Particle, inspection particle, reagent and inspection kit, and detection method

Abstract

To provide particles and inspection particles having improved redispersibility of particles, especially, improved redispersibility in a reactive functional group activation step.SOLUTION: A particle contains a polymer having a structure represented by the formula (1) and a polymer having a structure represented by the formula (2). The difference between a glass transition point at 90 degrees or more and 120 degrees of less on a DSC curve obtained during first heating in differential scanning calorimeter (DSC) measurement of the particle, and a glass transition point at 90 degrees or more and 120 degrees of less on a DSC curve obtained during second heating, has a relationship of -5°C or more and 5°C or less.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to particles, test particles, reagents and test kits, and a detection method.

Background Art

[0002] In recent years, research has been widely conducted on purifying or quantifying a target substance using test particles having a ligand with an affinity for the target substance added to the particle surface. Patent Documents 1 and 2 describe particles having a core-shell structure and containing a carboxy group, which is a reactive functional group, or a 2,3-dihydroxypropyl group, etc. in the shell. For particles used for such purposes, it is preferable that the aggregation rate with respect to the target substance is high and the sensitivity is improved by uniformly adding the ligand to the particle surface. When adding the ligand to the particles, it is necessary to purify by sedimenting the particles with a centrifuge or the like, removing the supernatant, and then redispersing the particle sediment. In order for the ligand to bind uniformly to the particle surface, it is required to redisperse the particles uniformly in the redispersion step. Patent Document 3 shows a method of controlling the surface charge and increasing the redispersibility by adding a surfactant or a dispersion aid when preparing the particles. However, in the method of controlling the surface charge with an additive, it becomes difficult to add an antigen (or antibody) to the particle surface, and aggregation due to an immune reaction may be inhibited. Therefore, depending on the type of antigen (or antibody) to be bound, complicated and cumbersome preparation is required, and a method for improving the redispersibility of the particles is demanded.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] According to the studies of the present inventors, in the structure such as the particles described in Patent Document 1 and Patent Document 2, when centrifugal sedimentation treatment is performed and redispersion is carried out, there are problems in redispersibility, such as the case where redispersion takes a long time or the particle size does not become equal to the initial particle size after redispersion. In particular, when a ligand is chemically bonded to the particle surface, it has been found that the particles are likely to aggregate in the reactive functional group activation step and redispersion is more difficult. In addition, Patent Document 3 describes a method using an additive to improve redispersibility, but complicated and cumbersome preparation may be required depending on the type of ligand to be bonded. Details of the particles are not described, and the reactive functional group activation step is not described either.

[0005] Therefore, an object of the present invention is to provide particles and test particles with improved redispersibility of the particles, particularly redispersibility in the reactive functional group activation step.

Means for Solving the Problems

[0006] The particle contains a polymer having a structure represented by the formula (1) and a polymer having a structure represented by the formula (2), and the difference between the glass transition point at 90 °C or higher and 120 °C or lower on the DSC curve obtained during the first heating in the differential scanning calorimetry (DSC) measurement of the particle and the glass transition point at 90 °C or higher and 120 °C or lower on the DSC curve obtained during the second heating has a relationship of -5.0 °C or higher and 5.0 °C or lower. It is a particle characterized by this.

Chemical formula

Chemical formula

[0007] According to the present invention, it is possible to provide particles and assay particles in which the redispersibility of the particles, particularly the redispersibility in the reactive functional group activation step, is improved. [Brief Description of the Drawings]

[0008]

Figure 1

[0009] Hereinafter, the present invention will be described in detail with reference to preferred embodiments. The present invention relates to particles containing a polymer having a structure represented by the following formula (1) and a polymer having a structure represented by the following formula (2), wherein the difference between the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the first temperature increase in the differential scanning calorimetry (DSC) measurement of the particles and the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the second temperature increase has a relationship of -5.0°C or higher and 5.0°C or lower. [Chemical Formula] (R 1 represents a group having an epoxy group, a hydroxy group or a carboxy group. R 1 may be different for each structural unit.) [Chemical Formula] (R 2represents a substituted or unsubstituted phenyl group or naphthyl group. However, in the case of substitution, the substituent is a methyl group or an ethyl group. R 2 may be different for each structural unit.)

[0010] The inventors have found that the difference in glass transition temperature is important for obtaining particles with good redispersibility, particularly in the reactive functional group activation step, and have discovered that it is particularly important that the difference is -5°C or more and 5°C or less.

[0011] The particles of the present invention are characterized in that the difference between the glass transition temperature at 90°C or more and 120°C or less on the DSC curve obtained during the first heating in differential scanning calorimetry (DSC) measurement and the glass transition temperature at 90°C or more and 120°C or less on the DSC curve obtained during the second heating is -5.0°C or more and 5.0°C or less, and more preferably -4.4°C or more and 4.4°C or less. Further, it is more preferably -2.8°C or more and 2.8°C or less. The smaller the difference between the glass transition temperatures obtained during the first and second heatings in DSC measurement, the more difficult it is for the particles to deform as particles. Since the particles are less likely to deform during centrifugation, the contact area between the particles becomes smaller, and it is considered that the redispersibility is improved because they can be dispersed with a small force. It is more preferable that the glass transition temperatures obtained during the first and second heatings in DSC measurement are 103°C or more and 111°C or less.

[0012] The particles of the present invention contain a polymer having a structure represented by the above formula (1). In particular, it is preferable that the formula (1) has a structure represented by formula (1-A). [Chemical formula] (R 31 and R 32 at least one of which is a hydroxy group, and the other represents a hydroxy group, a group represented by the following formula (1-B), or a group represented by the following formula (1-C).) [Chemical formula] (R10 represents a single bond or a methylene group. R 12 , R 13 , R 14 represents a hydrogen atom, a methyl group, a hydroxy group, or a hydroxymethyl group, and one or more of R 12 , R 13 and R 14 represent a hydroxy group. Y 1 represents a sulfur atom or an imino group. *1 indicates the bonding position.)

Chemical formula

[0013] Specific structures of the formula (1-A) are shown below, but are not limited thereto.

