JP2003329686A - Luminescent fine particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a luminescent fine particle suited for use as a molecular- recognition luminescent marker substance used in the sensitive measurements of target substances such as biological substances or environment-related substances, and being excellent in stability as to dispersion in biological solvents such as highly basic solutions or acidic solutions. <P>SOLUTION: The luminescent fine particle has a polymer having a polar functional group and physically and/or chemically bonded to the surface thereof. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention is suitable as a molecular recognition luminescent marker substance used in a highly sensitive measurement method of a target substance such as a biological substance, an environment-related substance, a high salt or acidic solution, and a biological solvent. And relates to luminescent fine particles having excellent dispersion stability.

[0002]

2. Description of the Related Art In recent years, as a highly sensitive method for measuring biologically-related substances such as proteins, viruses and nucleic acids contained in the living body and environment-related substances such as dioxins and metal ions, molecular recognition substances such as fluorescent substances have been used. A method using a molecular recognition body bound to a marker substance is widely used. However, conventionally used marker substances such as phosphors have low emission efficiency and are not always satisfactory in terms of sensitivity, and because they can only emit light of a certain wavelength, multiple samples can be detected at one time. It was difficult to use for complicated measurements such as

In recent years, semiconductor ultrafine particles (hereinafter, also referred to as semiconductor nanoparticles) have been attracting attention as molecular recognition luminescent fine particles used as a marker substance. Semiconductor nanoparticles are semiconductor ultrafine particles (ultrafine crystals) having a particle size equivalent to the exciton Bohr radius in bulk crystals, and the optical spectrum, that is, the absorption spectrum and the fluorescence spectrum, is adjusted by the particle size by the quantum confinement effect. It is possible to That is, it is possible to emit light of different wavelengths depending on the particle size.

The semiconductor nanoparticles usually have a particle size of 0.5 to 0.5.
The ultrafine particles have a size of 100 nm, preferably 0.5 to 50 nm, and more preferably 1 to 10 nm. The type of the semiconductor nanoparticles is, for example, I-, such as CuCl.
II-V such as Group VII compound semiconductors, CdS, CdSe, etc.
Examples thereof include III-V group compound semiconductors such as group I and InAs, and semiconductor crystals such as group IV semiconductors. Such semiconductor nanoparticles are synthesized by a colloidal chemical synthesis method, and the surface thereof is generally coated with a surfactant and / or a surface modifier and stabilized.

[0005] There are limited synthetic methods for obtaining semiconductor nanoparticles having high quantum efficiency and narrow particle size distribution, and the surface of the nanoparticles obtained by these methods is hydrophobic. There are many. However, if the surface is hydrophobic, the reactivity with the biologically relevant substances such as target proteins, viruses and nucleic acids decreases, and it becomes difficult to detect with high sensitivity. In addition, since semiconductor nanoparticles are likely to aggregate with each other due to Van der Waals forces, and aggregation also occurs during measurement, there is also a problem that the measurement must be terminated before aggregation occurs.

Therefore, when introduced into a living body for use, it is necessary to make the surface of the semiconductor nanoparticles hydrophilic and at the same time prevent the semiconductor nanoparticles from aggregating with each other. Examples of hydrophilicizing the surface of the semiconductor nanoparticles include WO00 / 1.
7656 "WATER-SOLUBLE THEOL-
A method as described in "CAPPED NANOCRYSTALS" has been reported. In this method, a functional group having a polarity such as a carboxyl group is grafted on the surface of the semiconductor nanoparticles to prevent the particles from aggregating due to the repulsion between the electric charges of the ions of the functional group.

However, according to this method, the long-term dispersibility is unstable even in an in vitro test in which the test conditions can be strictly controlled, and further, in vivo
In the o test, since it was in a living body with a high salt concentration, the electric charge of the functional group grafted on the particle surface was canceled by the ions existing in the living body, and the aggregation could not be prevented.

