JP2016057280A - Method and apparatus for detection or quantitative determination of biomolecule by competition method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detection or quantitative determination of biomolecules, the method that detects or quantitatively determines the biomolecules by a competition method with high sensitivity and high precision without due to non-uniformity or variations of components of a test kit and the variations of the flow of labeling reagent particles flowing through the test kit.SOLUTION: A test kit for detection or quantitative determination of biomolecules has test pieces for detection or quantitative determination of the biomolecules and labeling reagent particles for detection or quantitative determination of the biomolecules, in which the test pieces have a test area for capturing the biomolecules and the labeling reagent particles and materials competing with the biomolecules being a detection target are introduced on the surface of the labeling reagent particles. Also, a method for detection or quantitative determination of the biomolecules includes the steps of: dropping the mixture of the sample containing the biomolecules and the labeling reagent particles on the test pieces; measuring a measured value showing a labeling reagent particle weight connected in the test area by competing with the biomolecules; correcting the measured value showing the labeling reagent particle weight connected in the test area by the measured value showing the labeling reagent particle weight moving the test pieces; and detecting or quantitatively determining the biomolecules based on the corrected number value.SELECTED DRAWING: None

Description

  The present invention relates to a biomolecule detection or quantification method and a biomolecule detection or quantification apparatus.

As a method for detecting biomolecules such as nucleic acids and antigens in living bodies as target substances, there are a porous test piece in which a capturing substance for capturing biomolecules is immobilized on a carrier, and a detection method using labeled reagent particles. . In this method, the biomolecules and labeled reagent particles contained in the sample liquid are moved through the porous support of the test piece by capillary action. Then, for example, the labeling reagent particles are concentrated by bringing the capturing substance fixed in a line shape and the labeling reagent particles into contact with the porous support, and the line on which the capturing substance is fixed is colored with the labeling reagent particles. 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.

As a method for capturing a biomolecule with a capture substance immobilized on a porous test piece carrier and detecting the biomolecule, a sandwich method or a competitive method is known.
Among these, the competitive method is a method in which the capture substance immobilized on the porous support is contacted competitively with the labeling reagent particles and the target substance, and the color development degree derived from the labeling reagent particles in the area where the capture substance is immobilized. Based on this, the concentration of the target substance is quantified. In the competitive method, the labeled reagent particles and the target substance compete with each other and bind to the region where the capture substance is immobilized, so that the degree of color development derived from the labeled reagent particles increases as the concentration of the target substance decreases. Therefore, the detection method based on the competitive method can detect a very small amount of a low molecular weight biomolecule that cannot be detected by the sandwich method.
So far, a biomolecule detection method by a competitive method using various biomolecule detection test kits has been proposed (see, for example, Patent Documents 1 to 6).

International Publication No. 01/44811 Pamphlet International Publication No. 95/04283 Pamphlet Japanese Patent Laid-Open No. 7-270412 JP-A-7-159401 JP-A-6-102275 JP 2004-138550 A

As described above, an extremely small amount of a low molecular weight biomolecule that cannot be detected by the sandwich method can be detected with high sensitivity by the biomolecule detection method using the competitive method.
However, conventional biomolecule detection methods using the competitive method may cause errors in the quantified biomolecule concentration due to non-uniformity and variation in the components of the test kit and variations in the flow of labeled reagent particles flowing through the test kit. There is. Therefore, the conventional method for detecting biomolecules by the competitive method has a problem in the quantitative property of the detection target.

Therefore, the present invention detects or quantifies biomolecules with high sensitivity and high accuracy by a competitive method without depending on non-uniformity or variation in the components of the test kit, variation in the flow of labeled reagent particles flowing through the test kit, It is an object to provide a method for detecting or quantifying a biomolecule.
Furthermore, the present invention provides a biosensor for detecting or quantifying biomolecules with high sensitivity and high accuracy by a competitive method without being caused by non-uniformity or variation in components of the test kit, or variation in the flow of labeled reagent particles flowing through the test kit. It is an object to provide a molecule detection or quantification apparatus.

