JP2017150867A - Test kit for biomolecule detection, biomolecule detection method using this and labeling reagent for biomolecule detection to be used for these - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a test kit for biomolecule detection that can be used suitably for high-sensitivity detection of a biomolecule.SOLUTION: A test kit for biomolecule detection includes a test strip for biomolecule detection and a labeling reagent for biomolecule detection that consists of fluorescent silica nanoparticles. The test strip is formed by including a membrane: which is provided with a test area where a vaccine for capturing a biomolecule is fixed; and in which the labeling reagent transitions. The labeling reagent after capturing the biomolecule regarded as a target substance is fixed to the test area and a vaccine bindable to the biomolecule is introduced on each surface of the fluorescent silica nanoparticles.SELECTED DRAWING: None

Description

  The present invention relates to a biomolecule detection test kit, a biomolecule detection method using the same, and a biomolecule detection labeling reagent used therefor. In particular, the present invention relates to a test kit for testing dysentery amoeba, a method for testing dysentery amoeba using the same, and a labeling reagent for testing dysentery amoeba used therein.

Of the world population, about 500 million people are said to carry Entamoeba histolytica . Shigella amoeba is an anaerobic amoeba that is a pathogen that infests humans and causes gastrointestinal infectious diseases such as amoeba dysentery. About 10% of carriers of dysentery amoeba have developed amoeba dysentery, colitis, and liver abscess, and 40,000 to 110,000 people die each year.
In the early stages of dysentery, it is mild. However, in severe patients, ulcers occur in the large intestine, rectum and liver, and strawberry jelly-like mucous bloody stool is excreted several to several tens of times per day. In addition, symptoms such as intermittent diarrhea, gas accumulation in the intestine, and convulsive abdominal pain are also seen, and death may occur due to weakness. In addition, dysentery amoeba protozoa may reach the liver via the portal vein and develop extraintestinal amoebiasis.
Therefore, a method for rapidly detecting dysentery amoeba is required from the viewpoint of prevention of infection of dysentery amoeba and diagnosis of severity of amoeba dysentery.

  Conventional methods for testing dysentery amoeba include testing for dysentery by genetic testing, ELISA, and culture. However, in any of these methods, it usually takes two days or more until the test result is obtained, and it is not quick.

On the other hand, in recent years, fine particles of about several nm to 1 μm have been applied to the detection of various pathogenic bacteria and attracting attention. For example, 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 above labeling particles can be used as a labeling reagent that can be used for detection, quantification, etc. of biomolecules such as influenza nucleoprotein by binding biomolecules (antibodies, etc.) capable of binding to specific target molecules to the surface. It can be used (see, for example, Patent Document 1). However, a method for testing dysentery amoeba using fluorescent silica nanoparticles has not been developed yet.

International Publication No. 2012/147774 Pamphlet

An object of the present invention is to provide a test kit for detecting biomolecules that can be suitably used for detecting biomolecules with high sensitivity.
Moreover, this invention makes it a subject to provide the detection method of a biomolecule which can detect a biomolecule with high sensitivity.
Furthermore, this invention makes it a subject to provide the labeling reagent for a biomolecule detection which can be used suitably for the said test kit and a detection method.

The present inventors have conducted intensive studies in view of the above problems.
Usually, the major constituent proteins of pathogens are considered suitable as serum diagnostic antigens. However, an antibody against the antigen is not always useful for preventing or curing a disease. On the other hand, proteins useful as vaccines and fragments thereof can induce the production of antibodies useful for disease prevention and cure. However, vaccines are not necessarily the major antigens of pathogens. Usually, an antibody that is developed and used for diagnostic purposes only needs to have an interaction property with an antigen, so it does not matter whether the antibody itself is effective for prevention or cure. Thus, among all antibodies to pathogens in patient sera, the fraction may be minor. With a normal antibody detection system, it is not a good idea to detect minor antibodies in serodiagnosis.
In contrast, the present inventors have found that the detection sensitivity can be increased several hundred times by using fluorescent silica nanoparticles. Therefore, a practical detection system can be constructed even with a minor antibody as a target. It is a great merit that an antigen protein that has already been produced as a vaccine or whose production method has been established can be applied as it is as an antigen for diagnosis.
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 a labeling reagent for biomolecule detection composed of fluorescent silica nanoparticles,
The test piece includes a membrane to which the labeling reagent migrates, provided with a test region to which a vaccine for capturing biomolecules is fixed, and the labeling reagent for capturing the biomolecule as a target substance is immobilized on the test region. And
A biomolecule detection test kit in which a vaccine having a binding property to a biomolecule is introduced on the surface of the fluorescent silica nanoparticles.
(2) The test kit according to (1), wherein the vaccine specifically binds to an antigen derived from Shigella amoeba and a specific IgM antibody.
(3) The test kit according to (1) or (2), wherein the vaccine is a dysentery amoeba vaccine.
(4) The test kit according to any one of (1) to (3), wherein an average particle diameter of the fluorescent silica nanoparticles is 60 nm or more and 300 nm or less.
(5) The test kit according to any one of (1) to (4), wherein the membrane is further provided with a reference region to which a labeling reagent that does not capture a biomolecule as a target substance is immobilized. .

