detection of a multi-stranded nucleic acid, the detection composition may be combined with a suitable aqueous solvent.

  As such an aqueous solvent, all those that can be generally used as a reagent for nucleic acid detection can be applied as they are. For example, TBE buffer, TE buffer, TAE buffer, SSC solution, HEPES buffer, HEPSO buffer, TAPS buffer, Tris Examples include buffers, MOPS buffers, and CAPS buffers. Such an aqueous solvent can be present at 0.01% to 90% by weight relative to the total weight of the composition for detection.

  When the nucleic acid probe of the present invention is used for nucleic acid detection, it is generally used in an amount of 0.01 to 100 μg / ml, preferably 1 to 50 μg / ml, depending on the concentration of the target multi-stranded nucleic acid.

The multi-stranded nucleic acid fluorescence detection method of the present invention is a multi-stranded nucleic acid fluorescence detection method for detecting multi-stranded nucleic acid using fluorescence, wherein the compound (nucleic acid probe) interacts with the multi-stranded nucleic acid (reciprocal). Action step), photoexcitation of the fluorescent semiconductor nanoparticles of the A portion in the nucleic acid probe to cause excitation energy transfer to the multi-stranded nucleic acid-binding fluorescent molecule of the B portion in the nucleic acid probe (photoexcitation step), the excitation energy Detecting the fluorescence emitted from the B as a result of the movement (fluorescence detection step).
According to this multi-stranded nucleic acid fluorescence detection method, fluorescence is emitted from the multi-stranded nucleic acid-binding fluorescent molecule of the B portion by transfer of excitation energy by photoexcitation of the fluorescent semiconductor nanoparticles of the A portion in the nucleic acid probe. By detecting this fluorescence, a multi-stranded nucleic acid can be easily detected.

  The target multi-strand nucleic acid corresponds to the above-described multi-strand nucleic acid, and is generally preferably a multi-strand nucleic acid in solution. This multi-stranded nucleic acid takes a form solubilized in the sample solution, and can easily interact with the present nucleic acid probe solubilized in the solution. Thereby, the multi-stranded nucleic acid in the solution can be easily detected.

  Further, the multi-stranded nucleic acid may be a multi-stranded nucleic acid containing at least one single-stranded nucleic acid immobilized on a solid support. Here, the single-stranded nucleic acid immobilized on the solid support is a nucleic acid such as a single-stranded DNA or RNA selected according to a specific purpose, and is hybridized with a complementary nucleic acid present in the sample. Multi-stranded nucleic acids can be formed. Therefore, the multi-stranded nucleic acid here is immobilized on a solid support by hybridizing the single-stranded nucleic acid and the nucleic acid in the sample. The nucleic acid probe can also interact with such an immobilized multi-stranded nucleic acid, whereby a specific one of the multi-stranded nucleic acids formed on the solid support can be easily detected. .

It should be noted that such a multi-stranded nucleic acid containing an immobilized single-stranded nucleic acid only needs to contain at least one immobilized single-stranded nucleic acid, and whether or not other nucleic acid strands constituting the multi-stranded nucleic acid are immobilized. Does not matter.
In addition, normal conditions can be applied as they are as conditions for hybridization between an immobilized single nucleic acid and another nucleic acid chain, which are performed before the nucleic acid probe interacts.

  The solid support on which the single-stranded nucleic acid is immobilized may take any form such as a plate or a particle as long as the single-stranded nucleic acid can be immobilized. The substrate may be a glass substrate, a quartz substrate, or a non-fluorescent substrate. A quartz substrate, a methylcellulose membrane, etc. can be mentioned. In particular, as the substrate, a so-called DNA chip on which a single-stranded nucleic acid is immobilized can be preferably mentioned.

  For the interaction between the nucleic acid probe of the present invention and these multi-stranded nucleic acids, the conditions usually used for the binding of multi-stranded nucleic acids may be applied as they are. Specifically, the detection composition containing the nucleic acid probe is mixed with the multi-stranded nucleic acid. What is necessary is just to add to a multi-stranded nucleic acid in the environment where the solubility of a nucleic acid is maintained. Such conditions are as described above, and can be easily set as appropriate by those skilled in the art.