The present inventors have conducted intensive studies in view of the above problems. As a result, in the biomolecule detection or quantification method by the competitive method using the biomolecule detection or quantification test piece and the biomolecule detection or quantification test kit having the labeling reagent particles for biomolecule detection or quantification, the test piece The amount of labeled reagent particles that compete with and bind to biomolecules in the test area provided in the test area is corrected by the amount of labeled reagent particles that have moved the test piece. It has been found that the influence of the variation in the flow of the labeled reagent particles can be eliminated. As a result, it was found that a biomolecule can be detected or quantified with high sensitivity and high accuracy by a competitive method.
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 or quantification method by a competitive method using a biomolecule detection or quantification test kit,
The biomolecule detection or quantification test kit has a test piece for biomolecule detection or quantification, and labeled reagent particles for biomolecule detection or quantification,
The test piece has a test region for capturing biomolecules and labeled reagent particles,
A substance that competes with the biomolecule to be detected is introduced on the surface of the labeling reagent particle,
A mixture of a sample containing a biomolecule and the labeling reagent particles is dropped onto the test piece,
Measure the measured value indicating the amount of labeled reagent particles that compete with the biomolecule and bind to the test region,
The measured value indicating the amount of labeled reagent particles bound to the test area is corrected with the measured value indicating the amount of labeled reagent particles moved from the test piece,
Detect or quantify biomolecules based on the corrected values,
Biomolecule detection or quantification method.
(2) The biomolecule detection or quantification method according to (1), wherein the test piece is further provided with a reference region that captures labeled reagent particles that have not bound to the test region due to competition with the biomolecule. .
(3) The method for detecting or quantifying a biomolecule according to (1) or (2), wherein the labeling reagent particles are labeling reagent particles made of fluorescent silica nanoparticles.
(4) The biomolecule detection according to (3) above, wherein the measured value indicating the amount of labeled reagent particles bound to the test region is the emission intensity of the test region bound with the labeling reagent particles made of fluorescent silica nanoparticles. Or a quantitative method.
(5) The measured value indicating the amount of labeled reagent particles that have moved the test piece is the emission intensity of the test area and the emission intensity of the reference area to which the labeling reagent particles composed of fluorescent silica nanoparticles that have not bonded to the test area are bound. The biomolecule detection or quantification method according to item (3) or (4), wherein
(6) The luminescent intensity of the test region is divided by the total value of the luminescent intensity of the test region and the luminescent intensity of the reference region, and the measured value indicating the amount of labeled reagent particles bound to the test region is corrected. A method for detecting or quantifying the described biomolecule.
(7) A reference substance having binding properties to the reference region is introduced on the surface of the labeling reagent particle,
Due to the binding between the reference substance and the reference region, the labeled reagent particles that have not been bound to the test region are captured in the reference region.
The method for detecting or quantifying a biomolecule according to any one of (2) to (6).
(8) In the labeling reagent particle, the labeling reagent particle and a substance that competes with a biomolecule are bound to each other through the reference substance introduced on the surface of the labeling reagent particle. A method for detecting or quantifying biomolecules.
(9) The reference substance is at least one substance selected from the group consisting of bovine serum albumin, casein, mussel hemocyanin, human serum albumin, ovalbumin, globulin, avidin, and streptavidin (7 ) Or the biomolecule detection or quantification method according to item (8).
(10) The method for detecting or quantifying a biomolecule according to any one of (1) to (9), wherein the labeling reagent particles have an average particle size of 30 nm to 500 nm.

(11) A biomolecule detection or quantification device,
A light irradiation unit that irradiates light to a test region of a test piece for biomolecule detection or quantification in which a biomolecule and a labeled reagent particle compete and bind to each other;
A detection unit for detecting a measurement value indicating the amount of labeled reagent particles bound to the test region;
An apparatus for detecting or quantifying biomolecules, comprising:
(12) irradiating light to a reference region that captures labeled reagent particles that have not bound to the test region due to competition with a biomolecule;
A detection unit for detecting a measurement value indicating the amount of labeled reagent particles bound to the reference region;
The biomolecule detection or quantification device according to (11), further comprising:
(13) The labeling reagent particles are labeling reagent particles composed of fluorescent silica nanoparticles,
The detection unit for detecting the measurement value indicating the amount of labeled reagent particles bound to the test region and the detection unit for detecting the measurement value indicating the amount of labeled reagent particle bound to the reference region are the test region or reference region to which the labeling reagent particles are bound. The amount of labeled reagent particles bound is calculated from the measured emission intensity,
The biomolecule detection or quantification device according to (11) or (12).
(14) The method according to any one of (11) to (13), further including a correction unit that corrects the measurement value indicating the amount of labeled reagent particles bound to the test region with the measurement value indicating the amount of labeled reagent particles moved from the test piece. The biomolecule detection or quantification apparatus according to any one of the above.

The biomolecule detection or quantification method of the present invention is a biomolecule with high sensitivity and high accuracy by a competitive method without depending on non-uniformity or variation in the components of the test kit, or variation in the flow of labeled reagent particles flowing through the test kit. Can be detected or quantified.
Furthermore, the biomolecule detection or quantification device of the present invention is highly sensitive and highly accurate by the competitive method, without causing non-uniformity or variations in the components of the test kit and variations in the flow of labeled reagent particles flowing through the test kit. Molecules can be detected or quantified.

It is a figure which shows typically the test piece for a biomolecule detection or fixed_quantity | quantitative_assay which can be used by this invention, Fig.1 (a) is a top view, FIG.1 (b) is sectional drawing. It is a graph which shows the result of the detection test of the biomolecule performed in the Example (the correlation between the common logarithm of the progesterone concentration and the emission intensity of the test region of the test piece for biomolecule detection or quantification). Results of the biomolecule detection test conducted in the examples (the common logarithm of progesterone concentration and the luminescence intensity of the test area of the test piece for biomolecule detection or quantification are the luminescence intensity of the test area and the luminescence intensity of the reference area. It is a graph which shows the correlation with the value divided by the total value.

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 or quantification method of the present invention uses a biomolecule detection or quantification test kit (hereinafter also simply referred to as “test kit”) to detect biomolecules by a competitive method. The test kit for detecting or quantifying biomolecules has a test piece (hereinafter also referred to as “test strip”) and labeled reagent particles for detecting or quantifying biomolecules (hereinafter also simply referred to as “labeled reagent particles”).
Hereinafter, the configuration of the present invention will be described in detail focusing on preferred embodiments thereof.

[Competitive Law]
In the present invention, biomolecules are detected or quantified by a competitive method. Here, the “competitive method” is a method of competing between a measurement target substance (target substance) and labeled reagent particles, and capturing the measurement target substance and labeled reagent particles with a capture substance fixed to the porous support. It is. In the competitive method, when the amount of the labeling reagent particles is larger than that of the substance to be measured, the binding of the labeling reagent particles to the capture substance is superior. Therefore, the smaller the amount of the substance to be measured, the higher the degree of labeling with the labeling reagent particles in the region where the capture substance is immobilized. On the other hand, when the measurement target substance is present in a large amount, the binding of the measurement target substance to the capture substance is dominant, and therefore the degree of labeling in the region where the capture substance is fixed decreases. The competition method is based on such a mechanism, and measures the degree of labeling in the area where the capture substance is immobilized, and indirectly uses the phenomenon that the degree of labeling decreases as the concentration of the target substance increases. Quantify the concentration of the substance to be measured.

