JP7738411B2 - Particles and their manufacturing method - Google Patents

Description

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

In the fields of medicine and clinical testing, highly sensitive detection of trace amounts of biological components from blood, extracted organ tissue, etc. is necessary to investigate the causes of disease. Among the biological component detection methods, immunoassay is widely used.
One type of immunoassay is the latex agglutination method, which utilizes an antigen-antibody reaction. The latex agglutination method involves mixing latex particles with a liquid sample containing a target substance and measuring the degree of agglutination of the latex particles to detect and quantify the target substance. The latex particles used for detection are bound to a target-binding substance that specifically binds to the target substance. For example, when detecting an antigen in a liquid sample such as a biological sample as the target substance, an antibody can be used as the target-binding substance.

In latex agglutination, a target substance is captured by a target-binding substance bound to latex particles. The captured target substance is then further bound by target-binding substances on other latex particles, cross-linking multiple latex particles and resulting in agglutination. In other words, the amount of a target substance in a liquid sample, such as a biological sample, can be quantified by assessing the degree of agglutination of the latex particles. The degree of agglutination can be quantified by assessing the change in the amount of light transmitted through or scattered by the liquid sample.

While the latex agglutination method allows for the simple and rapid quantitative evaluation of target substances, it has the problem of being unable to detect small amounts of target substances in liquid samples such as biological samples.

In order to improve the detection sensitivity of target substances in liquid samples, it is possible to replace the method of detecting light transmitted through or scattered by a liquid sample with a detection method that utilizes more sensitive luminescence characteristics. Specifically, Patent Documents 1 and 2 propose sample testing methods that utilize fluorescence depolarization.

One problem with fluorescence measurement is the need for a washing step known as B/F (Bound/Free) separation, which separates the analyte from any unreacted luminescent material before measurement. However, fluorescence polarization deconvolution does not require this washing step. This makes sample testing easier than with latex agglutination, which uses fluorescence measurement. Furthermore, the measurement process basically involves simply mixing the luminescent material that reacts specifically with the analyte with the sample to be measured, and measurement can be performed using the same testing system as latex agglutination.

Patent Document 1 proposes an apparatus that can be used for fluorescence depolarization, which has been improved for clinical purposes, and describes that a single molecule such as fluorescein can be used as the luminescent material.
Furthermore, Patent Document 2 proposes an immunoassay method that includes preparing dye-labeled particles by supporting a dye having a long luminescence lifetime on an insoluble carrier, reacting the obtained dye-labeled particles with a sample liquid, and then determining the degree of luminescence polarization.
In Patent Document 2, the particle size of the fluorescently labeled particles is increased by using an insoluble carrier instead of the conventional antigen as the fluorescently labeled substance, and a dye with a long emission lifetime is used for the fluorescent label. This balances the decrease in the rotational Brownian motion of the fluorescently labeled particles that accompanies an increase in particle size with the long emission lifetime, making it possible to measure polymeric substances.

Special Publication No. 3-52575 Patent No. 2893772

The luminescent material disclosed in Patent Document 1 is a low molecular weight substance, and while it can be applied to measurements of low molecular weight drugs, low molecular weight antigens, etc., it has the problem that it cannot, in principle, be applied to measurements of high molecular weight substances such as proteins.
Furthermore, while the fluorescently labeled particles described in Patent Document 2 can be applied to the measurement of polymeric substances, the large size of the fluorescently labeled particles causes scattering of excitation light by the fluorescently labeled particles, which leads to depolarization, making it difficult to achieve high sensitivity in measurements.
The present invention has been made in consideration of the above-mentioned problems in the prior art, and aims to provide particles that can be applied to the measurement of both low molecular weight substances and high molecular weight substances, and that enable highly sensitive specimen testing using fluorescence polarization depolarization, as well as a method for producing the same.

A particle according to one embodiment of the present invention is a particle for specimen testing, characterized in that the particle comprises silica particles, the average particle size of the particle is 50 nm or more and 215 nm or less, the refractive index of the particle is 1.4 or less, and the particle carries a luminescent rare earth complex therein.
In addition, a method for producing particles according to another aspect of the present invention is characterized by comprising the steps of: (1) obtaining silica particles having a refractive index of 1.4 or less by condensation polymerization of a silica particle precursor in an acidic or basic solution in which template molecules are dissolved, and then removing the template molecules; and (2) obtaining luminescent dye-carrying particles by allowing the silica particles obtained in step 1 to carry a luminescent rare earth complex in a solution in which the luminescent rare earth complex is dissolved.

The present invention provides particles that can be applied to the measurement of both low-molecular-weight and high-molecular-weight substances, enabling highly sensitive specimen testing using the fluorescence polarization depolarization method, as well as a method for producing the same.

1 is a schematic diagram showing the structure of a particle according to one embodiment of the present invention. FIG. 10 is a diagram showing the results of calculation of transmittance by optical simulation.

The following describes in detail preferred embodiments of the present invention, but does not limit the scope of the present invention.

The particles according to the present invention are particles for use in specimen testing, the particles comprising silica particles, preferably having a surface coated with a hydrophilic compound, an average particle size of 50 nm to 215 nm, a refractive index of 1.4 or less, and carrying a luminescent rare earth complex therein.
Hereinafter, one embodiment of the particles according to the present invention will be described in detail with reference to the drawings.

FIG. 1 is a schematic diagram showing the structure of a particle according to one embodiment of the present invention.
Fig. 1 shows a luminescent particle 1 as a particle according to one embodiment of the present invention. The luminescent particle 1 has a silica particle 2 as a core particle, and the outer surface of the silica particle 2 is covered with a hydrophilic layer 4 made of a hydrophilic compound (coated with a hydrophilic compound). The silica particle 2 has pores 3 therein, and a luminescent rare earth complex 5 is supported in the pores 3. The refractive index and thickness of the hydrophilic layer 4 are set so as to be measurable by fluorescence depolarization spectroscopy.