(6) A biomolecule detection method using a biomolecule detection test kit,
The biomolecule detection test kit has a biomolecule detection test piece and a biomolecule detection labeling reagent composed of fluorescent silica nanoparticles,
The test piece comprises a membrane to which the labeling reagent migrates, provided with a test region to which a vaccine for capturing biomolecules is fixed,
A vaccine having a binding property to a biomolecule is introduced on the surface of the fluorescent silica nanoparticles,
A method for detecting a biomolecule, comprising detecting a biomolecule by immobilizing a labeling reagent that has captured a biomolecule by binding of a vaccine to the test region and detecting the label of the immobilized labeling reagent.
(7) The method for detecting a biomolecule according to (6), wherein the vaccine specifically binds to an IgM antibody specific for an antigen derived from Shigella amoeba.
(8) The method for detecting a biomolecule according to (6) or (7), wherein the vaccine is a dysentery amoeba vaccine.
(9) The method for detecting a biomolecule according to any one of (6) to (8), wherein the biomolecule is an IgM antibody specific for an antigen derived from Shigella amoeba.
(10) The method for detecting a biomolecule according to any one of (6) to (9), wherein the fluorescent silica nanoparticles have an average particle size of 60 nm to 300 nm.
(11) The biomolecule according to any one of (6) to (10), wherein the membrane is further provided with a reference region on which a labeling reagent that does not capture a biomolecule as a target substance is immobilized. Detection method.

(12) A labeling reagent for detecting a biomolecule, comprising a fluorescent silica nanoparticle, wherein a vaccine having a binding property to a biomolecule is introduced on the surface of the fluorescent silica nanoparticle.
(13) The labeling reagent for biomolecule detection according to (12), wherein the vaccine specifically binds to an IgM antibody specific for an antigen derived from Shigella amoeba.
(14) The labeling reagent for biomolecule detection according to (12) or (13), wherein the vaccine is a dysentery amoeba vaccine.
(15) The labeling reagent for biomolecule detection according to any one of (12) to (14), wherein the fluorescent silica nanoparticles have an average particle size of 60 nm to 300 nm.

The test kit of the present invention can be suitably used for highly sensitive detection of biomolecules.
The biomolecule detection method of the present invention can detect biomolecules with high sensitivity.
Furthermore, the labeling reagent of the present invention can be suitably used for the test kit and the detection method.

FIG. 1 is a diagram schematically illustrating a preferred embodiment of a biomolecule detection or quantification test piece that can be used in the present invention, FIG. 1A is a plan view, and FIG. 1B is a developed cross-sectional view. FIG. 2 is a diagram schematically showing another preferred embodiment of a test piece for biomolecule detection or quantification that can be used in the present invention, FIG. 2 (a) is a plan view, and FIG. 2 (b) is a developed sectional view. is there. FIG. 3 is a diagram schematically showing still another preferred embodiment of a test piece for biomolecule detection or quantification that can be used in the present invention, FIG. 3A is a plan view, and FIG. 3B is a developed cross-sectional view. It is.

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 biomolecule detection test kit of the present invention has a test piece for biomolecule detection and a labeling reagent for biomolecule detection composed of fluorescent silica nanoparticles. And the detection method of the biomolecule of this invention detects a biomolecule using the said test kit.
Hereinafter, the configuration of the present invention will be described in detail focusing on preferred embodiments thereof.

