WO2009116326A1 - Near infrared light-emitting fluorescent nanoparticle and biological label using the same - Google Patents

Abstract

Provided is a fluorescent nanoparticle which is in a nano-size suitable for biological labels, emits near infrared light passing through a so-called biological window, and has a high light-emission accuracy. Also, provided is a biological label using the same. The near infrared light fluorescent nanoparticle as described above is an infrared light-emitting fluorescent nanoparticle having an average particle size of 2 to 50 nm and emitting near infrared light of a wavelength ranging from 700 to 2000 nm when excited by near infrared light of a wavelength ranging from 700 to 900 nm, characterized by at least a part of the composition thereof being represented by the following general formula (1) APO4 or (2) AF3 (wherein A represents at least one element selected from the group consisting of Y, Lu and La), being doped with at least one kind of rare earth metal, and being modified at the surface thereof with polyethylene glycol having a terminal functional group.

Description

Near-infrared emitting phosphor nanoparticle and biomaterial labeling agent using the same

The present invention relates to near-infrared light emitting phosphor nanoparticles dispersible in water, and a biological material labeling agent using the same.

As a means for labeling a biological substance, a method using a biological substance labeling agent in which a molecular labeling substance is bound to a fluorescent substance has been studied. In this case, there is a problem that light in the ultraviolet region with a short wavelength used as excitation light damages the cell, and there is a demand for a long-wavelength excitation / light emission phosphor with little damage.

On the other hand, in recent years, in vivo optical imaging targeting small animals has attracted attention, and various optical system devices for observing cells in a small animal's living body from outside without damaging the living body (non-invasively) are available. Has begun to be sold from. In this method, a fluorescent material with a label that selectively gathers at a site to be observed in the living body is injected into the living body, and the emitted light emitted from the outside is externally monitored.

Thus, in order to excite the fluorescent material in the living body and extract the emitted light to the outside, the excitation light and the emitted light need to pass through the living body. Ultraviolet light and visible light are not preferred because they are highly absorbed by the living body and hardly transmit. On the other hand, when the wavelength is 1000 nm or more, the absorption of water rises and the transmittance decreases, which is not preferable. However, the near-infrared region of 700 to 1000 nm is a region called “biological window” and “spectral region window” with a particularly high transmittance of the living body, and a fluorescent material that exhibits excitation and emission within this range is desired. It has been.

Fluorescent materials such as organic fluorescent dyes conventionally used in the above method have the disadvantages of being rapidly deteriorated when irradiated with excitation light and having a short lifetime, and have low luminous efficiency and insufficient sensitivity.

Therefore, in recent years, a method using semiconductor nanoparticles as the fluorescent substance has attracted attention. For example, a biological substance labeling agent in which a polymer having a polar functional group is physically and / or chemically adsorbed and bonded to the surface of a semiconductor nanoparticle has been studied (see, for example, Patent Document 1). In addition, a biological substance labeling agent in which organic molecules are bonded to the surface of Si / SiO ₂ type semiconductor nanoparticles has been studied (for example, see Patent Document 2).

However, biological substance labeling agents using these conventional semiconductor nanoparticles have unsolved problems in terms of light emission accuracy.

For example, the semiconductor nanoparticles disclosed in Patent Document 1 substantially including the effects thereof are (CdSe / ZnS type) semiconductor nanoparticles, which are generally called quantum dots and are larger than the size of Bohr excitons. In the case of having a small particle size, the band gap changes depending on the size, that is, the emission wavelength changes by changing the particle size with the same composition. While such a quantum dot fluorescent material has the advantage that the emission wavelength can be freely changed depending on the size, it has the disadvantage that the accuracy of particle size control leads to the accuracy of the emission wavelength.

On the other hand, since the emission wavelength of inorganic phosphor nanoparticles having a luminescent center of a rare earth element does not depend on particle size control, the problem of light emission accuracy can be solved. It is disclosed that it becomes an external light emitting material. (Non-patent document 1 and Non-patent document 2)
However, another method disclosed is characterized in that inorganic phosphor nanoparticles modified with an organic compound are stably dispersed in an organic solvent (for example, Non-Patent Document 1 and Non-Patent Document). 2). Therefore, in order to use it as a biomarker material, it is necessary to make it water-soluble.