The sample test using the light-emitting particles 1 is carried out by the fluorescence depolarization method.
The luminescent particle 1 has a luminescent rare-earth complex 5, which has a transition moment (transition dipole moment). Therefore, when the luminescent rare-earth complex 5 is excited by light polarized along the transition moment, the emitted fluorescence is also polarized along the transition moment.
In reality, the luminescent particles 1 (luminescent material) in the solvent rotate due to Brownian motion or the like, and the transition moment of the luminescent rare-earth complex 5 also changes over time. Therefore, the rotational motion of the luminescent material during the time that the fluorescence is emitted causes a part of the fluorescence polarization to be eliminated, and this is measured and evaluated as polarization anisotropy in the fluorescence depolarization method.

The rotational movement of the light-emitting material can be expressed by the following equation 1:
Q=3Vη/kT...(1)
Q: rotational relaxation time of the luminescent material V: volume of the luminescent material η: viscosity of the solvent k: Boltzmann's constant T: absolute temperature The rotational relaxation time Q of the luminescent material is the time required for the luminescent material to rotate through an angle θ (68.5°) such that cosθ = 1/e.
From Equation 1, it can be seen that the rotational relaxation time of the light-emitting material is proportional to the volume of the material, that is, the cube of the particle size.

On the other hand, the relationship between the luminescence lifetime and rotational relaxation time of a material and the degree of polarization in the fluorescence depolarization method can be expressed by the following formula 2.
p0/p=1+A(τ/Q)...(2)
p0: Degree of polarization when the material is assumed to be stationary (Q = ∞) p: Degree of polarization A: Constant τ: Luminescence lifetime of the material Q: Rotational relaxation time

Equations 1 and 2 show that in order to measure large changes in the degree of polarization, the relationship between the luminescence lifetime and rotational relaxation time of the luminescent material, i.e., the volume (particle size) of the luminescent material, is important, and the larger the particle size of the luminescent material, the longer the luminescence lifetime must be. In other words, with fluorescence depolarization, by setting the size (particle size) and luminescence lifetime of the luminescent material within an appropriate range, it is possible to sensitively detect changes in size due to aggregation of the luminescent material caused by antigen-antibody reactions, etc.

To experimentally evaluate the degree of polarization shown in Equation 2, polarized light is irradiated onto a sample as excitation light, and light emission is detected in a direction 90 degrees different from the direction of propagation of the excitation light. At this time, the detected light is separated into a polarized component parallel to the polarization direction of the excitation light and a polarized component perpendicular to the polarization direction of the excitation light, and the polarization anisotropy can be calculated using Equation 3 below.
r(t)=(I∥(t)−GI⊥(t))/(I∥(t)+2GI⊥(t))
...(3)
r(t): Polarization anisotropy at time t I∥(t): Emission intensity of the polarization component parallel to the polarization direction of the excitation light at time t I⊥(t): Emission intensity of the polarization component perpendicular to the polarization direction of the excitation light at time t G: Correction value, when irradiating with excitation light whose vibration direction differs by 90 degrees from the excitation light used in sample measurement, the ratio of the emission intensity of the polarization component parallel to the polarization direction of the irradiated excitation light to the emission intensity of the polarization component perpendicular to the polarization direction of the irradiated excitation light

1 is spherical, and the average particle size of the phosphor particles 1 is 50 nm or more and 215 nm or less. Here, the average particle size of the phosphor particles 1 is a value (number average particle size) determined by a dynamic scattering method.
If the average particle size of the light-emitting particles 1 is 50 nm or more, the change in size when they bind to a target substance and cause aggregation in a sample test can be made sufficiently large, making it possible to accurately determine polarization anisotropy.
Furthermore, if the average particle size of the phosphor particles 1 is 215 nm or less, the scattering of light per particle of the phosphor particles 1 can be reduced, and depolarization due to scattered light can be suppressed, allowing the polarization anisotropy to be determined with high accuracy.

The coefficient of variation of the particle size distribution of the phosphor particles 1, i.e., the value obtained by dividing the average particle size of the phosphor particles 1 by the standard deviation, is preferably 10% or less, and more preferably 5% or less. When the phosphor particles 1 have a uniform particle size, the measured value of polarization anisotropy becomes stable, and measurements with excellent reproducibility can be performed.
The shape of the phosphor particles 1 is not limited to spherical, and they may have shapes other than spherical as long as they have the above average particle size when measured by dynamic scattering.
Next, each component of the phosphor particle 1 will be described in detail below.

(Silica particles 2)
The luminescent particle 1 has a silica particle 2 as a core particle. This allows the particle to be larger than when a single molecule is used as the luminescent material, and both low-molecular-weight substances and high-molecular-weight substances can be used as the target substance.
The silica particles 2 shown in Fig. 1 are spherical. However, the shape of the silica particles 1 is not limited to spherical, and the silica particles 1 may have a shape other than spherical as long as the average particle size of the light-emitting particles 1 measured by a dynamic scattering method falls within the above-mentioned range. The average particle size of the silica particles may be 40 nm or more and 205 nm or less, or 46 nm or more and 204 nm or less.

The material forming the silica particles 2 is composed of a material containing silica as a main component. The silica particles 2 can be obtained by hydrolysis and condensation of silicon alkoxide as a main component.
The silica particles 2 preferably have an organic functional group. This changes the surface properties of the silica particles 2, allowing for a wider range of supported amounts of the luminescent rare earth complex 5 and types of materials that can be supported. Silica particles 2 having an organic functional group can be produced, for example, by adding and reacting bifunctional and trifunctional metal alkoxides during the synthesis of the silica particles 2.
The hydrolysis and condensation of the alkoxide may be carried out under basic conditions containing an aqueous solvent.
The silica particles 2 shown in FIG. 1 are porous silica particles having a plurality of pores 3 , but the silica particles 2 possessed by the luminescent particles 1 may be hollow silica particles having one pore 3 .

