JP2017181050A - Test kit for biomolecule detection, biomolecule detection method using the same, and labeling reagent particle for biomolecule detection used therein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a test kit for biomolecule detection which can be applied to a light emission detector not provided with a filter that allows only a wavelength region overlapping an excitation wavelength to pass through.SOLUTION: Provided is a test kit for biomolecule detection comprising a test piece for biomolecule detection and labeling reagent particles for biomolecule detection consisting of phosphorescent pigment-containing silica nano-particles. The test piece includes a membrane in which is provided a test area for capturing biomolecules and through which the labeling reagent particles migrate, the labeling reagent particles having captured biomolecules that are the target being fixed to the test area, and a capture substance having affinity to biomolecules is introduced into the surfaces of the phosphorescent pigment-containing silica nano-particles.SELECTED DRAWING: None

Description

  The present invention relates to a biomolecule detection test kit, a biomolecule detection method using the same, and a labeling reagent particle for biomolecule detection used therein.

As a technique for detecting a biomolecule such as an antigen in a living body, there is a detection method using a porous test piece in which a capturing substance for capturing a biomolecule is immobilized on a carrier and a labeling reagent for capturing the biomolecule. In this method, the biomolecule contained in the sample liquid is captured by the labeled particles, and the porous support is moved by capillary action. Then, for example, the biomolecule is concentrated by bringing the capture substance fixed in a line shape and the biomolecule into contact with the porous support, and the line on which the capture substance is fixed is colored with the labeling reagent. The presence or absence of a biomolecule can be determined by this color development.
The following three points can be cited as features of such a biomolecule detection method.
(1) The time required for determination is short and a quick inspection is possible.
(2) The measurement can be performed simply by dropping the sample, and the operation is simple.
(3) Determination is easy without requiring a special detection device.
Utilizing these characteristics, the detection method is used for pregnancy test drugs and influenza test drugs, and is used as a new POCT (Point Of Care Testing) method. Also, in food inspection, it is widely used as a test reagent for food allergens, for example, and is attracting more and more attention.

On the other hand, in recent years, fine particles of about several nm to 1 μm have been applied to various fields and attracted attention. For example, the materials and applications of the fine particles are diverse, such as porous silica particles used for adsorbents and catalysts, and silica particles used for resin reinforcing agents. Silica nanoparticles containing fluorescent dyes are expected to be used as new labeling particles, particularly in the field of biotechnology. Since silica nanoparticles containing a high concentration of dye also have a high molar extinction coefficient, they are expected to be applied as more sensitive labeling particles.
The labeling particles can be used as labeling reagents that can be used for detection, quantification, staining, etc. of target molecules by binding biomolecules (proteins, nucleic acids, etc.) capable of binding to specific target molecules to the surface. (For example, see Patent Documents 1 and 2).

JP 2014-153057 A International Publication No. 2012/147774 Pamphlet

It is an object of the present invention to provide a biomolecule detection test kit that can be applied to a luminescence detection apparatus that does not include a filter that transmits only a wavelength region that overlaps an excitation wavelength.
Another object of the present invention is to provide a biomolecule detection method and a biomolecule detection labeling reagent using the biomolecule detection test kit.

By using the labeling reagent particles composed of the fluorescent dye-containing silica nanoparticles described in Patent Documents 1 and 2, biomolecules can be detected with high sensitivity and high accuracy.
In the labeling reagent particles described in Patent Documents 1 and 2, as shown in FIG. 6, the wavelength region of the excitation light that irradiates the labeling reagent particles partially overlaps the light emitting region emitted from the labeling reagent particles. Therefore, in order to detect fluorescence, it is necessary to provide a filter that transmits only the wavelength region overlapping the excitation wavelength in the light emission detection device. However, since such a filter is expensive, in order to provide a biomolecule detection system at a low cost, it is desired to develop a biomolecule detection method that does not require the filter of the luminescence detection device. In addition, a method for detecting a biomolecule that does not require a filter of the luminescence detection device is desirable from the viewpoint of making the detection kit compact.
However, in the conventional biomolecule detection method using labeling reagent particles as described in Patent Documents 1 and 2, light is emitted using a luminescence detection device that does not include a filter that transmits only the wavelength region overlapping with the excitation wavelength. Even if it detects, since the background value by light emission, such as fluorescence intensity, rises, it becomes difficult to ensure sufficient accuracy to detect a very small amount of biomolecules.

The present inventors have conducted intensive studies in view of the above problems. Specifically, the possibility of detecting biomolecules using dyes whose excitation wavelength region and emission wavelength do not overlap each other as shown in FIG. 2 was examined. As a result, the present inventors have found that biomolecules can be detected by removing excitation light noise and detecting phosphorescence without providing the above-described filter in the luminescence detection apparatus.
The present invention has been completed based on these findings.

The above-described problems of the present invention have been solved by the following means.
(1) A biomolecule detection test kit having a test piece for biomolecule detection and labeled reagent particles for biomolecule detection comprising phosphorescent dye-containing silica nanoparticles,
The test piece includes a membrane provided with a test region for capturing a biomolecule, and the labeling reagent particle migrates, and the labeling reagent particle capturing the biomolecule as a target substance is immobilized on the test region,
A test kit for detecting a biomolecule, wherein a capture substance having a binding property to a biomolecule is introduced on the surface of the phosphor dye-containing silica nanoparticles.
(2) The phosphorescent dye-containing silica nanoparticles according to the above (1), wherein the phosphorescent dye-containing silica nanoparticles are ultraviolet-excited phosphorescent particles that contain ultraviolet-excited phosphorescent dye that absorbs ultraviolet rays and emits phosphorescence on the visible light region side. kit.
(3) The kit according to (2) above, wherein the emission lifetime of the ultraviolet-excited phosphorescent dye is 10 microseconds to 100 milliseconds.
(4) The excitation wavelength of the ultraviolet-excited phosphorescent dye is in the range of 230 nm to 350 nm, and the phosphorescent emission peak of the ultraviolet-excited phosphorescent particle is between 450 to 600 nm, as described in (2) or (3) above kit.
(5) The kit according to any one of (2) to (4), wherein the chromophore of the dye is a residue of a compound represented by the following general formula (1) or (2).

(In general formulas (1) and (2), R represents an alkyl group, an aromatic ring group, a heterocyclic group, or a halogen atom. In general formula (2), X represents an alkyl group, an aromatic ring group, or Represents a heterocyclic group.)
(6) The test kit according to any one of (1) to (5), wherein an average particle diameter of the phosphorescent dye-containing silica nanoparticles is 60 nm or more and 300 nm or less.
(7) The test according to any one of (1) to (6), wherein the membrane is further provided with a reference region on which labeled reagent particles not capturing a biomolecule as a target substance are immobilized. kit.

