WO2013011984A1

WO2013011984A1 - Fluorescent probe and method for producing same

Info

Publication number: WO2013011984A1
Application number: PCT/JP2012/068111
Authority: WO
Inventors: 健治小野; 誠澤田; 真悟渕; 竹田　美和
Original assignee: 国立大学法人名古屋大学
Priority date: 2011-07-21
Filing date: 2012-07-17
Publication date: 2013-01-24
Also published as: JPWO2013011984A1; JP6019535B2

Abstract

This fluorescent probe is formed of a glass powder that contains rare earth element ions, which are excited by light having a wavelength in the near infrared region and emit light having a wavelength in the near infrared region other than the above-mentioned wavelength, in glass. This method for producing a fluorescent probe sequentially comprises: a melting step wherein a compound containing a rare earth element and a glass starting material are melted; a cooling step wherein the molten material obtained in the melting step is cooled, thereby producing a glass composition that contains rare earth element ions; and a crushing step wherein the glass composition is crushed, thereby obtaining a powder.

Description

Fluorescent probe and method for producing the same

The present invention relates to a fluorescent probe that emits near-infrared light having a wavelength different from that of irradiated light when irradiated with near-infrared light, and a method for manufacturing the same.

Physiological imaging using a fluorescence method is one of imaging methods that can capture structural changes and dynamic changes of an object in a minimally invasive manner without using radiation. In the fluorescence method, a fluorescent probe is widely used, and a fluorescent probe is useful because a structural change or a dynamic change of a target object (target molecule) is visualized and observed based on emitted fluorescence. As such a fluorescent probe, for example, Patent Document 1 describes a fluorescent dye (organic fluorescent dye) made of an organic compound such as a fluorescent protein such as GFP or DsRed, a Cy series such as Cy2, or an Alexa series such as Alexa488. Yes. Patent Document 2 describes many fluorescent probes made of other organic compounds.

Most of the fluorescent probes made of these organic compounds are excited with light having a short wavelength of around 480 nm, so that the excitation light cannot reach the deep part of the living body and can only image the outer surface of the living body. In addition, since the organic compound emits light in the visible light range, it overlaps with various fluorescence of substances constituting the living body, and is obtained as an image in which fluorescence of the target object and other fluorescence are mixed. For this reason, it is necessary to devise a method for clearly grasping the target object, such as spectroscopic analysis. Furthermore, since organic fluorescent dyes tend to fade due to the principle of fluorescence generation, there is a drawback that the fluorescence signal of the target substance disappears when the irradiation time of excitation light is long.

In order to solve these problems, it is considered that one of the clues is to use light in the near-infrared light region that penetrates from the outer surface of the living body to a depth of about 20 mm as excitation light and use a fluorescent probe corresponding to it. It is done.

Compared to the visible light region, in the near-infrared light region having a wavelength of more than 800 nm, the living body emits blood components and the like because it has not only high light permeability but also extremely low light absorption of water and hemoglobin. Almost no autofluorescence is seen. For this reason, when light in the near-infrared light region is used, it is considered that it is easy to clearly distinguish between the fluorescence of the target object and the fluorescence of other substances. Therefore, it is considered that the use of a near-infrared light region fluorescent probe enables unprecedented good imaging.

In recent years, as described in Patent Document 3, semiconductor quantum dots have begun to be used as fluorescent probes. In this method, fluorescent probes corresponding to various wavelengths can be produced by changing the particle diameter of the quantum dots. Further, unlike organic fluorescent dyes, a fluorescent probe in the near-infrared light region can be produced.

However, quantum dots that emit fluorescence in the near-infrared light region have a disadvantage that they have a large particle size and are difficult to use in living organisms. Furthermore, since the quantum dot contains a biological harmful substance such as cadmium, there is a need to reduce toxicity.

Patent Document 4 discloses silicon dioxide nanoparticles containing rare earth element ions, and Patent Document 5 discloses YAG nanocrystals containing rare earth element ions. Since these techniques are different from quantum dots, it is possible to produce a fluorescent probe that emits light with a wide range of wavelengths without depending on the particle diameter.
The invention described in

Patent Document

4 or 5 is considered to be more useful in controlling the emission wavelength, fading stability, particle size, and the like than the inventions described in Patent Documents 1 to 3. However, when introducing rare earth ions into YAG nanocrystals or silicon dioxide nanocrystals, crystal growth is performed so that the rare earth ions are incorporated at specific positions in the crystal so that they emit light with high efficiency. Conditions must be controlled and advanced techniques are required in the manufacturing process. Also, advanced techniques are needed to control the size of nanocrystals. Furthermore, it is necessary to prevent aggregation of the nanocrystals, and the production management cost becomes enormous. Since the silicon dioxide nanoparticles of Patent Document 4 are based on a synthesis method from a solution, this problem is important. Although Patent Document 5 suggests that an amorphous material is used instead of a crystalline material, the specific raw materials and manufacturing methods thereof are not described, and a method similar to that of YAG nanocrystals is used. It is estimated that amorphous nanoparticles are produced from the solution.

