Target substance detection method and target substance test kit

Abstract

Provided are a target substance detection method with which it is possible to detect a target substance in a specimen with high sensitivity, and a target substance inspection kit. A target substance detection method for detecting a target substance in a sample liquid is characterized by comprising: a step for forming a first complex in a first liquid by mixing together a specimen liquid that could contain the target substance, a luminescent reagent having a first site that specifically binds to the target substance, and a first compound having a second site that specifically binds to a site of the target substance other than the site that specifically binds to the luminescent reagent and also having a third site that is different from the second site; a step for forming, in a second liquid, a second complex that contains the first complex and an aggregate in which a second compound having a fourth site that binds to the third site is aggregated by bonding of the third site and the fourth site to each other; and a step for measuring a value relating to fluorescence anisotropy of the second liquid.

Description

Method for detecting target substances and test kit for target substances

[0001] This disclosure relates to a method for detecting a target substance and a test kit for a target substance.

[0002] In the fields of medicine and clinical testing, the highly sensitive detection or quantification of trace amounts of biological components from blood or collected organ tissue is necessary to investigate the causes and presence of diseases. In recent years, immunoassay has become widely used as a method for testing biological components. Many immunoassays require a washing step called B / F (Bound / Free) separation. One type of immunoassay that does not require B / F separation is the latex agglutination method and fluorescence polarization method. In particular, fluorescence polarization method is a highly sensitive detection method due to its measurement principle.

[0003] Patent Document 1 discloses a method for quantifying high molecular weight substances using a material in which a dye having long-life luminescence properties is adsorbed onto latex particles as a luminescent material.

[0004] Japanese Patent Application Publication No. 03-188374

[0005] However, in the field of clinical testing, there is a need for detection methods and test kits for target substances that can detect them with even greater sensitivity.

[0006] One embodiment of the present disclosure is a detection method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, comprising the steps of: obtaining a first liquid containing a first complex having the target substance, a luminescent reagent having a first site that specifically reacts with the target substance, and a first compound having a second site that specifically reacts with a site different from the site that specifically reacts with the luminescent reagent in the target substance, and a third site different from the second site; obtaining a second liquid containing a second complex having the first complex and an aggregate in which a plurality of the second compound having the third sites are bound together via the third sites; and measuring a value relating to the fluorescence polarization anisotropy of the second liquid.

[0007] Another embodiment of the present disclosure is a method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, comprising the steps of: obtaining a liquid A comprising the target substance, a luminescent reagent having a site A that specifically reacts with the target substance, a site B that specifically reacts with a site different from the site of the target substance that specifically reacts with the luminescent reagent, and a first ion having a site that has repeating units including an ionic functional group; obtaining a liquid B comprising the liquid A and a second ion having a charge opposite to the charge of the ionic functional group; and measuring a value relating to the fluorescence polarization anisotropy of the liquid B.

[0008] Another embodiment of the present disclosure is a test kit for detecting at least one of the presence or absence and concentration of a target substance in a sample solution by obtaining a value relating to fluorescence polarization anisotropy, comprising: a first reagent comprising a luminescent reagent having a site A that specifically reacts with the target substance; a first ion having a site B that specifically reacts with a site in the target substance different from the site that specifically reacts with the luminescent reagent, and a site having repeating units including an ionic functional group; and a second reagent comprising a second ion having a charge opposite to the charge of the ionic functional group.

[0009] Another embodiment of the present disclosure is a test kit for detecting at least one of the presence or absence and concentration of a target substance in a sample solution by obtaining a value relating to fluorescence polarization anisotropy, comprising: a first reagent comprising a luminescent reagent having a site A that specifically reacts with the target substance; a compound A having a site B that specifically reacts with a site different from the site that specifically reacts with the luminescent reagent in the target substance, and a site C different from site B; a third reagent comprising a third ion having a site D that specifically reacts with site C, and a site having repeating units including an ionic functional group; and a second reagent comprising a second ion having a charge opposite to the charge of the ionic functional group.

[0010] One embodiment of the present disclosure is a method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, comprising: a first mixture acquisition step of obtaining a first mixture containing a target substance, a luminescent reagent having a first site that specifically reacts with the target substance, and a first compound having a second site that specifically reacts with a site different from the site that reacts with the luminescent reagent, and a third site different from the second site; a second mixture acquisition step of obtaining a second mixture obtained by mixing the first mixture with a second compound having a plurality of fourth sites that can form covalent bonds with the third site, and a third compound for forming the covalent bonds between the third site and the fourth site; and a measurement step of measuring a value relating to the polarization anisotropy of the second mixture.

[0011] Another embodiment of the present disclosure is a target substance test kit for detecting at least one of the presence or absence and concentration of a target substance in a sample solution by obtaining a value relating to polarization anisotropy, the test kit comprising: a luminescent reagent having a first site that specifically reacts with the target substance; a first compound having a second site that specifically reacts with a site in the target substance different from the site that specifically reacts with the luminescent reagent, and a third site different from the second site; a second compound having a plurality of fourth sites that can form covalent bonds with the third site; and a third compound for forming the covalent bonds between the third site and the fourth site.

[0012] One embodiment of the present disclosure is a method for detecting a target substance in a sample solution, comprising the steps of: obtaining a first liquid containing a first complex having the target substance; a luminescent reagent having a first site that specifically reacts with the target substance; and a first compound having a second site that specifically reacts with a site different from the site that specifically reacts with the luminescent reagent and a third site different from the second site; obtaining a second liquid containing a second complex having an aggregate of a second compound having a plurality of fourth sites that bind to the third site and a third compound having a plurality of fifth sites that bind to the fourth site, and the first complex; and obtaining a value relating to the fluorescence polarization of the second liquid.

[0013] Another embodiment of the present disclosure is a target substance test kit for detecting at least one of the presence or absence and concentration of a target substance in a sample solution by obtaining a value relating to fluorescence polarization, comprising: a luminescent reagent having a first site that specifically reacts with the target substance; a first compound having a second site that specifically reacts with a site in the target substance different from the site that specifically reacts with the luminescent reagent, and a third site different from the second site; a second compound having a plurality of fourth sites that bind to the third site; and a third compound having a plurality of fifth sites that bind to the fourth site.

[0014] One embodiment of the present disclosure is a detection method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, comprising the steps of: obtaining a first liquid containing a first complex having the target substance, a luminescent reagent having a first site that specifically reacts with the target substance, and a first compound having a second site that specifically reacts with a site different from the site that specifically reacts with the luminescent reagent, and a third site having a temperature-responsive polymer; obtaining a third liquid containing a second complex having the first complex and an aggregate of the second compounds by changing the temperature of a second liquid containing the first complex and a plurality of second compounds having the third site; and obtaining a value relating to the fluorescence polarization of the third liquid.

[0015] Another embodiment of the present disclosure is a target substance test kit for detecting at least one of the presence or absence and concentration of a target substance in a sample solution by obtaining a value relating to fluorescence polarization, the test kit comprising: a luminescent reagent having a first site that specifically reacts with the target substance; and a first reagent containing a first compound having a second site that specifically reacts with a site in the target substance different from the site that specifically reacts with the luminescent reagent, and a third site having a temperature-responsive polymer.

[0016] Another embodiment of the present disclosure is a target substance test kit for detecting at least one of the presence or absence and concentration of a target substance in a sample solution by obtaining a value relating to fluorescence polarization, the test kit comprising: a luminescent reagent having a first site that specifically reacts with the target substance; a second reagent containing a third compound having a second site that specifically reacts with a site in the target substance different from the site that specifically reacts with the luminescent reagent and a fourth site different from the second site; and a third reagent containing a fourth compound having a fifth site that specifically reacts with the fourth site and a third site having a temperature-responsive polymer.

[0017] According to this disclosure, a method for detecting a target substance that can detect the target substance in a sample solution with high sensitivity, and a test kit for the target substance can be provided.

[0018] This is a schematic diagram illustrating an example of luminescent particles and a target substance in one embodiment of the present disclosure. This is a schematic diagram illustrating an example of a first ion in one embodiment of the present disclosure. This is a schematic diagram illustrating an example of complex A in one embodiment of the present disclosure. This is a schematic diagram illustrating an example of a third ion in one embodiment of the present disclosure. This is a schematic diagram illustrating another example of complex A in one embodiment of the present disclosure. This is a schematic diagram illustrating an example of complex B in one embodiment of the present disclosure. This is a diagram illustrating a step of a method for detecting a target substance according to one aspect of the present disclosure. This is a diagram illustrating luminescent particles and a target substance. This is a diagram illustrating compound 1a. This is a diagram illustrating the step of obtaining the mixture of 1a. This is a diagram illustrating compound 2a. This is a diagram illustrating the case where the step of obtaining the mixture of 1a includes a sub-step. This is a diagram illustrating the step of obtaining the mixture of 2a. This is a diagram illustrating an example where compound 4a is used in the step of obtaining the mixture of 2a. This is a diagram illustrating the case where the reaction used to form the crosslinked structure is an oxidation reaction. This is a diagram illustrating the step of obtaining the mixture of 2a when the reaction used to form the crosslinked structure is an oxidation reaction. This is a diagram illustrating the case where the reaction used to form the crosslinked structure is an aminoacyltransferase reaction. This figure illustrates the process of obtaining the 2a mixed solution when the reaction used to form the crosslinked structure is an aminoacyltransferase reaction. This figure illustrates the test kit according to the present disclosure. This figure illustrates the process of the method for detecting a target substance according to one embodiment of the present disclosure. This figure illustrates the process of the method for detecting a target substance according to one embodiment of the present disclosure. This figure illustrates the process of obtaining the 1b liquid. This figure illustrates the process of obtaining the 2b liquid. This figure illustrates the flow from detection to measurement of the target substance according to an embodiment of the present disclosure. This figure illustrates the case in which the test kit for a target substance according to the present disclosure is composed of three types of solutions. This figure illustrates the case in which the test kit for a target substance according to the present disclosure is composed of two types of solutions. This figure illustrates the process of the method for detecting a target substance according to one embodiment of the present disclosure. This figure illustrates the process of the method for detecting a target substance according to one embodiment of the present disclosure. This figure illustrates the luminescent particles and the target substance. This figure illustrates the compound 1c. This figure illustrates the process of obtaining the liquid 1c.This diagram illustrates the case where the process of obtaining the liquid in the first c includes sub-processes. This diagram illustrates the process of obtaining the liquid in the third c. This diagram illustrates the test kit according to this disclosure. This diagram illustrates the case where the test kit according to this disclosure includes the reagent in the second c and the reagent in the third c.

[0019] The method for detecting a target substance according to the present disclosure is a method for detecting a target substance in a sample solution, comprising the steps of: mixing a sample solution that may contain the target substance; a luminescent reagent having a first site that specifically binds to the target substance; and a first compound having a second site that specifically binds to a site in the target substance different from the site that specifically binds to the luminescent reagent, and a third site different from the second site, to form a first complex in a first liquid; forming a second complex in a second liquid, comprising an aggregate formed by the binding of a second compound having a fourth site that binds to the third site, the third site and the fourth site together, and the first complex; and measuring a value relating to the fluorescence anisotropy of the second liquid.

[0020] Here, as will be illustrated later, in some embodiments, the "third site" and the "fourth site" may be the same site. Also, the "aggregate formed by bonding of the second compound via the third site" may be referred to as a crosslinked body (crosslinked structure) containing multiple second compounds based on bonding via the third site.

[0021] Furthermore, the target substance testing kit of this disclosure is a target substance testing kit that detects a target substance in a sample solution by obtaining a value relating to fluorescence polarization, and comprises: a luminescent reagent having a first site that specifically binds to the target substance; a first reagent containing a first compound having a second site that specifically binds to a site in the target substance different from the site that specifically binds to the luminescent reagent, and a third site different from the second site; and a second reagent containing a second compound having a fourth site that binds to the third site.

[0022] <First Embodiment> The first embodiment, which is an example of one embodiment of the present disclosure, will be described in detail below, but this will not limit the scope of the present disclosure.

[0023] A method for detecting a target substance according to this embodiment is a detection method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, comprising the steps of: obtaining a first liquid containing a first complex having the target substance, a luminescent reagent having a first site that specifically reacts with the target substance, and a first compound having a second site that specifically reacts with a site different from the site that specifically reacts with the luminescent reagent in the target substance, and a third site different from the second site; obtaining a second liquid containing a second complex having the first complex and an aggregate in which a plurality of second compounds having the third sites are bound together via the third sites; and measuring a value relating to the fluorescence polarization anisotropy of the second liquid.

[0024] Here, the value relating to fluorescence polarization anisotropy can also be referred to as the value relating to fluorescence anisotropy, and may be, for example, fluorescence polarization degree or polarization anisotropy. Furthermore, the detection of a target substance may be defined as the detection of at least one of the presence or absence of the target substance or its concentration.

[0025] In the method for detecting a target substance of this disclosure, "sample solution that may contain the target substance" corresponds to "sample solution" and "target substance in the sample solution" in this embodiment. "Specifically binds" corresponds to "specifically reacts". "Luminescent reagent having a first site that specifically binds to the target substance" corresponds to "luminescent reagent having a first site that specifically reacts with the target substance". "Third site" corresponds to "third site", and "fourth site" corresponds to "multiple third sites".

[0026] The formation of the aggregate by the binding through the third site may be formed by at least one type of bond among ionic bonds, covalent bonds such as thiol bonds and peptide bonds, affinity bonds, and hydrophobic bonds. Therefore, the third site may be a site capable of forming these bonds (a site having ionic properties, a thiol group, an amino group, a group having an antibody or an antigen, a group having biotin or avidin, a hydrophobic functional group).

[0027] Further, the "plural second compounds having the third site" that form the aggregate may be the first compound unreacted with the target substance, or may be a compound added to the first liquid between the step of obtaining the first liquid and the step of obtaining the second liquid, or may be both of them. That is, the second compound may be the first compound, a compound different from the first compound, or both of them.

[0028] In the method for detecting a target substance of the present embodiment, after the first complex is formed in the step of obtaining the first liquid, an aggregate (which can also be referred to as a cross-linked structure, a cross-linked body, or an aggregate) is formed through the third site of the first complex and the third site of the plural second compounds, and the second complex is formed. A step of adding a third compound for forming the aggregate may be provided between the step of obtaining the first liquid and the step of obtaining the second liquid. For example, when the bond through the third site is an ionic bond, the third site can be an anionic site, and the third compound can be a cation. For example, the third site can be assumed to contain an ion derived from at least one of alginic acid, carboxymethyl cellulose, pectin, carrageenan, cellulose sulfate, chondroitin sulfate, DNA, and polyvinylphosphonic acid. Further, for example, the cation is Ca ２＋ , Ba ２＋ , Pb ２＋ , Cu ２＋ , Cd ２＋ , Sr ２＋ , Co ２＋ , Ni ２＋ , Zn ２＋ , Mn ２＋ , Al ３＋The third compound may also include a cationic polymer containing at least one ion derived from at least one of primary amines, secondary amines, tertiary amines, imidazolium groups, pyrrolidinium groups, pyridinyl groups, quaternary ammonium salts, and quaternary phosphonium salts. The third compound may be the same compound as the second compound.

[0029] Here, the second complex is larger than the first complex because it is an aggregate containing the first complex. Therefore, in the step of obtaining the first liquid, it is possible to react the target substance with the first compound, which has a relatively small molecular weight, with high reactivity. Furthermore, in the step of obtaining the second liquid, it is possible to obtain the second complex, which has a large molecular weight. This makes it possible to significantly change (increase) the value related to fluorescence polarization anisotropy.

[0030] The size of the first complex is not particularly limited, as long as it is large enough that the value of fluorescence polarization anisotropy <r> does not saturate. If the luminescent reagent has luminescent particles, if the size is roughly the same as the luminescent particles, the value of fluorescence polarization anisotropy <r> can be significantly changed when the second liquid is obtained. As a result, the amount of change in the value of fluorescence polarization anisotropy <r> before and after the reaction can be increased, thus improving the measurement sensitivity.

[0031] Therefore, specifically, the size of the first complex is preferably about the same size as the luminescent particles when the luminescent reagent has luminescent particles, and more specifically, it is preferably the particle size of the luminescent particles + 20 nm or less. Furthermore, the size of the second complex is preferably at least twice the size of the luminescent particles (it may also be at least twice the size of the first complex). Considering the magnitude of sedimentation and anisotropy changes of the second complex, the second complex is preferably 200 nm to 6 μm, and more preferably 1 μm to 6 μm. Here, the size of the complex can be the diameter (average particle diameter) determined, for example, by dynamic light scattering, similar to the luminescent particles described later.

[0032] The magnitude of the value <r> related to fluorescence polarization anisotropy depends on the rotational relaxation time and luminescence lifetime of the luminescent reagent. For example, in the case of luminescent particles using a europium complex, if the size of the second complex is 1 μm or larger, the value <r> related to fluorescence polarization anisotropy of the second complex will theoretically exceed the maximum value.

[0033] In the following embodiments, luminescent particles are used as the luminescent reagent. Below, examples of each step (from the first liquid acquisition step to the measurement step) of a method for detecting a target substance when the aggregate is formed by ionic bonding (the bonding via the third site is ionic bonding) will be described.

[0034] The method for detecting a target substance according to this embodiment is a method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, and is characterized by comprising the steps of: obtaining a liquid A containing the target substance, a luminescent reagent having a site A that specifically reacts with the target substance, a site B that specifically reacts with a site different from the site of the target substance that specifically reacts with the luminescent reagent, and a site having repeating units including an ionic functional group; obtaining a liquid B containing the liquid A and a second ion having a charge opposite to the charge of the ionic functional group; and measuring a value relating to the fluorescence polarization anisotropy of the liquid B.

[0035] In this disclosure, "luminescent reagent having a first site that specifically binds to a target substance" corresponds to "luminescent reagent having site A that specifically reacts with a target substance" in this embodiment. "Second site" corresponds to "site B". "Third site" and "fourth site" both correspond to "site having repeating units containing an ionic functional group". "First compound" and "second compound" both correspond to "first ion". "Third compound" corresponds to "second ion having a charge opposite to the charge of the ionic functional group". "First liquid" corresponds to "liquid A", and "second liquid" corresponds to "liquid B".

[0036] <Step to obtain the first liquid> Figure 1A is a schematic diagram showing an example of luminescent particles and target substance, Figure 1B is a schematic diagram showing an example of the first ion, and Figure 1C is a schematic diagram showing an example of composite A (first composite). In this embodiment, in the step to obtain the first liquid (also referred to as liquid A) (hereinafter sometimes referred to as the first liquid acquisition step), a first liquid is obtained by mixing a sample solution containing the target substance 3, luminescent particles 2, and the first ion 4.

[0037] The composite A shown in 1 (hereinafter sometimes referred to as composite A1) is a composite formed by the binding of the target substance 3, the luminescent particle 2, and the first ion 4 in the sample solution. As shown in Figure 1A, the luminescent particle 2 has a base particle 7 having a substrate 9 containing a luminescent molecule 8 and a hydrophilic layer 10 present on the surface of the substrate 9, and a site A (shown as 11 in Figures 1A to 1E; hereinafter sometimes referred to as site A11) that specifically reacts with site X of the target substance 3. Here, "specifically reacts" may also mean "specifically binds".

[0038] As shown in Figure 1B, the first ion 4 has a site B (shown as 14 in Figures 1A to 1E; hereafter sometimes referred to as site B14) that specifically reacts with a site Y different from the site X that reacts with the light-emitting particle 2 of the target substance 3, and an ionic site 12. Here, the ionic site 12 is a site having a repeating unit having an ionic functional group (a site having a polymerization site of a unit having an ionic functional group), and can also be called a site E having a repeating unit having an ionic functional group. The repeating unit can also be called a repeating unit.

[0039] In this embodiment, in the first liquid acquisition step, as shown in Figure 1C, a composite of luminescent particles 2, target substance 3, and first ions 4 is formed, which is composite A (a sandwich structure of luminescent particles - target substance - first ions) indicated by 1.