[Biomolecule]
In the present invention, the biomolecule as a detection target substance (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. .
As described above, in the present invention, biomolecules are detected or quantified by a competitive method. In the present invention, the biomolecule has no particular problem as long as the biomolecule can be recognized by detection or quantification by a competitive method. For example, the molecular weight of the biomolecule as the detection target substance in the present invention is preferably 40 or more, and more preferably 80 or more.
In general, the biomolecule detection method by the sandwich method is not suitable for detection of low molecular weight biomolecules. This is because it is necessary to bind two types of antibodies to biomolecules in the sandwich method. If the biomolecule is small, when one antibody binds to the biomolecule, the other antibody cannot bind due to the steric hindrance of the other antibody. Therefore, when detecting or quantifying a low molecular weight biomolecule, the competitive method is a relatively superior method compared to the sandwich method. Therefore, from the viewpoint that the detection or quantification method of the present invention is superior to the sandwich method, the biomolecule preferably has a low molecular weight. For example, the molecular weight of the biomolecule as the detection target substance in the present invention is preferably 1,000 or less, and more preferably 500 or less. However, such a molecular weight range does not indicate that a large molecular weight is not suitable for the competitive method.

In the present invention, the sample containing biomolecules is not particularly limited, but is a clinical sample (for example, blood, plasma, serum, lymph, urine, saliva, pancreatic juice, gastric juice, sputum, wipes collected from mucous membranes such as nose and throat) Body fluids such as liquids and stool), food samples (eg liquid beverages, semi-solid foods, solid foods, etc.), environmental sampling samples (eg soil, rivers, seawater and other natural samples, factory production lines and clean rooms) Sampling sample, wiped sample, etc.) by an air sampler installed in
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 detection or quantification of biomolecules]
Although there is no restriction | limiting in particular in the shape of the test piece contained in the test kit used by this invention, It is preferable that it is a planar test piece, and it is more preferable that it is a test piece for lateral flow.
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 competitively capturing the biomolecule and the labeled reagent particle, and the absorption pad are arranged in this order. It is preferable that the structures are connected in series so that capillarity occurs between them. And each constituent member is preferably backed by a backing sheet with an adhesive.
Hereinafter, a preferred embodiment of the present invention will be described with reference to FIG. 1 for a test piece having the above shape and structure. However, the present invention is not limited to this.

(Sample pad)
The sample pad 2 is a component that drops a mixture of a sample containing biomolecules and labeled reagent particles. 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 and labeling reagent particles 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 capture substance (hereinafter referred to as the “first capture agent” in the present specification, which has a specific binding property to each of the biomolecule and the labeling reagent particle and can form a complex with the biomolecule and the labeling reagent particle competitively) A trapping substance ”is also introduced into a predetermined area of the test area 10. Thereby, the biomolecule and the labeling reagent particle are captured in the test region 10 competitively.

  As shown in FIG. 1, the membrane 3 may further be provided with a reference region 11 that captures labeled reagent particles that have not bound to the test region 10 due to competition with biomolecules. The reference region 11 has a specific binding property to the labeling reagent particles, and a capture substance that can form a complex with the labeling reagent particles (hereinafter also referred to as “second capture substance” in this specification) is the membrane 3. It can be provided by fixing to a predetermined area. Note that the second capture substance is preferably a substance that has a specific binding property to the labeling reagent particles but does not have a binding property to the biomolecule that is the detection target material.

The shape of the test region 10 and the reference region 11 is not particularly limited as long as each capture substance is locally immobilized, and examples thereof include a line shape, a circular shape, and a belt shape. In the present invention, each of the test region 10 and the reference region 11 is preferably in a line shape, and more preferably in a line shape having a width of 0.5 to 1.5 mm.
In addition, as shown in FIG. 1, it is preferable to provide the reference area | region 11 downstream from the test area | region 10 with respect to the direction where a biomolecule and a labeling reagent particle move by capillary action.

The first capture substance used in the test region 10 has specific binding properties for each of the biomolecule and the labeling reagent particle. Therefore, the labeled reagent particles and the biomolecules that have moved from the sample pad 2 by capillary action compete with each other and bind to the test region 10 where the first capture substance is immobilized.
The first capture substance used in the test region 10 is not particularly limited as long as it has specific binding properties for the labeling reagent particles and the biomolecule. Examples of the combination of the first capture substance and the biomolecule include an antibody and its antigen combination, an antigen and its antibody combination, and an oligonucleotide that hybridizes with the nucleic acid under stringent conditions with a nucleic acid (DNA, RNA, etc.) A combination of a receptor and its ligand, a combination of a ligand and its receptor, a combination of a lectin and a sugar chain, and a combination of an aptamer and a molecule that specifically binds to the aptamer.
In the present specification, “stringent conditions” include, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W., et al. Russell., Cold Spring Harbor Laboratory Press], for example, 6 × SSC (composition of 1 × SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% Examples include conditions in which a solution containing SDS, 5 × Denhart and 100 mg / mL herring sperm DNA is incubated with a probe at 65 ° C. for 8 to 16 hours and hybridized.

