Functional infrared flourescent particle

The present invention relates to infrared fluorescent particles (also referred to as particles made of infrared phosphor) suitable for use in the fields of biochemistry and biochemistry such as imaging, and in particular, binds to a test substance. It relates to infrared fluorescent particles capable of being excited and emitting light with wavelengths in the near infrared region.

Currently, there is a technique called imaging for observing the accumulation of fluorescent substances in a living body at a specific site from outside the body, and fluorescent substances are expected for such applications. In imaging applications, in particular, not only safety and stability are required of fluorescent materials, but also excitation light and fluorescence are required to have high transparency to biological materials. Therefore, fluorescent substances such as organic phosphors or quantum dots that emit light in the ultraviolet region to the visible region still have problems in terms of fluorescence stability, toxicity and transparency.

<p> The present invention has been made to solve the above-mentioned problems. Therefore, an object of the present invention is to provide infrared fluorescent particles which are preferable in terms of transmission of excitation light and fluorescence to a biological substance and which can be used for applications such as imaging.</p>

<p> In order to solve the above problems, the present invention comprises a functional group or substance capable of binding to a test substance, and when irradiated with excitation light having a wavelength in the infrared region, fluorescence in the wavelength in the infrared region is emitted. Provided are infrared fluorescent particles that are emitted.</p><p> When the infrared fluorescent particles of the present invention are irradiated with excitation light having a wavelength in the infrared region (particularly near-infrared region), fluorescence having a wavelength in the infrared region (particularly near-infrared region) is emitted. Further, since the infrared fluorescent particles of the present invention have "functional groups or substances capable of binding to the test substance", the infrared fluorescent particles can be bonded to the test substance. As a result, infrared fluorescent particles can be used as a fluorescent probe for imaging. As described above, since the infrared fluorescent particles of the present invention have various useful functions, the infrared fluorescent particles of the present invention can also be referred to as "functional infrared fluorescent particles".</p>

<p> Since light in the infrared region has high transparency to biological substances and the like, the infrared fluorescent particles of the present invention can be used to reduce the influence of light emission, absorption or scattering by the test substance and substances existing around it. it can. Therefore, the background can be kept low and the sensitivity can be substantially increased. Further, in the infrared fluorescent particles made of a metal oxide, not only the fluorescence intensity is not substantially lowered by irradiation with light and stable fluorescence intensity can be obtained, but also the toxicity of the infrared fluorescent particles themselves is low. Therefore, the infrared fluorescent particles of the present invention made of metal oxides are particularly useful for imaging applications of test substances in samples containing various biological substances and for imaging specific tissues in the living body. ..</p>

Mode for carrying out the invention

Hereinafter, the infrared fluorescent particles of the present invention will be described in detail.

As used herein, the term "infrared fluorescent particle" means a particle that emits energy of light having a wavelength in the infrared region when irradiated with excitation light having a wavelength in the infrared region. Therefore, when irradiating the excitation light, if the light energy is radiated in a very short time, it emits light as "fluorescence", but if the light energy is radiated for a long time, it emits light as "phosphorescence". Thus, the "infrared fluorescent particles" of the present invention substantially mean particles that emit "fluorescence" or "phosphorescence".

Further, in the present specification, "capable of binding" of "functional group or substance capable of binding to a test substance" means that "functional group or substance" is physically or chemically added to "test substance". It means to combine. Therefore, the term "capable of binding" also includes an embodiment in which a "test substance" is bound to a "functional group or substance" due to, for example, "adsorption" or "Coulomb force".

Further, the "test substance" used in the present specification generally means a substance to be measured, but is not necessarily limited to the substance to be measured. A substance that is not the substance to be measured but is simply bound to the infrared fluorescent particles of the present invention for various purposes is also included in the "test substance".

