CN118318006A - Sample inspection particles - Google Patents

Abstract

Provided are particles capable of performing high-sensitivity sample inspection by a fluorescence depolarization method, and a method for producing the same. Specifically, there is provided a particle comprising a particle matrix, wherein the particle matrix comprises: a polymer comprising styrene units and organosilane units, and a europium complex, and wherein the particle matrix comprises siloxane bonds at least on the surface of the particle matrix.

Description

Sample inspection particles

Technical Field

The present invention relates to particles for sample inspection and a method for producing the same.

Background technique

In the field of medicine and clinical examination, high sensitivity detection or quantification of trace biological components from, for example, blood or collected organ parts is required to explore the cause and presence of, for example, a disease. Among the inspection techniques for biological components, immunoassays are widely used. In many immunoassays, a washing step called bound/free (B/F) separation is required. As an immunoassay that does not require B/F separation, a latex agglutination method using an antigen-antibody reaction is known. In the latex agglutination method, latex particles, each of which is loaded with, for example, antibodies that specifically bind to a target substance, are mixed with a liquid that may contain the target substance, and the degree of agglutination of the latex particles is measured.

In the latex agglutination method, the target substance is captured by an antibody that binds to latex particles and is specific to the target substance, and a plurality of latex particles are cross-linked via the captured target substance, resulting in agglutination of the latex particles. That is, the amount of the target substance in a liquid sample, such as a biological sample, can be quantified by evaluating the degree of agglutination of the latex particles. The degree of agglutination can be quantified by measuring and evaluating the change in the amount of light that passes through or is scattered by the liquid sample.

The latex agglutination method can detect/quantitatively evaluate an antigen as a target substance in a sample in a simple and rapid manner, but has a problem of detection limit, that is, the antigen cannot be detected when the amount of the antigen in a liquid sample such as a biological sample is low.

In order to improve the detection sensitivity of the target substance, it is necessary to measure the degree of agglutination with higher sensitivity. That is, it is conceivable to replace the system for measuring the change in the amount of light transmitted through or scattered by the liquid sample with a detection/quantification method utilizing the luminescence characteristics with higher sensitivity. Specifically, for example, a sample inspection method utilizing a fluorescence depolarization method has been proposed (Patent Documents 1 and 2).

In Patent Document 1, it is proposed to improve an apparatus for a fluorescence depolarization method for clinical use.

In the fluorescence depolarization method, the B/F separation required in the general fluorescence measurement method is not required. Therefore, the use of the fluorescence depolarization method enables a simple sample inspection as in the latex agglutination method. In addition, it is believed that using the fluorescence depolarization method, the measurement can be performed by the same test system as in the latex agglutination method, and only the luminescent material that specifically reacts with the measured substance is mixed during the measurement process. However, in Patent Document 1, it is proposed to use a single molecule such as fluorescein as a luminescent material, which is only applicable to drugs, low molecular weight antigens, etc. in principle.

Patent document 2 solves the problem of patent document 1, that is, the problem that the fluorescence depolarization method is only applied to drugs, low molecular weight antigens, etc. In patent document 2, in order to apply the fluorescence depolarization method to macromolecules such as proteins, it is proposed to use a material obtained by adsorbing a dye having a long-life luminescence property onto latex particles as a luminescent material. In patent document 2, based on the principle of the fluorescence depolarization method, it is proposed to quantify high molecular weight substances by balancing the reduction of the rotational Brownian motion of the substance in the liquid due to the increase in particle size and the length of the luminescence lifetime.

Citation list

Patent Literature

PTL 1: Japanese Patent Publication No. H3-52575

PTL 2: Japanese Patent No. 2893772

SUMMARY OF THE INVENTION

technical problem

However, in Patent Document 2, the fluorescent substance is carried on the latex particles after the particles are synthesized, so the interaction between the fluorescent substances adsorbed near the particle surface makes it difficult to stably determine the polarization anisotropy of the inspection particles. In addition, in Patent Document 2, bovine serum albumin (BSA) as a biomolecule is carried on the surface of the particles to suppress nonspecific adsorption, so there is a risk of batch-to-batch differences due to the wide particle size distribution and BSA as a protein.

Therefore, in Patent Document 2, highly sensitive detection of the target substance is still far from being achieved.

The present invention has been made in view of such background art, and an object of the present invention is to provide a particle capable of high-sensitivity sample inspection by a fluorescence depolarization method and a method for producing the same.

Technical solutions

According to one embodiment of the present invention, there is provided a particle comprising a particle matrix,

wherein the particle matrix comprises: a polymer containing styrene units and organosilane units; and a europium complex represented by the following formula (1), and

wherein the particle matrix comprises siloxane bonds at least on the surface of the particle matrix, Eu(A) _x (B) _y (C) _z …(1)

In formula (1), (A) represents a ligand represented by the following formula (2), (B) represents a ligand represented by the following formula (3) or (4), and (C) represents a ligand represented by the following formula (5):

[Chemical formula 1]

In formulae (2) to (5), ^R1 and ^R2 each independently represent an alkyl group, a perfluoroalkyl group, a phenyl group, or a thiophene group and each group may each have a substituent, ^R3 represents a hydrogen atom or a methyl group, ^R4 and ^R5 each independently represent an alkyl group or a phenyl group and each group may each have a substituent, ^R6 represents an alkyl group, a phenyl group, or a triphenylene group and each group may each have a substituent, and ^R7 and ^R8 each independently represent an alkyl group or a phenyl group and each group may each have a substituent; in formula (4), the bond represented by the dotted line may or may not exist; the substituents are each independently one of the following: a methyl group, a fluorine group, a chlorine group, or a bromine group; the alkyl groups each independently have 2 or more and 12 or less carbon atoms; and x, y, and z satisfy the following equation:

x=3;

y = 1 or 2;

z＝0 or 1; and

x+y+z=4 or 5.

Furthermore, it is preferred that the particle further comprises a layer containing a hydrophilic polymer.

Further features of the present invention will become apparent from the following description of exemplary embodiments with reference to the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the structure of a particle according to an embodiment of the present invention.

2 shows the results of fluorescence polarization measurements of samples of Examples 1, 2, 3, 4, and 7, and is an explanatory diagram of the relationship between the values of polarization anisotropy <r> of the samples determined thereby and their particle diameters.

3 is an explanatory diagram of the relationship between the luminescence intensity of the samples of Examples 1, 2, 3, 4, 5, 6 and 7 and Comparative Examples 1, 2, 3 and 4 and their europium contents.

FIG. 4 shows the evaluation results of the affinity particles 3 and is an explanatory diagram of the results of quantification of the CRP antigen concentration using the fluorescence depolarization method.

Implementation Description

Preferred embodiments of the present invention are described in detail below. However, the embodiments are not intended to limit the scope of the present invention.

An example of the particle of this embodiment will be described in detail with reference to FIG. 1 .

As shown in FIG. 1 , the particle according to this embodiment includes a particle substrate 1 containing a europium complex 3 and a hydrophilic layer 2 coating the surface thereof.

The particles according to this embodiment have a small particle size distribution, and the surfaces of the particles are coated with a hydrophilic layer.

The europium complex 3 is present inside the particles.

