JP2016014096A - Ultraviolet excited fluorescent particles, and detection method, image display method, image display screen, and image display unit using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide: ultraviolet excited fluorescent particles (fluorescent labeled particles) which enable fluorescence detection that is hardly affected by excitation light by suitably using a wavelength difference between excited light and fluorescent light of an ultraviolet excited fluorescent dye and, moreover, which can maintain excitation efficiency by the ultraviolet light at a high level; and a detection method using the same; and also to provide ultraviolet excited fluorescent particles which enable a medium to display good fluorescent images with irradiation light not interfering with visibility, and a display screen, a display unit, and a display method as image display applications using the same.SOLUTION: Provided are ultraviolet excited fluorescent particles containing an ultraviolet excited fluorescent dye in a silica particle, the dye absorbing ultraviolet light and emitting fluorescent light in a visible light region.

Description

  The present invention relates to ultraviolet-excited fluorescent particles, a detection method using the same, an image display method, an image display screen, and an image display device.

  Biotechnology is increasingly being applied to medical diagnostics. For example, immunochromatography is mentioned. This is an immunoassay method for determining the presence or absence of a colored portion that has captured a test substance. The procedure is as follows. First, the test substance is captured by the labeled particles. This moves through the support composed of the membrane by capillary action. Furthermore, the capture substance immobilized in a line on the support comes into contact with the test substance, where the test substance is captured and gradually concentrated. As a result, the line on which the capture substance is immobilized is strongly colored. Thereby, the presence or absence of the test substance can be accurately determined.

  Alternatively, flow cytometry and immunoassay are also used for various clinical tests and biochemical analyses. In the former, a fine nozzle is provided in the detector, and the attribute of each labeled particle passing therethrough is detected by fluorescence. Thereby, identification and quantification of a target substance can be performed. In the latter, a trace amount substance can be detected and quantified using an immune reaction or the like using a predetermined kit.

  By the way, dyes used as labels applied to the above-described clinical tests and the like include those that are discriminated by color development (absorption) and those that are discriminated by fluorescence emission. Unlike fluorescence detection, fluorescence emission has advantages such as being able to detect in the dark, and being able to measure fluorescence intensity with high accuracy, so that it is suitable for quantitative inspection. On the other hand, the fluorescent dye has a small difference (Stokes shift) between the excitation light and the fluorescence wavelength, and the detection sensitivity is improved by cutting the excitation light using a special optical filter. A technology has been developed in which a chain of excitation light and fluorescence wavelengths is expanded by FRET (Fluorescence resonance energy transfer), and a specific optical filter adapted thereto is applied (see Patent Document 1). ).

JP 2013-200316 A International Publication No. 2007/013601 Pamphlet Special table 2012-532211 (WO2011 / 002704)

  There is an example in which a fluorescent luminescent dye excited by ultraviolet rays is used as a labeling reagent (Patent Document 2). This receives excitation light having a wavelength of 300 to 400 nm and emits fluorescence having a longer wavelength. The difference is as large as 100 nm or more. Moreover, since the ultraviolet rays used as excitation light are not visible, they are unlikely to become an obstacle in visual inspection. As a result, it is expected that the detection sensitivity is greatly improved as compared with a conventional fluorescent dye having a Stokes shift of several tens of nm or less (see FIG. 1). On the other hand, research on application of such ultraviolet-excited fluorescent dyes to labeling reagents for clinical examinations has only just begun, and its applicability includes many unknowns.

  In view of the above points, the first object of the present invention is to suitably detect the wavelength difference between the excitation light and fluorescence of the ultraviolet excitation fluorescent dye, and to detect fluorescence that is not easily affected by the excitation light. In addition, the present invention is to provide ultraviolet excitation fluorescent particles (fluorescence-labeled particles) capable of maintaining the excitation efficiency by the ultraviolet rays at a high level and a detection method using the same.

Further, as a technique for authenticating an article using fluorescence, there is a technique in which particles having fluorescence are combined in a matrix to form a transparent structure (Patent Document 3). Here, the fluorescence on the particle surface is used. However, since only the particle surface is used, the fluorescence is weak, the particle diameter is small, and light does not easily scatter. Therefore, it is difficult to see the fluorescence from a direction other than the optical path, and it is difficult to use it for displaying images.
As a result of studying the application of the above-described ultraviolet-excited fluorescent particles, it was found that it was suitable for an ultraviolet irradiation type display device. A second object of the present invention is to provide ultraviolet-excited fluorescent particles capable of displaying a good fluorescent image on a medium without irradiating the irradiation light, and a display screen usable for image display using the same. A display device and a display method are provided.

（上記環Ａは芳香族環または複素芳香族環である。Ｘはイミノ基（ＮＲ^Ｎ）、酸素原子、硫黄原子、セレン原子、またはＢＲ^Ｎを表す。Ｒ^Ｃは水素原子または置換基を表す。Ｒ^Ｎは水素原子または置換基を表す。Ｒ^Ｍは脂肪族炭化水素基又は芳香族炭化水素基を表す。）
〔４〕上記式（１）が式（１−１）〜（１−３）のいずれかで表され、上記式（２）が式（２−１）〜（２−３）のいずれかで表され、上記式（３）が式（３−１）〜（３−３）のいずれかで表される〔３〕に記載の紫外励起蛍光粒子。

（式中、Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４は、それぞれ独立に、水素原子または置換基を示す。Ｘは式（１）と同義である。Ａｎ⁻は対アニオンを示す。Ｒ^Ｃ、Ｒ^Ｎ、Ｒ^Ｍは式（２）、式（３）と同義である。）
〔５〕上記紫外励起蛍光色素が、波長３００ｎｍ以上４７５ｎｍ未満の紫外線を吸収し、波長４００ｎｍ超７００ｎｍ以下かつ上記吸収波長よりも長い波長の蛍光を発光する〔１〕〜〔４〕のいずれか１項に記載の紫外励起蛍光粒子。
〔６〕紫外励起蛍光色素を含有するシリカ粒子を標識粒子として用いる検出方法であって、上記紫外励起蛍光色素に紫外線を照射し、これにより上記紫外励起蛍光色素から発せられる蛍光を検出して行う検出方法。
〔７〕上記紫外励起蛍光色素の発色団が含窒素複素芳香族化合物の残基である〔６〕に記載の検出方法。
〔８〕上記照射する紫外線が波長３００ｎｍ以上４７５ｎｍ未満であり、紫外励起蛍光色素から発せられる蛍光が波長４００ｎｍ超７００ｎｍ以下かつ上記照射波長よりも長い波長である〔６〕または〔７〕に記載の検出方法。
〔９〕〔１〕〜〔５〕のいずれか１項に記載の紫外励起蛍光粒子と、上記紫外励起蛍光粒子が移行するメンブレンとを有するイムノクロマトグラフィー用の検出キット。
〔１０〕上記メンブレンは、標的物質を捕捉した上記紫外励起蛍光粒子が固定化される検出領域を有する〔９〕に記載の検出キット。
〔１１〕上記メンブレンは、標的物質を捕捉していない上記紫外励起蛍光粒子が固定化される参照領域を有する〔９〕または〔１０〕に記載の検出キット。
〔１２〕〔１〕〜〔５〕のいずれか１項に記載の紫外励起蛍光粒子を用いる画像表示方法であって、上記紫外励起蛍光粒子を含む透明媒体に紫外線を照射し、照射された部分に存在する紫外励起蛍光粒子に含まれる紫外励起蛍光色素から発せられる蛍光とその散乱光により、上記透明媒体に画像を表示させる画像表示方法。
〔１３〕紫外線の照射が、レーザ、ＬＥＤ、およびランプのいずれかひとつにより行われる〔１２〕に記載の画像表示方法。
〔１４〕〔１〕〜〔５〕のいずれか１項に記載の紫外励起蛍光粒子を用いる画像表示装置であって、上記紫外励起蛍光粒子を含む透明媒体を具備する画像表示スクリーンと、画像表示スクリーンの所定の部分に紫外線を照射する紫外線光源とを備え、照射された部分に存在する蛍光粒子に含まれる蛍光色素から発せられる蛍光とその散乱光により、画像表示スクリーン上に画像を表示する画像表示装置。
〔１５〕シリカ粒子と透明媒体の屈折率差が０．１以上である〔１４〕に記載の画像表示装置。
〔１６〕ヘッドアップディスプレイまたはウエアラブルディスプレイである〔１４〕または〔１５〕に記載の画像表示装置。
〔１７〕〔１〕〜〔５〕のいずれか１項に記載の紫外励起蛍光粒子を含む透明媒体を具備する画像表示スクリーンであって、紫外線光源から照射された部分に存在する蛍光粒子に含まれる蛍光色素から発せられる蛍光とその散乱光により、画像表示スクリーン上に画像を表示することができる画像表示スクリーン。 The above problems have been solved by the following means.
[1] Ultraviolet-excited fluorescent particles containing in the silica particles an ultraviolet-excited fluorescent dye that absorbs ultraviolet rays and emits fluorescence in the visible light region side.
[2] The ultraviolet excited fluorescent particle according to [1], wherein the chromophore of the ultraviolet excited fluorescent dye is a residue of a nitrogen-containing heteroaromatic compound.
[3] The ultraviolet excitation fluorescent particle according to [1] or [2], wherein the chromophore of the ultraviolet excitation fluorescent dye is a residue of a compound represented by any one of the following formulas (1) to (3).