(Hole 3)
The refractive index of the silica constituting the silica particles 2 is generally about 1.46, and the refractive index of the luminescent particles 1 is 1.4 or less. In other words, when the silica particles 2 have voids 3 in a volume ratio of about 10% or more, the refractive index of the luminescent particles 1 can be set to 1.4 or less, thereby making it possible to suppress depolarization due to scattering of light in the luminescent particles 1.
Furthermore, when the silica particles 2 have voids 3 at a volume ratio of about 30% and the refractive index of the luminescent particles 1 is about 1.3, this is almost the same as the refractive index of water, 1.33. Therefore, it is possible to effectively suppress light scattering in the luminescent particles 1 when water is used as a medium, which is preferable.

One or more voids 3 are present inside the silica particle 2. A large number of voids 3 having a size of about 1 to 10 nm may be present inside the silica particle 2. Alternatively, one large void 3 having a size close to that of the silica particle 2 may be present, like a hollow structure. The greater the volume ratio of the voids 3 to the silica particle 2, the smaller the refractive index of the luminescent particle 1. In reality, there is an upper limit to the volume ratio of the voids 3 in the silica particle 2 that can be produced, so a realistic refractive index for the luminescent particle 1 is 1.2 or higher.
The pores 3 can be formed by using a surfactant, a block copolymer, or the like as a template when synthesizing the silica particles 2 .

(Hydrophilic layer 4)
Luminescent particles 1 have a hydrophilic layer 4 on the outer surface of silica particles 2. When luminescent particles 1 are in an aqueous medium, the hydrophilic layer 4 on the surface of luminescent particles 1 absorbs water, which is thought to serve as a light interference layer between the aqueous medium and silica particles 2. This makes it possible to further suppress light scattering in addition to the effect of silica particles 2 having pores 3. Furthermore, since luminescent particles 1 have a hydrophilic layer 4 on their surface, they can be made into particles that are highly effective in suppressing nonspecific adsorption without using BSA. This makes it possible to suppress quality variations from lot to lot.

The hydrophilic layer 4 is composed of a hydrophilic compound, that is, a molecule or polymer containing a hydrophilic group.
By immobilizing on the particle surface, as the hydrophilic group, molecules or polymers having a structure in which the electric charge is small or the electric charge is offset by a balance between cations and anions, nonspecific adsorption of impurities present in the sample to the particles can be reduced. For this reason, the hydrophilic compound constituting the hydrophilic layer 4 preferably contains at least one functional group or bond selected from the group consisting of a pyrrolidone group, an ether bond, a betaine group, and a hydroxyl group.

Specifically, the hydrophilic layer 4 can be primarily composed of, for example, polyethylene glycol, polyvinylpyrrolidone, sulfobetaine or phosphobetaine polymers, or polyglycidyl methacrylate in which the glycidyl group is ring-opened and a hydroxyl group is attached to the end of the molecule. Alternatively, monomolecules having hydrophilic groups may be directly attached to the surface of the silica particles 2 using a silane coupling agent or the like.

There are no limitations on the thickness of the hydrophilic layer 4, but it does not need to be thicker than the thickness required to exhibit hydrophilicity. If the hydrophilic layer 4 is too thick, it will resemble a hydrogel, and the thickness of the hydrated hydrophilic layer may change due to the influence of ions in the solvent. The thickness of the hydrophilic layer 4 is preferably 10 nm to 50 nm. The fluorescently labeled particles described in Patent Document 2 above have bovine serum albumin (BSA) supported on their surfaces to suppress nonspecific adsorption, which can lead to variations in the performance of the fluorescently labeled particles from lot to lot. On the other hand, the particles of this embodiment are coated with a hydrophilic compound (have a hydrophilic layer), thereby reducing nonspecific adsorption.

(Luminescent Rare Earth Complex 5)
In the present invention, the luminescent rare earth complex 5 is used as the luminescent dye because the wavelength and intensity of the luminescence are not easily affected by the surrounding environment and the luminescence has a long life.
Luminescent rare earth complex 5 is composed of a rare earth element and a ligand, and preferably contains at least one rare earth element selected from the group consisting of europium, terbium, neodymium, erbium, yttrium, lanthanum, cerium, samarium, gadolinium, dysprosium, thulium, ytterbium, and scandium.

As the rare earth element contained in the luminescent rare earth complex 5, europium and terbium can be suitably used, taking into consideration the luminescence lifetime and the fact that the luminescence wavelength range is in the visible light range.
For example, europium complexes generally have a luminescence lifetime of 0.1 to 1.0 ms. By appropriately adjusting this luminescence lifetime and the rotational relaxation time obtained from Equation 1, polarization anisotropy can be suitably detected. In the case of a europium complex in an aqueous dispersion, when the particle size of the luminescent particles 1 is approximately 50 to 300 nm, the polarization anisotropy expressed by Equation 3 becomes large before and after binding between the target substance and the luminescent particles 1.

At least one of the ligands in luminescent rare earth complex 5 has a light-harvesting function. The light-harvesting function refers to the ability to become excited by a specific wavelength and excite the central metal of the complex through energy transfer. Furthermore, a molecule with a transition moment is selected as the light-harvesting ligand. For example, luminescent rare earth complex 5 preferably includes at least one selected from bipyridine and phenanthroline as the light-harvesting ligand, although this is not limited to this.

Furthermore, it is preferable that the luminescent rare earth complex 5 has a ligand such as β-diketone, and the coordination of water molecules is suppressed, whereby the ligand such as β-diketone coordinated to the rare earth ion suppresses the deactivation process due to the transfer of energy to solvent molecules, etc., and strong fluorescent light is obtained.
The luminescent rare earth complex 5 may be a polynuclear complex as long as it has a transition moment.

