JP5790352B2 - Fluorescent substance-containing nanoparticles and biological substance detection method using the same - Google Patents

Description

  The present invention relates to a fluorescent substance-containing nanoparticle including a plurality of fluorescent substances having different excitation / emission characteristics, and a biological substance detection method using the same.

  Nanometer-order particles (hereinafter referred to as “nanoparticles”) have attracted attention in recent years because they exhibit different physical properties from the corresponding bulk bodies. In various fields including bioassay and medical diagnosis, Research has been done.

  For example, Patent Document 1 discloses a phosphor obtained by dispersing semiconductor ultrafine particles in a glass matrix, and describes that this phosphor has good water dispersibility and high light emission performance.

  In Patent Document 2, silica particles having communication holes in the presence of a functional compound such as a fluorescent dye compound are prepared, and then a specific tetraalkoxysilane is additionally contained to form a silica shell. Thus, it is described that the core-shell structured silica nanoparticles in which the functional compound is encapsulated in the communication hole are obtained by the production method including the step of confining the functional compound with the shell.

  However, the knowledge about the silica nanoparticle which includes both the semiconductor nanoparticle and the fluorescent dye has not been confirmed.

JP2005-105244 JP2009-196829

  In order to confirm the location of an antigen on a pathological section, when observing a tissue that has been subjected to fluorescent labeling using an antibody-modified fluorescent label, when using a fluorescent dye generally used as a fluorescent label The background noise caused by the autofluorescence of the tissue caused a problem that the signal-to-noise ratio of the signal of the fluorescent label was lowered and the diagnostic accuracy was deteriorated.

  One method for solving such a problem caused by autofluorescence is a method of observing only the label signal by performing time-resolved measurement using a long afterglow phosphor as a fluorescent label. However, simply using a long afterglow phosphor as a fluorescent label has a problem that it is difficult to achieve a sufficient S / N ratio because the afterglow intensity is low. Usually, in the case of visible excitation luminescence, although the autofluorescence is reduced compared to the ultraviolet excitation, the luminance is insufficient and a sufficient S / N ratio cannot be obtained.

  Accordingly, the present invention provides a fluorescent substance-encapsulating nanoparticle that can detect a biological substance in a tissue with high accuracy when used as a fluorescent labeling agent for pathological diagnosis. For the purpose.

The present inventor has found that the above-mentioned problems can be solved by using fluorescent substance-containing nanoparticles containing a plurality of fluorescent substances having different excitation / emission characteristics, and has reached the present invention.
That is, the present invention is shown in the following [1] to [ 45 ].

[1] viewed contains a first fluorescent material and a second fluorescent material having a fluorescent material and distinguishable excitation / emission characteristics of the first,
The first fluorescent substance and the second fluorescent substance are encapsulated by an organic polymer resin that is a polystyrene resin or a melamine resin, and
A fluorescent substance-encapsulated nanoparticle having a biomolecule recognizing substance and an aqueous dispersion modifying compound bonded to the surface .
[2] The phosphor-containing nanoparticle according to [1], wherein the first fluorescent material does not have an excitation spectrum in the ultraviolet region, and the second fluorescent material has an excitation spectrum in the ultraviolet region.

[3] The fluorescent substance-containing nanoparticle according to [2], wherein the inorganic fluorescent nanoparticle and the organic fluorescent dye are included.
[4] The phosphor-containing nanoparticle according to [3], wherein the inorganic phosphor nanoparticle includes a II-VI group compound or a III-V group compound.

[5] The phosphor-containing nanoparticle according to [3], wherein the inorganic phosphor nanoparticle contains simple silicon .

[6 ] The fluorescent substance-containing nanoparticle according to [1], wherein the first fluorescent substance is a fluorescent substance that is not a long afterglow phosphor, and the second fluorescent substance is a long afterglow phosphor.

[ 7 ] The fluorescent substance-containing nanoparticles according to [ 6 ], wherein the long afterglow phosphor and the organic fluorescent dye are included.
[ 8 ] The phosphor-containing nanoparticle according to [ 6 ] or [ 7 ], wherein the long afterglow phosphor is an inorganic particle.

[ 9 ] The fluorescent substance-containing nanoparticle according to [ 8 ], wherein the inorganic particles have a volume average particle diameter of 5 nm or more and 500 nm or less.
[ 10 ] The phosphor-containing nanoparticle according to [ 8 ], wherein the inorganic particles have a volume average particle size of 5 nm or more and 300 nm or less.

[ 11 ] The phosphor-containing nanoparticle according to any one of [ 6 ] to [ 10 ], wherein the long afterglow phosphor is an activated phosphor composed of a matrix and an activator.
[ 12 ] The fluorescent substance-containing nanoparticles according to [ 11 ], wherein the activator is a rare earth activator.

[ 13 ] The fluorescent substance-containing nanoparticles according to [ 11 ], wherein the activator is Mn ²⁺ .
[ 14 ] The fluorescent substance-containing nanoparticle according to [ 11 ], wherein the activator is Eu3 ⁺ .
[15] a fluorescent material containing nanoparticles according to any one of the said base is a _{Y 2 O 3 [11] ~} [14].

[ 16 ] The fluorescent substance-containing nanoparticle according to any one of [ 11 ] to [ 14 ], wherein the matrix is Zn ₂ SiO ₄ .

[17 ] A fluorescent labeling agent for pathological diagnosis comprising the fluorescent substance-containing nanoparticles according to any one of [1] to [ 16 ].

[ 18 ] (a) A tissue is labeled using a fluorescent substance-containing nanoparticle including a first fluorescent substance and a second fluorescent substance having excitation / emission characteristics distinguishable from the first fluorescent substance. And a process of
(B) For the tissue obtained in the step (a), a step of photographing an ultraviolet excitation image and a visible light excitation image in the same visual field;
(C) The ultraviolet excitation image obtained in the step (b) is compared with the visible light excitation image, and only pixels detected as bright spots in both the ultraviolet excitation image and the visible light excitation image are detected. A method for measuring a biological material in a tissue, comprising a step of recognizing an effective bright spot.
[19] The measurement method according to [18], wherein the first fluorescent material does not have an excitation spectrum in the ultraviolet region, and the second fluorescent material has an excitation spectrum in the ultraviolet region.
[20] The measurement method according to [19], wherein the fluorescent substance-containing nanoparticles include inorganic fluorescent nanoparticles and an organic fluorescent dye.
[21] The measurement method according to [20], wherein the inorganic phosphor nanoparticles include a II-VI group compound or a III-V group compound.
[22] The measurement method according to [20], wherein the inorganic phosphor nanoparticles include simple silicon.
[23] The above [18] to [22], wherein the fluorescent substance-containing nanoparticles are fluorescent substance-containing nanoparticles in which the first fluorescent substance and the second fluorescent substance are included by an organic polymer resin. The measuring method in any one.
[24] The measurement method according to [23], wherein the organic polymer resin is a polystyrene resin.
[25] The measurement method according to [23], wherein the organic polymer resin is a melamine resin.
[26] The phosphor-containing nanoparticle according to any one of [18] to [22], wherein the phosphor-containing nanoparticle is a phosphor-containing nanoparticle in which the first fluorescent material and the second fluorescent material are encapsulated with silica. The measuring method described.
[27] The measurement method according to any one of [18] to [26], wherein a biomolecule recognition substance is bonded to the surface of the fluorescent substance-containing nanoparticle.
[28] The measurement method according to any one of [18] to [27], wherein a modification compound for aqueous dispersion is bonded to a surface of the fluorescent substance-containing nanoparticle.

[ 29 ] (a) A tissue is labeled using a fluorescent substance-containing nanoparticle including a first fluorescent substance and a second fluorescent substance having excitation / emission characteristics distinguishable from the first fluorescent substance. And a process of
(B) For the tissue obtained in the step (a), a step of photographing an excitation image obtained at the time of excitation light irradiation and an afterglow image obtained by time-resolved fluorescence measurement in the same visual field;
(C) The excitation image obtained in the step (b) is compared with the afterglow image, and only pixels detected as bright spots in both the excitation image and the afterglow image are effective. viewing including the step of recognizing a bright spot,
A method for measuring a biological material in a tissue, wherein the first fluorescent material is a fluorescent material that is not a long afterglow phosphor, and the second fluorescent material is a long afterglow phosphor .
[30] The measurement method according to [29], wherein the fluorescent substance-containing nanoparticles include the long afterglow phosphor and the organic fluorescent dye.
[31] The measurement method according to [29] or [30], wherein the long afterglow phosphor is inorganic particles.
[32] The measurement method according to [31], wherein the inorganic particles have a volume average particle size of 5 nm or more and 500 nm or less.
[33] The measurement method according to [31], wherein the inorganic particles have a volume average particle size of 5 nm or more and 300 nm or less.
[34] The measurement method according to any one of [29] to [33], wherein the long afterglow phosphor is an activated phosphor composed of a matrix and an activator.
[35] The measuring method according to [34], wherein the activator is a rare earth activator.
[36] The measuring method according to [34], wherein the activator is Mn ²⁺ .
[37] The measurement method according to [34], wherein the activator is Eu ³⁺ .
[38] The measurement method according to any one of [34] to [37], wherein the matrix is Y ₂ O ₃ .
[39] The measurement method according to any one of [34] to [37], wherein the base material is Zn ₂ SiO ₄ .
[40] Any of [29] to [39], wherein the fluorescent substance-containing nanoparticles are fluorescent substance-containing nanoparticles in which the first fluorescent substance and the second fluorescent substance are included by an organic polymer resin. The measuring method of crab.
[41] The measurement method according to [40], wherein the organic polymer resin is a polystyrene resin.
[42] The measuring method according to [40], wherein the organic polymer resin is a melamine resin.
[43] The fluorescent substance-containing nanoparticles are any one of [29] to [39], wherein the fluorescent substance-containing nanoparticles are fluorescent substance-containing nanoparticles in which the first fluorescent substance and the second fluorescent substance are included in silica. Measuring method.
[44] The measurement method according to any one of [29] to [43], wherein a biomolecule recognition substance is bonded to the surface of the fluorescent substance-containing nanoparticle.
[45] The measurement method according to any one of [29] to [44], wherein a modified compound for aqueous dispersion is bonded to the surface of the fluorescent substance-containing nanoparticles.