Chemical formula

[0014] The structure represented by the formula (1-A) of the present invention is obtained by reacting the polymer obtained by polymerizing the monomer represented by the formula (X1) described below with the formula (X2) described below. Specific examples of the monomer represented by (X1) are not particularly limited, but glycidyl (meth) acrylate is preferred.

Chemical formula

Chemical formula

[0015] Among them, it is more preferable that Y1 in the formula (1-B) is S. This is because, in the examples described later, when Y1 is S, the difference in the glass transition points obtained during the first and second heating in the DSC measurement becomes smaller, and the redispersibility is improved. It is presumed that this is because the S atom has a denser structure in water than the N atom.

[0016] The particles of the present invention contain a polymer having a structure represented by the formula (2) and have a phenyl group or a naphthyl group which is substituted or unsubstituted at R2. The phenyl group and the naphthyl group are known to increase the glass transition point, and by containing the repeating unit structure represented by the formula (2) in the particles, the glass transition point of the particles is increased.

[0017] The particles of the present invention preferably have a mol ratio of the structure represented by the formula (1) to the structure represented by the formula (2) of 0.07 or more and 0.48 or less, as the redispersibility is further improved.

[0018] During the first heating in DSC measurement, since the endothermic peaks derived from the structure represented by formula (2) with a high glass transition point and the structure represented by formula (1) with a low glass transition point appear on the high-temperature side and the low-temperature side respectively, the structures represented by formula (1) and formula (2) mix within the particles during the first heating to form a gradient curve. Therefore, during the second heating in DSC measurement, the endothermic peak in the DSC measurement of the structure represented by formula (2) is affected by the endothermic peak of the structure represented by formula (1) and may be detected lower than the first time. Thus, by setting the molar ratio of the structure represented by formula (1) to the structure represented by formula (2) to 0.48 or less, it is possible to obtain particles with a small difference in glass transition points obtained during the first and second heatings in DSC measurement and that are difficult to deform, and the redispersibility can be improved. Also, by setting the molar ratio of the structure represented by formula (1) to the structure represented by formula (2) to 0.07 or more, the hydrophobicity of the particle surface is lowered and the hydrophobic interaction between particles is suppressed, so the redispersibility can be improved.

[0019] The molar ratio of the structure represented by formula (1) to the structure represented by formula (2) can be expressed as the ratio of the charged amounts when it has been confirmed that the polymerization addition rate is substantially 100%, but it can also be measured by FT-IR from the particles. Specifically, the peak height existing at 1500 - 1650 cm -1 derived from the C=C bond of the aromatic ring is designated as A, and the peak height existing at 1680 - 1750 cm -1 derived from the carbonyl group of the ester bond is designated as B, and the value of A / B can be calculated and converted to the molar ratio.

[0020] The particles of the present invention preferably have a core-shell structure in which the core is covered with a shell film (hereinafter simply referred to as "shell"). The particles of the present invention preferably have a core having a structure represented by formula (2) with a high glass transition temperature, covered with a shell containing a structure represented by formula (1) with a low glass transition temperature. Formula (1) has a hydrophilic structure. Further, it is more preferable that the content ratio of the structure represented by formula (1) in the shell of the particles is higher than the content ratio of the structure represented by formula (1) in the core. Having such a core-shell structure and the content ratio of the structure represented by formula (1) in the shell of the particles being higher than the content ratio of the structure represented by formula (1) in the core makes the particles less likely to deform during centrifugation and suppresses hydrophobic interactions, thus further improving the redispersibility.

[0021] The particles of the present invention may contain a cross-linking agent in the shell. By containing a cross-linking agent in the shell, a denser shell is formed, and the particles are less likely to deform during centrifugation, so it is considered that the redispersibility is improved.

[0022] The film thickness of the shell of the particles of the present invention is preferably 5 nm or more and 35 nm or less. More preferably, it is 10 nm or more and 25 nm or less. By making the film thickness of the shell 5 nm or more, the hydrophilicity of the particle surface is improved, and the hydrophobic interaction between particles is suppressed, so the redispersibility is improved. Also, by making the film thickness of the shell 35 nm or less, the particles are less likely to deform, and the redispersibility is improved.

[0023] Further, the particles of the present invention may have a structure other than the formula (1) and the formula (2). For example, there are structures obtained by polymerizing monomers such as styrenes, acrylates, and methacrylates, but it is not particularly limited. Also, the structure other than the formula (1) and the formula (2) may have a plurality of structures at the same time, but it is more preferable when the weight ratio of formula (1) in the particles is 9 wt% or more and 39 wt% or less.

[0024] The particles of the present invention preferably have a volume average particle diameter of 150 nm or more and 500 nm or less. By setting the particle diameter to 150 nm or more, the particles tend to be easily centrifugally sedimented. Further, by setting it to 500 nm or less, the force applied to the particles during centrifugation can be suppressed, and it is considered that the particles are less likely to be deformed and the redispersibility is improved.

[0025] The particles of the present invention preferably have a value of the ratio (Dv / Dn) of the volume average particle diameter (Dv) and the number average particle diameter (Dn) of 1.25 or less. Further, it is more preferable that the value of Dv / Dn is less than 1.15. It is known that the closer the value of Dv / Dn is to 1, the narrower the particle size distribution becomes. By setting the value of Dv / Dn to 1.25 or less, it is considered that the variation in the particle size is reduced and the contact area between the particles is reduced, thereby improving the redispersibility.

[0026] The method for producing the particles of the present invention is not particularly limited, but soap-free emulsion polymerization is preferred. By using soap-free emulsion polymerization, the particle size distribution becomes uniform and the redispersibility is improved. The two-stage swelling polymerization method in which the monomer forming the shell is swollen in the core and the shell is formed with a hydrophobic initiator or the like is known as an effective method for making the particle size distribution uniform, but the core is swollen. In order to further improve the redispersibility, it is considered preferable to react in a state where the particle density in the aqueous solution is high, so the shell is also preferably produced by soap-free emulsion polymerization.

[0027] In addition, when producing the particles of the present invention, a polymerization initiator may be used. The polymerization initiator is not particularly limited, but a hydrophilic polymerization initiator is preferred.

[0028] The test particles in the present invention have high affinity for a target substance selectively or specifically by a ligand added to the particle surface. In particular, it is preferably one in which a ligand is added to the particle surface by chemical bonding.