Also disclosed are water-solubilized inorganic phosphor nanoparticles that emit light in the visible light region by binding avidin via 6-aminohexylcarboxylic acid (eg, Non-Patent Document 3). Patent Document 2). However, those in which avidin is directly bonded to phosphor nanoparticles are described as having reduced emission luminance in an aqueous dispersion state, and are not sufficient for use as a biomarker material.

On the other hand, a biomarker material using YAG (yttrium, aluminum, garnet) with a rare earth element as a dopant as a near-infrared light emitting phosphor that emits light by near-infrared excitation is a near-infrared region that transmits through the "biological window". It is disclosed as a material that emits light (for example, see Patent Document 3). However, in the method in which avidin is directly bonded to the phosphor via the silane coupling agent described in the specification, as a result of our investigation, water dispersibility is not sufficient, and a desired light emission luminance cannot be obtained. In silane coupling, the surface becomes hydrophobic, which works disadvantageously for binding a biomolecule labeling substance such as hydrophilic avidin, and it is assumed that avidin cannot be sufficiently bonded.

Moreover, a biomarker material in which inorganic phosphor nanoparticles in which lanthanum phosphate is doped with a rare earth is surface-modified is exemplified (see, for example, Patent Document 4). Although a method for binding a biomarker material via polyethylene glycol having no functional group at the end has been described, as a result of our study, sufficient water dispersibility was not obtained and the emission luminance was not sufficient. Since polyethylene glycol does not have a functional group, it is presumed that the bonding with the inorganic phosphor nanoparticles becomes insufficient.
JP 2003-329686 A JP 2005-172429 A Japanese Patent Laid-Open No. 2007-230877 US2004 / 0014060 Publication Nano Letters Vol. 2,733-737 (2002) Chemistry of Materials Vol. 15, 46 04-4616 (2003) Angelwandte Chemie International Edition Vol. 43, 5954-5957 (2004)

The present invention has been made in view of the above-described problems and situations, and its solution is a nano-size suitable for a biological substance labeling agent, which emits near-infrared light that passes through a "biological window" It is to provide phosphor nanoparticles having high emission accuracy in a dispersed state. Moreover, it is providing the biological material labeling agent using the same.

As a result of intensive studies to solve the above problems, the present inventors have an average particle diameter of 2 to 50 nm, and when excited by near-infrared light having a wavelength in the range of 700 to 900 nm, the inventors have 700 to 2000 nm. Near-infrared emitting phosphor nanoparticles exhibiting near-infrared light emission having a wavelength within the range of at least a part of the composition represented by the following general formula (1) APO ₄ or (2) AF ₃
(Wherein A is at least one element selected from the group consisting of Y, Lu and La), which is doped with at least one rare earth element and whose surface is functional at the end. The present inventors have found a near-infrared light emitting phosphor nanoparticle characterized by being modified with a polyethylene glycol having a group, and further found that it is suitably used as a biological substance labeling agent.

By using polyethylene glycol having a functional group at the end for the surface modification of the near-infrared emitting phosphor nanoparticles, it can be expected that the functional group at the end of the polyethylene glycol and the nanoparticle surface form a coordination bond. It can be inferred that the bond between the particles and polyethylene glycol has become stronger. Therefore, after the surface treatment reaction of polyethylene glycol, the polyethylene glycol does not come off in the process of washing and removing unreacted polyethylene glycol, and it becomes possible to bind the polyethylene glycol to the particle surface at a higher density, and water dispersibility. Will greatly improve. As a result, it is considered that the nanoparticles of the present invention have high emission intensity even in an aqueous dispersion state.

Also, since the water dispersibility of the nanoparticles has been improved, it is easy to bind a labeling substance derived from a living body, and it can be assumed that it is suitable as a biomarker.

That is, the above-mentioned problem according to the present invention is solved by the following means.