International Publication WO2009 / 020197 JP 2008-149154 A International Publication No. WO2008 / 123291 Japanese translation of PCT publication No. 2003-522149 Japanese Patent Laid-Open No. 2007-230877

An object of the present invention is to provide a fluorescent probe using near infrared light and an efficient manufacturing method thereof.

The present inventors produced a glass composition in which ions of rare earth elements were dispersed in glass. And when near infrared light was irradiated to this glass composition, it discovered that the near infrared light different from the wavelength of irradiated light was emitted. Moreover, when the glass composition was crushed and atomized, the powder was found to be a fluorescent probe suitable for biological imaging using light in the near infrared light region as excitation light. That is, the present invention is shown below.
1. A fluorescent probe containing rare-earth element ions that are excited by light having a wavelength in the near-infrared light region and emit light having a wavelength in the near-infrared light region other than the wavelength, and comprising a glass powder containing the ion A fluorescent probe characterized by
2. 2. The fluorescent probe according to 1 above, wherein the near infrared light region has a wavelength in the range of 800 to 1200 nm.
3. 3. The fluorescent probe according to 1 or 2 above, wherein the rare earth element ion is at least one selected from Yb ion, Nd ion, Tm ion, Sm ion, Ho ion, Er ion, Dy ion and Pr ion.
4). 4. The fluorescent probe according to any one of 1 to 3 above, wherein the glass constituting the glass powder is at least one selected from boric acid glass, germanic acid glass, phosphoric acid glass and fluoride glass. .
5. The glass is Bi ₂ O ₃ —B ₂ O ₃ glass, Bi ₂ O ₃ —GeO ₂ glass, ZnO—B ₂ O ₃ glass, CaO—B ₂ O ₃ glass and CaO—P ₂ O _5. 5. The fluorescent probe as described in 4 above, which is at least one selected from a system glass.
6). 6. The fluorescent probe according to any one of 1 to 5 above, wherein a content ratio of the rare earth element is 0.4 to 2.0 at% with respect to a total amount of atoms constituting the fluorescent probe.
7). 7. The fluorescent probe according to any one of 1 to 6, wherein a lower limit value when measuring the maximum length of the powder is 0.1 μm and an upper limit value is 1.0 μm.
8). A melting step of melting a compound containing a rare earth element and a glass raw material, a cooling step of cooling a melt obtained by the melting step to produce a glass composition containing ions of rare earth elements, and the glass composition And a crushing step for crushing to obtain powder.
9. 9. The fluorescent probe according to 8 above, wherein the compound containing the rare earth element is at least one selected from compounds containing a rare earth element selected from Yb, Nd, Tm, Sm, Ho, Er, Dy and Pr. Production method.
10. 10. The method for producing a fluorescent probe according to 8 or 9, wherein the compound containing the rare earth element is an oxide.
11. 11. The fluorescent probe according to any one of the above 8 to 10, wherein the glass raw material contains a compound that forms at least one selected from boric acid glass, germanic acid glass, phosphoric acid glass, and fluoride glass. Production method.
12 12. The method for producing a fluorescent probe according to any one of 8 to 11, wherein the temperature in the melting step is 900 ° C. to 1350 ° C.
13. 13. The method for producing a fluorescent probe according to any one of 8 to 12, wherein in the crushing step, crushing with a pestle and mortar and / or crushing with a ball mill is performed.

The fluorescent probe of the present invention is made of glass powder in which ions of rare earth elements are dispersed in glass, and therefore emits near-infrared light different from the wavelength of the irradiated light when irradiated with near-infrared light. Utilizing this property, it can be suitably used for biological imaging using light in the near-infrared light region as excitation light. In addition, unlike organic dyes, the fluorescent probe of the present invention is chemically stable without fading, and there is no restriction on the particle size as in quantum dots. It is suitable as a fluorescent probe in the method. In particular, since the excitation light described above is excellent in permeability from the body surface to the inside of animals including humans, a desired site in a range from the surface to a depth of about 20 mm can be observed in more detail.

When the rare earth element ion in the present invention is at least one selected from Yb ion, Nd ion, Tm ion, Sm ion, Ho ion, Er ion, Dy ion and Pr ion, the near infrared light region Light having a wavelength different from that of the excitation light (fluorescence) can be easily obtained.
For example, in the case where both of Yb ions and Nd ions are included as rare earth element ions, light emission in the 1000 nm band (including 950 to 1050 nm) with high biological permeability can be obtained by irradiation with light having a wavelength of 808 nm. When Nd ions are used alone as rare earth element ions, light emission in the 900 nm band (including 860 to 930 nm) can be obtained by irradiation with light having a wavelength of 808 nm. It is done. Further, when Yb ions are used alone as rare earth element ions, light emission in the 1000 nm band (including 950 to 1050 nm) can be obtained by irradiation with light having a wavelength of 808 nm.