[0040] Furthermore, in the method for detecting a target substance of this embodiment, the first compound is a compound formed by the combination of a fourth compound having a second site and a fourth site different from the second site, and a fifth compound having a fifth site that specifically reacts with the fourth site and a third site, and the step of obtaining the first liquid can be a method for detecting a target substance comprising: a first sub-step of forming a subcomplex having the target substance, a luminescent reagent, and the fourth compound; and a second sub-step of mixing the liquid containing the subcomplex obtained in the first sub-step with the fifth compound to obtain a first complex having the subcomplex and the fifth compound.

[0041] In this case, the dissociation constant between the fourth and fifth sites can be made smaller than the dissociation constant between the second site and the target substance. For example, one of the fourth and fifth sites may be a site containing avidin, and the other may be a site containing biotin.

[0042] In this example, as shown in Figures 1D and 1E, the first ion 4 can be a compound formed by the specific reaction of compound A (shown as 41 in Figure 1D; hereafter sometimes referred to as compound A41) and the third ion 42. Compound A41 has a site B14 and a site C different from site B14 (shown as 15 in Figures 1D and 1E; hereafter sometimes referred to as site C15). The third ion 42 has a site D that specifically reacts with site C15 (shown as 16 in Figures 1D and 1E; hereafter sometimes referred to as site D16) and an ionic site 12. The sample solution containing the target substance 3 and the reagent solution may be mixed in two steps, and the first ion 4 may be formed in the mixture.

[0043] For example, the process of obtaining a first liquid (liquid A) has two sub-steps. In the first sub-step, the target substance 3, the luminescent particle 2, and compound A41 are mixed to form a complex C (shown as 17 in Figure 1E; hereafter sometimes referred to as complex C17), which is a complex (subcomplex) of the target substance 3, the luminescent particle 2, and compound A41. Then, in the second sub-step, the third liquid (also referred to as liquid C) containing the complex C17 formed in the first sub-step is mixed with a third ion 42 having repeating units containing an ionic functional group. This causes site C15 and site D16 to react, forming a complex A1, which is a complex of complex C17 and the third ion 42 (a complex of the target substance 3, the luminescent particle 2, and the first ion 4).

[0044] In such cases, the probability of reaction with the target substance 3 in the liquid is increased because compound A41, which has a smaller molecular weight than the first ion 4, reacts with the target substance 3, and then reacts with the third ion 42 to form the first ion 4. This is thought to be because the smaller molecular weight leads to faster Brownian motion in the liquid, thus increasing the probability of reaction with the target substance 3 within a specific time before reaching equilibrium. As a combination of sites C15 and D16, for example, one site can contain avidin and the other site can contain biotin. Furthermore, it is preferable that the dissociation constant of site C15 and site D16 is smaller than the dissociation constant of site B14 and site Y of the target substance 3 (it can also be expressed that the binding constant of site C15 and site D is larger than the binding constant of site B and site Y of the target substance 3).

[0045] The concentration of luminescent particles 2 in the first liquid obtained in the first liquid acquisition step (which may also be called the first composite formation step) is preferably 0.000001% by mass or more and 1% by mass or less, more preferably 0.00001% by mass or more and 0.01% by mass or less.

[0046] Here, it is desirable to add the first ion (including the first ion contained in complex A) in the first liquid so that its concentration is 10 to 10,000,000 times that of the estimated target substance 3. However, if the concentration of the estimated target substance in the first liquid is low, it is desirable to set the concentration of the first ion in the first liquid to a higher concentration than the above.

[0047] <Step to obtain the second liquid> In the step to obtain the second liquid (hereinafter sometimes referred to as the second liquid acquisition step; it may also be called the second composite formation step), the second liquid is obtained by mixing the first liquid with a second ion having a charge opposite to the charge of the ionic moiety of the first ion. The second ion forms ionic bonds with multiple ionic moieties of the first ion. Here, the charge of the ionic moiety of the first ion can also be said to be the charge of the ionic functional group contained in the repeating unit that constitutes the ionic moiety of the first ion. Here, the second liquid can also be referred to as liquid B.

[0048] The second ion is preferably a polyvalent ion, or an ion having a repeating unit of an ionic functional group having a charge opposite to that of the ionic functional group in the repeating unit containing the ionic functional group in the ionic moiety 12. By mixing the first liquid and the second ion, a cross-linked structure (aggregate) is formed, as shown in Figure 2, by the formation of ionic bonds between the compound having multiple ionic moieties 12 and the second ion 6 having a charge opposite to that of the ionic moieties 12. For example, if liquid B contains the target substance 3 and the unreacted first ion 4, the step of obtaining liquid B may include the step of forming a complex B (shown as 13 in Figure 2; hereafter sometimes referred to as complex B13) which contains complex A1, the unreacted first ion 4 and the second ion 6.

[0049] In this case, the compound having multiple ionic sites 12 contains complex A1, and an ionic bond is formed between the repeating ionic functional group of the first ion 4 in complex A1 and the second ion 6 of the compound having other ionic sites 12 (for example, the first ion 4 that exists in a free state unreacted with the target substance), thus forming a complex B13 that is larger than complex A1. In other words, it can be said that an aggregate (crosslinked structure) of multiple first ions 4, including the first ion of complex A1, is formed via the second ion 6.

[0050] The ratio of luminescent particles 2 in composite B to luminescent particles 2 in the second liquid depends on the amount of composite A1 formed in the first liquid acquisition step, i.e., the amount of target substance 3. When a value <r> related to the fluorescence polarization anisotropy of the second liquid is obtained in the measurement step described later, a change in the value related to fluorescence polarization anisotropy according to the amount of target substance 3 can be confirmed.

[0051] The detection method according to this embodiment makes it possible to achieve both high reactivity between the luminescent particles 2 and the target substance 3, and a large change in the value of fluorescence polarization anisotropy <r> obtained in the measurement step described later. This allows for highly sensitive detection of the target substance 3 even when it is present in trace amounts in the second liquid. In other words, as the composite B becomes a large structure, the change in the value of fluorescence polarization anisotropy <r> also increases, enabling highly sensitive detection of the target substance.

[0052] As described above, the ionic site 12 of the first ion 4 contained in complex A1 forms a cross-linked structure (aggregate) through ionic bonding with multiple unreacted first ions 4 and second ions 6. The formation of the cross-linked structure (also called ion aggregation reaction or ion cross-linking reaction) is thought to be determined by the balance of the number of ions in the liquid. When a metal ion is used as the second ion 6, it is preferable to add the second ion 6 at a concentration equal to or greater than the concentration of the first ion 4.

[0053] For example, when using alginate ions, which are obtained by the ionization of sodium alginate in liquid, as the first ion 4, and using a solution of alginate ions with a concentration of 1000 pM, it is preferable that the concentration of calcium ions be 5 mM or higher. At this time, the carboxylate group (carboxylate ion, COO) in sodium alginate is important. － ) becomes approximately 0.2 mM, and calcium ions (Ca ２＋ This means that it was added at approximately 25 times the normal concentration.

[0054] The concentration range of the second ion 6 in the second liquid (including the second ion 6 contained in complex B13) is preferably between 1 nM and 1000 mM. This range is thought to reduce the possibility of other proteins in the sample solution undergoing salt aggregation. However, as mentioned above, the appropriate concentration used in the reaction changes depending on the concentration of the first ion 4.

[0055] To increase the size of complex B, the second liquid may contain a fourth ion that has a site having the same charge as the ionic functional group of the repeating unit containing the ionic functional group of the first ion, and does not have a site that specifically reacts with the target substance such as site B. In such a case, for example, in the second liquid acquisition step, the fourth ion or an electrolyte that ionizes to generate the fourth ion may be added as an auxiliary. In this case, the fourth ion may have the same structure as the repeating unit containing the ionic functional group of the first ion, or it may have a different structure or molecular weight, as long as it has a charge with the same sign as the ionic functional group of the repeating unit containing the ionic functional group of the first ion.

[0056] While it is preferable to perform the second liquid acquisition step after the first liquid acquisition step, the first and second liquid acquisition steps may be performed simultaneously.

[0057] <Measurement Process> In the measurement process, the value <r> relating to the fluorescence polarization anisotropy of the second liquid is measured. Preferably, the temperature of the second liquid is 0 to 50°C and the viscosity is 0.5 to 50 mPa·s. Preferably, the measurement is performed when the concentration of luminescent particles in the second liquid is between 0.0001 mg / mL and 0.1 mg / mL, and the detection wavelength is preferably 500 to 700 nm. The measurement process may be performed simultaneously with the acquisition process of the second liquid. By performing the measurement process simultaneously with the acquisition process of the second liquid, it is possible to measure the value <r0> relating to the fluorescence polarization anisotropy immediately after the reaction and the value <r1> relating to the fluorescence polarization anisotropy after a certain reaction time. As will be described in detail later, by measuring the difference between <r1> and <r0> and the amount of change over time (dr / dt), and comparing it with a standard sample, it becomes possible to measure the concentration of the target substance in the sample liquid.

[0058] In the detection method according to this embodiment, the change in the value <r> related to fluorescence polarization anisotropy is increased by the formation of an aggregate containing luminescent particles via ionic bonding, thereby enabling highly sensitive measurement.

[0059] In this embodiment, the target substance in a liquid is quantitatively evaluated by observing the change in the rotational Brownian motion of the luminescent reagent, which results from the reaction of the luminescent reagent present in the liquid with the target substance, as a change in the value related to fluorescence polarization anisotropy. The principle of the fluorescence polarization method used in this case is described below. It should be noted that the following description is based on the premise of quantitative evaluation (measurement of the amount of target substance), but it goes without saying that it can also be used for qualitative evaluation (measurement of the presence or absence of target substance).

[0060] [Fluorescence Polarization Method] Even slight changes in the dispersion state in a liquid containing a luminescent reagent can be detected as changes in polarized emission characteristics. Specifically, when site A of the luminescent particles reacts with the target substance, an increase in the molecular weight of the reaction site with the target substance and the compound that binds to the luminescent particles via the target substance can be detected as a change in the value related to fluorescence polarization anisotropy, which indicates a change in the rotational Brownian motion of the luminescent particles.

[0061] Fluorescence polarization anisotropy means that there is anisotropy in the transition moment (transition dipole moment). Generally, if the luminescent molecule of a luminescent reagent is a luminescent dye with anisotropic transition moments, then if the excitation light is polarized along that transition moment, the emitted light will also be polarized along that transition moment. For example, if a luminescent reagent has a europium complex as its luminescent molecule, it exhibits fluorescence emission based on energy transfer from the ligand to the central metal ion. For this reason, the transition moment of polarized emission becomes complex, but the red emission around 610 nm, which originates from the electron transition from the lowest excited state 5D0 to 7F2, exhibits polarization anisotropy.

[0062] The principle of the fluorescence depolarization method is to measure the shift in the transition moment due to the rotational motion of the luminescent reagent during the time that polarized emission occurs. The rotational motion of the luminescent reagent can be expressed by the following equation (2): Q = 3Vη / kT ... (2) where, Q: rotational relaxation time of the luminescent reagent V: volume of the luminescent reagent η: viscosity of the solvent k: Boltzmann constant T: absolute temperature.

[0063] The rotational relaxation time of a luminescent reagent is the time required for the molecule to rotate by an angle θ (68.5°) such that cosθ = 1 / e.

[0064] From equation (2), it can be seen that the rotational relaxation time of the luminescent reagent is proportional to the volume of the luminescent reagent, i.e., the cube of its radius. On the other hand, the relationship between the luminescence lifetime of the luminescent reagent and the degree of polarization can be expressed by the following equation (3): p0 / p = 1 + A(τ / Q) ... (3) where, p0: degree of polarization when the luminescent reagent is stationary (Q = ∞) p: degree of polarization A: constant τ: luminescence lifetime of the luminescent reagent Q: rotational relaxation time.

[0065] From equations (2) and (3), it is clear that in order to measure a large change in polarization degree, the relationship between the luminescence lifetime of the luminescent reagent and the rotational relaxation time, i.e., the volume (particle size) of the luminescent reagent, is important, and the larger the particle size of the luminescent reagent, the longer the luminescence lifetime needs to be.

[0066] To experimentally determine the degree of polarization of the emission shown in equation (3), polarized light should be incident on the sample, and the emission should be detected at a 90-degree angle to the direction of propagation and vibration of the excitation light. At this time, the detected light should be separated into polarization components parallel and perpendicular to the polarization of the incident light and detected, and the polarization anisotropy should be evaluated using the formula shown in equation (4) below. r(t) = (I ∥ (t) - GI ⊥ (t) / (I ∥ (t) + 2GI ⊥ (t) ... (4) Here, r(t): Polarization anisotropy at time t I ∥ (t): Emission intensity of the emission component parallel to the excitation light at time t I ⊥ (t): Emission intensity of the emission component perpendicular to the excitation light at time t. G: Correction value, measured with excitation light whose vibration direction is 90 degrees different from the excitation light used for sample measurement. ⊥ / I ∥ It is the ratio of .

[0067] In other words, given the appropriate size and luminescence lifetime of the luminescent reagent, it is possible to sensitively read the change in the size of the luminescent reagent due to antigen-antibody reactions, etc., as a value related to fluorescence polarization anisotropy. In this specification, polarization anisotropy refers to the value of the degree of polarization corrected by G and 2G, and the degree of polarization is the value obtained by removing G and 2G from equation (4). In actual measurements, a correction value for G is necessary, so polarization anisotropy is determined. Alternatively, even without determining the correction value G, it is possible to compare the magnitude of relative polarization anisotropy if the measurement conditions are the same. The values ​​related to fluorescence polarization anisotropy in this disclosure include the degree of polarization and polarization anisotropy.

[0068] Furthermore, it is preferable that the luminescent reagent of this embodiment has a fluorescence polarization anisotropy value <r> (polarization anisotropy) of 0.01 or higher, which is determined by the following formula (1). (In equation (1), r is polarization anisotropy, I ＶＶ I is the emission intensity of the emission component whose vibration direction is parallel to the first polarization when excited with the first polarization. ＶＨ I is the emission intensity of the emission component whose vibration direction is perpendicular to the first polarization when excited with the first polarization. ＨＶI is the emission intensity of the emission component whose vibration direction is perpendicular to the second polarization when excited with a second polarization whose vibration direction is perpendicular to the first polarization. ＨＨ (where G is the emission intensity of the emission component whose vibration direction is parallel to the second polarization when excited with a second polarization whose vibration direction is orthogonal to the first polarization, and G is the correction value.)

[0069] [Target Substance Test Kit] Next, a target substance test kit using the above fluorescence polarization depolarization method will be described. The target substance test kit according to this disclosure is a test kit that detects at least one of the presence or absence and concentration of a target substance in a sample solution by obtaining a value related to fluorescence polarization anisotropy, and comprises a first reagent having a luminescent reagent having a site A that specifically reacts with the target substance, a site B that specifically reacts with a site in the target substance different from the site that reacts with the luminescent reagent, and a site having repeating units including an ionic functional group, and a second reagent having a second ion having a charge opposite to the charge of the first ion.

[0070] In the target substance testing kit of this disclosure, the "first site that specifically binds to the target substance" corresponds to "site A that specifically reacts with the target substance." The "second site that specifically binds to a site in the target substance different from the site that specifically binds to the luminescent reagent" corresponds to "site B that specifically reacts with a site in the target substance different from the site that specifically reacts with the luminescent reagent." The "first compound having a third site" and the "second compound having a fourth site" both correspond to "compound A having site C" and "a third ion having site D that specifically reacts with site C, and a site having a repeating unit including an ionic functional group." The "third compound" corresponds to "a second ion having a charge opposite to the charge of the ionic functional group."

[0071] Here, the concept that "the first reagent contains the first ion" includes both cases where the first ion is present in ionic form in a liquid or other medium, and cases where it is present in the form of a first electrolyte that generates the first ion. The same applies to the concept that the second reagent contains the second ion.

[0072] The first ion may be formed in a liquid by the reaction of compound A, which has a site B that specifically reacts with the target substance and a site C different from site B, with a third ion, which has a site D that specifically reacts with site C and a site having a repeating unit containing an ionic functional group. In this case, the ionic functional group of the third ion has the opposite charge to that of the second ion. In such a case, the detection kit for the target substance may contain the luminescent reagent, the first reagent containing compound A, and the third ion independently in the reagent. For example, the detection kit for the target substance may contain: a first reagent containing a luminescent reagent having a site A that specifically reacts with the target substance, a site B that specifically reacts with a site different from the site that specifically reacts with the target substance, and a site C different from site B; a third reagent containing a third ion having a site D that specifically reacts with site C and a site having a repeating unit containing an ionic functional group; and a second reagent containing a second ion having the opposite charge to that of the ionic functional group.

[0073] The target substance testing kit according to this embodiment may have, in addition to these reagents, a housing that encloses these reagents, and if the target substance is an antigen or antibody, it may also have a viscosity modifier for the antigen-antibody reaction. Examples of viscosity modifiers include, but are not limited to, polyvinyl alcohol, polyvinylpyrrolidone, sodium alginate, polysaccharides such as sucrose, and arginine.

[0074] Furthermore, the target substance test kit according to this embodiment may also include a positive control, a negative control, a serum diluent, etc., in addition to these reagents. As the medium for the positive control and negative control, serum, physiological saline, or a solvent may be used, in addition to serum or physiological saline that does not contain the target substance that can be measured.

[0075] Furthermore, the target substance testing kit according to this embodiment may also include a third substance, such as a solvent or blocking agent, in addition to these reagents. The third substance, such as a solvent or blocking agent, may also be included in combination of two or more types. Examples of solvents used in this embodiment include various buffer solutions such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer; however, the solvents included in the testing reagent in the first embodiment are not limited to these.

[0076] The luminescent reagent in the target substance testing kit according to this embodiment may be dispersed in a dispersion medium and exist in the form of a dispersion. The amount of luminescent reagent contained in the target substance testing kit according to this embodiment is preferably 0.000001% to 20% by mass, and more preferably 0.0001% to 1% by mass.

[0077] The target substance detection method and test kit according to this embodiment can measure the presence and concentration of the target substance, and are particularly suitable for use in detecting the target substance in a sample by fluorescence polarization. In the target substance detection method and test kit according to this disclosure, site A (first site) in the luminescent reagent and site B (second site) in the compound contained in the first reagent or in the first ion (first compound) may be sites containing ligands that react specifically with the target substance. Furthermore, site A (first site) in the luminescent reagent and site B (second site) in the first ion (first compound) may be sites containing antibodies, and the target substance may be an antigen.

[0078] <Luminescent Reagent> The luminescent reagent has luminescence properties and a site A that specifically reacts with the target substance. In this embodiment, "specifically reacts" is a concept that includes "specifically captures" and "specifically binds". The luminescent reagent may be a luminescent reagent having a site A that reacts with a specific site (site X) of the target substance and a site consisting of a luminescent molecule. Alternatively, the luminescent reagent may include luminescent particles having a site A that reacts with a specific site (site X) of the target substance and a substrate containing a luminescent molecule (a molecule that is excited and emits light when irradiated with light). Among these, it is preferable that the luminescent reagent be a luminescent particle, as described in this embodiment, because it is possible to measure the value related to fluorescence polarization anisotropy more sensitively when reacting with a trace amount of the target substance. Preferably, the luminescent particle has a substrate 9 containing a luminescent molecule 8, a hydrophilic layer 10 present on the surface of the substrate 9, and a site A 11 that specifically reacts with the target substance 3, as shown in the schematic diagram in Figure 1A. The state before the first portion 11 of the luminescent particle 2 is formed, that is, the state in which a hydrophilic layer 10 is formed on the surface of the substrate 9 containing the luminescent molecule 8, is sometimes referred to as the substrate particle 7. The luminescence described here includes phosphorescence and fluorescence. Furthermore, it is preferable that the luminescent particles have a small particle size distribution, and it is preferable that the surface of the luminescent particle 2 is covered with a hydrophilic layer 10, as will be described later. The following describes the parts of the luminescent particle and the method for producing the luminescent particle, using the example that the luminescent reagent is a luminescent particle as shown in the schematic diagram in Figure 1A.