[Biomolecule]
In the present invention, biomolecules derived from dysentery amoeba, acanthamoeba, hepatitis C virus, hepatitis B virus, rotavirus, pneumococci, etc. as detection targets (target substances) (hereinafter also simply referred to as “biomolecules”) There are no particular limitations, and examples include antigens, antibodies, nucleic acids, sugars, sugar chains, ligands, receptors, peptides, and other biologically active chemical substances. Among these, the present invention can be suitably used for detecting an antibody specific for a biomolecule derived from Shigella amoeba. Of the antibodies, IgM antibodies and IgG antibodies specific for Shigella amoeba-derived antigens are more preferred, and IgM antibodies are more preferred. IgM is usually produced early after reaction with the antigen, whereas IgG is produced behind IgM. Therefore, early diagnosis is possible by using IgM.
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 (hereinafter simply referred to as “labeling reagent”) comprising fluorescent silica nanoparticles. Biomolecules are detected by a specific molecule recognition reaction such as an antigen-antibody reaction using a biomolecule detection test kit (hereinafter also referred to simply as “test kit”).
Hereinafter, a test piece 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 test piece included in the test kit that can be preferably used in the first embodiment of the present invention is preferably a flat test piece as shown in FIGS. 1 and 2, and is a test piece for lateral flow. It is more preferable that
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 a biomolecule, a labeling reagent, 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. 1, the membrane 3 is provided with at least one test region 10. A vaccine (hereinafter also referred to as “capture substance”) capable of forming a complex with a biomolecule having a specific binding property with an antibody having a specific binding property to a biomolecule derived from Shigella amoeba has been tested in the test region 10. Has been introduced. When the labeling reagent that has moved from the sample pad 2 moves through the membrane 3, the labeling reagent that has captured the biomolecule as the target substance is immobilized on the test region 10.
In this test region 10, a complex composed of a capture substance-biomolecule-labeling reagent is formed, and the labeling reagent is concentrated. The biomolecule can be detected qualitatively or quantitatively depending on the amount of the labeling reagent has.

  As shown in FIG. 2, the membrane 3 is provided with a test region 20 for immobilizing the labeling reagent that has captured the biomolecule as the target substance, and captures the biomolecule as the target substance downstream of the test area 20. Therefore, a reference region 21 for immobilizing the labeling reagent that has not been immobilized in the test region 20 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.
As shown in FIG. 2, it is preferable to provide a reference region 21 downstream from the test region 20 in the direction in which the biomolecule and the labeling reagent move by capillary action.

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 0.3 μg or more, more preferably 0.5 μg or more, preferably 15 μg or less, and more preferably 3 μg or less. .
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 test piece included in the test kit that can be preferably used in the second embodiment of the present invention is preferably a flat test piece as shown in FIG. 3, and is a lateral flow test piece. It is more preferable.
The structure of the test piece is not particularly limited, but it has a sample pad, a member obtained by impregnating the labeling reagent (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. And it is a part which mixes the liquid and the labeling reagent containing the biomolecule which moved from the sample pad 2 by capillary action.
There is no restriction | limiting in particular in the quantity of the labeling reagent impregnated to the conjugate pad 5, It can set suitably.

(Membrane)
As shown in FIG. 3, 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 labeling reagent that has moved from the sample pad 2 moves through the membrane 3, the labeling reagent that has captured the biomolecule as the target substance is immobilized on the test region 20.
In this test region 20, a complex composed of a capture substance-biomolecule-labeling reagent is formed, and the labeling reagent is concentrated. The biomolecule can be detected qualitatively or quantitatively depending on the amount of the labeling reagent has.
Further, as shown in FIG. 3, a reference region 21 for immobilizing a labeling reagent that is not immobilized on the test region 20 because the biomolecule as a target substance is not captured downstream of the test region 20 on the membrane 3. 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.

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 0.3 μg or more, more preferably 0.5 μg or more, preferably 15 μg or less, and more preferably 3 μg or less. .
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]
The labeling reagent of the present invention comprises fluorescent silica nanoparticles. When fluorescent silica nanoparticles are used, the fluorescence intensity can be easily quantified by a fluorescence detection device, and biomolecules can be detected with high sensitivity and high accuracy. In addition, since biomolecules can be detected with high sensitivity, visual detection can also be realized. Furthermore, various functional groups can be introduced on the surface of the fluorescent silica nanoparticles, and the light emission in the test region is high-intensity. Therefore, when fluorescent silica nanoparticles are used, detection of biomolecules can be realized in a wide quantitative range.
Hereinafter, a labeling reagent composed of fluorescent 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 fluorescent silica nanoparticle, A fluorescent 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 fluorescent silica nanoparticles using a fluorescent dye as a fluorescent material will be specifically described. However, the present invention is not limited to this.
Silica particles containing a fluorescent dye are obtained by reacting a fluorescent dye with a silane coupling agent, and by using one or two or more silanes on 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 fluorescent dye having or added 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 and covalently bonds it. 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.