As described above, when the reference region 11 is provided on the membrane 3, the second capture substance for capturing only the labeling reagent particles is immobilized. The second capture substance has a specific binding property to the labeling reagent particle, but does not have a binding property to the biomolecule to be detected. Therefore, only the label reagent particles are bound to the reference region 11.
The second capture substance used for the reference region 11 can be appropriately selected from those having no binding property to biomolecules and having specific binding property to labeled reagent particles. Specifically, it can be appropriately selected from antibodies, antigens, nucleic acids, receptors, ligands, sugar chains, aptamers and the like.

By adjusting the amount of the first capture substance immobilized in the test region 10, the concentration range of the biomolecule to be quantified can be appropriately adjusted. For example, when the amount of the first capture substance immobilized is decreased, the range in which the biomolecule can be quantified shifts to the low concentration side. On the other hand, when the amount of the first capture substance immobilized is increased, the range in which the biomolecule can be quantified shifts to the high concentration side. However, if the fixed amount of the first capture substance is too small, sufficient line strength cannot be obtained, and the concentration range that can be quantified becomes narrow. Further, if the amount of the first capture substance immobilized in the test region 10 is more than the amount of the biomolecule, the biomolecule and the label reagent particle are all in the first state without competing between the biomolecule and the label reagent particle. It will be trapped by the trapping substance. Therefore, it is necessary to appropriately set the immobilization amount of the first capture substance in the test region 10 so that the biomolecule and the labeled reagent particle compete with each other and are captured by the first capture substance.
From the above viewpoint, when the shape of the test region 10 is a line, the amount of the first capture substance immobilized per unit length (cm) is preferably 0.001 μg or more, more preferably 0.01 μg or more, and 10 μg. The following is preferable, and 2 μg or less is more preferable. The amount of the second capture substance immobilized in the reference region 11 is not particularly limited as long as a sufficient amount of particles can be bound to detect the labeled reagent particles with a detection or quantification device. For example, when the shape of the reference region 11 is a line, the immobilized amount of the second capture substance per unit length (cm) is preferably 0.1 μg or more, more preferably 1 μg or more, preferably 20 μg or less, preferably 5 μg. The following is more preferable.
As a method for immobilizing each of the first capture substance and the second capture substance, a solution of each capture substance is applied to a predetermined region of the membrane 3, dropped or sprayed, and then dried and immobilized by physical adsorption. Is mentioned. Further, 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 immobilization of each capture substance.

(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 5 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 5).

[Labeling reagent particles]
As the labeling reagent particles used in the present invention, those usually applied to this kind of test kit can be used as appropriate. For example, labeling reagent particles composed of fluorescent silica nanoparticles, labeling reagent particles composed of light absorbing silica nanoparticles, labeling reagent particles composed of fluorescent latex nanoparticles, labeling reagent particles composed of light absorbing latex nanoparticles, labeling reagent particles composed of semiconductor fine particles, gold colloid Examples thereof include labeling reagent particles composed of particles and particles labeled with a radioactive substance.
The labeling reagent particles used in the present invention are preferably labeling reagent particles made of 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. 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, labeling reagent particles made 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.
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 substance that competes with the biomolecule to be detected is introduced on the surface of the fluorescent silica nanoparticles. By introducing a substance that competes with the biomolecule on the surface of the fluorescent silica nanoparticle, the biomolecule and the labeled reagent particle compete with each other and bind to the test region.
The substance that competes with the biomolecule is not particularly limited as long as it can compete with the biomolecule and can bind the labeling reagent particles to the test region 10, and is immobilized on the biomolecule to be detected or the test region 10. It can be appropriately selected depending on the first capture substance and the like. Note that the substance that competes with the biomolecule to be introduced onto the surface of the fluorescent silica nanoparticle may be the same kind of substance or a different kind of substance from the biomolecule to be detected.

When the reference region 11 is provided on the test piece 1 as described above, a reference substance having a binding property to the reference region is introduced onto the surface of the fluorescent silica nanoparticles. Due to the binding property between the reference substance and the second capture substance immobilized on the reference region 11, the labeled reagent particles that are not bound to the test region 10 due to competition with the biomolecule are captured in the reference region 11.
There is no particular limitation on the reference substance that can be preferably used in the present invention. Specifically, depending on the second capture substance immobilized on the reference region 11, protein (for example, bovine serum albumin (hereinafter also referred to as “BSA”), casein, mussel hemocyanin, human serum albumin, ovalbumin , Globulin, avidin, streptavidin, etc.), amino acids, tag peptides (eg, His tag, FLAG tag, etc.), antibodies, nucleic acids, sugar chains, and the like.

There is no particular limitation on the method for introducing the substance competing with the biomolecule and the reference substance onto the surface of the fluorescent silica nanoparticles, and the substance can be introduced according to a conventional method. For example, a substance that competes with a biomolecule by electrostatic attraction, van der Waals force, hydrophobic interaction, and the like, and a reference substance may be introduced on the surface of each fluorescent silica nanoparticle. Alternatively, a substance that competes with a biomolecule and a reference substance may be introduced onto the surface of each fluorescent silica nanoparticle by chemical bonding of a crosslinking agent or a condensing agent. In addition, when fluorescent silica nanoparticles aggregate when introducing a substance that competes with a biomolecule and / or a reference material on the surface of the fluorescent silica nanoparticle, the surface of the fluorescent silica nanoparticle is previously subjected to surface treatment by an alternate adsorption method. You may keep it.
The substance that competes with the biomolecule and the reference substance may be independently introduced onto the surface of the fluorescent silica nanoparticle, or the complex of the substance that competes with the biomolecule and the reference substance is introduced onto the surface of the fluorescent silica nanoparticle. It may be introduced. The fluorescent silica nanoparticles that can be preferably used in the present invention have a reference substance bonded to the surface thereof, and it is preferable that the fluorescent silica nanoparticles and a substance that competes with a biomolecule are bonded via the reference substance. . By setting it as such a structure, the efficiency of the fixation | immobilization of the substance which competes with the biomolecule to the fluorescence silica nanoparticle improves. Furthermore, non-specific adsorption of fluorescent silica nanoparticles to the test region can be suppressed, and the accuracy of the biomolecule quantification results can be further improved.