The excitation light irradiating the infrared fluorescent particles of the present invention and the generated fluorescence are highly transparent to the test substance and the substances existing around it, and are in the infrared region where light emission, absorption or scattering by those substances is small. Has a wavelength. Preferably, the peak wavelengths of the excitation light spectrum and the fluorescence spectrum are in the near infrared region range of 700 to 3000 nm. Light in the wavelength range shorter than such wavelengths not only absorbs and emits more light in the visible region by the test substance and the substances around it, but also scatters more, while more than such wavelengths. This is because light in a long wavelength range absorbs more infrared light from the sample. In particular, when the infrared fluorescent particles of the present invention are used for imaging a test substance in a sample containing a biological substance or imaging a specific part in the living body, water is often present in the surroundings, and thus excitation is performed. It is more preferable that the peak wavelengths of the optical spectrum and the fluorescence spectrum are in the near infrared region of 700 to 1300 nm, where light absorption by water is small. Further, considering that the larger the difference between the wavelength of the excitation light and the wavelength of the fluorescence, the easier it is to cut the influence of the excitation light in the fluorescence intensity measurement using the infrared fluorescent particles of the present invention, the excitation light spectrum. It is more preferable that the peak wavelength of the fluorescence spectrum is in the near-infrared region of 700 to 1100 nm, and the peak wavelength of the fluorescence spectrum is in the near-infrared region of 850 to 1200 nm.

If the difference between the peak wavelength of the excitation light spectrum and the peak wavelength of the fluorescence spectrum is 20 nm or less, it is difficult to separate the excitation light and the fluorescence by a filter or the like, and even if they can be separated, the respective lights overlap. It is preferable that the difference between the peak wavelength of the excitation light spectrum and the peak wavelength of the fluorescence spectrum is 20 nm or more because the existing portion must be cut and the light amount loss becomes large. More preferably, the difference between the peak wavelength of the excitation light spectrum and the peak wavelength of the fluorescence spectrum is 50 nm or more, and even more preferably 100 nm or more.

In the embodiment of the present invention, when the infrared fluorescent particles are used as a powder form, it is preferable that each infrared fluorescent particle has a uniform shape and size. Further, in the detection using such infrared fluorescent particles in a liquid, it is preferable that the infrared fluorescent particles can be uniformly dispersed in the liquid in order to suppress variations in the obtained results. Therefore, the upper limit of the diameter of the infrared fluorescent particles is preferably 5 μm or less, more preferably 500 nm or less, and further preferably 100 nm or less. On the other hand, the lower limit of the diameter of the infrared fluorescent particles is determined by whether or not it can be manufactured and whether or not a detectable fluorescence intensity can be obtained, and is generally preferably 2 nm or more. Based on the above, the infrared fluorescent particles preferably have a particle size of 2 nm to 5 μm. The "particle size" referred to here is, for example, when 100 particles are randomly selected from an image magnified by an electron microscope or an optical microscope, the diameter of each particle is read, and these are averaged. Refers to the particle size of. However, if the diameters are not uniform, the diameter of each particle shall be the average of the maximum and minimum diameters. It will be understood that the preferable particle size of the infrared fluorescent particles may change depending on the shape and type of the test substance or the infrared fluorescent particles.

The infrared fluorescent particles of the present invention may be formed from any material such as an inorganic material, an organic material, a composite material or a complex. In particular, infrared fluorescent particles formed from an inorganic material are preferable as the infrared fluorescent particles of the present invention because the decrease in fluorescence intensity due to irradiation with excitation light is small and the stability is excellent.

Further, in the embodiment of the present invention, infrared fluorescent particles which are preferable in terms of safety or environment are desirable. For example, metal oxide-based infrared fluorescent particles are generally highly stable and have low toxicity, and are therefore preferably used for the infrared fluorescent particles of the present invention. Examples of infrared fluorescent particles composed of metal oxides include compounds composed of transition metal elements, phosphorus elements and oxygen elements. Typical compounds are Y, Nd, Yb, and PO.₄, Lu / Nd / Yb / PO₄And La / Nd / Yb / PO₄(In the formula, Y: ittrium element, Nd: neodymium element, Yb: itterbium element, Lu: lutetium element, La: lanthanum element, P: phosphorus element, O: oxygen element) and the like can be mentioned.

Among the infrared fluorescent particles composed of metal oxides, the general formula A_1-xy Nd_x Yb_y PO₄(In the equation, A is at least one element selected from the group consisting of Y, Lu and La; 0 &lt;x 0.5; 0 &lt;y 0.5 and 0 &lt;x + y &lt;1). The compound represented is preferred. Furthermore, the above general formula A_1-xy Nd_x Yb_y PO₄Among the compounds represented by, those having an afterglow duration of 100 μs or more are particularly preferable. The "afterglow duration" here means the time obtained by measuring the time until the fluorescence intensity decreases to 1/10 after the excitation light irradiation is stopped.