(The ring A is an aromatic ring or a heteroaromatic ring. X represents an imino group (NR ^N ), an oxygen atom, a sulfur atom, a selenium atom, or BR ^N. R ^C represents a hydrogen atom or a substituent. .R ^N is .R ^M represents a hydrogen atom or a substituent is an aliphatic hydrocarbon group or an aromatic hydrocarbon group.)
[4] The above formula (1) is represented by any one of formulas (1-1) to (1-3), and the above formula (2) is any one of formulas (2-1) to (2-3). The ultraviolet-excited fluorescent particles according to [3], wherein the formula (3) is represented by any one of formulas (3-1) to (3-3).

(In the formula, R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom or a substituent. X has the same meaning as in formula (1). An ⁻ represents a counter anion. R ^C , R ^N and R ^M have the same ^{meanings as} in Formula (2) and Formula (3).)
[5] Any one of [1] to [4], wherein the ultraviolet excitation fluorescent dye absorbs ultraviolet light having a wavelength of 300 nm or more and less than 475 nm, and emits fluorescence having a wavelength of more than 400 nm to 700 nm or less and longer than the absorption wavelength. The ultraviolet excitation fluorescent particle according to item.
[6] A detection method using silica particles containing an ultraviolet-excited fluorescent dye as a labeling particle, which is performed by irradiating the ultraviolet-excited fluorescent dye with ultraviolet rays, thereby detecting fluorescence emitted from the ultraviolet-excited fluorescent dye Detection method.
[7] The detection method according to [6], wherein the chromophore of the ultraviolet excitation fluorescent dye is a residue of a nitrogen-containing heteroaromatic compound.
[8] The ultraviolet ray to be irradiated has a wavelength of 300 nm or more and less than 475 nm, and the fluorescence emitted from the ultraviolet excitation fluorescent dye has a wavelength of more than 400 nm and not more than 700 nm and longer than the irradiation wavelength, [6] or [7] Detection method.
[9] A detection kit for immunochromatography comprising the ultraviolet excited fluorescent particles according to any one of [1] to [5] and a membrane to which the ultraviolet excited fluorescent particles migrate.
[10] The detection kit according to [9], wherein the membrane has a detection region in which the ultraviolet-excited fluorescent particles capturing the target substance are immobilized.
[11] The detection kit according to [9] or [10], wherein the membrane has a reference region in which the ultraviolet-excited fluorescent particles that have not captured the target substance are immobilized.
[12] An image display method using the ultraviolet excited fluorescent particles according to any one of [1] to [5], wherein the transparent medium containing the ultraviolet excited fluorescent particles is irradiated with ultraviolet rays, and the irradiated portion An image display method for displaying an image on the transparent medium by fluorescence emitted from an ultraviolet excitation fluorescent dye contained in an ultraviolet excitation fluorescent particle present in the medium and its scattered light.
[13] The image display method according to [12], wherein the ultraviolet irradiation is performed by any one of a laser, an LED, and a lamp.
[14] An image display device using the ultraviolet excited fluorescent particles according to any one of [1] to [5], an image display screen including a transparent medium containing the ultraviolet excited fluorescent particles, and an image display An image having an ultraviolet light source for irradiating a predetermined part of the screen with ultraviolet light, and displaying an image on the image display screen by the fluorescence emitted from the fluorescent dye contained in the fluorescent particles present in the irradiated part and its scattered light Display device.
[15] The image display device according to [14], wherein the refractive index difference between the silica particles and the transparent medium is 0.1 or more.
[16] The image display device according to [14] or [15], which is a head-up display or a wearable display.
[17] An image display screen comprising a transparent medium containing the ultraviolet-excited fluorescent particles according to any one of [1] to [5], which is included in fluorescent particles present in a portion irradiated from an ultraviolet light source An image display screen capable of displaying an image on the image display screen by fluorescence emitted from the fluorescent dye and its scattered light.

In the present invention, ultraviolet rays refer to light (electromagnetic waves) having a wavelength of less than 475 nm, preferably 450 nm or less, and more preferably 400 nm or less. The lower limit is preferably 300 nm or more. The visible light region side only needs to be a longer wavelength side than the irradiated ultraviolet light, typically means light of more than 400 nm, preferably 450 nm or more. The upper limit is preferably 750 nm or less, and more preferably 700 nm or less.
In addition, the wavelength of light can be measured on a normal condition using a general fluorescence spectrophotometer.

In the first invention, the ultraviolet-excited fluorescent particles and the detection method suitably utilize the difference between the wavelength of the excitation light and the fluorescence of the ultraviolet-excited fluorescent dye, enable detection that is not easily affected by the excitation light, and that is based on the ultraviolet light. Excitation efficiency can be maintained at a high level. In the preferred embodiment, if necessary, the ultraviolet excitation fluorescent dye can be used at a high concentration, and at that time, high stability is exhibited and concentration quenching is suppressed.
In the second invention, the ultraviolet-excited fluorescent particles function as a light emitting element in a transparent medium, and exhibit good display performance suitable for a head-up display or a wearable display.

It is explanatory drawing which contrasted the excitation light wavelength and fluorescence wavelength of a general labeling reagent and ultraviolet excitation fluorescent dye. It is the perspective view which showed typically the test piece for immunochromatography which concerns on one Embodiment of this invention. It is an apparatus block diagram which shows typically the flow cytometer which concerns on one Embodiment of this invention. It is an apparatus block diagram which shows typically the display apparatus which concerns on one Embodiment of this invention.

  The ultraviolet-excited fluorescent particles of the present invention contain an ultraviolet-excited fluorescent dye that absorbs ultraviolet rays and emits fluorescence in the visible light region side in silica particles. Hereinafter, the present invention will be described in detail focusing on preferred embodiments thereof.

(Ultraviolet excitation fluorescent dye)
The ultraviolet excitation fluorescent dye is not particularly limited as long as it absorbs ultraviolet light and emits fluorescence in the visible light region side, but the chromophore is preferably a residue of a nitrogen-containing heteroaromatic compound. The nitrogen-containing heteroaromatic compound is preferably a five-membered ring compound or a six-membered ring compound, and more preferably a five-membered ring compound. The nitrogen-containing heteroaromatic compound is preferably an azole compound, and more preferably a diazole compound. Of these, an oxadiazole compound, a benzooxadiazole compound, a pyridooxadiazole compound, an imidazole compound, a benzimidazole compound, and a pyridoimidazole compound are preferable.

It is preferable that the chromophore of the ultraviolet excitation fluorescent dye is composed of a residue of a compound represented by any one of the following formulas (1) to (3).

The ring A is an aromatic ring or a heteroaromatic ring.
When A is an aromatic ring, it is preferably 6 to 24 carbon atoms, more preferably 6 to 14 carbon atoms, and particularly preferably 6 to 10 carbon atoms. Specifically, a benzene ring, a naphthalene ring, an anthracene ring, and a phenanthrene ring are preferable. When it is a heteroaromatic ring, it is preferably 3 to 12 carbon atoms, more preferably 3 to 8 carbon atoms, and particularly preferably 3 to 6 carbon atoms. Specific examples include a pyridine ring, a pyrazine ring, a pyrimidine ring, a pyridazine ring, a pyrrole ring, a pyrazole ring, and an imidazole ring, and a pyridine ring is preferable.
X is an imino group (NR ^N ), an alkenyl group (preferably 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms), an aryl group (preferably 6 to 22 carbon atoms, more preferably 6 to 14 carbon atoms, and 6 to 10 carbon atoms). particularly preferred), preferably an aralkyl group (7 to 23 carbon atoms, more preferably 7 to 15, particularly preferably 7 to 11), an oxygen atom, a sulfur atom, a selenium atom or BR ^{N,. RN} represents a hydrogen atom or a substituent. Preferably R ^N is the same as described below ^{R 1,} a hydrogen atom, an alkyl group (having 1 to 12 carbon atoms, more preferably 1 to 6, 1 to 3 is especially preferred) is more preferable.
R ^C is as defined ^{R N.
RM} represents an aliphatic hydrocarbon group (preferably having 1 to 12 carbon atoms, more preferably 2 to 5 carbon atoms) or an aromatic hydrocarbon group (preferably having 6 to 22 carbon atoms, more preferably 6 to 14 carbon atoms). The R ^M Of these, an alkyl group (preferably having 1 to 12 carbon atoms, more preferably 1-6, particularly preferably 1 to 3), preferably an aryl group (6 to 22 carbon atoms, more preferably 6 to 14, 6-10 are particularly preferred) and aralkyl groups (C7-23 are preferred, 7-15 are more preferred, and 7-11 are particularly preferred).

The formula (1) is represented by any one of formulas (1-1) to (1-3), the formula (2) is represented by any one of formulas (2-1) to (2-3), The formula (3) is preferably represented by any one of formulas (3-1) to (3-3).