  Hereinafter, an example of a method for preparing fluorescent silica nanoparticles in which a substance that competes with a biomolecule and a reference substance are introduced using BSA as a reference substance will be described. However, the present invention is not limited to this.

First, the silane coupling agent having a reactive functional group is hydrolyzed, the hydrolyzed silane coupling agent and the hydroxyl group present on the surface of the fluorescent silica nanoparticle are subjected to condensation polymerization, and the reactive functional group is converted to the fluorescent silica nanoparticle. Introduce to the surface.
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.

The reactive functional group introduced into the surface of the fluorescent silica nanoparticle is reacted with a linker molecule that forms a chemical bond therewith. Then, the linker molecule and BSA are bonded to form a complex of fluorescent silica nanoparticles and BSA.
When the reactive functional group is a thiol group, fluorescent silica nanoparticles into which the thiol group is introduced 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 activated esterified with carbodiimide and the amino group of BSA, thereby forming a complex of fluorescent 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 fluorescent 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 fluorescent silica nanoparticle and the substance which competes with a biomolecule are couple | bonded according to a conventional method. For example, BSA and a substance that competes with a biomolecule can be bound by electrostatic attraction, van der Waals force, hydrophobic interaction, or the like. Or BSA and the substance which competes with a biomolecule can be combined with the chemical bond of a crosslinking agent or a condensing agent. In addition, BSA and a substance that competes with a biomolecule may directly bind to form a complex, or BSA and a substance that competes with a biomolecule bind indirectly through another substance. A complex may be formed.

The amount of the substance that competes with the biomolecule on the surface of the fluorescent silica nanoparticle can be appropriately set without any particular limitation. For example, the introduction amount of the substance that competes with the biomolecule per 1 nm ² of the surface of the fluorescent silica nanoparticle is preferably 0.0001 or more and 0.1 or less, and more preferably 0.001 or more and 0.05 or less.

  There is no particular limitation on the amount of the reference substance bound to the surface of the fluorescent silica nanoparticles, and it can be set as appropriate. For example, the binding amount of the reference substance per 1 g of the fluorescent silica nanoparticles is preferably 1 mg or more and 200 mg or less, and more preferably 10 mg or more and 50 mg or less.

[Method for detecting or quantifying biomolecules]
In the biomolecule detection or quantification method of the present invention, a mixture of a biomolecule-containing sample and labeled reagent particles is dropped onto the test piece 1 and measured to indicate the amount of labeled reagent particles bound to the test region 10 in competition with the biomolecule. The measured value indicating the amount of labeled reagent particles bound to the test region is corrected with the measured value indicating the amount of labeled reagent particles moved from the test piece 1, and the biomolecule is detected or quantified based on the corrected value. To do.
Hereinafter, the biomolecule detection or quantification method having the above-described configuration will be described based on preferred embodiments. However, the present invention is not limited to this.

  A mixture of a liquid sample that can contain a biomolecule and the labeled reagent particles having the above-described configuration is dropped onto the sample pad 2 of the test piece 1 for biomolecule detection or quantification. The amount of the liquid sample dripped onto the sample pad 2 can be appropriately set according to the configuration of the test piece 1. In addition, the amount of biomolecule and labeling reagent particles dropped onto the sample pad 2 can be appropriately set according to the amount of the first capture substance immobilized in the test region 10 of the test piece 1.

The biomolecules that have moved from the sample pad 2 to the membrane 3 due to capillary action and the labeled reagent particles compete with each other, and are concentrated by binding to the first capture substance fixed on the test region 10 of the test piece 1. Is done. Then, the test region 10 is irradiated with light, and the label of the concentrated labeling reagent particles (the emission intensity of the dye contained in the fluorescent silica nanoparticles) is detected.
Here, the relationship between the emission intensity of the test region 10 and the amount of particles bound to the test region 10 is determined. The amount of labeled reagent particles bound to the test region 10 can be calculated from the relationship between the determined light emission intensity and the amount of particles and the actually measured light emission intensity of the test region 10. As a result, the biomolecule contained in the sample can be detected indirectly qualitatively or quantitatively.

There is no restriction | limiting in particular in the detection method of the emitted light intensity of the pigment | dye contained in fluorescent silica nanoparticle, You may detect visually and may detect using a general purpose fluorescence detector.
A general-purpose fluorescence detector includes 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.

  Next, the measured value indicating the amount of labeled reagent particles bound to the test region 10 is corrected with the measured value indicating the amount of labeled reagent particles moved from the test piece 1. This eliminates the effects of non-uniformity and variations in the components of the test kit and variations in the flow of labeled reagent particles flowing through the test kit.