Since the infrared fluorescent particles of the present invention can be adsorbed or bound to a specific part of a living body, not only the specific part can be detected but also the specific part can be imaged. Moreover, since the excitation light and fluorescence in the infrared region have high transparency to biological substances and the like, various kinds of excitation light and fluorescence can be obtained even in a deep part or around the test substance and infrared fluorescent particles. Detection or imaging is possible even in the presence of other substances.

For this reason, the infrared fluorescent particles of the present invention contain functional groups and substances that can be bonded to the test substance. Preferably, "functional groups or substances capable of binding to the test substance" are immobilized on the infrared fluorescent particles. The term "immobilization" as used herein generally means an embodiment in which "functional groups or substances capable of binding to the test substance" are present near the surface of the infrared fluorescent particles. It does not necessarily mean only the embodiment in which the "functional group or substance capable of binding to the test substance" is directly attached to the surface of the infrared fluorescent particles.

In the embodiment of the present invention, the "functional group capable of binding to the test substance" of the infrared fluorescent particle of the present invention is an amino group, a carboxyl group, an epoxy group, a thiol group, a nitro group, a succinimide group, or a maleimide. It is preferably at least one functional group selected from the group consisting of a group, a formyl group, a hydrazine group and a tosyl group. In this case, the infrared fluorescent particles of the present invention are bound or adsorbed to the test substance containing a functional group or the like having reactivity or affinity with these functional groups. It should be noted that the "functional group capable of binding to the test substance" exemplified above may be activated, and activation is possible by adding various catalysts, dehydrating agents, etc., which is a representative example. Examples thereof include addition of carbodiimide to a carboxyl group, acid anhydrideization of a carboxyl group, addition of a tertiary amine or alcohol to an epoxy group, and the like.

Further, in the embodiment of the present invention, the "substance capable of binding to the test substance" of the infrared fluorescent particle of the present invention is silica, hydroxyapatite, ligand, receptor, antigen, antibody, biotin, avidin, protein. It is preferably at least one substance selected from the group consisting of A, protein G, nucleic acids and sugar chains. In this case as well, the infrared fluorescent particles of the present invention are bound or adsorbed to the test substance through such a substance capable of binding to the test substance.

Since the "functional group or substance capable of binding to the test substance" as exemplified above is immobilized on the infrared fluorescent particles of the present invention, the test substance can be subjected to the functional group or substance. Infrared fluorescent particles will be bonded. The test substance may be any kind of substance, but is preferably a substance selected from the group consisting of biological tissues, microorganisms and cells. Since such a test substance often coexists with another substance as a living body or a mixture, other substances may exist around the test substance, and the infrared fluorescent particles of the present invention may be imaged or the like. When used in vivo, various bio-related substances may exist around the target test substance. Examples of such "substances existing around the test substance" include biological tissues other than the test substance, microorganisms, cells, body fluids such as blood, water, and the like.

Any method may be used as a method for introducing the "functional group capable of binding to the test substance" into the infrared fluorescent particles. For example, a method of reacting a silane coupling agent with the surface of infrared fluorescent particles is used. In this case, the functional group may be directly reacted with the surface of the infrared fluorescent particle, or the functional group may be reacted with the surface of the infrared fluorescent particle to which silica or the like is fixed in advance. Since the types of functional groups that can be attached with the silane coupling agent are limited, yet another substance is reacted with the functional groups introduced by the silane coupling agent to bring these functional groups into a more active state. Alternatively, another functional group may be introduced into the infrared fluorescent particles. A titanium coupling agent or silazane may be used instead of the silane coupling agent.

A substance having both a functional group adsorbed or bonded to the surface of an infrared fluorescent particle and a functional group to be introduced, for example, a dispersant such as polyethylene glycol having an amino group at both ends or polyethylene glycol having an amino group at one end and a carboxyl group at the other end is fixed. It may be transformed into. Similarly, infrared fluorescent particles may be formed so as to have a micelle or liposome shape in which a functional group appears on the outermost surface. Further, the infrared fluorescent particles may be coated with a polymer having a functional group (for example, polyallylamine or chitosan), or the functional group may be introduced into the coated polymer. Surfactants (eg, Tween, Triton, etc.) may also be added to the liquid used to improve the dispersibility of the infrared fluorescent particles in the liquid.