[0079] (Substrate) The substrate 9 of the substrate particle 7 may be mainly composed of a material that can contain the luminescent molecule 8. For example, if the luminescent molecule 8 is a europium complex, the substrate 9 is not particularly specified as long as it is a material that can stably incorporate the europium complex. It is preferably a polymer containing styrene units and organic silane units, and in particular, polymers obtained by polymerizing a composition containing a radically polymerizable organic silane with styrene as the main component are preferably used. By including styrene as the main component in the composition, it is possible to produce particles with a very uniform particle size distribution by the emulsion polymerization method described later. Furthermore, by using a polymer containing organic silane units, silanol groups (Si-OH) are generated in the polymer in an aqueous solvent, and siloxane bonds (Si-O-Si) are formed between them on the surface of the substrate 9. Through such siloxane bonds, substances that react with target substances 3 such as the hydrophilic layer 10 and ligands described later (which become site A11) can be imparted to the surface of the substrate 9. In this specification, luminescent particles before the hydrophilic layer is formed may be referred to as the substrate.

[0080] (Site A that specifically reacts with the target substance) The site A11 of the luminescent particle 2 that specifically reacts with the target substance 3 preferably contains a ligand that reacts with a specific target substance. The ligand described here is a substance that specifically reacts with (may bind to or capture) a specific target substance, and any substance that shows affinity to a specific target substance can be used. Furthermore, the concept that site A11 of the luminescent particle 2 contains a ligand that reacts with a specific target substance includes, for example, a substrate particle 7 on which a substance that reacts with the target substance 3 is immobilized, as described above.

[0081] Examples of combinations of target substance 3 and ligand (site A11) include antigens and antibodies, small molecules and their receptors, enzymes and substrates, and complementary nucleic acids. More specific examples of antigens and antibodies include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and all kinds of small molecules, along with antibodies that specifically bind to them. Other examples include receptors and small molecules, hormones, neurotransmitters, signaling molecules, membrane proteins, etc., that specifically bind to them. Furthermore, examples include parts or fragments of DNA, RNA, cDNA, etc. derived from bacteria, viruses, cells, etc., synthetic nucleic acids, primers, probes, etc., and nucleic acids that are complementary to them. In addition to the above, any combination known to have affinity can be used as a combination of target substance 3 and ligand (site A11). Typical ligands in this embodiment include antibodies, antigens, and nucleic acids.

[0082] (Hydrophilic layer) From the viewpoint of maintaining the uniformity and monodispersity of the luminescent particles 2, it is desirable not to apply anything to the surface of the substrate 9. However, from the viewpoint of detecting the target substance 3, it is preferable to suppress the nonspecific adsorption of substances other than the target substance 3 onto the luminescent particles 2. Therefore, it is preferable that a hydrophilic layer 10 is formed on the surface of the substrate 9 so that the surface of the substrate particles 7 becomes hydrophilic. As an alternative method to suppressing nonspecific adsorption, there is also a method of supporting proteins such as BSA on the surface of the substrate 9, but the method of forming a hydrophilic layer 10 on the surface of the substrate 9 is preferable because it is less prone to lot-to-lot variation.

[0083] The hydrophilic layer 10 may contain hydrophilic molecules, such as hydrophilic polymers. Hydrophilic polymers are polymers containing hydrophilic groups, and specific examples of hydrophilic groups include hydroxyl groups, ethers, pyrrolidones, and betaine structures. Specific examples of hydrophilic polymers include polyethylene glycol, polyvinylpyrrolidone, sulfobetaine polymers, phosphobetaine polymers, and polyglycidyl methacrylic acid, which is obtained by opening the glycidyl ring and modifying the molecule with a hydroxyl group. These can be the main components of the hydrophilic layer 10. Alternatively, the hydrophilic layer 10 may be formed by directly applying a single molecule having a hydrophilic group to the surface of the substrate 9 of the base particle 7 using a silane coupling agent or the like. In this specification, a more specific example of a hydrophilic polymer, a polymer having a pyrrolidone ring, may be abbreviated as "PVP".

[0084] The hydrophilic layer 10 preferably covers at least a portion of the surface of the substrate 9, and more preferably covers a large portion of the surface of the substrate 9. This makes it possible to suppress the nonspecific adsorption of the light-emitting particles 2, as described above. Also, as will be described later, if the hydrophilic layer 10 is formed during the synthesis of the substrate 9, the hydrophilic layer 10 may be included in a portion of the substrate 9, but it is preferable that the hydrophilic layer 10 mainly exists on the surface of the light-emitting particles 2 on the outside of the substrate 9. There is no limit to the thickness of the hydrophilic layer 10, but it does not need to be thicker than the thickness required to exhibit hydrophilicity. The thickness of the hydrophilic layer 10 is preferably between 1 nm and 15 nm. By keeping the thickness within this range, it becomes less likely to become like a hydrogel, and the thickness of the hydrophilic layer is less likely to become unstable due to hydration caused by ions in the solvent.

[0085] (Luminescent Molecules) The luminescent reagent preferably contains a rare earth complex. The luminescent molecules 8 contained in the substrate particles 7 of the luminescent particles 2 are preferably rare earth complexes, and more preferably europium complexes, considering factors such as luminescence lifetime (i.e., long lifetime) and the visible emission wavelength range. Europium complexes have the characteristics of having a long luminescence lifetime, with the wavelength and intensity of the emission being less affected by the surroundings. Europium complexes are composed of europium elements and ligands. Europium complexes generally have a luminescence lifetime of 0.1 to 1.0 ms. It is necessary to appropriately adjust this luminescence lifetime and the rotational relaxation time obtained from the above formula (2).

[0086] When the luminescent molecule 8 is a europium complex, at least one of the ligands constituting the europium complex is a ligand with a light-harvesting function. The light-harvesting function is the action of being excited at a specific wavelength and exciting the central metal of the complex by energy transfer. Furthermore, it is preferable that the ligands constituting the europium complex include ligands such as β-diketones to prevent coordination of water molecules. Ligands such as β-diketones that are coordinated to the europium ion suppress the deactivation process due to energy transfer to solvent molecules, etc., and strong luminescence can be obtained. The europium complex may also be a polynuclear complex.

[0087] Specific examples of europium complexes include tris(2-thenoyltrifluoroacetone)bis(triphenylphosphine oxide)europium(III) and tris(2-thenoyltrifluoroacetone)(triphenylphosphine oxide)(dibenzylsulfoxide)europium(II). I) (tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide)europium(III)) and tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III)) are examples.

[0088] Furthermore, when the Brownian rotation of the europium complex can be considered to have stopped in the medium, it is desirable that the polarization anisotropy represented by the aforementioned equation (4) be 0.10 or greater. The state in which the Brownian rotation can be considered to have stopped means that the rotational relaxation time of the luminescent reagent is sufficiently longer than the luminescence lifetime of the europium complex.

[0089] It is preferable for luminescent molecules to be incorporated into the substrate in large quantities, as this increases the luminescence intensity per particle. On the other hand, if luminescent molecules aggregate in the substrate, the interaction between ligands can affect the excitation efficiency of the europium complex, making it difficult to measure values ​​related to fluorescence polarization anisotropy while maintaining reproducibility. For example, if the particle size of luminescent particle 2 is 100 nm, it is preferable that each luminescent particle contains 1,000 to 3,000,000 luminescent molecules. Whether or not the europium complex exhibits non-aggregative luminescence behavior in the substrate can be determined from the excitation spectrum of the sample. Luminescent particles with strong emission not only enable high-sensitivity measurement, but also maintain emission even with small particle sizes, thus enabling faster biochemical reaction rates. Therefore, smaller particle sizes result in a larger diffusion coefficient of Brownian motion in the liquid, making it possible to detect reactions in a shorter time.

[0090] The diameter of luminescent particles can be determined by dynamic light scattering. When luminescent particles dispersed in a solution are irradiated with laser light and the scattered light is observed with a photon detector, the intensity distribution due to the interference of scattered light constantly fluctuates because the luminescent particles are constantly moving due to Brownian motion. Dynamic light scattering is a measurement method that observes this Brownian motion as fluctuations in scattered light intensity. The fluctuation of scattered light with respect to time is expressed by an autocorrelation function, and the translational diffusion coefficient is determined. From the determined diffusion coefficient, the Stokes diameter can be calculated, and the size of the luminescent particles dispersed in the solution can be derived.

[0091] The diameter of the luminescent particles is preferably such that the average particle diameter is 25 nm or more and 500 nm or less, and more preferably such that the average particle diameter is 50 nm or more and 300 nm or less. By setting the average particle diameter to 50 nm or more and 300 nm or less, the value related to the fluorescence polarization anisotropy after the aggregation reaction can be increased, and the amount of luminescent molecules such as europium complex that can be contained in each luminescent particle can be increased.

[0092] Furthermore, it is preferable that the luminescent particles have a small particle size distribution. In addition, although the figure shows an example in which both the substrate particles 7 and the substrate 9 are spherical, the shapes of the substrate particles 7 and the substrate 9 in this embodiment are not limited.

[0093] <First Ion> The first ion 4 has a site B that reacts with the target substance 3 and an ionic site 12. As mentioned above, the ionic site 12 has repeating units of an ionic functional group, so the first ion 4 can also be a polymer ion generated from a polymer electrolyte. The first reagent may contain a first electrolyte, and the first electrolyte may generate the first ion 4. The first electrolyte may be a polymer electrolyte. The first electrolyte may contain at least one of alginic acid, carboxymethylcellulose, pectin, carrageenan, cellulose sulfate, chondroitin sulfate, DNA, and polyvinylphosphonic acid. In the following description, "site B14 that reacts with the target substance 3" may be simply referred to as "site B14". The first ion 4 is electrically charged in liquid and may have a site B14 that reacts with the target substance 3 and an ionic site 12, as shown in the schematic diagram in Figure 1B. The site B14 that reacts with the target substance 3 has the same function as the site A11 on the luminescent particle 2 that reacts with the target substance 3, but it reacts with a different site (site Y) of the target substance 3 than the site (site X) that reacts with site A11 on the luminescent particle 2. In this embodiment, the site B14 that reacts with the target substance 3 can be an antibody or the like. When an antibody is used for the site B14 that reacts with the target substance 3, it may be a monoclonal antibody or a polyclonal antibody.

[0094] The first ion 4 reacts with the luminescent particle 2 via the target substance 3. The site B14 that reacts with the target substance 3 is, for example, a site consisting of a ligand that reacts with the target substance 3. It can also be said that the site A11 on the luminescent particle 2 and the site B14 on the first ion 4 both react specifically with different sites on the target substance 3, thus specifically reacting via the target substance 3. Therefore, for example, if both the site A11 that specifically reacts with the target substance 3 and the site B14 that specifically reacts with the target substance 3 are antibodies, they recognize different epitopes of the target substance 3. Note that the site B14 that specifically reacts with the target substance 3 has the function of reacting with the target substance 3, but it may include not only sites that contribute to the function of reacting with the target substance 3 but also sites that do not contribute.

[0095] The ionic portion 12 of the first ion 4 is a charged portion in the liquid (also called a charged portion) and is a portion having a repeating unit containing an ionic functional group. When the ionic functional group is an anion, the ionic functional group may be, for example, a carboxylate group, a sulfonate group, or a phosphonato group. The first ion 4 may contain a polymer having a repeating unit containing at least one of a carboxylate group, a sulfonate group, and a phosphonato group as the ionic functional group. When the first ion is generated from the first electrolyte, the first electrolyte may contain at least one of a carboxylate group, a sulfo group, and a phosphoryl group as the repeating unit. When the ionic functional group is a cation, the ionic functional group may contain, for example, ions derived from (or ionized from) primary amines, secondary amines, tertiary amines, imidazolium salts, pyrrolidinium salts, pyridinyl groups (pyridinyl nitrogen), quaternary ammonium salts, and quaternary phosphonium salts. Therefore, the ionic site 12 may be a site containing ions formed by the ionization in liquid of at least one of the polyelectrolytes (first electrolytes) such as alginic acid, carboxymethylcellulose, pectin, carrageenan, cellulose sulfate, chondroitin sulfate, DNA, polyvinylphosphonic acid, polyethyleneimine, polypyridine, polyimidazole, polypyrrole, chitosan, and polylysine. Furthermore, as mentioned above, the first ion 4 may be formed during the first liquid acquisition step.

[0096] <Second Ion> The second ion 6 forms an aggregate (crosslinked structure) by ionic bonding with the ionic site 12. The second ion 6 is an ion that has the opposite charge to the charge of the ionic functional group in the repeating unit of the ionic functional group contained in the first ion 4. Note that the term "first ion" as used here includes not only charged atoms but also charged compounds. The second reagent may contain a second electrolyte, and the second electrolyte may generate the second ion.

[0097] If the first ion 4 has a negative charge, the second ion 6 is Ca２＋ Ba ２＋ Pb ２＋ ,Cd ２＋ , Cd ２＋ , Sr ２＋ Co ２＋ Ni ２＋ , Zn ２＋ Mn ２＋ Al ３＋ Polyvalent ions such as, primary amines, secondary amines, tertiary amines, nitrogen-containing structures such as imidazolium groups, pyrrolidinium groups, pyridinyl groups, quaternary ammonium salts, or cationic polymers derived from quaternary phosphonium salts, and amination polysaccharides can be used. More specifically as cationic polymers, materials having structures such as polyethyleneimine, polypyridine, polypyrrole, chitosan, polylysine, and polyaluminum chloride can be selected. Among these, it is preferable that the second ion 6 is a polyvalent ion or an ion derived from a cationic polymer (a polymer having repeating units containing cationic functional groups).

[0098] Furthermore, if the first ion 4 has a positive charge, the second ion 6 can be anion obtained by ionizing alginic acid, carboxymethylcellulose, pectin, carrageenan, cellulose sulfate, chondroitin sulfate, DNA, polyvinylphosphonic acid, etc., in liquid.

[0099] [Method for Manufacturing Luminescent Particles] Next, an example of a method for manufacturing luminescent particles used in the target substance detection method and target substance testing kit according to this embodiment will be described. In the following example, a europium complex is used as the luminescent molecule, styrene and siloxane particles are used as the substrate, and a ligand is used to form a site (site A) that specifically reacts with the target substance to form luminescent particles.

[0100] A method for producing luminescent particles may include the steps of: preparing an emulsion by mixing a radical polymerizable monomer containing styrene and a radical polymerizable organosilane, a radical polymerization initiator, a polarizing luminescent europium complex, and a hydrophilic polymer with an aqueous medium; and heating the emulsion to polymerize the radical polymerizable monomer. Furthermore, the method may include the step of imparting ligand-binding functional groups, as described later, to the surface of the luminescent particles.

[0101] Here, the ligand-binding functional group capable of forming a site A on the surface of the substrate particle that specifically reacts with the target substance is preferably a functional group that can fix site A, and more preferably a functional group that can bind site A. Examples include carboxyl groups, amino groups, thiol groups, epoxy groups, maleimide groups, succinimidyl groups, and alkoxysilyl groups (silicon alkoxide structures).

[0102] (Radical Polymerizable Monomers) Luminescent particles are produced by polymerizing radical polymerizable monomers, which include at least styrene and radical polymerizable organosilanes. The radical polymerizable monomers may further include monomers selected from the group consisting of acrylate monomers and methacrylate monomers. Examples of monomers include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, and mixtures thereof. That is, one or more of these monomers can be used in addition to styrene and radical polymerizable organosilanes. A monomer having two or more double bonds in a single molecule, such as divinylbenzene, may also be used as a crosslinking agent.

[0103] By including radically polymerizable organic silanes in the radically polymerizable monomers, siloxane bonds are imparted to the substrate of the luminescent particles. Examples of radically polymerizable organic silanes include vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, or combinations thereof. Using radically polymerizable organic silanes forms an inorganic oxide skeleton within the substrate of the luminescent particles, improving the physical and chemical stability of the luminescent reagent. Furthermore, using radically polymerizable organic silanes increases the affinity between the substrate 9 and the hydrophilic layer 10 and ligand-binding functional groups.

[0104] Furthermore, the inclusion of a radically polymerizable organic silane in the radically polymerizable monomer imparts silanol groups to the surface of the substrate. These silanol groups form hydrogen bonds with hydrophilic polymers, such as PVP. As a result, hydrophilic polymers like PVP are more strongly adsorbed onto the surface of the substrate.

[0105] (Radical polymerization initiators) Radical polymerization initiators can be widely used from azo compounds, organic peroxides, etc. Specifically, examples include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionic acid) dimethyl, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), potassium persulfate (KPS), etc.

[0106] (Hydrophilic Polymers) Luminescent particles may contain hydrophilic polymers as a hydrophilic layer. Hydrophilic polymers are preferable to suppress nonspecific adsorption. Examples of hydrophilic polymers include hydrophilic polymers having repeating units such as ethers, betaines, and pyrrolidone rings. The hydrophilic layer is contained in the synthesized luminescent particles and is preferably mainly present on the surface of the substrate on which the luminescent particles are located. For example, by adding PVP when synthesizing the substrate, it is possible to give the surface of the substrate nonspecific adsorption suppression ability and ligand binding ability, and by forming site A on the substrate particles, luminescent particles with specific adsorption suppression ability can be obtained. In addition, since PVP added during the synthesis of the substrate is more hydrophilic than radical polymerizable monomers, it is present at the interface between the solvent and the substrate during polymerization during synthesis. During the polymerization of the substrate, PVP is partially incorporated, and through physical and chemical adsorption such as the interaction between pyrrolidone rings and styrene (radical polymerizable monomer), PVP is adsorbed on the outside to form a hydrophilic layer.

[0107] The molecular weight of PVP is preferably between 10,000 and 100,000, and more preferably between 40,000 and 70,000. If the molecular weight is less than 10,000, the hydrophilicity of the surface of the luminescent particles is weak, making nonspecific adsorption more likely. If the molecular weight is greater than 100,000, the hydrophilic layer becomes too thick, causing gelation and making it difficult to handle. In addition to PVP, a hydrophilic polymer may be added as a protective colloid during the synthesis of the substrate for the luminescent particles.

[0108] Furthermore, parameters A1 and A2 related to the luminescent particles preferably satisfy A2 - A1 ≤ 0.1. A1 and A2 are defined as follows: A1 is the absorbance of a mixture obtained by adding 30 μL of a dispersion of 0.1% by mass of luminescent particles to 60 μL of buffer mixed with 16 μL of 15-fold diluted human serum, and A2 is the absorbance after being left at 37°C for 5 minutes after the addition. The absorbance is measured with a light path of 10 mm and a wavelength of 572 nm. Luminescent particles in which A2 - A1 is 0.1 or less are preferred because they exhibit less non-specific adsorption of impurities in the serum.

[0109] (Aqueous medium) The aqueous medium (aqueous solution) used in the above-described method for producing luminescent particles preferably contains 80% by mass or more and 100% by mass or less of water. The aqueous solvent is preferably water or an organic solvent soluble in water, and examples include a solution of methanol, ethanol, isopropyl alcohol, or acetone mixed with water. If an organic solvent other than water is included in an amount greater than 20% by mass, dissolution of polymerizable monomers may occur during the production of luminescent particles.

[0110] Furthermore, it is preferable that the above-mentioned aqueous medium has a pH of 6 to 9 beforehand. If the pH is less than 6 or greater than 9, the alkoxy or silanol groups of the radically polymerizable organosilane may undergo condensation polymerization or react with other functional groups before polymer formation, which may cause the resulting particles to aggregate. In this embodiment, condensation polymerization of the alkoxide is not intentionally performed before polymerization. The pH is preferably adjusted using a pH buffer, but it may also be adjusted with an acid or a base.

[0111] In addition, surfactants, defoamers, salts, thickeners, etc. may be added to the aqueous medium in amounts of 10% or less.

[0112] When producing luminescent particles, it is preferable to first dissolve PVP in an aqueous medium whose pH has been adjusted to 6 to 9. The PVP content is preferably 0.01% to 10% by mass relative to the aqueous medium, and more preferably 0.03% to 5% by mass. If the PVP content is 0.01% by mass or more, the amount of luminescent particles adsorbed onto the substrate is small, and the effect is easily exhibited. If the content is 10% by mass or less, an excessive increase in the viscosity of the aqueous medium is suppressed, and sufficient stirring can be performed.