By using a liquid in which the light-emitting particles 1 according to this embodiment are dispersed, it is possible to measure polarization anisotropy in relation to the aggregation and dispersion behavior of the light-emitting particles 1 while suppressing the effects of light scattering. Therefore, a colloidal liquid in which the light-emitting particles 1 according to this embodiment are dispersed in an aqueous medium can be used, for example, as a highly sensitive antibody testing reagent using the fluorescence polarization depolarization method. A buffer solution may also be used as the aqueous medium. Furthermore, to improve the stability of the liquid in which the particles according to this embodiment are dispersed, surfactants, preservatives, sensitizers, etc. may be added to the aqueous medium.

(Method for producing particles)
The method for producing the phosphor particles 1 having a core-shell structure according to this embodiment includes the following steps.

A process (step 1) in which silica particle precursors are polycondensed in an acidic or basic solution in which template molecules are dissolved, and then the template molecules are removed to obtain silica particles 2 having a refractive index of 1.4 or less.
A step (step 2) of obtaining luminescent dye-supported particles by supporting the luminescent rare earth complex 5 on the silica particles 2 obtained in step 1 in a solution in which the luminescent rare earth complex 5 has been dissolved.
A step (step 3) in which the surfaces of the luminescent dye-carrying particles obtained in step 2 are coated with a hydrophilic compound to obtain luminescent particles 1.

(Process 1)
The silica particles 2 in FIG. 1 can be obtained by a hydrolysis-condensation reaction of a solution containing silicon alkoxide as a silica particle precursor as a main component.
The silicon alkoxide used as the silica particle precursor is preferably a tetraalkoxysilane, and tetraethyl orthosilicate is typically used. In order to change the surface properties of the silica particles 2, bifunctional and trifunctional metal alkoxides may be added and reacted during the synthesis of the silica particles 2.
The hydrolysis-condensation reaction of the alkoxide is preferably carried out under basic conditions containing an aqueous solvent, and the basic catalyst can be ammonia, sodium hydroxide, or an amino acid such as lysine.
The pores 3 of the silica particles 2 can be formed in the silica particles 2 by using a surfactant or a block copolymer as a template. Specifically, micelles formed in a liquid using a long-chain alkylammonium salt or a polyethylene oxide-polypropylene oxide block copolymer are used as a template, and a skeleton mainly composed of silica is precipitated on the surface by hydrolysis of an alkoxide.
The template molecule is preferably at least one selected from a surfactant and a block copolymer.
In step 1, the particle size can be controlled by appropriately changing the reaction temperature, reaction time, concentration of each component, or type of solvent.
A method of leaching out template molecules with a weakly acidic solution can be used to remove the template from the pores 3. For example, the particles packed in the semipermeable membrane can be immersed in a solution mixed with acetic acid, and the solvent can be exchanged several times to remove the template from the pores 3.

(Step 2)
An example of a method for supporting the luminescent rare earth complex 5 in the pores 3 is to immerse the silica particles 2 obtained in step 1 in a solution in which the luminescent rare earth complex 5 has been dissolved. The adsorption of the luminescent rare earth complex 5 can be controlled by balancing the concentration of the luminescent rare earth complex 5, its solubility in the solvent, and its affinity for the surface of the silica particles 2 formed by the pores 3.
When the luminescent rare earth complex 5 is hydrophobic, the surface of the silica particle 2 formed by the pores 3 may be surface treated with a molecule having a high affinity for the luminescent rare earth complex 5 .
For example, the surface may be treated with a silane coupling agent, followed by hydrophobic treatment using the functional groups of the silane coupling agent. Examples of the silane coupling agent that can be used include alkyltrialkoxysilane, phenyltrialkoxysilane, methacryloxyalkoxysilane, and vinylalkoxysilane.
Alternatively, the surface can be hydrophobized using a silazane such as hexamethyldisilazane. Solvents that can be used for the hydrophobization using silazane include polar solvents such as water, alcohols, dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide. Also, solvents such as chloroform, toluene, and benzene can be used, and mixtures of these can also be used.
The size of the pores 3 is preferably larger than that of the luminescent rare earth complexes 5, so that the luminescent rare earth complexes 5 can be adsorbed onto the surfaces of the silica particles 2 formed by the pores 3 by the above method.

(Step 3)
The following methods can be used to coat the surfaces of the luminescent dye-carrying particles obtained in step 2 with a hydrophilic compound and form the hydrophilic layer 4: a method of coating the surfaces of the luminescent dye-carrying particles with a hydrophilic polymer by seed polymerization or the like, a method of directly adsorbing a hydrophilic polymer onto the luminescent dye-carrying particles, and a method of directly providing hydrophilic groups onto the surfaces of the luminescent dye-carrying particles using a silane coupling agent.
When performing seed polymerization, the surface of the luminescent dye-carrying particles may be treated with methacryloxypropyltrimethoxysilane or the like, and then glycidyl methacrylate or the like may be polymerized in an aqueous solvent. Potassium persulfate, azo polymerization initiators, or the like may be used as the initiator. Seed polymerization can be performed by heating to 50 to 70°C under deoxygenated conditions with gentle stirring.
The glycidyl groups present in the obtained sample can be ring-opened with ethanolamine, tris(hydroxymethyl)aminomethane, mercaptopropanediol, or the like to provide hydroxyl groups. Furthermore, carboxylic acids can be provided to the particle surface by treating the glycidyl groups with mercaptosuccinic acid, or the like. By providing functional groups such as carboxylic acids or amines on the particle surface, it becomes possible to bind, for example, antibodies to the particles.
Alternatively, sulfobetaine monomers or the like can be used in place of the glycidyl methacrylate monomer.
Furthermore, styrene monomer and methacryloxypropyltrimethoxysilane may be used instead of glycidyl methacrylate monomer. The styrene monomer and methacryloxypropyltrimethoxysilane are reacted in a solution using polyvinylpyrrolidone as a dispersant. This allows the formation of a hydrophilic layer 4 on the surface of the luminescent dye-carrying particles.

