JP2009138120A - Near infrared ray-emitting fluorophor, biological substance-labeling agent using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a near infrared ray-emitting fluorophor which emits rear infrared having wavelengths in a range of 700 to 2,000 nm, when excited with near infrared rays having wavelengths in a range of 700 to 900 nm, to provide fluorophor nano particles having nano sizes suitable for biological substance-labeling agents, emitting near infrared rays passing through "biological window", and having high ray-emitting accuracy, and to provide a biological substance-labeling agent using the same. <P>SOLUTION: The near infrared ray-emitting fluorophor emitting near infrared rays having wavelengths in a range of 700 to 2,000 nm, when excited with near infrared rays having wavelengths in a range of 700 to 900 nm, is characterized in that at least one portion of the composition is represented by the following general formula (1): A<SB>1-x-y</SB>Nd<SB>x</SB>Yb<SB>y</SB>(PO<SB>3</SB>)<SB>3</SB>(wherein, A is an element selected from Y, Lu and La; 0<x≤0.5, 0<y≤0.5, 0<x+y<1). <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

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

  As means for labeling a biological substance, a method using a biological substance labeling agent in which a molecular labeling substance is bound to a marker substance has been studied. When a fluorescent material is used for the marker substance, it is a problem that light in the ultraviolet region with a short wavelength used as excitation light damages the cell, and a long-wavelength excitation / luminescence phosphor with little damage is desired. It has been.

  On the other hand, in recent years, in vivo optical imaging targeting small animals has attracted attention, and optical system devices that allow observation of cells in the living body of small animals from the outside without damaging the living body (non-invasively) are provided by various manufacturers. 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, it is necessary that the excitation light and the emitted light 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, 700 to 1000 nm in the near infrared region is a region having a specifically high biological transmittance called “biological window” and “spectral region window”, and a fluorescent material exhibiting excitation and emission within this range is desired. It has been.

  The marker substances such as organic fluorescent dyes conventionally used in the above method have the disadvantages that they are severely deteriorated when irradiated with excitation light and have a short lifetime, and the luminous efficiency is low and the sensitivity is not sufficient.

Therefore, in recent years, a method using semiconductor nanoparticles as the marker 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, there have been unsolved problems with respect to light emission accuracy and the like in the biomaterial labeling agents using these conventional semiconductor nanoparticles.

  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, near-infrared light-emitting phosphors that emit light by near-infrared excitation are generally used as latent image forming inks for preventing counterfeiting of credit cards and prepaid cards that are used as payment methods in place of cash in recent years. Yes. Known _{examples, A 1-xy Nd x Yb} y PO 4
(Wherein A is at least one selected from Y, Lu and La, and 0 <x ≦ 0.5, 0 <y ≦ 0.5, and x + y <1.0). (Patent Document 3) Both of these are excited by a near-infrared light emitting diode (center wavelength: 880 nm) and emit light at 980 nm, so that both excitation light and light emission pass through the “biological window” and have a preferable composition. I know that there is. However, when used as a latent image forming ink, the size of the phosphor particles is generally formed in the range of several microns to sub-micron, and is 100 nm or less which can be suitably used as a biomarker. The particles have not been used conventionally (see Patent Document 3).
JP 2003-329686 A JP 2005-172429 A Japanese Patent No. 3336572

  The present invention has been made in view of the above-mentioned problems and situations, and the problem to be solved is 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. It is providing the near-infrared light emission fluorescent substance which shows light emission of near-infrared light. Furthermore, the present invention is to provide phosphor nanoparticles having a nano-size suitable for a biological substance labeling agent, emitting near-infrared light passing through a “biological window” and having high emission accuracy. Moreover, it is providing the biological material labeling agent using the same.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have made the phosphor a specific composition and a specific particle size, thereby producing a near-infrared light-emitting phosphor, and further a near-infrared light-emitting phosphor nanoparticle. Was obtained, and the present invention was achieved.

  That is, the said subject which concerns on this invention is solved by the following means.