  Since the fluorescent substance-encapsulating nanoparticles of the present invention contain a plurality of fluorescent substances having different excitation / emission characteristics, the luminance can be improved by adding together the fluorescent emission obtained under different measurement conditions in the same field of view. Is possible. In addition, the S / N ratio is dramatically improved by enabling calculation processing to extract only a portion where fluorescence is confirmed at the same pixel as a signal through a plurality of light emission images obtained under a plurality of different measurement conditions in the same visual field. When used as a fluorescent labeling agent for pathological diagnosis, it is possible to detect a biological substance in a tissue with high accuracy.

[Fluorescent substance-containing nanoparticles]
The fluorescent substance-containing nanoparticle according to the present invention is a particle including a first fluorescent substance and a second fluorescent substance having excitation / luminescence characteristics distinguishable from the first fluorescent substance.

Fluorescent substance Examples of the fluorescent substance used in the present invention include fluorescent organic dyes and semiconductor nanoparticles. When excited by ultraviolet to visible light having a wavelength in the range of 200 to 700 nm, it preferably emits visible to near infrared light having a wavelength in the range of 400 to 900 nm.

More preferably, the emission wavelength of the phosphor contained in the same particle preferably has an emission peak of plus or minus 5 nm for observation.
In the fluorescent substance-containing nanoparticle according to the present invention, it is preferable that both the first fluorescent substance and the second fluorescent substance have the excitation wavelength and the emission wavelength in the above ranges, respectively. It is necessary to have excitation / emission characteristics distinguishable from the first fluorescent material. In a typical embodiment of the present invention, a fluorescent material generally used as an existing fluorescent labeling agent having general excitation / emission characteristics is used as the first fluorescent material, and the second fluorescent material is used. As the fluorescent material, a fluorescent material capable of emitting fluorescence under the condition that the first fluorescent material does not substantially emit fluorescence is used. Here, a fluorescent substance having “general excitation / emission characteristics” has a short fluorescence lifetime and often has excitation / emission characteristics in the visible to near infrared light region. , Fluorescent substances having a long fluorescence lifetime (that is, long afterglow phosphors), and fluorescent substances having an excitation spectrum in the ultraviolet region.

  Here, “having an excitation spectrum in the ultraviolet region” in this specification means that fluorescence having a wavelength in the range of 400 nm to 900 nm is emitted by excitation light having a wavelength of less than 400 nm. However, from the viewpoint of minimizing the denaturation of the biological substance labeled with the fluorescent substance-encapsulating nanoparticles according to the present invention at the time of fluorescence measurement, it has a wavelength in the range of 200 nm to less than 400 nm in practice. It is used to mean that the excitation light emits fluorescence having a wavelength in the range of 400 nm to 900 nm.

Hereinafter, specific examples of the fluorescent substance that can be the first fluorescent substance and / or the second fluorescent substance are shown.
Organic fluorescent dye In the present invention, an organic dye can be used as a fluorescent substance. Here, the organic fluorescent dye refers to an organic compound that emits fluorescence due to the presence of a conjugated π-electron system such as an aromatic ring or a heterocyclic ring, and examples thereof include fluorescent labeling agents widely used in the field of biochemistry.

  Organic fluorescent dyes include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, cascade dye molecules, coumarin dye molecules, and eosin dyes. Examples include molecules, NBD dye molecules, pyrene dye molecules, Texas Red dye molecules, and cyanine dye molecules.

  Specifically, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein, 6 -Carboxy-2 ', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2 ', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy Rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 633, Alexa Fluor 633 , Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, 3030 ODIPY 650/665 (all manufactured by Invitrogen), methoxy coumarin, eosin, NBD, pyrene, Cy5, Cy5.5, can be mentioned Cy7 like.

These organic fluorescent dyes may be used alone or in combination of two or more.
In many cases, the organic fluorescent dye used in the present invention has a visible region, specifically, a visible to near-infrared region, specifically when excited by excitation light having a wavelength in the range of 400 nm to 700 nm. Is a fluorescent substance that emits fluorescence having a wavelength in the range of 400 nm to 900 nm, and does not have an excitation spectrum in the ultraviolet region. Therefore, the organic fluorescent dye is usually used as a “first fluorescent substance”.

  However, some organic fluorescent dyes such as Alexa Fluor 350 (maximum absorption wavelength 346 nm) and coumarin dye molecules may excite and emit fluorescence by light in the ultraviolet region, specifically light having a wavelength of less than 400 nm. . Therefore, such an organic fluorescent dye can be used as a “second fluorescent material” by combining with a fluorescent material having a larger excitation wavelength, for example, a fluorescent material having an excitation wavelength in the range of 500 to 700 nm. .

Semiconductor nanoparticles In the present invention, in addition to the above “organic fluorescent dyes”, semiconductor nanoparticles can also be used as fluorescent substances. Here, as semiconductor nanoparticles, II-VI group compounds, III-V group compounds, and semiconductor nanoparticles each containing a group IV element as a component ("II-VI group semiconductor nanoparticles", " Any of “III-V semiconductor nanoparticles” and “Group IV semiconductor nanoparticles”) can be used.

  Specific examples include, but are not limited to, CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.

It is also possible to use semiconductor nanoparticles having a core / shell structure in which the semiconductor nanoparticles are used as a core and a shell is provided thereon. Hereinafter, as a notation method of the semiconductor nanoparticles having a shell in this specification, when the core is CdSe and the shell is ZnS, it is expressed as CdSe / ZnS. For example, CdSe / ZnS, CdS / ZnS, InP / ZnS, InGaP / ZnS, Si / SiO ₂ , Si / ZnS, Ge / GeO ₂ , Ge / ZnS, and the like can be used, but are not limited thereto.

These semiconductor nanoparticles may be used individually by 1 type, or may be used in combination of multiple types.
The semiconductor nanoparticles used in the present invention can be produced by a known method, or commercially available ones can also be used. Here, as the semiconductor nanoparticles, those subjected to surface treatment with an organic polymer or the like may be used as necessary. Examples thereof include CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like.

  Since the semiconductor nanoparticles described above can change the wavelength characteristics depending on the constituent components and the particle diameter, they can be used as the first fluorescent material and the second fluorescent material. For example, semiconductor nanoparticles having an excitation spectrum in the ultraviolet region may be used as the second fluorescent material, and another semiconductor nanoparticle having no excitation spectrum in the ultraviolet region or the organic fluorescent dye may be combined as the first fluorescent material. it can.

-Long afterglow phosphor In the present invention, in addition to the above "organic fluorescent dye" and "semiconductor nanoparticle", a long afterglow phosphor can also be used as a fluorescent substance. Here, “long afterglow” in this specification means that the fluorescence maintains a light emission intensity of 1/10 or more of that at the time of excitation even after 1 millisecond has elapsed since the end of the excitation light irradiation. . In addition, since the above-mentioned “organic fluorescent dye” and “semiconductor nanoparticle” are generally extinguished before one millisecond elapses after the irradiation of the excitation light, the phosphor is not a long afterglow phosphor. Applicable.

  Here, the composition of the long afterglow phosphor is not particularly limited, and various known compositions can be applied. From the viewpoint of stability, a phosphor composed of an inorganic compound, for example, an inorganic oxide phosphor An inorganic halide phosphor is preferable.