Fig. 1 is a schematic diagram illustrating an example of particles of this embodiment. The diameter of the particles shown in Fig. 1 is 25 nm or more and 500 nm or less.

The diameter of each particle can be determined by the dynamic light scattering method. When particles dispersed in a solution are irradiated with laser light and the resulting scattered light is observed with a photon detector, the intensity distribution due to interference of the scattered light constantly fluctuates because the particles constantly move their positions through Brownian motion.

Dynamic light scattering is a measurement method that observes the state of Brownian motion as fluctuations in the intensity of scattered light. The fluctuations of scattered light with respect to time are expressed as an autocorrelation function, from which the translational diffusion coefficient is determined. The Stokes diameter is determined from the determined diffusion coefficient, and the size of each particle dispersed in the solution can be derived.

From the viewpoint of maintaining the uniformity and monodispersity of the particles, it is desirable that the particles according to this embodiment do not provide anything on the surface of the particles. However, in order to be used for sample inspection, it is necessary to prevent non-specific adsorption of substances other than the target onto the particles, and therefore the particles need to be coated to maintain their surface hydrophilicity.

A method of supporting BSA on the surface of each particle is widely used as a technique for maintaining hydrophilicity, but this method may cause many variations. In view of this, the surface of the particle of this embodiment is coated with a hydrophilic polymer.

According to the particles of this embodiment, since the particles contain europium complexes, phosphorescence with a long life can be emitted. In the particles of this embodiment, the average particle size as the average value of the particle diameter is preferably 25 nm or more and 500 nm or less, more preferably 50 nm or more and 300 nm or less. When the average particle size is greater than 500 nm, the polarization anisotropy before agglutination becomes higher, resulting in a small difference in polarization isotropy with the agglutination reaction. In addition, when the average particle size is less than 25 nm, the change between the size before and after agglutination becomes smaller, making it difficult to capture the change in polarization anisotropy by phosphorescence fluorescence depolarization.

By reducing the particle size distribution of the particles and introducing a europium complex that indicates polarized luminescence into the particles, changes in polarized luminescence characteristics can be captured even when the dispersion state of the particles in the liquid changes slightly. Specifically, even if the concentration of the target substance in the solution is about nanograms to about picograms per milliliter, when the particles are agglomerated by the target substance, changes in the rotational Brownian motion of the particles can be captured as changes in polarization anisotropy.

"Polarized luminescence" refers to the following phenomenon: when a luminescent dye having anisotropy in transition moment (transition dipole moment) uses polarized light along its transition moment as excitation light, its luminescence is also polarized light along the transition moment. Europium complexes show fluorescent luminescence based on energy transfer from ligands to central metal ions, so the transition moment is complex, but the red luminescence near 610nm obtained by the electron transition from the lowest excited state 5D0 to 7F2 is emitted as polarized light.

The principle of fluorescence depolarization is to measure the shift of the transition moment based on the rotational motion of the luminescent material during the occurrence of polarized luminescence. The rotational motion of the luminescent material can be expressed by equation (6):

Q＝3Vη/kT … (6)

Where Q represents the rotational relaxation time of the material, V represents the volume of the material, η represents the viscosity of the solvent, "k" represents the Boltzmann constant, and T represents the absolute temperature.

The rotational relaxation time of a material is the period of time required for a molecule to rotate through an angle θ (68.5°), where cosθ = 1/e.

From equation (6), it is found that the rotational relaxation time of the luminescent material is proportional to the volume of the material (that is, when the luminescent material has a particle shape, the volume of the material is the cube of the particle size). At the same time, the relationship between the luminescence lifetime and the polarization degree of the luminescent material can be expressed by equation (7):

p0/p＝1+A(τ/Q) … (7)

Wherein p0 represents the degree of polarization when the material is stationary (Q=∞), "p" represents the degree of polarization, A is a constant, τ represents the luminescence lifetime of the material, and Q represents the rotational relaxation time.

From equations (6) and (7), it is found that the polarization degree is affected by the luminescence lifetime and the rotational relaxation time of the luminescent material, that is, by the volume of the luminescent material, and the volume depends on the particle size. Therefore, in other words, the polarization degree is affected by the balance between the particle size and the luminescence lifetime of the luminescent material.

When the polarization degree of the light emitting material represented by equation (7) is determined experimentally, it is appropriate to allow polarized light to enter the light emitting material and detect the light emission in the 90° direction relative to the traveling direction and vibration direction of the excitation light. In this case, it is appropriate to detect the detected light by dividing into polarized light components in parallel and perpendicular directions relative to the polarized light as the incident light, and to evaluate the polarization anisotropy by the formula represented by the following equation (8):

r(t)＝(I//(t)-GI⊥(t))/(I//(t)+2GI⊥(t)) … (8)

Wherein r(t) represents the polarization anisotropy at time "t", I//(t) represents the luminous intensity of the luminous component parallel to the excitation light at time "t", I⊥(t) represents the luminous intensity of the luminous component perpendicular to the excitation light at time "t", and G represents the correction value, that is, the ratio of I⊥/I// measured with excitation light whose vibration direction differs by 90° from the vibration direction of the excitation light used for sample measurement.

That is, when the particle size and the luminescence lifetime fall within an appropriate range, the size change of the luminescent material due to, for example, a reaction with a target substance can be sensitively read as a change in polarization anisotropy. That is, the r(t) of the unagglomerated luminescent material is observed to be low, and the r(t) of the agglomerated luminescent material is observed to be high. This is the principle of the fluorescence depolarization method.

Polarization anisotropy refers to the value of polarization degree corrected with G and 2G, and is a value obtained by removing G and 2G from equation (8). In actual measurement, the correction value G is required, thereby determining polarization anisotropy.

It is preferred that the particles according to an embodiment of the present invention have a polarization anisotropy <r> of 0.01 or more, which is determined by the following equation (9).

[Mathematical formula 1]

In equation (9),

<r> represents polarization anisotropy,

I _VV represents the luminous intensity of the luminous component having a vibration direction parallel to the vibration direction of the first polarized light beam when excited by the first polarized light beam,

I _VH represents the luminous intensity of the luminous component having a vibration direction perpendicular to the vibration direction of the first polarized light beam when excited by the first polarized light beam,

I _HV represents the luminous intensity of the luminous component perpendicular to the vibration direction of the second polarized light beam when excited by the second polarized light beam, the vibration direction of the second polarized light beam being perpendicular to the vibration direction of the first polarized light beam,

I _HH represents the luminous intensity of the luminous component having a vibration direction parallel to the vibration direction of the second polarized beam when excited by the second polarized beam, the vibration direction of the second polarized beam being perpendicular to the vibration direction of the first polarized beam, and

G represents the correction value.