When the glass in the present invention is at least one selected from boric acid glass, germanic acid glass, phosphoric acid glass and fluoride glass, it is excellent in the transparency of excitation light and fluorescence wavelengths. Luminous intensity can be obtained.
Further, when the content ratio of the rare earth element constituting the powder is 0.4 to 2.0 at% with respect to the total amount of atoms constituting the fluorescent probe, higher fluorescence intensity can be obtained.

According to the method for producing a fluorescent probe of the present invention, a fluorescent probe can be produced efficiently. After the glass raw material is melted by the melting process, the fluorescent probe is manufactured through the cooling process and the crushing process, and thus a manufacturing method with a high degree of freedom can be obtained. Moreover, since a glass composition can be selected widely as mentioned above, a general glass raw material can be handled freely according to a crushing characteristic and a light emission characteristic. For example, as a glass raw material, Bi ₂ O ₃ —B ₂ O ₃ glass, Bi ₂ O ₃ —GeO ₂ glass, ZnO—B ₂ O ₃ glass, CaO—B ₂ O ₃ glass, or CaO—P _{2 is used.} with the material for forming the O ₅ based glass, a melting temperature in the melting step, it is possible to reduce, it becomes possible to smoothly form the glass composition. Note that, in the method using nanocrystal growth from a solution, such as the method described in the cited document, the growth conditions differ depending on the crystal matrix composition, and thus the degree of freedom is small. Furthermore, since the glass composition can be easily atomized by general crushing, the method for producing the fluorescent probe of the present invention is very simple. In addition, regarding the prevention of aggregation, a general aggregation inhibitor may be used when the glass composition is produced and then crushed. As described above, the manufacturing cost and the management cost can be reduced by the manufacturing method of the fluorescent probe of the present invention.

It is an external appearance image of the glass composition (before crushing) produced with the manufacturing method of the fluorescent probe of the present invention. It is a graph which shows the particle size distribution of the fluorescent probe produced with the manufacturing method of the fluorescent probe of this invention. 2 is an image taken by irradiating visible light on the fluorescent probe powder obtained in Example 1. FIG. It is the image image | photographed by irradiating the near-infrared light to the fluorescent probe (powder) obtained in Example 1. 2 is a fluorescence spectrum obtained by irradiating the fluorescent probe (powder) obtained in Example 1 with near infrared light. It is the image image | photographed under natural light, after inject | pouring the fluorescent probe obtained in Example 1 into the tail vein of a mouse | mouth. It is the image image | photographed by injecting the near infrared light after inject | pouring the fluorescent probe obtained in Example 1 into the tail vein of a mouse | mouth. It is the image image | photographed under natural light, after integrating the fluorescent probe obtained in Example 1 in the lungs of a mouse | mouth. It is the image image | photographed by irradiating near-infrared light, after integrating the fluorescent probe obtained in Example 1 in the mouse | mouth lung. It is the image image | photographed by irradiating near-infrared light before inject | pouring the fluorescent probe obtained in Example 1 under the skin of a mouse | mouth. It is the image image | photographed by irradiating near infrared light, after inject | pouring the fluorescent probe obtained in Example 1 under the skin of a mouse | mouth. It is the fluorescence spectrum obtained by irradiating the near-infrared light to the fluorescent probe powder obtained in Example 4. It is the fluorescence spectrum obtained by irradiating the near-infrared light to the fluorescent probe powder obtained in Example 5. It is the fluorescence spectrum obtained by irradiating the near-infrared light to the fluorescent probe powder obtained in Example 6. It is the fluorescence spectrum obtained by irradiating the near-infrared light to the fluorescent probe powder obtained in Example 7.

1. Fluorescent probe The fluorescent probe of the present invention is a fluorescent probe containing rare earth element ions that are excited by light having a wavelength in the near infrared light region and emit light having a wavelength in the near infrared light region other than the wavelength, It consists of glass powder containing ions. The near-infrared wavelength region in the present invention means a wavelength range of about 750 nm to 2500 nm, and preferably a wavelength range of 800 nm to 1200 nm.
In this specification, Yb, Nd, Tm, Sm, Ho, Er, Dy and Pr are rare earth elements ytterbium (Yb), neodymium (Nd), thulium (Tm), samarium (Sm), holmium (Ho). ), Erbium (Er), dysprosium (Dy) and praseodymium (Pr).
The glass powder constituting the fluorescent probe of the present invention is a powder in which ions of rare earth elements are dispersed in glass. This means a state in which rare earth element ions are scattered and contained in the glass, and is not a state in which a specific chemical structure is formed by the rare earth element ions. In the fluorescent probe of the present invention, rare earth element ions exist as trivalent ions.