Examples of the long afterglow phosphor that can be suitably used in the present invention include an activated phosphor composed of a matrix and an activator. Here, the matrix is represented by metal oxides typified by Y ₂ O ₃ , Zn ₂ SiO ₄ , phosphates typified by Ca ₅ (PO ₄ ) ₃ Cl, and ZnS, SrS, CaS 2, etc. Preferred examples thereof include, for example, ZnS, Y ₂ O ₂ S, (Y, Gd) ₃ Al ₅ O ₁₂ , YAlO ₃ , BaAl ₂ Si ₂ O ₈ , Y ₃ Al ₅ O _12. Y ₂ SiO ₃ , Zn ₂ SiO ₄ , Y ₂ O ₃ , BaMgAl ₁₀ O ₁₇ , BaAl ₁₂ O ₁₉ , (Ba, Sr, Mg) O · aAl ₂ O ₃ , (Y, Gd) BO ₃ , YO ₃ , (Zn, Cd) S, SrGa ₂ S ₄ , SrS, SnO ₂ , Ca ₁₀ (PO ₄ ) ₆ (F, Cl) ₂ , (Ba, Sr) (Mg, Mn) Al ₁₀ O ₁₇ , (Sr, _{Ca, Ba, Mg) 10 (} PO 4) 6 Cl 2, (La, Ce) PO 4, CeMgAl 11 O 19, _{dMgB 5 O 10, Sr 2 P} 2 O 7, Sr 4 Al 14 O 25, BaMgAl 14 O 23, Ba 2 Mg 2 Al 12 O 22, Ba 2 Mg 4 Al 8 O 18, Ba 3 Mg 5 Al 18 O 35 , (Ba, Sr, Ca) (Mg, Zn, Mn) Al ₁₀ O ₁₇ . Of these, Y ₂ O ₃ and Zn ₂ SiO ₄ are preferably used from the viewpoint of emission intensity.

Further, as activators, ions of rare earth metals such as Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, and Yb, and ions of metals such as Ag, Al, Mn, and Sb In view of the emission wavelength and the emission lifetime, Mn ²⁺ and Eu ³⁺ are particularly preferably used.

The above matrix and activator are not particularly limited in elemental composition and can be partially replaced with elements of the same family, and the obtained inorganic phosphor preferably absorbs ultraviolet rays and emits visible light.
The specific compounds of the phosphor used as the long afterglow phosphor in the present invention are shown below, but are not limited thereto:
Zn ₂ GeO ₄ : Mn, Y ₂ O ₂ S: Eu ³⁺ ,
(Ba, Mg) ₂ SiO ₄ : Eu ³⁺ ,
Ca ₂ Y ₈ (SiO ₄ ) ₆ O ₂ : Eu ³⁺ ,
LiY ₉ (SiO ₄ ) ₆ O ₂ : Eu ³⁺ ,
(Ba, Mg) Al ₁₆ O ₂₇ : Eu ³⁺ ,
(Ba, Ca, Mg) ₅ (PO ₄ ) ₃ Cl: Eu ³⁺ ,
YVO ₄ : Eu ³⁺ ,
YVO ₄ : Eu ³⁺ , Bi ³⁺ ,
CaS: Eu ³⁺ ,
Y ₂ O ₃ : Eu ³⁺ ,
YAlO ₃ : Eu ³⁺ ,
YBO ₃ : Eu ³⁺ ,
(Y, Gd) BO ₃ : Eu ³⁺ .

  As a method for producing the long afterglow phosphor used in the present invention, a known method can be used. For example, a solid phase method, a liquid phase method, a hydrothermal method, a combustion method, or the like can be used. For example, the long-life phosphor can be obtained in the form of inorganic particles by mixing and firing the above-mentioned compound constituting the matrix and the above-described compound constituting the activator. The volume average particle size of the inorganic particles is preferably 5 nm or more and 500 nm or less so that the phosphor-containing nanoparticles encapsulating such inorganic particles have a nano-size size, and preferably 5 nm or more and 300 nm or less. It is more preferable.

  Since the above long afterglow phosphor has a longer fluorescence lifetime than the above-mentioned “organic fluorescent dye” and “semiconductor nanoparticle”, it can be used as a “second fluorescent substance” in the present invention. “Organic fluorescent dyes” or “semiconductor nanoparticles” can be combined as a “first fluorescent substance”.

Basic Structure of Fluorescent Substance-Incorporated Nanoparticle The fluorescent substance-encapsulating nanoparticle of the present invention is a nanoparticle including the above-mentioned “fluorescent substance”, and can be distinguished from the first fluorescent substance and the first fluorescent substance. And a second fluorescent material having excellent excitation / emission characteristics. Here, in a typical embodiment of the present invention, a fluorescent material generally used as an existing fluorescent labeling agent having general excitation / emission characteristics is used as the first fluorescent material. As the fluorescent material, a fluorescent material capable of emitting fluorescence under the condition that the first fluorescent material does not substantially emit fluorescence is used. Specifically, the first fluorescent material and the second fluorescent material are present in a state of being included in the base material.

  The fluorescent substance-encapsulating nanoparticles according to the first embodiment of the present invention include a fluorescent substance having no excitation spectrum in the ultraviolet region as the first fluorescent substance, and a fluorescence having an excitation spectrum in the ultraviolet area as the second fluorescent substance. It is the fluorescent substance inclusion | inner_cover nanoparticle containing a substance. As a typical example of the first embodiment, an organic fluorescent dye is used as the first fluorescent material, and an inorganic fluorescent nanoparticle is used as the second fluorescent material.

  The fluorescent substance-encapsulating nanoparticles according to the second embodiment of the present invention include a fluorescent substance that is not a long afterglow phosphor as the first fluorescent substance and a long afterglow phosphor as the second fluorescent substance. It is a fluorescent substance-containing nanoparticle. In the second embodiment, an organic fluorescent dye is used as the first fluorescent substance, and an activated fluorescent substance composed of inorganic particles, particularly a base material and an activator, is used as the second fluorescent substance. Here, as the first fluorescent substance, the semiconductor nanoparticles can be used instead of the organic fluorescent dye or together with the organic fluorescent dye.

  In the present invention, the “fluorescent substance-encapsulating nanoparticle” means a substance in which the fluorescent substance is dispersed inside the nanoparticle. In the “fluorescent substance-encapsulating nanoparticle”, even if the fluorescent substance is chemically bonded to the base material. It does not have to be bonded.

  The base material constituting the nanoparticles is not particularly limited, and examples thereof include organic polymer resins such as polystyrene resin, melamine resin and polylactic acid resin, and silica.

The fluorescent substance-containing nanoparticles used in the present invention can be prepared by a known method.
For example, silica nanoparticles encapsulating a fluorescent organic dye can be synthesized with reference to the synthesis of FITC-encapsulated silica particles described in Langmuir 8, Vol. 2921 (1992). By using a desired fluorescent organic dye instead of FITC, various fluorescent organic dye-encapsulated silica nanoparticles can be synthesized.

  Silica nanoparticles encapsulating semiconductor nanoparticles can be synthesized with reference to the synthesis of CdTe-encapsulated silica nanoparticles described in New Journal of Chemistry, Vol. 33, p. 561 (2009).

  Polystyrene nanoparticles encapsulating a fluorescent organic dye may be copolymerized using an organic dye having a polymerizable functional group described in US Pat. No. 4,326,008 (1982) or polystyrene described in US Pat. No. 5,326,692 (1992). It can be created using a method of impregnating nanoparticles with a fluorescent organic dye.

  Polymer nanoparticles encapsulating semiconductor nanoparticles can be prepared using the method of impregnating semiconductor nanoparticles into polystyrene nanoparticles described in Nature Biotechnology, Vol. 19, p. 631 (2001).

  The average particle diameter of the nanoparticles encapsulating the fluorescent material used in the present invention is not particularly limited, but those having a size of about 30 to 800 nm can be used. Further, the coefficient of variation indicating the variation in the particle diameter is not particularly limited, but 20% can be used. In the present invention, the average particle diameter is obtained when an electron micrograph is taken using a scanning electron microscope (SEM), the cross-sectional area is measured for a sufficient number of particles, and the measured value is defined as the area of a corresponding circle. Is obtained as the particle diameter. In the present application, the arithmetic average of the particle sizes of 1000 particles is defined as the average particle size. The coefficient of variation was also a value calculated from the particle size distribution of 1000 particles.

Fluorescent substance-encapsulated nanoparticles having a biological substance recognition substance bonded to the surface The fluorescent substance-encapsulated nanoparticles of the present invention are mainly used as a fluorescent labeling agent for pathological diagnosis. Therefore, the fluorescent substance-encapsulating nanoparticles of the present invention can be used to specifically label biological substances to be labeled (hereinafter sometimes referred to as “target biological substances”). A biomolecule recognition substance that functions as a substance recognition site is preferably bound to the surface. That is, the fluorescent substance-encapsulating nanoparticles of the present invention preferably have a biomolecule recognition substance on the surface. Here, in the present invention, the biological material recognition site is a site that specifically binds and / or reacts with the target biological material. In the present specification, among the fluorescent substance-containing nanoparticles according to the present invention, fluorescent substance-encapsulated nanoparticles in which a biomolecule recognition substance is bound to the surface are particularly referred to as “biomolecule recognition substance-modified fluorescent substance-containing nanoparticles”. Sometimes called.

  The biomolecule recognition substance used in the present invention is not particularly limited as long as it is a substance that specifically binds and / or reacts with the target biomaterial, and examples thereof include nucleotide chains, proteins, and antibodies. It is done.

Among these, for antibodies, various antibodies including antibodies constituting antibody drugs can be used. Here, in the present invention, the term “antibody” is used to include any antibody fragment or derivative, and includes Fab, Fab ′ ₂ , CDR, humanized antibody, multifunctional antibody, single chain antibody (ScFv), and the like. Including various antibodies.