When using a hydrophilic silane coupling agent, polyethylene glycol or a silane coupling agent having a betaine group can be used. In this case, if a silane coupling agent having succinic acid as a functional group or aminopropylalkoxysilane is simultaneously reacted, a scaffold for binding antibodies, etc. to the particles can be simultaneously formed on the surface of the luminescent particle 1.

When directly adsorbing a hydrophilic polymer onto the surface of luminescent dye-carrying particles, polyvinylpyrrolidone or the like can be used. Polyvinylpyrrolidone has a high affinity for the silica surface and is easily adsorbed. Furthermore, by using a polyvinylpyrrolidone-polyacrylic acid copolymer, carboxylic acid can be added to the hydrophilic layer 4.

By binding various types of antibodies or other ligands to the luminescent particles 1 according to this embodiment, they can be used as specimen testing particles. To bind a ligand to the luminescent particles 1, it is sufficient to appropriately select the optimal method for binding the target ligand using the functional groups present in the hydrophilic layer 4.

(Affinity particles)
As a method for using the light-emitting particle 1 according to this embodiment, for example, an affinity particle having the light-emitting particle 1 according to this embodiment and a ligand bound to the light-emitting particle 1 can be provided.

A ligand is a compound that specifically binds to a receptor possessed by a specific target substance. The site at which a ligand binds to a target substance is fixed, and the ligand has a selective or specific high affinity. Examples of combinations of a ligand and a target substance include, but are not limited to, antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones or neurotransmitters and their receptors, nucleic acids and nucleic acid-recognizing receptors, etc. For example, when used to detect antigens or antibodies in a sample, antibodies or antigens can be used as the ligand. Examples of nucleic acids include deoxyribonucleic acid and the like.
In the present invention, affinity particles refer to particles that have a selective or specific high affinity for a target substance. The ligand is preferably at least one selected from the group consisting of an antibody, an antigen, and a nucleic acid.

The method for bonding the reactive functional group possessed by the phosphor particle 1 of this embodiment to the ligand through a chemical reaction can be any conventionally known method as long as it achieves the objectives of the present invention. Furthermore, when bonding the ligand through an amide bond, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate.

When the affinity particles have an antibody (antigen) as a ligand and the target substance is an antigen (antibody), a testing system based on the immuno-latex agglutination assay, which is widely used in fields such as clinical testing and biochemical research, can be preferably applied.

(In vitro diagnostic test reagents)
The test reagent for in vitro diagnosis according to the present invention, i.e., a test reagent for use in in vitro diagnosis to detect a target substance in a specimen, comprises the affinity particles described above and a dispersion medium for dispersing the affinity particles. The amount of the affinity particles contained in the test reagent is preferably 0.001% by mass or more and 20% by mass or less, and more preferably 0.01% by mass or more and 10% by mass or less.
The test reagent may contain a third substance such as a solvent or a blocking agent in addition to the affinity particles, as long as the object of the present invention can be achieved. Two or more types of third substances such as solvents or blocking agents may be combined. Examples of solvents include various buffer solutions such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvents contained in the test reagent are not limited to these.

(Test kit)
A test kit according to the present invention for use in detecting a target substance in a specimen in in vitro diagnosis comprises the test reagent and a housing containing the test reagent.
The test kit may further contain a sensitizer that promotes particle aggregation during an antigen-antibody reaction, for example. Examples of sensitizers include, but are not limited to, polyvinyl alcohol, polyvinylpyrrolidone, polyalginic acid, etc.
The test kit may also include a positive control, a negative control, a serum dilution solution, etc. As the medium for the positive control and the negative control, serum not containing the target substance to be measured, physiological saline, or a solvent may be used.
The kit according to the present invention can be applied to conventionally known methods to measure the concentration of a target substance, and can be particularly suitably used for detecting a target substance in a specimen by latex agglutination.

(Detection method)
The detection method according to the present invention is a method for detecting a target substance in a specimen by in vitro diagnosis, and includes the steps of: mixing the detection reagent described above with a specimen that may contain the target substance to obtain a mixed solution; irradiating the mixed solution with polarized light; and detecting the polarized light emitted from the mixed solution.
As mentioned above, in the process of detecting the polarized light emitted from the mixed liquid, the polarized components of the emitted light are separated into an emission component parallel to the excitation light and an emission component perpendicular to the excitation light and then detected, and the polarization anisotropy can be calculated using equation (3).
By optically detecting the agglutination reaction between the affinity particles and the target substance occurring in the mixed solution, the target substance in the sample can be detected, and the concentration of the target substance can be measured.
The detection reagent and the sample are preferably mixed at a pH ranging from 3.0 to 11.0. The mixing temperature is preferably 20°C to 50°C, and the mixing time is preferably 1 to 20 minutes. A solvent is preferably used for detection.
The concentration of the affinity particles in the mixed solution is preferably 0.001% by mass or more and 5% by mass or less, and more preferably 0.01% by mass or more and 1% by mass or less.

The present invention will be explained in more detail below using examples. However, the present invention is not limited to these examples.