(Particle Matrix 1)

FIG. 1 shows an example of spherical particles, which include a particle matrix 1. FIG. 1 shows an example in which both the particles and the particle matrix 1 are spherical, but the shape of the particles and the particle matrix 1 of this embodiment is not limited thereto. The particle matrix 1 is not particularly limited as long as the particle matrix 1 is a material capable of stably incorporating a europium complex, but the matrix is preferably a polymer containing a styrene unit and an organosilane unit. In particular, for example, a polymer obtained by polymerizing a composition containing styrene as a main component and a free radical polymerizable organosilane is appropriately used. When the composition contains styrene as a main component, particles having a uniform particle size distribution can be prepared by an emulsion polymerization method described later. In addition, when a polymer containing an organosilane unit is used, in an aqueous medium, a silanol group (Si-OH) is generated in the polymer, and a siloxane bond (Si-O-Si) is formed on the surface of the particle matrix with each other, through which a hydrophilic layer or ligand described later can be provided. The particles according to this embodiment preferably have a ligand binding functional group capable of binding a ligand to the outside of the particle matrix.

(Hydrophilic layer 2)

The hydrophilic layer 2 is formed by incorporating a hydrophilic polymer or a hydrophilic molecule on the outside of the particle matrix 1. The hydrophilic polymer or the hydrophilic molecule is a polymer or a molecule containing a hydrophilic group, and specific examples of the hydrophilic group include molecules or polymers each having a hydroxyl group, an ether, a pyrrolidone or a betaine structure. Specific examples of the hydrophilic polymer include polyethylene glycol, polyvinyl pyrrolidone, a polymer of sulfobetaine, a polymer of phosphobetaine, and polyglycidyl methacrylate (a molecule thereof has an end modified by opening the glycidyl group with a hydroxyl group), and these hydrophilic polymers can each be used as a main component of the hydrophilic layer 2. Alternatively, the hydrophilic layer 2 can be formed by directly providing a single molecule having a hydrophilic group on the surface of the particle matrix 1 using a silane coupling agent or the like. The thickness of the hydrophilic layer 2 is not limited, but it is not necessary to set the thickness to be greater than the thickness that can exhibit hydrophilicity. When the hydrophilic layer 2 is too thick, there is a risk that the hydrophilic layer becomes hydrogel-like and hydrates due to the influence of ions in the solvent, thereby making its thickness unstable. The thickness of the hydrophilic layer 2 is appropriately 1 nm or more and 15 nm or less.

(Europium complex 3)

The particles of this embodiment include europium complex 3 as a luminescent dye. Europium complex 3 is characterized in that the wavelength and intensity of its luminescence are hardly affected by the surrounding environment, so the luminescence has a long life. Europium complex 3 includes europium element and ligand. Considering the luminescence lifetime, visible light emission wavelength region, etc., the luminescent dye needs to be a europium complex. Europium generally has a luminescence lifetime of from 0.1ms to 1.0ms. It is necessary to appropriately adjust the luminescence lifetime and rotational relaxation time obtained from equation (6). In the case where europium is in an aqueous dispersion, when the diameter of the particle is from about 50nm to about 300nm, the polarization anisotropy represented by equation (8) changes significantly before and after aggregation.

At least one of the constituent ligands of the europium complex 3 is a ligand having a light-collecting function. "Light-collecting function" refers to the action of exciting the central metal of the complex by energy transfer at a specific wavelength. In addition, it is preferred that the constituent ligands of the europium complex 3 include ligands such as β-diketones to prevent the coordination of water molecules. Ligands coordinated to europium ions, such as β-diketones, inhibit the deactivation process caused by energy transfer to solvent molecules, etc., thereby providing strong fluorescence luminescence.

The europium complex 3 may be a polynuclear complex.

The europium complex 3 is preferably represented by formula (1):

Eu(A) _x (B) _y (C) _z …(1)

In formula (1), (A) represents a ligand represented by the following formula (2), (B) represents a ligand represented by the following formula (3) or (4), and (C) represents a ligand represented by the following formula (5):

[Chemical formula 2]

In formulae (2) to (5), ^R1 and ^R2 each independently represent an alkyl group, a perfluoroalkyl group, a phenyl group, or a thiophene group and each group may each have a substituent, ^R3 represents a hydrogen atom or a methyl group, ^R4 and ^R5 each independently represent an alkyl group or a phenyl group and each group may each have a substituent, ^R6 represents an alkyl group, a phenyl group, or a triphenylene group and each group may each have a substituent, and ^R7 and ^R8 each independently represent an alkyl group or a phenyl group and each group may each have a substituent; the bond represented by the dotted line in formula (4) may or may not exist; the substituents are each independently one of the following: a methyl group, a fluorine group, a chlorine group, or a bromine group; the alkyl groups each independently have 2 or more and 12 or less carbon atoms; and x, y, and z satisfy the following equation:

x=3;

y = 1 or 2;

z = 0 or 1; and

x+y+z=4 or 5.

A preferred specific example of the ligand represented by the formula (2) is 2-thenoyltrifluoroacetone.

A preferred specific example of the ligand represented by the formula (3) is triphenylphosphine oxide.

A preferred specific example of the ligand represented by the formula (5) may be dibenzyl sulfoxide.

Preferred specific examples of the ligand represented by the formula (4) may include ligands represented by the following formulae (10) and (11).

[Chemical formula 3]

[Chemical formula 4]

In addition, specific examples of the europium complex include [tris(2-thienoyltrifluoroacetone)(bis(triphenylphosphine oxide))europium(III)], [tris(2-thienoyltrifluoroacetone)(triphenylphosphine oxide)(dibenzyl sulfoxide)europium(III)], and [tris(2-thienoyltrifluoroacetone)(phenanthroline)europium(III)].

When the Brownian rotational motion of the europium complex 3 can be regarded as stationary in the medium, the polarization anisotropy represented by equation (8) is preferably 0.08 or more. The state where the Brownian rotational motion can be regarded as stationary refers to a state where the rotational relaxation time of the particle is sufficiently longer than the luminescence lifetime of the europium complex 3.

The europium complex 3 is preferably incorporated in the particle matrix 1 in as much amount as possible because the luminescence intensity per particle becomes stronger. Meanwhile, when the molecules of the europium complex 3 are aggregated in the particle matrix 1, it is difficult to measure the polarization anisotropy while maintaining reproducibility due to factors such as the interaction between ligands affecting the excitation efficiency of the europium complex 3. Whether the europium complex 3 in the particle matrix 1 exhibits non-aggregated luminescence behavior can be determined based on the excitation spectrum of the sample.

Particles with strong luminescence not only enable highly sensitive measurements but also increase biochemical reaction rates because they maintain luminescence even when their particle diameter is reduced. As the particle size becomes smaller, the diffusion coefficient of Brownian motion in the liquid becomes larger, so the reaction can be detected in a shorter time.

When using a liquid in which the particles of the embodiment are dispersed, the change in the anisotropy of polarized luminescence can be detected with high sensitivity corresponding to the aggregation/dispersion behavior of the particles. Therefore, by using a fluorescence depolarization method, the dispersion obtained by dispersing the particles of the embodiment in an aqueous medium can be used as a high-sensitivity inspection reagent. A buffer solution can be used as an aqueous medium. In addition, a surfactant, a preservative, a sensitizer, etc. can be added to an aqueous medium to enhance the stability of the liquid in which the particles according to the embodiment are dispersed.

(Method for producing granules)

One example of a method for producing the particles of this embodiment is described below.

The method for producing particles of this embodiment includes the step of mixing a radical polymerizable monomer including at least styrene and a radical polymerizable organosilane, a radical polymerization initiator, a polarized luminescent europium complex, and a hydrophilic polymer with an aqueous medium to prepare an emulsion (first step).