  Specifically, an anti-HER2 antibody that specifically binds to HER2, which is a protein present on the cell surface, an anti-ER antibody that specifically binds to an estrogen receptor (ER) present in the cell nucleus, and actin that forms a cytoskeleton And an anti-actin antibody that specifically binds to. Of these, anti-HER2 antibody and anti-ER antibody combined with fluorescent substance-encapsulated nanoparticles are preferable because they can be used for selection of breast cancer dosage.

  Further, the “target biological substance” (that is, the biological substance to be labeled with the fluorescent substance-encapsulating nanoparticles of the present invention) is not particularly limited, but in many cases, it is labeled through an antigen-antibody reaction. Therefore, those that function as an antigen for the biomolecule recognition substance can be mentioned.

  In the present invention, the term “antigen” refers to a biological substance, in particular, a molecule or molecular fragment. Examples of such “molecule” or “molecular fragment” include nucleic acids (single-stranded, DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid) etc., or nucleoside, nucleotide and their modified molecules), protein (polypeptide, oligopeptide etc.), amino acid (modified) Amino acids are also included), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules or complexes thereof, such as tumor markers, signaling substances, hormones, etc. There may be, and it is not specifically limited. For example, when an antibody drug used as an anticancer agent is used as an antibody, cancer growth control factors, metastasis control factors, growth control factor receptors, metastasis control factor receptors, and the like are suitable target antigens. In addition to antigens related to cancer, inflammatory cytokines such as TNF-α (Tumor Necrosis Factor α) and IL-6 (Interleukin-6) receptor, virus-related molecules such as RSV F protein, etc. Can be a biological material.

For example, cell growth factors such as HER2 and their receptors, and estrogen receptor (ER) are preferably used as the “target biological substance”.
The mode of binding between the biomolecule recognition substance and the fluorescent substance-encapsulating nanoparticles is not particularly limited, and examples thereof include covalent bonding, ionic bonding, hydrogen bonding, coordination bonding, 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.

  Moreover, there may be an organic molecule for linking these between the biological substance recognition site and the fluorescent substance-containing nanoparticle. For example, a polyethylene glycol chain can be used to suppress non-specific adsorption with a biological substance, and for example, SM (PEG) 12 manufactured by Thermo Scientific can be used.

When the biological substance recognition site is bound to the fluorescent substance-containing silica nanoparticles, the same procedure can be applied regardless of whether the fluorescent substance is a fluorescent organic dye or a semiconductor nanoparticle.
For example, a silane coupling agent that is a compound widely used for bonding an inorganic substance and an organic substance can be used. This silane coupling agent is a compound having an alkoxysilyl group that gives a silanol group by hydrolysis at one end of the molecule and a functional group such as a carboxyl group, an amino group, an epoxy group, an aldehyde group at the other end, Bonding with an inorganic substance through an oxygen atom of the silanol group.

  Specifically, mercaptopropyltriethoxysilane, glycidoxypropyltriethoxysilane, aminopropyltriethoxysilane, a silane coupling agent having a polyethylene glycol chain (for example, PEG-silane no. SIM6492.7 manufactured by Gelest), etc. Can be given. When using a silane coupling agent, you may use individually by 1 type and may use 2 or more types together.

  Silane coupling agents having hydrophilic functional groups such as the polyethylene glycol chain introduced to suppress non-specific adsorption with biological materials and silane coupling agents having a polyethylene glycol chain are used for aqueous dispersion. It also functions as a modifying compound. Therefore, in one embodiment of the fluorescent substance-containing nanoparticles of the present invention, the aqueous dispersion modifying compound is bonded to the surface.

A known procedure can be used for the reaction procedure of the organic fluorescent dye-encapsulated silica nanoparticles and the silane coupling agent.
For example, the obtained fluorescent dye-encapsulated silica nanoparticles are dispersed in pure water, aminopropyltriethoxysilane is added, and the mixture is reacted at room temperature for 12 hours. After completion of the reaction, fluorescent substance-encapsulated silica nanoparticles whose surface is modified with an aminopropyl group can be obtained by centrifugation or filtration. Subsequently, by reacting an amino group with a carboxyl group in the antibody, the antibody can be bound to the fluorescent organic dye-containing silica nanoparticles via an amide bond. If necessary, a condensing agent such as EDC (1-Ethyl-3- [3-Dimethylaminopropyl] carbohydrate Hydrochloride: Pierce) may be used.

If necessary, a linker compound having a site capable of directly binding to organic fluorescent dye-encapsulated silica nanoparticles modified with an organic molecule and a site capable of binding to a molecular target substance can be used.
As a specific example, sulfo-SMCC (Sulfosuccinimidyl 4 [N-maleimidemethyl] -cyclohexane-1-carboxylate: manufactured by Pierce, Inc.) having both a site selectively reacting with an amino group and a site reacting selectively with a mercapto group. ), The amino group of the organic fluorescent dye-encapsulated silica nanoparticles modified with aminopropyltriethoxysilane and the mercapto group in the antibody are bonded to each other to produce antibody-bound organic fluorescent dye-encapsulated silica nanoparticles.

  When the biological substance recognition site is bound to the fluorescent substance-encapsulated polystyrene nanoparticles, the same procedure can be applied regardless of whether the fluorescent substance is a fluorescent organic dye or a semiconductor nanoparticle. That is, by impregnating fluorescent nanoparticles or semiconductor nanoparticles with polystyrene nanoparticles having a functional group such as an amino group, fluorescent substance-containing polystyrene nanoparticles having a functional group can be obtained. Thereafter, EDC or sulfo-SMCC is used. In this way, antibody-bound fluorescent substance-encapsulated polystyrene nanoparticles can be produced.

[Fluorescent labeling agent for pathological diagnosis]
Since the fluorescent substance-containing nanoparticles described above include the first fluorescent substance and the second fluorescent substance having different excitation / emission characteristics, when used for the tissue labeling process, A first emission image including fluorescence emitted from the first fluorescent material and a second emission image including fluorescence emitted from the second fluorescent material but not including fluorescence from the first fluorescent material are captured. Then, by performing arithmetic processing based on these luminescent images, it is possible to provide pathological diagnosis data from which the influence of the background due to the autofluorescence of the tissue is removed.

  Therefore, in the present invention, a fluorescent labeling agent for pathological diagnosis including such fluorescent substance-containing nanoparticles is also provided. Here, the fluorescent labeling agent for pathological diagnosis of the present invention may be the above-described fluorescent substance-encapsulating nanoparticles themselves, may be in a form dissolved or dispersed in an appropriate aqueous solvent such as PBS, or the above-described fluorescent substance-encapsulating agent. In addition to the nanoparticles, other components such as adjuvants such as stabilizers and pharmaceutically acceptable fillers may be included as constituents as required.

[Measurement method of biological material]
The biological material measurement method according to the present invention will be described below.
The method for measuring a biological material according to the present invention relates to a method for measuring a biological material in a tissue using the fluorescent substance-containing nanoparticles described above as a fluorescent labeling agent for pathological diagnosis. Specifically,
(A) a step of labeling the tissue using the fluorescent substance-containing nanoparticles described above;
(B) About the tissue obtained in the step (a), the first emission image including the fluorescence emitted from the first fluorescent substance and the fluorescence emitted from the second fluorescent substance are included in the same visual field. Taking a second emission image that does not include fluorescence from the first fluorescent material;
(C) The first light emission image and the second light emission image obtained in the step (b) are compared, and brightness is obtained in both the first light emission image and the second light emission image. The present invention relates to a method for measuring a biological material in a tissue, including a step of recognizing only pixels detected as dots as effective bright spots.

Here, in the embodiment using a fluorescent material having an excitation spectrum in the ultraviolet region as the second fluorescent material, the steps (b) and (c) are respectively
(B-1) For the tissue obtained in the step (a), a step of taking an ultraviolet excitation image and a visible light excitation image in the same visual field, and
(C-1) A pixel detected as a bright spot in both the ultraviolet excitation image and the visible light excitation image by comparing the ultraviolet excitation image obtained in the step (b) with the visible light excitation image Can be performed as a process of recognizing only the effective bright spot. In this case, the visible light excitation image corresponds to the first emission image, and the ultraviolet excitation image corresponds to the second emission image.

In the embodiment using a long afterglow phosphor as the second phosphor, the steps (b) and (c) are respectively
(B-2) For the tissue obtained in the step (a), in the same field of view, a step of photographing an excitation image obtained at the time of excitation light irradiation and an afterglow image obtained by time-resolved fluorescence measurement; and
(C-2) The excitation image obtained in the step (b) is compared with the afterglow image, and only pixels detected as bright spots in both the excitation image and the afterglow image are detected. This can be performed as a step of recognizing an effective bright spot. In this case, the excitation image corresponds to the first light emission image, and the afterglow image corresponds to the second light emission image.

1. Step (a) In the measurement method of the present invention, as the step (a), a step of labeling the tissue using the fluorescent substance-containing nanoparticles described above is performed. Specifically, the fluorescent substance-encapsulating nanoparticles having a substance that recognizes a biological substance to be labeled (that is, a biomolecule recognition substance) bound to the surface react with the tissue, and this tissue is reacted through an antigen-antibody reaction. This is a step of labeling the “biological substance to be labeled” existing above. Here, the method for labeling the tissue is not particularly limited, and the labeling can be performed using a conventionally known method. For example, a labeling process can be performed on a pathological section tissue by a technique similar to that of conventionally known cell staining. However, the measurement method of the present invention is applicable not only to a pathological section tissue but also to various tissues.