[0113] Next, a radical polymerizable monomer containing styrene and a radical polymerizable organic silane is added to the above aqueous medium to form an emulsion. The mass ratio of styrene to radical polymerizable organic silane is 6:4 to 100:1. Furthermore, a europium complex is mixed into the prepared emulsion. At this time, if the solubility of the europium complex is low, a water-insoluble organic solvent may be added. The mass ratio of the europium complex to the radical polymerizable monomer is 1:1000 to 1:10. If the mass ratio of styrene to radical polymerizable organic silane is less than 6:4, the specific gravity of the entire particle will increase, and particle sedimentation may become significant. In addition, in order to improve the adhesion between PVP and luminescent particles, it is desirable to set the mass ratio of styrene to radical polymerizable organic silane to 100:1 or higher.

[0114] The mass ratio of the weight of the aqueous medium to the total amount of radically polymerizable monomers is preferably between 5:5 and 9.5:0.5. When the mass ratio of the weight of the aqueous medium to the total amount of radically polymerizable monomers is 5:5 or higher, the aggregation of the generated luminescent particles can be suppressed. Furthermore, when the mass ratio of the weight of the aqueous medium to the total amount of radically polymerizable monomers is 9.5:0.5 or lower, a sufficient amount of luminescent particles can be generated.

[0115] The radical polymerization initiator is used after being dissolved in water, a buffer, etc. The amount of the radical polymerization initiator relative to the total mass of styrene and radically polymerizable organosilane can be used in an emulsion between 0.5% and 10% by mass.

[0116] The step of heating the emulsion only needs to be performed so that the entire emulsion is heated uniformly. The heating temperature can be arbitrarily set between 50°C and 80°C, and the heating time between 2 hours and 24 hours. By heating the emulsion, the radical polymerizable monomers are polymerized.

[0117] The surface or hydrophilic layer of the substrate may have functional groups capable of forming a site A that reacts with a target substance. Such functional groups are not particularly limited as long as they can bind antibodies, antigens, enzymes, etc., but may include, for example, carboxyl groups, amino groups, thiol groups, epoxy groups, maleimide groups, succinimidyl groups, silicon alkoxide groups, etc., or groups containing these functional groups. For example, it is possible to impart functional groups to the surface of the substrate by mixing a silane coupling agent having functional groups for forming site A with the synthesized substrate. Specifically, a carboxyl group can be imparted to the surface of the substrate by preparing an aqueous solution of a silane coupling agent having carboxyl groups and mixing it with a dispersion of the synthesized substrate. At this time, a dispersant such as polysorbate 20 may be added to the reaction solution. The reaction temperature can be arbitrarily set between 0°C and 80°C, and the reaction time between 1 hour and 24 hours. In order to suppress the rapid condensation reaction of the silane coupling agent, it is preferable to set the reaction time to 3 to 14 hours at or below room temperature of about 25°C. Depending on the ligand-binding functional group, an acid or alkali catalyst can be added to accelerate the reaction that forms on the substrate surface.

[0118] By forming a substrate particle containing a luminescent molecule and attaching a site A that reacts with various target substances such as antibodies, it can be used as a luminescent particle for sample testing. For example, the optimal method for attaching the desired antibody or other substance can be selected by utilizing the functional groups present in the hydrophilic layer.

[0119] (Introduction of site A) The chemical reaction for chemically bonding a functional group capable of forming site A with a substance having site A (e.g., a ligand) can be carried out using conventionally known methods to the extent that the objective of this embodiment can be achieved. For example, when forming an amide bond with a ligand, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate.

[0120] (Method for producing an electrolyte that generates the first ion) An example of a method for producing an electrolyte that generates the first ion used in this embodiment will be described. The method for producing an electrolyte that generates the first ion can be any method in which an ionic site, that is, a site having repeating units containing an ionic functional group (charged site), is attached to a compound having a site B that reacts with a target substance, as long as the activity of the ligand is maintained. When the ligand is an antibody and sodium alginate is used as the electrolyte that generates the first ion, an ionic site can be attached to the antibody by utilizing the amino group present in the antibody. By mixing a molecule having both a thiol group and an N-hydroxysuccinimide active ester group with the antibody and reacting them, the amino group of the antibody and the N-hydroxysuccinimide active ester group form a covalent bond. N-Succinimidyl 3-(2-pyridyldithio)propionate (3-(2-pyridyldithio)propionic acid N-succinimidyl) can be used as such a reagent. Alternatively, an amide bond can be formed with the antibody using a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide].

[0121] <Second Embodiment> An example of one embodiment of the present disclosure will be described in detail below, but this will not limit the scope of the present disclosure.

[0122] The method for detecting a target substance according to this embodiment is a detection method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, comprising the steps of: obtaining a liquid 1a containing a composite of 1a having the target substance, a luminescent reagent having a 1a site that specifically reacts with the target substance, and a compound 1a having a 2a site that specifically reacts with a site different from the site that specifically reacts with the luminescent reagent in the target substance, and a 3a site different from the 2a site; obtaining a liquid 2a containing a composite of 2a having the composite of 1a and an aggregate in which a plurality of compounds 2a having 3a sites are bonded via the 3a sites; and measuring a value relating to the fluorescence polarization anisotropy of the liquid 2a.

[0123] In the method for detecting a target substance of this disclosure, “sample solution that may contain the target substance” corresponds to “sample solution” in this embodiment. “Specifically binds” corresponds to “specifically reacts.” “First / second liquid” corresponds to “mixture of 1a / 2a.” “First compound” corresponds to “compound 1a.” “Second compound having a fourth site” corresponds to “compound 2a having a plurality of 4a sites capable of forming covalent bonds with site 3a” and “compound 3a for forming the covalent bond between site 3a and site 4a.”

[0124] The formation of aggregates via bonding through site 3a may be by thiol bonds or peptide bonds. Therefore, site 3a may be a site capable of forming these bonds (a group having a thiol group or an amino group). Furthermore, the "multiple compounds of 2a having site 3a" that form the aggregates described herein may be compounds of 1a that have not reacted with the target substance, compounds added to liquid 1a after the step of obtaining liquid 1a, or both.

[0125] In the target substance detection method of this embodiment, after the first a complex is formed in the step of obtaining the first a liquid, an aggregate (which can also be called a crosslinked body or aggregate) is formed via the third a portion of the first a complex and the third a portions of the multiple second a compounds, thereby forming the second a complex. Here, since the second a complex has the structure of an aggregate containing the first a complex, it is larger than the first a complex. Therefore, in the step of obtaining the first a liquid, it is possible to react the target substance with the first a compound, which has a relatively small molecular weight, with high reactivity, and in the step of obtaining the second a liquid, it is also possible to obtain the second a complex, which has a large molecular weight, so that the value related to fluorescence polarization anisotropy can be greatly changed (increased).

[0126] The size of the 1a complex is not particularly limited as long as it is large enough that the value of fluorescence polarization anisotropy <r> does not saturate. If the luminescent reagent has luminescent particles, if the size is about the same as the luminescent particles, the value of fluorescence polarization anisotropy <r> can be significantly changed when the liquid 2a is obtained. As a result, the amount of change in the value of fluorescence polarization anisotropy <r> before and after the reaction can be increased, thus improving the measurement sensitivity.

[0127] Therefore, specifically, the size of the first a complex is preferably about the same size as the luminescent particles when the luminescent reagent has luminescent particles, and more specifically, it is preferably the particle size of the luminescent particles + 20 nm or less. Furthermore, the size of the second a complex is preferably at least twice the size of the luminescent particles (it may also be at least twice the size of the first a complex). Considering the magnitude of sedimentation and anisotropy changes of the second a complex, the second a complex is preferably 200 nm to 6 μm, and more preferably 1 μm to 6 μm.

[0128] The magnitude of the value <r> related to fluorescence polarization anisotropy depends on the rotational relaxation time and luminescence lifetime of the luminescent reagent. For example, in the case of luminescent particles using a europium complex, if the size of the 2a complex is 1 μm or larger, the value <r> related to fluorescence polarization anisotropy of the 2a complex will theoretically exceed the maximum value.

[0129] In the following embodiments, examples of each step (from the step of obtaining the liquid in 1a to the measurement step) of a method for detecting a target substance when luminescent particles are used as a luminescent reagent and the aggregate is formed by covalent bonds such as thiol bonds and peptide bonds will be described.

[0130] [Method for detecting target substance] The method for detecting a target substance according to this embodiment is a method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, and comprises the following steps (see Figure 3): A step of obtaining a first a mixture containing the target substance, a luminescent reagent having a first a site that specifically reacts with the target substance, and a first a compound having a second a site that specifically reacts with a site different from the site that reacts with the luminescent reagent, and a third a site different from the second a site (the first a mixture acquisition step shown in S1001a); A step of obtaining a second a mixture by mixing the first a mixture with a second a compound having multiple fourth a sites that can form covalent bonds with the third a site, and a third a compound for forming covalent bonds between the third a site and the fourth a site (the second a mixture acquisition step shown in S1002a); and A measurement step of measuring a value related to the polarization anisotropy of the second a mixture (the measurement step shown in S1003a).

[0131] <Step to obtain the mixture of 1a> In this embodiment, the step to obtain the mixture of 1a involves mixing a sample solution containing the target substance 3a, luminescent particles 2a, and compound 4a of 1a to obtain the mixture of 1a. Figures 4A to 4E illustrate the step to obtain the mixture of 1a. Although it is referred to as the mixture of 1a here, it can of course also be called the liquid of 1a. Furthermore, the step to obtain the mixture of 1a can also be called the step to form the composite of 1a.

[0132] As shown in Figure 4A, the target substance 3a in the sample solution binds to the luminescent particle 2a via the first a portion 11a. The luminescent particle 2a is not particularly limited as long as it has the first a portion 11a and is capable of emitting light, but in this example, the luminescent particle 2a has a substrate 9a containing a luminescent molecule 8a and a hydrophilic layer 10a formed on its surface. The target substance 3a binds to the first a compound 4a via the second a portion 12a. In the figure, the portion of the target substance 3a that binds to the first a portion 11a is shown as Xa, and the portion that binds to the second a portion 12a is shown as Yb.

[0133] The luminescent component of the luminescent particle 2a may be a rare earth complex. As shown in Figure 4B, the compound 4a of the first a has a second a site 12a that specifically reacts with the target substance 3a, and a third a site 13a that is different from the second a site 12a.

[0134] Figure 4C shows an example of the process for obtaining the first mixture. The luminescent particles 2a and the compound 4a of the first mixture form a so-called sandwich structure with the target substance 3a in between, thereby preparing a mixture in which the composite 1a of the first mixture is formed.

[0135] The compound 6a of 2a is not particularly limited as long as it contains multiple portions 14a of 4a, but in the example shown in Figure 4D, the compound 6a of 2a has a hydrophilic polymer 15a that contains multiple portions 14a of 4a. Furthermore, the mixing acquisition step of 1a may also include the sub-step shown in Figure 4E. The sub-step will be described later.

[0136] <Step to obtain the mixture of 2a> Figures 5A and 5B illustrate the step to obtain the mixture of 2a, which includes the composite 5a of 2a. Although referred to as the mixture of 2a here, it can also be called the liquid of 2a. Furthermore, the step to obtain the mixture of 2a can also be called the step to form the composite of 2a.

[0137] In the second a mixture acquisition step, as shown in Figure 5A, the second a mixture is prepared such that the first a complex 1a and the second a compound 6a have a covalent bond 30a. As shown in Figure 5A, the fourth a portion 14a of the second a compound 6a is added to the first a mixture to form a covalent bond 30a with the third a portion 13a of the first a complex 1a. At this time, the formation of the covalent bond 30a may be promoted by using the third a compound 16a to promote the enzyme substrate reaction that contributes to the formation of the covalent bond 30a. The covalent bond 30a between the third a portion 13a and the fourth a portion 14a, and the covalent bond 30a between the fourth a portions 14a of the second a compound 6a, form a cross-linked aggregate (which can also be called an aggregate) and form the second a complex 5a. In this case, if the presence of multiple types of compounds promotes the enzyme substrate reaction that contributes to the formation of the covalent bond 30a, then some of the multiple types of compounds that contribute to the formation of the covalent bond 30a may be included in the mixture of 1a, and the other types of compounds may be added as compound 16a of 3a to prepare the mixture of 2a, or multiple types of compounds may be added sequentially to prepare the mixture of 2a.

[0138] As shown in Figure 5B, the mixture of 2a may also contain compound 4a 31a, which is different from compound 2a 6a and has the site 3a 13a. By including compound 4a 31a, the crosslinking reaction is promoted together with compound 2a 6a (in this case, the third compound of this disclosure corresponds to compound 2a 6a in this embodiment), and the aggregate 17a (crosslinked structure) of the complex 2a 5a can be made larger. With the above, a mixture containing the complex 2a 5a for detecting the target substance 3a is prepared.

[0139] The size of the second composite 5a is preferably at least twice the size of the first composite 1a. Specifically, the size of the first composite 1a is preferably 5 nm to 500 nm, and the size of the second composite 5a is preferably 200 nm to 6000 nm.

[0140] In the method for detecting a target substance according to the embodiment described above, (A) the third a portion 13a and the fourth a portion 14a have thiol groups, the covalent bond 30a is a disulfide bond, and the second a compound 6a includes any of those selected from the group consisting of peroxidases, oxidizing agents, and compounds having phenol groups, or (B) the third a portion 13a has a glutamine residue or a lysine residue, the fourth a portion 14a has a glutamine or lysine residue, the covalent bond 30a is an amide bond formed between the glutamine residue and the lysine residue, and the second a compound 6a may include transglutaminase.

[0141] If (A) is satisfied, compound 6a of 2a has a hydrophilic polymer compound having a thiol group, and compound 16a of 3a contains a compound having a phenol group, and the compound having a phenol group may include one or more selected from the group consisting of tyramine, glycyltyrosine, phenol, and pyrogallol. Furthermore, such a hydrophilic polymer compound may be any of the group consisting of sodium alginate, gelatin, polyethylene glycol, and carboxymethylcellulose.

[0142] If (B) is satisfied, compound 6a of 2a is a protein, and the protein can be casein. In addition, a calcium-independent transglutaminase derived from microorganisms can be used as compound 16a of 3a.

[0143] The sites 11a and 2a (12a) are sites containing an antibody or a fragment thereof, and the target substance 3a can be an antigen.

[0144] In the fluorescence polarization method according to this embodiment, by utilizing the covalent bond 30a, an aggregate 17a (crosslinked structure) containing the luminescent reagent is formed as a result, and the design is such that the polarization anisotropy <r> is large, thus enabling highly sensitive measurement. The target substance detection method of this embodiment will be further explained with reference to Figures 4A to 4E, and Figures 5A and 5B.

[0145] <Luminescent Reagent> The luminescent reagent has a first a site that reacts with the target substance. The luminescent reagent may be a luminescent molecule having a first a site that reacts with a specific site (site Xa) of the target substance and a site consisting of a luminescent molecule, or it may be a luminescent particle having a substrate containing a luminescent molecule (a molecule that is excited and emits light when irradiated with light) and a first a site that specifically reacts with the target substance. Among these, the luminescent reagent is preferably a luminescent particle because it can more sensitively detect polarization anisotropy when reacting with trace amounts of the target substance. Preferably, the luminescent particle has a substrate 9a containing a luminescent molecule 8a, a first a site 11a that specifically reacts with the target substance 3a, and a hydrophilic layer 10a present on the surface of the substrate 9a, as shown in Figure 4A. Note that luminescence includes phosphorescence and fluorescence. Furthermore, it is preferable that the luminescent particle 2a has a small particle size distribution, and it is preferable that the surface of the luminescent particle 2a is covered with the hydrophilic layer 10a, as will be described later. Here, "specifically reacts" may also mean "specifically binds". In the following, we will describe the various parts of the luminescent particle 2a and the method for manufacturing the luminescent particle 2a, using the example that the luminescent reagent is a luminescent particle 2a as shown in the schematic diagram in Figure 4A.

[0146] (Substrate) The substrate 9a of the luminescent particle 2a can be any material that can contain the luminescent molecule 8a. For example, if the luminescent molecule 8a is a europium complex, the substrate 9a is not particularly specified as long as it is a material that can stably incorporate the europium complex. The substrate 9a is preferably a polymer containing styrene units and organic silane units, and a polymer obtained by polymerizing a composition containing a radically polymerizable organic silane with styrene as the main component is particularly suitable. By including styrene as the main component in the composition, it is possible to produce particles with a very uniform particle size distribution by the emulsion polymerization method described later. Furthermore, by using a polymer containing organic silane units, silanol groups (Si-OH) are generated in the polymer in an aqueous solvent, and siloxane bonds (Si-O-Si) can be formed between them on the surface of the substrate 9a of the particles. Through such siloxane bonds, which are bonding functional groups, substances that react with target substances 3a such as the hydrophilic layer 10a and the 1a portion 11a described later can be imparted to the surface of the substrate 9a.

[0147] (First a portion that specifically reacts with the target substance) The first a portion 11a of the luminescent particle 2a that specifically reacts with the target substance 3a preferably includes a portion that reacts with a specific target substance. The portion that reacts with the target substance is a substance that specifically reacts with (may bind to or capture) a specific target substance, and any substance that shows affinity to a specific target substance can be used. Furthermore, the concept that the first a portion 11a of the luminescent particle 2a includes a portion that reacts with a specific target substance includes, for example, a substrate 9a on which a substance that reacts with the target substance 3a is immobilized, as described above.

[0148] Examples of combinations of a target substance and a site that specifically reacts with it include antigens and antibodies, small molecules and their receptors, enzymes and substrates, and complementary nucleic acids. More specific examples of antigens and antibodies include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and all kinds of small molecules, along with antibodies that specifically bind to them. Other examples include receptors and small molecules, hormones, neurotransmitters, signaling molecules, membrane proteins, etc., that specifically bind to them. Furthermore, examples include parts or fragments of DNA, RNA, cDNA, etc. derived from bacteria, viruses, cells, etc., synthetic nucleic acids, primers, probes, etc., and nucleic acids that are complementary to them. In addition to the above, any combination known to have affinity can be used as a combination of a target substance and a site that reacts with it. In this embodiment, the site that reacts with the target substance is typically one of antibodies, antigens, or nucleic acids.

[0149] (Hydrophilic layer) From the viewpoint of maintaining particle uniformity and monodispersity, it is desirable that nothing be applied to the surface of the substrate 9a. However, from the viewpoint of detecting the target substance, it is preferable to suppress the nonspecific adsorption of substances other than the target substance 3a to the luminescent particles 2. Therefore, it is preferable that a hydrophilic layer 10a is formed on the surface of the substrate 9a so that the surface of the luminescent particles 2a becomes hydrophilic. As a method for forming the hydrophilic layer 10a on the surface of the substrate 9a, there is also a method of supporting a protein such as BSA on the surface of the substrate 9a, but the method of forming the hydrophilic layer 10a on the surface of the substrate 9a is preferable because it is less likely to cause lot-to-lot variation.

[0150] The hydrophilic layer 10a may include, for example, hydrophilic polymers or hydrophilic molecules. Hydrophilic polymers and hydrophilic molecules are polymers or molecules containing hydrophilic groups, and specific examples of hydrophilic groups include molecules and polymers having hydroxyl groups, ethers, pyrrolidones, betaine structures, etc. Specific examples of hydrophilic polymers include polyethylene glycol, polyvinylpyrrolidone, sulfobetaine polymers, phosphobetaine polymers, and polyglycidyl methacrylic acid, which has a glycidyl group opened and a hydroxyl group modified at the end of the molecule. These can be the main components of the hydrophilic layer 10a. Alternatively, the hydrophilic layer 10a may be formed by directly applying a single molecule having a hydrophilic group to the surface of the substrate 9a of the luminescent particle 2a using a silane coupling agent or the like. In this specification, a more specific example of a hydrophilic polymer, a polymer having a pyrrolidone ring, may be abbreviated as "PVP".