  Specific examples of the fluorescent dye having or added with the active group are represented by 5- (and -6) -carboxytetramethylrhodamine-NHS ester (trade name, manufactured by emp Biotech GmbH) and the following formulae, respectively. Examples thereof include fluorescent dye compounds having an NHS ester group such as DY550-NHS ester or DY630-NHS ester (both trade names, manufactured by Dyomics GmbH).

  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 fluorescent silica nanoparticles is not particularly limited, but is preferably 20 nm or more, more preferably 30 nm or more, still more 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. Is 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 diameter 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 average area of the circle diameter corresponding to the value obtained by dividing the total occupied area by the number of selected fluorescent silica nanoparticles (100) (average circle equivalent diameter) was obtained. Is.
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 fluorescent silica nanoparticles having a desired average particle size, ultrafiltration is performed using an ultrafiltration membrane such as YM-10 or YM-100 (both trade names, manufactured by Millipore), and the particle size is reduced. This is possible by removing particles that are too large or too small, or by centrifuging at an appropriate gravitational acceleration and collecting only the supernatant or precipitate.
The fluorescent silica nanoparticles are preferably monodispersed as a particulate material. The coefficient of variation of the particle size distribution of the fluorescent silica nanoparticles, that is, the so-called CV value is not particularly limited, but 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.

A vaccine having a binding property to a biomolecule is introduced on the surface of the fluorescent silica nanoparticles.
There is no restriction | limiting in particular as a method of introduce | transducing a vaccine on the surface of a fluorescent silica nanoparticle, According to a conventional method, it can introduce | transduce. For example, the vaccine may be introduced on the surface of the fluorescent silica nanoparticles by electrostatic attraction, van der Waals force, hydrophobic interaction or the like. Or you may introduce | transduce a vaccine into the surface of a fluorescent silica nanoparticle by the chemical bond of a crosslinking agent or a condensing agent. In addition, when the fluorescent silica nanoparticles are aggregated when a vaccine to be introduced on the surface of the fluorescent silica nanoparticles is introduced, the surface of the fluorescent silica nanoparticles may be subjected to surface treatment in advance by an alternate adsorption method.

  Hereinafter, a method for preparing fluorescent silica nanoparticles in which a vaccine is introduced on the particle surface will be described. However, the present invention is not limited to this.

First, a silane coupling agent having a reactive functional group is hydrolyzed, the hydrolyzed silane coupling agent and a hydroxyl group present on the surface of the fluorescent silica nanoparticle are subjected to polycondensation, and the reactive functional group is converted to a phosphorescent dye silica nanoparticle. Introduce to the surface of the particles.
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 group is preferably 0.002 to 0.2 pieces / nm ² in the fluorescent silica particle surface, and more preferably 0.002 to 0.1 pieces / nm ² . 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.

And the reactive functional group introduce | transduced on the surface of the fluorescent silica nanoparticle and a vaccine are combined according to a conventional method. For example, the reactive functional group and the vaccine can be bound by electrostatic attraction, van der Waals force, hydrophobic interaction, or the like. Or a reactive functional group and a vaccine can be combined with the chemical bond of a crosslinking agent or a condensing agent. The reactive functional group and the vaccine may be directly bonded to form a complex, or the reactive functional group and the vaccine may be indirectly bonded via another substance to form a complex. May be.
From the viewpoint of further preventing non-specific adsorption of the functional molecule-containing silica nanoparticles to which the biomolecule is bound, the fluorescent silica nanoparticles to which the vaccine is bound are optional blocking agents such as polyethylene glycol (PEG) and serum albumin (BSA). May be included. Moreover, you may contain antiseptic | preservatives, such as sodium azide.