1. Near-infrared light having an average particle size of 2 to 50 nm and emitting near-infrared light having a wavelength in the range of 700 to 2000 nm when excited by near-infrared light having a wavelength in the range of 700 to 900 nm A luminescent phosphor nanoparticle, the composition of which at least part is at least one selected from the group consisting of general formula (1) APO ₄ or (2) AF ₃ (wherein A is Y, Lu and La) A near-infrared emitting phosphor characterized in that at least one rare earth element is doped and the surface thereof is modified with a polyethylene glycol having a functional group at its terminal. Nanoparticles.

2. 2. The near-infrared light emitting phosphor nanoparticles according to the above 1, wherein the rare earth element to be doped is any one of praseodymium, neodymium, holmium, erbium, ytterbium, or a combination thereof.

3. 3. The near-infrared light emitting phosphor nanoparticles according to 1 or 2, wherein the functional group of polyethylene glycol having a functional group at the terminal is an amino group.

4. 4. A biological material labeling agent, wherein the near-infrared light emitting phosphor nanoparticles according to any one of 1 to 3 and a molecular labeling material are bonded via an organic molecule.

5. 5. The biological substance labeling agent according to 4 above, wherein the molecular labeling substance is a nucleotide chain.

6. 6. The biological material labeling agent according to 4 or 5 above, wherein the organic molecule that binds the near-infrared light emitting phosphor nanoparticles and the molecular labeling material is biotin and avidin.

By the means of the present invention, a near-infrared emitting phosphor that emits near-infrared light having a wavelength in the range of 700 to 2000 nm when excited by near-infrared light having a wavelength in the range of 700 to 900 nm. Can be provided. Furthermore, it is a nano-size suitable for a biological substance labeling agent, can emit near-infrared light that passes through a “biological window”, and can provide phosphor nanoparticles with high emission accuracy. Moreover, the biological material labeling agent using the same can be provided.

The near-infrared light emitting phosphor nanoparticles of the present invention have an average particle size of 2 to 50 nm, and when excited by near infrared light having a wavelength in the range of 700 to 900 nm, A near-infrared phosphor nanoparticle that emits near-infrared light having a wavelength, wherein at least a part of the composition is represented by the following general formula (1) APO ₄ or (2) AF ₃ (where A is A polyethylene glycol having at least one element selected from the group consisting of Y, Lu, and La, doped with at least one rare earth element, and having a functional group at its terminal. It is characterized by being modified. This feature is a technical feature common to the inventions according to claims 1 to 6.

As an embodiment of the present invention, the doped rare earth element is preferably any one or a combination of praseodymium, neodymium, holmium, erbium, ytterbium.

Furthermore, it is preferable that the functional group of polyethylene glycol having a functional group at the terminal is an amino group.

Further, the near-infrared light emitting phosphor nanoparticles of the present invention can be used as a biological material labeling agent by binding with a molecular labeling substance via an organic molecule. In the biological substance labeling agent, the molecular labeling substance is preferably a nucleotide chain. The organic molecules are preferably biotin and avidin.

Hereinafter, the present invention, its components, and the best mode for carrying out the present invention will be described in detail.

(Near-infrared emitting phosphor nanoparticles)
The near-infrared light emitting phosphor nanoparticles of the present invention have an average particle size of 2 to 50 nm, and when excited by near infrared light having a wavelength in the range of 700 to 900 nm, A near-infrared phosphor nanoparticle that emits near-infrared light having a wavelength, and at least a part of the composition is represented by the following general formula (1) APO ₄ or (2) AF ₃ (where A is Y , Lu and La, and at least one rare earth element is doped.

Moreover, as a preferable embodiment, at least one of Pr and Tb is contained as a co-activator.

In addition, when the near-infrared light emitting phosphor nanoparticles finally formed are particles of 50 nm or less, when the number of metal elements in the constituent elements is 4 or more, or a co-activator of 10 atom% or less When it is contained, the emission intensity is remarkably increased as compared with particles produced by a conventional solid phase method, and when there are three kinds of metal elements or when no coactivator is contained.

As a production method for producing the near-infrared light emitting phosphor nanoparticles of the present invention, for example, Nano Letters Vol. 2, 733-737 (2002) or Chemistry of Materials Vol. 15, 4604-4616 (2003) can be applied.