Furthermore, the method for producing particles of this embodiment includes a step (second step) of heating the emulsion to polymerize the radical polymerizable monomer.

The particles of this embodiment are particles obtained by copolymerizing compounds having double bonds each having radical polymerizability.

In addition, the manufacturing method of the particles of this embodiment can include the step (third step) of providing the ligand binding functional group described later on the surface of the particles. In this article, the ligand binding functional group refers to a functional group capable of binding a ligand. Specifically, any one of a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimide group or an alkoxysilyl group (silane oxide structure) can be used.

(Free radical polymerizable monomer)

The manufacture of particles is carried out by polymerizing free radical polymerizable monomers, and the free radical polymerizable monomers include at least styrene and free radical polymerizable organosilanes. The free radical polymerizable monomers may further include monomers selected from the following: acrylic monomers, and methacrylic monomers. Examples of monomers for making particles may include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate and mixtures thereof. That is, in addition to styrene and free radical polymerizable organosilanes, one or more of these monomers may also be used. In addition, monomers having two or more double bonds per molecule, such as divinylbenzene, may be used as crosslinking agents.

A free radical polymerizable organosilane is included in the free radical polymerizable monomer to provide a siloxane bond on the particle matrix 1. Examples of the free radical polymerizable organosilane may include vinyl trimethoxysilane, vinyl triethoxysilane, p-phenylvinyl trimethoxysilane, 3-methacryloxypropyl methyl dimethoxysilane, 3-methacryloxypropyl trimethoxysilane, 3-methacryloxypropyl methyl diethoxysilane, 3-methacryloxypropyl triethoxysilane, 3-acryloxypropyl trimethoxysilane, and combinations thereof. The free radical polymerizable organosilane is used to form a skeleton of an inorganic oxide in the particle matrix 1 to improve the physical and chemical stability of the particles. In addition, the use of the free radical polymerizable organosilane enhances the affinity between the particle matrix 1 and each of the hydrophilic polymer and the ligand binding functional group.

In addition, the radical polymerizable organic silane is included in the radical polymerizable monomer to provide silanol groups on the surface of the particle matrix 1. The silanol groups and the hydrophilic polymer such as PVP form hydrogen bonds. Therefore, the hydrophilic polymer such as PVP is more strongly adsorbed to the surface of the particle matrix 1.

(Free Radical Polymerization Initiator)

Compounds selected from a wide range of, for example, azo compounds and organic peroxides may each be used as a radical polymerization initiator. Specific examples thereof may include: 2,2′-azobis(isobutyronitrile), 2,2′-azobis(2,4-dimethylvaleronitrile), 2,2′-azobis(2-methylbutyronitrile), 4,4′-azobis(4-cyanovaleric acid), 2,2′-azobis(2-methylpropionamidine) dihydrochloride, 2,2′-azobis(2-methylpropionic acid) dimethyl ester, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), and potassium persulfate (KPS).

(Hydrophilic polymer)

The particles may include a hydrophilic polymer as a hydrophilic layer 2. The hydrophilic polymer preferably inhibits nonspecific adsorption. Examples of hydrophilic polymers include hydrophilic polymers each containing a unit having an ether, betaine or pyrrolidone ring. Preferably, the layer containing the hydrophilic polymer is contained in the synthetic particles and is mainly present on the particle surface outside the particle matrix. In this article, the polymer with a pyrrolidone ring is sometimes abbreviated as "PVP". When PVP is added during the synthesis of the particles, the particles can be simultaneously given nonspecific adsorption inhibition ability and ligand binding ability. The PVP added during the synthesis has a higher hydrophilicity than each free radical polymerizable monomer, and therefore exists at the interface between the solvent and the particle matrix being polymerized during the synthesis. By allowing part of the PVP to participate in the polymerization, or by physical/chemical adsorption, such as the interaction between the pyrrolidone ring and styrene (free radical polymerizable monomer), the particle matrix adsorbs PVP to its outside.

The molecular weight of PVP is preferably 10000 to 100000, more preferably 40000 to 70000. When the molecular weight is less than 10000, the hydrophilicity of the particle surface is weak, so nonspecific adsorption is likely to occur. When the molecular weight is greater than 100000, the hydrophilic layer becomes thick and forms a gel, which becomes difficult to handle.

In addition to PVP, another hydrophilic polymer can be added as a protective colloid when synthesizing the particles.

In addition, the particles satisfy A2-A1≤0.1.

Definitions of A1 and A2 are as follows. That is, for a mixture obtained by adding 30 μL of a 0.1 wt% particle dispersion to 60 μL of a buffer solution mixed with 16 μL of human serum diluted 15 times, the absorbance of the mixture immediately after the addition is represented by A1, and the absorbance of the mixture after standing at 37° C. for 5 minutes after the addition is represented by A2. The absorbance is measured at an optical path of 10 mm and a wavelength of 572 nm.

Particles showing A2-A1 equal to or less than 0.1 have little nonspecific adsorption to impurities in serum and are therefore preferred.

(Aqueous medium)

The aqueous medium (aqueous solution) used in the method for producing the above-mentioned particles preferably contains 80% by weight or more and 100% by weight or less of water in the medium. The aqueous medium is preferably water or a water-soluble organic solvent, examples of which include solutions obtained by mixing water with methanol, ethanol, isopropanol or acetone. When an organic solvent other than water is added in an amount greater than 20% by weight, dissolution of the polymerizable monomer may occur during particle production.

In addition, it is preferred to adjust the pH of the aqueous medium in advance to 6 or more and 9 or less. When the pH value is less than 6 or greater than 9, the alkoxy group or silanol group of the radical polymerizable organosilane is likely to undergo polycondensation or react with another functional group before forming a polymer, thereby causing aggregation of the particles to be obtained. In this embodiment, polycondensation of the alkoxide is not intentionally performed before polymerization.

The above pH is preferably adjusted using a pH buffer, but may also be adjusted using an acid or a base.

In addition to the above, a surfactant, a defoaming agent, a salt, a thickener, etc. may be used by adding at a ratio of 10% or less relative to the aqueous medium.

In the production of the particles according to this embodiment, preferably, PVP is first dissolved in an aqueous medium whose pH has been adjusted to 6 to 9. The content of PVP is preferably 0.01% by weight or more and 10% by weight or less, and more preferably 0.03% by weight to 5% by weight relative to the aqueous medium. When the content is less than 0.01% by weight, the amount of adsorption on the particle matrix is small, and the effect thereof is not exhibited. In addition, when the content is greater than 10% by weight, the viscosity of the aqueous medium is likely to increase, thereby preventing sufficient stirring.

Subsequently, a radical polymerizable monomer including styrene (A) and a radical polymerizable organosilane (B) is added to the above aqueous medium to prepare an emulsion. The weight ratio between styrene (A) and the radical polymerizable organosilane (B) is 6:4 to 100:1. In addition, the prepared emulsion is mixed with a europium complex. At this time, when the solubility of the europium complex is low, an organic solvent insoluble in water may be added. The weight ratio between the europium complex and the radical polymerizable monomer is 1:1000 to 1:10.