In the formula, R ¹ , R ² , R ³ and R ⁴ each independently represent a hydrogen atom or a substituent. R ¹ , R ² , R ³ and R ⁴ are each independently a hydrogen atom, a halogen atom, an alkyl group (preferably having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, particularly preferably 1 to 3 carbon atoms), and alkenyl. Group (C2-C12 is preferable, C2-C6 is more preferable), alkynyl group (C2-C12 is preferable, C2-C6 is more preferable), alkoxy group (C1-C12 is preferable, 6 is more preferable, and 1 to 3 is particularly preferable), a hydroxyl group, a cyano group, a sulfonic acid group, and an aromatic hydrocarbon group (6 to 22 carbon atoms are preferable, 6 to 14 are more preferable, and 6 to 10 are particularly preferable). ), A heterocyclic group (C1-C12 is preferable and 2-5 are more preferable).
X is synonymous with Formula (1).
R ^M has the same meaning as described above.
An ⁻ represents a counter anion, and is preferably a halide ion, CF ₃ SO ₃ ⁻ , BF ₄ ⁻ , or PF ₆ ⁻ . )

R ² and R ³ are each an aromatic group (preferably 6 to 22 carbon atoms, more preferably 6 to 14 carbon atoms) or a heteroaromatic group (preferably 1 to 12 carbon atoms, more preferably 2 to 5 carbon atoms). preferable. The ring structure forming the heteroaromatic group includes thiophene or a derivative thereof, furan or a derivative thereof, pyrrole or a derivative thereof, imidazole or a derivative thereof, oxazole or a derivative thereof, thiazole or a derivative thereof, pyrazole or a derivative thereof, and pyridine or a derivative thereof. Residue. Examples of the aromatic group include a phenyl group and a naphthyl group. R ² and R ³ may further have a substituent, for example, an alkyl group (preferably having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, particularly preferably 1 to 3 carbon atoms), an alkoxy group (having 1 to carbon atoms). 12 is preferable, 1 to 6 is more preferable, and 1 to 3 is particularly preferable, an alkenyl group (preferably having 2 to 12 carbon atoms, more preferably 2 to 5), and an acyl group (preferably having 1 to 12 carbon atoms are preferable). -6 is more preferable, and 2-3 is particularly preferable.

R ¹ or R ⁴ may constitute a bonding part. For example, the bonding portion functions as a connecting portion between the chromophore and the silane coupling agent when introduced into the silica particles. In this respect, the bonding portion preferably has an alkoxysilyl group (preferably having 1 to 12 carbon atoms, more preferably 1 to 6 and particularly preferably 1 to 3).
The temporary bonding part for deriving the bonding part includes hydroxyl group, thiol group, amino group, carbamoyl group, carboxyl group, isocyanate group, isothiocyanate group, epoxy group, halogenated alkyl group, triazine group, carbodiimide group, activity It may have an esterified carbonyl group, maleimide group, or succinimide group.

The coupling _{portion, - (CH 2) n -} , - NHCOO -, - CONH -, - CH 2 NH -, - CH 2 NR -, - COO -, - SO 2 NH -, - HN-C (= NH ) —NH—, —O—, —S—, —NR—, — (CH ₂ —CH ₂ —O) _n —, —CH═CH—, —C≡C—, —Ar—, —CO—Ar. You may have the coupling group which concerns on -NR- or these combination. R is an alkyl group (preferably having 1 to 6 carbon atoms). n is an integer of 1-10. Ar is an arylene group (preferably having 6 to 10 carbon atoms).

What is represented by the following formula | equation (I) can be used for the coupling group in a coupling | bond part.
-[(CHRa) p-X] r- (CHRb) q-[(CHRa) p-X] r-
(I)
In the formula, X is a direct bond, —NHCOO—, —CONH—, —COO—, —SO ₂ NH—, —HN—C (═NH) —NH—, —O—, —S—, —NR—, —CH═CH—, —C≡C—, —Ar—, —CO—Ar—NR—, or a combination thereof is represented. Ra and Rb are each independently a hydrogen atom, an aliphatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring, or an aromatic hydrocarbon group. A group substituted by any one charged group selected from the group consisting of a sulfonic acid group, a hydroxyl group, a quaternary amine group and a carboxyl group, if necessary. Ar represents an arylene group. p, q, and r each independently represent an integer of 0-20. r + q ≧ 1.

  Examples of the compound having the chromophore of formula (1) are listed below, but the present invention is not construed as being limited by this illustration.

  The method for synthesizing the ultraviolet-excited fluorescent dye may be a conventional method. For example, it can be synthesized according to Scheme 1 below. Here, an example is shown in which an active esterified carbonyl group is used as a reactive group, and -COO- is used as a functional group of the spacer portion that binds to the reactive group. For the active esterified carbonyl group, N-hydroxy-succinimide ester or maleimide ester can be used. By using N-hydroxy-succinimide and using DCC (N, N'-dicyclohexylcarbodiimide) as a condensing agent, the organic EL dye and the target molecule are bonded by an amide bond via the N-hydroxy-succinimide ester.

Ｄｙｅは蛍光色素の発色団である。Ｒはアルコキシシリル基又はアルコキシシリルアルキル基である。

Dye is a chromophore of a fluorescent dye. R is an alkoxysilyl group or an alkoxysilylalkyl group.

  These dyes can be usually purchased. For example, fluoride yellow, fluoride red, etc. manufactured by IST can be used.

  In the present invention, the excitation light wavelength of the ultraviolet excitation fluorescent dye is in the ultraviolet region, and the fluorescence wavelength is on the visible light region side. Its definition is as shown above.

In a preferred embodiment of the present invention, it is preferable that a larger amount of the ultraviolet excitation fluorescent dye is incorporated into the ultraviolet excitation fluorescent particle in the ultraviolet excitation fluorescent particle. In order to incorporate silica in a high concentration, it is preferable to use a dye having a traction group (traction group) that is introduced into the ultraviolet excitation fluorescent particles. The traction group is not particularly limited, but is preferably a nitrogen-containing group, and examples thereof include a —NR ^N — group, ═N—, and —NR ^N ₂ group. Or an oxygen-containing group is mentioned, -C = O group, -O- group, etc. are mentioned. Of these, a nitrogen-containing group is preferable.
On the other hand, a dye compound such as fluorescein that does not have a polar group or an amino group derivative (including pyridine / pyrrole) in the dye skeleton hardly reacts with, for example, 3-aminopropyltriethoxysilane (APS), It may be difficult to form a chemical bond with the particle network (does not chemically bond with —O—Si—O—). If it does so, it may become easy to leak a pigment from particles. In consideration of these points, it is preferable to select a dye species to be immobilized in the silica particles at the design stage.

[Immunochromatography]
The ultraviolet excited fluorescent particles of the present invention are preferably applied to immunochromatography. In the immunochromatographic test strip (test strip), it is preferable that the sample pad, the conjugate pad, the membrane, and the absorption pad are connected in series so that a capillary phenomenon occurs.

  FIG. 2 shows a test piece for immunochromatography suitably applied to the present invention. As shown in the figure, a test strip 10 including a membrane 8c having the above-described test region and reference region is included in a housing 6 (a housing upper portion 6a and a housing lower portion 6b), and a long test body 100 is included. I am doing. A detection opening 61 and a sample introduction opening 62 are provided in the housing upper part 6a. In the fluorescence observation, the irradiation light can be sent to the internal test strip 10 through the detection opening 61, and the fluorescence emitted therefrom can be collected and detected and observed. On the other hand, the test solution S can be supplied to the test strip 10 through the sample introduction opening 62 to perform a measurement test. As described above, the immunochromatographic test strip 10 of this embodiment includes the sample pad 8a, the conjugate pad 8b, the antibody-immobilized membrane 8c, and the absorption pad 8d. Furthermore, it is preferable that each said structural member is backed by the backing sheet (not shown) with an adhesive.

(Sample pad)
The sample pad 8a is a component that drops a sample containing a target substance. The material, dimensions, etc. are not particularly limited, and general materials applicable to this type of product can be used.

(Conjugate pad)
The conjugate pad 8b is a constituent member impregnated with the fluorescently labeled particles 2. The target substance contained in the sample that has moved from the sample pad 8a by capillary action is captured and labeled by the fluorescently labeled particles in a specific molecular recognition reaction such as an antigen-antibody reaction.
The content of the fluorescently labeled particles per unit area (cm ² ) in the conjugate pad 8b is not particularly limited, but is preferably 1 μg to 100 μg. Examples of the impregnation method include a method in which the dispersion liquid of the fluorescent labeling particles is applied, dropped or sprayed, and then dried.

(Antibody immobilized membrane)
Wherein the antibody immobilization part in the antibody immobilized membrane 8c, determine the presence or absence of the target substance, ie, providing a test line n _t of the target substance capture antibody is immobilized for determining the positive-negative. The antibody-immobilized membrane 8c preferably includes a control line _nr on which an antibody for capturing fluorescently labeled particles is immobilized.
The membrane 8c is a structural member in which the target substance 1 labeled with the fluorescently labeled particles 2 moves by capillary action, and the antibody immobilization is performed in which a sandwich type immune complex forming reaction consisting of immobilized antibody-target substance-fluorescent labeled particles is performed. Part (determination part). The shape of the antibody immobilization part (determination part) in the membrane is not particularly limited as long as the capture antibody is locally immobilized, and includes a line shape, a circular shape, a belt shape, etc. It is preferable that there is a line shape having a width of 0.5 to 1.5 mm.