[0008]

In view of the above situation, the present invention is suitable as a marker substance for molecular recognition luminescent fine particles used in a highly sensitive measurement method of a target substance such as a biological substance and an environment-related substance. It is an object of the present invention to provide luminescent fine particles having excellent dispersion stability in a biological solvent including a salt or an acidic solution.

[0009]

The present invention is a luminescent fine particle composed of semiconductor nanoparticles in which a polymer having a polar functional group is physically and / or chemically bonded to the surface. The present invention is described in detail below.

The luminescent fine particles of the present invention are composed of semiconductor nanoparticles characterized in that a polymer having a polar functional group is physically and / or chemically bonded to the surface. The semiconductor nanoparticles are not particularly limited, and include, for example, CuC.
I-VII group compound semiconductors such as I, II-VI group semiconductors such as CdS and CdSe, III-V group compound semiconductors such as InAs, semiconductor crystals such as IV group semiconductors, metal oxides such as TiO ₂ , phthalocyanines, azo compounds And the like, or a composite material thereof. As such a composite material, for example, CdS is core-CdS.
e for shell, CdSe for core-CdS for shell, CdS
Is a core-ZnS shell, CdSe is a core-ZnS shell, CdSe nanocrystals are a core-ZnS shell, C
Examples thereof include those having a core-shell structure in which dSe nanocrystals have a core-ZnSe as a shell and Si has a core-SiO ₂ as a shell.

The semiconductor nanoparticles may be those whose surface is chemically or physically modified as long as the object of the present invention is not impaired. It may be one to which an additive such as an agent or an antioxidant is added. Such semiconductor nanoparticles can be prepared by a colloidal chemical method such as a reverse micelle method (Liano).
s, P.S. et al. Chem. Phys.
Lett. , 125, 299 (1986)) and the hot soap method (Peng, X. et al., J.
Am. Chem. Soc. , 119, 7019
(1997)) and the like.

The preferable lower limit of the particle size of the semiconductor nanoparticles is 0.5 nm, and the upper limit thereof is 100 nm. 0.5 nm
If it is less than 1, it becomes an atom or molecule itself, and 1
If it exceeds 00 nm, it may become a bulk property. A more preferable lower limit is 0.5 nm and an upper limit is 50 n
m, more preferably the lower limit is 1 nm and the upper limit is 10 nm. The shape of the semiconductor nanoparticles is not particularly limited, and examples thereof include spherical shape, rod shape, plate shape, thin film shape, fiber shape, tube shape and the like. Of these, spherical shape is preferable.

In the luminescent fine particles of the present invention, a polymer having a polar functional group is physically and / or chemically bonded to the surface of the semiconductor nanoparticles. By physically and / or chemically bonding a polymer having a polar functional group,
Agglomeration of particles can be prevented by electrical repulsion between polar functional groups of the polymer. The polymer having a polar functional group preferably has a functional group that is ionized under acidic conditions and / or basic conditions. In general, in vivo, depending on the site, both acidic and basic conditions are possible, but by having both a functional group that ionizes under acidic conditions and a functional group that ionizes under basic conditions at the same time,
Since the polar functional group on the surface can be ionized under acidic condition or basic condition, aggregation of particles can be prevented by electrostatic repulsion. Examples of such a functional group include a carboxyl group, a sulfonyl group, a phosphoric acid group, an ammonium group and the like. Further, the polymer having the polar functional group is preferably a natural product because it is used for in vivo measurement.

Proteins are particularly preferable as the polymer capable of satisfying such various requirements. By binding a protein and a semiconductor nanoparticle, the luminescent fine particle of the present invention can exhibit stable dispersion stability in a living body, and can also exhibit the function originally possessed by the protein. It can be used to measure target substances such as substances and environment-related substances. For example, if a protein having cell membrane permeability is used as the protein,
It can also be used for analysis of target substances existing in cells without destroying the cells. The protein is preferably an immunoreactive inactive protein.
If an immunoreactive inactive protein is used in a living body, it can be measured without being eliminated by the immune system of the living body.