When the weight ratio between styrene (A) and radical polymerizable organosilane (B) is less than 6:4, the overall specific gravity of the particles is likely to increase, resulting in significant sedimentation of the particles. In addition, in order to increase the adhesion between PVP and the luminescent particles, it is desirable that the weight ratio between styrene (A) and radical polymerizable organosilane (B) is set to 100:1 or greater.

The weight ratio between the weight of the aqueous medium and the total amount of the radical polymerizable monomers is preferably 5:5 to 9.9:0.1. When the weight ratio between the weight of the aqueous medium and the total amount of the radical polymerizable monomers is less than 5:5, the particles to be produced may significantly aggregate. In addition, when the weight ratio between the weight of the aqueous medium and the total amount of the radical polymerizable monomers is greater than 9.9:0.1, although there is no problem in the production of particles, the yield thereof may be reduced.

The radical polymerization initiator is used by being dissolved in water, a buffer, etc. The radical polymerization initiator can be used in an amount of 0.5 to 10% by mass in the emulsion relative to the total weight of styrene (A) and the radical polymerizable organosilane (B).

In the step of heating the emulsion, it is only necessary to uniformly heat the entire emulsion. The heating temperature can be set arbitrarily between 50° C. and 80° C., and the heating time can be set arbitrarily between 2 hours and 24 hours. By heating the emulsion, the free radical polymerizable monomer is polymerized.

The particles of this embodiment may have a ligand binding functional group on its surface. The ligand binding functional group is not particularly limited as long as the functional group can bind antibodies, antigens, enzymes, etc. However, for example, the functional group may be a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimide group or a silane oxide group, or include any of these functional groups. For example, a silane coupling agent with a ligand binding functional group and a synthesized particle may be mixed to provide a functional group on the particle surface. Specifically, an aqueous solution of a silane coupling agent with a carboxyl group may be prepared and mixed with a dispersion of synthetic particles to provide a carboxyl group on the particle surface. At this point, a dispersant such as Tween 20 may be added to the reaction solution. The reaction temperature may be arbitrarily set between 0°C and 80°C, and the reaction time may be arbitrarily set between 1 hour and 24 hours. In order to suppress the sudden condensation reaction of the silane coupling agent, it is appropriate to set the temperature to be equal to or lower than room temperature of about 25°C, and to set the reaction time to about 3 hours to about 14 hours. Depending on the ligand binding functional groups, the reaction with the particle surface can be promoted by adding acid or base catalysts.

The particles of this embodiment can be used as particles for sample detection by binding a ligand such as any of various antibodies thereto. It is only necessary to select the best technique for binding an antibody of interest or the like by utilizing the functional groups present on the hydrophilic layer 2 .

(Ligand/Affinity Particles)

In this embodiment, an affinity particle including the particle according to this embodiment and a ligand bound to a ligand binding functional group may be provided.

The particles of this embodiment preferably include a ligand specific for the target substance. By including the ligand, the particles of this embodiment can detect/quantify the target substance based on the fluorescence depolarization method. In this embodiment, "ligand" refers to a compound that specifically binds to a specific target substance.

The ligand binds to the target substance at a predetermined site and has a selectively or specifically high affinity. Any substance that shows interaction with a certain substance can be used as a target substance. Examples of target substances may include antigens, antibodies, low molecular weight compounds, various receptors, enzymes, matrices, nucleic acids, cytokines, hormones, neurotransmitters, transmitters and membrane proteins. Examples of antigens include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances and any low molecular weight compounds. Examples of nucleic acids include DNA, RNA or cDNA derived from bacteria, viruses or cells, parts or fragments thereof, synthetic nucleic acids, primers and probes. Examples of low molecular weight compounds include cytokines, hormones, neurotransmitters, transmitters, membrane proteins and their receptors. Any compound that shows affinity for a specific substance can be used as a ligand. Examples of combinations of ligands and target substances, or target substances and ligands may include the following. That is, the examples may include: antigens and antibodies; low molecular weight compounds and their receptors; enzymes and matrices; and nucleic acids that complement each other. In addition, the example may include antibodies and any of the following substances specific to them: allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and any low-molecular-weight compounds. In addition, the example may include receptors and any of the following substances specific to them: low-molecular-weight compounds, cytokines, hormones, neurotransmitters, transmitters, and membrane proteins. In addition, the example may include DNA, RNA, or cDNA derived from bacteria, viruses, or cells, parts or fragments thereof, synthetic nucleic acids, primers, or probes, and nucleic acids complementary thereto. In addition to the above, any combination known to have affinity may be used as a combination of a target substance and a ligand. A typical example of a ligand in this embodiment is any one of an antibody, an antigen, and a nucleic acid.

In this embodiment, methods known so far can be applied to the chemical reaction of chemically bonding the ligand-binding functional group included in the particles according to this embodiment to the ligand, as long as the purpose of this embodiment can be achieved. In addition, when the ligand is amide-bonded, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be appropriately used.

The affinity particles in this embodiment can be preferably applied to the latex immunoagglutination measurement method widely used in the fields of clinical trials, biochemical research, etc.

(Test reagents for in vitro diagnosis)

The test reagent for in vitro diagnosis, i.e., the test reagent for the target substance in the test sample detected by in vitro diagnosis in this embodiment, includes the affinity particles according to the embodiment and the dispersion medium for dispersing the affinity particles. The affinity particles of the embodiment are preferably incorporated in the test reagent of the embodiment in an amount of 0.000001% by mass to 20% by mass, more preferably 0.0001% by mass to 1% by mass. In addition to the affinity particles according to the embodiment, the test reagent according to the embodiment may also include a third substance, such as an additive or a blocker, as long as the purpose of the embodiment can be achieved. The reagent may include a combination of two or more third substances, such as an additive and a blocker. Examples of the dispersion medium to be used in the embodiment include various buffer solutions, such as phosphate buffer solution, glycine buffer solution, Goode buffer solution, tris buffer solution (Tris buffer solution) and ammonia buffer solution, but the dispersion medium included in the test reagent of the embodiment is not limited thereto.

When the test reagent in this embodiment is used to detect an antigen or an antibody in a sample, the antibody or the antigen can be used as a ligand.

(Test kit)

The test kit for detecting the target substance in the sample by the in vitro diagnosis of this embodiment includes the above-mentioned reagent and the shell of the encapsulated reagent. The kit according to this embodiment may include a sensitizer for promoting particle agglutination during antigen-antibody reaction. Examples of sensitizers include polyvinyl alcohol, polyvinyl pyrrolidone and polyalginic acid, but are not limited thereto. In addition, the test kit according to this embodiment may include a positive control, a negative control, a serum diluent, etc. As a medium for the positive control or the negative control, serum, saline or solvent that does not contain a measurable target substance can be used. The test kit according to this embodiment can be used for detecting the method of the target substance according to this embodiment in the same manner as the kit of the prior art for detecting the target substance in the sample by in vitro diagnosis. In addition, the concentration of the target substance can also be measured by methods known so far, and in particular, the test kit is suitable for detecting the target substance in the sample by latex agglutination method.