The amount of antibody immobilization in the antibody immobilization part (test region) n _t is not particularly limited, but when the shape is a line, 0.5 μg to 5 μg per unit length (cm) is preferable. Examples of the immobilization method include a method in which an antibody solution is applied, dropped or sprayed and then dried and immobilized by physical adsorption. After the above-described antibody immobilization, the whole antibody-immobilized membrane is preferably subjected to a so-called blocking treatment in order to prevent the influence of nonspecific adsorption on the measurement. For example, a method of immersing in a buffer solution containing a blocking agent such as albumin, casein, polyvinyl alcohol or the like for an appropriate period of time and then drying may be mentioned. Examples of the commercially available blocking agent include skim milk (manufactured by DIFCO), 4% block ace (manufactured by Meiji Dairies), and the like.
The membrane 8c further has a reference region _nr in which the ultraviolet excitation fluorescent particles 2 in which the target substance is not captured are captured. Accordingly, the presence / absence and amount of the target substance can be fixed as compared with the fluorescence / absorption in the test region n _t . In order to fulfill this function, the test labeling particles 2 are composed of ultraviolet-excited fluorescent particles 2a and a binding substance 2b. The binding substance 2b has binding properties with the target substance.

(Absorption pad)
The absorption pad 8d is a constituent member that absorbs the test solution (target substance) S and the fluorescently labeled particles 2 that have moved through the membrane by capillary action and always generates a constant flow.

There are no particular restrictions on the material of each of these constituent members, and members used for immunochromatographic test strips can be used, but glass such as Glass Fiber Conjugate Pad (trade name, manufactured by MILLIPORE) is used as the sample pad and conjugate pad. A fiber pad is preferable, a nitrocellulose membrane such as Hi-Flow Plus 120 membrane (trade name, manufactured by MILLIPORE) is preferable as the membrane, and a cellulose membrane such as Cellulose Fiber Sample Pad (trade name, manufactured by MILLIPORE) is preferable as the absorbent pad. Is preferred.
Examples of the backing sheet with an adhesive include AR9020 (trade name, manufactured by Adhesives Research).

(Binding substance)
In the present embodiment, the test binding substance 2b is used integrally with the ultraviolet excitation fluorescent particle 2a (fluorescent labeling particle 2). Details of the material will be described in detail in the section of UV-excited fluorescent particles described later.

(Capturing substance for testing)
In the membrane used in this embodiment, the test capturing substance 4 is immobilized on the material of the membrane. The capture substance 4 has a binding ability to the target substance 1 so as to capture a complex including the ultraviolet excitation fluorescent particles 2a, the binding substance 2b, and the target substance 1. Since the capturing substance 4 has the binding ability as described above, a complex composed of the fluorescently labeled particles 2 and the target substance 1 can be captured. As a result, a line emitting fluorescence from the fluorescently labeled particles 2 is formed in the test region n _t .

(Reference capture material)
In the present embodiment, the reference capturing substance 5 is fixed to the reference region n _{r of the} membrane. This binds directly to the binding substance without using the target substance 1. Therefore, when fluorescently labeled particles 2 that are mixed with the transferred test solution S and are not bound to the target substance 1 are transferred, they are directly captured (see FIG. 2). As a result, a line exhibiting fluorescence by the fluorescently labeled particles is formed in the reference region _nr . The reference capture substance 5 is not particularly limited, and examples thereof include biomolecules capable of binding to the binding substance, and specific examples include antibodies.

(Material)
There is no restriction | limiting in particular as a material of each structural member employable for the plane test piece (test strip) 10 of this embodiment, The normal member used for the test strip for immunochromatography can be used. As the sample pad and the conjugate pad, for example, a glass fiber pad such as Glass Fiber Conjugate Pad (trade name, manufactured by MILLIPORE) is preferable. As the membrane, a nitrocellulose membrane such as Hi-Flow Plus 120 membrane (trade name, manufactured by MILLIPORE) is preferable. A cellulose membrane such as Cellulose Fiber Sample Pad (trade name, manufactured by MILLIPORE) is preferable as the absorbent pad. When using the said backing sheet with an adhesive, AR9020 (A brand name, the product made by Adhesives Research) etc. are mentioned.

(Introduction of labeled particles into the conjugate pad)
In the immunochromatography of this embodiment, it is preferable to introduce into the conjugate pad fluorescently labeled particles to which a binding substance is bound as the fluorescently labeled particles. The content of the fluorescent labeling particles per unit area (cm ² ) in the conjugate pad is not particularly limited, but is preferably 20 μg / cm ^{2 to 2} mg / cm ^{2, and} more preferably 20 to 200 μg / cm ² . If the content is too large, the number of analyte bindings per particle decreases, and the detection sensitivity decreases. Examples of the method of inclusion include a method in which the dispersion liquid of the fluorescent labeling particles is applied, dropped or sprayed and then dried. At this time, the fluorescently labeled particles are contained and once dried, the fluorescently labeled particles may be contained, or the fluorescently labeled particles may be mixed in advance and the mixed colloid may be contained.

[Detection method]
In immunochromatography, the detection device is used to detect and measure the fluorescence of the labeled particles through the detection unit. From the viewpoint of suitably detecting the fluorescence emitted by the fluorescently labeled particles, the excitation light source (light irradiator) preferably emits excitation light having a wavelength of 300 nm or more, and more preferably emits excitation light having a wavelength of 350 nm or more. Moreover, it is preferable to emit excitation light having a wavelength of less than 475 nm, more preferably 450 nm or less, and particularly preferably 400 nm or less. Examples of the excitation light source include a UV lamp and a UV LED. More preferably, the irradiation side includes a filter for transmitting only light having a specific wavelength from the excitation light source. Specifically, light having a wavelength longer than the excitation wavelength is cut. Furthermore, it is more preferable that the detection side includes a filter that removes the excitation light and transmits only fluorescence from the viewpoint of accurately detecting fluorescence.
In addition, when there is no filter, since excitation light cannot be visually observed by exciting using a wavelength shorter than the maximum excitation wavelength, it is preferable.

In a preferred embodiment of the present invention, the fluorescence wavelength of the fluorescent labeling particles is preferably more than 400 nm, preferably 450 nm or more, and more preferably 500 nm or more. The upper limit is preferably 750 nm or less, more preferably 700 nm or less, further preferably 650 nm or less, and particularly preferably 600 nm or less. In particular, the above range is preferable in consideration of the light receiving sensitivity characteristic and price of a widely used detector (light sensor).
In addition, as for the wavelength said here, a peak refers to the peak top or the whole vicinity, and shall mean a peak top (apex) in a narrow sense.

  The difference between the fluorescence wavelength emitted by the fluorescent particles and the wavelength of the excitation light is preferably 30 nm or more, more preferably 50 nm or more, further preferably 80 nm or more, and further preferably 100 nm or more, It is especially preferable that it is 130 nm or more. Although there is no upper limit in particular, it is practical that it is 300 nm or less.

  In addition, the preferable range of the wavelength of excitation light and fluorescence described here and the relationship between both are the same in the embodiment used as a display described later.

  The light receiving device is particularly preferably a photomultiplier tube, a CCD detector, or a CMOS detector that receives the fluorescence and detects the fluorescence, thereby detecting fluorescence of intensity or wavelength that cannot be visually confirmed. Since the fluorescence intensity can be measured, the target substance can be quantified, and highly sensitive detection and quantification are possible. As a characteristic in the light receiving sensitivity of the light receiver, it is preferable to apply a light receiving region (light receiving peak) having a high sensitivity at a wavelength of 400 to 700 nm from the viewpoint of availability of a highly sensitive product, and to a wavelength of 500 to 600 nm. It is more preferable to apply one having a highly sensitive light receiving region (light receiving peak).

<Flow cytometry>
As an example, flow cytometry is performed using an apparatus having a detection unit as shown in FIG. As is apparent from the figure, in this apparatus, each particle passes through the nozzle, and the fluorescence emission of the particle can be detected at this portion. Specifically, light of a plurality of wavelengths can be sequentially applied at the exit of the detection unit, and fluorescence emission therefrom can be detected. Examples of the flow cytometry apparatus include two light sources EL-1 and EL-2. In this embodiment, excitation light of 409 nm and 440 nm is applied. Some detectors have two to four channels, and fluorescence at 530 nm, 585 nm, 661 nm, 670 nm, etc. is often detected.

  The flow cytometer is not particularly limited, and a conventionally used flow cytometer can be used as it is. Prior to irradiation with excitation light, a sheath liquid (for example, IsoFlow sheath liquid (trade name, manufactured by Beckman Coulter, Inc.), Dako sheath fluid (trade name, manufactured by Dako Japan, etc.)), target biomolecule, and fluorescently labeled particles It is preferable that a sample solution containing etc. is introduced into the flow cell. Here, it is preferable that a laminar flow is formed in which the sheath liquid and the sample liquid do not mix. As the sample liquid flows from the flow cell, the sample liquid flows while forming a sample core surrounded by a sheath liquid flow, and a laser is irradiated when passing through the irradiation region.