The amount of the labeled reagent particles that have moved the test piece 1 can be determined by a conventional method.
For example, the reference region 11 may be provided downstream of the test region 10 in the test piece 1, and labeled reagent particles that have not been bound to the test region 10 due to competition with biomolecules may be bound to the reference region 11. Then, the reference region 11 is irradiated with light, the light emission intensity of the reference region 11 is measured, and the amount of the labeled reagent particles that have moved the test piece is calculated from the total value of the light emission intensity of the test region 10 and the light emission intensity of the reference region 11. can do.
Or the density | concentration of the labeling reagent particle | grains in the mixture dripped at the test piece 1 can be determined, and the amount of the labeling reagent particle which moved the test piece 1 can also be calculated from the quantity of the mixture actually dripped at the test piece 1.

  As a specific method for correcting the amount of labeled reagent particles bound to the test region 10 with the total amount of labeled reagent particles that have moved the test piece 1, the emission intensity of the test region 10 is changed to the emission intensity of the test region 10 and the reference region. A method of correcting the light emission intensity of the test region 10 by dividing by the total value of the light emission intensity of 11 is mentioned.

[Biomolecular detection or quantification device]
The biomolecule detection or quantification apparatus of the present invention includes a light irradiation unit that irradiates light to the test region 10 of the test piece 1 and a detection unit that detects a measurement value indicating the amount of labeled reagent particles bound to the test region 10. .
The biomolecule detection or quantification device of the present invention is coupled to the reference region 11 and a light irradiation unit that irradiates light to the reference region 11 that captures the labeled reagent particles that are not bound to the test region 10 due to competition with the biomolecule. A detection unit for detecting a measurement value indicating the amount of the labeled reagent particles may be further provided.

  The biomolecule detection or quantification apparatus of the present invention can be suitably used for the above-described biomolecule detection or quantification method using the labeling reagent particles made of fluorescent silica nanoparticles. In the biomolecule detection or quantification apparatus of the present invention, a detection unit for detecting a measurement value indicating the amount of labeled reagent particles bound to the test region and a detection unit for detecting a measurement value indicating the amount of labeling reagent particle bound to the reference region Measures the emission intensity of the test region 10 or the reference region 11 to which the labeling reagent particles are bound, and calculates the amount of bound labeling reagent particles from the measured emission intensity.

  The biomolecule detection or quantification apparatus of the present invention further corrects the measured value indicating the amount of the labeled reagent particles bound to the test region 10 with the measured value indicating the amount of the labeled reagent particles moved from the test piece 1 (central). It is preferable to have correction means such as a computer in which a recording medium such as an arithmetic processing unit), a memory, or a hard disk is built. Furthermore, the biomolecule detection or quantification apparatus of the present invention calculates the concentration of the biomolecule based on the numerical value corrected by the correction means, and the biomolecule such as a computer incorporating a recording medium such as a CPU, a memory, a hard disk or the like. It is preferable to provide a concentration calculation means.

  In the biomolecule detection or quantification apparatus of the present invention, the light irradiation unit and the detection unit may be integrated as in the general-purpose fluorescence detector described above.

  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 Particles Consisting of Fluorescent Silica Nanoparticles Introduced with Substances Competing with Biomolecules via Reference Substances (1) Preparation of Fluorescent Silica Nanoparticles Fluorescent Silica Nanos Containing Fluororhodamine Carboxyrhodine 6G Particles 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 thiol group into fluorescent silica nanoparticles 1 g of the fluorescent silica nanoparticles obtained above was dispersed in 150 mL of a mixture 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 (hereinafter referred to as “thiol group-introduced fluorescent silica nanoparticles A”) were obtained.
About 500 mg of obtained thiol group-introduced fluorescent 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 fluorescent silica nanoparticles A was 0.046 / nm ² .

(3) Binding of thiol group-introduced fluorescent silica nanoparticles A and BSA By the following method, the thiol group-introduced fluorescent silica nanoparticles A were bound to the thiol group-introduced fluorescent silica nanoparticles A as a reference substance labeled with BSA.
420 μL of DMF was added to 80 μL of a dispersion of thiol group-introduced fluorescent silica nanoparticles A (concentration 25 mg / mL, dispersion medium: distilled water), 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. DMF500 μL was added again to disperse the thiol group-introduced fluorescent silica nanoparticles A. To this, 1 mg of 3-maleimidobenzoic acid was added as a linker molecule and mixed for 30 minutes. In this way, a thioether bond was formed between the maleimide group of the linker molecule and the thiol group of the thiol group-introduced fluorescent silica nanoparticle A.

The reaction mixture was centrifuged at 15000 × g for 10 minutes at a gravitational acceleration. After removing the supernatant, 88.6 μL of distilled water was added to disperse the particles. Subsequently, 0.5 M 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 μL of 3-dimethylaminopropyl) carbodiimide) was added and mixed. To this, 6.0 μL of BSA (10 mg / mL, Sigma Aldrich) was added and mixed for 60 minutes.
Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. To this, 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.

Quantification of BSA bound to thiol group-introduced fluorescent silica nanoparticles A was performed using 200 μL of the obtained colloid as a sample. For quantification of BSA, PierceBCA Protein Assay Kit (trade name, manufactured by Thermo Fisher Scientific) was used.
As a result, the binding amount of BSA per 1 g of thiol group-introduced fluorescent silica nanoparticles A was 14.6 mg.