(1) Preparation of Silica Particles 1 Silica particles 1 were prepared as core particles by modifying the Stöber method.
Specifically, ethanol, pure water, hexadecyltrimethylammonium bromide, and L-lysine were first added to a round-bottom, four-neck separable flask and stirred for 30 minutes using a mechanical stirrer. Next, the sample was heated to 70°C in an oil bath while still stirring, and then tetraethyl orthosilicate was added all at once and stirred vigorously. The amounts of the reagents charged were 10 mL of ethanol, 90 mL of pure water, 790 mg of hexadecyltrimethylammonium bromide, 900 mg of L-lysine, and 895 mg of tetraethyl orthosilicate.
After stirring overnight and cooling the sample, the precipitate was collected by centrifugation and the product was washed with a mixed solvent of ethanol and pure water. The washed sample was packed into a semipermeable membrane, and the semipermeable membrane packed with the sample was immersed in a 0.7% by volume acetic acid solution to remove the template. The semipermeable membrane that had been immersed overnight was immersed again in new acetic acid solution three times, and then washed with a mixed solvent of ethanol and pure water to obtain silica particles 1.
The resulting silica particles 1 had an average particle size of 46 nm. Electron microscope observation confirmed the presence of regular pores in the resulting silica particles with an average particle size of 46 nm. Furthermore, nitrogen adsorption measurement of a dried sample revealed that the porosity of silica particles 1 was approximately 30%.
The presence or absence of pores and the porosity were evaluated using an electron microscope (product name: S5500, manufactured by Hitachi High-Technologies) and a nitrogen adsorption analyzer (product name: Tristar, manufactured by Shimadzu Corporation). The average particle size was evaluated by dynamic light scattering using a Zetasizer Nano S (manufactured by Malvern Instruments).

(2) Preparation of Silica Particles 2 Silica particles 2 were prepared in the same manner as in the preparation of silica particles 1 in (1) above, except that the amounts of the solvents used were changed to 20 mL of ethanol and 80 mL of pure water. The average particle size of the obtained silica particles 2 was 95 nm.

(3) Preparation of Silica Particles 3 In the preparation of silica particles 1 in (1) above, the amounts of the solvents charged were changed to 50 mL of ethanol and 50 mL of pure water. Otherwise, silica particles 3 were synthesized in the same manner as silica particles 1. The average particle size of the obtained silica particles 3 was 204 nm.

(4) Preparation of Silica Particles 4 Tetraethyl orthosilicate was dissolved in a mixed solvent of ethanol and pure water and stirred for 30 minutes. After stirring, ammonia water was added at a volume ratio of 5% to the solvent, and the mixture was vigorously stirred for 5 hours. The product was washed with a mixed solution of ethanol and pure water to obtain silica particles 4. The average particle size of the obtained silica particles 4 was 100 nm.

Example 1
The silica particles 1 obtained in (1) above were dried at 60°C and then dispersed in octanol. The octanol solution in which the europium complex had been dissolved was mixed with the solution in which the silica particles 1 had been dispersed, and the mixture was stirred overnight. The europium complex used was (1,10-phenanthroline)tris[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedionato]europium(III) (hereinafter abbreviated as Eu(TTA) ₃ phen).
After stirring, the sample was centrifuged and washed with a toluene solvent. After washing, the sample was dispersed in a mixed solvent of ethanol and pure water in which polyvinylpyrrolidone K-30 had been dissolved, and stirred overnight to allow polyvinylpyrrolidone K-30 to be adsorbed onto the sample surface. A VOC-free silane coupling agent X12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added to the obtained sample, and a carboxylic acid treatment was performed. The obtained sample was washed with pure water to obtain luminescent particles.

Example 2
The silica particles 2 obtained in (2) above were dried at 60° C. and then dispersed in octanol. The octanol solution in which Eu(TTA) ₃ phen was dissolved was mixed with the solution in which 95 nm silica particles were dispersed, and the mixture was stirred overnight.
After stirring, the sample was centrifuged and washed with a toluene solvent. After washing, 3-glycidyloxypropyltrimethoxysilane was added to the ethanol solvent and stirred at 50°C overnight. The pH of the stirred sample was adjusted to 10 using triethylamine, and the sample was then added to an aqueous solution of mercaptosuccinic acid and mercaptopropanediol and stirred at 60°C overnight to provide hydroxyl and carboxyl groups to the sample surface. The obtained sample was washed with pure water to obtain luminescent particles.

Example 3
Luminescent particles were obtained in the same manner as in Example 1, except that the type of core particles used was changed to the silica particles 3 obtained in (3) above.

Example 4
The silica particles 3 obtained in (3) above were dried at 60°C and then dispersed in octanol. The octanol solution in which the terbium complex had been dissolved was mixed with the solution in which the silica particles 3 had been dispersed, and the mixture was stirred overnight. The terbium complex used was tris(acetylacetonato)(1,10-phenanthroline)terbium(III) (hereinafter abbreviated as Tb(AcAc) ₃ phen).
After stirring, the sample was centrifuged and washed with toluene solvent. After washing, a hydrophilic silane coupling agent synthesized by reacting polyethylene glycol with a molecular weight of 400 and 3-(triethoxysilyl)propyl isocyanate in an ethanol solvent in a pyridine solution under an argon atmosphere was added, and the mixture was stirred overnight at 50°C. Furthermore, a VOC-free silane coupling agent X12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added, and a carboxylic acid treatment was carried out. The obtained sample was washed with pure water to obtain luminescent particles.

Example 5
The silica particles 3 obtained in (3) above were dried at 60°C and then dispersed in a toluene solution containing phenyltrimethoxysilane, and the surfaces of the pores of the silica particles 3 were modified with phenyl groups. The toluene solution containing Eu(TTA) ₃ phen was mixed with the solution containing the silica particles 3 dispersed therein, and the mixture was stirred overnight.
After stirring, the sample was centrifuged and washed with toluene. After washing, the sample was dispersed in an aqueous solution of polyvinylpyrrolidone-polyacrylic acid copolymer and stirred overnight to allow the copolymer to adsorb onto the sample surface. The obtained sample was washed with pure water to obtain luminescent particles.

(Comparative Example 1)
Polystyrene particles were prepared by emulsion polymerization.
Specifically, pure water, styrene monomer, sodium p-styrenesulfonate, styrene monomer dissolved in Eu(TTA) ₃ phen, and polyvinylpyrrolidone-polyacrylic acid copolymer were first added to a round-bottom, four-neck separable flask. The resulting mixture was then stirred for 30 minutes with nitrogen bubbling using a mechanical stirrer. The sample was then heated to 70°C in an oil bath while stirring, and potassium persulfate (manufactured by Aldrich) was added as a catalyst. The styrene polymerization reaction was then carried out for 8 hours in a nitrogen atmosphere.
After cooling the sample, the precipitate was collected by centrifugation and the luminescent particles were washed with pure water. The luminescent particles containing polystyrene as the main component obtained above had an average particle size of 100 nm.