  By setting the flow cytometer to a predetermined flow condition, only one fluorescently labeled particle labeled with a target biomolecule can be present in each detection zone. Thereby, detection data of only one fluorescent labeling particle can be acquired. The flow cytometry can simultaneously measure a plurality of targets in one measurement operation. This is a so-called multiplexing method. The flow rate at the time of measurement is not particularly limited, but for quantification of the target biomolecule, it can be set to 100 to 1000 fluorescently labeled particles / second, and for detection or sorting of target cells. , 100-1000 cells / second. For example, when FACSCalibur (trade name; manufactured by Becton Dickinson) is used as the flow cytometer, the difference between the sheath pressure (pressure for flowing the sheath liquid) and the sample pressure (pressure for flowing the sample liquid) is 1 to 2 kpsi. There can be certain conditions.

  In each detection region, fluorescence emission from fluorescently labeled particles irradiated by a laser through the detection region using any one or more fluorescent channels (eg, FL1, FL2, FL3) such as a photomultiplier tube Can be collected. Moreover, you may collect the light (forward scattered light or side scattered light) refracted by each silica particle using arbitrary scattered light condensing channels, such as a photomultiplier tube. The flow cytometer is a data collection / analysis means (hardware or software) that records, stores and analyzes data detected by fluorescent channels or the like from fluorescence emission or scattered light derived from each target biomolecule that has passed through the detection area. ). Commercially available products such as FACSDiVa, EXPO32, Summit, AppSan, LysisII, and CellQuest (all of which are trade names) may be used as the data collection and analysis means.

  As flow cytometers, FACSCalibur (trade name; manufactured by Becton Dickinson), PERFLOW Ana (trade name; manufactured by Furukawa Electric Co., Ltd.), EPICS ALTRA (trade name; manufactured by Beckman Coulter, Inc.), CyAn (trade name; Dako) Commercially available flow cytometers such as JSAN (trade name; manufactured by Bay Bioscience) may be used. Regarding the use of fluorescently labeled particles for flow cytometry, reference can be made to, for example, WO2007 / 097377 pamphlet and JP2009-222718A.

<Display device>
The ultraviolet-excited fluorescent particles of the present invention can be applied to various applications such as displays by mixing and dispersing in a transparent medium such as a resin. For example, when a screen in the form of a plate is formed and ultraviolet light is irradiated to a predetermined place, only the irradiated portion emits fluorescence, so that it can be used as an image display. Here, the base material of the fluorescent particles is silica, and usually has a refractive index difference from the resin of the transparent medium, and thus shows a Tyndall phenomenon in which light is dispersed in the optical path. Therefore, it can be seen from other than the optical path direction, which is preferable. Moreover, it is more preferable that the average particle diameter of the ultraviolet excitation fluorescent particles (display element particles) is 100 nm or more because a Tyndall phenomenon is likely to occur.

  FIG. 4 is an example of a display device according to a preferred embodiment using the ultraviolet excited fluorescent particles of the present invention. The display device 40 according to the present embodiment includes a display 41 and a light source 42. The display 41 is formed of a fluorescent display plate in which ultraviolet excitation fluorescent particles (display element particles) are dispersed in a plate-shaped transparent medium. By irradiating the display 41 with ultraviolet rays 44 from a light source, a predetermined image 43 can be displayed there as a fluorescence emission image. In this example, a character (display image) 43 “10” is displayed.

  The material of the transparent medium is not particularly limited, but a normal transparent resin can be used. For example, polyolefin resin (polyethylene resin, polypropylene resin), polyester resin, polyacrylic resin (polyacrylate resin, polymethacrylate resin), nylon resin And cellulose resin.

In the present invention, from the viewpoint of improving the display characteristics as described above, it is preferable that the refractive index of the material of the transparent medium including the silica material constituting the ultraviolet excitation fluorescent particles is high. As will be described later, the refractive index of silica is about 1.40 to 1.46, but the refractive index of the transparent medium is preferably 1.45 or more, and more preferably 1.5 or more. The upper limit is practically 1.8 or less. The refractive index here is a value at a wavelength of 589 nm and 23 ° C.
The difference in refractive index between the ultraviolet excited fluorescent particles (silica particles) and the transparent medium is preferably 0.05 or more, and more preferably 0.1 or more. The upper limit is practically 0.4 or less. The refractive index here is a value at a wavelength of 589 nm and 23 ° C. The large difference is preferable because the fluorescence visibility of the fluorescent particles from other than the light incident position is increased.
The thickness of the transparent medium is not particularly limited, but is preferably about 0.02 to 1 mm. As for the density | concentration of the ultraviolet excitation fluorescent particle in a transparent medium, about 0.001-1 mass% is preferable.
Typical examples of the refractive index of each transparent material are listed below.
[Table A]
――――――――――――――――――――――――――――
Material Refractive index ――――――――――――――――――――――――――――
Polyvinylidene fluoride 1.42
Cellulose acetate 1.46-1.50
Polymethyl methacrylate 1.49
Polypropylene 1.49
Nylon 1.53
Polyethylene 1.53
Polyvinyl chloride 1.53
Polycarbonate 1.59
Polystyrene 1.6
Polyvinylidene chloride 1.61
Polyethylene terephthalate 1.66
Polymethylmethacrylate 1.67-1.76
――――――――――――――――――――――――――――

Examples of image display displays and display devices using them include head-up displays with a screen placed inside the windshield of an automobile or aircraft, wearable devices with a small screen on glasses, etc., screens placed on window glass, etc. Display devices for advertisements and the like.
As the light source, a method of scanning with an oscillating mirror using an ultraviolet laser, a method of combining a lens and a liquid crystal shutter using a light source such as an ultraviolet LED and an ultraviolet lamp, and the like are possible.
In the present embodiment, the relationship between the wavelength of ultraviolet light (excitation light) irradiated from the light source and the wavelength of fluorescence emitted from the fluorescent particles and the difference between them is the same as that described above for the labeled particles.
As a conventional technique of the projection type overhead display, for example, JP-A-2005-82103 can be cited. In this method, an image generated by a liquid crystal projector is projected onto a light-scattering screen, and further reflected by a windshield using an optical system for visual recognition. A liquid crystal projector and screen are required, and the optical system becomes complicated. On the other hand, the present invention directly generates a visible image on a screen (provided on a part of the windshield), and the optical system becomes simple and can be viewed from a wide angle.

<Other uses>
The fluorescent particles of the present invention can be applied to other various immunoassays, biochips, cell detection / sorting systems, or cell detection / sorting methods.

[Target substance (specimen)]
In the present invention, examples of target substances to be detected and quantified include antigens, antibodies, DNA, RNA, sugars, sugar chains, ligands, receptors, peptides, chemical substances, and the like. In the present invention, the sample containing the target substance is not particularly limited, and examples thereof include liquid samples such as urine and blood.

[Ultraviolet excited fluorescent particles]
The ultraviolet excitation fluorescent particles used in the present invention are composed of silica particles and an ultraviolet excitation fluorescent dye. There is no particular limitation on the method for preparing the ultraviolet-excited fluorescent particles, and particles obtained by any arbitrary preparation method may be used. Examples thereof include the sol-gel method described in Journal of Colloid and Interface Science, 159, 150-157 (1993). In the present invention, it is particularly preferable to use particles obtained according to the method for preparing fluorescent silica particles described in International Publication No. 2007 / 074722A1.

Specifically, the ultraviolet-excited fluorescent particles may be one kind or a product obtained by reacting the ultraviolet-excited fluorescent dye with a silane coupling agent and covalently bonding, ionic bonding, or other chemical bonds or adsorptions. It can be prepared by condensation polymerization of two or more silane compounds to form a siloxane bond. As a result, ultraviolet-excited fluorescent particles in which an organosiloxane component and a siloxane component are bonded by siloxane are obtained.
Preferred embodiments of the method for preparing the ultraviolet-excited fluorescent particles include a temporary bond (N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxy group). Silane having a UV-excited fluorescent dye (precursor) having a methyl group, an epoxy group, a cyano group or the like) and a substituent (for example, amino group, hydroxyl group, thiol group) that reacts with the active group. An ultraviolet-excited fluorescent dye having a bond portion obtained by reacting with a coupling agent. Ultraviolet-excited fluorescent particles can be prepared by condensation-polymerizing one or two or more silane compounds with the ultraviolet-excited fluorescent dye, which is the product, to form a siloxane bond.

  Examples of the case where γ-aminopropyltriethoxysilane (APS), a UV-excited fluorescent dye using a silane compound and tetraethoxysilane (TEOS) are used as the silane coupling agent are shown below.

  Specific examples of the silane coupling agent having a substituent (a compound that converts a temporary bond part of an ultraviolet excitation dye into a bond part) include γ-aminopropyltriethoxysilane (APS), 3- [2- (2-amino Examples include silane coupling agents having an amino group such as ethylamino) ethylamino] propyl-triethoxysilane, N-2 (aminoethyl) 3-aminopropylmethyldimethoxysilane, and 3-aminopropyltrimethoxysilane. Of these, APS is preferable.

  The silane compound to be polycondensed is not particularly limited, but TEOS, γ-mercaptopropyltrimethoxysilane (MPS), γ-mercaptopropyltriethoxysilane, γ-aminopropyltriethoxysilane (APS), 3-thio Mention may be made of cyanatopropyltriethoxysilane, 3-glycidyloxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane. it can. Among these, TEOS is preferable from the viewpoint of forming the siloxane component inside the silica particles, and MPS or APS is preferable from the viewpoint of forming the organosiloxane component inside the silica particles.