(8) A method for detecting biomolecules using a test kit for detecting biomolecules,
The biomolecule detection test kit has a biomolecule detection test piece and a labeling reagent particle for biomolecule detection composed of phosphorescent dye-containing silica nanoparticles,
The test piece includes a membrane provided with a test region for capturing a biomolecule, and the labeling reagent particle migrates, and the labeling reagent particle capturing the biomolecule as a target substance is immobilized on the test region,
A capture substance having binding properties to biomolecules is introduced on the surface of the phosphorescent dye-containing silica nanoparticles,
Biomolecule detection method.
(9) The phosphorescent dye-containing silica nanoparticles according to (8), wherein the phosphorescent dye-containing silica nanoparticles are ultraviolet-excited phosphorescent particles that contain ultraviolet-excited phosphorescent dye that absorbs ultraviolet rays and emits phosphorescence on the visible light region side. Biomolecule detection method.
(10) The method for detecting a biomolecule according to (9), wherein the emission lifetime of the ultraviolet-excited phosphorescent dye is 10 microseconds or more and 100 milliseconds or less.
(11) The excitation wavelength of the ultraviolet-excited phosphorescent dye is in the range of 230 nm to 350 nm, and the phosphorescent emission peak of the ultraviolet-excited phosphorescent particle is between 450 to 600 nm, as described in (9) or (10) above Biomolecule detection method.
(12) The biomolecule detection according to any one of (9) to (11), wherein the chromophore of the dye is a residue of the compound represented by the general formula (1) or (2). Method.
(13) The biomolecule detection method according to any one of (8) to (12), wherein an average particle diameter of the phosphorescent dye-containing silica nanoparticles is 60 nm or more and 300 nm or less.
(14) The phosphor molecule according to any one of (8) to (13), wherein phosphorescence measurement is started before 100 milliseconds elapses after irradiation of excitation light to the phosphorescent dye-containing silica nanoparticles is stopped. Detection method.
(15) Any one of the above (8) to (14), wherein phosphorescence measurement is started 100 milliseconds after elapse of 10 microseconds or more after irradiation of excitation light to the phosphorescent dye-containing silica nanoparticles is stopped. A method for detecting a biomolecule according to 1.
(16) After stopping the irradiation of excitation light to the phosphorescent dye-containing silica nanoparticles, phosphorescence is measured within the phosphorescence emission duration of the phosphorescent dye-containing silica nanoparticles according to any one of (8) to (15) The detection method of the biomolecule of description.
(17) The living body according to any one of (8) to (16), wherein the membrane is further provided with a reference region on which labeled reagent particles not capturing a biomolecule as a target substance are immobilized. Molecular detection method.

(18) A labeling reagent for detecting biomolecules, comprising silica nanoparticles containing phosphorescent dyes.
(19) The phosphorescent dye-containing silica nanoparticles according to the above (18), wherein the phosphorescent dye-containing silica nanoparticles are ultraviolet-excited phosphorescent particles that contain ultraviolet-excited phosphorescent dye that absorbs ultraviolet rays and emits phosphorescence on the visible light region side. Labeling reagent for biomolecule detection.
(20) The labeling reagent for biomolecule detection according to (19), wherein the emission lifetime of the ultraviolet excited phosphorescent dye is 10 microseconds or more and 100 milliseconds or less.
(21) The excitation wavelength of the ultraviolet-excited phosphorescent dye is in the range of 230 nm to 350 nm, and the phosphorescent emission peak of the ultraviolet-excited phosphorescent particle is between 450 to 600 nm, according to (19) or (20) above Labeling reagent for biomolecule detection.
(22) The biomolecule detection according to any one of (19) to (21), wherein the chromophore of the dye is a residue of the compound represented by the general formula (1) or (2) Labeling reagent.
(23) The labeling reagent for biomolecule detection according to any one of (18) to (22), wherein an average particle diameter of the phosphorescent dye-containing silica nanoparticles is 60 nm to 300 nm.

The biomolecule detection test kit of the present invention can also be applied to a luminescence detection apparatus that does not include a filter that transmits only the wavelength region overlapping the excitation wavelength.
In addition, the biomolecule detection method of the present invention can detect a biomolecule even by using a luminescence detection apparatus that does not include a filter that transmits only the wavelength region overlapping with the excitation wavelength.
Furthermore, the biomolecule-detecting labeling reagent comprising the phosphorescent dye silica nanoparticles of the present invention can be suitably used in the above method.

It is an example of the relationship between the elapsed time after excitation light irradiation with respect to the reagent for biomolecule detection used by this invention, and emitted light intensity. It is a graph which shows typically the wavelength range and intensity of excitation light with which the labeling reagent for biomolecule detection used in the present invention is irradiated, and the wavelength range and intensity of luminescence emitted from the reagent. FIG. 3 is a diagram schematically illustrating a preferred embodiment of a biomolecule detection test piece that can be used in the present invention, FIG. 3A is a plan view, and FIG. 3B is a developed cross-sectional view. FIG. 4 is a diagram schematically showing another preferred embodiment of a test piece for detecting biomolecules that can be used in the present invention, FIG. 4A is a plan view, and FIG. 4B is a developed cross-sectional view. FIG. 5 is a diagram schematically showing still another preferred embodiment of a test piece for detecting biomolecules that can be used in the present invention, FIG. 5 (a) is a plan view, and FIG. 5 (b) is a developed sectional view. . It is a graph which shows typically the wavelength range and intensity | strength of the excitation light irradiated to the labeling reagent for biomolecule detection used with the conventional method, and the wavelength range and intensity | strength of light emission emitted from a reagent.

In the present specification, the “substance” includes a compound or a chemically synthesized molecule, as well as a biomolecule (protein, peptide, nucleic acid, etc.). These may be of artificial origin or of natural origin.
In this specification, the term “bond” or “linkage” generally refers to continuous integration from a state in which a plurality of objects are separated, and chemical bonds such as covalent bonds, ionic bonds, and hydrogen bonds. In addition to this, it means chemical adsorption and physical adsorption, as well as physical connection states such as fitting, screwing and occlusion. Here, “coupled” or “linked” means that a plurality of units may be coupled directly or indirectly through another unit.
Furthermore, “detection” in the present specification is a concept including not only qualitative detection and quantitative detection but also various other measurements, identification, analysis, evaluation, and the like.

The test kit for detecting a biomolecule of the present invention has a test piece for detecting a biomolecule and labeling reagent particles for detecting a biomolecule comprising silica nanoparticles containing phosphorescent dye. The biomolecule detection method of the present invention detects a biomolecule using the biomolecule detection test kit.
Hereinafter, the configuration of the present invention will be described in detail focusing on preferred embodiments thereof.

[Biomolecule]
In the present invention, the biomolecule as a detection target (target substance) is not particularly limited, and examples thereof include antigens, antibodies, nucleic acids, sugars, sugar chains, ligands, receptors, peptides, and other biologically active chemical substances. Of these, the present invention can be suitably used for antigen detection.

In the present invention, the sample containing a biomolecule is not particularly limited, but is collected from a clinical specimen (eg, blood, plasma, serum, lymph, urine, saliva, pancreatic juice, gastric juice, sputum, nose, throat, etc.) Body fluids such as wiping fluids, feces, etc.), food samples (eg, liquid beverages, semi-solid foods, solid foods, etc.), environmental sampling samples (eg, natural samples such as soil, rivers, seawater, production lines in factories, Sampling sample by an air sampler installed in a clean room, wiping sample, etc.).
Moreover, if a sample is a liquid, it can also be used as it is. When the sample is semi-solid or solid, it can be used after being subjected to a treatment such as dilution or extraction.