(4) Binding of thiol group-introduced fluorescent silica nanoparticles A and progesterone via BSA By the following method, progesterone was bound to the thiol group-introduced fluorescent silica nanoparticles A as a substance that competes with biomolecules via BSA. .
200 μL of the colloid remaining after BSA quantification was centrifuged at 15000 × g for 10 minutes, and the supernatant was removed. To this, 500 μL of 10 mM KH ₂ PO ₄ (pH 7.5) was added to disperse the particles. The dispersion was centrifuged for 10 minutes at a gravitational acceleration of 15000 × g and the supernatant was removed. Subsequently, 74.6 μL of distilled water was added to disperse the particles. Further, 100 μL of 0.5M MES (pH 6.0), 230.4 μL of 50 mg / mL NHS, and 75 μL of 19.2 mg / mL EDC were added and mixed well.
To this was added 20 μL of progesterone-11α-hemisuccinate (0.05 mg / mL) and mixed for 60 minutes.

The reaction solution was centrifuged for 10 minutes at a gravitational acceleration of 15000 × g, and the supernatant was collected.
As a result of quantitative measurement of progesterone contained in the collected supernatant by ELISA, the decrease from the initial concentration was 0.13 μg. That is, it was found that 0.13 μg of progesterone was bound to the fluorescent silica nanoparticles. When this was calculated per thiol group-introduced fluorescent silica nanoparticle A per 1 nm ² , the amount of progesterone bound was 0.017 per 1 nm ² .

After collecting the supernatant, 400 μL of 10 mM KH ₂ PO ₄ (pH 7.5) was added to the sediment of the particles to disperse the particles. Subsequently, centrifugation was performed at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. Subsequently, 400 μL of 10 mM KH ₂ PO ₄ (pH 7.5) was added to disperse the particles to obtain a colloid of fluorescent silica nanoparticles to which progesterone was bonded via BSA.

Preparation Example 2 Preparation of Test Strip for Biomolecule Detection or Quantification A test strip for biomolecule detection or quantification was prepared by the following method.
Rabbit-derived anti-progesterone polyclonal antibody (Sigma) as a test region (test line) 10 having a width of about 1 mm at a position about 8 mm from the end of the membrane 3 (length 25 mm, trade name: Hi-Flow Plus180 membrane, manufactured by MILLIPORE) A solution containing 1 mg / mL of Aldrich ((50 mM KH ₂ PO ₄ , pH 7.0) + 5% sucrose) was applied at a coating amount of 0.75 μL / cm.
In addition, as a reference region (control line) 11 having a width of about 1 mm, a solution containing 1 mg / mL of rabbit-derived anti-BSA polyclonal antibody (Millipore) ((50 mM KH ₂ PO ₄ , pH 7.0) sugar free) Was applied at a coating amount of 0.75 μL / cm.
Thereafter, the membrane 3 was dried at 50 ° C. for 30 minutes to produce a membrane 3. The distance between the test line and the control line was 5 mm.

In the order of sample pad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE) 2, membrane 3 and absorption pad (Cellulose Fiber Sample Pad (CFSP), manufactured by MILLIPORE) 4, a backing sheet (trade name: AR9020, (Made by Adhesives Research)). The membrane 3 was arranged so that the test line was located on the sample pad 2 side and the control line was located 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 biomolecule detection or quantification.

Test Example 1 Progesterone Quantification A solution of progesterone (Sigma Aldrich) was prepared at the concentrations shown in Table 1.
Subsequently, 100 μL of the progesterone solution and 2 μL of the colloid (2.5 mg / mL) of the fluorescent silica nanoparticles prepared in Preparation Example 1 were mixed, and the mixture was dropped onto the sample pad 2 of the test strip 1.
After 15 minutes, the following detection device was used to digitize the emission intensity (T) of the test line and the emission intensity (C) of the control line. Further, a value (T / (T + C)) obtained by dividing the light emission intensity (T) of the test line by the total value of the light emission intensity (T) of the test line and the light emission intensity (C) of the control line was calculated.
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 532 nm, and has a mechanism for irradiating the sample with the laser diode and transmitting the reflected light through an optical filter that transmits only light having a wavelength of 550 nm or more and then receiving the light with the PMT. .

Based on the results shown in Table 1, a graph was prepared by plotting the common logarithm of progesterone concentration on the horizontal axis and the emission intensity (T) of the test line on the vertical axis. The result is shown in FIG. The horizontal axis represents the common logarithm of progesterone concentration, and the vertical axis represents the test line emission intensity (T) divided by the sum of the test line emission intensity (T) and the control line emission intensity (C) (T / ( A graph plotting T + C)) was prepared. The result is shown in FIG.
And the determination coefficient was calculated | required about each graph shown in FIG. The coefficient of determination is the regression log of the linear equation using the least squares method with the horizontal axis as the common logarithm of progesterone concentration (ng / mL) and the vertical axis as the luminescence intensity (au) or (T / (T + C)) of the test line. Calculated by creating The results are shown in Table 2.

  As can be seen from FIGS. 2 and 3 and Table 2, the value of the coefficient of determination is closer to 1 when the (T / (T + C)) value is used on the vertical axis than when the T value is used on the vertical axis. The variation in the measured values of the test line emission intensity and the control line emission intensity was small.

Test Example 2 Accuracy Evaluation of Measurement Value of Progesterone Amount The test was carried out 10 times in the same manner as in Test Example 1 for a solution having a progesterone concentration of 5 ng / mL. Then, the variation coefficient CV value (standard deviation / average value) of the emission intensity (T) of the test line and the value obtained by dividing the emission intensity of the test line by the total value of the emission intensity of the test line and the emission intensity of the control line (T / (T + C)) variation coefficient CV value (standard deviation / average value) was calculated. The results are shown in Table 3.