Specific examples of such a protein include albumin, myoglobin, casein and the like. Of these, albumin is preferable because it is also excellent in terms of binding to the molecular recognition substance described below.

The semiconductor nanoparticles and the polymer having the polar functional group are physically and / or chemically bonded. The form of the bond is not particularly limited, and examples thereof include chemical adsorption, physical adsorption, coordination, hydrogen bond, ionic bond, covalent bond and the like. From the viewpoint of stability of binding, it is preferable that they are bound by a binding mode having a strong binding force. Note that, in addition to direct bonding, indirect bonding, that is, other organic molecules that mediate bonding between the surface of the semiconductor nanoparticles and the polymer may be present as long as the object of the present invention is not impaired. Good. Further, it is preferable that a plurality of the polymers having the polar functional group are bound so as to surround the surface of the semiconductor nanoparticles. By increasing the coverage of the surface of the polymer semiconductor nanoparticles having polar functional groups, the dispersion stability of the luminescent particles of the present invention is further improved.

The method for producing the luminescent fine particles of the present invention is not particularly limited.
Examples include a method of immersing in an albumin solution. By this method, albumin is physically adsorbed on the surface of the semiconductor nanoparticles.

Since the polymer having a polar functional group is physically and / or chemically bonded to the surface of the luminescent fine particles of the present invention, the particles are caused by electrical repulsion between polar functional groups of the polymer. Aggregation can be prevented. In particular, when a protein is used as a polymer having a polar functional group, it is possible to maintain a stable dispersed state in a living body and to penetrate into a cell without destroying the cell through a cell membrane. . Further, the luminescent fine particles of the present invention,
Since semiconductor nanoparticles are used, they emit fluorescence of high brightness (photoluminescence) upon irradiation with excitation light, and can be suitably used for highly sensitive measurement of trace components. Further, by selectively using the luminescent fine particles of the present invention composed of semiconductor nanoparticles having different particle sizes according to the purpose, the emission spectrum can be arbitrarily adjusted and designed, and application to more precise measurement can be achieved. .

The molecule-recognizing luminescent particles obtained by adsorbing and / or binding a molecule-recognizing substance to the luminescent particles of the present invention are suitable for use in a highly sensitive method for measuring a target substance such as a biological substance or an environment-related substance. You can Such molecular recognition luminescent fine particles are also one aspect of the present invention.

The above-mentioned molecular recognition substance is not particularly limited as long as it specifically reacts with the target substance, and examples thereof include proteins such as antigens and antibodies, and cyclic compounds such as DNA, cyclodextrin and crown ether.

The method for producing the molecular recognition luminescent fine particles of the present invention is not particularly limited, and examples thereof include a physical adsorption method and a chemical bonding method. For example, when the molecular recognition substance is a protein, the amino group of the protein and the luminescent fine particle are bonded directly or by a condensation reagent by treating the luminescent fine particle with an aminosilane derivative or the like. When the molecular recognition substance is a PCR product of DNA, it can be electrostatically bound by coating the luminescent fine particles with poly L-lysine. When the molecular recognition substance is an oligonucleotide, the luminescent fine particles previously coated with alkylaminoasilane are protected with a linker having a photosensitizing protective group, deprotected by light irradiation, and the oligonucleotide chain is synthesized by repeating the synthesis. How to do