[Test specimen for biomolecule detection]
In the present invention, a biomolecule detection test strip (hereinafter, also referred to as “test strip” or “test strip”) and a biomolecule detection labeling reagent particle (hereinafter simply referred to as “label”). It is preferable to detect a detection target substance such as an antigen by an antigen-antibody reaction using a biomolecule detection test kit (hereinafter also referred to as “reagent particles”).
Hereinafter, test kits that can be preferably used in the present invention will be described.

[Test specimen that can be preferably used in the first embodiment of the present invention]
The shape of the test piece included in the test kit that can be preferably used in the first embodiment of the present invention is not particularly limited, but is preferably a flat test piece, and is a lateral flow test piece. Is more preferable.
Further, the structure of the test piece is not particularly limited, but the sample addition member (sample pad), the membrane having the test region for capturing the detection target substance, and the absorption pad are mutually in this order, and the capillary phenomenon occurs mutually. The structure is preferably connected in series so as to occur. And each constituent member is preferably backed by a backing sheet with an adhesive.
Hereinafter, the test piece having the above shape and structure will be described with reference to FIGS. However, the present invention is not limited to this.

(Sample pad)
The sample pad 2 is a constituent member for dropping a sample containing biomolecules, labeled reagent particles, and a complex thereof. The material, dimensions, etc. of the sample pad 2 are not particularly limited, and general materials applied to this type of product can be used.

(Membrane)
The membrane 3 is a structural member for capturing biomolecules that have moved from the sample pad 2 by capillary action.
As shown in FIG. 3, the membrane 3 is provided with at least one test region 10. A capture substance having specific binding property to the biomolecule and capable of forming a complex with the biomolecule is introduced into the test region 10. The labeled reagent particles that have moved from the sample pad 2 migrate through the membrane 3, whereby the labeled reagent particles that have captured the biomolecules as the target substance are immobilized on the test region 10.
In this test region 10, a complex composed of a capture substance-biomolecule-labeled reagent particles is formed, and the labeled reagent particles are concentrated. The biomolecule can be detected qualitatively or quantitatively depending on the amount of the labeling reagent particles have.

  As shown in FIG. 4, the membrane 3 is provided with a test region 20 for immobilizing labeled reagent particles that have captured a biomolecule as a target substance, and a biomolecule as a target substance is placed downstream of the test area 20. You may further provide the reference area | region 21 which fix | immobilizes the labeling reagent particle | grains which were not fix | immobilized in the test area | region 20 since it has not captured. By providing such a reference region 21, it can be confirmed whether the sample dropped on the sample pad 2 moves to the membrane 3 by capillary action and further moves beyond the test region 20. The reference area 21 may not be provided.

  The shape of the test region and the reference region is not particularly limited as long as the capture substance is locally immobilized, and examples thereof include a line shape, a circular shape, and a belt shape. In the present invention, the test region and the reference region are each preferably in a line shape, and more preferably in a line shape having a width of 0.5 to 1.5 mm.

The capture substance is not particularly limited as long as it can capture biomolecules by specific binding such as antigen-antibody reaction and form a complex composed of capture substance-biomolecule-labeled reagent particles in the test region. .
The capture substance for capturing the labeling reagent particles used in the reference region can be appropriately selected from those having binding properties to the labeling reagent particles. Specifically, it can be appropriately selected from antibodies, antigens, nucleic acids, receptors, ligands, sugar chains, aptamers and the like.

There is no particular limitation on the amount of the trapping substance introduced in the test region, and it can be set as appropriate. For example, when the shape of the test region is a line, the amount of the capture substance immobilized per unit length (cm) is preferably 0.001 μg or more, more preferably 0.01 μg or more, preferably 10 μg or less, and preferably 2 μg or less. More preferred.
Examples of the method for immobilizing the capture substance include a method in which a solution of the capture substance is applied to a predetermined region of the membrane 3, dropped or sprayed, dried, and immobilized by physical adsorption. In order to prevent the influence of nonspecific adsorption on the measurement, the entire membrane 3 may be subjected to a so-called blocking process after the capture substance is immobilized.

(Absorption pad)
The absorption pad 4 is a constituent member for absorbing a solution that has moved through the membrane 3 by capillary action and generating a constant flow.

There is no restriction | limiting in particular as a material of each of these structural members, The member normally used for this kind of test piece can be used. For example, a glass fiber pad such as Glass Fiber Conjugate Pad (trade name, manufactured by MILLIPORE) can be preferably used as the sample pad 2. As the membrane 3, a nitrocellulose membrane such as Hi-Flow Plus180 membrane (trade name, manufactured by MILLIPORE) can be preferably used. As the absorbent pad 4, a cellulose membrane such as Cellulose Fiber Sample Pad (trade name, manufactured by MILLIPORE) can be preferably used.
Examples of the pressure-sensitive adhesive backing sheet 6 include AR9020 (trade name, manufactured by Adhesives Research).

  As a method for preparing the test piece, in order of the sample pad 2, the membrane 3, and the absorption pad 4, in order to easily cause capillary action between the members, the members adjacent to both ends of each member are about 1 to 5 mm. The test strip 1 can be manufactured by superimposing (preferably on the backing sheet 6).

[Test specimen that can be preferably used in the second embodiment of the present invention]
The shape of the test piece included in the test kit that can be preferably used in the second embodiment of the present invention is not particularly limited, but is preferably a flat test piece, and is a test piece for lateral flow. Is more preferable.
The structure of the test piece is not particularly limited, but there are a sample pad, a member obtained by impregnating the labeled reagent particles (hereinafter also referred to as “conjugate pad”), and a test region for capturing the detection target substance. It is preferable that the membrane and the absorption pad have a structure in which the membrane and the absorption pad are connected in series so that capillary action occurs in this order. And each constituent member is preferably backed by a backing sheet with an adhesive.
Hereinafter, the test piece having the above shape and structure will be described with reference to FIG. However, the present invention is not limited to this.

(Sample pad)
The sample pad 2 is a constituent member that drops biomolecules. The material, dimensions, etc. of the sample pad 2 are not particularly limited, and general materials applied to this type of product can be used.

(Conjugate pad)
The conjugate pad 5 is a component obtained by impregnating the labeling reagent particles. And it is a part which mixes the liquid containing the biomolecule moved from the sample pad 2 by capillary action and the labeling reagent particles.
There is no restriction | limiting in particular in the quantity of the labeling reagent particle impregnated to the conjugate pad 5, It can set suitably.

(Membrane)
As shown in FIG. 5, the membrane 3 is provided with at least one test region 20. A capture substance that has a specific binding property to the biomolecule and can form a complex with the biomolecule is introduced into the test region 20. When the labeled reagent particles that have moved from the sample pad 2 migrate through the membrane 3, the labeled reagent particles that have captured the biomolecule as the target substance are immobilized on the test region 20.
In this test region 20, a complex composed of a capture substance-biomolecule-labeled reagent particles is formed, and the labeled reagent particles are concentrated. The biomolecule can be detected qualitatively or quantitatively depending on the amount of the labeling reagent particles have.
Further, as shown in FIG. 5, the reference region for immobilizing labeled reagent particles that are not immobilized on the test region 20 because the biomolecules as the target substance are not captured downstream of the test region 20 on the membrane 3. 21 may be further provided. By providing such a reference region 21, it can be confirmed whether the sample dropped on the sample pad 2 moves to the membrane 3 by capillary action and further moves beyond the test region 20. The reference area 21 may not be provided.