1. 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, At least a part of the composition is represented by the following general formula (1).
Formula _{(1): A 1-xy} Nd x Yb y (PO 3) 3
(Wherein A is an element selected from Y, Lu and La; 0 <x ≦ 0.5; 0 <y ≦ 0.5 and 0 <x + y <1)
2. 2. The near-infrared light-emitting phosphor according to 1 above, wherein the near-infrared light-emitting phosphor is a near-infrared light-emitting phosphor nanoparticle having an average particle diameter of 2 to 50 nm.

  3. 3. The near infrared light emitting phosphor as described in 1 or 2 above, wherein the surface of the near infrared light emitting phosphor is hydrophilized.

  4). 4. A biological material labeling agent comprising the near-infrared light emitting phosphor nanoparticles described in 2 or 3 bound to a molecular labeling material 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.

  A near-infrared light 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 by the above means of the present invention. 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 of the present invention 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. It is an external light-emitting phosphor, and at least a part of the composition is represented by the general formula (1). This feature is a technical feature common to the inventions according to claims 1 to 6.

  As an embodiment of the present invention, an embodiment in which the near-infrared light-emitting phosphor is a near-infrared light-emitting phosphor nanoparticle having an average particle diameter of 2 to 50 nm is preferable. Moreover, it is preferable that the surface of the near-infrared light emitting phosphor is subjected to a hydrophilic treatment.

  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.

  In the present application, the “nanoparticle” means a particle having an average particle diameter (diameter) of less than 100 nm, and a preferable average particle diameter in the present invention is 2 to 50 nm.

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

(Near infrared phosphor)
The near-infrared light-emitting phosphor of the present invention 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. It is an external light-emitting phosphor, and at least a part of the composition is represented by the following general formula (1).
Formula _{(1): A 1-xy} Nd x Yb y (PO 3) 3
(Wherein A is an element selected from Y, Lu and La; 0 <x ≦ 0.5; 0 <y ≦ 0.5 and 0 <x + y <1)
As raw materials for producing the near-infrared light emitting phosphor of the present invention, oxides or halides of various elements contained in the general formula (1) can be used. For example, neodymium oxide, neodymium chloride, neodymium nitrate, ytterbium oxide, ytterbium chloride, ytterbium nitrate, lanthanum oxide, lanthanum chloride, lanthanum nitrate, yttrium oxide, yttrium chloride, yttrium nitrate, pradocem chloride, terbium chloride, orthophosphoric acid, ammonium phosphate , Ammonium dihydrogen phosphate and the like can be used.

(Near-infrared emitting phosphor nanoparticles)
The near-infrared light emitting phosphor nanoparticles of the present invention preferably have an average particle size of 2 to 50 nm. Moreover, as a preferable aspect, at least one element 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 four or more, or 10 atom (atom)% or less. When the co-activator is contained, the emission intensity is significantly higher than particles produced by the conventional solid-phase method, and when the number of metal elements is three or when no co-activator is contained. Become.

  In addition, the raw material for manufacturing the near-infrared light emission fluorescent nanoparticle of this invention is the same as the raw material of the said near-infrared light emission fluorescent substance.

  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 are photographed.

(Method for producing near-infrared emitting phosphor nanoparticles)
The nano-sized near-infrared light-emitting phosphor that is a feature of the present invention is achieved by a production method in which a raw material is dissolved in a solvent and dried and fired by passing through a spray firing furnace.

  In the spray / dry pyrolysis method, the raw material solution is generally atomized by a nozzle and ultrasonic waves into fine droplets, and the solvent of the droplets is evaporated at a high temperature and the obtained solid particles are heated at a high temperature. This is a method of decomposing and obtaining fine particles of a target compound (hereinafter also simply referred to as “particles”). The particle size of the phosphor can be controlled by the droplet size and the raw material solution concentration.

  Further, at this time, the phosphoric acid flux is simultaneously sprayed as a phosphor raw material, thereby preventing an increase in size due to aggregation of particles. Since the phosphor particles are collected in a state of being wrapped in the flux, even if the particles after spray firing are aggregated, the flux part is stuck, and the internal particles are kept as a single unit. By dissolving and removing the flux, nano particles could be achieved.