A method for preparing a section that can be used as a tissue to which the measurement method of the present invention can be applied is not particularly limited, and a method prepared by a known method can be used.
For example, when a paraffin-embedded section that is widely used as a pathological section is used as a tissue, step (a) may be performed in the following procedure.

(1) Deparaffinization treatment step A pathological section is immersed in a container containing xylene to remove paraffin. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, xylene may be exchanged during the immersion.

  Next, the pathological section is immersed in a container containing ethanol to remove xylene. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, ethanol may be exchanged during the immersion.

  The pathological section is immersed in a container containing water and ethanol is removed. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, water may be exchanged during the immersion.

(2) Activation treatment process The activation process of the target biological substance is performed according to a known method.
The activation conditions are not particularly defined, but as the activation liquid, 0.01 M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, 0.1 M Tris-HCl buffer, etc. are used. be able to. As the heating device, an autoclave, a microwave, a pressure cooker, a water bath, or the like can be used. The temperature is not particularly limited, but can be performed at room temperature. The temperature can be 50 to 130 ° C. and the time can be 5 to 30 minutes.

  Next, the section after the activation treatment is immersed in a container containing PBS and washed. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, PBS may be replaced during the immersion.

(3) Labeling with fluorescent substance-encapsulating nanoparticles A PBS dispersion of the fluorescent substance-encapsulating nanoparticles is prepared, placed on a pathological section, and reacted with a biological substance to be detected. The fluorescent substance-encapsulating nanoparticles used at this time are preferably used in the form of “biomolecule recognition substance-modified fluorescent substance-encapsulating nanoparticles” by previously binding a biological substance to the surface.

  Here, when labeling a plurality of “target biological substances”, PBS dispersions of fluorescent substance-containing nanoparticles bound with biomolecule recognition substances corresponding to each biological substance are prepared, respectively. Can be placed on a pathological section and each can react with a target biological substance. At this time, even if the first fluorescent substance and / or the second fluorescent substance constituting the fluorescent substance-containing nanoparticles are changed for each biomolecule recognition substance so that a plurality of “target biological substances” can be distinguished from each other. Good.

  When placed on a pathological section, each fluorescent substance-containing nanoparticle PBS dispersion may be mixed in advance, or may be placed separately and sequentially. The mixing ratio of the fluorescent substance-containing nanoparticles containing the first fluorescent substance and the fluorescent substance-containing nanoparticles containing the second fluorescent substance is not particularly limited, but the ratio of the two is sufficient for the effect of the present invention to be exhibited. May be 1: 1 to 5: 1.

  The temperature is not particularly limited, but can be performed at room temperature. The reaction time is preferably 30 minutes or more and 24 hours or less. In addition, it is preferable to dripping well-known blocking agents, such as BSA containing PBS, before dyeing | staining with a fluorescent substance inclusion nanoparticle.

Next, the stained section is immersed in a container containing PBS to remove unreacted fluorescent substance-containing nanoparticles. The temperature is not particularly limited, but can be performed at room temperature. The immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, PBS may be replaced during the immersion.
Hematoxylin-eosin staining may be performed for tissue morphology observation.
A cover glass is placed on the section and sealed. A commercially available mounting agent may be used as needed.

2. Step (b) In the measurement method of the present invention, as the step (b), the first luminescence containing the fluorescence emitted from the first fluorescent substance in the same field of view for the tissue obtained in the step (a). A step of capturing a second light-emitting image including the image and the fluorescence emitted from the second fluorescent material but not including the fluorescence from the first fluorescent material is performed.

  In the present invention, the first luminescent image and the second luminescent image can be acquired using a fluorescence microscope, and can also be acquired in the form of an image including three-dimensional information using a confocal microscope. When the first light emission image and the second light emission image are taken, an excitation light source and a fluorescence detection optical filter corresponding to the absorption maximum wavelength and the fluorescence wavelength of the fluorescent material constituting the fluorescent material-containing nanoparticles are used. select.

  Here, in an embodiment in which a fluorescent material having an excitation spectrum in the ultraviolet region is used as the second fluorescent material, the step (b) is the same as the ultraviolet excitation image in the same field for the tissue obtained in the step (a). This can be performed as a step of capturing a visible light excitation image. In this case, the first emission image is taken using visible light as excitation light, and the second emission image is taken using ultraviolet light as excitation light in the same field of view. Become. Here, the visible light used as the excitation light is specifically light in the range of 400 nm to 700 nm, and the ultraviolet light is light in the range of less than 400 nm, more practically in the range of 200 nm to less than 400 nm. It is. The first emission image and the second emission image are acquired in the range from the visible region to the near infrared region, more specifically in the wavelength range from 400 nm to 900 nm, as long as they are not affected by the excitation light. can do.

  In an embodiment using a fluorescent material having an excitation spectrum in the ultraviolet region as the second fluorescent material, the step (b) is performed in the same field of view with the excitation light irradiation for the tissue obtained in the step (a). This can be performed as a step of photographing the obtained excitation image and the afterglow image obtained by time-resolved fluorescence measurement. In this case, after taking the first emission image in the presence of excitation light, the excitation light is extinguished and a certain time elapses (for example, after the excitation light is extinguished and after 100 micron to 10 milliseconds), The second light emission image is taken with the same field of view as the first light emission image.

3. Step (c) In the measurement method of the present invention, as the step (c), the first light emission image obtained in the step (b) is compared with the second light emission image, and the first light emission. A step of recognizing only pixels detected as bright spots in both the image and the second light emission image as effective bright spots is performed.

In particular,
(i) Measure the number of bright spots and / or emission luminance for each of the first emission image and the second emission image obtained in the step (b),
(ii) Light emission was recognized in both the first light emission image and the second light emission image by comparing each pixel on the first light emission image with each corresponding pixel on the second light emission image. The pixel is counted as an effective bright spot and added, and a process for obtaining the analytical bright spot data is performed by performing a calculation process that does not count the bright pixel as a bright spot.

  By obtaining such a process, background noise due to tissue autofluorescence can be removed, so that bright spot data with a high S / N ratio can be obtained and diagnostic accuracy can be improved. is there.

And the expression level of the target biological substance can be measured based on the number of bright spots or light emission luminance in the analytical bright spot data obtained by such a process.
The measurement of the number of bright spots or emission luminance can be performed using commercially available image analysis software, for example, G-Count, a total bright spot automatic measurement software manufactured by G. Angstrom.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.
The afterglow time of each phosphor was measured by 1/10 afterglow time using PTI-3000 (manufactured by Otsuka Electronics Co., Ltd.).

[Synthesis Example 1-1: Synthesis of Cy5-encapsulated silica nanoparticles]
“Nanoparticles A1” were produced by the methods of the following steps (1) to (4).
Step (1): 1 mg (0.00126 mmol) of N-hydroxysuccinimide ester derivative of Cy5 (manufactured by GE Healthcare) and 400 μL (1.796 mmol) of tetraethoxysilane were mixed.
Step (2): Ethanol 40 mL and 14% aqueous ammonia 10 mL were mixed.
Step (3): The mixture prepared in Step (1) was added to the mixture prepared in Step (2) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
Step (4): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
When the obtained silica nanoparticle A1 was observed with a scanning electron microscope (SEM; Hitachi S-800 type), the average particle size was 110 nm and the coefficient of variation was 12%.

[Synthesis Example 1-2: Synthesis of Cy5-encapsulated polystyrene nanoparticles]
“Nanoparticles A2” were produced by the methods of the following steps (1) to (3).
Step (1): Cy5 N-hydroxysuccinimide ester derivative (GE Healthcare) 1 mg (0.00126 mmol), dichloromethane 60 μL, ethanol 120 μL Step (2): Polystyrene having amino group as surface functional group The mixture prepared in step (1) was added to 1.5 mL of a nanoparticle (particle size: 100 nm) aqueous dispersion (manufactured by micromod) while vigorously stirring. Stirring was performed for 12 hours from the start of addition.
Step (3): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. Washing with ethanol and pure water was performed once by the same procedure.
When SEM observation of the obtained polystyrene nanoparticles A2 was performed, the average particle size was 100 nm and the coefficient of variation was 6%.

[Synthesis Example 1-3: Formation of fluorescent organic dye-encapsulated melamine nanoparticles]
“Nanoparticles A3” were produced by the following method.
450 g of water and 1 mg (0.00126 mmol) of Cy5 (manufactured by GE Healthcare) are warmed to 70 ° C. Resin (15 mg of Madurit SMW818) is stirred in 50 g of water and added at 70 ° C. The solution remains clear. The temperature is raised again to 70 ° C., 2 μl of 98-100% formic acid is added and the mixture is stirred for a further 20 minutes at this temperature. After about 1 minute, the batch showed a slight turbidity. Then, when refinement | purification by ultrafiltration (30 kilodalton membrane) was performed, the melamine nanoparticle A3 was obtained.
It was confirmed by measurement with a scanning electron microscope that the average particle size of the obtained particle group was about 46 nm.