The ions of rare earth elements contained in the fluorescent probe of the present invention are excited by light having a wavelength corresponding to the excitation band, and emit light when relaxing from the excited state to the ground state. In the fluorescent probe of the present invention, light having a wavelength corresponding to the excitation band of rare earth element ions is in the near-infrared light region, and the wavelength of emitted light is in the near-infrared light region and is different from the excitation light. Since it is a wavelength, it can be said that it is a fluorescent probe using light of a wavelength other than the excitation light. Moreover, since the rare earth element ions are contained in the glass having excellent transparency to excitation light and fluorescence, sufficient emission intensity in the fluorescent probe can be obtained without reducing the emission intensity of the rare earth element ions. Can do.

The rare earth element ion used in the fluorescent probe of the present invention is an ion of at least one element selected from 17 elements from scandium to lutetium. Only one type of ion in the powder may be used, or a combination of two or more types may be used. In the present invention, since fluorescence with high light transmittance to a living body can be obtained, Yb ion, Nd ion, Tm ion, Sm ion, Ho ion, Er ion, Dy ion and Pr ion are preferable. Yb ions and Nd ions are preferred. Yb ions and Nd ions may be used in combination, or may be used alone.
For example, in a fluorescent probe containing Yb ions and Nd ions as the emission center, two ions are excited by receiving light including the vicinity of 808 nm, and emit light in the near infrared light region of 850 nm to 1100 nm. In this way, a fluorescent probe that emits light at a wavelength different from that of the excitation light and is suitable for observation of a living body can be obtained.

The lower limit of the content ratio of the rare earth element in the powder constituting the fluorescent probe of the present invention is preferably 0.05 at% with respect to the total amount of atoms constituting the fluorescent probe from the viewpoint of emission intensity. More preferably, it is 0.4 at%, More preferably, it is 0.7 at%. Moreover, an upper limit is 10 at% normally, Preferably it is 7 at%, More preferably, it is 3.4 at%, More preferably, it is 2.0 at%. If the content of rare earth element ions is too high, concentration quenching may occur. Therefore, the content ratio may be appropriately set in accordance with the type of rare earth element ions and the like within a range where concentration quenching does not occur.

The glass constituting the glass powder in the present invention is an amorphous solid composed of an inorganic compound exhibiting a glass transition phenomenon. This amorphous solid preferably has the same degree of rigidity as a crystal pulverized by a known method. From the viewpoint of an industrial process, a lower melting point is preferable. In addition, a lower limit is 340 degreeC normally. Specific examples of the glass include boric acid glass, germanic acid glass, phosphoric acid glass, and fluoride glass. One kind of the glass may be used, or two or more kinds may be combined. Among these, since high emission intensity can be obtained, Bi ₂ O ₃ —B ₂ O ₃ glass, Bi ₂ O ₃ —GeO ₂ glass, ZnO—B ₂ O ₃ glass, CaO— are more preferable. B ₂ O ₃ glass and CaO—P ₂ O ₅ glass. More preferred is Bi ₂ O ₃ —B ₂ O ₃ based glass.

The form of the fluorescent probe of the present invention is a powder, and the shape is a lump such as a sphere, an ellipsoid, a polyhedron (cube, cuboid, octahedron, etc.), etc .; Linear body, curved body) and the like. Specifically, the size of the fluorescent probe is such that the upper limit of the maximum length measured with an electron microscope, a laser scatterometer or the like is preferably 10 μm, more preferably 5 μm, and even more preferably 1 μm or less. However, the lower limit is usually 0.1 μm. Since the emission intensity of the fluorescent probe depends on the volume of the powder, sufficient emission intensity can be obtained if the lower limit of the maximum length is 0.1 μm. In addition, it is preferable that the fluorescent probe is small because it is easy to move in the capillaries of animals including humans. The shape preferable for biological imaging is a shape having as few pointed parts as possible, and a shape close to a polyhedron or a sphere is more preferable.

2. Method for Producing Fluorescent Probe The method for producing a fluorescent probe of the present invention was obtained by melting a compound containing a rare earth element (hereinafter also simply referred to as “rare earth compound”) and a glass raw material, and the melting step. It comprises a cooling step of cooling the melt to produce a glass composition containing rare earth element ions, and a crushing step of crushing the glass composition to obtain a powder.
In the manufacturing method of the present invention, unlike the technique in which the rare earth element must be taken into a specific position in the crystal, the ions of the rare earth element are simply taken into the glass composition by the melting step and the cooling step. Does not require manufacturing techniques.

The melting step is a step of melting a mixture containing a rare earth compound and a glass raw material. By this melting process, both the rare earth compound and the glass raw material are melted and mixed to produce a melt.

The rare earth compound is a compound that forms a molten mixture with a glass raw material in a melting step and generates ions of rare earth elements in the glass composition after the cooling step. Examples of such compounds include oxides, hydroxides, sulfates, carbonates, nitrates, phosphates and halides containing rare earth elements. Such rare earth compounds may be used alone or in combination of two or more. Among these, a compound containing a rare earth element selected from Yb, Nd, Tm, Sm, Ho, Er, Dy, and Pr is preferable because fluorescence with high light transmittance to a living body is obtained, and oxidation of these elements is preferable. The product is particularly preferred. Specifically, when producing a fluorescent probe in which Yb ions or Nd ions are dispersed in glass, Yb ₂ O ₃ or Nd ₂ O ₃ is preferably used as the rare earth compound.