  The shape of the test region and the reference region is not particularly limited as long as the capture substance is locally immobilized, and examples thereof include a line shape, a circular shape, and a belt shape. In the present invention, the test region and the reference region are each preferably in a line shape, and more preferably in a line shape having a width of 0.5 to 1.5 mm.

The capture substance is not particularly limited as long as it can capture an antigen by specific binding such as an antigen-antibody reaction and form a complex composed of a capture substance-biomolecule-labeled reagent particles in the test region.
The capture substance for capturing the labeling reagent particles used in the reference region can be appropriately selected from those having binding properties to the labeling reagent particles. Specifically, it can be appropriately selected from antibodies, antigens, nucleic acids, receptors, ligands, sugar chains, aptamers and the like.

There is no particular limitation on the amount of the trapping substance introduced in the test region, and it can be set as appropriate. For example, when the shape of the test region is a line, the amount of the capture substance immobilized per unit length (cm) is preferably 0.001 μg or more, more preferably 0.01 μg or more, preferably 10 μg or less, and preferably 2 μg or less. More preferred.
Examples of the method for immobilizing the capture substance include a method in which a solution of the capture substance is applied to a predetermined region of the membrane 3, dropped or sprayed, dried, and immobilized by physical adsorption. In order to prevent the influence of nonspecific adsorption on the measurement, the entire membrane 3 may be subjected to a so-called blocking process after the capture substance is immobilized.

(Absorption pad)
The absorption pad 4 is a constituent member for absorbing a solution that has moved through the membrane 3 by capillary action and generating a constant flow.

There is no restriction | limiting in particular as a material of each of these structural members, The member normally used for this kind of test piece can be used. For example, as the sample pad 2 and the conjugate pad 5, glass fiber pads such as Glass Fiber Conjugate Pad (trade name, manufactured by MILLIPORE) can be preferably used. As the membrane 3, a nitrocellulose membrane such as Hi-Flow Plus180 membrane (trade name, manufactured by MILLIPORE) can be preferably used. As the absorbent pad 4, a cellulose membrane such as Cellulose Fiber Sample Pad (trade name, manufactured by MILLIPORE) can be preferably used.
Examples of the pressure-sensitive adhesive backing sheet 6 include AR9020 (trade name, manufactured by Adhesives Research).

  As a method for preparing the test piece, in order of the sample pad 2, the conjugate pad 5, the membrane 3, and the absorption pad 4, the members adjacent to both ends of each member in order to easily cause capillary action between the members. The test strip 1 can be manufactured by superimposing them on the backing sheet 6 (preferably on the backing sheet 6).

[Labeling reagent particles]
The labeling reagent particles of the present invention are composed of phosphorescent dye-containing silica nanoparticles. Among them, phosphorescent dye-containing silica nanoparticles having an excitation wavelength in the ultraviolet region and an emission wavelength in the visible light region are preferable, and an ultraviolet excitation phosphorescent pigment that absorbs ultraviolet rays and emits phosphorescence in the visible light region side is contained in the silica particles. Ultraviolet excited phosphor particles are more preferred. The emission lifetime of the ultraviolet excitation phosphor is preferably 10 microseconds or more and 100 milliseconds or less. Moreover, it is preferable that the excitation wavelength of an ultraviolet excitation phosphorescence dye exists in the range of 230 nm-350 nm, and the phosphorescence emission peak of an ultraviolet excitation phosphor particle exists between 450-600 nm. By using such phosphorescent dye-containing silica nanoparticles, light can be detected after the excitation light is cut, so that noise caused by the excitation light is eliminated, and biomolecules can be detected with high sensitivity and high accuracy. In addition, various functional groups can be introduced on the surface of the silica nanoparticles, and light emission in the test region has high luminance, so that detection of biomolecules can be realized in a wide quantitative range.
Hereinafter, labeling reagent particles made of phosphorescent dye-containing silica nanoparticles will be described. However, the present invention is not limited to this.

  There is no restriction | limiting in particular in the preparation method of a phosphorescent dye containing silica nanoparticle, A phosphorescent dye containing silica nanoparticle can be obtained by arbitrary arbitrary preparation methods. For example, Journal of Colloid and Interface Science, 159, p. Reference can be made to the sol-gel method described in 150-157 (1993) and the colloidal silica particle preparation method described in International Publication No. 2007/074722.

A preparation example of phosphorescent dye-containing silica nanoparticles will be specifically described. However, the present invention is not limited to this.
Silica particles containing a phosphorescent dye are prepared by reacting a phosphorescent dye with a silane coupling agent, and by combining one or two or more silanes with a product obtained by covalent bond, ionic bond or other chemical bond or adsorption. It can be prepared by condensation polymerization of a compound to form a siloxane bond. As a result, silica particles in which the organosiloxane component and the siloxane component are bonded by siloxane are obtained. Examples include N-hydroxysuccinimide (NHS) ester groups, maleimide groups, isocyanate groups, isothiocyanate groups, aldehyde groups, paranitrophenyl groups, diethoxymethyl groups, epoxy groups, cyano groups and the like. A product obtained by reacting a covalently bonded phosphorescent dye with a silane coupling agent having a substituent (for example, an amino group, a hydroxyl group, or a thiol group) that reacts with the active group. It can be prepared by condensation-polymerizing one or two or more silane compounds to form a siloxane bond.

  A case where aminopropyltriethoxysilane (APS) is used as the silane coupling agent and tetraethoxysilane (TEOS) is used as the silane compound is illustrated below.

(UV-excited luminescent dye)
The ultraviolet-excited luminescent dye is not particularly limited as long as it absorbs ultraviolet light and emits light in the visible light region, but the chromophore is preferably a residue of a nitrogen-containing heteroaromatic compound.
The chromophore of the ultraviolet excitation luminescent dye is preferably composed of a residue of a compound represented by the following general formula (1) or (2).

  In the general formulas (1) and (2), R is an alkyl group (preferably having 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms). , For example, methyl, ethyl, propyl, isopropyl, tert-butyl, pentyl, tert-pentyl, hexyl, octyl, tert-octyl, dodecyl, tridecyl, tetradecyl, pentadecyl, etc., aromatic ring groups (preferably carbon 6 to 24, more preferably 6 to 14 carbon atoms, more preferably 6 to 10 carbon atoms, such as phenyl, p-chlorophenyl, mesityl, tolyl, xylyl, naphthyl, anthryl, azulenyl, acenaphthenyl, fluorenyl, phenanthryl, indenyl, Pyrenyl, biphenylyl, etc.), heterocyclic groups ( Preferably, it is a heterocyclic group having 0 to 20 carbon atoms, and the ring-constituting hetero atom is preferably an oxygen atom, a nitrogen atom, or a sulfur atom, and a 5-membered or 6-membered ring may be condensed with a benzene ring or a heterocyclic ring. The ring may be a saturated ring, an unsaturated ring, or an aromatic ring, and examples thereof include 2-pyridyl, 4-pyridyl, 2-imidazolyl, 2-benzimidazolyl, 2-thiazolyl, 2-oxazolyl, and the like. Or a halogen atom (for example, fluorine atom, chlorine atom, bromine atom).