There is no particular limitation on the amount of the vaccine introduced to the surface of the fluorescent silica nanoparticles, and it can be set as appropriate. For example, the introduction amount of the vaccine per 1 nm ² of the surface of the fluorescent silica nanoparticles is preferably 0.001 or more and 0.1 or less, and more preferably 0.003 or more and 0.015 or less.

[vaccine]
Next, the vaccine used in the present invention will be described.
A “vaccine” is a protein or fragment thereof that can induce the production of antibodies useful for the prevention or cure of disease. However, vaccines are not necessarily the major antigens of pathogens. Usually, an antibody developed or used for diagnostic purposes only needs to have an interaction property with an antigen, so it does not matter whether the antibody itself is effective for prevention or cure.
The vaccine used in the present invention is different in that respect, and was originally developed for the purpose of treatment and prevention. Thus, among all antibodies to pathogens in patient sera, the fraction may be minor. With a normal antibody detection system, it is not a good idea to detect minor antibodies in serodiagnosis.
However, by using fluorescent silica nanoparticles, the detection sensitivity can be increased several hundred times as shown in the examples described later. Therefore, according to the present invention, a practical detection system can be constructed even with a minor antibody as a target. It is a great merit that an antigen protein that has already been produced as a vaccine or whose production method has been established can be applied as it is as an antigen for diagnosis. In addition, there is a possibility that the antigen recognized by the produced antibody is different from the early stage to the recovery stage of infection.

Antigens are proteins and sugar chains contained in pathogens, and humans and animals produce antibodies by immune reaction against them. In general, when using antibodies or antigens for diagnostic purposes, many screening points are selected that exist at high concentrations. This is because it is more advantageous in sensitivity and efficiency to target many things.
On the other hand, the reaction with the vaccine is different from the reaction with the antigen. When an antibody that interacts with a vaccine exists, it is not necessary to newly develop the antibody for diagnostic use. However, antibodies that interact with vaccines are not necessarily produced from immunization against a large number of antigens.
According to the present invention, as shown in the examples described later, a biomolecule at a concentration that cannot be detected by colloidal gold particles can be detected by using a labeling reagent for detecting biomolecules composed of fluorescent silica nanoparticles combined with a vaccine. can do. By supplementing the vaccine antibody reaction system with fluorescent silica nanoparticles in this way, a vaccine that has been developed as an existing therapeutic use and not sufficient for a diagnostic use can be used effectively.

  As a specific example of the vaccine used in the present invention, there is a vaccine for dysentery amoeba described in JP-A-2009-22238.

[Method for detecting biomolecules]
Next, a biomolecule detection method using a biomolecule detection test kit having a biomolecule detection test piece having the above-described configuration and a labeling reagent composed of fluorescent 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 labeling reagent 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.
Then, the complex of the biomolecule that has moved from the sample pad 2 to the membrane 3 by capillary action and the fluorescent silica nanoparticles is concentrated by binding with the vaccine introduced on the test region (test line) of the test piece 1. . Then, the test area is irradiated with light, and the label of the concentrated labeling reagent 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 of the present invention is 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, a liquid sample that can contain the biomolecule and the labeling reagent are mixed. For example, when a biomolecule is detected by an immunochromatography method, the labeling reagent is contained in the sample pad 2 or the membrane 3, and the biomolecule is contained when a liquid sample that can contain the biomolecule passes through these members. A liquid sample that can be mixed and the labeling reagent may be mixed. Alternatively, the conjugate pad 5 containing the labeling reagent is provided upstream of the membrane 3, and when the liquid sample that can contain a biomolecule passes through this member, the liquid sample that can contain the biomolecule and the label You may mix with a reagent. 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 fluorescent silica nanoparticles is concentrated by binding with the vaccine introduced on the test region (test line) of the test piece 1. The Then, the test area is irradiated with light, and the label of the concentrated labeling reagent 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 method of detecting the label | marker of a labeling reagent, You may detect visually and may detect using a general purpose phosphorescent dye detector.
A general-purpose fluorescence detector typically comprises an excitation light source and a filter. 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 said filter is a filter which permeate | transmits only the light of a specific wavelength from an excitation light source, and selects suitably from the fluorescence wavelength of the said fluorescent microparticle, and a fluorescence wavelength. The fluorescence detector may include a photomultiplier tube or a CCD detector that receives fluorescence. Thereby, it is possible to detect fluorescence of intensity or wavelength that cannot be visually confirmed, and to measure the fluorescence intensity.
The wavelength of the excitation light to be irradiated is not particularly limited, but is preferably 300 nm or more, more preferably 400 nm or more, and particularly preferably 500 nm or more. Moreover, 700 nm or less is preferable, 600 nm or less is more preferable, and 550 nm or less is especially preferable.
The fluorescence wavelength is preferably 350 nm or more, more preferably 450 nm or more, and particularly preferably 530 nm or more. Moreover, 800 nm or less is preferable, 750 nm or less is more preferable, and 580 nm or less is particularly preferable.
In the fluorescence detector, the light irradiation unit and the detection unit may be integrated.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