When albumin is used as the polymer having a polar functional group used in the luminescent fine particles of the present invention, the method for producing the molecular recognition luminescent fine particles of the present invention using biotin and avidin is preferable. is there. That is, since biotin has excellent binding properties to albumin, when albumin is used as the polymer having a polar functional group in the luminescent fine particles of the present invention, biotin can be easily bound to the albumin. it can. On the other hand, biotin can easily bind to proteins such as antigens and antibodies used as molecular recognition substances, DNA, and the like. Luminescent fine particles of the present invention having biotin on the surface,
Antigens with biotin on the surface, proteins such as antibodies, and D
A molecular recognition substance such as NA can be easily bound via avidin. As described above, albumin is used as the polymer having a polar functional group used in the luminescent fine particles of the present invention, and the molecule-recognizing luminescent fine particles (Av) in which the luminescent fine particles and the molecular recognition substance are bound via biotin and avidin.
idin-Biotin-Albumin-Quant
um Dots: hereinafter, also referred to as ABA-QDs) is preferable because it is easy to produce and has a wide range of applications. One embodiment of such molecular recognition luminescent fine particles of the present invention is shown in FIGS.
It was shown to.

The molecule-recognizing luminescent fine particles of the present invention can be used as a target substance for various bio-related substances, environment-related substances and the like by selecting a molecule-recognizing substance. The target substance to be measured by the molecular recognition luminescent particles of the present invention is not particularly limited as long as it can produce an antibody or a receptor, and examples thereof include proteins such as antigens / antibodies and abnormal prions, dioxins and the like. Examples thereof include endocrine disrupting substances, viruses such as AIDS virus, peptides, nucleic acids, metal ions and the like.

The molecular recognition luminescent fine particles of the present invention are excellent in dispersion stability because they are composed of the luminescent fine particles of the present invention. Therefore, when used in an in vitro method such as detection of a protein by an ELISA method, detection of DNA or RNA by an immunoscreening method or a hybridization method, aggregation does not easily occur, and a stable test can be performed for a long time. In addition, because it has excellent dispersion stability in vivo, it can measure target substances in vivo with high sensitivity, and can be applied to in vivo tests such as tracking of drugs and biological components and analysis of drug action mechanism. can do.
Furthermore, by using it as a carrier for affinity chromatography, it can be applied to separation and concentration of target substances.
A detection kit for a target substance using the molecular recognition luminescent fine particles of the present invention is also one of the present invention.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

(Example 1) 11-Mercaptoundecanoic acid sodium salt surface-modified luminescent fine particles (MUA-QD)
1 mg and bovine serum albumin (Sigma: BSA) 1
0 mg was dissolved in 0.1 mL of distilled water in advance, and
2- [N-morpholino] ethanesulfonic acid (MES)
3.4 mL of buffer (pH 4.8) was added and mixed thoroughly. Then, 10 mg / mL 1-dissolved in distilled water
0.5 mL of a solution of ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Pierce, EDC) was added, and the mixture was reacted with stirring at room temperature for 2 hours. Unreacted B
SA was removed by centrifuging the reaction solution with Microcon YM-100 (manufactured by Millipore) at 15,000 × g for 5 minutes. After centrifugation, washing twice with PBS buffer (pH 7.2), and then dissolving with 0.5 mL of PBS buffer, luminescent fine particles (BSA-bound MUA-QD) in which bovine serum albumin was bonded to the surface of the semiconductor element Got

The BSA-bound MUA-QD obtained was 0,
It was dissolved in 10 mM phosphate buffer (pH 7.2) containing sodium chloride at concentrations of 0.25, 0.5, 1, 2, and 4 M, and left standing at room temperature for 3 days. After stirring well, 10
It was made with mM phosphate buffer (pH 7.4).
Electrophoresis was performed on a 5% agarose gel at 135 V for 50 minutes. MUA-QD without ABS as a control
Electrophoresis was performed in the same manner. The obtained electrophoresis image is shown in FIG. From FIG. 3, BSA-bound MUA-
With QD, a broad electrophoretic image was observed even after treatment with 4 M sodium chloride, indicating that the particles were sufficiently dispersed. On the other hand, it was found that MUA-QD was not electrophoresed at all even when treated with 0.25 M sodium chloride, and aggregated.