(Inspection Method)

The method for detecting a target substance in a sample by in vitro diagnosis of the embodiment includes a step of mixing the affinity particles according to the embodiment with a sample that may contain the target substance. In addition, the mixing of the affinity particles according to the embodiment with the sample is preferably carried out in the range of pH 3.0 to pH 11.0. In addition, the mixing temperature is in the range of 20°C to 50°C, and the mixing time is in the range of 1 minute to 60 minutes. In addition, the detection method preferably uses a solvent. In addition, in the detection method according to the embodiment, the concentration of the affinity particles according to the embodiment is preferably 0.000001% by mass to 1% by mass in the reaction system, and more preferably 0.00001% by mass to 0.001% by mass. In the method for detecting a target substance in a sample according to the embodiment, the agglutination reaction caused by the mixing of the affinity particles according to the embodiment with the sample is preferably detected by a fluorescence depolarization method. Specifically, the method includes the steps of: mixing an inspection reagent with a sample to provide a mixed liquid; irradiating the mixed liquid with polarized light; and respectively detecting the polarized light components of the luminescence of the affinity particles in the mixed liquid.

By optically detecting the above-mentioned agglutination reaction occurring in the mixed liquid, the target substance in the sample is detected, and the concentration of the target substance can be measured.

Example

The present invention is specifically described below by way of examples. However, the present invention is not limited to these examples.

(1) Preparation of particles 1-12

Polyvinyl pyrrolidone (PVP-K30, manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in a 2-morpholineethanesulfonic acid (MES) buffer solution (manufactured by Kishida Chemical Co., Ltd.) having a pH of 7 to prepare a solvent A.

[Tris(2-thenoyltrifluoroacetone)(bis(triphenylphosphine oxide))europium(III)] (manufactured by Central Techno Corporation, hereinafter referred to as “Eu(TTA) ₃ (TPPO) ₂ ”), [Tris(2-thenoyltrifluoroacetone)(triphenylphosphine oxide)(dibenzyl sulfoxide)europium(III)] (manufactured by Central Techno Corporation, hereinafter referred to as “Eu(TTA) ₃ (TPPO)(DBSO)”), or [Tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III)] (manufactured by Central Techno Corporation, hereinafter referred to as “Eu(TTA) ₃ Phen”) used as a europium complex, as well as a styrene monomer (manufactured by Kishida Chemical Co., Ltd.) and 3-methacryloxypropyltrimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter referred to as “MPS”) are mixed to prepare reaction liquid B.

The reaction solution B is added to a four-necked flask containing solvent A, and the mixture is stirred with a mechanical stirrer set to 300 rpm. After stirring for 15 minutes under nitrogen flow conditions, the temperature of the prepared oil bath is set to 70°C, and nitrogen flow is then performed for 15 minutes. After the mixed solution is heated and stirred, an aqueous solution dissolved with potassium persulfate (hereinafter referred to as "KPS") (manufactured by Sigma-Aldrich) or ammonium persulfate (hereinafter referred to as "APS") (manufactured by Kishida Chemical Co., Ltd.) is added to the reaction solution, and emulsion polymerization is performed for 20 hours. After the polymerization reaction, the resulting suspension is ultrafiltered with about 4L of ion exchange water using an ultrafiltration membrane with a molecular weight cutoff of 100K to wash the product. Thereby a dispersion of particles 1 to 12 is obtained. The mass ratios of the respective reagents used to prepare particles 1 to 12 are shown in Table 1.

In the preparation of particle 9, ethanol was added to solvent A at a mass ratio of 0.1.

[Table 1]

Take an aliquot of each dispersion of particles 1 to 12 obtained by emulsion polymerization and add it to an aqueous solution in which 1% by mass Tween 20 (manufactured by Kishida Chemical Co., Ltd.) is dissolved. After stirring for 10 minutes, a silane coupling agent X12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) is added, and the mixture is stirred overnight. After stirring, the dispersion is centrifuged, the supernatant is removed, and the precipitate is redispersed with pure water. The centrifugation and redispersion operations are performed 3 or more times to wash the product. The washed precipitate is redispersed in pure water. Thus, ligand binding functional groups are introduced into particles 1 to 8. The mass ratio between particles, pure water and loaded X12-1135 is set to 1:300:2.

(Example 1)

A particle dispersion was prepared in which a sample corresponding to Synthetic Particle 1 was diluted with pure water to a concentration of 0.002 mg/mL.

(Example 2)

A particle dispersion was prepared in which a sample corresponding to Synthetic Particle 2 was diluted with pure water to a concentration of 0.002 mg/mL.

(Example 3)

A particle dispersion was prepared in which a sample corresponding to Synthetic Particle 3 was diluted with pure water to a concentration of 0.002 mg/mL.

(Example 4)

A particle dispersion was prepared in which a sample corresponding to Synthetic Particle 4 was diluted with pure water to a concentration of 0.002 mg/mL.

(Example 5)

A particle dispersion was prepared in which a sample corresponding to Synthetic Particle 5 was diluted with pure water to a concentration of 0.002 mg/mL.

(Example 6)

A particle dispersion was prepared in which a sample corresponding to the synthetic particle 6 was diluted with pure water to a concentration of 0.002 mg/mL.

(Example 7)

A particle dispersion was prepared in which a sample corresponding to Synthetic Particle 7 was diluted with pure water to a concentration of 0.002 mg/mL.

(Example 8)

A particle dispersion was prepared in which a sample corresponding to the synthetic particle 12 was diluted with pure water to a concentration of 0.002 mg/mL.

(Manufacturing CRP antibody-modified affinity particles)

Take an aliquot of 0.25 mL of the particle dispersion corresponding to 1.2 wt % of the synthetic particles 3, and replace the solvent with 1.6 mL of MES buffer solution at pH 6.0. 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and sodium N-hydroxysulfosuccinimide sulfonate are added to the particle MES buffer solution at 0.5 wt %, and the mixture is reacted at 25° C. for 1 hour. After the reaction, the dispersion is washed with MES buffer solution at pH 5.0, anti-CRP antibodies are added at 100 μg/mL, and the anti-CRP antibodies are bound to the particles at 25° C. for 2 hours. After binding, the particles are washed with tris buffer solution at pH 8. After the reaction, the particles are washed with a phosphate buffer solution to provide affinity particles 3 modified with CRP antibodies (sometimes referred to as "affinity particles 3") at a concentration of 0.3 wt %.

The binding of the antibody to the particles was confirmed by measuring the decrease in the antibody concentration in the buffer solution to which the antibody was added using the BCA assay.

The obtained affinity particles 3 were observed before and after mixing with the CRP antigen diluted with the MES buffer solution. The affinity particles 3 fixed at 0.0001 mg/mL and the CRP antigen concentrations of 0 pg/mL to 1000 pg/mL were used for the study. The observations were performed at room temperature. The method for measuring fluorescence polarization will be described later.

(Comparative Example 1)

A particle dispersion was prepared in which a sample corresponding to the synthetic particle 8 was diluted with pure water to a concentration of 0.002 mg/mL.

(Comparative Example 2)

A particle dispersion was prepared in which a sample corresponding to Synthetic Particle 9 was diluted with pure water to a concentration of 0.002 mg/mL.

(Comparative Example 3)

A particle dispersion was prepared in which a sample corresponding to the synthetic particle 10 was diluted with pure water to a concentration of 0.002 mg/mL.