[Synthesis Example 2-1: Synthesis of InP / ZnS QD]
“Inorganic phosphor nanoparticles a” were prepared by the following process.
Indium myristate 0.1 mmol,
Stearic acid 0.1 mmol,
Trimethylsilylphosphine 0.1 mmol,
0.1 mmol of dodecanethiol,
Zinc undecylenate 0.1mmol
Was placed in a three-necked flask together with 8 ml of octadecene and heated at 300 ° C. for 1 hour while refluxing in a nitrogen atmosphere. After lowering to room temperature, 0.01 g of mercaptopropionic acid was added and stirred for 2 hours. As a result, an InP / ZnS semiconductor nanoparticle solution having an emission peak wavelength of 670 nm and a concentration of 3.0 InPM was obtained as inorganic phosphor nanoparticle a. .

[Synthesis Example 2-2: Synthesis of CdSe / ZnS QD]
“Inorganic phosphor nanoparticles b” were produced by the method of the following steps.
Se powder (0.7896 g) was added to trioctylphosphine (TOP, 7.4 g) and the mixture was heated to 150 ° C. (under a nitrogen stream) to make a TOP-Se stock solution.

  Separately, CdO (0.450 g) and stearic acid (8 g) were heated to 150 ° C. in a three-necked flask under an argon atmosphere. After CdO dissolved, the solution was cooled to room temperature. To the solution is added trioctylphosphine oxide (TOPO, 8 g) and 1-heptadecyl-octadecylamine (HDA, 12 g) and the mixture is again heated to 150 ° C., where the TOP-stock solution is quickly added. . Thereafter, the temperature of the chamber was heated to 220 ° C., and further increased to 250 ° C. over 120 minutes at a constant rate. Thereafter, the temperature was lowered to 100 ° C., zinc acetate dihydrate was added and stirred for dissolution, and then a trioctylphosphine solution of hexamethyldisilylthiane was added dropwise, and stirring was continued for several hours to complete the reaction. After the temperature was lowered to room temperature, 0.01 g of mercaptopropionic acid was added and stirred for 1 hour 20 hours, then washed and redispersed in pure water. A 0 CdM CdSe / ZnS semiconductor nanoparticle solution was obtained.

[Synthesis Example 2-3: Synthesis of Si / SiO ₂ Semiconductor Nanoparticles] “Inorganic phosphor nanoparticles c” were produced by the following method.
A mixed gas of Si semiconductor nanoparticle monosilane gas 0.5 liter / min and nitrogen gas 19.45 liter / min was introduced into a reaction tube made of quartz glass held at a temperature of 700 ° C. and a pressure of 50 KPa, and reacted for 0.5 hour. At this time, powder formed in the lower part of the reaction tube.

The reaction tube was replaced with nitrogen gas to atmospheric pressure. The powder was heated at 1100 ° C. for 1 hour while rotating the reaction tube.
Thereafter, air was introduced at 800 ° C. at 20 liter / min for 10 minutes when the temperature was lowered, and then nitrogen replacement was performed again to cool the reaction tube.

After cooling the reaction tube to room temperature, 100 ml of pure water and 0.5 mg of mercaptoundecanoic acid are added to 1 mg of powder and stirred for 10 minutes at 40 ° C. Inorganic phosphor nanoparticles with Si core / SiO ₂ shell composition c was obtained.

[Synthesis Example 3-1: Synthesis of (Y, Gd) BO ₃ : Eu ³⁺ Encapsulated Nanoparticles]
First, liquids A to D having the following compositions were prepared:
Liquid A: Low molecular weight gelatin 15% solution 1000 cc;
Liquid B: a liquid obtained by dissolving 0.156 mol of yttrium nitrate hexahydrate and 0.90 mol of gadolinium nitrate in 500 cc of water;
C liquid: a solution obtained by dissolving 0.0065 mol of europium nitrate in 50 cc of water;
Liquid D: A liquid obtained by dissolving 0.123 mol of boric acid in 500 cc of water.

  The solution A was vigorously stirred at 60 ° C., and the solutions B, C and D, which were also kept at 60 ° C., were simultaneously added at a constant rate over 4 minutes. The white precipitate formed in the liquid A was filtered, dried, and then baked in the atmosphere at 1400 ° C. for 2 hours to obtain nanoparticles d.

[Synthesis Example 3-2: Synthesis of Zn ₂ SiO ₄ : Mn ²⁺ Encapsulating Nanoparticles]
First, liquids A to D having the following compositions were prepared:
Liquid A: Low molecular gelatin 15% aqueous solution 1000 cc;
Liquid B: A solution prepared by dissolving 35.33 g of zinc nitrate hexahydrate and 1.79 g of manganese nitrate hexahydrate in pure water to make 500 cc;
Liquid C: Liquid in which 18.25 g of 28% ammonia water was mixed with pure water to make 500 cc;
Liquid D: Liquid obtained by mixing 12.52 g of a colloidal silica 30R25 particle size 20 nm 30% solution 12.52 g manufactured by Clariant Japan with pure water to make 200 cc.

  While liquid A was vigorously stirred at room temperature, liquid B and liquid C were added at a constant rate over 30 minutes. As a result, a white precipitate was formed. Subsequently, after adding D liquid in A liquid over 4 minutes, solid-liquid separation was performed by the pressure filtration method. Next, the recovered cake was dried at 100 ° C. for 24 hours to obtain a dried precursor. The obtained precursor was calcined at 700 ° C. for 3 hours in the air, and further calcined at 1200 ° C. for 3 hours in an atmosphere of 100% nitrogen to obtain nanoparticles e.

[Synthesis Example 4-1: Synthesis of Fluorescent Organic Dye and Inorganic Phosphor Nanoparticle Encapsulated Silica Nanoparticle]
“Nanoparticles A4” were produced by the following steps (1) to (3).
Step (1): Cy5 N-hydroxysuccinimide ester derivative (manufactured by GE Healthcare) 1 mg (0.00126 mmol), inorganic phosphor nanoparticles a 0.00126 μmol, and tetraethoxysilane 400 μL (1.796 mmol) were mixed. .
Step (2): The mixture prepared in Step (1) was added to a mixture of 40 mL of ethanol and 10 mL of 14% aqueous ammonia at room temperature. Stirring was performed for 12 hours from the start of addition.
Step (3): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
When the obtained silica nanoparticle A4 was observed with a scanning electron microscope (SEM; Hitachi S-800 type), the average particle size was 110 nm and the coefficient of variation was 12%.

[Synthesis Example 4-2: Synthesis of Fluorescent Organic Dye, Inorganic Phosphor Nanoparticle-Incorporated Polystyrene Nanoparticle]
“Nanoparticles A5” were produced by the methods of the following steps (1) to (3).
Step (1): 1 mg (0.00126 mmol) of N-hydroxysuccinimide ester derivative of Cy5 (manufactured by GE Healthcare) and inorganic phosphor nanoparticles b0.00126 μmol dispersed in ethanol, dissolved in 60 μL of dichloromethane, and 120 μL of ethanol I let you.
Step (2): The liquid mixture prepared in Step (1) was added to 1.5 mL of a polystyrene nanoparticle aqueous dispersion (manufactured by micromod) having a surface functional group amino group and vigorously stirred. Stirring was performed for 12 hours from the start of addition.
Step (3): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. Washing with ethanol and pure water was performed once by the same procedure.
When SEM observation of the obtained polystyrene nanoparticles A5 was performed, the average particle size was 100 nm and the coefficient of variation was 6%.

[Synthesis Example 4-3: Synthesis of fluorescent organic dye and inorganic phosphor melamine nanoparticles]
“Nanoparticles A6” were produced by the method of the following step.
450 g of water, 1 mg (0.00126 mmol) of Cy5 (manufactured by GE Healthcare), and 0.00126 μmol of inorganic phosphor nanoparticles c are warmed to 70 ° C. Resin (15 mg of Madurit SMW818) is stirred in 50 g of water and added at 70 ° C. The solution remains clear. The temperature is raised again to 70 ° C., 2 μl of 98-100% formic acid is added and the mixture is stirred for a further 20 minutes at this temperature. After about 1 minute, the batch showed a slight turbidity. Then, when refinement | purification by ultrafiltration (30 kilodalton membrane) was performed, the melamine nanoparticle A6 was obtained. It was confirmed by measurement with a scanning electron microscope that the average particle size of the obtained particle group was about 46 nm.

[Synthesis Example 5-1: Synthesis of fluorescent organic dye, silica nanoparticles encapsulating inorganic phosphor nanoparticles]
“Nanoparticles A7” were produced by the methods of the following steps (1) to (3).
Step (1): 1 mg (0.00126 mmol) of N-hydroxysuccinimide ester derivative of Cy5 (manufactured by GE Healthcare), 0.00126 μmol of nanoparticles d dispersed in ethanol, and 400 μL (1.796 mmol) of tetraethoxysilane Were mixed.
Step (2): The mixture prepared in Step (1) was added to a mixture of 40 mL of ethanol and 10 mL of 14% aqueous ammonia at room temperature. Stirring was performed for 12 hours from the start of addition.
Step (3): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
When the obtained silica nanoparticle A7 was observed with a scanning electron microscope (SEM; Hitachi S-800 type), the average particle size was 110 nm and the coefficient of variation was 12%.