When prepared as described above, spherical or nearly spherical silica particles can be produced. The nearly spherical silica particles specifically have a shape in which the ratio of the major axis to the minor axis is 2 or less.
In order to obtain silica particles having a desired average particle size, ultrafiltration is performed using an ultrafiltration membrane such as YM-10, YM-100 (both trade names, manufactured by Millipore), and the particle size is large. It is possible to remove particles that are too small or too small, or to centrifuge at an appropriate gravitational acceleration and collect only the supernatant or precipitate.

  In the embodiment used for detecting the labeling substance, the labeling particle can be obtained by introducing a binding substance into the fluorescent labeling particle. At this time, the binding substance that is adsorbed or bonded to the surface of the ultraviolet-excited fluorescent particles has binding properties with the target substance. By utilizing this property, if the target substance is present in the sample solution, it can be bound to the sample liquid and labeled in the system. The binding substance is preferably a biomolecule. Biomolecules include antigens, antibodies, DNA, RNA, sugars, sugar chains, ligands, receptors, proteins or peptides. Here, a ligand refers to a substance that specifically binds to a protein, such as a substrate that binds to an enzyme, a coenzyme, a regulatory factor, a hormone, or a neurotransmitter, as well as a low molecular weight molecule or ion. Also includes high molecular weight materials.

<Ultraviolet transmittance>
In the ultraviolet-excited fluorescent particles of the present invention, the advantage of using silica as the particle material is exhibited in the high transmittance of ultraviolet rays. Below, the transmittance | permeability (average) value of the ultraviolet-ray (300 nm-400 nm) of the polystyrene and silica which comprise latex particle is compared.
Polystyrene 50-80% (25 ° C. 300-400 nm)
Silica 90% (25 ° C 300-400nm)
Thus, it is understood that silica has a high transmittance of ultraviolet rays, and therefore, the good performance of ultraviolet excitation fluorescent dyes using ultraviolet rays as excitation light is effectively exhibited.

<Density>
Latex particles have a density of 1 g / cm ³ and are low in density. Therefore, if the particle size is reduced, separation by centrifugation becomes impossible, and in particular, the particles may not be used as label particles or display element particles. On the other hand, since silica has a relatively high density (for example, about 2 g / cm ³ ), it tends to settle even if the particle size is small. When the density is increased, separation by centrifugation is facilitated, and labor during preparation is reduced, so that silica particles can be suitably used as labeled particles or display element particles.

<Refractive index>
In addition, the low refractive index as described below also has an advantage of using silica particles without causing light scattering due to the influence of surrounding water in biological detection applications. Alternatively, it is preferable for the display device to easily cause a Tyndall phenomenon.
[Refractive index] 589nm 23 ℃
Polystyrene beads 1.59
Silica particles 1.40-1.46
Water 1.33
(National Astronomical Observatory of Science Chronology)
Reflected light intensity R = (n ₀ −n _i ) ²
n ₀ Water refractive index n _i Particle refractive index R (polystyrene beads) = 7.93 × 10 ⁻³
R (silica particle) = 6.57 × 10 ^{−4 to} 2.17 × 10 ⁻³

<Average particle size>
-Immunochromatography The average particle diameter (primary particles) of the ultraviolet-excited fluorescent particles is preferably 1 nm or more, more preferably 20 nm or more, and particularly preferably 200 nm or more for use in immunochromatography. The upper limit is preferably 1 μm or less, more preferably 700 nm or less, further preferably 500 nm or less, and particularly preferably 400 nm or less.
-Flow cytometry The fluorescent labeling particle | grains which have the ultraviolet excitation fluorescent particle of this invention can be used conveniently also as such a reagent of flow cytometry. In this case, the average particle size (primary particles) of the fluorescently labeled particles is preferably 300 nm or more, more preferably 500 nm or more, and particularly preferably 800 nm or more from the viewpoint of the detection ability (sensitivity) of the flow cytometer. Although there is no upper limit in particular, 1200 nm or less is practical.
-Biochip When using for a biochip, it is preferable that the average particle diameter (primary particle) of an ultraviolet excitation fluorescent particle is 3 nm or more. The upper limit is preferably 30 nm or less, more preferably 20 nm or less, and particularly preferably 10 nm or less. For the use of fluorescent particles for immunoassays and biochips, for example, WO2007 / 074722 can be referred to. When applied to cell detection, the average particle diameter of the fluorescent particles is preferably 10 nm or more from the viewpoint of improving the sensitivity. The upper limit is preferably 200 nm or less, more preferably 100 nm or less, and particularly preferably 50 nm or less. Regarding the use of fluorescent particles for cell detection and the like, for example, WO2007 / 097377 and WO2008 / 018586 can be referred to.
-Display device From the viewpoint of causing light scattering, the average particle size of the fluorescent particles is preferably 50 nm or more, more preferably 100 nm or more, and 150 nm in any of the primary particle size and the secondary particle size from the viewpoint of causing light scattering. The above is particularly preferable. In addition, the upper limit is preferably 20000 nm or less, more preferably 10,000 nm or less, and particularly preferably 5000 nm or less in both the primary particle diameter and the secondary particle diameter from the viewpoint of display resolution.

In the present invention, unless otherwise specified, the average particle diameter is based on the total projected area of 100 fluorescent particles randomly selected from an image of a transmission electron microscope (TEM), a scanning electron microscope (SEM), or the like. The area occupied by the fluorescent particles is obtained by an image processing apparatus, and the average value of the diameters of the circles (average circle equivalent diameter) corresponding to the value obtained by dividing the total occupied area by the number of selected fluorescent particles (100) is obtained. It is a thing. The average particle size obtained with this electron microscope is usually measured as the average particle size of particles composed of primary particles.
On the other hand, the average particle diameter measured by the dynamic light scattering method includes not only primary particles but also secondary particles formed by aggregation of primary particles. An example of the particle size measuring device by the dynamic light scattering method is Zetasizer Nano (trade name; manufactured by Malvern). This method measures the time fluctuation of the light scattering intensity by the light scatterer such as fine particles, calculates the Brownian motion velocity of the light scatterer from the autocorrelation function, and derives the particle size distribution of the light scatterer from the result. Is.

  The ultraviolet-excited fluorescent particles are preferably monodispersed as a particulate substance, and the variation coefficient of particle size distribution, so-called CV value, is not particularly limited, but is preferably 10% or less, more preferably 8% or less.

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not construed as being limited thereto. In addition, when it is set as a part and% in the display of a compounding quantity and a density | concentration, it shall be based on a mass unless otherwise indicated.
(Comparative example)
5.8 mg of 5- (and -6) -carboxytetramethylrhodamine succinimidyl ester (trade name, manufactured by emp Biotech GmbH) was dissolved in 1 ml of dimethylformamide (DMF). 2.6 μl of APS was added thereto and reacted at room temperature (23 ° C.) for 1 hour. To 400 μl of the obtained reaction solution, 128 ml of ethanol, 400 μl of TEOS (tetraethoxysilane), 28.8 ml of distilled water, and 400 μl of 28% by mass ammonia water were added and reacted at room temperature for 24 hours. The reaction solution was centrifuged for 30 minutes at a gravitational acceleration of 18000 × g, and the supernatant was removed. 4 ml of distilled water was added to the precipitated silica particles to disperse the particles, and the mixture was again centrifuged for 30 minutes at a gravitational acceleration of 18000 × g. This washing operation was further repeated twice to remove unreacted TEOS, ammonia and the like contained in the labeled silica nanoparticle dispersion, thereby obtaining 94.2 mg of silica nanoparticles having an average particle diameter of 172 nm. Yield about 88%.

  To 500 μl of the colloid of the composite particles of silica particles / alginic acid containing 20 mg / ml rhodamine containing the concentration, 100 μl of 0.5 M MES (2-Morpholine ethanesulfide acid) buffer (pH 6.0), 461 μl of 50 mg / ml NHS, 19 150 μl of 2 mg / ml EDC and 1.089 ml of distilled water were added and mixed for 30 minutes. The reaction solution was centrifuged at a gravitational acceleration of 15,000 × G for 30 minutes, and after removing the supernatant, 1 ml of distilled water was added to disperse the particles. Similarly, centrifugation was repeated twice and dispersion in distilled water was repeated to wash the particles. Finally, the particles were dispersed in 1 ml of 50 mM KH 2 PO 4 buffer (pH 8.0). To this, 1 ml of 1 mg / ml anti-hCG antibody (Anti-hCG mouse IgG1, manufactured by Medix Biochemical) dissolved in 50 mM KH 2 PO 4 buffer (pH 8.0) was added and mixed for 2 hours. Subsequently, 100 μl of a 100 mg / ml BSA solution was added and further mixed for 1 hour. The reaction solution was centrifuged at a gravitational acceleration of 15,000 × G for 30 minutes, and after removing the supernatant, 1 ml of 50 mM KH 2 PO 4 buffer (pH 8.0) was added to disperse the particles. Similarly, the particles are washed twice by repeating centrifugation and dispersion in 50 mM KH 2 PO 4 buffer (pH 8.0), and finally dispersed in 1 ml of 50 mM KH 2 PO 4 buffer (pH 8.0). Thus, a colloid of rhodamine-containing silica particles (fluorescently labeled particles for comparison) modified with an anti-hCG antibody was obtained (yield 10 mg / ml × 1 ml).