[0029] In addition, the ligand in the present invention refers to a compound that specifically binds to a receptor possessed by a specific target substance. The site where the ligand binds to the target substance is determined and has selective or specifically high affinity. Examples include antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones and neurotransmitters and their receptors, nucleic acids, etc., but the ligand in the present invention is not limited to these. Examples of nucleic acids include deoxyribonucleic acid. The test particles in the present invention have selective or specifically high affinity (affinity) for the target substance. It is preferable that the ligand in the present invention is any one of an antibody, an antigen, and a nucleic acid.

[0030] The reagent in the present invention has the test particles in the present invention and a dispersion medium for dispersing the test particles. The reagent in the present invention may contain a third substance such as a solvent or a blocking agent in addition to the test particles in the present invention within the range capable of achieving the object of the present invention. Two or more kinds of third substances such as a solvent and a blocking agent may be combined and contained. Examples of the dispersion medium used in the present invention include various buffer solutions such as phosphate buffer, glycine buffer, Good buffer, Tris buffer, ammonia buffer, etc., but the dispersion medium contained in the reagent in the present invention is not limited to these.

[0031] The test kit in the present invention has the above reagent and a housing that encloses the above reagent. As the kit in the present invention, in addition to the reagent in the present invention (hereinafter, reagent 1), a reaction buffer solution (hereinafter, reagent 2) may be provided. A sensitizer may be contained in both or either one of reagent 1 and reagent 2. Further, the kit in the present invention may include a positive control, a negative control, a serum diluent, etc. in addition to reagent 1 and reagent 2. As the medium for the positive control and the negative control, in addition to serum and physiological saline that do not contain a measurable target substance, a solvent may be used.

[0032] The inspection method using the particles in the present invention is not particularly limited, but an inspection method using an antibody (antigen) as a ligand and an antigen (antibody) as a target substance is preferred. Specifically, immunochromatography, immunofluorescence analysis, chemiluminescent immunoassay, latex agglutination method, etc. can be mentioned, and among them, it can be preferably applied to the latex agglutination measurement method widely used in the fields of clinical inspection, biochemical research, etc.

[0033] As the latex agglutination method in the present invention, it is a method for detecting a target substance in a sample, and includes a step of mixing the sample that may contain the target substance with the test reagent to obtain a mixed solution, a step of irradiating the mixed solution with light, and a step of detecting at least one of transmitted light or scattered light from the light irradiated on the mixed solution. Examples of the method for redispersing the particles include a redispersion method by stirring and a redispersion method by vibration. Examples of the redispersion method by stirring include a stirring method for forming a vortex such as a vortex mixer and a method for directly stirring the precipitate. Examples of the redispersion method by vibration include an ultrasonic dispersion method.

[0034] By adding a ligand to the surface using the particles of the present invention, it can be used as an inspection particle for a specific target. When adding the ligand to the surface by chemical bonding, it is necessary to activate the reactive functional groups on the particle surface. This step is defined as the reactive functional group activation step of the present invention. The method for activating the reactive functional groups is not particularly limited, and general methods can be used. When the reactive functional group is a carboxyl group, it can be activated using a condensing agent and a condensation aid. Examples of the condensing agent include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, etc. In particular, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is preferred. Examples of the condensation aid include sodium N-hydroxysulfosuccinimide, N-hydroxysuccinimide, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, ethyl cyano(hydroxyimino)acetate. In particular, sodium N-hydroxysulfosuccinimide is preferred.

[0035] [Method for Measuring Volume Average Particle Size and Particle Size Distribution in Aqueous Dispersion of Particles] The method for measuring the volume average particle size (Dv) in the aqueous dispersion of the particles in the present invention will be described. The Dv of the particles present in the aqueous dispersion in the present invention is measured by the dynamic light scattering method. For example, it is measured at 25 °C using a Zetasizer (Zetasizer ultra: manufactured by Malvern Panalytical). The Dv of the core of the present invention is measured for the aqueous dispersion of the core obtained in Step 1. Also, the Dv of the particles having the core and shell of the present invention is measured for the aqueous dispersion of the particles obtained in Step 3. In addition, the particle size distribution of the particles having the core and shell of the present invention is calculated by measuring the number average particle size (Dn) by the dynamic light scattering method and taking the ratio (Dv / Dn) of the Dv to the Dn.

[0036] [Method for Measuring Film Thickness of Shell of Particles] The method for calculating the film thickness of the shell of the particles in the present invention will be described. The film thickness of the shell is calculated by subtracting the volume average particle size of the core from the volume average particle size of the particles obtained by measuring the aqueous dispersion of the particles obtained in Step 3 and dividing the result by 2.

[0037] [Method for Measuring Glass Transition Point of Particles] The glass transition point in the present invention is defined as the temperature of the intersection of a straight line obtained by extending the baseline on the low-temperature side (a straight line in the range of 5 to 10 °C before the value of the time derivative of thermal decomposition starts to increase) in DSC measurement to the high-temperature side and a tangent line drawn at a point where the gradient of the curve of the step-like change part of the glass transition becomes maximum (see Figure 1). Hereinafter, the method for measuring the glass transition point of the particles in the present invention will be described. The glass transition point of the particles in the present invention is measured using particles in a freeze-dried state weighed so as to be 10 mg or more and 15 mg or less in an aluminum pan. The measuring device and measuring conditions are as follows. · Measuring device: Differential scanning calorimeter: DSC7020AS-3D (trade name, manufactured by Hitachi High-Tech Science Corporation) · Aluminum pan: GAA-0068 (manufactured by Hitachi High-Tech Science Corporation) · Temperature conditions: Heat up from 30 °C to 250 °C at 10 °C / min, then cool down from 250 °C to 30 °C at 10 °C / min, and then heat up from 30 °C to 250 °C at 10 °C / min again. · Analysis software: TA7000 Standard Analysis (Hitachi High-Tech Science Corporation)

Example

[0038] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

[0039] [Production Example 1] (Synthesis of Particle 1) (Step - 1) 23.52 g of styrene (St: Kishida Chemical Co., Ltd.), 0.43 g of divinylbenzene (DVB: Kishida Chemical Co., Ltd.) and 800.00 g of ion-exchanged water were weighed into a 2 L four-neck separable flask and mixed to form a mixed solution. Then, while stirring the mixed solution at 100 rpm, it was maintained at 70 °C, and nitrogen flow was carried out at a flow rate of 200 ml / min to deoxygenate the inside of the four-neck separable flask. Next, a solution prepared separately by dissolving 1.02 g of V-50 (Fuji Film Wako Pure Chemical Corporation) in 30.00 g of ion-exchanged water was added to the mixed solution to initiate soap-free emulsion polymerization. By reacting for 48 hours from the start of polymerization, a dispersion of particles (core 1) serving as the core composed of a copolymer of St and DVB was obtained. A part of it was sampled and evaluated using dynamic light scattering (Zetasizer ultra: Malvern Panalytical). As a result, the volume average particle size was 274 nm.