[Synthesis Example 5-2: Synthesis of Fluorescent Organic Dye, Inorganic Phosphor Nanoparticle-Incorporated Polystyrene Nanoparticle]
“Nanoparticles A8” were produced by the methods of the following steps (1) to (3).
Step (1): 1 mg (0.00126 mmol) of N-hydroxysuccinimide ester derivative of Cy5 (manufactured by GE Healthcare) and inorganic phosphor nanoparticles b0.00126 μmol dispersed in ethanol, dissolved in 60 μL of dichloromethane, and 120 μL of ethanol Step (2): The mixture prepared in Step (1) was added to a vigorously stirred 1.5 mL polystyrene nanoparticle aqueous dispersion (made by micromod) with a surface functional group amino group. did. Stirring was performed for 12 hours from the start of addition.
Step (3): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. Washing with ethanol and pure water was performed once by the same procedure.
When SEM observation of the obtained polystyrene nanoparticles A8 was performed, the average particle size was 100 nm and the coefficient of variation was 6%.

[Synthesis Example 5-3: Fluorescent Organic Dye, Inorganic Phosphor Melamine Nanoparticle]
“Nanoparticles A9” were produced by the following method.
450 g of water, 1 mg (0.00126 mmol) of Cy5 (manufactured by GE Healthcare), and inorganic fluorescent nanoparticles a0.00126 μmol are warmed to 70 ° C. Resin (15 mg of Madurit SMW818) is stirred in 50 g of water and added at 70 ° C. The solution remains clear. The temperature is raised again to 70 ° C., 2 μl of 98-100% formic acid is added and the mixture is stirred for a further 20 minutes at this temperature. After about 1 minute, the batch showed a slight turbidity. Then, when refinement | purification by ultrafiltration (30 kilodalton membrane) was performed, the melamine nanoparticle A9 was obtained. It was confirmed by measurement with a scanning electron microscope that the average particle size of the particle group obtained after the generation was about 46 nm.

[Example 1: Binding of antibody to nanoparticles]
Antibody binding was performed on the nanoparticles A1 to A9 by the following procedure.
Specifically, the operations of steps (1) to (12) were performed to bind the antibodies, thereby producing “antibody-binding particles 1 to 9”.
Step (1): 1 mg of nanoparticles A1 to A9 were dispersed in 5 mL of pure water. 100 μL of aminopropyltriethoxysilane aqueous dispersion was added and stirred at room temperature for 12 hours.
Step (2): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
Step (3): Ethanol was added to disperse the sediment, followed by centrifugation again. When washing with ethanol and pure water was performed once in the same procedure, amino group-modified nanoparticles B1 to B9 respectively corresponding to the nanoparticles A1 to A9 were obtained. When FT-IR measurement of the obtained nanoparticles B1 to B9 was performed, absorption derived from an amino group could be observed, and it was confirmed that the amino group could be modified.
Step (4): The amino group-modified nanoparticles B1 to B9 obtained in Step (3) are adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid). did.
Step (5): SM (PEG) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleidopropionamidyl) -dodecaethyleneglycol] ester) is mixed with the solution prepared in step (4) to a final concentration of 10 mM, The reaction was carried out for 1 hour.
Step (6): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
Step (7): PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The washing | cleaning by the same procedure was performed 3 times. Finally, it was redispersed using 500 μL PBS.
Step (8): 1 M dithiothreitol (DTT) was added to 100 μL of anti-HER2 antibody dissolved in 100 μL of PBS and allowed to react for 30 minutes.
Step (9): Excess DTT was removed from the reaction mixture with a gel filtration column to obtain a reduced anti-HER2 antibody solution.
Step (10): Each particle dispersion obtained in step (7) and the reduced anti-HER2 antibody solution obtained in step (9) were mixed in PBS and allowed to react for 1 hour.
Step (11): 4 μL of 10 mM mercaptoethanol was added to stop the reaction.
Step (12): The reaction mixture was centrifuged at 10,000 G for 60 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The washing | cleaning by the same procedure was performed 3 times. Finally, when 500 μL of PBS was redispersed, antibody-bound particles 1 to 9 corresponding to the nanoparticles A1 to A9, respectively, were obtained.

[Example 2: Pathological staining experiment]
Evaluation experiment: (1) Tissue staining using antibody-binding particles 1-9 Human breast tissue was immunostained using antibody-binding particles 1-9 prepared in Example 1 above.

  As a stained section, a tissue array slide (CB-A712) manufactured by Cosmo Bio was used. Observe the HER2 staining concentration in advance by DAB staining, and prepare two types of lots: (1) a lot with a high HER2 expression level and (2) a lot with a low HER2 expression level. Staining was performed using the binding particles. Here, such tissue staining was performed for each of the antibody-binding particles 1 to 9.

  Here, for each antibody-bound particle, each of a tissue array slide of a lot with a high HER2 expression level (hereinafter “slide 1”) and a tissue array slide of a lot with a low HER2 expression level (hereinafter “slide 2”), The operations described in (1) to (10) below were performed.

(1): A pathological section was immersed in a container containing xylene for 30 minutes. The xylene was changed three times during the process.
(2): A pathological section was immersed in a container containing ethanol for 30 minutes. The ethanol was changed three times during the process.
(3): The pathological section was immersed in a container containing water for 30 minutes. The water was changed three times along the way.
(4): A pathological section was immersed in 10 mM citrate buffer (pH 6.0) for 30 minutes.
(5): The autoclave process was performed at 121 degreeC for 10 minutes.
(6): The section after autoclaving was immersed in a container containing PBS for 30 minutes.
(7) 1% BSA-containing PBS was placed on the tissue and left for 1 hour.
(8) 10 μL of antibody-bound particles diluted to 0.05 nM with PBS containing 1% BSA was mixed and placed on the tissue and left for 3 hours.
(9): The stained sections were each immersed for 30 minutes in a container containing PBS.
(10): Aquex made by Merck Chemicals was dropped, and then a cover glass was placed and enclosed.

Evaluation experiment: (2) Bright spot measurement of tissue stained with antibody-binding particles 1 to 6 The tissue section stained by the above evaluation experiment (1) is irradiated with excitation light to fluoresce, and the tissue section is subjected to Olympus. Images were acquired using a DSU confocal microscope. The excitation wavelength was switched to 633 nm and 365 nm to obtain a detection wavelength of 660 nm.
The number of bright spots and light emission luminance were measured using G-count, a bright spot measurement software manufactured by Zeon Angstrom. The measurement method was implemented by the following two methods.

・ Measurement method 1
The tissue stained with the antibody-binding particles 1 to 6 was observed with the excitation wavelength set to 633 nm and the detection wavelength set to 660 nm.

  The number of bright spots was determined by measuring the bright spots of 30 cells for each of 8 spots in the tissue array slide, and calculating the average value. The luminous intensity was obtained by adding the fluorescence intensity of the entire field of view for each of the 8 spots and calculating the average value.

・ Measurement method 2
The tissues stained with the antibody-binding particles 1 to 6 were observed with two excitation wavelengths of 633 nm and 450 nm and a detection wavelength of 660 nm.

  For the number of bright spots, two images with different excitation wavelengths were superimposed in the same field of view, and the pixel value that was the bright spot in the two images at the same time was taken as the bright spot, and the luminous intensity of the bright spot was added. Otherwise, a process of not counting as bright spots was performed.

  The bright spot of each 30 cells was measured for 8 spots in the tissue array slide, and the average value was obtained. The luminous intensity was obtained by adding the fluorescence intensity of the entire field of view for each of the 8 spots and calculating the average value.

  The results obtained by the measurement method 2 are shown in Table 1 below together with the results obtained by the measurement method 1. Here, in Table 1, antibody binding particles 1 to 6 are represented as particles 1 to 6, respectively.

以上のように、紫外励起画像の観察を行わない計測方法１であっても、本発明の抗体結合粒子４〜６は、無機蛍光体ナノ粒子を内包しない抗体結合粒子１〜３と比べて輝度が高く、輝点観察のS/N比が改善された。さらに本発明の測定方法による計測方法２を用いることによって、計測方法１と比べてS/N比が飛躍的に改善されることが示された。

As described above, even in the measuring method 1 in which the ultraviolet excitation image is not observed, the antibody-binding particles 4 to 6 of the present invention have a brightness higher than that of the antibody-binding particles 1 to 3 that do not encapsulate the inorganic phosphor nanoparticles. The S / N ratio for bright spot observation was improved. Furthermore, it was shown that the S / N ratio was dramatically improved by using the measuring method 2 according to the measuring method of the present invention as compared with the measuring method 1.

・ Measurement method 3
For the antibody-binding particles 1 to 3 and 7 to 9, the excitation wavelength is 254 nm, the detection wavelength is 510 to 540 nm for particles 1, 3, 7, and 9, and 600 to 700 nm for particles 2 and 8, respectively. Set and observe. After irradiating the sample with excitation light, an observation image was acquired 1 msec after ending the excitation light irradiation.