[0151] Preferably, the hydrophilic layer 10a covers at least a portion of the surface of the substrate 9a, and more preferably, it covers a large portion of the surface of the substrate 9a. This makes it possible to suppress the nonspecific adsorption of luminescent particles 2a, as described above. Also, as will be described later, if the hydrophilic layer 10a is formed during the synthesis of the substrate 9a, the hydrophilic layer 10a may be included in a portion of the substrate 9a, but it is preferable that the hydrophilic layer 10a mainly exists on the outer particle surface of the substrate 9a. There is no limit to the thickness of the hydrophilic layer 10a, but it does not need to be thicker than the thickness required to exhibit hydrophilicity. The thickness of the hydrophilic layer 10a is preferably 1 nm to 15 nm. By keeping the thickness within this range, it is less likely to become like a hydrogel, and the thickness of the hydrophilic layer 10a is less likely to become unstable due to hydration caused by ions in the solvent.

[0152] (Luminescent Molecules) The luminescent molecules 8a contained in the luminescent particles 2a are preferably rare-earth complexes, and more preferably europium complexes, considering factors such as luminescence lifetime (i.e., long lifetime) and the visible emission wavelength range. Europium complexes have the characteristics of having a long luminescence lifetime, with the wavelength and intensity of the emission being less affected by the surroundings. Europium complexes are composed of europium elements and ligands. Europium complexes generally have a luminescence lifetime of 0.1 to 1.0 ms. It is necessary to appropriately adjust this luminescence lifetime and the rotational relaxation time obtained from formula (2a), which will be described later.

[0153] When the luminescent molecule 8a is a europium complex, at least one of the ligands constituting the europium complex is a ligand with a light-harvesting function. The light-harvesting function is the action of being excited at a specific wavelength and exciting the central metal of the complex by energy transfer. Furthermore, it is preferable that the ligands constituting the europium complex include ligands such as β-diketones to prevent coordination of water molecules. Ligands such as β-diketones that are coordinated to the europium ion suppress the deactivation process due to energy transfer to solvent molecules, etc., and strong luminescence can be obtained. The europium complex may be a polynuclear complex.

[0154] Furthermore, specific examples of europium complexes include tris(2-thenoyltrifluoroacetone)bis(triphenylphosphineoxide)europium(III), tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide)europium( Examples include tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide)europium(III) and tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III).

[0155] Furthermore, when the Brownian rotation of the europium complex can be considered to have stopped in the medium, it is desirable that the polarization anisotropy represented by equation (3a), described later, is 0.10 or higher. The state in which the Brownian rotation can be considered to have stopped means that the rotational relaxation time of the luminescent particle 2a is sufficiently longer than the luminescence lifetime of the europium complex.

[0156] It is preferable that the luminescent molecules 8a are incorporated into the substrate 9a in large quantities, as this increases the luminescence intensity per particle. On the other hand, if the luminescent molecules 8a aggregate in the substrate 9a, the interaction between ligands may affect the excitation efficiency of the europium complex, making it difficult to measure polarization anisotropy while maintaining reproducibility. If the particle size of the luminescent particles 2a is 100 nm, then luminescent particles 2a containing approximately 1,000 to 3,000,000 luminescent molecules 8a per particle are preferably used. Whether the europium complex exhibits non-aggregative luminescence behavior in the substrate 9a can be determined from the excitation spectrum of the sample. Luminescent particles 2a with strong luminescence not only enable high-sensitivity measurement, but also maintain luminescence even with small particle sizes, thus enabling faster biochemical reaction rates. Therefore, smaller particle sizes of luminescent particles 2a result in a larger diffusion coefficient of Brownian motion in the liquid, making it possible to detect the reaction in a shorter time.

[0157] The diameter of the luminescent particle 2a can be determined by dynamic light scattering. When a laser beam is shone on particles dispersed in a solution and the scattered light is observed with a photon detector, the intensity distribution due to the interference of the scattered light constantly fluctuates because the particles are constantly moving due to Brownian motion. Dynamic light scattering is a measurement method that observes this Brownian motion as fluctuations in the intensity of scattered light. The fluctuation of scattered light with respect to time is expressed by an autocorrelation function, and the translational diffusion coefficient is determined. From the determined diffusion coefficient, the Stokes diameter can be found, and the size of the luminescent particle 2 dispersed in the solution can be derived.

[0158] The diameter of the luminescent particles 2a is preferably such that the average particle size is 25 nm or more and 500 nm or less, and more preferably 50 nm or more and 300 nm or less. By setting the average particle size to 50 nm or more and 300 nm or less, the polarization anisotropy after the aggregation reaction is increased, and the amount of luminescent molecules 8a such as europium complex that can be contained in each luminescent particle can be increased.

[0159] Furthermore, it is preferable that the luminescent particles 2a have a small particle size distribution. Also, although Figure 4A shows an example where both the luminescent particles 2a and the substrate 9a are spherical, the shapes of the luminescent particles 2a and the substrate 9a in this embodiment are not limited.

[0160] <Compound 1a> Compound 1a 4a has, as shown in the schematic diagram of Figure 4B, a second a site 12a that specifically reacts with a site Y different from the site Xa that specifically reacts with the luminescent particle 2a of the target substance 3a, and a third a site 13a different from the second a site 12a. The second a site 12a that reacts with the target substance 3a has the function of reacting with the target substance 3a, similar to the first a site 11a of the luminescent particle 2a that reacts with the target substance 3a, but it reacts with a site (site Ya) different from the site (site Xa) to which the first a site 11a of the luminescent particle 2a reacts.

[0161] The first a portion 11a is, for example, a site where affinity bonding occurs with the target substance 3a. The first a portion 11a of the luminescent particle 2a and the second a portion 12a of the compound 4a of the first a portion both react specifically with different sites on the target substance 3a, thus reacting specifically via the target substance 3a. The second a portion 12a has the function of reacting with the target substance 3a, but it may include not only sites that contribute to the function of reacting with the target substance 3a but also sites that do not contribute.

[0162] The 3a site 13a of compound 4a of the 1a is a site that forms a covalent bond 30a with the 4a site 14a of compound 6a of the 2a, which will be described later, upon specific stimuli or chemical reactions, thereby forming an aggregate 17a (crosslinked structure).

[0163] The 3a portion 13a and the 4a portion 14a described later can also be called binding sites, and if they are functional groups, they can also be called binding functional groups. The 2a portion 12a has the function of reacting with the target substance 3a, but it may include not only sites that contribute to the function of reacting with the target substance 3a but also sites that do not contribute.

[0164] When the 3a portion 13a and the 4a portion 14a are bonding functional groups, their chemical structure can be selected depending on the reaction used to form the crosslinked structure. For example, when the reaction used to form the crosslinked structure is an oxidation reaction, it is preferable that the 3a portion 13a and the 4a portion 14a (described later) contain thiol groups. Since thiol groups form disulfide bonds in oxidation reactions, they can form bonds with compound 4a of 1a and compound 6a of 2a. In particular, an enzyme (compound 16a of 3a) can be preferably used as a catalyst to oxidize the thiol group used in the formation of the bond between compound 4a of 1a and compound 6a of 2a. For example, horseradish peroxidase (hereinafter abbreviated as HRP) functions as an oxidation catalyst when oxidized and activated by hydrogen peroxide. This property is used to oxidize the thiol group, form a disulfide bond, and form the crosslinked structure.

[0165] Furthermore, for example, if the reaction used to form the crosslinked structure is an aminoacyltransferase reaction, it is preferable that site 13a of 3a and site 14a of 4a (described later) are a non-repeating combination selected from glutamine residues and lysine residues. Since glutamine residues and lysine residues are isopeptidized by the aminoacyltransferase reaction, this reaction can be used to form crosslinked structures between compound 4a of 1a and compound 6a of 2a. In particular, for the formation of the crosslinked structure between compound 4a of 1a and compound 6a of 2a, the aminoacyltransferase reaction of the glutamine residues and lysine residues can be suitably used with an enzyme (compound 16a of 3a) as a catalyst. Transglutaminase functions as an isopeptide crosslinking catalyst when a substrate containing glutamine residues and lysine residues is present. This property can be used to form a crosslinked structure between glutamine residues and lysine residues via isopeptide bonds.

[0166] If the reaction used to form the cross-linked structure is an aminoacyltransferase reaction, compound 4a of 1a may be formed by linking site 12a of 2a with a peptide tag having a binding functional group. Alternatively, if the reaction used to form the cross-linked structure is an aminoacyltransferase reaction, compound 4a of 1a may be formed by linking site 12a of 2a with compound 6a of 2a, which is selected later when the reaction used to form the cross-linked structure is an aminoacyltransferase reaction.

[0167] <Compound 2a> When the reaction used to form the crosslinked structure is an oxidation reaction, compound 2a 6a has multiple 4a portions 14a that can form covalent bonds 30a with the 3a portion 13a, as shown in Figure 4D. Because compound 2a 6a has multiple 4a portions 14a, a crosslinked structure is formed by the covalent bond 30a between compound 1a 4a and compound 2a 6a, based on the bond between the 3a portion 13a and the 4a portion 14a. The hydrophilic polymer 15a of compound 2a 6a has portions that connect with multiple 4a portions 14a, and the hydrophilic polymer 15a also allows for dispersion in a liquid. The ratio of the hydrophilic polymer 15a to the 4a moieties 14a in compound 6a of 2a is not particularly limited as long as there are multiple 4a moieties 14a in the hydrophilic polymer 15a. However, a larger number of 4a moieties 14a increases the likelihood of aggregation and precipitation in the solution during the mixed solution acquisition step of 2a described later. Therefore, it is preferable that the hydrophilic polymer 15a has four or more 4a moieties 14a, the molecular weight of the polymer is 10,000 or more, and it may also have repeating units having 4a moieties 14a. Suitable hydrophilic polymers 15a include sodium alginate, gelatin, carboxymethylcellulose, polyoxazoline, polyethylene glycol, and the like.

[0168] The case where the reaction used to form the crosslinked structure is an oxidation reaction will be explained using Figure 6A. In this case, it is preferable that compound 4a of 1a has a thiol group at site 13a of 3a, and compound 6a of 2a has multiple thiol groups at site 14a of 4a. Thus, when site 13a of 3a and site 14a of 4a are the same site and form a covalent bond 30a, compound 6a of 2a can also be a hydrophilic polymer compound having multiple sites 13a of 3a.

[0169] As shown in Figure 6B, compound 16a of the third a forms a crosslinked structure via disulfide bonds between compound 4a of the first a and compound 6a of the second a, and also enables the formation of a crosslinked structure via disulfide bonds between the compounds 6a of the second a. Compound 16a of the third a is a peroxidase (HRP is exemplified in the example in Figure 6B), an oxidizing agent (H is exemplified in the example in Figure 6B). ２ O ２ It is preferable that the compound includes a compound having a phenol group (for example, thiamine in the example of Figure 6B). The peroxidase is oxidized by hydrogen peroxide and functions as an oxidation catalyst. This property is used to oxidize the thiol group, forming a crosslinked structure by a disulfide bond between the 3a portion 13a and the 4a portion 14a. This allows the 1a compound 4a to be crosslinked to a part of the large aggregate 17a made up of the 2a compound 6a.

[0170] When the reaction used to form the crosslinked structure is an aminoacyltransferase reaction, it is preferable that compound 6a of 2a has multiple sites 14a of 4a (glutamine residue or lysine residue) that can form an amide bond with site 13a of 3a (glutamine residue or lysine residue), as shown in Figure 7A. Compound 6a of 2a can also be a hydrophilic protein having multiple sites 14a of 4a. When compound 16a of 3a is added, as shown in Figure 7B, the catalytic action of compound 16a of 3a enables the formation of a crosslinked structure by isopeptide bonds between compound 4a of 1a and compound 6a of 2a, as well as the formation of a crosslinked structure by isopeptide bonds between the compounds 6a of 2a themselves. This allows compound 4a of 1a to be crosslinked to a part of the large crosslinked structure (assembly 17a) of compound 6a of 2a. As an example of a hydrophilic protein which is compound 6a of 2a having multiple sites 14a of 4a and multiple sites 13a of 3a, casein, gelatin, collagen, keratin, etc., can be suitably used.

[0171] In this embodiment, compound 6a of 2a has multiple binding functional groups (the 4a portion 14a) and a hydrophilic polymer 15a. In Figure 4D, compound 6a of 2a has multiple 4a portions 14a, which are binding functional groups, and also has a hydrophilic polymer 15a. In the step of obtaining the mixed solution of 2a, as shown in Figure 7B, compound 6a of 2a is bound by the binding functional groups to form a composite 7a of compound 6a of 2a, and further compound 6a of 2a is bound via the 4a portion 14a to form an aggregate 17a, which is a larger measurement material. The binding of compound 6a of 2a and the crosslinking reaction between the 4a portion 14a and compound 6a of 2a may occur simultaneously. Compound 6a of 2a is water-soluble due to the effect of the hydrophilic polymer 15a, but it is preferable that it aggregates after the step of obtaining the mixed solution of 2a and precipitates in the solution.

[0172] <Compound 3a> When forming a crosslinked structure using an oxidation reaction, as described above, there are no particular limitations as compound 3a 16a, but peroxidase, oxidizing agents, and compounds having a phenol group can be suitably used. In this case, compounds having a phenol group can include tyramine, glycyltyrosine, phenol, and pyrogallol.

[0173] When forming a crosslinked structure using an aminoacyltransferase reaction, as described above, transglutaminase can be used as compound 16a of 3a. Transglutaminase (EC 2.3.2.13; protein-glutamine:gamma-glutamyltransferase; protein-glutamine:amine γ-glutamyltransferase, CAS 80146-85-6) is not limited in origin as long as it functions as an isopeptide crosslinking catalyst for substrates having glutamine residues and lysine residues, but species of the genera Streptoverticillium, Streptomyces, Actinomadura, Bacillus (e.g., Bacillus circulans, Bacillus subtilis) Microbial transglutaminases derived from species of the genera Corynebacterium, Clostridium, Enterobacter, Micrococcus, Providencia, or their isolates (e.g., subtilis) are calcium-independent transglutaminases that do not require calcium to induce changes in the enzyme's three-dimensional structure and to enable enzymatic activity, and are therefore suitably used in this disclosure. Commercially available calcium-independent transglutaminases derived from S. mobarensis, such as ACTIVA® (Ajinomoto Co., Inc.), are also suitable for this disclosure. The calcium-independent transglutaminase contained in ACTIVA has multiple sites 14a of 4a and multiple sites 13a of 3a. When casein, gelatin, collagen, and keratin, which are exemplified as hydrophilic proteins, are used as compound 6a of 2a, their reactivity (substrate specificity) to endogenous binding functional groups of antibodies, albumin, and other plasma proteins is extremely small and negligible compared to their reactivity (substrate specificity) to compound 6a of 2a. Therefore, they are particularly suitable as compound 6a of 2a in this disclosure.

[0174] Next, we will describe in more detail each step (from the first a) of the method for detecting a target substance when using luminescent particles 2a as the luminescent reagent in this embodiment.

[0175] [Step to obtain the first a mixture] In the step to obtain the first a mixture, a first a mixture is obtained which has a first a complex 1a comprising a target substance 3a in the sample solution, a luminescent particle 2a having a first a site 11a that specifically reacts with the target substance 3a, a second a site 12a that specifically reacts with a site (site Ya) different from the site (site Xa) of the target substance 3a, and a first a compound 4a having a third a site 13a different from the second a site 12a.

[0176] In the first a mixture acquisition step, as shown in Figure 4C, it is sufficient for a complex (a sandwich structure of luminescent particles - target substance - compound of first a) to be formed between the luminescent particles 2a, the target substance 3a, and the compound of first a. For example, this could be a step of mixing a sample solution containing the target substance 3a with the luminescent particles 2a and compound of first a 4a, or the sample solution and a reagent solution containing the luminescent particles 2a and compound of first a 4a may be mixed in two steps, and the complex may be formed in the mixture.

[0177] The first step (1a) for obtaining the mixed liquid may have multiple sub-steps. For example, Figure 4E shows an example in which the first step (1a) for obtaining the mixed liquid has two sub-steps. This example will be explained.

[0178] As a sub-step of the first a, a first a subcompound 20a having a second a portion 12a that reacts with the target substance 3a and a fifth a portion 18a that is different from the second a portion 12a, and not having a third a portion 13a that reacts with the fourth a portion 14a, is mixed with the target substance 3a and luminescent particles 2a to form a subcompound 23a of the first a subcompound 20a, the target substance 3a, and luminescent particles 2a.

[0179] Then, as a sub-step 2a, a mixture containing the sub-compound 23a formed in the sub-step 1a is mixed with the sub-compound 2a 21a having a portion 19a of 6a that reacts with portion 18a of 5a and a portion 13a of 3a (which reacts with portion 14a of 4a). By reacting the portion 18a of 5a and the portion 19a of 6a to form compound 4a of 1a, a complex 1a of 1a is formed, which is a complex of luminescent particles 2a, target substance 3a, and compound 4a of 1a.

[0180] In such cases, the probability of reaction with the target substance 3a in liquid is increased by having the first a subcompound 20a, which has a smaller molecular weight than the first a compound 4a, react with the target substance 3a, followed by the reaction of the second a subcompound 21a to form the first a compound 4a. This is because the smaller molecular weight leads to faster Brownian motion in liquid, thus increasing the probability of reaction with the target substance 3a within a specific time before reaching equilibrium. Examples of combinations of the fifth a site 18a and the sixth a site 19a include a site containing avidin and a site containing biotin. Furthermore, it is preferable that the dissociation constant of the fifth a site 18a and the sixth a site 19a is smaller than the dissociation constant of the second a site 12a and the target substance 3a (the binding constant of the fifth a site 18a and the sixth a site 19a may be larger than the binding constant of the second a site 12a and the target substance 3a).

[0181] The concentration of luminescent particles 2a in the mixture of 1a is preferably 0.000001% to 1% by mass, more preferably 0.00001% to 0.01% by mass. Here, it is desirable that the concentration of compound 4a of 1a in the mixture of 1a be about 10 to 10,000,000 times the concentration of the estimated target substance 3a. If the concentration of the estimated target substance 3a in the mixture of 1a is low, it is desirable to use a higher dilution ratio.

[0182] In this embodiment, compound 4a of 1a can be used in large quantities to form the composite 1a of 1a with the luminescent particles 2a obtained through the mixture acquisition step of 1a. Compound 4a of 1a itself reacts in the mixture acquisition step of 2a due to the effect of site 13a of 3a, regardless of whether it reacts with the luminescent reagent and target substance in the mixture acquisition step of 1a, and is incorporated into the composite 5a of 2a. On the other hand, in the mixture of 1a, by being present in large quantities in a free state in the liquid, it is possible to increase the probability of reaction with the target substance present in trace amounts. As a result, even if the target substance is present in trace amounts, the polarization anisotropy <r> of the mixture of 2a to be measured can be made higher.

[0183] The concentration of compound 4a in the mixture of 1a should preferably be 10 to 1,000,000 times the concentration of the target substance 3a. If the concentration of the target substance 3a is low, it is desirable to use a higher dilution ratio.

[0184] When using an aminoacyltransferase reaction to form a crosslinked structure, as shown in Figure 7A, in the first a mixture acquisition step, a mixture is obtained of the target substance 3a in the sample solution, a luminescent particle 2a having a first a site 11a that specifically reacts with the target substance 3a, a first a compound 4a having a second a site 12a that specifically reacts with a site (site Ya) different from the site (site Xa) of the target substance 3a, and a third a site 13a different from the second a site 12a, and a second a compound 6a having multiple fourth a sites 14a that can form a covalent bond 30a with the third a site 13a.

[0185] The holding time from the first mixture acquisition step (1a) to the second mixture acquisition step (2a) described later is the time for the first a site 11a and the second a site 12a to react with the target substance 3a. It is desirable that this holding time be approximately 2.3 times or more the reaction time constant under the process conditions (temperature, pH, ligand concentration) so that 90% or more of the reaction proceeds.