The glass raw material is a material capable of forming glass by being cooled after being made into a melt by heating. As this glass raw material, a well-known glass forming compound can be used, and examples thereof include SiO ₂ , GeO ₂ , P ₂ O ₅ , B ₂ O _{3 and the} like. In order to control various physical properties of the glass, modified oxides such as Bi ₂ O ₃ , ZnO, and CaO can be used in combination. Specifically, when the glass constituting the glass powder is Bi ₂ O ₃ —B ₂ O ₃ glass, Bi ₂ O ₃ and H ₃ BO ₃ are preferably used.
When a raw material for forming Bi ₂ O ₃ —B ₂ O ₃ glass is used as the glass raw material, the melting point is relatively low and the vitrification range is relatively wide, which is advantageous for the production of a fluorescent probe.
Considering the production cost, it is preferable to use a glass raw material that lowers the melting point of the glass to be formed. Moreover, it is preferable to adjust the ratio of each oxide in glass so that the glass from which an appropriate crushing characteristic is obtained can be obtained.

The mixing ratio of the rare earth compound and the glass raw material in the melting step is appropriately selected according to the purpose of use of the fluorescent probe. As described above, since the preferable content ratio of the rare earth element is 0.05 to 10 at% with respect to the total amount of atoms constituting the fluorescent probe of the present invention, the amount of the rare earth compound used is that of the rare earth compound and the glass raw material. The total amount is preferably 0.1 to 10 mol%, more preferably 1 to 5 mol%. When the amount of the rare earth compound used is within the above range, concentration quenching is suppressed, and a fluorescent probe excellent in emission characteristics can be obtained.

In the melting step, the method for preparing the melt and the melting conditions are not particularly limited.
The melting temperature in the melting step is appropriately selected depending on the kind of the rare earth compound and the glass raw material, but is preferably 800 ° C. to 1500 ° C., more preferably 900 ° C. to 1350 ° C., further preferably 1000 ° C. to 1250 ° C. It is.
Further, the melting time in the melting step is preferably 5 to 60 minutes, more preferably 10 to 15 minutes.

The cooling step is a step of cooling the melt obtained in the melting step to obtain a glass composition containing rare earth element ions. The method for cooling the melt and the cooling conditions are not particularly limited. For example, air cooling by being left in an air atmosphere can be mentioned. The cooling rate can be, for example, several kelvins per second.

The crushing step is a step of crushing the glass composition obtained in the cooling step to obtain a powder. The powder obtained in this crushing process can be used as a fluorescent probe. The pulverization method of the glass composition is not particularly limited, and a known method of pulverizing and atomizing a lump such as glass can be applied. Examples include crushing with a pestle and mortar, crushing with a ball mill, and the like. These pulverization methods can also be performed in combination.

Hereinafter, an example of the method for producing the fluorescent probe of the present invention will be shown.

A fluorescent probe made of a powder of Bi ₂ O ₃ —B ₂ O ₃ glass containing Yb ions and Nd ions is Yb ₂ O ₃ powder, Nd ₂ O ₃ powder, Bi ₂ O ₃ powder and H ₃ BO _3. The powder is mixed at a predetermined ratio, heated to melt the mixture, then the melt is cooled, and further, the cooled product (glass composition) is pulverized. In the case of melting the mixture, an alumina crucible or the like is used, and the melting temperature is set at, for example, 1000 ° C. to 1250 ° C. Cooling may be performed by pouring a melt (melt) into a stainless or carbon mold and air cooling. In addition, when melting is performed at a temperature exceeding 1000 ° C., Bi ions are reduced. In this case, when Sb ₂ O ₃ powder is used, reduction of Bi ions can be suppressed. When the total amount of the rare earth compound and the glass raw material is 100 mol%, if the amount of Yb ₂ O ₃ used is 5.0 mol% or less and the amount of Nd ₂ O ₃ used is 6.0 mol% or less, 1000 A glass composition can be produced by cooling the melt at 0C. As a raw material of the fluorescent probe, Yb ₂ O ₃ is 1.0 mol%, Nd ₂ O ₃ is 1.0 mol%, Bi ₂ O ₃ is 48.5 mol%, B ₂ O ₃ is 48.5 mol%, Sb ₂ O _3. FIG. 1 shows an appearance image of a glass composition (before crushing) prepared with 1.0 mol%.

Next, a fluorescent probe can be produced by crushing the obtained glass composition by the method exemplified above. By changing the crushing conditions, the particle size distribution of the finally obtained fluorescent probe can be controlled, for example, as shown in FIGS.