  For the number of bright spots, two images with different excitation wavelengths were superimposed in the same field of view, and the pixel value that was the bright spot in the two images at the same time was taken as the bright spot, and the luminous intensity of the bright spot was added. Otherwise, a process of not counting as bright spots was performed.

  The bright spot of each 30 cells was measured for 8 spots in the tissue array slide, and the average value was obtained. The luminous intensity was obtained by adding the fluorescence intensity of the entire field of view for each of the 8 spots and calculating the average value.

  The results obtained by the measurement method 3 are shown in Table 2 below in comparison with the results by the measurement method 1. Here, in Table 2, antibody binding particles 1 to 3 and 7 to 9 are represented as particles 1 to 3 and 7 to 9, respectively.

以上のように、本発明の抗体結合粒子は７〜９は、紫外励起残光画像の観察を行わない計測方法１を用いたときには、長残光蛍光体を含まない抗体結合粒子１〜３と同等の輝度及びS/N比を示すに留まったものの、紫外励起残光画像の観察を行った計測方法３を用いたときには、抗体結合粒子１〜３と比べて輝度が高く、輝点観察のS/N比も著しく改善された。

As described above, antibody binding particles 7 to 9 of the present invention are antibody binding particles 1 to 3 that do not contain a long afterglow phosphor when the measurement method 1 that does not observe an ultraviolet excitation afterglow image is used. Although the luminance and S / N ratio are only shown, when the measurement method 3 in which the ultraviolet excitation afterglow image is observed is used, the luminance is higher than those of the antibody-binding particles 1 to 3, and the bright spot observation is performed. The S / N ratio was also significantly improved.

  On the other hand, with respect to slides 1 and 2 labeled with antibody-binding particles 1 to 2, when measuring method 3 was used, no bright spots were observed regardless of the amount of HER2 expression in the tissue used for the slide.

  As described above, it was shown that the S / N ratio was dramatically improved by using the measurement method of the present invention.

Claims (45)

A first fluorescent substance and a second fluorescent material having a fluorescent material and distinguishable excitation / emission characteristics of the first look-containing,
The first fluorescent substance and the second fluorescent substance are encapsulated by an organic polymer resin that is a polystyrene resin or a melamine resin, and
A fluorescent substance-encapsulated nanoparticle having a biomolecule recognizing substance and an aqueous dispersion modifying compound bonded to the surface .   The phosphor-containing nanoparticle according to claim 1, wherein the first fluorescent material does not have an excitation spectrum in the ultraviolet region, and the second fluorescent material has an excitation spectrum in the ultraviolet region.   The fluorescent substance-containing nanoparticle according to claim 2, wherein the fluorescent substance-containing nanoparticle includes an inorganic fluorescent nanoparticle and an organic fluorescent dye.   The phosphor-containing nanoparticle according to claim 3, wherein the inorganic phosphor nanoparticle comprises a II-VI group compound or a III-V group compound.   The phosphor-containing nanoparticle according to claim 3, wherein the inorganic phosphor nanoparticle contains simple silicon.   The phosphor-containing nanoparticle according to claim 1, wherein the first fluorescent material is a fluorescent material that is not a long afterglow phosphor, and the second fluorescent material is a long afterglow phosphor. The phosphor-containing nanoparticle according to claim 6, wherein the long afterglow phosphor and the organic fluorescent dye are encapsulated. The phosphor-containing nanoparticle according to claim 6 or 7, wherein the long afterglow phosphor is an inorganic particle. The phosphor-containing nanoparticle according to claim 8 , wherein the inorganic particles have a volume average particle diameter of 5 nm or more and 500 nm or less. The phosphor-containing nanoparticle according to claim 8 , wherein the inorganic particles have a volume average particle diameter of 5 nm or more and 300 nm or less. The phosphor-containing nanoparticle according to any one of claims 6 to 10 , wherein the long afterglow phosphor is an activated phosphor composed of a matrix and an activator. The fluorescent substance-containing nanoparticle according to claim 11 , wherein the activator is a rare earth activator. The phosphor-containing nanoparticle according to claim 11 , wherein the activator is Mn ²⁺ . The phosphor-containing nanoparticle according to claim 11 , wherein the activator is Eu ³⁺ . Phosphor containing nanoparticles according to any one of claims 11 to 14, wherein the maternal is Y ₂ O _3. Phosphor containing nanoparticles according to any one of claims 11 to 14 wherein the matrix is a Zn ₂ SiO _4. Pathology for fluorescent labeling agent containing the fluorescent material containing nanoparticles according to any one of claims 1-16. (A) a step of labeling a tissue using a fluorescent substance-containing nanoparticle including a first fluorescent substance and a second fluorescent substance having excitation / emission characteristics distinguishable from the first fluorescent substance ; ,
(B) For the tissue obtained in the step (a), a step of photographing an ultraviolet excitation image and a visible light excitation image in the same visual field;
(C) The ultraviolet excitation image obtained in the step (b) is compared with the visible light excitation image, and only pixels detected as bright spots in both the ultraviolet excitation image and the visible light excitation image are detected. A method for measuring a biological material in a tissue, comprising a step of recognizing an effective bright spot.   The measurement method according to claim 18, wherein the first fluorescent material does not have an excitation spectrum in the ultraviolet region, and the second fluorescent material has an excitation spectrum in the ultraviolet region.   The measurement method according to claim 19, wherein the fluorescent substance-containing nanoparticles include inorganic fluorescent nanoparticles and an organic fluorescent dye.   The measurement method according to claim 20, wherein the inorganic phosphor nanoparticles include a II-VI group compound or a III-V group compound.   The measurement method according to claim 20, wherein the inorganic phosphor nanoparticles contain simple silicon.   The measurement according to any one of claims 18 to 22, wherein the fluorescent substance-containing nanoparticles are fluorescent substance-containing nanoparticles in which the first fluorescent substance and the second fluorescent substance are encapsulated by an organic polymer resin. Method.   The measurement method according to claim 23, wherein the organic polymer resin is a polystyrene resin.   The measurement method according to claim 23, wherein the organic polymer resin is a melamine resin.   The measurement method according to any one of claims 18 to 22, wherein the fluorescent substance-containing nanoparticles are fluorescent substance-containing nanoparticles in which the first fluorescent substance and the second fluorescent substance are included in silica.   27. The measurement method according to any one of claims 18 to 26, wherein a biomolecule recognition substance is bonded to the surface of the fluorescent substance-containing nanoparticle.   The measurement method according to any one of claims 18 to 27, wherein a modification compound for aqueous dispersion is bonded to a surface of the fluorescent substance-containing nanoparticle. (A) a step of labeling a tissue using a fluorescent substance-containing nanoparticle including a first fluorescent substance and a second fluorescent substance having excitation / emission characteristics distinguishable from the first fluorescent substance ; ,
(B) For the tissue obtained in the step (a), a step of photographing an excitation image obtained at the time of excitation light irradiation and an afterglow image obtained by time-resolved fluorescence measurement in the same visual field;
(C) The excitation image obtained in the step (b) is compared with the afterglow image, and only pixels detected as bright spots in both the excitation image and the afterglow image are effective. viewing including the step of recognizing a bright spot,
A method for measuring a biological material in a tissue, wherein the first fluorescent material is a fluorescent material that is not a long afterglow phosphor, and the second fluorescent material is a long afterglow phosphor .   30. The measurement method according to claim 29, wherein the fluorescent substance-containing nanoparticles include the long afterglow phosphor and an organic fluorescent dye.   The measurement method according to claim 29 or 30, wherein the long afterglow phosphor is an inorganic particle.   The measurement method according to claim 31, wherein the volume average particle diameter of the inorganic particles is 5 nm or more and 500 nm or less.   The measurement method according to claim 31, wherein the volume average particle diameter of the inorganic particles is 5 nm or more and 300 nm or less.   The measurement method according to any one of claims 29 to 33, wherein the long afterglow phosphor is an activated phosphor composed of a matrix and an activator.   The measurement method according to claim 34, wherein the activator is a rare earth activator.   The activator is Mn ²⁺ The measurement method according to claim 34.   The activator is Eu ³⁺ The measurement method according to claim 34.   The mother body is Y ₂ O _Three The measurement method according to any one of claims 34 to 37.   The base is Zn ₂ SiO _Four The measurement method according to any one of claims 34 to 37.   The measurement according to any one of claims 29 to 39, wherein the fluorescent substance-containing nanoparticles are fluorescent substance-containing nanoparticles in which the first fluorescent substance and the second fluorescent substance are included by an organic polymer resin. Method.   The measurement method according to claim 40, wherein the organic polymer resin is a polystyrene resin.   The measurement method according to claim 40, wherein the organic polymer resin is a melamine resin.   The measurement method according to any one of claims 29 to 39, wherein the fluorescent substance-containing nanoparticles are fluorescent substance-containing nanoparticles in which the first fluorescent substance and the second fluorescent substance are included in silica.   44. The measurement method according to claim 29, wherein a biomolecule recognition substance is bonded to the surface of the fluorescent substance-containing nanoparticle.   45. The measurement method according to any one of claims 29 to 44, wherein an aqueous dispersion modifying compound is bonded to the surface of the fluorescent substance-containing nanoparticle.