  As the photoexcitation conditions in the photoexcitation step, the above-described photoexcitation conditions of the fluorescent semiconductor nanoparticle part in the nucleic acid probe can be applied as they are, and damage to near ultraviolet light, particularly biological samples, from the viewpoint of detecting signal fluorescence in the visible range. It is preferable to carry out with near-ultraviolet light of 300 to 400 nm from the viewpoint of reducing the above. Such light irradiation can be easily performed with an inexpensive ultraviolet light source.

The fluorescence detection step detects fluorescence emitted from the multi-stranded nucleic acid-binding fluorescent molecule of the nucleic acid probe as a result of the transfer of photoexcitation energy in the photoexcitation step. Since the emitted fluorescence is light having a wavelength different from the wavelength of the excitation light, it is not necessary to reduce noise by attaching an excitation light cut filter.
The detection and detection of the fluorescent substance from the nucleic acid probe and the detection of the target substance based thereon can usually be applied with the conditions and means used for this purpose as they are. Those skilled in the art can easily select such conditions and means as appropriate. At this time, the fluorescence to be detected corresponds to the fluorescence from the multi-stranded nucleic acid-binding fluorescent molecule of the nucleic acid probe, but if other fluorescent dyes are present in the detection composition, these fluorescences are detected. Fluorescence from the dye may be included. Usually, means used for this purpose can be applied as it is to fluorescence detection, and examples thereof include a CCD camera.

  The multi-stranded nucleic acid fluorescence detection method of the present invention can be used as a qualitative or quantitative analysis method for a target RNA containing a specific base sequence expected to be contained in a gene mixture such as DNA or RNA. In addition, this method is useful for use in the field of clinical diagnosis such as genetic diagnosis, and is also preferably used for qualitative or quantitative determination of microorganisms in environments such as food, indoors, soil, rivers, and the ocean. be able to.

  The following examples further illustrate the present invention, but the scope of the present invention is not limited to these examples. Further,% in the examples is based on weight (mass) unless otherwise specified.

(1) Synthesis of compound I-1 (synthesis of zinc oxide nanoparticles)
Dehydrated ethanol (250 ml) was added to zinc acetate dihydrate (5.49 g, 25 mmol), and the mixture was gently refluxed for 2 hours while distilling off the solvent with a Dean-Steark dehydrator. The solvent distilled off was 150 ml. 150 ml of dehydrated EtOH was again added to the white turbid reaction solution and heated to reflux, and the clarified reaction solution was cooled with water to room temperature. Tetramethylammonium hydroxide (25% methanol solution, 11.4 ml, 28 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 4 hours.

  Subsequently, 3-aminopropyltrimethoxysilane (4.7 ml, 25 mmol) and water (1.5 ml, 83.3 mmol) were added, and the mixture was stirred at 60 ° C. for 4 hours. A white solid precipitates 7 minutes after the start of the reaction. The reaction solution was water-cooled to room temperature, and the solid was suction filtered and washed with ethanol. When the obtained white powder was dried under reduced pressure, zinc oxide nanoparticles having an aminated surface were obtained. Yield 6.0g

200 mg of the particles were dissolved in 10 ml of distilled water, and gel filtration was performed using a column filled with Sephadex G25. All subsequent reactions used the desalted aqueous solution.
The nanoparticles synthesized by this formulation show a transparent and good dispersion state even at 10 wt%. The peak pattern of the hexagonal (wurtzite) zinc oxide sample belonging to the powder X-ray diffraction space group P6 ₃ mc coincided with the peak pattern, and the particle size was 3 nm.

(Dye synthesis)
The dye I-1d was synthesized by the method shown below.