  In the general formula (2), X is an alkyl group (preferably having 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, for example methyl. , Ethyl, propyl, isopropyl, tert-butyl, pentyl, tert-pentyl, hexyl, octyl, tert-octyl, dodecyl, tridecyl, tetradecyl, pentadecyl and the like, and an aromatic ring group (preferably having 6 to 24 carbon atoms). , More preferably 6 to 14 carbon atoms, more preferably 6 to 10 carbon atoms, such as phenyl, p-chlorophenyl, mesityl, tolyl, xylyl, naphthyl, anthryl, azulenyl, acenaphthenyl, fluorenyl, phenanthryl, indenyl, pyrenyl, biphenylyl, etc. Or a heterocyclic group (preferably Is a heterocyclic group having 0 to 20 carbon atoms, and the ring-constituting hetero atom is preferably an oxygen atom, a nitrogen atom or a sulfur atom, and may be a 5-membered or 6-membered ring fused with a benzene ring or a heterocycle, This ring may be a saturated ring, an unsaturated ring, or an aromatic ring, and examples thereof include 2-pyridyl, 4-pyridyl, 2-imidazolyl, 2-benzimidazolyl, 2-thiazolyl, 2-oxazolyl and the like. .

  Specific examples of the silane coupling agent having a substituent include γ-aminopropyltriethoxysilane (APS), 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane, N-2 ( Examples thereof include silane coupling agents having an amino group such as (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, APS, 3-thiocyanatopropyltriethoxysilane, 3-glycidyl. Mention may be made of oxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane. 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 prepared. Here, the “spherical silica particles” specifically have a shape in which the ratio of the major axis to the minor axis is 2 or less.

The average particle diameter of the silica nanoparticles is not particularly limited, but is preferably 20 nm or more, more preferably 30 nm or more, further preferably 60 nm or more, preferably less than 1000 nm, more preferably 600 nm or less, further preferably 500 nm or less, and 300 nm or less. Particularly preferred. When the particle size is too small, the detection sensitivity is lowered, and when the particle size is too large, the porous support (membrane) used for the test piece becomes clogged.
In the present invention, the average particle size is calculated from the total projected area of 100 labeled reagent silica particles randomly selected from an image of a transmission electron microscope (TEM), a scanning electron microscope (SEM), or the like. The occupying area was obtained by an image processing apparatus, and the average value of the diameters of the circles corresponding to the value obtained by dividing the total occupied area by the number of selected silica nanoparticles (100) (average equivalent circle diameter) was obtained. is there.
The average particle diameter is an average particle diameter of particles composed only of primary particles, unlike the “particle size by dynamic light scattering method” described later, which is a concept including secondary particles formed by aggregation of primary particles.
In order to obtain silica nanoparticles having a desired average particle diameter, ultrafiltration is performed using an ultrafiltration membrane such as YM-10, YM-100 (both trade names, manufactured by Millipore), and the particle diameter 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.
The silica nanoparticles are preferably monodispersed as a particulate material. There is no particular limitation on the coefficient of variation of the particle size distribution of the silica nanoparticles, so-called CV value, but it is preferably 10% or less, more preferably 8% or less.

In the present specification, the “particle size by the dynamic light scattering method” is measured by the dynamic light scattering method and differs from the average particle size described above in that not only the primary particles but also the primary particles are aggregated. It is a concept including the secondary particles, and serves as an index for evaluating the dispersion stability of the composite 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.

  On the surface of the phosphorescent dye-containing silica nanoparticles, a capture substance having a binding property to a biomolecule such as an antigen is introduced. The capture substance introduced into the surface of the silica nanoparticles is not particularly limited as long as it has specific binding properties for biomolecules.

  There is no restriction | limiting in particular as a method of introduce | transducing a capture | acquisition substance into the surface of a phosphorescent pigment containing silica nanoparticle, According to a conventional method, it can introduce | transduce. For example, a trapping substance may be introduced onto the surface of phosphorescent dye-containing silica nanoparticles by electrostatic attraction, van der Waals force, hydrophobic interaction, or the like. Or you may introduce | transduce a capture | acquisition substance on the surface of a phosphorescent dye containing silica nanoparticle by the chemical bond of a crosslinking agent or a condensing agent. In addition, when the phosphorescent dye-containing silica nanoparticles are aggregated when the trapping substance to be introduced to the surface of the phosphorescent dye-containing silica nanoparticles is introduced, the surface of the phosphorescent dye-containing silica nanoparticles is subjected to a surface treatment in advance by an alternate adsorption method. May be.

  Hereinafter, a method for preparing phosphorescent dye-containing silica nanoparticles will be described. However, the present invention is not limited to this.

First, a silane coupling agent having a reactive functional group is hydrolyzed, and the hydrolyzed silane coupling agent and the hydroxyl group present on the surface of the phosphor nanoparticle containing phosphorescent dye are subjected to polycondensation to phosphorylate the reactive functional group. It introduce | transduces into the surface of a pigment | dye silica nanoparticle.
Specific examples of the silane coupling agent having a reactive functional group include 3-mercaptopropyltrimethoxysilane, MPS, APS, 3-thiocyanatopropyltriethoxysilane, 3-glycidyloxypropyltriethoxysilane, 3-isothione. Oceanatopropyltriethoxysilane, 3- [2- (2-aminoethylamino) ethylamino] propyltriethoxysilane, (-chloropropyltrimethoxysilane, vinylmethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3- Examples include acryloxypropyltrimethoxysilane, N-phenyl-3-aminopropyltrimethoxysilane, 3-ureidopropyltriethoxysilane, and bis (triethoxysilylpropyl) tetrasulfide.

The reactive functional group is preferably at least one reactive functional group selected from a thiol group, amino group, carboxyl group, halogen group, vinyl group, epoxy group and isocyanate group, and more preferably a thiol group.
When the reactive functional group is a thiol group, the density of the thiol groups in the phosphorescent pigment-containing silica particles surface is preferably 0.002 to 0.2 pieces / nm ^2, is 0.002 to 0.1 pieces / nm ² More preferred. The amount B of the thiol group present on the surface of the pigment-containing silica particle can be measured using DNTB (5,5′-dithiobis (2-nitrobenzoic acid)) as a reagent. As a method for quantifying a thiol group using DNTB, for example, the method of Archives of Biochemistry and Biophysics, 82, 70 (1959) can be used. As an example of a specific method, 20 μL of 10 mM DNTB solution dissolved in phosphate buffer (pH 7.0) and 2.5 mL of silica particle colloid prepared to 200 mg / mL are mixed, and after 4 hours, 412 nm The amount of thiol groups present on the particle surface can be determined from a calibration curve prepared using γ-mercaptopropyltrimethoxysilane (MPS) as a standard substance.

The reactive functional group introduced into the surface of the silica nanoparticles is reacted with a linker molecule that forms a chemical bond with the reactive functional group. Then, the linker molecule and bovine serum albumin (BSA) are bound to form a complex of phosphorescent dye-containing silica nanoparticles and BSA.
When the reactive functional group is a thiol group, phosphorescent dye-containing silica nanoparticles having a thiol group introduced therein and a linker molecule having a maleimide group and a carboxyl group are allowed to coexist in an aprotic solvent. As a result, a thioether bond is formed between the thiol group and the maleimide group, and particles having linker molecules bonded thereto are produced. Subsequently, particles to which a linker molecule is bonded, carbodiimide, and BSA having an amino group are allowed to coexist in an aqueous solvent. As a result, an amide bond is formed between the carboxyl group of the linker molecule active esterified with carbodiimide and the amino group of BSA, thereby forming a complex of phosphorescent dye silica nanoparticles and BSA.
Even when the reactive functional group is an amino group, a carboxyl group, a vinyl group, an epoxy group, or an isocyanate group, a complex of BSA and phosphorescent dye-containing silica nanoparticles can be formed by a conventional method. For these methods, for example, the descriptions in JP2009-274923A, JP2009-162537A, JP2010-10000542, and the like can be referred to.