As an ion source of a rare earth element, when using a _Yb 2 _{O 3} and _Nd 2 _{O 3,} the composition of the usage, as well as the glass raw material of _Yb 2 _{O 3} and _Nd 2 _{O 3,} as desired emission characteristics can be obtained, as appropriate , You may change. For example, when the total of the rare earth compound and the glass raw material is 100 mol%, Yb ₂ O ₃ is 5.0 mol%, Nd ₂ O ₃ is 2.9 mol%, Bi ₂ O ₃ is 43.9 mol%, B ₂ A fluorescent probe produced with 48.1 mol% of O ₃ gives sufficient emission characteristics. Further, the amount of _Yb 2 _{O 3} and _Nd 2 _{O 3,} respectively, are fixed to 5.0 mol% and 3.0mol%, _Bi 2 _{O 3} and _B 2 _{O 3} usage, respectively, 91.9Mol % And 0 mol%, 82.4 mol% and 9.5 mol%, 73.2 mol% and 18.8 mol%, 64.5 mol% and 27.3 mol%, 55.2 mol% and 33.7 mol%, or 36.6 mol % And 55.4 mol% can also be changed.

The fluorescent probe of the present invention is suitable for biological imaging, and a method for introducing the fluorescent probe into the living body is exemplified below.
(1) A method of directly injecting a fluorescent probe with a syringe (2) A method of directly injecting a dispersion containing a fluorescent probe and a liquid with a syringe (3) A fluorescent probe with endocytosis or phagocytosis (4) A method of injecting cells labeled with a fluorescent probe on a cell membrane by a syringe (5) A method of injecting an antibody or a chemical substance labeled with a fluorescent probe with a syringe; and Excitation light is irradiated from outside the living body, and light emission from the fluorescent probe is observed. At that time, since the wavelength of the excitation light and the emission wavelength are different, an optical filter that removes the excitation light may be used. As a light source for excitation light, a commercially available light source may be used, and a semiconductor light emitting device such as a light emitting diode or a laser diode is preferable. The light emission of the rare earth ion-dispersed glass fluorescent probe may be performed using an existing two-dimensional detector such as a Si-based CCD camera or an InGaAs camera.
Since the fluorescent probe of the present invention is chemically stable, even if it is introduced into a desired site in a living body, it does not cause alteration or decomposition. Strength can be obtained.

Hereinafter, the embodiment of the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1
(1) Preparation of fluorescent probe As a rare earth element compound, 1 mol% Yb ₂ O ₃ powder (Kanto Chemical Co., Ltd. material research reagent) and 4 mol% Nd ₂ O ₃ powder (Kanto Chemical Co., Ltd. material research reagent) As a glass raw material, 47 mol% Bi ₂ O ₃ powder (material research reagent manufactured by Kanto Chemical Co., Inc.), H ₃ BO ₃ powder (special grade reagent manufactured by Nacalai Tesque) in an amount of 47 mol% in terms of B ₂ O ₃ , 1 mol% of Sb ₂ O ₃ powder (reagent manufactured by Wako Pure Chemical Industries, Ltd.) and (rare earth element compound and glass raw material are combined to make 100 mol%) are mixed in a zipper bag, and fluorescent A mixed powder as a raw material of the probe was prepared. Next, the mixed powder was put into an alumina crucible and heated at 1250 ° C. for 10 minutes in an air atmosphere to be melted. Subsequently, the melt (melt) was poured out into a stainless steel mold and air-cooled at room temperature (about 20 ° C.) to obtain a glass composition in which rare earth ions were dispersed. Thereafter, the obtained glass composition was crushed with a pestle, a mortar and a ball mill to prepare a powder mixture (fluorescent probe) having a maximum length of 0.1 μm or more and 1 μm or less as measured by a laser scatterometer. The content ratio of the rare earth elements (the total content of Yb and Nd) was 2.0 at% with respect to the total of all atoms constituting the fluorescent probe.
FIGS. 3 and 4 show images of the fluorescent probe when the fluorescent probe obtained as described above is irradiated with visible light and near-infrared light including a wavelength of 808 nm, respectively. When near-infrared light including a wavelength of 808 nm was irradiated, it was confirmed that it was excited to emit fluorescence and reflected the powder shape of the fluorescent probe. Further, it was confirmed by fluorescence spectrum measurement that the wavelength of fluorescence was 850 to 1100 nm (see FIG. 5).

(2) Evaluation of biological imaging 0.1 g of the fluorescent probe was put into 1 ml of a phosphate buffer (pH 7.4, 10 mM) and stirred to obtain a dispersion in which the fluorescent probe was suspended. The obtained dispersion was filled into a 1 ml syringe with a 26 gauge needle, and 200 μl was injected from the tail vein of the mouse. FIG. 6 and FIG. 7 show images of a mouse tail imaged under natural light, and fluorescence emitted from a fluorescent probe contained in the mouse tail (wavelength 850˜) when excited by irradiation with near infrared light including 808 nm. The image which image | photographed 1100 nm light) is shown.