(Preparation Example 1) Preparation of Labeling Reagent Consisting of Fluorescent Silica Nanoparticles with Vaccine Bonded to the Particle Surface (1) Preparation of Fluorescent Silica Nanoparticles Fluorescent silica nanoparticles containing carboxyrhodamine 6G as a fluorescent molecule were prepared by the following method.
31 mg of 5- (and-6) -carboxyrhodamine 6G • succinimidyl ester (trade name, manufactured by emp Biotech GmbH) was dissolved in 10 mL of dimethylformamide (DMF). To this, 12 μL of APS (manufactured by Shin-Etsu Silicone) was added 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.
After completion of the reaction, centrifugation was performed at a gravity acceleration of 18000 × g for 30 minutes, and the supernatant was removed. Distilled water (4 mL) was added to the precipitated 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 and ammonia contained in the dispersion.
As a result, 1.65 g of fluorescent silica nanoparticles containing fluorescent molecules having an average particle size of 271 nm were obtained (yield: about 94%).

(2) Introduction of vaccine to fluorescent silica nanoparticle surface 1 g of the fluorescent silica nanoparticle was dispersed in 150 mL of a mixed solution of water / ethanol = 1/4. To this, 1.5 mL of MPS (manufactured by Wako Pure Chemical Industries, Ltd.) was added. Subsequently, 20 mL of 28% aqueous ammonia was added and mixed at room temperature for 4 hours.
After completion of the reaction, centrifugation was performed at a gravity acceleration of 18000 × g for 30 minutes, and the supernatant was removed. Distilled water (10 mL) was added to the precipitated fluorescent silica nanoparticles to disperse the fluorescent silica nanoparticles, and centrifuged again at a gravity acceleration of 18000 × g for 30 minutes. This washing operation was further repeated twice to remove unreacted MPS, ammonia and the like contained in the dispersion. As a result, fluorescent silica nanoparticles into which thiol groups were introduced (average particle size 270 nm, hereinafter referred to as “thiol group-introduced fluorescent silica nanoparticles A”) were obtained.

To 40 μL of the thiol group-introduced fluorescent silica nanoparticle A dispersion prepared in Preparation Example 1 (concentration 25 mg / mL, dispersion medium: distilled water), DMF 460 μL was added and centrifuged at 15000 × 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 to disperse the thiol group-introduced fluorescent silica nanoparticles A. To this, 1 mg of 3-maleimidobenzoic acid as a linker molecule was added and mixed for 30 minutes, thereby forming a thioether bond between the maleimide group of the linker molecule and the thiol group of the thiol group-introduced fluorescent silica nanoparticle A. The reaction solution was centrifuged at 15000 × g for 10 minutes, and the supernatant was removed. 82.5 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 was added 12.4 μL of dysentery amoeba vaccine (1.3 mg / mL, manufactured by Tokai University) and mixed for 10 minutes.

Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. 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, Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) was used. As a result, the binding amount of the vaccine was 10 mg per 1 g of the fluorescent molecule-containing silica nanoparticles to which the antibody was bound.

(Preparation Example 2) Preparation of a labeling reagent comprising colloidal gold particles with the vaccine bound to the particle surface Add 100 μL of Shigella amoeba vaccine (1.0 mg / ml, manufactured by Tokai University) to 0.5 mL of colloidal gold (particle size 40 nm) Allowed to stand at room temperature for 10 minutes. Subsequently, 100 μL of a phosphate buffer (pH 7.5) containing 1% by weight of bovine serum albumin was added, and the mixture was allowed to stand at room temperature for 10 minutes. Thereafter, the mixture was centrifuged at 8,000 × G for 15 minutes, the supernatant was removed, and 100 μL of phosphate buffer (pH 7.5) containing 1% by weight of bovine serum albumin was added to disperse the particles.