(Fluorescence measurement of labeled particles)
Recombinant hCG (manufactured by Rohto Pharmaceutical Co., Ltd.) (100 μL) was mixed with fluorescently labeled particles (10 mg / ml), dropped onto the sample pad portion of the test strip, and left for 5 minutes. The test strip has the form shown in the figure, and was produced based on Japanese Patent No. 5100541.

  The filter on the excitation light source side was not used. The microchannel chip or test strip was irradiated with a UV LED (18 mW), and fluorescence was visually observed. The wavelength of the excitation light was 375 nm.

(Example)
A labeling agent was prepared in the same manner except that emp Biotech used in the above Comparative Example was changed to, for example, Fluoride Yellow UV-excited fluorescent dye manufactured by IST. The detection of hCG was obtained as in the comparative example. As a result, excellent effects according to the present invention were confirmed as follows. In the comparative example, a 532 nm LED was used as the excitation light source.

＋ ： 目視確認が可能
＋＋： より明瞭に目視確認が可能
± ： 目視確認が困難

+: Visual check is possible
++: Visual confirmation is possible more clearly
±: Visual confirmation is difficult

  As described above, according to the present invention, even a very small amount of specimen (test substance) can be detected with high sensitivity without being affected by excitation light.

DESCRIPTION OF SYMBOLS 1 Target substance 2 Fluorescent label particle | grains 2a Ultraviolet excitation fluorescent particle 2b Binding substance 4 Capturing substance for test 5 Capturing substance for reference 6 Case 61 Detection opening part 62 Sample introduction opening part 6a Case upper part 6b Case lower part 8a Sample Pad 8b Conjugate pad 8c Antibody-immobilized membrane 8d Absorption pad 10 Test strip 100 Long specimen _nt Test line n _r Control line L Lateral flow direction S Test solution 40 Display device 41 Display 42 Light source 43 Display image 44 Ultraviolet light

（上記環Ａは芳香族環または複素芳香族環である。Ｘはイミノ基（ＮＲ^Ｎ）、酸素原子、硫黄原子、セレン原子、またはＢＲ^Ｎを表す。Ｒ^Ｃは水素原子または置換基を表す。Ｒ^Ｎは水素原子または置換基を表す。Ｒ^Ｍは脂肪族炭化水素基又は芳香族炭化水素基を表す。Ａｎ ⁻ は対アニオンを表す。）
〔４〕上記式（１）が下記式（１−１）〜（１−３）のいずれかで表され、上記式（２）が下記式（２−１）〜（２−３）のいずれかで表され、上記式（３）が下記式（３−１）〜（３−３）のいずれかで表される〔３〕に記載の紫外励起蛍光粒子。

（式中、Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４は、それぞれ独立に、水素原子または置換基を示す。Ｘは式（１）と同義である。Ａｎ⁻は対アニオンを示す。Ｒ^Ｃ、Ｒ^Ｎ、Ｒ^Ｍは式（２）、式（３）と同義である。）
〔５〕上記紫外励起蛍光色素が、波長３００ｎｍ以上４７５ｎｍ未満の紫外線を吸収し、波長４００ｎｍ超７００ｎｍ以下かつ上記吸収波長よりも長い波長の蛍光を発光する〔１〕〜〔４〕のいずれか１項に記載の紫外励起蛍光粒子。
〔６〕紫外励起蛍光色素を粒子内部に含有するシリカ粒子を標識粒子として用いる検出方法であって、
上記紫外励起蛍光色素が、発色団が含窒素複素芳香族化合物である有機色素の残基であり、かつ、該紫外励起蛍光色素に紫外線を照射し、これにより該紫外励起蛍光色素から発せられる可視光領域側の蛍光を検出して行う検出方法。
〔７〕上記紫外励起蛍光色素が、上記シリカ粒子の内部で、該シリカ粒子と化学結合を介して結合している〔６〕に記載の検出方法。
〔８〕上記照射する紫外線が波長３００ｎｍ以上４７５ｎｍ未満であり、上記紫外励起蛍光色素から発せられる蛍光が波長４００ｎｍ超７００ｎｍ以下かつ上記照射波長よりも長い波長である〔６〕または〔７〕に記載の検出方法。
〔９〕〔１〕〜〔５〕のいずれか１項に記載の紫外励起蛍光粒子と、上記紫外励起蛍光粒子が移行するメンブレンとを有するイムノクロマトグラフィー用の検出キット。
〔１０〕上記メンブレンは、標的物質を捕捉した上記紫外励起蛍光粒子が固定化される検出領域を有する〔９〕に記載の検出キット。
〔１１〕上記メンブレンは、標的物質を捕捉していない上記紫外励起蛍光粒子が固定化される参照領域を有する〔９〕または〔１０〕に記載の検出キット。
〔１２〕〔１〕〜〔５〕のいずれか１項に記載の紫外励起蛍光粒子を用いる画像表示方法であって、
上記紫外励起蛍光粒子を含む透明媒体に紫外線を照射し、照射された部分に存在する紫外励起蛍光粒子に含まれる紫外励起蛍光色素から発せられる蛍光とその散乱光により、上記透明媒体に画像を表示させる画像表示方法。
〔１３〕紫外線の照射が、レーザ、ＬＥＤ、およびランプのいずれかひとつにより行われる〔１２〕に記載の画像表示方法。
〔１４〕〔１〕〜〔５〕のいずれか１項に記載の紫外励起蛍光粒子を用いる画像表示装置であって、
上記紫外励起蛍光粒子を含む透明媒体を具備する画像表示スクリーンと、画像表示スクリーンの所定の部分に紫外線を照射する紫外線光源とを備え、照射された部分に存在する蛍光粒子に含まれる蛍光色素から発せられる蛍光とその散乱光により、画像表示スクリーン上に画像を表示する画像表示装置。
〔１５〕シリカ粒子と透明媒体の屈折率差が０．１以上である〔１４〕に記載の画像表示装置。
〔１６〕ヘッドアップディスプレイまたはウエアラブルディスプレイである〔１４〕または〔１５〕に記載の画像表示装置。
〔１７〕〔１〕〜〔５〕のいずれか１項に記載の紫外励起蛍光粒子を含む透明媒体を具備する画像表示スクリーンであって、
紫外線光源から照射された部分に存在する蛍光粒子に含まれる蛍光色素から発せられる蛍光とその散乱光により、画像表示スクリーン上に画像を表示することができる画像表示スクリーン。
The above problems have been solved by the following means.
[1] Ultraviolet- excited fluorescent particles containing an ultraviolet- excited fluorescent dye that absorbs ultraviolet rays and emits fluorescence in the visible light region side, inside the silica particles ,
Ultraviolet-excited fluorescent particles, wherein the ultraviolet-excited fluorescent dye is a residue of an organic dye whose chromophore is a nitrogen-containing heteroaromatic compound, and functions as a light-emitting element of the organic dye in the ultraviolet-excited fluorescent particle.
[2] the ultraviolet excitation fluorescent dye, the inside of the silica particles, ultraviolet excitation fluorescent particles according to you are bonded via chemical bonds with the silica particles (1).
[3] The ultraviolet excitation fluorescent particle according to [1] or [2], wherein the chromophore of the ultraviolet excitation fluorescent dye is a residue of a compound represented by any one of the following formulas (1) to (3).

(The ring A is an aromatic ring or a heteroaromatic ring. X represents an imino group (NR ^N ), an oxygen atom, a sulfur atom, a selenium atom, or BR ^N. R ^C represents a hydrogen atom or a substituent. .R ^N is .R ^M represents a hydrogen atom or a substituent an represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group ^- represents a counter anion)..
[4] the above formula (1) is represented by any one of the following formulas (1-1) to (1-3), any of the above formula (2) is the following formula (2-1) to (2-3) represented by either the formula (3) is an ultraviolet excitation fluorescent particles according to the represented by (3) in any one of the following formulas (3-1) to (3-3).