[0186] The concentration of luminescent particles 2a in the first mixture is preferably 0.000001% to 1% by mass, more preferably 0.00001% to 0.01% by mass. The concentration of compound 4a of the first mixture in the first mixture is preferably 10 to 1,000,000 times the concentration of the target substance 3a. If the concentration of the target substance 3a is low, it is desirable to use a higher concentration. The mixing of compound 6a of the second mixture, which has multiple 4a sites 14a capable of forming covalent bonds 30a with 3a sites 13a, in the first mixture can be separated as a sub-step of the first mixture acquisition step and performed immediately before or after the second mixture acquisition step described later.

[0187] [Step to obtain the second a mixture] In the step to obtain the second a mixture, the second a mixture is obtained by mixing the first a complex 1a, the second a compound 6a having multiple fourth a sites 14a capable of forming a covalent bond 30a with the third a site 13a, and the third a compound 16a for forming a covalent bond 30a between the third a site 13a and the fourth a site 14a.

[0188] When the first complex 1a, the second compound 6a, and the third compound 16a for forming a covalent bond 30a between the third site 13a and the fourth site 14a are mixed, a crosslinked structure (assembly 17a) is formed by the covalent bond 30a between the third site 13a of the first complex 1a and multiple fourth sites 14a of the second compound 6a, which exist in a free state (as individual compounds) in the mixture and are capable of forming a covalent bond 30a with the third site 13a, as shown in the schematic diagram in Figure 5B. This forms a large second complex 5a including the crosslinked structure.

[0189] The mixture of 2a may contain compound 16a of 3a to promote the covalent bond 30a between the 3a portion 13a and the 4a portion 14a. In such a case, the compound 16a of 3a, which promotes the formation of the covalent bond 30a, is contained in the liquid, and the covalent bond between the 3a portion 13a and the 4a portion 14a may be promoted by mixing the liquid containing compound 16a of 3a with the mixture of 1a, or the covalent bond between the 3a portion 13a and the 4a portion 14a may be promoted by mixing the mixture of 1a, the liquid containing compound 6a of 2a, and the liquid containing compound 16a of 3a, which promotes the formation of the covalent bond 30a between the 3a portion 13a and the 4a portion 14a. Furthermore, in the step of obtaining the mixture of 2a, compound 31a of 4a having the 3a portion 13a may be included in order to form a larger aggregate 17a.

[0190] When an oxidation reaction is used to form a cross-linked structure, for example, as shown in Figure 6B, the 3a site 13a can be a thiol group, and the 3a compound 16a may be, for example, a substance related to an enzyme-substrate reaction. A substance related to an enzyme-substrate reaction is any substance related to an enzyme-substrate reaction, and includes enzymes, substrates, or substances generated by the reaction of an enzyme and a substrate. Substances related to the reaction between peroxidase and a substrate include peroxidase, oxidizing agents, and compounds having a phenol group.

[0191] The reaction between HRP and thiol groups generates hydrogen peroxide through the self-oxidation reaction of the thiol groups by oxygen in the solution, so the crosslinking reaction proceeds even if only HRP is mixed. However, the reaction rate is slow, so in order to form large aggregates 17a (crosslinked structures) in a short time, it is better to directly add hydrogen peroxide to the solution in the second a mixture acquisition step. In such an example, compound 16a of third a may contain multiple types of substances related to the enzyme substrate reaction, and compound 16a of third a may contain HRP and hydrogen peroxide, which is a compound that promotes the covalent bond between site 13a of third a and site 14a of fourth a when it coexists with HRP. In such a case, multiple types of substances related to the enzyme substrate reaction may be added to the first a mixture separately, and as a result, a second a mixture containing compound 16a of third a containing multiple types of substances related to the enzyme substrate reaction may be formed. A radical transfer agent may also be used as a compound that promotes covalent bonding. Radical transfer agents form radicals using an HRP catalyst, converting thiol groups into sulfur radicals. The formed sulfur radicals react with the thiol groups to create a disulfide cross-linked structure. Examples of such radical transfer agents include water-soluble phenolic organic compounds such as phenol, tyramine, dopamine, glycyltyrosine, and pyrogallol.

[0192] When an aminoacyltransferase reaction is used to form a crosslinked structure, as shown in Figure 7B, in the second a mixture acquisition step, a mixture of the first a mixture and the third a compound 16a for forming a covalent bond 30a between the third a site 13a and the fourth a site 14a is obtained. The catalytic action of the third a compound 16a induces the formation of an isopeptide crosslinked structure between the first a compound 4a and the second a compound 6a, as well as the formation of a crosslinked structure by isopeptide bonds between the second a compounds 6a, and the first a compound 4a is crosslinked to a large aggregate 17a made up of the second a compounds 6a.

[0193] The proportion of luminescent particles 2a contained in the composite 5a of 2a in the mixture of 2a depends on the proportion of the composite 1a of 1a formed in the mixture of 1a, i.e., the amount of target substance 3a. As a result, when the polarization anisotropy <r> of the mixture of 2a is obtained in the measurement process described later, a change in the value related to polarization anisotropy according to the amount of target substance 3a can be confirmed.

[0194] This makes it possible to achieve both high reactivity with the target substance 3a and a large polarization anisotropy <r> obtained in the measurement step described later, even when the target substance 3a is present in trace amounts in the mixture of 2a, and to detect the target substance 3a with high sensitivity. In other words, as the composite 5a of 2a becomes a large structure, the amount of change in polarization anisotropy <r> also increases, so the target substance 3a can be detected with high sensitivity.

[0195] Naturally, the composite 5a of the second a is larger than the luminescent particles 2a, preferably at least twice the size of the luminescent particles 2a. Considering the sedimentation of aggregates and the magnitude of the change in anisotropy, the composite 5a of the second a is preferably 200 nm to 6000 nm in size, and more preferably 1 μm to 6 μm in size. The magnitude of the polarization anisotropy <r> depends on the rotational relaxation time and luminescence lifetime of the luminescent particles 2a. For example, in the case of luminescent particles 2a using a europium complex, if the size of the composite 5a of the second a is 1 μm or more, the polarization anisotropy <r> of the composite 5a of the second a will theoretically exceed the maximum value. The step of obtaining the second a mixture is preferably performed after the step of obtaining the first a mixture, but the steps of obtaining the first a mixture and the second a mixture may be performed simultaneously.

[0196] [Measurement Step] In the measurement step, the polarization anisotropy <r> of the mixture of 2a is measured. The measurement conditions are preferably such that the liquid is in a liquid at a temperature of 0 to 50°C and the viscosity of the liquid is 0.5 to 50 mPa·s. The concentration of the luminescent reagent is preferably measured at 0.0001 mg / mL to 0.1 mg / mL, and the detection wavelength is preferably 500 to 700 nm. The measurement step may be performed simultaneously with the mixture acquisition step of 2a. By measuring simultaneously with the mixture acquisition step of 2a, it is possible to measure the polarization anisotropy <r0> immediately after the reaction and the polarization anisotropy <r1> after a certain reaction time. By measuring the difference between <r1> and <r0> and the amount of change with respect to time (dr / dt), and comparing it with a standard sample, it is possible to measure the concentration of target substance 3 in the sample liquid.

[0197] In this embodiment, the target substance 3a in the liquid is quantitatively evaluated by capturing the change in the rotational Brownian motion of the luminescent reagent, which occurs as a result of the reaction of luminescent particles 2a present in the liquid with the target substance 3a, as a change in the value related to fluorescence polarization anisotropy. The principle of the fluorescence polarization method used in this case is described below. It should be noted that the following description is based on the premise of quantitative evaluation (measurement of the amount of target substance 3a), but it goes without saying that it can also be used for qualitative evaluation (measurement of the presence or absence of target substance 3a).

[0198] [Fluorescence Polarization Method] Even slight changes in the dispersion state in a liquid containing a luminescent reagent can be detected as changes in polarized emission characteristics. Specifically, when the site of the luminescent particle that reacts with the target substance reacts with the target substance, the molecular weight of the reaction site with the target substance and the compound that binds to the luminescent particle via the target substance increases. This change in the rotational Brownian motion of the luminescent particle can be detected as a change in the value related to fluorescence polarization anisotropy.

[0199] Fluorescence polarization anisotropy means that there is anisotropy in the transition moment (transition dipole moment). Generally, if the luminescent molecule of a luminescent reagent is a luminescent dye with anisotropic transition moments, then if the excitation light is polarized along that transition moment, the emitted light will also be polarized along that transition moment. For example, if a luminescent reagent has a europium complex as its luminescent molecule, it exhibits fluorescence emission based on energy transfer from the ligand to the central metal ion. For this reason, the transition moment of polarized emission becomes complex, but the red emission around 610 nm, which originates from the electron transition from the lowest excited state 5D0 to 7F2, exhibits polarization anisotropy.

[0200] The principle of fluorescence polarization is to measure the shift in the transition moment due to the rotational motion of the luminescent reagent during the time that polarized emission occurs. The rotational motion of the luminescent reagent can be expressed by the following equation (2a): Q = 3Vη / kT ... (2a) where, Q: rotational relaxation time of the luminescent reagent V: volume of the luminescent reagent η: viscosity of the solvent k: Boltzmann constant T: absolute temperature.

[0201] The rotational relaxation time of a luminescent reagent is the time required for the molecule to rotate by an angle θ (68.5°) such that cosθ = 1 / e.

[0202] From equation (2a), it can be seen that the rotational relaxation time of the luminescent reagent is proportional to the volume of the luminescent reagent, i.e., the cube of its radius. On the other hand, the relationship between the luminescence lifetime of the luminescent reagent and the degree of polarization can be expressed by the following equation (3a): p0 / p = 1 + A(τ / Q) ... (3a) where, p0: degree of polarization when the luminescent reagent is stationary (Q = ∞) p: degree of polarization A: constant τ: luminescence lifetime of the luminescent reagent Q: rotational relaxation time.

[0203] From equations (2a) and (3a), it is clear that in order to measure a large change in polarization degree, the relationship between the luminescence lifetime of the luminescent reagent and the rotational relaxation time, i.e., the volume (particle size) of the luminescent reagent, is important, and the larger the particle size of the luminescent reagent, the longer the luminescence lifetime needs to be.

[0204] To experimentally determine the degree of polarization of the emission shown in equation (3a), polarized light should be incident on the sample, and the emission should be detected at a 90-degree angle to the direction of propagation and vibration of the excitation light. At this time, the detected light should be separated into polarization components parallel and perpendicular to the polarization of the incident light and detected, and the polarization anisotropy should be evaluated using the formula shown in equation (4a) below. r(t) = (I∥(t) - GI⊥(t)) / (I∥(t) + 2GI⊥(t)) ... (4a) where, r(t): Polarization anisotropy at time t I∥(t): Emission intensity of the emission component parallel to the excitation light at time t I⊥(t): Emission intensity of the emission component perpendicular to the excitation light at time t G: Correction value, the ratio of I⊥ / I∥ measured with excitation light whose vibration direction is 90 degrees different from the excitation light used for sample measurement.

[0205] In other words, given the appropriate size and luminescence lifetime of the luminescent reagent, it is possible to sensitively read the change in the size of the luminescent reagent due to antigen-antibody reactions, etc., as a value related to fluorescence polarization anisotropy. In this specification, polarization anisotropy refers to the value of the degree of polarization corrected by G and 2G, and the degree of polarization is the value obtained by removing G and 2G from equation (4a). In actual measurements, a correction value for G is necessary, so polarization anisotropy is determined. Alternatively, even without determining the correction value G, it is possible to compare the magnitude of relative polarization anisotropy if the measurement conditions are the same. The values ​​related to fluorescence polarization anisotropy in this disclosure include the degree of polarization and polarization anisotropy.

[0206] Furthermore, it is preferable that the luminescent reagent of this embodiment has a fluorescence polarization anisotropy value <r> (polarization anisotropy) of 0.01 or higher, which is determined by the following formula (1a). (In equation (1a), r is polarization anisotropy, I ＶＶ I is the emission intensity of the emission component whose vibration direction is parallel to the first polarization when excited with the first polarization. ＶＨ I is the emission intensity of the emission component whose vibration direction is perpendicular to the first polarization when excited with the first polarization. ＨＶ I is the emission intensity of the emission component whose vibration direction is perpendicular to the second polarization when excited with a second polarization whose vibration direction is perpendicular to the first polarization. ＨＨ(where G is the emission intensity of the emission component whose vibration direction is parallel to the second polarization when excited with a second polarization whose vibration direction is orthogonal to the first polarization, and G is the correction value.)

[0207] (Method for producing luminescent particles) Next, a test kit for target substances using the fluorescence polarization method described above will be explained. The method for detecting target substances in this embodiment and the method for producing luminescent particles 2a, which will be described later, include a step (Aa) of preparing an emulsion by mixing a radical polymerizable monomer containing at least styrene and a radical polymerizable organosilane, a radical polymerization initiator, a polarizing luminescent europium complex, and a hydrophilic polymer with an aqueous medium, and a step (Ba) of heating the emulsion to polymerize the radical polymerizable monomer, and may further include a step (Ca) of imparting the first a portion 11a, which will be described later, to the surface of the luminescent particles 2a. Here, the functional group capable of forming the first a portion 11a is a functional group to which the first a portion 11a can be attached, and specifically, any of the following can be used: a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, or an alkoxysilyl group (silicon alkoxide structure).

[0208] (Radical Polymerizable Monomers) The luminescent particles 2a are produced by polymerizing a radical polymerizable monomer, which comprises at least styrene and a radical polymerizable organosilane. The radical polymerizable monomer may further include monomers selected from the group consisting of acrylate monomers and methacrylate monomers. Examples of monomers include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, and mixtures thereof. That is, one or more of these monomers can be used in addition to styrene and a radical polymerizable organosilane. A monomer having two or more double bonds in a single molecule, such as divinylbenzene, may also be used as a crosslinking agent.

[0209] By including radically polymerizable organic silanes in the radically polymerizable monomer, siloxane bonds are imparted to the substrate 9a. Examples of radically polymerizable organic silanes include vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, or combinations thereof. By using radically polymerizable organic silanes, an inorganic oxide skeleton is formed within the substrate 9a, which improves the physical and chemical stability of the luminescent particles 2. Furthermore, by using radically polymerizable organic silanes, the hydrophilicity of the substrate 9a and the functional groups for introducing the hydrophilic layer 10a and the 1a moiety 11a is increased.

[0210] Furthermore, the inclusion of a radically polymerizable organic silane in the radically polymerizable monomer imparts silanol groups to the surface of the substrate 9a. The silanol groups form hydrogen bonds with hydrophilic polymers, such as PVP. As a result, hydrophilic polymers such as PVP are more strongly adsorbed onto the surface of the substrate 9a.

[0211] (Radical polymerization initiators) Radical polymerization initiators can be widely used from azo compounds, organic peroxides, etc. Specifically, examples include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionic acid) dimethyl, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), potassium persulfate (KPS), etc.

[0212] (Hydrophilic Polymer) The luminescent particle 2a may contain a hydrophilic polymer as a hydrophilic layer 10a. The hydrophilic polymer preferably suppresses nonspecific adsorption. Examples of hydrophilic polymers include hydrophilic polymers having repeating units such as ethers, betaines, and pyrrolidone rings. The hydrophilic layer 10a is contained in the synthesized luminescent particle 2a and preferably exists mainly on the surface of the substrate 9a on which the luminescent particle 2a is located. For example, by adding PVP when synthesizing the substrate 9a, it is possible to give the surface of the substrate 9a nonspecific adsorption suppression ability and ligand binding ability, and by forming a first a portion 11a on the luminescent particle 2a that specifically reacts with the target substance 3a, it is possible to obtain luminescent particle 2a having specific adsorption suppression ability. Furthermore, since the PVP added during the synthesis of the substrate 9a is more hydrophilic than radical polymerizable monomers, it is present at the interface between the solvent and the substrate 9a during polymerization during synthesis. During polymerization of the substrate 9a, some PVP is incorporated, and through physical and chemical adsorption such as the interaction between the pyrrolidone ring and styrene (a radically polymerizable monomer), PVP is adsorbed on the outside to form a hydrophilic layer 10a. In this specification, polymers having a pyrrolidone ring may be abbreviated as "PVP".

[0213] The molecular weight of PVP is preferably between 10,000 and 100,000, and more preferably between 40,000 and 70,000. If the molecular weight is less than 10,000, the hydrophilicity of the surface of the luminescent reagent is weak, making it prone to nonspecific adsorption. If the molecular weight is greater than 100,000, the hydrophilic layer 10a becomes too thick, causing gelation and making it difficult to handle. In addition to PVP, a hydrophilic polymer may be added as a protective colloid during the synthesis of the substrate 9a.

[0214] Furthermore, parameters A1 and A2 related to the luminescent particles 2a preferably satisfy A2 - A1 ≤ 0.1. A1 and A2 are defined as follows: A1 is the absorbance of a mixture obtained by adding 30 μL of a dispersion of 0.1% by mass of a luminescent reagent to 60 μL of buffer mixed with 16 μL of 15-fold diluted human serum, and A2 is the absorbance after being left at 37°C for 5 minutes after the addition. The absorbance is measured with a light path of 10 mm and a wavelength of 572 nm. Luminescent particles 2a in which A2 - A1 is 0.1 or less are preferred because they exhibit less non-specific adsorption of impurities in the serum.

[0215] (Aqueous medium) The aqueous medium (aqueous solution) used in the method for producing the luminescent particles 2a described above preferably contains 80% by mass or more and 100% by mass or less of water. The aqueous medium is preferably water or an organic solvent that is soluble in water, and examples include a solution of methanol, ethanol, isopropyl alcohol, or acetone mixed with water. If an organic solvent other than water is included in an amount greater than 20% by mass, there is a risk that polymerizable monomers will dissolve during particle production.

[0216] Furthermore, it is preferable that the above-mentioned aqueous medium is pre-adjusted to a pH of 6 to 9. If the pH is less than 6 or greater than 9, the alkoxide or silanol groups of the radically polymerizable organic silane may undergo condensation polymerization or react with other functional groups before polymer formation, potentially causing the resulting particles to aggregate. In this embodiment, condensation polymerization of the alkoxide is not intentionally performed before polymerization.

[0217] The pH adjustment described above is preferably done using a pH buffer, but it may also be done with an acid or a base. In addition, surfactants, defoamers, salts, thickeners, etc. may be added to the aqueous medium in a proportion of 10% or less.

[0218] When producing the luminescent particles 2a, it is preferable to first dissolve the PVP in an aqueous medium whose pH has been adjusted to 6 to 9. The PVP content is preferably 0.01% to 10% by mass relative to the aqueous medium, and more preferably 0.03% to 5% by mass. If it is less than 0.01% by mass, the amount adsorbed to the substrate 9a will be small and the effect will not be exhibited. If it is more than 10% by mass, the viscosity of the aqueous medium will increase, and it may not be possible to stir it sufficiently.

[0219] Next, a radical polymerizable monomer containing styrene (A) and a radical polymerizable organic silane (B) is added to the aqueous medium to form an emulsion. The mass ratio of styrene (A) to radical polymerizable organic silane (B) is 6:4 to 100:1. Furthermore, the europium complex is mixed into the prepared emulsion. At this time, if the solubility of the europium complex is low, a water-insoluble organic solvent may be added. The mass ratio of the europium complex to the radical polymerizable monomer is 1:1000 to 1:10.

[0220] If the mass ratio of styrene (A) to radically polymerizable organic silane (B) is less than 6:4, the overall specific gravity of the particles will increase, and particle sedimentation may become significant. Furthermore, in order to improve the adhesion between PVP and luminescent particles 2a, it is desirable to set the mass ratio of styrene (A) to radically polymerizable organic silane (B) to 100:1 or higher.

[0221] The mass ratio of the aqueous medium to the total amount of radically polymerizable monomers is preferably between 5:5 and 9.5:0.5. If the mass ratio is less than 5:5, the aggregated particles produced may become significant. If the mass ratio is greater than 9.5:0.5, particle formation will not be a problem, but the amount produced may be reduced.

[0222] The radical polymerization initiator is used after being dissolved in water, a buffer, etc. The amount of the radical polymerization initiator relative to the total mass of styrene (A) and radically polymerizable organosilane (B) can be used in an emulsion between 0.5% and 10% by mass.