As the raw material for producing the near-infrared light emitting phosphor nanoparticles of the present invention, halides, nitrates and the like of various elements contained in the general formula (1) can be used. For example, neodymium chloride, neodymium nitrate, ytterbium chloride, ytterbium nitrate, lanthanum chloride, lanthanum nitrate, yttrium chloride, yttrium nitrate, pradocem chloride, erbium chloride, and the like can be used.

As the phosphoric acid source, orthophosphoric acid or the like can be used, and as the fluoride source, sodium fluoride or the like can be used.

In the present invention, the average particle diameter of the near-infrared light emitting phosphor nanoparticles must originally be determined in three dimensions, but it is difficult because it is too fine, and in reality it must be evaluated with a two-dimensional image. It is preferable to obtain by averaging a large number of images taken by changing the shooting scene of the electron micrograph using an electron microscope (TEM). Therefore, in the present invention, the average particle diameter is a diameter obtained by taking an electron micrograph using a TEM, measuring a cross-sectional area of a sufficient number of particles, and setting the measured value as an area of a corresponding circle. Obtained as the diameter, the arithmetic average was taken as the average particle diameter. The number of particles photographed with a TEM is preferably 20 or more, and more preferably 100 particles.
(Hydrophilic treatment of aggregates of near-infrared emitting phosphor nanoparticles)
The near-infrared light emitting phosphor nanoparticles described above can be obtained in an organic solvent dispersed state. However, when used as a biological material labeling agent, it is necessary to perform a hydrophilic treatment in order to exhibit water dispersibility. As a hydrophilic treatment method, there is a method of chemically and / or physically binding a surface modifier to the particle surface.

As a method of hydrophilization treatment, for example, there is a method of chemically and / or physically binding a surface modifier to the particle surface after removing the lipophilic group on the surface with pyridine or the like. As the surface modifier, those having a carboxyl group / amino group as a hydrophilic group are preferably used, and specific examples include mercaptopropionic acid, mercaptoundecanoic acid, aminopropanethiol and the like.

In the present invention, it is preferable to use polyethylene glycol having a functional group at the terminal as the surface modifier.

Examples of the functional group include a carboxyl group, a thiol group, and an amino group. Among them, those having an amino group can be preferably used.

Further, the number average molecular weight of polyethylene glycol is preferably 1000 to 5000. If the molecular weight is lower than this, the hydrophilic effect is not sufficiently exhibited. If the molecular weight is higher than this, the particle size of the bonded nanoparticles becomes large and the movement of the particles in the cell is prevented. It is not preferred as a labeling agent. The number average molecular weight here is obtained by converting a GPC retention time of a target polymer by creating a calibration curve for the retention time using polystyrene with a known molecular weight in gel permeation chromatography (GPC). It is.

Specifically, examples of the polyethylene glycol having an amino group at its terminal include NOF SUNBRIGHT MEPA-20H (number average molecular weight 2000), SUNBRIGHT HO-034PA (number average molecular weight 3400), and the like.

(Biological substance labeling agent)
The biological material labeling agent according to the present invention is obtained by binding a near-infrared light emitting phosphor nanoparticle surface-treated with the above-described polyethylene glycol having a functional group at a terminal and a molecular labeling substance via an organic molecule. .

(Molecular labeling substance)
The biological substance labeling agent according to the present invention can label a biological substance by specifically binding and / or reacting with the target biological substance.

Examples of the molecular labeling substance include nucleotide chains, antibodies, antigens, and cyclodextrins.

(Organic molecule)
In the biological material labeling agent according to the present invention, the near-infrared light emitting phosphor nanoparticles subjected to a hydrophilic treatment and the molecular labeling substance are bound by an organic molecule. The organic molecule is not particularly limited as long as it is an organic molecule capable of binding a near-infrared emitting phosphor nanoparticle and a molecular labeling substance. It is also preferable to use avidin together with biotin. The form of the bond is not particularly limited, and examples thereof include a covalent bond, an ionic bond, a hydrogen bond, a coordinate bond, physical adsorption, and chemical adsorption. A bond having a strong bonding force such as a covalent bond is preferable from the viewpoint of bond stability.