(Comparative Example 2)
The silica particles 4 obtained in (4) above were dried overnight at 60°C and dispersed in a toluene solvent. A toluene solution in which Eu(TTA) ₃ phen had been dissolved was mixed with the liquid in which the silica particles 4 had been dispersed, and the mixture was stirred overnight. After stirring, the sample was washed with a toluene solvent. After washing, the sample was dispersed in an aqueous solution in which polyvinylpyrrolidone-polyacrylic acid copolymer had been dissolved, and the mixture was stirred overnight, thereby allowing the polyvinylpyrrolidone-polyacrylic acid copolymer to be adsorbed onto the sample surface. The obtained sample was washed with pure water, and luminescent particles were obtained.

(Preparation of biotin-modified affinity particles)
Biotin was bound to each of the luminescent particles obtained in the examples and comparative examples.
Specifically, 0.8 mL of a 1% by weight particle dispersion was first taken, and the solvent was replaced with 1.6 mL of MES buffer solution at pH 5.4. Water-soluble carbodiimide (WSC) and sodium N-hydroxysulfosuccinimide (sulfo-NHS) were added to the particle dispersion in MES buffer solution at 0.5% by weight, respectively, and the mixture was allowed to react at 25°C for 1 hour. After the reaction, the dispersion was washed with MES buffer solution, and an amino-functionalized biotin molecule (Amin-PEG2-Biotin, manufactured by Thermo Fisher Scientific) was added. The mixture was allowed to react at 25°C for 2 hours to bind biotin to the particles. After binding, the particles were washed with MES buffer solution, and ethanolamine (manufactured by Tokyo Chemical Industry Co., Ltd.) was added, followed by a 30-minute reaction at 25°C. After the reaction, the particles were washed with MES buffer solution to obtain biotin-modified affinity particles at a concentration of 2% by weight.

(Evaluation of luminescent particles)
The measurements of the luminescent particles obtained in the examples and comparative examples were carried out as follows.
The thickness of the hydrophilic layer was evaluated by observation with an electron microscope (product name: S5500, manufactured by Hitachi High-Technologies) and measurement by dynamic light scattering using a Zetasizer Nano S (manufactured by Malvern Instruments).
The concentration of the suspension in which the luminescent particles were dispersed was evaluated using a gravimetric analyzer (trade name: Thermoplus TG8120, manufactured by Rigaku).
The fluorescence spectrum was measured with an excitation light wavelength of 330 nm and polarizers inserted in the optical paths on the excitation and emission sides. The excitation side of the polarizer was fixed, and the emission side was set parallel or perpendicular to the excitation side. A spectrofluorometer (product name: F-4500, manufactured by Hitachi High-Tech Science) was used.
The transmittance of the light-emitting particles was evaluated using the absorbance of light at a wavelength of 330 nm in the ultraviolet-visible absorption spectrum. The light path length was set to 10 mm, and the transmittance was measured for a dispersion liquid in which the particles were dispersed in pure water to a particle concentration of 0.002% by volume.
To evaluate the transmittance of the luminescent particles in an aggregated state, a dispersion was prepared by adding avidin to a phosphate buffer solution (pH 7.4) containing dispersed biotin-conjugated affinity particles. The transmittance was also evaluated using a Hitachi High-Tech Science double-beam spectrophotometer U-2810.
The transmittance was evaluated as A when it was 90% or more, and as B when it was less than 90%.

(Performance evaluation)
Table 1 shows the materials used in the examples and comparative examples and the evaluation results of transmittance.
The silica particles 1 to 3 used in the examples were found to have approximately 30% voids by volume as a result of nitrogen adsorption measurements, and the refractive index was approximately 1.3 based on the volume ratio of the silica component to the voids. The product obtained in Comparative Example 1 had a refractive index of approximately 1.6, since the refractive index of polystyrene was 1.59. Silica particles 4 used in Comparative Example 2 had a refractive index of at least 1.4, since the refractive index of silica was 1.45.

The luminescent particles (particles) obtained in the examples all have silica particles as core particles, and therefore are larger than those using monomolecular luminescent materials, making them suitable for measuring both low-molecular-weight and high-molecular-weight target substances.
The luminescent particles obtained in Examples 1, 2, 3, and 5 and Comparative Examples 1 and 2 emitted red light when excited with light having a wavelength of 330 nm, confirming that a europium complex was adsorbed to the produced particles. Furthermore, the luminescent particles obtained in Example 4 emitted green light when excited with light having a wavelength of 330 nm, confirming that a terbium complex was adsorbed to the produced particles.

In all of the luminescent particles obtained in the Examples and Comparative Examples, the particle dispersions at a concentration of 0.002% by volume were stable.
The thickness of the hydrophilic layer of the products obtained in the examples and comparative examples was 10 nm or less.
The transmittance in the non-aggregated state was 95% or more for all of the luminescent particles obtained in the Examples, and 90% or more for the luminescent particles obtained in Comparative Example 2. On the other hand, the luminescent particles obtained in Comparative Example 1 had a transmittance of 80% or less, which revealed that significant multiple scattering occurred due to the particles in the dispersion.
After aggregation, the transmittance of all the luminescent particles obtained in the examples remained at 90% or more, but that of the luminescent particles obtained in Comparative Example 2 was 80%, and multiple scattering of incident light by the particles became evident.