[0223] The process of heating the emulsion described above only needs to ensure that the entire emulsion is heated uniformly. The heating temperature can be arbitrarily set between 50°C and 80°C, and the heating time between 2 hours and 24 hours. By heating the emulsion, the radical polymerizable monomers are polymerized.

[0224] The surface of the substrate 9a or the hydrophilic layer 10a may have a functional group capable of forming a first a molar 11a that specifically reacts with the target substance 3a. Such a functional group is not particularly limited as long as it is a functional group capable of binding antibodies, antigens, enzymes, etc., but may be, for example, a carboxyl group, amino group, thiol group, epoxy group, maleimide group, succinimidyl group, silicon alkoxide group, etc., or may contain these functional groups. For example, it is possible to impart a functional group to the particle surface by mixing a silane coupling agent having a functional group for introducing the first a molar 11a with the synthesized particles. Specifically, a carboxyl group can be imparted to the particle surface by preparing an aqueous solution of a silane coupling agent having a carboxyl group and mixing it with the synthesized particle dispersion. At this time, a dispersant such as polysorbate 20 may be added to the reaction solution. The reaction temperature can be arbitrarily set between 0°C and 80°C, and the reaction time between 1 hour and 24 hours. To suppress the rapid condensation reaction of the silane coupling agent, it is preferable to set the reaction time to 3 to 14 hours at or below room temperature of about 25°C. Depending on the functional group used to introduce the 1a moiety 11a, an acid or alkali catalyst may be added to accelerate the reaction on the particle surface.

[0225] By forming the first a portion 11a of various antibodies, etc., on the luminescent particle 2a, it can be used as a particle for sample testing. The optimal method for binding the target antibody, etc., using the functional groups present in the hydrophilic layer 10a should be selected.

[0226] (Introduction of the 1a portion) The chemical reaction for chemically bonding the functional group for forming the 1a portion 11a on the luminescent particle 2a to a substance containing the 1a portion 11a can be carried out using conventionally known methods to the extent that the objectives of this disclosure can be achieved. Furthermore, when forming the 1a portion 11a with an amide bond, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate.

[0227] (Method for producing compound 1a) An example of a method for producing compound 4a of compound 1a used in this embodiment will be described.

[0228] The method for producing compound 4a of 1a is a method that can confer site 13a of 3a to a substance that specifically reacts with TSH (thyroid-stimulating hormone), and any method is acceptable as long as the activity of the reaction of site 12a of 2a, which specifically reacts with the target substance 3a, is maintained. When site 12a of 2a is an antibody and a thiol group is conferred as site 13a of 3a, an amino group present in the antibody can be used. By mixing a molecule having both a thiol group and an N-hydroxysuccinimide active ester group with the antibody and reacting them, the amino group of the antibody and the N-hydroxysuccinimide active ester group form a covalent bond. As such a reagent, N-succinimidyl 3-(2-pyridyldithio)propionate (hereinafter abbreviated as SPDP) can be used. Alternatively, an amide bond can be formed with the antibody using a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide].

[0229] When using the aminoacyltransferase reaction to form a cross-linked structure, any method is acceptable for linking a peptide tag having a functional group that binds to the antibody as a site that specifically reacts with the target substance 3a, as long as the ligand activity is maintained. One example is to react a synthetic oligopeptide with an N-hydroxysuccinimide active ester group with the amino group of the antibody.

[0230] When using aminoacyltransferase reactions to form cross-linked structures, any method is acceptable for linking the antibody to a hydrophilic protein having multiple binding functional groups, as long as the ligand's activity is maintained. Click chemistry, such as strain-promoted azide-alkyne cycloaddition or strain-promoted reverse electron-demanded Diels-Alder reaction, can be used. Alternatively, biotin-modified antibodies and biotin-modified hydrophilic proteins can be linked via avidin or streptavidin.

[0231] (Method for producing compound 2a) An example of a method for producing compound 2a 6a used in the embodiment will be described. Compound 2a 6a can be produced by any method as long as the part 4a 14a can be attached to the hydrophilic polymer 15a. When the hydrophilic polymer 15a is sodium alginate and a thiol group is attached as part 4a 14a, compound 2a 6a can be obtained by activating the carboxyl group of sodium alginate using a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] and reacting it with an aminoethanethiol.

[0232] (Test reagent for in vitro diagnostics) The test reagent for in vitro diagnostics in this embodiment, that is, the test reagent used for detecting a target substance in a sample by in vitro diagnostics, comprises a luminescent reagent such as luminescent particles according to this embodiment and a dispersion medium for dispersing the luminescent reagent. The amount of the luminescent reagent according to this embodiment contained in the test reagent in this embodiment is preferably 0.000001% to 20% by mass, and more preferably 0.0001% to 1% by mass. The test reagent according to this embodiment may also contain a third substance such as a solvent or a blocking agent in addition to the luminescent reagent according to this embodiment, to the extent that the objectives of this disclosure can be achieved. Two or more types of third substances such as solvents or blocking agents may be included in combination. Examples of solvents used in this embodiment include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvent contained in the test reagent in this embodiment is not limited to these. When the test reagent in this embodiment is used for detecting an antigen or antibody in a sample, the ligand can be an antibody or an antigen.

[0233] [Target Substance Test Kit] The present disclosure also provides the following test kit, as illustrated in Figure 8: A target substance test kit 100a for detecting at least one of the presence or absence and concentration of a target substance in a sample solution by obtaining a value relating to polarization anisotropy, comprising: a luminescent reagent having a first a site 11a that specifically reacts with the target substance (the test kit 100a does not contain the target substance); a first a compound 4a having a second a site 12a that specifically reacts with a site in the target substance different from the site that specifically reacts with the luminescent reagent, and a third a site 13a different from the second a site 12a; a second a compound 6a having a plurality of fourth a sites 14a that can form covalent bonds with the third a site 13a; and a third a compound 16a for forming covalent bonds between the third a site 13a and the fourth a site 14a.

[0234] In the target substance testing kit of this disclosure, "specifically binds" corresponds to "specifically reacts" in this embodiment. "First compound" corresponds to "compound 1a". "Second compound having a fourth site" corresponds to "compound 2a having a plurality of sites 4a capable of forming a covalent bond with site 3a" and "compound 3a for forming a covalent bond between site 3a and site 4a".

[0235] The test kit 100a according to this embodiment can be used in the method for detecting a target substance according to this embodiment, in the same way as kits used for detecting a target substance in a sample by conventional in vitro diagnostics. Furthermore, the concentration of the target substance can also be measured by conventionally known methods, and it is particularly suitable for use in detecting a target substance in a sample by fluorescence polarization method.

[0236] The target substance testing kit 100a of this embodiment comprises a first reagent 101a and a second reagent 102a. The first reagent 101a comprises a luminescent reagent containing luminescent particles 2a and a first compound 4a, and the second reagent 102a may include a second compound 6a. In this case, the third compound 16a may contain multiple types of compounds for forming a covalent bond between the third site 13a and the fourth site 14a, and one of the first reagent 101a or the second reagent 102a may contain different types of compounds from among the multiple types of compounds. In addition to these reagents, the kit may have a housing that encloses these reagents, and if the target substance is an antigen or antibody, it may also have a viscosity modifier for use during the antigen-antibody reaction. Examples of viscosity modifiers include, but are not limited to, polyvinyl alcohol, polyvinylpyrrolidone, and sodium alginate.

[0237] Furthermore, the target substance test kit 100a of this embodiment may also include a positive control, a negative control, a serum diluent, etc., in addition to these reagents. As the medium for the positive control and negative control, in addition to serum, physiological saline, etc., that do not contain the target substance that can be measured, a solvent may be used. The concentration of the target substance can also be measured, and it is particularly suitable for use in detecting the target substance in a sample by fluorescence polarization method.

[0238] Furthermore, the target substance testing kit 100a of this embodiment may also contain a third substance, such as a solvent or blocking agent, in addition to these reagents. The third substance, such as a solvent or blocking agent, may also be included in combination of two or more types. Examples of solvents used in this embodiment include various buffer solutions such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer; however, the solvents included in the testing reagents of this embodiment are not limited to these.

[0239] The luminescent reagent in the target substance testing kit 100a of this embodiment may be dispersed in a dispersion medium and exist in the form of a dispersion. The amount of luminescent particles 2a contained in the target substance testing kit 100a of this embodiment is preferably 0.000001% to 20% by mass, and more preferably 0.0001% to 1% by mass.

[0240] The target substance testing kit 100a of this embodiment may (A) have a thiol group in each of the third a and fourth a sites, and the compound of third a is selected from the group consisting of peroxidases, oxidizing agents, and compounds having a phenol group, or (B) have at least one of two different glutamine residues and lysine residues in each of the third a and fourth a sites, and the covalent bond is an amide bond formed between the glutamine residue and the lysine residue, and the compound of third a may include transglutaminase.

[0241] If (A) is satisfied, the compound of 2a is a hydrophilic polymer compound having a thiol group, and the compound having a phenol group may contain one or more selected from the group consisting of tyramine, glycyltyrosine, phenol, and pyrogallol. In addition, the hydrophilic polymer compound may be any of the group consisting of sodium alginate, gelatin, polyethylene glycol, and carboxymethylcellulose. If (B) is satisfied, the compound of 2a contains a protein, and the protein may be casein.

[0242] <Third Embodiment> In this embodiment, the target substance in the sample solution is quantitatively evaluated by detecting the change in the rotational Brownian motion of the luminescent particles as a result of their reaction with the target substance, and this change in polarization anisotropy. The principle of fluorescence polarization is described below. Although the following description is based on the premise of quantitative evaluation (measurement of the amount of target substance), it goes without saying that each aspect of this disclosure using fluorescence polarization can be used not only for quantitative evaluation but also for qualitative evaluation (presence or absence of target substance).

[0243] (Fluorescence Polarization Method) By encapsulating a europium complex that exhibits polarized emission as a coloring material within the luminescent reagent, even slight changes in the dispersion state of particles in liquid can be detected as changes in polarized emission properties. When an antigen-antibody reaction occurs, if the luminescent particles aggregate or bind to a large aggregate via the antigen, a change in the rotational Brownian motion of the luminescent particles occurs. This change in rotational Brownian motion can be detected as a value related to fluorescence polarization (change in polarization anisotropy or fluorescence polarization degree).

[0244] Polarization anisotropy means that there is anisotropy in the transition moment (transition dipole moment). Polarized emission generally means that in the case of luminescent dyes with anisotropic transition moments, if the excitation light is polarized along that transition moment, the emitted light will also be polarized along that transition moment. In the case of europium complexes, since fluorescence emission is based on energy transfer from ligands to the central metal ion, the transition moment of polarized emission is complex, but the red emission around 610 nm, which originates from the electron transition from the lowest excited state 5D0 to 7F2, exhibits polarization anisotropy.

[0245] The principle of fluorescence polarization is to measure the shift in the transition moment due to the rotational motion of the light-emitting material during the time that polarized emission occurs. The rotational motion of the light-emitting material can be expressed by equation (2b): Q = 3Vη / kT ... (2b) where, Q: rotational relaxation time of the material V: volume of the material η: viscosity of the solvent k: Boltzmann constant T: absolute temperature.

[0246] The rotational relaxation time of a material is the time required for the molecule to rotate by an angle θ (68.5°) such that cosθ = 1 / e.

[0247] From equation (2b), it can be seen that the rotational relaxation time of the luminescent material is proportional to the volume of the material, i.e., the cube of the particle radius. On the other hand, the relationship between the luminescence lifetime and the degree of polarization of the material in fluorescence depolarization can be expressed by equation (3b). p0 / p = 1 + A(τ / Q) ... (3b) Here, p0: degree of polarization when the material is stationary (Q = ∞) p: degree of polarization A: constant τ: luminescence lifetime of the material Q: rotational relaxation time.

[0248] From equations (2b) and (3b), it is clear that in order to measure a large change in polarization degree, the relationship between the luminescence lifetime of the luminescent material and the rotational relaxation time, 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 needs to be.

[0249] To experimentally determine the degree of polarization of the emission shown in equation (3b), polarized light should be incident on the sample, and the emission should be detected at a 90-degree angle to the direction of propagation and vibration of the excitation light. At this time, the detected light should be separated into polarization components parallel and perpendicular to the polarization of the incident light and detected, and the degree of polarization should be evaluated using the equation shown in equation (4b). r(t) = (I∥(t) - GI⊥(t)) / (I∥(t) + 2GI⊥(t)) ... (4b) Here, r(t): Polarization anisotropy at time t I∥(t): Emission intensity of the emission component parallel to the excitation light at time t I⊥(t): Emission intensity of the emission component perpendicular to the excitation light at time t G: Correction value, the ratio of I⊥ / I∥ measured with excitation light whose vibration direction is 90 degrees different from the excitation light used for the sample measurement.

[0250] In other words, within the appropriate particle size and luminescence lifetime range, it is possible to sensitively read changes in particle size of luminescent materials due to antigen-antibody reactions, etc., as a value of polarization anisotropy. Polarization anisotropy is the value of the degree of polarization corrected for G and 2G, and the degree of polarization is the value obtained by removing G and 2G from equation (4b). In actual measurements, a correction value for G is necessary, so polarization anisotropy is determined.

[0251] Furthermore, it is preferable that the luminescent particles of this disclosure have a polarization anisotropy <r> of 0.01 or more, as determined by the following formula (1b). In equation (1b), <r>...polarization anisotropy I ＶＶ ...The emission intensity of the emission component whose vibration direction is parallel to the first polarization when excited by the first polarization I ＶＨ ...The emission intensity of the emission component whose vibration direction is perpendicular to the first polarization when excited by the first polarization I ＨＶ ...When excited with a second polarization whose vibration direction is perpendicular to the first polarization, the emission intensity of the emission component whose vibration direction is perpendicular to the second polarization is I. ＨＨ ...When excited with a second polarization whose vibration direction is perpendicular to the first polarization, the emission intensity G of the emission component whose vibration direction is parallel to the second polarization is a correction value.

[0252] In the fluorescence polarization method according to this embodiment, the design is such that the polarization anisotropy <r> is maximized when particles aggregate via the antigen, thus enabling highly sensitive measurements.

[0253] In this disclosure, instead of polarization anisotropy <r>, the degree of fluorescence polarization, which indicates the change in plane polarization, can be used as an indicator. The degree of fluorescence polarization can be expressed in terms of polarization degree milli P (also called milli-polarization units; hereafter abbreviated as mP). The degree of fluorescence polarization (mP) can be calculated using the measured polarization fluorescence intensity as shown in the following formula (5b): mP = (I∥ - I⊥) / (I∥ + I⊥) ... (5b) where, mP: degree of fluorescence polarization I∥: emission intensity of the emission component parallel to the polarization direction of the excitation light I⊥: emission intensity of the emission component perpendicular to the polarization direction of the excitation light.

[0254] (Method for detecting target substance) The method for detecting target substance according to this embodiment is a method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, and as shown in Figure 9A, comprises the following steps: A step of obtaining a liquid of 1b containing a complex of 1b having a target substance, a luminescent reagent having a 1b site that specifically reacts with the target substance, and a compound of 1b having a 2b site that specifically reacts with a site different from the site that specifically reacts with the luminescent reagent in the target substance, and a 3b site different from the 2b site (S1001b); A step of obtaining a liquid of 2b containing a complex of 2b having a complex of 1b and an aggregate in which a compound of 2b having a plurality of 3b sites is bound together via the 3b site (S1002b); A step of measuring a value related to the fluorescence polarization of the liquid of 2b (S1003b).

[0255] Here, the aggregate can also be called a crosslinked structure, a crosslinked body, or an aggregate. Furthermore, S1001 may be called the step of forming the first b composite, and S1002 may be called the step of forming the second b composite.

[0256] <Embodiment 3-1> The following embodiment 3-1, which is an example of the method for detecting a target substance of the above embodiment, is a method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, and as shown in Figure 9B, it comprises the following steps: A step of obtaining a liquid 1b containing a complex of 1b having a target substance, a luminescent reagent having a 1b site that specifically reacts with the target substance, and a compound 1b having a 2b site that specifically reacts with a site different from the site that specifically reacts with the luminescent reagent in the target substance, and a 3b site different from the 2b site (S2001b); A step of obtaining a liquid 2b containing a complex of 2b having an aggregate of a compound 2b having a plurality of 4b sites that bind to a 3b site, and a compound 3b having a plurality of 5b sites that bind to a 4b site, and the complex of 1b (S2002b); A step of obtaining a value relating to the fluorescence polarization of the liquid 2b (S2003b).

[0257] In the method for detecting a target substance of this disclosure, "sample solution that may contain a target substance" corresponds to "sample solution" in this embodiment. "Specifically binds" corresponds to "specifically reacts". "First compound" corresponds to "compound 1b". "Second compound having a fourth site" corresponds to "compound 2b having multiple sites 4b that bind to site 3b" and "compound 3b having multiple sites 5b that bind to site 4b".

[0258] In Embodiment 3-1, the "complex of 2b having an aggregate of a compound of 2b having multiple 4b sites that bind to the 3b site, and a compound of 3b having multiple 5b sites that bind to the 4b site, and a complex of 1b" is an example of the "complex of 2b having a complex of 1b, and an aggregate of a compound of 2b having multiple 3b sites that are bound together via the 3b site" in this embodiment.

[0259] Below, an example of the measurement method according to the embodiment of Section 3-1 will be described in more detail with reference to Figures 10A and 10B. In the embodiment of Section 3-1, luminescent particles are used as the luminescent reagent, but the luminescent reagent is not limited to particles and may be a molecule in which a luminescent dye is bound to a site that specifically reacts with the target substance.

[0260] In the step of obtaining the liquid 1b of the first b in Figure 10A, a reaction product (complex 21b of the first b) is obtained in which a luminescent particle 2b having a site 11b of the first b that specifically reacts with the target substance 3b in the sample liquid, and a compound 4b of the first b that has a site 12b of the second b that specifically reacts with the target substance 3b and a site (site Yb) of the target substance 3b that reacts with a site (site Xb) different from the site (site Yb) of the target substance 3b that reacts with the luminescent particle 2b, and a site 13b of the third b that is different from the site 12b of the second b, are specifically bound. In Figure 10A, the site of the target substance 3b that binds with the site 11b of the first b is shown as Xb, and the site that binds with the site 12b of the second b is shown as Yb. At this time, there is no particular specification regarding the mode of binding between the target substance 3b and the compound 4b of the first b, but it can be binding by an antigen-antibody reaction in which the target substance 3b is an antigen and the compound 4b of the first b contains an antibody.

[0261] In the step of obtaining the liquid 5b of the second b in Figure 10B, compound 17b of the second b, which has multiple sites 14b of the fourth b that react with site 13b of the third b, and compound 19b of the third b, which has multiple sites 15b of the fifth b that react with site 14b of the fourth b, are mixed to form an affinity complex 6b (which can also be called an aggregate of compound 17b of the second b and compound 19b of the third b) consisting of compound 17b of the second b and compound 19b of the third b. At this time, site 13b of the third b and site 15b of the fifth b may be the same site. Next, by combining the affinity complex 6b with the complex 21b of the first b obtained in the step of obtaining the liquid 1b of the first b, liquid 5b of the second b containing the measurement substance (also called complex 7b of the second b) can be obtained. Furthermore, when the second complex 7b is formed, the first complex 21b and the second compound 17b may bond first, then the third compound 19b may bond to the second compound 17b, and subsequently an aggregate of the second compound 17b and the third compound 19b may be formed. Alternatively, the aggregate of the second compound 17b and the third compound 19b may bond to the composite formed by the bonding of the first complex 21b, the second compound 17b, and the third compound 19b.

[0262] The proportion of luminescent particles 2b contained in the second b complex 7b depends on the proportion of the first b complex 21b in the first b liquid 1b, i.e., the amount of target substance 3b. As a result, by measuring the fluorescence polarization value (polarization anisotropy or fluorescence polarization degree; may also be called the fluorescence anisotropy value) of the second b liquid 5b, it is possible to confirm the change in the fluorescence polarization value in accordance with the amount of target substance 3b present.

