JP2009138119A - Near infrared ray-emitting fluorophor nano particle, biological substance-labeling agent using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide fluorophor nano particles which have a nano size suitable for a biological substance-labeling agent, emit rear infrared ray passing through "biological window", and have high ray-emitting accuracy, and to provide a biological substance-labeling agent using the same. <P>SOLUTION: The near infrared ray-emitting fluorophor nano particles having an average particle diameter of 2 to 50 nm and 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>4</SB>(wherein, A is an element selected from Y, Lu and La; 0<x≤0.5, 0<y≤0.5, x+y<1.0), and the fluorophor nano particles are dried, calcinated and produced by passing their raw materials through a spray calcination oven in a state dissolved in a solvent. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to near-infrared emitting phosphor nanoparticles 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, in _{A 1-xy Nd x Yb y} PO 4 ( wherein, A is at least one selected from Y, Lu and La, 0 <x ≦ 0.5,0 < y ≦ 0. 5, x + y <1.0) (see 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 are found to be preferable compositions.

However, when used as a latent image forming ink, the size of phosphor particles is generally formed in the range of several microns to submicron, and stable emission accuracy is obtained. Particles of 100 nm or less that can be suitably used as an agent have not been conventionally used (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-described problems and circumstances, 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” and emits light. It is to provide phosphor nanoparticles with high accuracy. 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 produced phosphor nanoparticles having a specific particle size as phosphor nanoparticles having a specific composition by a specific manufacturing method. The present inventors have found that body nanoparticles can be obtained, and have reached the present invention.

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

1. Near-red light having an average particle diameter 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. Externally emitting phosphor nanoparticles, at least part of the composition of which is represented by the following general formula (1), and dried and fired by passing through a spray firing furnace with the raw material dissolved in a solvent The near-infrared light emitting phosphor nanoparticles characterized by being manufactured.
Formula _{(1): A 1-xy} Nd x Yb y PO 4
(In the formula, A is an element selected from Y, Lu and La, and 0 <x ≦ 0.5, 0 <y ≦ 0.5, and x + y <1.0.)
2. 2. The near-infrared light-emitting phosphor nanoparticle according to 1, wherein the surface thereof is hydrophilized.

  3. A biological material labeling agent, wherein the near-infrared light emitting phosphor nanoparticles according to any one of the above 1 and 2 are bound to a molecular labeling material via an organic molecule.

  4). 4. The biological substance labeling agent according to 3 above, wherein the molecular labeling substance is a nucleotide chain.

  5). 5. The biological material labeling agent according to 3 or 4 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 above-mentioned means of the present invention, it is possible to provide phosphor nanoparticles having a nano-size suitable for a biological substance labeling agent, emitting near-infrared light that passes through a “biological window” and having 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 diameter of 2 to 50 nm, and when excited by near-infrared light having a wavelength in the range of 700 to 900 nm, are in the range of 700 to 2000 nm. A near-infrared phosphor nanoparticle that emits near-infrared light having a wavelength of at least a portion of which the composition is represented by the general formula (1) and the raw material is dissolved in a solvent. It is characterized by being manufactured by drying and firing by passing through a spray firing furnace. This feature is a technical feature common to the inventions according to claims 1 to 5.

  As an embodiment of the present invention, it is preferable that the near-infrared light emitting phosphor nanoparticles of the present invention have a hydrophilic surface.

  Moreover, the near-infrared light emitting phosphor nanoparticle 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 emitting phosphor nanoparticles)
The near-infrared light emitting phosphor nanoparticles of the present invention are characterized in that 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 4
(In the formula, 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.)
The near-infrared light emitting phosphor nanoparticles of the present invention exhibit properties of emitting near infrared light having a wavelength in the range of 700 nm to 2000 nm when excited by near infrared light having a wavelength in the range of 700 nm to 900 nm. It is necessary to make the average particle size of the nanoparticles 2 to 50 nm in order to have the above. 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.

  As raw materials for producing the near-infrared light-emitting phosphor nanoparticles 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.

  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.

[Hydrophilic treatment of near-infrared 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 emitting phosphor particles are simply referred to as “phosphors”.

The phosphors: Nd ₀ . ₁ Yb ₀ . ₁ Y ₀ . ₈ PO ₄ was produced.

Example 1 Method for Producing Phosphor 1 115 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, and a solution A is obtained.

  After the liquid A was reacted at a drying step of 200 ° C. and a baking step of 1200 ° C. using a spray baking apparatus described in JP-A No. 2003-277745, the obtained powder was placed in hot water at 80 ° C. for 10 minutes. Soaked for hours. After cooling, it was washed with 1N nitric acid, and then washed with water to obtain phosphor 1.

<Example 2>: Production method of phosphor 2 Example 2 except that 230 g of ammonium dihydrogen phosphate, 44 g of neodymium nitrate, 41 g of ytterbium nitrate, and 30.6 g of yttrium nitrate were dissolved in dilute nitric acid to give 500 ml. Similarly, phosphor 2 was obtained.

<Comparative example 1> Production method of phosphor 3 The following powder raw materials 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 were placed in a mortar and mixed sufficiently, and then made of alumina. After filling the crucible with the lid, it was put in an electric furnace, heated from room temperature to 1200 ° C. at a constant heating rate over 2 hours, and then fired at 1200 ° C. for 5 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 3.

  The phosphors 1, 2 and 3 formed as described above were observed with a TEM, and the particle size was measured for 100 particles to determine the average particle size.

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

  The results are shown in Table 1.

  From the results shown in Table 1, the phosphor obtained in the present invention has a maximum (maximum) emission wavelength in the range of 970 to 980 nm and high emission intensity. At the same time, the average particle diameter is 2 to 50 nm. You can see that it is in range.

<Example 3>
Phosphor 1: 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 (5)

Near-red light having an average particle diameter 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. Externally emitting phosphor nanoparticles, at least part of the composition of which is represented by the following general formula (1), and dried and fired by passing through a spray firing furnace with the raw material dissolved in a solvent The near-infrared light emitting phosphor nanoparticles characterized by being manufactured.
Formula _{(1): A 1-xy} Nd x Yb y PO 4
(In the formula, A is an element selected from Y, Lu and La, and 0 <x ≦ 0.5, 0 <y ≦ 0.5, and x + y <1.0.) The near-infrared-emitting phosphor nanoparticle according to claim 1, wherein the surface thereof is hydrophilized. A biological material labeling agent comprising the near-infrared light emitting phosphor nanoparticles according to any one of claims 1 to 2 and a molecular labeling substance bonded via an organic molecule. The biological substance labeling agent according to claim 3, wherein the molecular labeling substance is a nucleotide chain. The biological material labeling agent according to claim 3 or 4, wherein the organic molecule that binds the near-infrared light emitting phosphor nanoparticle and the molecular labeling substance is biotin and avidin.