(Comparative Example 4)

A particle dispersion was prepared in which a sample corresponding to the synthetic particle 11 was diluted with pure water to a concentration of 0.002 mg/mL.

(Product Evaluation)

The products of Examples and Comparative Examples were evaluated as described below.

The shape of the product was evaluated using an electron microscope (S-5500, manufactured by Hitachi High-Technologies Corporation).

The average particle size of the product was evaluated by using dynamic light scattering (Zetasizer Nano S, manufactured by Malvern).

The concentration of the suspension in which each product was dispersed was evaluated with a mass spectrometer (Thermo plus TG8120, manufactured by Rigaku Corporation).

The fluorescence spectrum of each product is measured using an excitation light of 340nm and a polarizer placed on the light path of each side of the excitation side and the luminescent side. The polarizer on the excitation light side is fixed, and the polarizer on the luminescent side is placed in a direction parallel to or perpendicular to the excitation light side polarizer. The equipment used is a fluorescence spectrophotometer F-4500 (manufactured by Hitachi High-Technologies Corporation). The peak wavelength of the observation light for analyzing polarized luminescence is set to 611nm. The obtained data of polarized luminescence is analyzed with the above equation (8) to determine polarization anisotropy "r".

The evaluation of antigen-antibody reaction using CRP was performed using a microplate reader. Nivo (manufactured by PerkinElmer Co., Ltd.) was used as an apparatus. A filter with a central wavelength of 355 nm and a half-width of 40 nm was used on the excitation light side, and a filter with a central wavelength of 615 nm and a half-width of 8 nm was used on the luminescent side. D400 was used as a dichroic mirror. The measurement time was set to 1000 milliseconds, and changes in polarization anisotropy were observed within 30 minutes from the start of the reaction. The measurement temperature was fixed at 37 ° C.

Each product was evaluated for nonspecific agglutination inhibition as described below.

60 μl of a human serum solution diluted 15 times with a buffer solution was added to each particle dispersion (3 mg/mL) prepared by the same method as in Examples 1-5 and Comparative Examples 1-3, and the mixture was kept at 37°C for 5 minutes. The absorbance at 527 nm was measured before and after incubation, and the change in absorbance before and after incubation was measured 3 times. Table 2 shows the average value of these 3 times. The evaluation was as follows: When the change in the "absorbance × 10000" value was less than 1000, it was determined that nonspecific agglutination was inhibited; when the amount was 1000 or more, it was determined that nonspecific agglutination occurred.

(Performance Evaluation)

According to the results of the nonspecific agglutination inhibition evaluation, in each of Examples 1 to 8 and Comparative Examples 1 to 4, the change in absorbance was equal to or less than a specific value (the change in the "absorbance × 10000" value was 1000 or less), and thus the particles were considered to be able to inhibit nonspecific adsorption.

The structures and physical properties of the granular materials of Examples 1 to 8 and Comparative Examples 1 to 4 are shown in Table 2 (the particle size in Table 2 represents the average particle diameter of the particles).

[Table 2]

The diameters (average particle diameters) of the particles of Examples 1, 2, 3, 4, 5, 6, 7 and 8 were 89 nm, 95 nm, 125 nm, 163 nm, 155 nm, 156 nm, 109 nm and 115 nm, respectively. The luminescence intensities of the particles of Examples 1, 2, 3, 4, 5, 6, 7 and 8 were 3132 cps, 3175 cps, 1221 cps, 2070 cps, 1656 cps, 1212 cps, 1877 cps and 3585 cps, respectively. In addition, the amounts of europium element present in the particles of Examples 1, 2, 3, 4, 5, 6, 7 and 8 were 2424 ppm, 2108 ppm, 1978 ppm, 1952 ppm, 1411 ppm, 1170 ppm, 2209 ppm and 3895 ppm, respectively. For each of the particles of Examples 1, 2, 3, 4, 5, 6, 7 and 8 having a high europium concentration of about 2000 ppm, the relationship between the polarization anisotropy "r" value of the particles determined by polarization measurement results and their particle size is shown in FIG. 2 .

As can be seen from Figure 2, there is a very high correlation between particle size and polarization anisotropy.

According to Table 2, the particles of Comparative Examples 1, 2, 3 and 4 do not have a europium concentration greater than 1000ppm, and their luminous intensities are 1099cps, 212cps, 836cps and 997cps, respectively, which are lower than the values of the embodiments. The relationship between the europium concentration in the particles and their luminous intensity is shown in Figure 3. In Figure 3, the black circles represent the results of the embodiments, and the black triangles represent the results of the comparative examples. As can be seen from Figure 3, as the europium concentration in the particles becomes higher, the luminous intensity increases more. In addition, the particles of all embodiments show a higher luminous intensity than the particles of the comparative examples. It is also confirmed that the solubility of the europium complexes used in the particles of the comparative examples in the particle matrix is relatively low, and their concentrations cannot be increased any further.

Meanwhile, when a europium complex having high solubility to mix the europium complex with the particle matrix is used, the particles themselves cannot be synthesized. Only when the complex of the example of the present invention is used, particles showing strong luminescence can be synthesized.

Table 3 shows the results of quantification of antigen-antibody reactions using the CRP antibody evaluated against affinity particle 3 produced in the "Preparation of CRP antibody-modified affinity particles" section by using the fluorescence depolarization method.

[table 3]

As can be seen from Table 3, when the slope of the polarization degree change Δr obtained by dividing the change amount of polarization anisotropy by time in 30 minutes is determined, the change amount of polarization anisotropy before and after the antigen-antibody reaction changes according to the CRP antibody concentration. The CRP antigen concentration used is very small, and is in the region (ng/mL level) that cannot be detected by general latex agglutination method. For the particles carrying BSA thereon, due to wide particle size distribution, the numerical deviation prediction of polarization anisotropy, therefore, in the case of detecting low concentration of target substance, it is difficult to accurately measure the rate of change. In addition, it has been confirmed that even under the state of low concentration with 0.001mg/ml, the particles also show sufficiently strong luminescence, reaching the degree to which luminescence can be detected and polarization anisotropy changes can be captured.

Therefore, it is revealed that the particles according to the embodiments of the present invention are each a polarized luminescent material having high precision.

Therefore, the particles according to the embodiments of the present invention can be used to provide particles for sample inspection of the fluorescence depolarization method with extremely high detection sensitivity. In particular, the particles according to the embodiments of the present invention each have a high luminescence intensity, and thus each excels in the effect of lowering the detectable lower limit of polarization anisotropy. Therefore, the particles according to the embodiments of the present invention are each suitable for detecting a target substance at a low concentration.

According to the particles of this embodiment, the change in anisotropy of polarized luminescence can be detected with high sensitivity in response to the aggregation/dispersion behavior of the particles. That is, the particles of this embodiment are suitable for use in the fluorescence depolarization method. According to the particles of this embodiment, the target substance can be detected and quantified with high sensitivity by the fluorescence depolarization method.

In addition, when a hydrophilic layer is formed on the surface of the particle, nonspecific adsorption to the particle of this embodiment is effectively suppressed, so there is no need to use a nonspecific adsorbent such as BSA. Therefore, by using the particle of this embodiment based on the fluorescence depolarization method, the target substance can be detected with high sensitivity.