Specifically, when the near-infrared emitting phosphor nanoparticles are hydrophilized with mercaptoundecanoic acid, avidin and biotin can be used as organic molecules. In this case, the carboxyl group of the nanoparticle subjected to hydrophilic treatment is preferably covalently bonded to avidin, and avidin is further selectively bonded to biotin, and biotin is further bonded to the biological material labeling agent to Become.

Near-infrared light emitting phosphor nanoparticles having an amino group exposed on the surface may use a bivalent cross-linking agent such as EMCS (N- (6-maleimidocaproyloxy) succinimide) or NHS ester (N-hydroxysuccinimide). By forming an amide bond with a carboxyl group activated with a ruthenium ester, etc., it can be linked to avidin or streptavidin, avidin or streptavidin fusion protein, biotin, antibody or antigen. The avidin or streptavidin to which these near-infrared emitting phosphor nanoparticles are bound, a fusion protein of avidin or streptavidin, biotin, antibody, and antigen can be used as a detection antibody or a detection enzyme in the sandwich method.

In addition, when near-infrared emitting phosphor nanoparticles are incorporated into functional beads, they can be used for detection of biomolecules, concentration measurement, etc. by flow cytometry. The functional beads at this time are the surfaces of polymer beads such as polystyrene beads, polypropylene beads, crosslinked acrylic beads, polylactic acid beads, magnetic beads, glass beads, metal beads, etc. with a size of 0.1 μm to 100 μm. This refers to chemical modification that specifically adsorbs or binds to the derived substance. The method for dispersing near-infrared light emitting phosphor nanoparticles in such functional beads is not particularly limited. For example, in the case of functional beads using polymer beads, near-infrared light emitting phosphor nanoparticles are previously contained in a solvent. Is dispersed and the beads are swollen in the solvent, whereby the near-infrared light emitting phosphor nanoparticles can be incorporated into the functional beads.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

In the following, the near-infrared light emitting phosphor nanoparticles are simply referred to as “phosphors”. As a notation of the inorganic material doped with rare earth, for example, lanthanum phosphate doped with neodymium is represented as LaPO ₄ : Nd.

Example 1 Method for Producing LaPO ₄ : Nd (Phosphor 1) Modified with Polyethylene Glycol Having an Amino Group at the Terminal Using Lanthanum Chloride and Neodymium Chloride, Chemistry of Materials Vol. 15, 4604-4616 (2003), LaPO ₄ : Nd nanoparticles were synthesized.

To the LaPO ₄ : Nd nanoparticle powder (10 mg), polyethylene glycol having an amino group (SUNBRIGHT MEPA-20H: 0.5 g, manufactured by NOF Corporation) was dispersed in 1 ml of 0.1 M NaOH aqueous solution and heated until boiling. After cooling to room temperature, ultrasonic irradiation was performed for 2 minutes.

The obtained phosphor was added with 0.4 ml of 0.1 M Tris Buffer (tris (hydroxymethyl) aminomethane hydrochloride, pH 9) and 0.65 ml of ethanol, stirred, and then centrifuged (19400 g for 10 minutes). ). After removing the supernatant, 0.15 ml of 0.1M Tris Buffer (pH 9) was added and redispersed. Washing with Tris Buffer and ethanol was repeated three times to obtain phosphor 1.
Example 2 Method for Producing LaF ₃ : Nd (Phosphor 2) Modified with Polyethylene Glycol Having an Amino Group at the Terminal Using Lanthanum Chloride and Neodymium Chloride, Nano Letters Vol. 2, 733-737 (2002), LaF ₃ : Nd nanoparticles were synthesized.