  Compound I-1a (0.52 g, 1.0 mmol) and Compound I-1b (0.40 g, 1.0 mmol) are dissolved in 20 ml of dimethylformamide, and triethylamine (0.42 ml, 3.0 mmol) is added to the mixture at 50 ° C. For 2 hours. Crystals formed by adding 50 ml of ethyl acetate to the reaction solution were filtered. The crystals were recrystallized from methanol to obtain dye I-1c (280 mg, 48%).

The dye I-1c (0.1 g) was dissolved in 10 ml of methanol, 3 ml of 1% aqueous lithium hydroxide solution was added, and the mixture was stirred at room temperature for 3 hours. Isopropyl alcohol and ethyl acetate were added to the reaction solution to precipitate crystals and filtered. The crystal was purified by gel filtration using Sephadex LH-20 (manufactured by Amersham) to obtain dye I-1d (090 mg).
¹ H NMR (300MHz, DMSO-d6) δ: 8.70 (2H, dd), 8.14 (1H, d), 7.94 (2H, m), 7.72 (2H, m), 7.30 (2H, dd), 6.77 (1H , s), 4.70 (2H, bs), 3.97 (3H, s), 3.40-3.20 (4H, m), 3.00 (6H, s), 2.20-1.80 (8H, m)

(Active dye esterification)
Compound I-1d (7.0 mg, 1 × 10 ⁻⁵ mol), N-hydroxysuccinimide (3.4 mg, 3 × 10 ⁻⁵ mol) was dissolved in 1 ml of 0.01 M MES buffer (pH 6.0). -Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (5.8 mg, 3 × 10 ⁻⁵ mol) was added and stirred at room temperature for 1 hour.

(Linking of nanoparticles and pigment)
200 μl of the above active esterification reaction solution, 1 ml of nanoparticle gel filtration eluate, and 1300 μl of 0.01 M HEPES buffer (pH 7.2) were mixed and stirred at room temperature for 3 hours. The reaction solution was subjected to gel filtration (elution with water) using a column packed with Sephadex G25 to obtain an aqueous dispersion of compound I-1.

(2) Synthesis of Compound I-2 to I-6 Except that the dye structure was changed, synthesis was performed under the same conditions as I-1, and then dyes I-2 to I-6 were synthesized.

[Example 3]
DNA staining of agarose electrophoresis gel using Compound I-1 (Preparation of staining solution)
Compound I-1 was diluted with 0.5 × TBE buffer (pH 8.47) to prepare an absorbance at 500 nm of 0.08. 400 μl of this solution was weighed and further diluted with 40 ml of 0.5 × TBE buffer (pH 8.47) to obtain a staining solution.
For comparison, Sybr Safe, an intercalator manufactured by Molecular Probes, was prepared at the same concentration as described above.
(Electrophoresis gel staining)
A gel obtained by running 5 μl / lane of Smart Ladder (0.2-10 kbp) manufactured by Nippon Gene for 30 minutes was incubated with the staining solution at room temperature for 30 minutes.

(DNA detection)
A cooled CCD camera system equipped with an ultraviolet illuminator; a gel stained with DNA was imaged using LAS-1000 manufactured by Fujifilm. The results are shown in FIG.
In FIG. 1, the left gel is the result of staining with compound I-1 of the present invention.
The middle gel is the result of staining with I-1d, which is a partial structure of the compound of the present invention, that is, only with an intercalating dye.
The gel on the right is stained with the intercalating dye SYBR Safe sold by Molecular Probes.

As is clear from FIG. 1, it can be seen that the compound I-1 of the present invention can detect DNA with higher sensitivity and higher SN ratio than existing intercalators.
Therefore, it is clear that by using the nucleic acid probe according to the present invention, a multi-stranded nucleic acid can be detected with high sensitivity and ease without using an excitation light cut filter.

It is a photograph of the gel which shows DNA dyeing | staining using the various compounds (left lane: I-1, center lane: I-1d, right lane: SYBER Safe) based on the Example of this invention.