The present invention is not limited to the above-described embodiments, and various changes and modifications may be made without departing from the spirit and scope of the present invention. The scope of the present invention is determined by the claims.

This application claims priority based on Japanese Patent Application No. 2021-192077 filed on November 26, 2021 and Japanese Patent Application No. 2022-185946 filed on November 21, 2022, the entire contents of which are incorporated herein by reference.

[reference numerals list]

1 Particle Matrix

2 Hydrophilic layer

3 Europium complex

Claims (17)

1. A particle comprising a matrix of particles,

Wherein the particulate matrix comprises: a polymer comprising styrene units and organosilane units, and a europium complex represented by the following formula (1),

Wherein the particulate substrate contains siloxane bonds at least on the surface of the particulate substrate:

Eu(A)_x(B)_y(C)_z…(1)

in the formula (1), (a) represents a ligand represented by the following formula (2), (B) represents a ligand represented by the following formula (3) or (4), and (C) represents a ligand represented by the following formula (5):

[ chemical formula 1]

In the formulas (2) to (5), R ¹ and R ² each independently represent an alkyl group, a perfluoroalkyl group, a phenyl group, or a thiophene group and each group may each have a substituent, R ³ represents a hydrogen atom or a methyl group, R ⁴ and R ⁵ each independently represent an alkyl group or a phenyl group and each group may each have a substituent, R ⁶ represents an alkyl group, a phenyl group, or a triphenylyl group and each group may each have a substituent, and R ⁷ and R ⁸ each independently represent an alkyl group or a phenyl group and each group may each have a substituent; in formula (4), a bond represented by a dotted line may or may not be present; each substituent is independently one of the following: a methyl group, a fluoro group, a chloro group, or a bromo group; the alkyl groups each independently have 2 or more and 12 or less carbon atoms; and x, y, and z satisfy the following equations:

x＝3；

y=1 or 2;

z=0 or 1; and is also provided with

X+y+z=4 or 5.

2. The particle according to claim 1, wherein the particle has a polarization anisotropy < r > of 0.01 or greater as determined by the following equation (9):

[ mathematics 1]

In the equation (9) for the case of the vehicle,

< R > represents polarization anisotropy of the light,

I _VV denotes the luminous intensity of a luminous component having a vibration direction parallel to the vibration direction of the first polarized light beam when excited by the first polarized light beam,

I _VH denotes the light emission intensity of a light emission component having a vibration direction perpendicular to the vibration direction of the first polarized light beam when excited by the first polarized light beam,

I _HV denotes the luminous intensity of a luminous component having a vibration direction perpendicular to the vibration direction of the first polarized light beam when excited by a second polarized light beam, which vibration direction is perpendicular to the vibration direction of the first polarized light beam,

I _HH denotes the luminous intensity of a luminous component having a vibration direction parallel to the vibration direction of the second polarized light beam when excited by the second polarized light beam, which is perpendicular to the vibration direction of the first polarized light beam.

3. The particle of claim 1 or 2, wherein the particle comprises a layer comprising a hydrophilic polymer outside the particle matrix.

4.A particle according to claim 3, wherein the hydrophilic polymer is polyvinylpyrrolidone.

5. The particle according to any one of claim 1 to 4,

Wherein the particles have ligand binding functional groups capable of binding ligands to the outside of the particle matrix, and

Wherein the ligand binding functional group has at least one functional group selected from the group consisting of: carboxylic acid groups, amino groups, thiol groups, epoxy groups, maleimide groups, and succinimide groups.

6. The particle according to any one of claims 1 to 5, wherein the average particle diameter of the particle is 50nm or more and 300nm or less.

7. The particle of any one of claims 1 to 6, wherein the particle satisfies the formula:

A2-A1≤0.1

wherein, for a mixture obtained by adding 30. Mu.L of 0.1 wt% particle dispersion to 60. Mu.L of buffer solution mixed with 16. Mu.L of human serum diluted 15 times, the absorbance of the mixture immediately after the addition is represented by A1, and the absorbance of the mixture after standing at 37℃for 5 minutes after the addition is represented by A2, the absorbance being measured at an optical path of 10mm and a wavelength of 572 nm.

8. A method of making a particle, the method comprising:

a first step of mixing a radical polymerizable monomer including styrene and a radical polymerizable organosilane, a radical polymerization initiator, a europium complex represented by the following formula (1), a hydrophilic polymer and an aqueous medium to prepare an emulsion; and

A second step of polymerizing the radical polymerizable monomer by stirring the emulsion:

Eu(A)_x(B)_y(C)_z…(1)

In the formula (1), (a) represents a ligand represented by the following formula (2), (B) represents a ligand represented by the following formula (3) or (4), and (C) represents a ligand represented by the following formula (5):

[ chemical formula 2]

In the formulas (2) to (5), R ¹ and R ² each independently represent an alkyl group, a perfluoroalkyl group, a phenyl group, or a thiophene group and each group may each have a substituent, R ³ represents a hydrogen atom or a methyl group, R ⁴ and R ⁵ each independently represent an alkyl group or a phenyl group and each group may each have a substituent, R ⁶ represents an alkyl group, a phenyl group, or a triphenylyl group and each group may each have a substituent, and R ⁷ and R ⁸ each independently represent an alkyl group or a phenyl group and each group may each have a substituent; in formula (4), a bond represented by a dotted line may or may not be present; each substituent is independently one of the following: a methyl group, a fluoro group, a chloro group, or a bromo group; the alkyl groups each independently have 2 or more and 12 or less carbon atoms; and x, y, and z satisfy the following equations:

x＝3；

y=1 or 2;

z=0 or 1; and is also provided with

X+y+z=4 or 5.

9. A method for producing an inspection reagent comprising the step of dispersing the particles according to claim 1 in a dispersion medium.

10. The method for producing an inspection reagent according to claim 9, wherein the average particle diameter of the particles is 50nm or more and 300nm or less.

11. An affinity particle comprising:

The particle of claim 5; and

A ligand bound to the ligand binding functional group.

12. The affinity particle of claim 11, wherein the ligand comprises one selected from the group consisting of: antibodies, antigens, proteins, and nucleic acids.

13. An in vitro diagnostic test agent comprising:

Affinity particles according to claim 11 or 12; and

A dispersion medium for dispersing the affinity particles.

14. The test reagent for in vitro diagnosis according to claim 13, wherein the test reagent is used for detecting an antigen or an antibody in a sample by an agglutination method.

15. An in vitro diagnostic test kit comprising:

The in vitro diagnostic test agent according to claim 13 or 14; and

A housing containing the inspection reagent.

16. A method of detecting a target substance in a sample, the method comprising the step of mixing the in vitro diagnostic test reagent according to claim 13 or 14 with the sample.

17. A method for detecting a target substance in a sample by fluorescence depolarization, the detection method comprising the steps of:

Mixing the in vitro diagnostic test reagent according to claim 13 or 14 with a test sample to provide a mixed liquid;

Illuminating the mixed liquid with polarized light; and

Detecting polarized light emitted by excitation of the polarized light illuminating the mixed liquid.