  And BSA couple | bonded with a silica nanoparticle and the capture | acquisition substance are couple | bonded according to a conventional method. For example, BSA and the trapping substance can be bound by electrostatic attraction, van der Waals force, hydrophobic interaction, or the like. Alternatively, BSA and the trapping substance can be bonded by chemical bonding of a crosslinking agent or a condensing agent. Note that BSA and the capture substance may be directly bonded to form a complex, or BSA and the capture substance may be indirectly bonded via another substance to form a complex.

The amount of the trapping substance introduced into the surface of the phosphor pigment-containing silica nanoparticles can be appropriately set without any particular limitation. For example, the amount of the trapping substance introduced per 1 nm ² of the surface of the phosphorescent dye-containing silica nanoparticles is preferably 0.0001 mol or more and 0.1 mol or less, and preferably 0.001 mol or more and 0.05 mol or less.

[Method for detecting biomolecules]
Next, a biomolecule detection method using a biomolecule detection test kit having a biomolecule detection test piece and a labeling reagent particle composed of phosphorescent dye-containing silica nanoparticles will be described based on a preferred embodiment. . However, the present invention is not limited to this.

First, in the first embodiment of the present invention, a mixture of a liquid sample that can contain a biomolecule and the labeled reagent particles of the present invention is dropped onto the sample pad 2 of the test piece 1. The amount of the liquid sample dripped onto the sample pad 2 can be appropriately adjusted according to the configuration of the test piece 1.
And the composite of the biomolecule that has moved from the sample pad 2 to the membrane 3 by capillary action and the phosphorescent dye-containing silica nanoparticles is bonded to the capture substance introduced onto the test region (test line) of the test piece 1. Concentrated. Then, the test area is irradiated with light, and the label of the concentrated labeling reagent particles is detected. Biomolecules can be detected depending on the presence or absence of the detected label or the degree of labeling.

In the second embodiment of the present invention, the labeling reagent particles of the present invention are contained in a dry state in a member used for a biomolecule assay. Then, before the biomolecule is detected by a specific reaction such as an antigen-antibody reaction, the liquid sample that can contain the biomolecule and the labeling reagent particles are mixed. For example, when biomolecules are detected by immunochromatography, the labeling reagent particles are included in the sample pad 2 or the membrane 3, and when the liquid sample that can contain biomolecules passes through these members, the biomolecules are detected. A liquid sample that may be contained and the labeling reagent particles may be mixed. Alternatively, the conjugate pad 5 containing the labeling reagent particles is provided upstream of the membrane 3, and when the liquid sample that can contain the biomolecule passes through this member, the liquid sample that can contain the biomolecule and Labeling reagent particles may be mixed. In these cases, a specific reaction such as an antigen-antibody reaction is performed after a liquid sample that can contain a biomolecule passes through the member.
Then, the complex of the biomolecule that has moved from the conjugate pad 5 to the membrane 3 by capillary action and the silica nanoparticle containing phosphorescent dye is bound to the capture substance introduced onto the test region (test line) of the test piece 1. To concentrate. Then, the test area is irradiated with light, and the label of the concentrated labeling reagent particles is detected. Biomolecules can be detected depending on the presence or absence of the detected label or the degree of labeling.

There is no restriction | limiting in particular in the detection method of the label | marker of label | marker reagent particle | grains, You may detect visually and may detect using a general purpose phosphorescent dye detector.
Further, as described above, in the present invention, an ultraviolet excited phosphorescent dye having a light emission lifetime of 10 microseconds to 100 milliseconds is preferably used. Therefore, it is preferable to start the measurement of phosphorescence 100 milliseconds after the irradiation of excitation light to the phosphorescent dye-containing silica nanoparticles is stopped, and after the irradiation of excitation light to the phosphorescent dye-containing silica nanoparticles is stopped, More preferably, the phosphorescence measurement is started after elapse of 100 seconds or more before elapse of 100 seconds. Furthermore, it is preferable to measure phosphorescence within the phosphorescence emission duration of the phosphorescent dye-containing silica nanoparticles after the irradiation of excitation light to the phosphorescent dye-containing silica nanoparticles is stopped.

  A general-purpose emission detector comprises an excitation light source. Examples of the excitation light source include a mercury lamp, a halogen lamp, a xenon lamp, a laser diode, and a light emitting diode. The emission detector may be provided with a filter that transmits only light of a specific wavelength from the excitation light source, but it is preferable not to provide a filter from the viewpoint of downsizing and cost reduction of the detector. The light emission detector may include a photomultiplier tube or a CCD detector that receives light emission. As a result, it is possible to detect light emission with an intensity or wavelength that cannot be visually confirmed, and to measure the light emission intensity.

The wavelength of the excitation light to be irradiated is not particularly limited, but is preferably 230 nm or more and less than 400 nm, and particularly preferably 250 nm or more and less than 330 nm.
The emission wavelength is preferably 450 nm or more, particularly preferably 480 nm or more.

  Hereinafter, the present invention will be described in more detail based on examples. The present invention is not limited to these examples.

(Preparation Example 1) Preparation of phosphorescent dye-containing labeled silica nanoparticles (1) Preparation of phosphorescent dye-containing silica nanoparticles A succinimidyl ester of a phosphorescent dye having the following structural formula (excitation wavelength: 293 nm, emission wavelength: 500-600 nm, 31 mg (manufactured by AST Corporation) was dissolved in 10 mL of dimethylformamide (DMF). 12 μL of APS (manufactured by Shin-Etsu Silicone) was added thereto and reacted at room temperature (23 ° C.) for 1 hour to obtain a phosphorescent dye-APS complex (5 mM).

600 μL of the obtained phosphorescent dye-APS complex solution was mixed with 140 mL of ethanol, 6.5 mL of TEOS (manufactured by Shin-Etsu Silicone), 20 mL of distilled water and 15 mL of 28% by mass ammonia water, 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 phosphorescent pigment-containing silica nanoparticles, the particles were dispersed, and centrifuged again at a gravity acceleration of 18000 × g for 30 minutes. This washing operation was further repeated twice to remove unreacted TEOS, ammonia and the like contained in the dispersion liquid of phosphorescent dye-containing silica nanoparticles, thereby obtaining 2.33 g of phosphorescent dye-containing silica nanoparticles having an average particle diameter of 250 nm. Yield about 90%.