[0040] (Step - 2) The solid content concentration of the dispersion obtained in Step 1 was adjusted with ion-exchanged water so that it became 2.0%. The liquid volume of the adjusted dispersion was 1261.52 g. A part of this dispersion was sampled, and the filtrate from which the particles were removed was evaluated for the contents of St and DVB using gas chromatography. As a result, the total content of St and DVB was 4 ppm. Next, 12.23 g of glycidyl methacrylate (GMA: Kishida Chemical Co., Ltd.) was added, and while stirring at 100 rpm, the temperature was lowered to 70 °C, and nitrogen flow was carried out at a flow rate of 200 ml / min to deoxygenate the inside of the four-neck separable flask. Then, a solution prepared separately by dissolving 0.30 g of V-50 in 10.00 g of ion-exchanged water was added to the mixed solution to initiate the formation of the shell film. By continuously stirring for 17 hours after the start of the reaction, a dispersion containing mother particles (mother particle 1) having a core-shell structure was obtained. After the dispersion was slowly cooled to room temperature, a part of it was sampled and evaluated for the polymerization conversion rate using gas chromatography. As a result, it was confirmed that it was substantially 100%.

[0041] (Step - 3) An aqueous solution prepared in advance by dissolving mercaptosuccinic acid (MSA: Wako Pure Chemical Industries, Ltd.) and 3-mercapto-1,2-propanediol (MPD: Wako Pure Chemical Industries, Ltd.) in an aqueous dispersion containing mother particle 1 (the molar ratio of 3-mercapto-1,2-propanediol to mercaptosuccinic acid is 6:4 (mole fraction), and the total number of moles of MSA and MPD is equal to the number of moles of the glycidyl methacrylate) was added, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. Next, while stirring the above at 200 rpm, the temperature was raised to 70 °C, and this state was maintained for another 18 hours to obtain a dispersion of particle 1 having a core-shell structure. Particle 1 was separated from the dispersion by a centrifuge, and the operation of redispersing particle 1 in ion-exchanged water was repeated 5 times to purify particle 1, and finally, it was stored in the state of an aqueous dispersion prepared so that particle 1 was 1.0 wt%. The results of evaluating the particle physical properties of particle 1 and the weight ratio of formula (1) in particle 1 are shown in Table 1.

[0042] [Table 1]

[0043] [Production Example 2] (Synthesis of Particle 2) (Step - 1) A dispersion of the core particle (core 2) was prepared under the same conditions as in Step 1 of Production Example 1. The volume average particle size was 270 nm. (Step - 2) Using the dispersion obtained in the above Step - 1, except that the amount of GMA was changed from 12.23 g to 5.15 g and the amount of V - 50 was changed from 0.30 g to 0.13 g, a dispersion of mother particle (mother particle 2) was obtained by the same experimental operation as in Step - 2 of Production Example 1. When the polymerization conversion rate was evaluated using gas chromatography, it was confirmed that it was substantially 100%. (Step - 3) A dispersion of particle 2 having a core-shell structure was obtained by the same experimental operation as in Step - 3 of Production Example 1, except that mother particle 2 was used. The results of evaluating the particle physical properties of particle 2 and the weight ratio of formula (1) in particle 2 are shown in Table 1.

[0044] [Production Example 3] (Synthesis of Particle 3) (Step - 1) 25.35 g of styrene (St: Kishida Chemical Co., Ltd.), 0.46 g of divinylbenzene (DVB: Kishida Chemical Co., Ltd.) and 1491.02 g of ion - exchanged water were weighed into a 2 - L four - neck separable flask to form a mixed solution. After that, while stirring this mixed solution at 140 rpm, it was maintained at 70 °C, and the inside of the four - neck separable flask was deoxygenated by performing a nitrogen flow at a flow rate of 200 ml / min. Next, a solution prepared separately by dissolving 1.10 g of V - 50 (Fuji Film Wako Pure Chemical Corporation) in 30.00 g of ion - exchanged water was added to the mixed solution to initiate soap - free emulsion polymerization. By reacting for 48 hours from the start of polymerization, a dispersion of particles (core 3) composed of a copolymer of St and DVB was obtained. A part of it was sampled, and when the volume - average particle diameter of core 3 was evaluated using dynamic light scattering (Zetasizer ultra: Malvern Panalytical), the volume - average particle diameter was 219 nm. (Step - 2) Using the dispersion obtained in the above Step - 1, except that the amount of GMA was changed from 12.23 g to 13.18 g and the amount of V - 50 was changed from 0.30 g to 0.32 g, a dispersion of mother particles 3 was obtained by the same experimental operation as in Step - 2 of Production Example 1. When the polymerization conversion rate was evaluated using gas chromatography, it was confirmed that it was substantially 100%. (Step - 3) Except for using the mother particles 3, a dispersion of particles 3 having a core - shell structure was obtained by the same experimental operation as in Step - 3 of Production Example 1. The results of evaluating the particle physical properties of particle 3 and the weight ratio of the formula (1) in particle 3 are shown in Table 1.