According to the image shown in FIG. 7, the emission of the fluorescent probe in the tail vein of the mouse could be clearly observed (see the emission line between the two arrows). Since light having a wavelength of 808 nm is one of excitation maximum wavelengths with relatively high biological permeability, the fluorescent probe in the tail vein could be sufficiently excited. Since the fluorescence of the fluorescent probe is light with a wavelength of 850 to 1100 nm, the light penetrates the living body and can be easily observed from outside the mouse's tail even though the fluorescent probe is in the tail vein. . Furthermore, no luminescence around the tail vein of the mouse was observed, and the tail vein was observed with minimal invasiveness and no background luminescence.

Example 2
A fluorescent probe dispersion prepared by the same method as in Example 1 was injected into the body of a mouse by the same method as in Example 1, and the fluorescent probe was accumulated in the lung. FIG. 8 and FIG. 9 show images obtained by photographing the part including the lung after incising only the skin of the mouse. FIG. 8 is an image taken under natural light, and FIG. 9 is an image taken of fluorescence (light with a wavelength of 850 to 1100 nm) emitted from a fluorescent probe by being excited by irradiation with near infrared light including 808 nm. It is an image.

From the image shown in FIG. 8, the accumulation of the fluorescent probe is not clear, but according to the image shown in FIG. 9, the emission of the fluorescent probe accumulated in the lung was clearly observed. Even when only the hair was removed without incising the skin, the same fluorescence observation could be performed by irradiation with near infrared light. Thus, imaging of the lung with minimal invasiveness and no background light emission was possible. Finally, accumulation in the lung was confirmed by laparotomy.

Example 3
A fluorescent probe dispersion prepared by the same method as in Example 1 was injected under the skin of the mouse by the same method as in Example 1. 10 and 11 show images taken at the same position before and after injection of the fluorescent probe. In either case, 808 nm excitation light was irradiated.

As shown in FIG. 10, no luminescence was observed in the image before the fluorescent probe was injected under the skin of the mouse. On the other hand, as shown in FIG. 11, after the fluorescent probe was injected under the skin of the mouse, the fluorescence at 850 to 1100 nm was clearly observed only from the injection part under the skin. Thus, only the fluorescent probe existing under the skin could be observed with minimal invasiveness.

In Examples 1 to 3, the atomized fluorescent probe is directly used, but the surface of the fluorescent probe can be coated with a polymer or labeled with an antibody or a drug. When labeling a fluorescent probe, a method of directly binding an antibody or the like to a functional group (for example, a hydroxyl group) on the surface of the fluorescent probe, or a surface of the fluorescent probe with a silane coupling agent (for example, 3-amino A method of binding an antibody or the like after coating with propyltrimethoxysilane) or the like can be applied.

Example 4 (Production of fluorescent probe)
As a rare earth element compound, 1 mol% Yb ₂ O ₃ powder (Kanto Chemical Co., Ltd. material research reagent), 1 mol% Nd ₂ O ₃ powder (Kanto Chemical Co., Ltd. material research reagent), and a glass raw material, 54 mol% ZnO powder (material research reagent manufactured by Kanto Chemical Co., Inc.) and H ₃ BO ₃ powder (special grade reagent manufactured by Nacalai Tesque) in an amount of 44 mol% in terms of B ₂ O ₃ and (rare earth element compound and glass) The raw materials were combined to make 100 mol%) to prepare a mixed powder that was a raw material for the fluorescent probe. Next, the mixed powder was put into an alumina crucible, and a fluorescent probe was produced in the same manner as in Example 1 except that the mixture was heated and melted at 1250 ° C. for 10 minutes in an air atmosphere. The content ratio of the rare earth elements (the total content of Yb and Nd) was 0.8 at% with respect to the total of all atoms constituting the fluorescent probe.
By measuring the fluorescence spectrum of the fluorescent probe obtained as described above, it was confirmed that the wavelength of fluorescence was 870 to 1100 nm (see FIG. 12).

Example 5 (Production of fluorescent probe)
As a rare earth element compound, 1 mol% Er ₂ O ₃ powder (Kanto Chemical Co., Ltd. material research reagent), and as a glass raw material, 49 mol% Bi ₂ O ₃ powder (Kanto Chemical Co., Ltd. material research reagent), B ₂ H ₃ BO ₃ powder (special grade reagent manufactured by Nacalai Tesque) in an amount of 49 mol% in terms of O ₃ , and 1 mol% Sb ₂ O ₃ powder (reagent manufactured by Wako Pure Chemical Industries, Ltd.) and (rare earth element compound and The glass raw materials were combined to make 100 mol%) to prepare a mixed powder that was a raw material for the fluorescent probe. Next, the mixed powder was put into an alumina crucible, and a fluorescent probe was produced in the same manner as in Example 1 except that the mixture was heated and melted at 1250 ° C. for 10 minutes in an air atmosphere. The content ratio of Er, which is a rare earth element, was 0.4 at% with respect to the total of all atoms constituting the fluorescent probe.
By measuring the fluorescence spectrum of the fluorescent probe obtained as described above, it was confirmed that the wavelength of fluorescence was 1440 to 1640 nm (see FIG. 13).