(2) Introduction of thiol group 1 g of the particles obtained above was dispersed in 150 mL of a mixed solution of water / ethanol = 1/4. To this, 1.5 mL of MPS (Wako Pure Chemical Industries, Ltd.) was added. Subsequently, 20 mL of 28% aqueous ammonia was added and mixed at room temperature for 4 hours.
The reaction solution was centrifuged for 30 minutes at a gravitational acceleration of 18000 × g, and the supernatant was removed. Distilled water (10 mL) was added to the precipitated silica nanoparticles to disperse the particles, and the mixture was again centrifuged for 30 minutes at a gravitational acceleration of 18000 × g. This washing operation is further repeated twice to remove unreacted MPS, ammonia and the like contained in the dispersion of phosphorescent dye-containing silica nanoparticles having a thiol group, and phosphorescent dye-containing silica nanoparticles (hereinafter referred to as thiol groups) into which thiol groups have been introduced. Group-introduced phosphorescent dye-containing silica nanoparticles A) were obtained.
With respect to 500 mg of the obtained thiol group-introduced phosphorescent dye-containing silica nanoparticles A, quantitative analysis of the thiol group introduced using DNTB was performed. As a result, the density of thiol groups on the particle surface of the thiol group-introduced phosphor dye-containing silica nanoparticles A was 0.046 / nm ² .

(3) Bonding of Phosphor Dye-Containing Silica Nanoparticles and Antibody Via Thiol Group Addition of DMF 460 μL to 40 μL of thiol group-introduced phosphor dye-containing silica nanoparticles A dispersion (concentration 25 mg / mL, dispersion medium: distilled water), 15000 Centrifugation was performed at a gravitational acceleration of × g for 10 minutes. The supernatant was removed, 500 μL of DMF was added and centrifuged, and the supernatant was removed. Again, 500 μL of DMF was added, and the thiol group-introduced phosphor dye-containing silica nanoparticles A were dispersed. By adding 1 mg of N- (4-aminophenyl) maleimide as a linker molecule and mixing for 30 minutes, a thioether bond is formed between the maleimide group of the linker molecule and the thiol group of the thiol group-introduced phosphor dye-containing silica nanoparticles. Formed.
This reaction solution was centrifuged for 10 minutes at a gravitational acceleration of 15000 × g, and after removing the supernatant, 90.6 μL of distilled water was added to disperse the particles. Subsequently, 0.5 μM MES (2-morpholinoethanesulfonic acid) (pH 6.0) 100 μL, 50 mg / mL NHS (N-hydroxysuccinimide) 230.4 μL, 19.2 mg / mL EDC (1-ethyl-3- ( 75 [mu] L of 3-dimethylaminopropyl) carbodiimide) was added and mixed. To this, 4.0 μL of anti-influenza A nucleoprotein antibody (6.2 mg / ml, derived from mouse, HyTest) was added and mixed for 10 minutes.
Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. Then, 400 μL of 10 mM KH ₂ PO ₄ (pH 7.5) was added to disperse the particles. Subsequently, centrifugation was performed at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. Again, 400 μL of 10 mM KH ₂ PO ₄ (pH 7.5) was added, and the particles were dispersed to obtain a colloid.
Using this colloid as a sample, protein quantification was performed. For protein quantification, PierceBCA Protein Assay Kit (trade name, manufactured by Thermo Fisher Scientific) was used. As a result, the amount of antibody bound per 1 g of phosphorescent dye-containing silica nanoparticles was 7.9 mg.

Subsequently, 10 μL of 10% BSA was added to the colloid and mixed for 10 minutes. Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. Then, 500 μL of 10 mM KH ₂ PO ₄ (pH 7.5) was added to disperse the particles, followed by centrifugation at 15000 × g for 10 minutes, and the supernatant was removed. Again, 400 μL of 10 mM KH ₂ PO ₄ (pH 7.5) was added to disperse the particles to obtain a colloid in which phosphorescent dye-containing silica nanoparticles A bound with an anti-influenza A influenza nucleoprotein antibody were dispersed.

Preparation Example 2 Preparation of Fluorescent Dye-Containing Labeled Silica Nanoparticles 31 mg of 5- (and -6) -carboxyrhodamine 6G succinimidyl ester (trade name, manufactured by emp Biotech GmbH) as a fluorescent molecule is dissolved in 10 mL of DMF. did. 12 μL of APS (manufactured by Shin-Etsu Silicone) was added thereto and reacted at room temperature (23 ° C.) for 1 hour to obtain 5- (and-6) -carboxyrhodamine 6G-APS complex (5 mM).
600 μL of the obtained 5- (and-6) -carboxyrhodamine 6G-APS complex solution was mixed with 140 mL of ethanol, 6.5 mL of TEOS (manufactured by Shin-Etsu Silicone), 20 mL of distilled water and 15 mL of 28% by mass ammonia water. The reaction was performed 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 fluorescent molecule-containing silica nanoparticles, the particles were dispersed, and centrifuged again at a gravity acceleration of 18000 × g for 30 minutes. This washing operation was further repeated twice to remove unreacted TEOS, ammonia, and the like contained in the dispersion liquid of fluorescent molecule-containing silica nanoparticles, to obtain 1.65 g of fluorescent molecule-containing silica nanoparticles having an average particle diameter of 271 nm. Yield about 94%.
Thereafter, the same operation as in Preparation Example 1 was performed to obtain a colloid in which fluorescent molecule-containing silica nanoparticles bound with anti-influenza A influenza nucleoprotein antibody were dispersed.

(Preparation Example 3) Preparation of immunochromatographic test strip Test area (test line) with a width of about 1 mm at a position of about 6 mm from the end of membrane 3 (length 25 mm, trade name: Hi-Flow Plus180 membrane, manufactured by MILLIPORE) 20, a solution ((50 mM KH ₂ PO ₄ , pH 7.0) + 5% sucrose) containing 1 mg / mL of rabbit-derived anti-influenza A nucleoprotein antibody (polyclonal antibody, manufactured in-house) was applied at 0.75 μL / cm The amount was applied.

Subsequently, as a control line 21 having a width of about 1 mm, a solution ((50 mM KH ₂ PO ₄ , pH 7.0) containing an anti-mouse IgG antibody derived from goat (AKP Goat anti-mouse IgG Antibody, BioLegend) at 1 mg / mL. ) Sugar Free) was applied at a coating amount of 0.75 μL / cm and dried at 50 ° C. for 30 minutes. The distance between the test line 20 and the control line 21 was 3 mm.

  The antibody-immobilized membrane 3, the sample pad (trade name: Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE) 2 and the absorption pad (trade name: Cellulose Fiber Sample Pad (CFSP), manufactured by MILLIPORE) 4 in this order. And a backing sheet (trade name: AR9020, manufactured by Adhesives Research) 6. The membrane 3 was configured such that the test line 20 was on the sample pad 2 side and the control line 21 was on the absorption pad 4 side. Subsequently, it was cut into strips having a width of 5 mm and a length of 60 mm to produce a test strip 1 for detecting biomolecules.

(Test example) Detection of influenza nucleoprotein Influenza nucleoprotein solution having the composition shown in Table 1 was prepared. Subsequently, 100 μL of the same mixed solution and 2 μL of the colloid of Preparation Example 1 (2.5 mg / mL) were dropped onto the sample pad 2 of the test strip 1. The same test was performed on the colloid of Preparation Example 2. After 15 minutes, the line emission intensity was measured by the following detector and digitized.
The results are shown in Table 1.