Similarly, any method may be used as a method for immobilizing the "substance capable of binding to the test substance" on the infrared fluorescent particles. For example, the sol-gel method can be used to immobilize silica. Further, an adhesion method as described in Japanese Patent Application Laid-Open No. 2004-031792 can also be preferably used. Specifically, for example, the surface of the infrared fluorescent particles is neutralized by adding an acid after adding a preferable amount of sodium silicate to a water suspension in which the infrared fluorescent particles are dispersed. A specific amount of silica can be adhered and formed in the vicinity. Further, another deposition method can be used to immobilize a calcium phosphate-based compound such as hydroxyapatite. Specifically, for example, infrared fluorescent particles are dispersed in water, a calcium salt aqueous solution and a phosphate aqueous solution are added thereto, the pH is adjusted, and a calcium phosphate-based compound is precipitated near the surface of the infrared fluorescent particles. After that, hydrothermal treatment is performed. As a result, the calcium phosphate-based compound can be adhered and formed near the surface of the infrared fluorescent particles.

Since it can be considered that various functional groups are present on the surface of metal oxides and the like, substances such as antigens, antibodies, biotins, avidin, nucleic acids and / or sugar chains are composed of infrared phosphors. The substance may be bonded to the surface of the metal oxide particles simply by being mixed with the metal oxide particles, and the infrared fluorescent particles of the present invention can be obtained by such a simple method.

Furthermore, it is possible to bind to the test substance by shifting from the solution condition having high solubility to the "substance capable of binding to the test substance" to the solution condition having low solubility thereof. It is also possible to deposit "a substance" on the surface of infrared fluorescent particles. In addition, a certain functional group is fixed to the infrared fluorescent particle in advance, and then a "functional group or substance capable of binding to the test substance" is bonded to the functional group, or A "functional group or substance capable of binding to a test substance" is previously bonded to a substance or the like having a certain functional group and having a property of being immobilized on the surface of infrared fluorescent particles, and then By immobilizing such a substance on the surface of infrared fluorescent particles, more reliable immobilization can be performed.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to such Examples.

&lt;&lt; Synthesis of infrared fluorescent particles &gt;&gt; (Example 1) Infrared fluorescent particles before "functional groups or substances capable of binding to a test substance" are immobilized according to Example 1 of Japanese Patent Application Laid-Open No. 3336572 (hereinafter, also referred to as "infrared fluorescent particles A"). Was synthesized. Specifically, Nd₂O₃: 3.5g, Yb₂O₃: 4.0g, Y₂O₃18.0g and H₃PO₄: A raw material consisting of 60.0 g is thoroughly mixed, filled in an alumina-made crucible with a lid, placed in an electric furnace, and heated from room temperature to about 700 ° C over 2 hours at a constant heating rate, and then. , 700 ° C for 6 hours. Immediately after the completion of firing, it was taken out from the electric furnace and allowed to cool in air. Next, boiling water at 100 ° C was added to the crucible and boiled. The fluorescent particles obtained as a result were taken out from the crucible, washed with 1N nitric acid, washed with water, and dried. By the above operation, the general formula Nd_0.1Yb_0.1Y_0.8PO₄Infrared fluorescent particles A represented by. In the infrared fluorescent particle A, "a functional group or a substance capable of binding to a test substance" is not immobilized. In the infrared fluorescent particle A, when the excitation light having a peak wavelength of about 810 nm was irradiated, the peak wavelength of the fluorescence spectrum of about 980 nm was obtained.

Next, 5 parts by weight of the obtained infrared fluorescent particles A were dispersed in water, and 1 part by weight of tetraethoxysilane and 5 parts by weight of aqueous ammonia were added and stirred to precipitate silane on the particle surface. The supernatant was removed by centrifugation. Further, after adding water and stirring, the washing step of centrifuging to remove the supernatant was repeated 5 times, and finally dried at 100 ° C. By the above operation, infrared fluorescent particles on which silica is fixed were obtained.