  As can be seen from Table 3, the coefficient of variation when the T value was adopted was 16%, whereas the coefficient of variation when the T / (T + C) value was adopted was 5%. That is, it can be seen that when the T / (T + C) value is adopted, the variation in the measured value is small and the accuracy of the measured value is high.

The light emission intensity (T) of the test line affects the non-uniformity and variation of the components of the test kit and the variation in the flow of the labeled reagent particles that move through the membrane by capillary action. Therefore, as shown in Tables 2 and 3, the correlation between the emission intensity of the test line and the progesterone concentration is low.
On the other hand, the sum of the emission intensity of the test line and the emission intensity of the control line is used as an amount indicating the flow of the labeled reagent particles that have moved through the membrane 3, and the value of the emission intensity T of the test line is corrected with this value. Thus, even if there were non-uniformities and variations in the components of the test kit and variations in the flow of the labeled reagent particles, a high correlation between the emission intensity of the test line and the biomolecule concentration was obtained. That is, according to the present invention, the detection accuracy of biomolecules can be improved.

1 Test Strip 2 Sample Pad 3 Membrane 4 Absorption Pad 5 Backing Sheet 10 Test Area 11 Reference Area

Claims (14)

A biomolecule detection or quantification method using a competition method using a biomolecule detection or quantification test kit,
The biomolecule detection or quantification test kit has a test piece for biomolecule detection or quantification, and labeled reagent particles for biomolecule detection or quantification,
The test piece has a test region for capturing biomolecules and labeled reagent particles,
A substance that competes with the biomolecule to be detected is introduced on the surface of the labeling reagent particle,
A mixture of a sample containing a biomolecule and the labeling reagent particles is dropped onto the test piece,
Measure the measured value indicating the amount of labeled reagent particles that compete with the biomolecule and bind to the test region,
The measured value indicating the amount of labeled reagent particles bound to the test area is corrected with the measured value indicating the amount of labeled reagent particles moved from the test piece,
Detect or quantify biomolecules based on the corrected values,
Biomolecule detection or quantification method.   The method for detecting or quantifying a biomolecule according to claim 1, further comprising a reference region that captures labeled reagent particles that are not bound to the test region due to competition with the biomolecule.   The method for detecting or quantifying a biomolecule according to claim 1 or 2, wherein the labeling reagent particles are labeling reagent particles made of fluorescent silica nanoparticles.   The method for detecting or quantifying a biomolecule according to claim 3, wherein the measured value indicating the amount of labeled reagent particles bound to the test region is the emission intensity of the test region bound to the labeling reagent particles made of fluorescent silica nanoparticles.   The measured value indicating the amount of labeled reagent particles that have moved the test piece is the total value of the luminescence intensity of the test region and the luminescence intensity of the reference region to which the labeling reagent particles composed of the fluorescent silica nanoparticles not bound to the test region are bound. The method for detecting or quantifying a biomolecule according to claim 3 or 4, wherein:   6. The biomolecule according to claim 5, wherein the luminescence intensity of the test region is divided by the total value of the luminescence intensity of the test region and the luminescence intensity of the reference region, and the measured value indicating the amount of labeled reagent particles bound to the test region is corrected. Detection or quantification method. A reference substance having a binding property to the reference region is introduced on the surface of the labeling reagent particle;
Due to the binding between the reference substance and the reference region, the labeled reagent particles that have not been bound to the test region are captured in the reference region.
The method for detecting or quantifying the biomolecule according to any one of claims 2 to 6.   The detection of a biomolecule according to claim 7, wherein in the labeling reagent particle, the labeling reagent particle and a substance that competes with the biomolecule are bound via the reference substance introduced onto the surface of the labeling reagent particle. Or a quantitative method.   9. The reference substance according to claim 7 or 8, wherein the reference substance is at least one substance selected from the group consisting of bovine serum albumin, casein, mussel hemocyanin, human serum albumin, ovalbumin, globulin, avidin, and streptavidin. A method for detecting or quantifying the described biomolecule.   The method for detecting or quantifying a biomolecule according to any one of claims 1 to 9, wherein the labeling reagent particle has an average particle size of 30 nm to 500 nm. A biomolecule detection or quantification device comprising:
A light irradiation unit that irradiates light to a test region of a test piece for biomolecule detection or quantification in which a biomolecule and a labeled reagent particle compete and bind to each other;
A detection unit for detecting a measurement value indicating the amount of labeled reagent particles bound to the test region;
An apparatus for detecting or quantifying biomolecules, comprising: Irradiating light to a reference region that captures labeled reagent particles that did not bind to the test region due to competition with biomolecules, a light irradiation unit;
A detection unit for detecting a measurement value indicating the amount of labeled reagent particles bound to the reference region;
The biomolecule detection or quantification device according to claim 11, further comprising: The labeling reagent particle is a labeling reagent particle composed of fluorescent silica nanoparticles,
The detection unit for detecting the measurement value indicating the amount of labeled reagent particles bound to the test region and the detection unit for detecting the measurement value indicating the amount of labeled reagent particle bound to the reference region are the test region or reference region to which the labeling reagent particles are bound. The amount of labeled reagent particles bound is calculated from the measured emission intensity,
The biomolecule detection or quantification apparatus according to claim 11 or 12. 14. The correction device according to claim 11, further comprising a correction unit that corrects a measured value indicating the amount of labeled reagent particles bound to the test region with a measured value indicating the amount of labeled reagent particles moved from the test piece. Biomolecule detection or quantification device.

Images