[0045] [Production Example 4] (Synthesis of Particle 4) (Step - 1) A dispersion of core particles (core 4) was prepared under the same conditions as in Step 1 of Production Example 1. The volume - average particle diameter was 270 nm. (Step - 2) Using the dispersion obtained in the above Step-1, a dispersion of the mother particles 4 was obtained by the same experimental operation as in Step-2 of Production Example 1, except that the amount of GMA was changed from 12.23 g to 7.51 g and the amount of V-50 was changed from 0.30 g to 0.18 g. When the polymerization conversion rate was evaluated using gas chromatography, it was confirmed that it was substantially 100%. (Step-3) A dispersion of the particles 4 having a core-shell structure was obtained by the same experimental operation as in Step-3 of Production Example 1, except that the mother particles 4 were used. Table 1 shows the results of evaluating the particle physical properties of the particles 4 and the weight ratio of the formula (1) in the particles 4.

[0046] [Production Example 5] (Synthesis of particles 5) (Step-1) In Step-1 of Production Example 1, a dispersion of particles (core 5) was obtained by the same experimental operation, except that the amount of DVB used was changed from 0.43 g to 1.47 g. A part of it was sampled and evaluated using dynamic light scattering (Zetasizer ultra: manufactured by Malvern Panalytical), and the volume average particle diameter was 253 nm. (Step-2) In Step-2 of Production Example 1, using the dispersion of core 5, 1.16 g of DVB was added simultaneously with GMA, and a dispersion of mother particles 5 was obtained by the same experimental operation, except that the amount of V-50 was changed from 0.30 g to 0.13 g. When the polymerization conversion rate was evaluated using gas chromatography, it was confirmed that it was substantially 100%. (Step-3) A dispersion of the particles 5 having a core-shell structure was obtained by the same experimental operation as in Step-3 of Production Example 1, except that the mother particles 5 were used. Table 1 shows the results of evaluating the particle physical properties of the particles 5 and the weight ratio of the formula (1) in the particles 5.

[0047] [Production Example 6] (Synthesis of particles 6) (Step-1) A dispersion of the core particles (core 6) was prepared under the same conditions as in Step 1 of Production Example 1. The volume average particle diameter was 271 nm. (Process - 2) A dispersion of mother particles 6 was obtained by the same experimental operation as in Process - 2 of Production Example 1. (Process - 3) An aqueous solution in which 2 - amino - 2 - hydroxymethyl - 1,3 - propanediol hydrochloride (Tris: Tokyo Chemical Industry) was dissolved from MPD was subjected to the same experimental operation as in Process - 3 of Production Example 1, except that the total number of moles of MSA and Tris was changed from an equal multiple to twice the number of moles of the glycidyl methacrylate, to obtain a dispersion of particles 6 having a core - shell structure. Table 1 shows the results of evaluating the particle physical properties of particles 6 and the weight ratio of the formula (1) in particles 6.

[0048] [Comparative Production Example 1] (Synthesis of Comparative Particles 1) St, GMA, DVB, V - 50 and ion - exchanged water were used in the following formulation: St / GMA / DVB / V - 50 / H20 = 1.2 / 1.8 + 0.3 / 0.04 / 0.06 / 110 (g). After nitrogen substitution, a polymerization reaction was carried out at 70 °C for 24 hours. 0.3 g of GMA was added 2 hours after the start of polymerization to obtain a dispersion of comparative mother particles 1. The obtained comparative mother particles 1 were precipitated by centrifugation (15,000 rpm, 15 min, 4 °C), and after decantation of the supernatant, they were redispersed in 200 ml of water. The above operation was repeated 3 times to wash the comparative mother particles 1, and finally they were dispersed in water. To introduce an amino group into 0.25 g of this washed mother particle 1, NH 4OH (55.3 mmol; equivalent to 50 times the amount of GMA unit) was added and the pH was adjusted to 11 with 1N HCl. The reaction was carried out at 70 °C for 24 hours with stirring using a stirrer to open the epoxy group of GMA. Next, an example of immobilization on the comparative mother particle 1 obtained above using ethylene glycol diglycidyl ether (EGDE; Wako Pure Chemical Industries, Ltd.) is shown. An excess of EGDE was charged so as to be 100 times the amount (mol) of the amino groups of about 62.5 mg of comparative mother particle 2, and the mixture was stirred at 30 °C for 24 hours at pH 11 (prepared with 1N NaOH) to form a covalent bond between the epoxy group of EGDE and the amino groups on the comparative mother particle 1. An excess amount of EGDE was added to prevent the epoxy groups at both ends of one molecule of EGDE from being simultaneously immobilized on the comparative mother particle 1. As a result, a dispersion of particles (EGDE particles) in which EGDE was bound to the comparative mother particle 1 was obtained. After the reaction, the product was washed three times with water by centrifugation. To introduce amino groups into 0.25 g of the washed EGDE particles, NH 4 OH (55.3 mmol; equivalent to 50 times the amount of GMA unit) was added and the pH was adjusted to 11 with 1N HCl. The reaction was carried out at 70 °C for 24 hours with stirring. Then, after centrifugal purification using ion-exchanged water under the conditions of 4 °C, 27000 G, and 20 minutes for three times, the product was redispersed in methanol so that the solid content ratio became 1 wt%. Next, 0.88 g of succinic anhydride (Tokyo Chemical Industry Co., Ltd.) was added to dispersion liquid 1 weighed so that the amount of particles became 0.20 g, and the mixture was shaken at 30 °C for 5 hours to react the primary amine of the particles with amino groups introduced at the EGDE terminal and succinic anhydride, thereby obtaining comparative particle 1. Table 1 shows the results of evaluating the particle physical properties of comparative particle 1.

[0049] [Comparative Production Example 2] (Synthesis of Comparative Particle 2) A core produced under the same conditions as in Step - 1 of Production Example 1 was prepared in 500 g of water to a solid content of 5.0 g. To this, an organic solvent (Shellsol TK: 0.1 g, manufactured by Shell Chemicals), 4.66 g of GMA were added in sequence and stirred. After that, 2 g of AIBN (azobisisobutyronitrile) was added and slowly stirred at 75 °C for 24 hours to form a polymer part. Next, after cooling this reaction solution, it was filtered through a 500 - mesh wire net. By washing these particles with distilled water using centrifugation, a dispersion of Comparative Particle 2 having a core - shell structure was obtained. Table 1 shows the results of evaluating the particle physical properties of Comparative Particle 2.