(Example 2) After dispersing 5 parts by weight of the infrared fluorescent particles A of Example 1 in water / ethyl alcohol (volume ratio 1/1), 1 part by weight of a silane coupling agent having an amino group was mixed, and the mixture was stirred for 1 hour. The supernatant was removed by centrifugation and then dried at 120 ° C. As a result, infrared fluorescent particles having an fixed amino group were obtained.

(Example 3) The same operation as in Example 2 was carried out except that the silane coupling agent having an epoxy group was used instead of the silane coupling agent having an amino group. As a result, infrared fluorescent particles having an fixed epoxy group were obtained.

(Example 4) 1 part by weight of the infrared fluorescent particles on which the epoxy group was fixed obtained in Example 3 was dispersed in 20 parts by weight of a 5 wt% ethanolamine aqueous solution, stirred overnight, and then repeatedly washed with water and acetone to obtain a hydroxyl group. Infrared fluorescent particles were obtained. Next, 1 part by weight of the infrared fluorescent particles on which the hydroxyl group was fixed was dispersed in 20 parts by weight of pyridine, 0.2 part by weight of tosyl loride was added, and the mixture was stirred overnight, and then washed with toluene was repeated 4 times. By the above operation, infrared fluorescent particles having a tosyl group fixed were obtained.

(Example 5) After dispersing 1 part by weight of the infrared fluorescent particles obtained in Example 2 in water, 100 weight by weight of 10 mg / ml water-soluble carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride). After adding the parts and stirring, the supernatant was removed by centrifugation. Then, the washing operation of adding water, stirring, centrifuging, and removing the supernatant was repeated three times. After adding 0.06 part by weight of PBS buffered saline (PBS) and streptavidin and reacting at 37 ° C for 2 hours, the supernatant is removed by centrifugation, and then the washing step with PBS is performed 5 times. Repeated. As a result, infrared fluorescent particles on which streptavidin was immobilized were obtained.

(Example 6) After 1 part by weight of the infrared fluorescent particles on which the epoxy group was fixed obtained in Example 3 was dispersed in 100 parts by weight of PBS, 0.06 part by weight of streptavidin was added and the mixture was stirred overnight. Then, washing with PBS was repeated 3 times. As a result, infrared fluorescent particles on which streptavidin was immobilized were obtained.

(Example 7) After dispersing 1 part by weight of the infrared fluorescent particles having the tosyl group fixed in Example 4 in 100 parts by weight of PBS, 0.01 part by weight of streptavidin was added and the mixture was stirred overnight. Then, washing with PBS was repeated 3 times. As a result, infrared fluorescent particles on which streptavidin was immobilized were obtained.

(Example 8) The same operation as in Example 2 was performed except that the silica-fixed infrared fluorescent particles obtained in Example 1 were used instead of the infrared fluorescent particles A in Example 1. That is, 5 parts by weight of the silica-fixed infrared fluorescent particles obtained in Example 1 are dispersed in water / ethyl alcohol (volume ratio 1/1), and 1 part by weight of a silane coupling agent having an amino group is mixed. After stirring for 1 hour, the supernatant was removed by centrifugation and then dried at 120 ° C. As a result, infrared fluorescent particles having an fixed amino group were obtained.

(Example 9) The same operation as in Example 5 was performed except that the infrared fluorescent particles obtained in Example 8 were used instead of the infrared fluorescent particles obtained in Example 2. That is, after the infrared fluorescent particles on which the amino group was fixed obtained in Example 8 were dispersed in water, 10 mg / ml of water-soluble carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) was dispersed. Salt) 100 parts by weight was added, and the mixture was stirred and then centrifuged to remove the supernatant. Then, the washing operation of adding water, stirring, centrifuging, and removing the supernatant was repeated three times. Then, 0.06 part by weight of PBS buffer and streptavidin were added and reacted at 37 ° C. for 2 hours, and then the supernatant was removed by centrifugation, and then the washing step with PBS was repeated 5 times. As a result, infrared fluorescent particles on which streptavidin was immobilized were obtained.

(Comparative example 1) Infrared fluorescent particles with nothing fixed on the surface were obtained. That is, the infrared fluorescent particles A obtained in the process of Example 1 were used.