<Detection device>
A detection unit comprising a light source, an optical filter, and a photomultiplier tube (PMT), the detection unit having a mechanism that linearly moves at a constant speed by a motor, and a recording mechanism that records the received light intensity of the PMT every 50 μsec. A detection device was prepared. The detection unit is a laser diode having a light source of 293 nm, and has a mechanism for receiving light by the PMT after irradiating the sample with the laser diode and transmitting light through an optical filter that transmits only light having a wavelength of 420 nm or more.

  The measurement was performed according to the following procedure. First, the PMT is turned on from the PC via the function generator in advance, and after 0.001 μsec, the sample is irradiated with a laser having a wavelength of 293 nm via the function generator, and the sample emits light for 10 μsec via the PMT. And received light. This operation was repeated, data was accumulated in the oscilloscope, and all data was output to the PC.

From the results in Table 1, when the particles produced in Preparation Example 1 were used, the absolute value of the emission intensity was smaller than when the particles produced in Preparation Example 2 were used. However, it can be seen that the detection concentration range of the sample is not changed, and the same sensitivity as in the conventional method can be obtained.
Furthermore, the same results were obtained with the sample of Preparation Example 1 even when measurement was performed excluding an optical filter that transmits only light having a wavelength of 420 nm or more.

1 Test Strip 2 Sample Pad 3 Membrane 4 Absorption Pad 6 Backing Sheet 10 Test Area 20 Test Area 21 Reference Area

Claims (23)

A biomolecule detection test kit having a test piece for biomolecule detection and labeled reagent particles for biomolecule detection composed of phosphorescent dye-containing silica nanoparticles,
The test piece includes a membrane provided with a test region for capturing a biomolecule, and the labeling reagent particle migrates, and the labeling reagent particle capturing the biomolecule as a target substance is immobilized on the test region,
A test kit for detecting a biomolecule, wherein a capture substance having a binding property to a biomolecule is introduced on the surface of the phosphor dye-containing silica nanoparticles.   The kit according to claim 1, wherein the phosphorescent dye-containing silica nanoparticles are ultraviolet-excited phosphorescent particles containing an ultraviolet-excited phosphorescent dye that absorbs ultraviolet rays and emits phosphorescence in a visible light region side.   The kit according to claim 2, wherein the emission lifetime of the ultraviolet-excited phosphorescent dye is 10 microseconds or more and 100 milliseconds or less.   The kit according to claim 2 or 3, wherein the excitation wavelength of the ultraviolet excitation phosphorescent dye is in the range of 230 nm to 350 nm, and the phosphorescence emission peak of the ultraviolet excitation phosphor particles is between 450 to 600 nm. The kit according to any one of claims 2 to 4, wherein the chromophore of the dye is a residue of a compound represented by the following general formula (1) or (2).

(In general formulas (1) and (2), R represents an alkyl group, an aromatic ring group, a heterocyclic group, or a halogen atom. In general formula (2), X represents an alkyl group, an aromatic ring group, or Represents a heterocyclic group.)   The test kit according to any one of claims 1 to 5, wherein an average particle size of the phosphorescent dye-containing silica nanoparticles is 60 nm or more and 300 nm or less.   The test kit according to any one of claims 1 to 6, wherein a reference region to which labeled reagent particles that do not capture a biomolecule as a target substance are immobilized is further provided on the membrane. A biomolecule detection method using a biomolecule detection test kit,
The biomolecule detection test kit has a biomolecule detection test piece and a labeling reagent particle for biomolecule detection composed of phosphorescent dye-containing silica nanoparticles,
The test piece includes a membrane provided with a test region for capturing a biomolecule, and the labeling reagent particle migrates, and the labeling reagent particle capturing the biomolecule as a target substance is immobilized on the test region,
A capture substance having binding properties to biomolecules is introduced on the surface of the phosphorescent dye-containing silica nanoparticles,
Biomolecule detection method.   The biomolecule detection method according to claim 8, wherein the phosphorescent dye-containing silica nanoparticles are ultraviolet-excited phosphorescent particles containing an ultraviolet-excited phosphorescent dye that absorbs ultraviolet rays and emits phosphorescence in a visible light region side. .   The method for detecting a biomolecule according to claim 9, wherein the emission lifetime of the ultraviolet-excited phosphorescent dye is 10 microseconds or more and 100 milliseconds or less.   The method for detecting a biomolecule according to claim 9 or 10, wherein the excitation wavelength of the ultraviolet excitation phosphorescent dye is in the range of 230 nm to 350 nm, and the phosphorescence emission peak of the ultraviolet excitation phosphor particles is between 450 to 600 nm. The method for detecting a biomolecule according to any one of claims 9 to 11, wherein the chromophore of the dye is a residue of a compound represented by the following general formula (1) or (2).

(In general formulas (1) and (2), R represents an alkyl group, an aromatic ring group, a heterocyclic group, or a halogen atom. In general formula (2), X represents an alkyl group, an aromatic ring group, or Represents a heterocyclic group.)   The method for detecting a biomolecule according to any one of claims 8 to 12, wherein an average particle diameter of the phosphorescent dye-containing silica nanoparticles is 60 nm or more and 300 nm or less.   The method for detecting a biomolecule according to any one of claims 8 to 13, wherein measurement of phosphorescence is started before 100 milliseconds have elapsed after irradiation of excitation light to the phosphorescent dye-containing silica nanoparticles is stopped.   The measurement of phosphorescence according to any one of claims 8 to 14, wherein phosphorescence measurement is started 100 milliseconds after elapse of 10 microseconds or more after irradiation of excitation light to the phosphorescent dye-containing silica nanoparticles is stopped. Detection method.   The biomolecule detection according to any one of claims 8 to 15, wherein phosphorescence is measured within the phosphorescence emission duration of the phosphorescent dye-containing silica nanoparticles after the irradiation of excitation light to the phosphorescent dye-containing silica nanoparticles is stopped. Method.   The method for detecting a biomolecule according to any one of claims 8 to 16, further comprising a reference region on the membrane to which a labeled reagent particle that does not capture a biomolecule as a target substance is immobilized.   A labeling reagent for detecting biomolecules, comprising phosphorescent dye-containing silica nanoparticles.   19. The biomolecule detection label according to claim 18, wherein the phosphorescent dye-containing silica nanoparticles are ultraviolet-excited phosphorescent particles that contain ultraviolet-excited phosphorescent dye that absorbs ultraviolet rays and emits phosphorescence on the visible light region side in the silica particles. reagent.   The labeling reagent for detecting a biomolecule according to claim 19, wherein the emission lifetime of the ultraviolet-excited phosphorescent dye is 10 microseconds or more and 100 milliseconds or less.   The labeling reagent for detecting a biomolecule according to claim 19 or 20, wherein the excitation wavelength of the ultraviolet excitation phosphorescent dye is in the range of 230 nm to 350 nm, and the phosphorescence emission peak of the ultraviolet excitation phosphor particles is between 450 to 600 nm. The labeling reagent for biomolecule detection according to any one of claims 19 to 21, wherein the chromophore of the dye is a residue of a compound represented by the following general formula (1) or (2).

(In general formulas (1) and (2), R represents an alkyl group, an aromatic ring group, a heterocyclic group, or a halogen atom. In general formula (2), X represents an alkyl group, an aromatic ring group, or Represents a heterocyclic group.)   23. The labeling reagent for biomolecule detection according to any one of claims 18 to 22, wherein the phosphorescent dye-containing silica nanoparticles have an average particle diameter of 60 nm to 300 nm.

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