[0050] [Comparative Production Example 3] (Synthesis of Comparative Particle 3) (Step - 1) In Step - 1 of Production Example 1, a dispersion of particles (Comparative Core Particle 3) was obtained by the same experimental operation except that the polymerization time was changed from 48 hours to 3 hours. When a part of it was sampled and evaluated using dynamic light scattering (Zetasizer ultra: manufactured by Malvern Panalytical), the volume - average particle diameter was 233.5 nm. (Step - 2) A dispersion of Comparative Mother Particle 3 was obtained by the same experimental operation as in Step - 2 of Production Example 1. (Step - 3) A dispersion of Comparative Particle 3 having a core - shell structure was obtained by the same experimental operation as in Step - 3 of Production Example 1 except that the above - mentioned Comparative Mother Particle 3 was used. Table 1 shows the results of evaluating the particle physical properties of Comparative Particle 3 and the weight ratio of Formula (1) in Comparative Particle 3.

[0051] [Evaluation 1] Redispersibility evaluation of particles having activated carboxy groups (particle - size criterion) (Reactive functional group activation step of particles) For the particles prepared in Production Examples 1 to 6 and Comparative Production Examples 1 to 3, 180 μL of a 1.7 wt% aqueous suspension was taken into a 1.5 mL microtube, and 90 μL of a 5.0% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 90 μL of a 5.0% aqueous solution of N-hydroxysulfosuccinimide sodium were added thereto as condensing agents, and the mixture was stirred at room temperature for 30 minutes to obtain particles having carboxyl groups activated (hereinafter referred to as activated particles). (Redispersion step) The dispersion of the activated particles prepared above was sedimented by a centrifuge at a force of 20000 G for 5 minutes, the supernatant was removed, and 250 μL of a Tris-HCl buffer solution with a pH of 8.0 was newly added. Six samples of the activated particle sediment with the buffer solution added were prepared, and ultrasonic dispersion treatment was performed 16 seconds × 15 times at a mode of High and a liquid temperature of 4 °C using an ultrasonic disruptor Bioruptor II (TYPE6) manufactured by Sonic Bio Co., Ltd.

[0052] (Redispersion evaluation) After visually confirming the state of the particles in the microtube after the ultrasonic dispersion treatment, the particle sizes of the respective particles were measured with a Zetasizer ULTRA manufactured by Spectris Co., Ltd., and the average value of the six samples of the particle size change before and after activation was calculated and evaluated as follows. A: Activated particle size after ultrasonic dispersion / Particle size before activation = 1.05 or less, and at the time of ultrasonic dispersion treatment for 16 seconds × 6 times, a uniform solution without a precipitate was visually observed. B: Activated particle size after ultrasonic dispersion / Particle size before activation = 1.05 or less C: Activated particle size after ultrasonic dispersion / Particle size before activation > 1.05 and ≤ 1.1 D: Activated particle size after ultrasonic dispersion / Particle size before activation > 1.1 and ≤ 2.0 E: Visually, a precipitate remained.

[0053] The evaluation results of the activated particles of Particles 1 to 6 and Comparative Particles 1 to 3 are shown in Table 2. For all of Particles 1 to 6, the evaluation results were C or higher. On the other hand, for Comparative Particles 1 to 3, the evaluation results were all E. As shown in Table 1 for Comparative Particle 1 and Comparative Particle 2, the weight ratios of the formula (1) in the particles were 64% and 63% respectively. Compared with Particles 1 to 6, the proportion of the structure represented by the formula (1) with a lower glass transition point was larger, so the particles were soft and easily deformed. Therefore, the redispersibility decreased. For Comparative Particle 3, since the polymerization time was short, the cross-linking of the core particles was insufficient and St remained in the solution. Therefore, as shown in Table 1, the glass transition point during the first heating was lower than that during the second heating, the particles were soft and easily deformed, and the redispersibility decreased.

[0054] [Evaluation 2] Redispersibility evaluation of particles having activated carboxyl groups (time-based) (Reactive functional group activation step of particles) For the particles prepared in Production Examples 1 to 6 and Comparative Production Examples 1 to 3, 180 μL of a 1.7 wt% water-suspension was taken into a 1.5 mL microtube, and 90 μL of a 5.0% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 90 μL of a 5.0% aqueous solution of N-hydroxysulfosuccinimide sodium were added thereto as a condensing agent, and the mixture was stirred at room temperature for 30 minutes to obtain activated particles. (Redispersion step) The dispersion of the activated particles prepared above was sedimented by a centrifuge at a force of 20000G for 5 minutes, the supernatant was removed, and 250 μL of a Tris-HCl buffer solution with a pH of 8.0 was newly added. Six samples of the activated particle sediment with the buffer solution added were prepared, and ultrasonic dispersion treatment was carried out at a mode of High and a liquid temperature of 4 °C for 16 seconds using an ultrasonic disruptor Bioruptor II (TYPE6) manufactured by Sonic Bio Co., Ltd. The state of the particles in the microtube after the ultrasonic dispersion treatment was visually observed to confirm whether there was no precipitate and the solution in which the particles were dispersed was uniformly turbid. When there was a precipitate or the turbidity of the solution in which the particles were dispersed was non-uniform, additional ultrasonic dispersion treatment was carried out for 16 seconds, visual observation was performed, and this operation was repeated until there was no precipitate and the solution in which the particles were dispersed was uniformly turbid.

[0055] (Redispersibility evaluation) The average value of six measurements of the time when it was visually confirmed that the solution in which particles were dispersed without precipitates in the microtube was uniformly turbid was calculated, and the evaluation was performed as follows. A: 30 seconds or less B: 45 seconds or less C: 60 seconds or less D: 61 seconds or more

[0056] The evaluation results of the activated particles of Particles 1 to 6 and Comparative Particles 1 to 3 are shown in Table 2. For all of Particles 1 to 6, the evaluation results were C or higher. When the average redispersion time of the particles is short and the redispersibility is high, the damage to the ligand is small when the ligand-added particles are redispersed by ultrasonic irradiation. When the damage to the ligand is small, the reactivity is high when it becomes a test particle, so the sensitivity is high. On the other hand, all of Comparative Particles 1 to 3 were rated D. Regarding Comparative Particle 1 and Comparative Particle 2, as shown in Table 1, the weight ratios of the formula (1) in the particles are 64% and 63% respectively, and compared with Particles 1 to 6, the proportion of the structure represented by the formula (1) with a low glass transition point is large, so the particles are soft and easily deformed. Therefore, the redispersibility decreases. Regarding Comparative Particle 3, since the polymerization time is short, the cross-linking of the core particles is insufficient and St remains in the solution. Therefore, as shown in Table 1, the glass transition point at the first temperature rise is lower than the glass transition point at the second temperature rise, the particles are soft and easily deformed, and the redispersibility decreases.