As the flux, it is preferable to add 1.5 to 10 times the stoichiometric ratio of the raw material phosphate. When the flux is small, the phosphor is fused. If it is excessive, the concentration of the raw material becomes dilute, and the reaction takes time and the yield decreases. [Hydrophilic treatment of near-infrared emitting phosphor nanoparticle aggregates]
Although the near-infrared light emitting phosphor nanoparticles described above are obtained as an aggregate, the surface of the aggregate is generally hydrophobic. For example, when used as a biological material labeling agent, the dispersion is water-dispersed as it is. The surface of the nanoparticle is preferably subjected to a hydrophilization treatment because the properties are poor and the particles are aggregated. As a method for the 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.

Specifically, for example, 10 ⁻⁵ g of near-infrared light emitting phosphor nanoparticles are dispersed in 10 ml of pure water in which 0.2 g of mercaptoundecanoic acid is dissolved, and stirred at 40 ° C. for 10 minutes to form the surface of the shell. By processing, the surface of the near-infrared-emitting phosphor nanoparticles can be modified with a carboxyl group.

[Biological substance labeling agent]
The biological material labeling agent according to the present invention is obtained by binding the above-mentioned hydrophilic-treated near-infrared light emitting phosphor nanoparticles, a molecular labeling substance and 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. For example, among proteins, albumin, myoglobin, casein, and the like are also a kind of protein. 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 coordination 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, avidin and biotin can be used as organic molecules when the near-infrared emitting phosphor nanoparticles are hydrophilized with mercaptoundecanoic acid. 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.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this. In the following, the near-infrared light emitting phosphor nanoparticles are simply referred to as “phosphors”.

  The composition analysis of the obtained phosphor was performed by X-ray diffraction (XRD) (manufactured by PANalytical), and the identification of the composition was compared with the obtained diffraction peak as a reference database. Specifically, using the analysis software X'pert High Score manufactured by PANalytical, using ICDD PDF2 as a reference database, a score indicating the degree of coincidence was obtained, and those having a score of 80 or more were determined as the matrix composition of the phosphor. Identified.

<Example 1> Phosphor 1: Nd ₀ . ₁ Yb ₀ . ₁ Y ₀ . ₈ (PO ₃ ) ₃ production method 59.8 g of ammonium dihydrogen phosphate, 3.5 g of neodymium oxide, 4.0 g of ytterbium oxide, and 18.0 g of yttrium oxide were mixed in a mortar, and then covered with an alumina lid. After filling the crucible, it was put into an electric furnace, heated from room temperature to 700 ° C. at a constant heating rate over 2 hours, and then fired at 700 ° C. for 3 hours. Immediately after the completion of firing, the product was taken out of the electric furnace and cooled in air. Next, water was put in the crucible and immersed in hot water at 80 ° C. for 10 hours. After cooling, it was washed with 1N nitric acid, and then washed with water to obtain phosphor 1. As a result of XRD analysis, the phosphor matrix composition was Y (PO ₃ ) ₃ .

Example 2 Phosphor 2: Nd ₀ . ₁ Yb ₀ . ₁ Y ₀ . ₈ (PO ₃ ) ₃ Production Method The composition of Example 1 was used except that the firing conditions were raised from room temperature to 1000 ° C. over 2 hours at a constant heating rate, and then fired at 1000 ° C. for 5 hours. Thus, phosphor 2 was obtained.

As a result of XRD analysis, the phosphor matrix composition was Y (PO ₃ ) ₃ .

<Comparative Example 1> Phosphor 3: Nd ₀ . ₁ Yb ₀ . ₁ Y ₀ . ₈ PO ₄ Production Method After putting 23 g of ammonium dihydrogen phosphate, 3.5 g of neodymium oxide, 4.0 g of ytterbium oxide, and 18.0 g of yttrium oxide into a mortar, mixing them well, and then filling the crucible with an alumina lid. The phosphor 3 was subjected to the same operation as in Example 1 except that it was put in an electric furnace and heated from room temperature to 700 ° C. at a constant heating rate over 2 hours and then fired at 700 ° C. for 5 hours. Got. As a result of XRD analysis, the matrix composition of the powder was YPO ₄ .

<Comparative Example 2> Phosphor 4: Nd ₀ . ₁ Yb ₀ . ₁ Y ₀ . ₈ PO ₄ Production Method The composition of Example 1 was used except that the composition of Comparative Example 1 was used, and the firing conditions were raised from room temperature to 1200 ° C. at a constant heating rate over 2 hours and then fired at 1200 ° C. for 5 hours. The same operation was performed to obtain phosphor 4. As a result of XRD analysis, the powder matrix composition was YPO ₄ .