(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 a strip shape having a width of 5 mm and a length of 60 mm to produce a test strip 1 for detecting biomolecules having the configuration shown in FIG.

(Test Example) Detection of IgM antibody specific to Shigella amoeba antigen A solution of IgM antibody specific to Shigella amoeba antigen was prepared with the composition shown in Table 1. Subsequently, 100 μL of IgM antibody solution specific for Shigella amoeba antigen and 2 μL of the colloid (2.5 mg / mL) of the labeling reagent prepared in Preparation Example 1 or 2 were mixed, and the mixture was used as a sample pad 2 of test strip 1. It was dripped in. After 15 minutes, a visual inspection was performed.
The results are shown in Table 1. In Table 1 below, “−” means undetectable and “+” means detectable. A “negative sample” is a buffer solution that does not contain IgM antibodies.

As shown in Table 1, when a labeling reagent composed of colloidal gold particles was used, the antibody concentration could be detected only at a high concentration of 1000 ng / mL.
On the other hand, according to the present invention, the antibody can be detected in a low concentration range of 1 ng / mL.

1 Test Strip 2 Sample Pad 3 Membrane 4 Absorption Pad 5 Conjugate Pad 6 Backing Sheet 10 Test Area 20 Test Area 21 Reference Area

Claims (15)

A biomolecule detection test kit having a test piece for biomolecule detection and a labeling reagent for biomolecule detection composed of fluorescent silica nanoparticles,
The test piece includes a membrane to which the labeling reagent migrates, provided with a test region to which a vaccine for capturing biomolecules is fixed, and the labeling reagent for capturing the biomolecule as a target substance is immobilized on the test region. And
A biomolecule detection test kit in which a vaccine having a binding property to a biomolecule is introduced on the surface of the fluorescent silica nanoparticles.   The test kit according to claim 1, wherein the vaccine specifically binds to an antigen derived from Shigella amoeba and a specific IgM antibody.   The test kit according to claim 1 or 2, wherein the vaccine is a dysentery amoeba vaccine.   The test kit according to any one of claims 1 to 3, wherein an average particle diameter of the fluorescent silica nanoparticles is 60 nm or more and 300 nm or less.   The test kit according to any one of claims 1 to 4, further comprising a reference region in which a labeling reagent that does not capture a biomolecule as a target substance is immobilized 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 biomolecule detection labeling reagent composed of fluorescent silica nanoparticles,
The test piece comprises a membrane to which the labeling reagent migrates, provided with a test region to which a vaccine for capturing biomolecules is fixed,
A vaccine having a binding property to a biomolecule is introduced on the surface of the fluorescent silica nanoparticles,
A method for detecting a biomolecule, comprising detecting a biomolecule by immobilizing a labeling reagent that has captured a biomolecule by binding of a vaccine to the test region and detecting the label of the immobilized labeling reagent.   The method for detecting a biomolecule according to claim 6, wherein the vaccine specifically binds to an IgM antibody specific for an antigen derived from Shigella amoeba.   The method for detecting a biomolecule according to claim 6 or 7, wherein the vaccine is a dysentery amoeba vaccine.   The method for detecting a biomolecule according to any one of claims 6 to 8, wherein the biomolecule is an IgM antibody specific for an antigen derived from Shigella amoeba.   The method for detecting a biomolecule according to any one of claims 6 to 9, wherein an average particle diameter of the fluorescent silica nanoparticles is 60 nm or more and 300 nm or less.   The biomolecule detection method according to any one of claims 6 to 10, wherein a reference region to which a labeling reagent that has not captured a biomolecule as a target substance is immobilized is further provided on the membrane.   A labeling reagent for detecting a biomolecule, comprising a fluorescent silica nanoparticle, wherein a vaccine having a binding property to a biomolecule is introduced on the surface of the fluorescent silica nanoparticle.   The labeling reagent for biomolecule detection according to claim 12, wherein the vaccine specifically binds to an IgM antibody specific for an antigen derived from Shigella amoeba.   The labeling reagent for biomolecule detection according to claim 12 or 13, wherein the vaccine is a Shigella amoeba vaccine. The biomolecule detection labeling reagent according to any one of claims 12 to 14, wherein the fluorescent silica nanoparticles have an average particle diameter of 60 nm or more and 300 nm or less.

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