Example 6 (Production of fluorescent probe)
As a rare earth element compound, 1 mol% Sm ₂ O ₃ powder (Kanto Chemical Co., Ltd. material research reagent), as a glass raw material, 54.5 mol% ZnO powder (Kanto Chemical Co., Ltd. material research reagent), and B H ₃ BO ₃ powder (special grade reagent manufactured by Nacalai Tesque) in an amount of 44.5 mol% in terms of ₂ O ₃ is mixed with (rare earth element compound and glass raw material are combined to 100 mol%), and the fluorescent probe A mixed powder as a raw material was prepared. Next, the mixed powder was put into an alumina crucible, and a fluorescent probe was produced in the same manner as in Example 1 except that the mixture was heated and melted at 1250 ° C. for 10 minutes in an air atmosphere. The content ratio of Sm which is a rare earth element was 0.6 at% with respect to the total of all atoms constituting the fluorescent probe.
By measuring the fluorescence spectrum of the fluorescent probe obtained as described above, it was confirmed that the wavelength of fluorescence was 750 to 1050 nm (see FIG. 14).

Example 7 (Production of fluorescent probe)
As a rare earth element compound, Pr ₆ O ₁₁ powder (material research reagent manufactured by Kanto Chemical Co., Inc.) in an amount of 1 mol% in terms of Pr ₂ O ₃ and 54.5 mol% ZnO powder (manufactured by Kanto Chemical Co., Ltd.) as a glass raw material. Material research reagent), and H ₃ BO ₃ powder (special grade reagent manufactured by Nacalai Tesque) in an amount of 44.5 mol% in terms of B ₂ O ₃ and (rare earth element compound and glass raw material are combined to make 100 mol%) ) Were mixed to prepare a mixed powder as a raw material of the fluorescent probe. Next, the mixed powder was put into an alumina crucible, and a fluorescent probe was produced in the same manner as in Example 1 except that the mixture was heated and melted at 1250 ° C. for 10 minutes in an air atmosphere. The content ratio of Pr, which is a rare earth element, was 0.6 at% with respect to the total of all atoms constituting the fluorescent probe.
By measuring the fluorescence spectrum of the fluorescent probe obtained as described above, it was confirmed that the wavelength of fluorescence was 760 to 1100 nm (see FIG. 15).

The fluorescent probe of the present invention is suitable for biological imaging using near infrared light.

Claims

A fluorescent probe containing rare-earth element ions that are excited by light having a wavelength in the near-infrared light region and emit light having a wavelength in the near-infrared light region other than the wavelength, and comprising a glass powder containing the ion A fluorescent probe characterized by
The fluorescent probe according to claim 1, wherein the near-infrared light region has a wavelength in a range of 800 to 1200 nm.
3. The fluorescent probe according to claim 1, wherein the rare earth element ion is at least one selected from Yb ion, Nd ion, Tm ion, Sm ion, Ho ion, Er ion, Dy ion and Pr ion.
The fluorescence according to any one of claims 1 to 3, wherein the glass constituting the glass powder is at least one selected from boric acid glass, germanic acid glass, phosphoric acid glass and fluoride glass. probe.
The glass is Bi 2 O 3 —B 2 O 3 glass, Bi 2 O 3 —GeO 2 glass, ZnO—B 2 O 3 glass, CaO—B 2 O 3 glass and CaO—P 2 O 5. The fluorescent probe according to claim 4, wherein the fluorescent probe is at least one selected from a system glass.
The fluorescent probe according to any one of claims 1 to 5, wherein a content ratio of the rare earth element is 0.4 to 2.0 at% with respect to a total amount of atoms constituting the fluorescent probe.
The fluorescent probe according to any one of claims 1 to 6, wherein a lower limit when the maximum length of the powder is measured is 0.1 µm, and an upper limit is 1.0 µm.
A melting step of melting a compound containing a rare earth element and a glass raw material;
A cooling step of cooling the melt obtained by the melting step to produce a glass composition containing ions of rare earth elements;
A crushing step of crushing the glass composition to obtain a powder, and a method for producing a fluorescent probe.
The fluorescent probe according to claim 8, wherein the rare earth element-containing compound is at least one selected from compounds containing a rare earth element selected from Yb, Nd, Tm, Sm, Ho, Er, Dy, and Pr. Manufacturing method.
The method for producing a fluorescent probe according to claim 8 or 9, wherein the compound containing the rare earth element is an oxide.
The fluorescent probe according to any one of claims 8 to 10, wherein the glass raw material contains a compound forming at least one selected from boric acid glass, germanic acid glass, phosphoric acid glass and fluoride glass. Manufacturing method.
The method for producing a fluorescent probe according to any one of claims 8 to 11, wherein a temperature in the melting step is 900 ° C to 1350 ° C.
The method for producing a fluorescent probe according to any one of claims 8 to 12, wherein in the crushing step, crushing with a pestle and mortar and / or crushing with a ball mill is performed.