  With respect to the phosphors 1, 2, 3 and 4 formed as described above, emission spectra at an excitation light of 810 nm were measured. The results are summarized in Table 1.

From the results shown in Table 1, infrared emission fluorescence is obtained when the matrix composition according to the present invention is Y (PO ₃ ) ₃ (Examples 1 and 2) when firing at a lower temperature and in a shorter time than that of the matrix composition YPO _4. It turns out that a body is obtained.

Example 3 Phosphor 5: Nd ₀ . ₁ Yb ₀ . ₁ Y ₀ . ₈ (PO ₃ ) ₃ Production Method 115 g of ammonium dihydrogen phosphate, 44 g of neodymium nitrate, 41 g of ytterbium nitrate, and 30.6 g of yttrium nitrate are dissolved in dilute nitric acid to make 200 ml.

After the liquid A was reacted at a drying step of 200 ° C. and a baking step of 700 ° C. using a spray baking apparatus described in Japanese Patent Application Laid-Open No. 2003-277745, the obtained powder was placed in hot water at 80 ° C. for 10 minutes. Soaked for hours. After cooling, the phosphor 5 was washed with 1N nitric acid and then washed with water. As a result of XRD analysis, the phosphor matrix composition was Y (PO ₃ ) ₃ .

<Comparative Example 3> Phosphor 6: Nd ₀ . ₁ Yb ₀ . ₁ Y ₀ . ₈ PO ₄ Production Method Phosphor as in Example 1 except that 115 g of ammonium dihydrogen phosphate, 44 g of neodymium nitrate, 41 g of ytterbium nitrate, 30.6 g of yttrium nitrate were dissolved in dilute nitric acid to make 200 ml. 6 was obtained. As a result of XRD analysis, the powder matrix composition was YPO ₄ .

<Comparative Example 4> Phosphor 7: Nd ₀ . ₁ Yb ₀ . ₁ Y ₀ . ₈ PO ₄ Production Method Example 1 except that 115 g of ammonium dihydrogen phosphate, 44 g of neodymium nitrate, 41 g of ytterbium nitrate, 30.6 g of yttrium nitrate were dissolved in dilute nitric acid to make 200 ml, and the baking process was 1200 ° C. In the same manner as in Example 1, a phosphor 7 was obtained. As a result of XRD analysis, the powder matrix composition was YPO ₄ .

  The phosphors 5, 6 and 7 formed as described above were observed with a TEM, and the particle size of 100 particles was measured to obtain the average particle size.

  In addition, an emission spectrum at excitation light of 810 nm was measured. The relative light emission intensity with the light emission peak intensity of the phosphor 1 as 100 is shown.

  The above results are summarized in Table 2.

From the results shown in Table 2, the phosphor of Y (PO ₃ ) _{3 based} on the present invention has an average particle diameter in the range of 2 to 50 nm by spray firing. Furthermore, it can be seen that nanoparticles exhibiting high emission intensity can be obtained by low-temperature firing as compared with a phosphor whose base material is YPO ₄ .

<Example 4>
Phosphor 2: 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 resulting 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.

  This confirmed the labeling (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)

A near-infrared light 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, the composition of which A near-infrared light emitting phosphor characterized in that at least a part is represented by the following general formula (1).
Formula _{(1): A 1-xy} Nd x Yb y (PO 3) 3
(Wherein A is an element selected from Y, Lu and La; 0 <x ≦ 0.5; 0 <y ≦ 0.5 and 0 <x + y <1) The near-infrared light-emitting phosphor according to claim 1, wherein the near-infrared light-emitting phosphor is a near-infrared light-emitting phosphor nanoparticle having an average particle diameter of 2 to 50 nm. The near-infrared light-emitting phosphor according to claim 1 or 2, wherein a surface of the near-infrared light-emitting phosphor is hydrophilized. A biological material labeling agent comprising the near-infrared light emitting phosphor nanoparticles according to claim 2 and a molecular labeling substance bonded 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 molecule that binds the near-infrared light emitting phosphor nanoparticles and the molecular labeling substance is biotin and avidin.