The polarization anisotropy was calculated using equation (3) from the results of fluorescence spectrum measurements before and after adding sodium chloride to the dispersion of luminescent particles obtained in the example to a concentration of 8% by mass. The polarization anisotropy changed by 0.01 or more before and after the addition of sodium chloride. This confirmed that it is possible to capture the particle aggregation reaction from changes in the polarization anisotropy value. On the other hand, with the luminescent particles obtained in the comparative example, no change in the polarization anisotropy value could be confirmed due to low transmittance and the significant impact of depolarization due to scattering.

Silica particles were prepared in the same manner as Silica Particles 1, except that the average particle size was 20 nm or less. The polarization anisotropy of the luminescent particles obtained using these silica particles was also evaluated before and after the addition of sodium chloride, but no change in the value was observed. This is thought to be because the size of the aggregated particles was not large enough to allow polarization anisotropy to be observed.

(Optical simulation)
The transmittance of a hypothetical particle dispersion was calculated by optical simulation. In the optical simulation, the scattering coefficient was calculated from the refractive index, particle size, and particle concentration using Mie scattering theory. Next, the transmittance was calculated from the product of the calculated scattering coefficient and the optical path length. The refractive index, particle size, and particle number density were each set to values in accordance with the examples and comparative examples. Specifically, the calculation was performed using multiple refractive index values selected from the range of 1.3 to 1.6, multiple particle size values selected from the range of 50 to 300 nm, and a particle concentration of 0.002% by volume.

(Material evaluation by optical simulation)
The results of the transmittance calculations performed by optical simulation are shown in FIG.
The horizontal axis of the coordinate system in Fig. 2 represents particle size, and the vertical axis represents refractive index. The values shown on the coordinate system plane in Fig. 2 represent calculated transmittances for light with a wavelength of 330 nm, assuming that at least two or more particles are aggregated.
Optical simulation results show that when the refractive index is 1.4 or less and the particle size is 200 nm or less, the transmittance is 90% or more. On the other hand, when the refractive index is greater than 1.4, the transmittance is high when the particle size is about 50 nm, but when the particle size is 100 nm or more, the transmittance drops significantly with increasing particle size, and the degree of scattering increases significantly.
The refractive index of 1.46 is assumed to be for silica particles without pores, and the transmittance was already 86% when the particle size was 100 nm.
Furthermore, the refractive index of 1.6 is assumed to be for polystyrene particles, and in this case, even if the particle size was 50 nm, the transmittance was below 80%.

The particles obtained in the above examples have a porosity of approximately 30%, and therefore the refractive index is thought to be approximately 1.33. In other words, as shown in Figure 2, the transmittance exceeds 90%, which is consistent with the transmittance measurement results obtained in the examples.
From the results of optical simulations, it has been found that particles that can suppress multiple scattering of light in particles and can be used for measurement have a refractive index of 1.4 or less and a particle size of 200 nm or less.

From the above, it can be seen that the luminescent particles (particles) obtained in the examples are able to suppress multiple scattering of light in liquid, and that the impact of depolarization due to scattering is small in polarization anisotropy measurements.

As a result, the particles according to the examples can be provided as specimen testing particles for fluorescence polarization depolarization, which have high detection sensitivity. In particular, the particles according to the examples are excellent at reducing noise in the detection signal, making them suitable for detecting low concentrations of target substances.

1 Luminescent particle 2 Silica particle 3 Hole 4 Hydrophilic layer 5 Luminescent rare earth complex

Claims (17)

Particles for specimen testing using a fluorescence depolarization method ,
the particles comprise silica particles;
The average particle size of the particles is 50 nm or more and 215 nm or less,
The refractive index of the particles is 1.4 or less,
the particles support a luminescent rare earth complex therein ;
Particles characterized in that a dispersion of 0.002% by volume of the particles dispersed in pure water has a transmittance of 90% or more for light with a wavelength of 330 nm . 2. The particle according to claim 1, wherein the surface of the silica particle is coated with a hydrophilic layer made of a hydrophilic compound. The particles described in claim 2, wherein the hydrophilic compound contains at least one functional group or bond selected from the group consisting of a pyrrolidone group, an ether bond, a betaine group, and a hydroxyl group. 4. The particle according to claim 2, wherein the thickness of the hydrophilic layer is 10 nm or more and 50 nm or less. 5. The particle according to claim 1, wherein the luminescent rare earth complex contains at least one rare earth element selected from the group consisting of europium, terbium, neodymium, erbium, yttrium, lanthanum, cerium, samarium, gadolinium, dysprosium, thulium, ytterbium , and scandium. 6. The particle according to claim 1, wherein the luminescent rare earth complex contains at least one selected from the group consisting of bipyridine and phenanthroline as a ligand. The particles according to any one of claims 1 to 6 , wherein the silica particles are porous silica particles. The silica particles according to any one of claims 1 to 7 , wherein the silica particles are hollow silica particles. The silica particles according to any one of claims 1 to 8 , wherein the silica particles have an organic functional group. 10. The particles according to claim 1, wherein the coefficient of variation of the particle size distribution of the particles is 10% or less. 11. The particles according to claim 1, wherein the coefficient of variation of the particle size distribution of the particles is 5% or less. 12. An affinity particle comprising the particle according to claim 1 and a ligand bound to the particle. The affinity particle according to claim 12 , wherein the ligand is at least one selected from the group consisting of an antibody, an antigen, and a nucleic acid. 14. An in vitro diagnostic test reagent comprising the affinity particles according to claim 12 or 13 and a dispersion medium for dispersing the affinity particles. The test reagent according to claim 14 , wherein the ligand is an antibody or an antigen and is used to detect the antigen or antibody in a sample. 16. An in vitro diagnostic test kit comprising: the test reagent according to claim 14 or 15 ; and a housing containing the test reagent. A method for detecting a target substance in a specimen using a fluorescence polarization depolarization method, comprising:
a step of mixing the test reagent according to claim 14 or 15 with a specimen to obtain a mixed solution;
irradiating the mixed liquid with polarized light;
and detecting polarized light emitted from the mixed liquid.