(In the formula, R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom or a substituent. X has the same meaning as in formula (1). An ⁻ represents a counter anion. R ^C , R ^N and R ^M have the same ^{meanings as} in Formula (2) and Formula (3).)
[5] Any one of [1] to [4], wherein the ultraviolet excitation fluorescent dye absorbs ultraviolet light having a wavelength of 300 nm or more and less than 475 nm, and emits fluorescence having a wavelength of more than 400 nm to 700 nm or less and longer than the absorption wavelength. The ultraviolet excitation fluorescent particle according to item.
[6] A detection method using silica particles containing ultraviolet-excited fluorescent dye inside the particles as labeled particles ,
The ultraviolet excitation fluorescent dye, a visible chromophore is a residue of an organic dye is a nitrogen-containing heteroaromatic compound, and ultraviolet rays are irradiated to the ultraviolet excitation fluorescent dye, thereby emitted from the ultraviolet excitation fluorescent dye A detection method performed by detecting fluorescence in the light region side .
[7] the ultraviolet excitation fluorescent dye, in the interior of the silica particles, the detection method described in that linked via a chemical bond with the silica particles [6].
[8] ultraviolet rays the radiation is less than or more wavelength 300 nm 475 nm, according to a wavelength longer than or less and the irradiation wavelength fluorescence wavelength 400nm ultra 700nm emitted from the ultraviolet excitation fluorescent dye [6] or [7] Detection method.
[9] A detection kit for immunochromatography comprising the ultraviolet excited fluorescent particles according to any one of [1] to [5] and a membrane to which the ultraviolet excited fluorescent particles migrate.
[10] The detection kit according to [9], wherein the membrane has a detection region in which the ultraviolet-excited fluorescent particles capturing the target substance are immobilized.
[11] The detection kit according to [9] or [10], wherein the membrane has a reference region in which the ultraviolet-excited fluorescent particles that have not captured the target substance are immobilized.
[12] An image display method using the ultraviolet-excited fluorescent particles according to any one of [1] to [5] ,
Irradiating ultraviolet radiation on a transparent medium including over SL ultraviolet excitation fluorescent particles, the fluorescence and the scattered light emitted from the ultraviolet excitation fluorescent dye contained in the ultraviolet excitation fluorescent particles present in irradiated portion, an image on the transparent medium The image display method to be displayed.
[13] The image display method according to [12], wherein the ultraviolet irradiation is performed by any one of a laser, an LED, and a lamp.
[14] An image display device using the ultraviolet-excited fluorescent particles according to any one of [1] to [5] ,
Comprising an image display screen comprising a transparent medium including on SL ultraviolet excitation fluorescent particles, an ultraviolet light source for irradiating ultraviolet rays to a predetermined portion of the image display screen, a fluorescent dye contained in the fluorescent particles present in irradiated portion An image display device that displays an image on an image display screen by fluorescence emitted from the light and its scattered light.
[15] The image display device according to [14], wherein the refractive index difference between the silica particles and the transparent medium is 0.1 or more.
[16] The image display device according to [14] or [15], which is a head-up display or a wearable display.
[17] An image display screen comprising a transparent medium containing the ultraviolet-excited fluorescent particles according to any one of [1] to [5] ,
By fluorescence and scattered light emitted from the fluorescent dye contained in the fluorescent particles present in the irradiated portion from the ultraviolet light source, an image display screen capable of displaying an image on the image display on the screen.

(Ultraviolet excitation fluorescent dye)
Although ultraviolet excitation fluorescent dye is not particularly limited as long as it emits fluorescence in a visible light region side by absorbing ultraviolet light, in the present invention, Ru residues der of the chromophore nitrogen-containing heteroaromatic compound. The nitrogen-containing heteroaromatic compound is preferably a five-membered ring compound or a six-membered ring compound, and more preferably a five-membered ring compound. The nitrogen-containing heteroaromatic compound is preferably an azole compound, and more preferably a diazole compound. Of these, an oxadiazole compound, a benzooxadiazole compound, a pyridooxadiazole compound, an imidazole compound, a benzimidazole compound, and a pyridoimidazole compound are preferable.

To 500 μl of the colloid of the composite particles of silica particles / alginic acid containing 20 mg / ml rhodamine containing the concentration, 100 μl of 0.5 M MES (2-Morpholine ethanesulfide acid) buffer (pH 6.0), 461 μl of 50 mg / ml NHS, 19 150 μl of 2 mg / ml EDC and 1.089 ml of distilled water were added and mixed for 30 minutes. The reaction solution was centrifuged at a gravitational acceleration of 15,000 × G for 30 minutes, and after removing the supernatant, 1 ml of distilled water was added to disperse the particles. Similarly, centrifugation was repeated twice and dispersion in distilled water was repeated to wash the particles. Finally, the particles were dispersed in 1 ml of 50 mM K H ₂ P O ₄ buffer (pH 8.0). To this, 1 ml of 1 mg / ml anti-hCG antibody (Anti-hCG mouse IgG1, manufactured by Medix Biochemica) dissolved in 50 mM K H ₂ PO ₄ buffer (pH 8.0) was added and mixed for 2 hours. Subsequently, 100 μl of a 100 mg / ml BSA solution was added and further mixed for 1 hour. The reaction solution was centrifuged at a gravitational acceleration of 15,000 × G for 30 minutes, and after removing the supernatant, 1 ml of 50 mM K H ₂ P O ₄ buffer (pH 8.0) was added to disperse the particles. In the same manner, the particles were washed twice by repeating centrifugation and dispersion in 50 mM K H ₂ P O ₄ buffer (pH 8.0), and finally, 50 mM K H ₂ P O ₄ buffer (pH 8). 0.0) A colloid of rhodamine-containing silica particles (fluorescently labeled particles for comparison) that was dispersed in 1 ml and modified with anti-hCG antibody was obtained (yield 10 mg / ml × 1 ml).

Claims (17)

  Ultraviolet-excited fluorescent particles containing in the silica particles an ultraviolet-excited fluorescent dye that absorbs ultraviolet rays and emits fluorescence in the visible light region side.   The ultraviolet excitation fluorescent particle according to claim 1, wherein the chromophore of the ultraviolet excitation fluorescent dye is a residue of a nitrogen-containing heteroaromatic compound. The ultraviolet excitation fluorescent particle according to claim 1 or 2, wherein the chromophore of the ultraviolet excitation fluorescent dye is a residue of a compound represented by any one of the following formulas (1) to (3).

(The ring A is an aromatic ring or a heteroaromatic ring. X represents an imino group (NR ^N ), an oxygen atom, a sulfur atom, a selenium atom, or BR ^N. R ^C represents a hydrogen atom or a substituent. .R ^N is .R ^M represents a hydrogen atom or a substituent is an aliphatic hydrocarbon group or an aromatic hydrocarbon group.) The formula (1) is represented by any one of formulas (1-1) to (1-3), the formula (2) is represented by any one of formulas (2-1) to (2-3), The ultraviolet excitation fluorescent particle according to claim 3, wherein the formula (3) is represented by any one of formulas (3-1) to (3-3).

(In the formula, R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom or a substituent. X has the same meaning as in formula (1). An ⁻ represents a counter anion. R ^C , R ^N and R ^M have the same ^{meanings as} in Formula (2) and Formula (3).)   The ultraviolet ray according to any one of claims 1 to 4, wherein the ultraviolet excitation fluorescent dye absorbs ultraviolet rays having a wavelength of 300 nm or more and less than 475 nm, and emits fluorescence having a wavelength of more than 400 nm and less than 700 nm and longer than the absorption wavelength. Excited fluorescent particles.   A detection method using silica particles containing an ultraviolet-excited fluorescent dye as a labeling particle, wherein the ultraviolet-excited fluorescent dye is irradiated with ultraviolet rays, thereby detecting fluorescence emitted from the ultraviolet-excited fluorescent dye.   The detection method according to claim 6, wherein the chromophore of the ultraviolet excitation fluorescent dye is a residue of a nitrogen-containing heteroaromatic compound.   The detection method according to claim 6 or 7, wherein the ultraviolet ray to be irradiated has a wavelength of 300 nm or more and less than 475 nm, and the fluorescence emitted from the ultraviolet excitation fluorescent dye has a wavelength longer than 400 nm and shorter than 700 nm and longer than the irradiation wavelength.   The detection kit for immunochromatography which has the ultraviolet excitation fluorescent particle of any one of Claims 1-5, and the membrane which the said ultraviolet excitation fluorescent particle transfers.   The detection kit according to claim 9, wherein the membrane has a detection region in which the ultraviolet excitation fluorescent particles capturing the target substance are immobilized.   11. The detection kit according to claim 9, wherein the membrane has a reference region in which the ultraviolet excitation fluorescent particles not capturing a target substance are immobilized.   It is an image display method using the ultraviolet excitation fluorescent particle of any one of Claims 1-5, Comprising: The ultraviolet medium which irradiates an ultraviolet-ray to the transparent medium containing the said ultraviolet excitation fluorescent particle, and exists in the irradiated part An image display method for displaying an image on the transparent medium by fluorescence emitted from an ultraviolet excitation fluorescent dye contained in the fluorescent particles and scattered light thereof.   The image display method according to claim 12, wherein the ultraviolet irradiation is performed by any one of a laser, an LED, and a lamp.   An image display device using the ultraviolet-excited fluorescent particles according to any one of claims 1 to 5, wherein the image display screen includes a transparent medium containing the ultraviolet-excited fluorescent particles, and a predetermined portion of the image display screen. And an ultraviolet light source that irradiates ultraviolet rays, and an image display device that displays an image on an image display screen by fluorescence emitted from a fluorescent dye contained in fluorescent particles present in the irradiated portion and its scattered light.   The image display device according to claim 14, wherein a difference in refractive index between the silica particles and the transparent medium is 0.1 or more.   The image display device according to claim 14, wherein the image display device is a head-up display or a wearable display.   An image display screen comprising a transparent medium comprising the ultraviolet excitation fluorescent particles according to any one of claims 1 to 5, wherein the screen is emitted from a fluorescent dye contained in fluorescent particles present in a portion irradiated from an ultraviolet light source. An image display screen capable of displaying an image on the image display screen by the fluorescent light and its scattered light.

Images