(Comparative example 2) An organic infrared phosphor having nothing fixed on the surface was prepared. Specifically, IRDye800 Conjugated Streptavidin, which is an infrared fluorescent organic dye, was used as it was.

(Comparative example 3) A visible fluorophore with nothing fixed on the surface was prepared. Specifically, Shin Loihi Color Base SW-13 containing a visible fluorescent pigment was used as it was.

Using the infrared fluorescent particles of Examples 1 to 9 and the materials of Comparative Examples 1 to 3, the adsorption or binding property of the biological substance was examined.

&lt;&lt; Confirmation test of binding amount and adsorption amount &gt;&gt; (Confirmation test of nucleic acid adsorption amount) The following tests were carried out to confirm the amount of nucleic acid (λDNA) adsorbed on the infrared fluorescent particles of Example 1 and Comparative Example 1 described above. The case where the infrared fluorescent particles of Example 1 are used will be described, but the same operation will be performed even when the infrared fluorescent particles of Comparative Example 1 are used. (A) reagent (B) The infrared fluorescent particles of Example 1 were dispersed in sterile water to prepare a 0.2 mg / ml dispersion. (B) As a biological sample for isolating nucleic acid, λDNA (Nacalai Tesque) was diluted with sterile water to prepare a 10 μg / 100 μl λDNA solution. (C) As a solution for nucleic acid extraction, buffer A [7M guanidine hydrochloride (Nacalai Tesque), 50 mM Tris-HCl (Sigma), pH 7.5], which is a buffer solution containing a chaotropic substance, was used. (D) As a cleaning solution, buffer A [7M guanidine hydrochloride (Nacalai Tesque), 50 mM Tris-HCl (Sigma), pH 7.5], which is a buffer solution containing a chaotropic substance, was used. (E) A 70 wt% ethanol solution and an acetone solution were used as reagents for removing high-concentration salts. (F) Sterilized water was used as an eluent for recovering the nucleic acid bound to the infrared fluorescent particles of Example 1.

(B) Test operation (1) To 100 μl of the λDNA solution, 1,000 μl of the nucleic acid extraction solution was injected and mixed. (2) After that, 20 μl of the dispersion liquid of the infrared fluorescent particles of Example 1 was added. (3) The mixture was left at room temperature for 10 minutes while mixing about every 2 minutes. (4) Infrared fluorescent particles were collected at the bottom of the tube by centrifugation. (5) The solution was aspirated with a pipette and discharged. (6) 1cc of a cleaning solution containing guanidine hydrochloride was injected into the tube. (7) After sufficiently mixing with the infrared fluorescent particles of Example 1, centrifugation was performed again, and the solution was discarded in the same manner as described above. (8) The cleaning operation was repeated again. (9) Infrared fluorescent particles to which nucleic acids were bound were washed with 1 cc of 70 wt% ethanol by the same method as described above, and a high concentration of guanidine hydrochloride was removed. (10) Again, the cells were washed with 1 cc of 70 wt% ethanol and 1 cc of acetone. (11) The above tube was placed in a heat block at about 56 ° C. and left for about 10 minutes to completely evaporate and remove acetone in the tube and infrared fluorescent particles. (12) 100 μl of sterilized water was added to the infrared fluorescent particles to which the nucleic acid was bound by the above method, the tube was placed in a heat block at about 56 ° C, and the mixture was left for 10 minutes while mixing every 2 minutes. (13) Then, the solution to be collected by centrifugation was aspirated with a pipette and transferred to another new tube. Normally, the recovered amount was about 70 μl. (14) With respect to the nucleic acid thus recovered, the absorbance (OD 260 nm) was measured by using an absorbance meter (manufactured by JASCO Corporation, V-570) to determine the concentration of the nucleic acid. Then, the amount of nucleic acid adsorbed was obtained by multiplying this concentration by the recovery volume to determine the amount of nucleic acid recovered.