A phosphor 2 was obtained in the same manner as in Example 1 except that the LaF ₃ : Nd nanoparticle powder (10 mg) was used.
(Example 3) Method for producing LaPO ₄ : Nd, Yb (phosphor 3) modified with polyethylene glycol having an amino group at the end Example 1 except that 10 mol% ytterbium chloride was added to neodymium chloride. The same operation was performed to obtain phosphor 3.
Example 4 Method for Producing LaPO ₄ : Er (Phosphor 4) Modified with Polyethylene Glycol Having an Amino Group at the Terminal The same operation as in Example 1 was performed except that erbium chloride was used instead of neodymium chloride The phosphor 4 was obtained.
(Example 5) Method for producing LaPO ₄ : Pr (phosphor 5) modified with polyethylene glycol having an amino group at the terminal The same operation as in Example 1 was conducted except that praseodymium chloride was used instead of neodymium chloride. The phosphor 5 was obtained.
(Example 6) Method for producing LaF ₃ : Ho (phosphor 6) modified with polyethylene glycol having an amino group at the end The same operation as in Example 2 was conducted except that holmium chloride was used instead of neodymium chloride. The phosphor 5 was obtained.
(Comparative Example 1) Production method of LaPO ₄ : Nd (phosphor 7) modified with polyethylene glycol having no functional group at the terminal As polyethylene glycol, polyethylene glycol having no functional group at the terminal (polyethylene glycol dimethyl ether manufactured by Aldrich) A phosphor 3 was obtained in the same manner as in Example 1 except that the number average molecular weight 2000) was used.

The particle size of the phosphor formed as described above was observed by TEM, the particle size was measured for 100 particles, and the average particle size was obtained.

Further, an emission spectrum at 810 nm of excitation light was measured in a state of being dispersed in 1 ml of 0.1 M Tris Buffer (pH 9). Table 1 shows the relative fluorescence intensity when the emission peak wavelength of each phosphor and the emission peak intensity of the phosphor 1 are defined as 100.

As described above, the phosphors 1 to 6 (Examples 1 to 6) modified with polyethylene glycol having a functional group at the terminal of the present invention are phosphors modified with polyethylene glycol having no functional group at the terminal. 7, it can be seen that the emission intensity in the infrared region by the excitation light in the infrared region is sufficiently high even in the water dispersion state.
<Example 7>
10 ^-5 g of phosphor 1 was dispersed in 10 ml of pure water in which 0.2 g of mercaptoundecanoic acid was dissolved, stirred at 40 ° C. for 10 minutes, and the shell surface was treated to modify the surface with a carboxyl group. A phosphor 8 was obtained.

Phosphor 8: 25 mg of avidin was added to an aqueous dispersion of 1.0 × 10 ⁻⁵ mol / L and stirred at 40 ° C. for 10 minutes to prepare avidin-conjugated nanoparticles.

The obtained avidin-conjugated nanoparticle solution was mixed and stirred with a biotinylated oligonucleotide having a known base sequence to prepare an oligonucleotide labeled with a nanoparticle.

When the above labeled (labeled) oligonucleotide is dropped and washed on a DNA chip on which oligonucleotides having various base sequences are immobilized, the oligonucleotide has a complementary base sequence to the labeled (labeled) oligonucleotide. Only the spot of was emitted with excitation light of 810 nm.

From this, it was possible to confirm the labeling of the oligonucleotide with the nanoparticles. That is, it can be seen from this result that a biological substance labeling agent using the near-infrared light emitting phosphor nanoparticles of the present invention can be provided.

Claims (6)

Near-infrared light having an average particle size of 2 to 50 nm and emitting near-infrared light having a wavelength in the range of 700 to 2000 nm when excited by near-infrared light having a wavelength in the range of 700 to 900 nm A luminescent phosphor nanoparticle, the composition of which is at least partly selected from the group consisting of general formula (1) APO ₄ or (2) AF ₃ (wherein A is selected from the group consisting of Y, Lu and La) Near-infrared light emission, characterized in that it is doped with at least one rare earth element and the surface thereof is modified with polyethylene glycol having a functional group at its terminal. Phosphor nanoparticles. 2. The near-infrared light emitting phosphor nano according to claim 1, wherein the rare earth element to be doped is any one of praseodymium, neodymium, holmium, erbium, ytterbium, or a combination thereof. particle. The near-infrared phosphor nanoparticle according to claim 1 or 2, wherein the functional group of polyethylene glycol having a functional group at the terminal is an amino group. A near-infrared light emitting phosphor nanoparticle according to any one of claims 1 to 3 and a molecular labeling substance bonded together via an organic molecule. . The biological substance labeling agent according to claim 4, wherein the molecular labeling substance is a nucleotide chain. The biological material labeling agent according to claim 4 or 5, wherein the organic molecules that bind the near-infrared light emitting phosphor nanoparticles and the molecular labeling substance are biotin and avidin.