[0057] [Evaluation 3] Evaluation of non-specific adsorption to particles (Milk evaluation) For Particle 1, a dispersion was prepared by dispersing it in a phosphate buffer solution to a concentration of 0.1 wt%. Next, 60 μL of a milk-like liquid composed of triolein, lecithin, free fatty acids, bovine albumin, and Tris buffer solution was added to 30 μL of each dispersion, and the absorbance at a wavelength of 572 nm was measured for the dispersion immediately after stirring. The absorbance measurement was performed using a spectrophotometer Biospectrometer manufactured by Eppendorf. Then, after allowing these dispersions to stand at 37 °C for 5 minutes, the absorbance at a wavelength of 572 nm was measured again, and the change in absorbance ΔABS×10000 was calculated. The evaluation was based on the value of ΔABS×10000 as follows. A: ΔABS×10000 is 30 or less B: ΔABS×10000 is greater than 30 and 50 or less C: ΔABS×10000 is greater than 50 and 100 or less D: ΔABS×10000 is greater than 100 and 500 or less E: ΔABS×10000 is 500 or more The evaluation result was A. It was confirmed that the particles of the present invention are excellent in the ability to suppress non-specific adsorption.

[0058] [Evaluation 4] Preparation of test particles by sensitizing particles with an antibody and evaluation of the latex agglutination sensitivity of the test particles (Preparation of test particles by sensitizing particles with an antibody) Regarding Particle 1, 180 μL of a 1.7 wt% water-suspension was placed in a 1.5 mL microtube, and 90 μL of a 5.0% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 90 μL of a 5.0% aqueous solution of N-hydroxysulfosuccinimide sodium were added thereto, and the mixture was stirred at room temperature for 30 minutes to obtain a particle dispersion having carboxyl groups activated (activated particle dispersion). After centrifugal washing, 270 μL of a phosphate buffer-physiological saline (hereinafter referred to as PBS) at pH 7.2 was added, and the particles with activated carboxyl groups were dispersed by ultrasonic waves. There, 5 μL of a 15.0 mg / mL dispersion of monoclonal mouse anti-human C-reactive protein (hereinafter referred to as CRP antibody), clone C5 (Funakoshi Corporation) was added, and the mixture was stirred at room temperature for 3 hours to obtain test particles by sensitizing the particles with the antibody. After centrifugally washing these test particles, 1 mL of PBS was added and stored in a dispersed state.

[0059] (Evaluation of latex agglutination sensitivity of test particles) A standard serum for CRP was diluted with PBS to a concentration of 0.75 mg / dL to obtain a CRP sample solution. A mixed solution (hereinafter referred to as R1) was prepared by mixing 1 μL of the CRP sample solution and 50 μL of a buffer solution (PBS containing 0.01% Tween 20), and the mixture was kept warm at 37°C. Next, 50 μL of a dispersion of each of the affinity particles that had been sufficiently dispersed again by ultrasonic waves before use (particle concentration 0.1 wt%, referred to as R2) was mixed with R1. The absorbance at a wavelength of 572 nm was measured for the mixed solution (volume 101 μL) immediately after stirring. The absorbance measurement was performed using a spectrophotometer Biospectrometer manufactured by Eppendorf. Then, after allowing this mixed solution to stand at 37°C for 5 minutes, the absorbance at a wavelength of 572 nm was measured again, and the value of the change in absorbance ΔABS×10000 was calculated.

[0060] As a result of the calculation, a value of 10000 or more was obtained, and the evaluation result was good. It was confirmed that the particles of the present invention have sufficient ability as test particles. For particles 2 to 6, good results are also expected for Evaluation 3 and Evaluation 4.

[0061] [Table 2]

[0062] As described above, particles 1 to 6 of the present invention have higher redispersibility than Evaluation 1. On the other hand, Comparative Examples 1 to 3 have low redispersibility.