Further, BSA-coupled MUA-QD and MUA-
QD and 10 mM MES buffer (pH 4.0 and p
H5.5) and 10 mM phosphate buffer (pH 7.
It melt | dissolved in 4) and it left still at room temperature for 12 hours. After thorough stirring, electrophoresis was performed on a 0.5% agarose gel prepared with 10 mM phosphate buffer (pH 7.4) at 135 V for 50 minutes. The obtained electrophoresis image is shown in FIG. From FIG. 4, BSA-bound MUA-QD showed the same mobility even at pH 4 as at pH 7.4, indicating that it was sufficiently dispersed even under acidic conditions. On the other hand, M
In UA-QD, the pH of the sample migrates at pH 5.5, but
It was found that the mobility was inferior to that in the case of 7.4 and some aggregation was observed, and further that the aggregation was complete at pH 4.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, it is suitable as a marker substance for molecular recognition luminescent fine particles used in a highly sensitive measurement method of a target substance such as a bio-related substance and an environment-related substance, and particularly, dispersion stability in vivo It is possible to provide excellent luminescent fine particles.

[Brief description of drawings]

FIG. 1 is a schematic view showing one embodiment of the molecular recognition luminescent fine particles of the present invention.

FIG. 2 is a schematic view showing one embodiment of the molecular recognition luminescent fine particles of the present invention.

FIG. 3 shows BSA-bound MUA-QD and M produced in the examples.
It is an electrophoresis image after treating UA-QD with sodium chloride of different concentration.

FIG. 4 shows BSA-bound MUA-QD and M prepared in the examples.
It is an electrophoretic image after processing UA-QD with different pH.

[Explanation of symbols]

1 Luminescent particles 2 Semiconductor nanoparticles 3 albumin 4 biotin 5 Avidin 6 Molecular recognition substance (oligo DNA) 7 Molecular recognition substance (IgG antibody)

Continued front page    (71) Applicant 502166891             Shinya Maenozono             7-3-1 Hongo, Bunkyo-ku, Tokyo (71) Applicant 000002174             Sekisui Chemical Co., Ltd.             2-4-4 Nishitenma, Kita-ku, Osaka-shi, Osaka (72) Inventor Kenichi Hanaki             1-11-4-1001 Nishi-Waseda, Shinjuku-ku, Tokyo (72) Inventor Kenji Yamamoto             1-8-18 West, Bunkyo-ku, Tokyo (72) Inventor Yukio Yamaguchi             7-3-1 Hongo, Bunkyo-ku, Tokyo (72) Inventor Shinya Maenozono             7-3-1 Hongo, Bunkyo-ku, Tokyo (72) Inventor Yoichiro Komori             2-4-4 Nishitenma, Kita-ku, Osaka-shi, Osaka             Sekisui Chemical Co., Ltd. F term (reference) 4H045 AA10 BA70 CA40 DA70 EA20                       EA50 EA61 FA50

Claims (9)

[Claims] 1. A luminescent fine particle, wherein a polymer having a polar functional group is physically and / or chemically bonded to the surface. 2. The luminescent fine particle according to claim 1, wherein the polymer having a polar functional group has a functional group which is ionized under acidic conditions and / or basic conditions. 3. The luminescent fine particle according to claim 1 or 2, wherein the polymer having a polar functional group is a natural polymer. 4. The luminescent fine particle according to claim 1, 2 or 3, wherein the polymer having a polar functional group is a protein. 5. The luminescent fine particle according to claim 4, wherein the protein is an immunoreactive inactive protein. 6. The luminescent fine particle according to claim 4 or 5, wherein the protein is albumin. 7. A molecule-recognizing luminescent particle, which is obtained by adsorbing or binding a molecule-recognizing substance to the luminescent particle according to claim 1. 8. The molecular recognition luminescent particles according to claim 7, wherein the luminescent particles and the molecular recognition substance are bound to each other via biotin and avidin. 9. A detection kit for a target substance, which comprises the molecular recognition luminescent fine particle according to claim 7 or 8.