Claims (12)

The compound represented by the following general formula (I).
A-L-B (I)
(In the formula (I), A represents a fluorescent semiconductor nanoparticle having a particle diameter of 1 to 20 nm, B represents a multi-stranded nucleic acid-binding fluorescent molecule, and L represents a divalent linking group adsorbed or bound to the surface of A. Represents.)   The compound according to claim 1, wherein A in the general formula (I) is a metal oxide.   2. A is an oxide of a metal selected from Y, Eu, Tb, Tm, Ba, Ca, Mg, Al, Mn, Zn, Si, Sr, Ga, Yb, Cr, Ce, Pb and W. Compound.   The compound according to claim 3, wherein A in the general formula (I) is zinc oxide. The compound according to any one of claims 1 to 4, wherein B in the general formula (I) is represented by the following general formula (II).
(Wherein X represents an oxygen atom, a sulfur atom, C (R ³ ) (R ⁴ ), N (R ³ ), wherein R ³ and R ⁴ represent an alkyl group having 1 to 3 carbon atoms,
R ¹ and R ² are each independently an alkyl group having 1 to 20 carbon atoms or a — (R ⁵ O) _k —R ⁶ group having or not having a nitrogen atom in a substituted or unsubstituted main chain, Here, R ⁵ represents an alkyl group having 2 to 3 carbon atoms, R ⁶ represents an alkyl group having 1 to 2 carbon atoms, k represents an integer of 1 to 3, provided that R ¹ and R ² At least one has a group substituted with a functional group capable of being linked to the linking group L;
V and W are each independently a hydrogen atom, a halogen atom, an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an aryl group having 6 to 10 carbon atoms, or a heteroaryl group having 1 to 5 carbon atoms Represents
p and q represent an integer of 0 to 4;
n represents an integer of 0 to 3,
M represents a counter ion that is necessary to neutralize the charge of the molecule and may be bound to R ¹ or R ² ;
m represents a number necessary for charge neutralization. ) The compound according to any one of claims 1 to 5, wherein L in the general formula (I) is a hydrolyzate of the compound represented by the general formula (III).
Q- (R ⁷ ) ₄ (III)
(In the formula, Q represents a Si or Ti atom, and R ⁷ represents an organic group. R ⁷ may be the same or different, but at least one of R ⁷ is a functional group for linking to B. Group.) The compound according to any one of claims 1 to 5, wherein L in the general formula (I) is a hydrolyzate of the compound represented by the general formula (IV).
E- (CH ₂ ) _r —Si (OR ⁸ ) ₃ (IV)
(In the formula (IV), E represents an amino group, a carboxyl group or a mercapto group, r represents an integer of 2 to 5, and R ⁸ represents an alkyl group having 3 or less carbon atoms.)   The compound according to claim 7, wherein L in the general formula (I) is a hydrolyzate of 3-aminopropyltrialkoxysilane. A nucleic acid probe represented by the following general formula (I).
A-L-B (I)
(In the formula (I), A represents a fluorescent semiconductor nanoparticle having a particle diameter of 1 to 20 nm, B represents a multi-stranded nucleic acid-binding fluorescent molecule, and L represents a divalent linking group adsorbed or bound to the surface of A. Represents.) A multi-strand nucleic acid fluorescence detection method for detecting multi-strand nucleic acid using fluorescence,
Interacting the compound of any one of claims 1 to 8 with a multi-stranded nucleic acid;
Photoexciting the fluorescent semiconductor nanoparticles of A in the general formula (I) to cause excitation energy transfer to the multi-stranded nucleic acid-binding fluorescent molecule of B in the general formula (I);
Detecting fluorescence emitted from the B as a result of the excitation energy transfer;
A multi-stranded nucleic acid fluorescence detection method comprising   The multi-stranded nucleic acid fluorescence detection method according to claim 10, wherein the multi-stranded nucleic acid is a multi-stranded nucleic acid in a solution.   The multi-stranded nucleic acid fluorescence detection method according to claim 10, wherein the multi-stranded nucleic acid is a multi-stranded nucleic acid containing at least one single-stranded nucleic acid immobilized on a solid support.