(Confirmation test of binding amount of streptavidin and biotinylated HRP) 20 ng / ml biotinylated HRP (Horse Radish Peroxidase) 100 μl in 5 μg of streptavidin-immobilized infrared fluorescent particles obtained in Examples 5, 6, 7, and 9. Was added and stirred for 30 minutes, and 100 μl of tetramethylbenzidine (TMB) was further added and allowed to stand for 30 minutes. After stopping the reaction with 200 μl of 1N sulfuric acid, the intensity of color development was measured as the absorbance at a wavelength of 450 nm using a spectrophotometer (manufactured by JASCO Corporation, V-570), and based on comparison with a sample of known concentration, The amount of streptavidin and biotinylated HRP bound was determined.

(Results of confirmation test of binding amount and adsorption amount) Table 1 shows the results of the adsorption amount or the binding amount of the infrared fluorescent particles obtained in Examples 1 to 9 and the materials of Comparative Examples 1 to 3.

<tables num="1"><img file="JP4902183B2_D0001.tif" /></tables>

Based on the results in Table 1, the infrared fluorescent particles based on Examples 1 to 9 of the present invention in which silica, amino groups, epoxy groups, etc. are immobilized are comparative examples in which these substances or functional groups are not immobilized. It was found that various specific test substances can be bound or adsorbed as compared with the materials 1 and 3. From this, it can be understood that the infrared fluorescent particles of the present invention can be bound to a biological substance and can be bound to a specific site of a living body or a part thereof.

Fluorescence spectrum measurement&gt;&gt; Next, various lasers and Si photodiodes were combined, and the fluorescence intensity of infrared fluorescent particles to which λDNA, streptavidin or biotinylated HRP was bound or adsorbed was measured.

As a light source, a laser of 810 nm was used for Examples 1 to 9, and light other than the excitation light was cut by a filter to obtain excitation light. Further, Comparative Example 1 was 810 nm, Comparative Example 2 was 780 nm, and Comparative Example. For No. 3, a 532 nm laser was used, and light other than the excitation light was cut with a filter to obtain the excitation light. Further, a Si photodiode was used for fluorescence detection to cut the excitation light of each Example and Comparative Example, and the excitation light of Examples 1 to 9 was around 980 nm, Comparative Example 1 was around 980 nm, and Comparative Example 2 was 810 nm. In the vicinity, in Comparative Example 3, a filter that transmits a wavelength region near 590 nm was installed in front.

Fluorescence measurement samples were prepared by dispersing or dissolving the infrared particles obtained in Examples 1 to 9 and the materials of Comparative Examples 1 to 3 in water, dropping them on a membrane filter and drying them. Then, the sample was irradiated with excitation light.

As a result, strong fluorescence was observed in both Examples 1 to 9 and Comparative Examples 1 to 3.

Further, when a thin cowhide was placed on the sample and the same fluorescence measurement was performed, fluorescence was observed in Examples 1 to 9 and Comparative Example 1 although the fluorescence intensity decreased by about two orders of magnitude. In Comparative Example 2, the fluorescence intensity decreased by about 2 to 3 orders of magnitude, but fluorescence was also observed in this case. Fluorescence could not be observed in Comparative Example 3.

Further, when each sample was irradiated with a laser of 810 nm, no decrease in fluorescence intensity was observed in each of the particles of Examples 1 to 9 and Comparative Examples 1 and 3, but the organic fluorescent dye of Comparative Example 2 showed about 5 The fluorescence intensity decreased by nearly 1/3 after a minute of irradiation.

From the above results, the fluorescence of Examples 1 to 9 and Comparative Example 1 of the metal oxide-based infrared fluorescent particles has higher transparency than the fluorescence of Comparative Example 3 of the fluorescent substance in the visible region, and is organic. It was also found that the deterioration of the fluorescence intensity due to light irradiation was smaller than that of the fluorescence of Comparative Example 2 using the fluorescent dye. In addition, since the fluorescence intensity can be observed with a combination of a laser and a photodiode, it was found that an image can be obtained by scanning this.

Light in the infrared region (particularly in the near-infrared region) related to the infrared fluorescent particles of the present invention has high transparency to biological substances and the like. In addition, the infrared fluorescent particles of the present invention can be adsorbed or bound to a specific substance. Therefore, the infrared fluorescent particles of the present invention can be used for applications such as imaging of these specific substances or objects having the specific substances. In addition, the infrared fluorescent particles of the present invention can also be used as a reagent for detection and quantitative analysis of a specific substance by utilizing the property of adsorbing or binding to a specific substance.