[0063] The disclosure of this embodiment includes the following configurations. [Configuration 1] The particles contain a polymer having a structure represented by the following formula (1) and a polymer having a structure represented by the following formula (2). The difference between the glass transition point at 90 °C or higher and 120 °C or lower on the DSC curve obtained during the first heating in the differential scanning calorimetry (DSC) measurement of the particles and the glass transition point at 90 °C or higher and 120 °C or lower on the DSC curve obtained during the second heating is -5.0 °C or higher and 5.0 °C or lower. The particles are characterized by this. [Chemical formula] (R 1 represents a group having an epoxy group, a hydroxy group, or a carboxy group. R 1 may be different for each structural unit.) [Chemical formula] (R 2 represents a substituted or unsubstituted phenyl group or naphthyl group. However, in the case of substitution, the substituent is a methyl group or an ethyl group. R 2 may be different for each structural unit.) [Constitution 2] The particles according to Constitution 1, wherein the molar ratio of the structure represented by the formula (1) to the structure represented by the formula (2) is 0.07 or more and 0.48 or less. [Constitution 3] The particles according to Constitution 1 or 2, wherein the particles have a core-shell structure. [Constitution 4] The particles according to Constitution 3, wherein the shell contains a polymer having a structure represented by the formula (1), and the core contains a polymer having a structure represented by the formula (2). [Constitution 5] The particles according to Constitution 4, wherein the content ratio of the structure represented by the formula (1) in the shell is higher than the content ratio of the structure represented by the formula (1) in the core. [Constitution 6] The particles according to any one of Constitutions 3 to 5, wherein the film thickness of the shell is 5 nm or more and 35 nm or less. [Constitution 7] The difference between the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the first heating in differential scanning calorimetry (DSC) measurement and the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the second heating is -4.4°C or higher and 4.4°C or lower. The particle according to any one of Configurations 1 to 6. [Configuration 8] The difference between the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the first heating in differential scanning calorimetry (DSC) measurement and the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the second heating is -2.8°C or higher and 2.8°C or lower. The particle according to Configuration 7. [Configuration 9] The volume average particle diameter of the particle is 150 nm or more and 500 nm or less. The particle according to any one of Configurations 1 to 8. [Configuration 10] The content of the structure represented by the formula (1) in the particle is 9% by weight or more and 39% by weight or less. The particle according to any one of Configurations 1 to 9. [Configuration 11] The formula (1) is the formula (1-A). The particle according to Configuration 1. [Chemical formula] (R 31 and R 32 represent that at least one of them is a hydroxy group, and the other represents a hydroxy group, a group represented by the following formula (1-B), or a group represented by the following formula (1-C).) [Chemical formula] (R 10 represents a single bond or a methylene group. R 12 、R 13 、R 14 represent a hydrogen atom, a methyl group, a hydroxy group, or a hydroxymethyl group, and one or more of R 12 、R 13 and R 14 represent a hydroxy group. Y 1represents a sulfur atom or an imino group. *1 indicates the bonding position.) [Chemical formula] (R 15 represents a hydrogen atom, a methyl group, a hydroxy group, or a carboxy group.) Y 2 represents a sulfur atom or an imino group. Y 3 represents a single bond or a methylene group. *2 indicates the bonding position.) [Configuration 12] Y in the formula (1-B) above 1 The particle according to Configuration 11, wherein Y is a sulfur atom.) [Configuration 13] An inspection particle, characterized in that a ligand is added to the surface of the particle according to any one of Configurations 1 to 12.) [Configuration 14] A reagent, characterized in that the inspection particle according to Configuration 13 is dispersed in an aqueous solution.) [Configuration 15] An inspection kit, characterized by comprising the reagent according to Configuration 14 and a housing that encloses the reagent.) [Method 1] A method for detecting a target substance in a sample, characterized by mixing the reagent according to Configuration 14 and a sample that may contain the target substance.) [Method 2] A method for detecting a target substance in a sample by latex agglutination method, comprising the steps of: mixing a sample that may contain the target substance with the reagent according to Configuration 14 to obtain a mixture; irradiating the mixture with light; and detecting at least one of transmitted light or scattered light from the light irradiated on the mixture.)

Claims

1. The particles contain a polymer having a structure represented by the following formula (1) and a polymer having a structure represented by the following formula (2), and the difference between the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the first heating in the differential scanning calorimetry (DSC) measurement of the particles and the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the second heating is -5.0°C or higher and 5.0°C or lower. The particles are characterized by this. 【Chemical 1】 (R １ represents a group having an epoxy group, a hydroxy group or a carboxy group. R １ may differ for each structural unit.) [Chemical Formula 2] (R ２ represents a substituted or unsubstituted phenyl group or naphthyl group, provided that in the case of substitution, The substituent is a methyl group or an ethyl group. R ２ may be different for each structural unit.)

2. The particles according to claim 1, wherein the molar ratio of the structure represented by the formula (1) to the structure represented by the formula (2) is 0.07 or more and 0.48 or less.

3. The particles according to claim 1, wherein the particles have a core-shell structure.

4. The particles according to claim 3, wherein the shell contains a polymer having a structure represented by the formula (1), and the core contains a polymer having a structure represented by the formula (2).

5. The particles according to claim 4, wherein the content ratio of the structure represented by the formula (1) in the shell is higher than the content ratio of the structure represented by the formula (1) in the core.

6. The particles according to claim 3, wherein the film thickness of the shell is 5 nm or more and 35 nm or less.

7. The particles according to claim 1, wherein the difference between the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the first heating in the differential scanning calorimetry (DSC) measurement and the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the second heating is -4.4°C or higher and 4.4°C or lower.

8. The particles according to claim 7, wherein the difference between the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the first heating in the differential scanning calorimetry (DSC) measurement and the glass transition point at 90°C or higher and 120°C or lower on the DSC curve obtained during the second heating is -2.8°C or higher and 2.8°C or lower.

9. The particles according to claim 1, wherein the volume average particle diameter of the particles is 150 nm or more and 500 nm or less.

10. The particles according to claim 1, wherein the content of the structure represented by the formula (1) in the particles is 9% by weight or more and 39% by weight or less.

11. The particles according to claim 1, wherein the formula (1) is represented by the formula (1-A). [Chemical Formula 3] (R ３１ and R ３２ in which at least one is a hydroxy group and the other is a hydroxy group, a group represented by the following formula (1-B), or a group represented by the following formula (1-C). ) [Chemical Formula 4] (R １０ represents a single bond or a methylene group. R １２ 、R １３ 、R １４ represents a hydrogen atom, a methyl group, a hydroxy group, or a hydroxymethyl group, and one or more of R １２ 、R １３ and R １４ represent a hydroxy group. Y １ represents a sulfur atom or an imino group. *1 indicates the bonding position.) 【Chemical Formula 5】 (R １５ represents a hydrogen atom, a methyl group, a hydroxy group, or a carboxy group. Y ２ represents a sulfur atom or an imino group. Y ３ represents a single bond or a methylene group. *2 indicates the bonding position.)

12. Y in the formula (1-B) above １ The particle according to claim 11, wherein Y is a sulfur atom.

13. Inspection particles, characterized in that a ligand is added to the surface of the particles according to any one of claims 1 to 12.

14. A reagent, wherein the inspection particles according to claim 13 are dispersed in an aqueous solution.

15. An inspection kit, comprising the reagent according to claim 14 and a housing containing the reagent.

16. A method for detecting a target substance in a sample, the method comprising mixing the reagent according to claim 14 and a sample that may contain the target substance.

17. A method for detecting a target substance in a sample by latex agglutination, the method comprising: obtaining a mixed solution by mixing the reagent according to claim 14 and a sample that may contain the target substance; irradiating the mixed solution with light; and detecting at least one of transmitted light or scattered light from the light irradiated on the mixed solution.

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