[0263] While it is possible to perform the steps of obtaining the first liquid 1b and the second liquid 5b simultaneously, it is preferable to perform the first liquid 1b step before the second liquid 5b step. This is because performing the first liquid 1b step and the second liquid 5b step simultaneously may reduce the reaction efficiency of the first liquid 1b step.

[0264] The equilibrium dissociation constant (K) of the bond between site 3b 13b and site 4b 14b, and between site 4b 14b and site 5b 15b. ＤThe ratio (K) is preferably 10 nM or less. Also, the equilibrium dissociation constant (K) of the binding between the target substance 3b and site 12b of the second b is also specified. Ｄ The equilibrium dissociation constant (K) of the bond between site 13b of the third b and site 14b of the fourth b, and between site 14b of the fourth b and site 15b of the fifth b is greater than the equilibrium dissociation constant (K) of the bond between site 13b of the third b and site 14b of the fourth b and site 15b of the fifth b. Ｄ It is preferable that the ratio is smaller. This is because the affinity complex 6b is formed quickly, and the efficiency of forming the second complex 7b, which includes the luminescent particles 2b - target substance 3b - compound 4b of the first b - affinity complex 6b (an aggregate of the compound of the second b and the compound of the third b), in the step of obtaining the liquid 5b of the second b is increased.

[0265] According to this disclosure, there is no particular designation for the binding mode between site 13b of 3b and site 14b of 4b, and between site 14b of 4b and site 15b of 5b. However, site 13b of 3b and site 15b of 5b may be biotin-containing sites, and site 14b of 4b may be a site that specifically binds to biotin in avidin. That is, compound 17b of 2b may contain at least one of avidin, streptavidin, neutraavidin, and an immobilization carrier of these avidins, while compound 19b of 3b may be a protein having multiple biotin-containing sites. In this case, the immobilization carrier (carrier A16b) contained in compound 17b of 2b may be a nanoparticle. Furthermore, the protein (carrier B18b) contained in compound 19b of 3b may be any of albumin, gelatin, casein, or globulin.

[0266] The size of the affinity complex 6b is at least larger than the luminescent particle 2b, preferably 40 μm or less. The preferred size of the second complex 7b containing the affinity complex 6b is at most twice the size of the first complex 21b, and not exceeding 400 times, i.e., between 200 nm and 40,000 nm.

[0267] (Luminescent Particles) An example of luminescent particles in this embodiment will be described in detail with reference to Figures 10A and 10B. The luminescent particle 2b in this embodiment, which is an example of a luminescent reagent, has a base particle 20b consisting of a substrate 9b, a luminescent molecule 8b, a hydrophilic layer 10b, and a first b portion 11b that specifically reacts with the target substance 3b, which will be described later.

[0268] The luminescent molecule 8b contained in the substrate 9b is, in particular, a molecule that is excited and emits light upon irradiation with light, and molecules that emit light through chemical reactions, such as luminol, are undesirable. The luminescence includes phosphorescence and fluorescence. More preferably, in this embodiment, the substrate 9b contains a rare earth complex as the luminescent molecule 8b that is luminescent and has a long luminescence lifetime. Preferred examples of rare earth complexes include those of europium, terbium, neodymium, erbium, yttrium, lanthanum, cerium, samarium, gadolinium, dysprosium, thulium, ytterbium, and scandium. Since the value related to fluorescence polarization (polarization anisotropy or fluorescence polarization degree) depends on the change in rotational motion of the luminescent material during the luminescence time, it is preferable to use a rare earth complex with a long luminescence lifetime as the luminescent substance. Even more preferably, the substrate 9b contains a europium complex.

[0269] With respect to the luminescent particles 2b, from the viewpoint of maintaining particle uniformity and monodispersity, it is desirable not to apply anything to the surface of the substrate particles 20b other than the first b portion 11b that specifically reacts with the target substance. However, in order to use it in the measurement method according to this embodiment, it is necessary to prevent nonspecific adsorption of substances other than the target substance onto the substrate particles 20b, so it is preferable to have a hydrophilic layer 10b on the surface of the luminescent particles 2b in order to maintain hydrophilicity on the surface.

[0270] As a method for maintaining hydrophilicity in the hydrophilic layer 10b on the surface, a commonly used method is to support bovine serum albumin (BSA) on the surface of the substrate 9b, but this method may result in lot-to-lot variations. Therefore, it is preferable that the luminescent particles 2b include a hydrophilic layer 10b made of a hydrophilic, non-protein polymer. The concentration of the luminescent particles 2b in the reaction solution is preferably 0.000001% to 1% by mass, more preferably 0.00001% to 0.01% by mass.

[0271] The luminescent particles 2b used in this embodiment exhibit long-lived luminescence by containing a europium complex. Preferably, the luminescent particles 2b used in this embodiment have an average particle size of 25 nm to 500 nm, and more preferably, an average particle size of 50 nm to 300 nm. If the average particle size exceeds 500 nm, the value related to fluorescence polarization (polarization anisotropy or fluorescence polarization degree) before the reaction between the luminescent particles 2b and the target substance 3b becomes high, resulting in a high value related to fluorescence polarization during the process of obtaining the first liquid 1b, and a small difference between this value and the value related to fluorescence polarization after the process of obtaining the second liquid 5b. Also, if the average particle size is less than 25 nm, the amount of europium complex that can be contained per particle decreases, so the luminescence intensity of the luminescent particles 2 becomes weaker when compared with larger particles in the same number of moles. In this specification, the average particle size is the number-average particle size, and the average particle size can be measured by dynamic light scattering.

[0272] Dynamic light scattering is a measurement method that observes the Brownian motion of particles as fluctuations in scattered light intensity. When laser light is shone on particles dispersed in a solution and the scattered light is observed with a photon detector, the intensity distribution due to interference of scattered light is constantly fluctuating because the particles are constantly moving due to Brownian motion. The fluctuation of scattered light with respect to time is represented by an autocorrelation function, and the translational diffusion coefficient is determined. From the determined diffusion coefficient, the Stokes diameter can be found, and the size of the particles dispersed in the solution (also called particle size or particle diameter) can be derived.

[0273] The luminescent particles 2b preferably have a small particle size distribution, and preferably have a polydispersity index pdi of 0.1 or less. This is because fluorescence polarization measurement is based on the principle of measuring the size of the luminescent particles 2b, and if the size of the luminescent particles 2b varies, problems such as reduced measurement accuracy and a narrower measurement range may occur. An example of luminescent particles 2b is a polymer containing polystyrene and siloxane bonds, with a hydrophilic polymer on the surface of the particles. The base particles 20b may also have a hydrophilic layer 10b on its surface containing a hydrophilic polymer that includes one of ether, betaine, or pyrrolidone rings. By including a hydrophilic polymer on the surface of the luminescent particles, nonspecific adsorption is suppressed. In this way, by suppressing nonspecific adsorption with a hydrophilic polymer rather than a protein, the particle size distribution is suppressed, which is advantageous for measurements based on polarization anisotropy or fluorescence polarization degree.

[0274] (Substrate) Figure 10A shows an example where the luminescent particles are spherical, and the luminescent particles 2b include the substrate 9b. In Figure 10A, both the luminescent particles 2b and the substrate 9b are shown as spherical, but the shapes of the luminescent particles and the substrate 9b in this embodiment are not limited. The substrate 9b is not particularly specified as long as it is a material that can stably incorporate the europium complex, but it is preferably a polymer containing styrene units and organic silane units, and in particular polymers obtained by polymerizing a composition containing radically polymerizable organic silane with styrene as the main component are preferably used. By including styrene as the main component in the composition, it is possible to produce luminescent particles 2b with a very uniform particle size distribution by the emulsion polymerization method described later. Furthermore, by using a polymer containing organic silane units, silanol groups (Si-OH) are generated in the polymer in an aqueous solvent, forming siloxane bonds (Si-O-Si) on the surface of the substrate 9b, and through this, the hydrophilic layer 10b and functional groups described later can be imparted. In this embodiment, it is preferable that the light-emitting particle 2b has a functional group on the surface of the substrate particle 20b that can bond the first b portion 11b.

[0275] (Hydrophilic Layer) In this embodiment, it is preferable that the luminescent particles 2b have a small particle size distribution and that the surface of the particles is coated with a hydrophilic material. The hydrophilic layer 10b can be composed of a hydrophilic polymer or hydrophilic molecule on the outside of the substrate 9b. The hydrophilic polymer or hydrophilic molecule is not particularly specified as long as it is a polymer or molecule containing a hydrophilic group, and examples of hydrophilic groups include molecules and polymers having hydroxyl groups, ethers, pyrrolidones, betaine structures, etc. Examples of hydrophilic polymers include polyethylene glycol, polyvinylpyrrolidone, sulfobetaine polymers, phosphobetaine polymers, and polyglycidyl methacrylic acid, which has a glycidyl group opened and a hydroxyl group modified at the end of the molecule, and these can be the main components of the hydrophilic layer 10b. Alternatively, the hydrophilic layer 10b may be formed by directly applying a single molecule having a hydrophilic group to the surface of the substrate 9b using a silane coupling agent or the like. There is no limit to the thickness of the hydrophilic layer 10b, but it does not need to be thicker than the thickness required to exhibit hydrophilicity. If the hydrophilic layer 10b is too thick, it may become like a hydrogel, and the thickness of the hydrophilic layer 10 may become unstable due to hydration caused by ions in the solvent. The thickness of the hydrophilic layer 10b is preferably between 1 nm and 15 nm.

[0276] (Europium complex as a luminescent molecule) The europium complex, exemplified as the luminescent molecule 8b, has the characteristics of having a long emission lifetime, with the wavelength and intensity of the emission being less affected by the surroundings. The europium complex is composed of the europium element and a ligand. Considering the emission lifetime and the visible emission wavelength range, the luminescent dye is preferably a rare earth complex, especially a europium complex. Europium generally has an emission lifetime of 0.1 to 1.0 ms. It is necessary to appropriately adjust this emission lifetime and the rotational relaxation time obtained from formula (2b). In the case of europium in an aqueous dispersion, if the diameter of the luminescent particles 2b is about 50 to 300 nm, the polarization anisotropy <r> can change significantly before and after the step of obtaining the liquid 5b of 2b.

[0277] At least one of the ligands constituting the europium complex has a light-harvesting function. Light-harvesting function refers to the action of exciting the central metal of the complex by energy transfer when excited at a specific wavelength. Furthermore, it is preferable that the ligands constituting the europium complex include ligands such as β-diketones to prevent coordination of water molecules. Ligands such as β-diketones that coordinate to the europium ion suppress the deactivation process due to energy transfer to solvent molecules, etc., and strong luminescence is obtained.

[0278] Europium complexes may be polynuclear complexes. Specific examples of europium complexes include tris(2-thenoyltrifluoroacetone)bis(triphenylphosphine oxide)europium(III) and tris(2-thenoyltrifluoroacetone)(triphenylphosphine oxide)(dibenzylsulfoxide)europium(I Examples include (II) (tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide)europium(III)) and tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III)).

[0279] When the Brownian rotation of the europium complex can be considered to have stopped in the medium, it is desirable that the polarization anisotropy represented by equation (4b) is 0.10 or greater. The state in which the Brownian rotation can be considered to have stopped means that the rotational relaxation time of the particles is sufficiently longer than the luminescence lifetime of the europium complex.

[0280] It is preferable for the europium complex to be incorporated in large quantities into the substrate 9b, as this increases the emission intensity per particle. On the other hand, if the europium complex aggregates in the substrate 9b, the interaction between ligands affects the excitation efficiency of the europium complex, making it difficult to measure polarization anisotropy or fluorescence polarization degree while maintaining reproducibility. Whether the europium complex exhibits non-aggregated emission behavior in the substrate 9b can be determined from the excitation spectrum of the sample.

[0281] The luminescent particles 2b, which possess strong light emission, not only enable highly sensitive measurements but also maintain their luminescence even when their particle size is reduced, thus accelerating biochemical reaction rates. For example, smaller particle sizes result in a larger diffusion coefficient for Brownian motion in a liquid, making it possible to measure reactions in a shorter time.

[0282] (First b site that specifically reacts with the target substance) The luminescent particle 2b has a first b site 11b that specifically reacts with the target substance 3b (site Xb). Specifically reacting with site Xb means specifically interacting with site Xb, or specifically binding to site Xb (it may also be said that site Xb is specifically captured). Mechanisms for specifically binding to the target substance include, for example, electrostatic interactions, van der Waals interactions, and hydrogen bonding interactions.

[0283] Site 11b of 1b can be anything that exhibits affinity to a specific substance. Examples of combinations of site 11b of 1b and site Xb, or site Xb and site 11b of 1b, include antigens and antibodies, small molecules and their receptors, enzymes and substrates, and complementary nucleic acids. Examples of antibodies and their specific substances include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and all kinds of small molecules. Examples of receptors and their specific substances include small molecules, cytokines, hormones, neurotransmitters, signaling molecules, and membrane proteins. Examples of DNA, RNA, cDNA, parts or fragments thereof, synthetic nucleic acids, primers, probes, etc. derived from bacteria, viruses, cells, etc., and nucleic acids that are complementary to them. In addition to the above, any combination known to have affinity can be used as the combination of site Xb and site 11b of 1b. In this embodiment, the first b portion 11b is typically an antibody, an antigen, or a nucleic acid.

[0284] In this disclosure, examples of target substances 3b to be measured include antigens, antibodies, small molecule compounds, various receptors, enzymes, substrates, nucleic acids, cytokines, hormones, neurotransmitters, signaling molecules, membrane proteins, etc. Antigens include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and all kinds of small molecule compounds. Nucleic acids include DNA, RNA, cDNA derived from bacteria, viruses, cells, etc., parts or fragments thereof, synthetic nucleic acids, primers, probes, etc. Small molecule compounds include cytokines, hormones, neurotransmitters, signaling molecules, membrane proteins, etc., and their receptors, etc.

[0285] (Compound 1b having a site 2b and a site 3b different from site 2b) In this embodiment, compound 1b 4b has a site 2b 12b that specifically reacts with a site (site Yb) different from the site luminescent particle 2b of the target substance 3b, and a site 3b 13b different from site 2b 12b. A preferred example of compound 1b 4b is a compound having an antibody that binds to site Yb via site 2b 12b and biotin as the site 3b 13b different from site 2b 12b.

[0286] Compound 4b of the first b reacts specifically with site Yb of the target substance 3b in Figure 10A. Therefore, the luminescent particle 2b and compound 4b of the first b can react specifically via the target substance 3b.

[0287] The third b site 13b can be any substance that can bind to the fourth b site 14b in liquid by an affinity reaction, as shown in Figure 10B. For example, when the third b site 13b contains biotin, iminobiotin, desthiobiotin, or a biotin derivative, the fourth b site 14b that reacts with the third b site 13b can be any site that has a binding affinity for biotin, such as avidin, streptavidin, neutraavidin, or a biotin-binding site of an avidin carrier. When the third b site 13b is biotin, the fourth b site 14b is preferably a biotin-binding site of streptavidin. On the other hand, when the third b site 13b is avidin, the fourth b site 14b is preferably a site containing biotin. In particular, from the viewpoint of increasing the specific reactivity of compound 4b of the first b with the target substance 3b, the molecular weight of compound 4b of the first b can be reduced, so a preferred site 13b of the third b is a site containing biotin (for example, a group having biotin).

[0288] The dissociation constant (K) of the bond between site 13b of the third b and site 14b of the fourth b. ＤThe ) is 10 nM or less. This allows for the rapid formation of affinity complex 6b and facilitates the formation of the luminescent particle 2b - target substance 3b - compound 4b of the first b - affinity complex 6b in the step of obtaining the liquid 5b of the second b. Dissociation constant of biotin and avidin binding (K Ｄ ) is 1 fM and can form a stable bond very quickly, making it a particularly preferred combination of bonding sites.

[0289] The compound 4b of the first b preferably has at least one of the sites 13b of the third b. For example, if site 12b of the second b is an antibody, at least one biotin molecule should be chemically attached to the antibody molecule. This is because at least one biotin molecule is present, allowing it to bind to the affinity complex 6b. The number of sites 13b of the third b can be increased within a range that does not affect the binding affinity of site 12b of the second b to the target substance 3b. Typically, it is preferable to label the antibody with one to eight biotin molecules, and in particular, one to four molecules are preferred as they do not affect the affinity reaction.

[0290] In this embodiment, the compound 4b of the first b, which binds to the target substance 3b, can be used in large quantities to form a reaction product (complex 21b of the first b) with the luminescent particles 2b obtained through the step of obtaining the liquid 1b of the first b. The compound 4b of the first b may be incorporated into the affinity complex 6b in the step of obtaining the liquid 5b of the second b, regardless of whether or not it reacts with the target substance 3b in the step of obtaining the liquid 1b of the first b. By being present in large quantities in a free state in the liquid during the step of obtaining the liquid 1b of the first b, it becomes possible to increase the probability of reaction with the target substance 3b, which is present in trace amounts. As a result, even if the target substance 3b is present in trace amounts, the polarization anisotropy <r> or fluorescence polarization degree mP of the liquid 5b of the second b to be measured can be made higher.

[0291] The concentration of compound 4b in the first b should preferably be approximately 10 to 1,000,000 times the concentration of the target substance 3b. If the concentration of the target substance 3b is low, it is desirable to use a higher concentration.

[0292] (Compound 2b and Compound 3b) In this embodiment, as shown in Figure 10B, in the step of obtaining liquid 5b of 2b, the affinity complex 6b can be quickly formed by mixing compound 17b of 2b, which has multiple 4b sites 14b that react with 3b sites 13b, and compound 19b of 3b, which has multiple 5b sites 15b that react with 4b sites 14b, in liquid.

[0293] At this time, the formation of affinity complex 6b is due to an affinity reaction occurring between the 4th b site 14b of compound 2b 17b and the 5th b site 15b of compound 3b 19b. At this time, the equilibrium dissociation constant (K) of the bond between the 4th b site 14b and the 5th b site 15b is Ｄ The concentration is 10 nM or less. This allows for the rapid formation of the affinity complex 6b. The formed affinity complex 6b has a fourth b site 14b of compound 2b 17b, which can undergo an affinity reaction with the third b site 13b of compound 1b 4b. Therefore, in the step of obtaining liquid 2b 5b, the formation of the luminescent particle 2b - target substance 3b - compound 1b 4b - affinity complex 6b becomes possible. Note that the fifth b site 15b may be the same site as the third b site 13b.

[0294] An example of a preferred site combination is one in which site 4b 14b is the site that binds to biotin in avidin, and site 5b 15b is the site that contains biotin. The dissociation constant (K) of the binding of biotin to avidin. Ｄ ) is 1 fM and can form a stable bond very quickly, so it is particularly preferred as a combination of binding sites. Of course, site 14b of 4b may be a site containing biotin, and site 15b of 5b may be a site that binds to biotin in avidin. Another example is that site 14b of 4b may be a site containing avidin, and site 15b of 5b may be an anti-avidin antibody. Alternatively, site 14b of 4b may be a site consisting of an anti-avidin antibody, and site 15b of 5b may be avidin.

[0295] Compound 17b of the second b has multiple sites 14b of the fourth b that react with site 13b of the third b, and contains carrier A16b. Carrier A16b is not necessarily required if the molecule has multiple sites 14b of the fourth b that react with site 13b of the third b. Examples of compound 17b of the second b that do not require carrier A16b are avidin, streptavidin, and neutraavidin. These are preferred because they have four binding sites with biotin. Compound 17b of the second b only needs to have two or more sites that can bind to compound 19b of the third b, which will be described later. If site 13b of the third b is a site containing biotin, compound 17b of the second b is preferably avidin, and site 14b of the fourth b is preferably a site within avidin that binds to biotin. On the other hand, if site 13b of the third b is a site containing avidin, it is preferable that site 14b of the fourth b is a site containing biotin.

[0296] Preferred examples of compound 17b of 2b include avidin, streptavidin, neutraavidin, and a carrier on which avidin is immobilized (hereinafter sometimes referred to as an avidin-immobilized carrier). Here, an avidin-immobilized carrier (hereinafter sometimes referred to as an avidin-immobilized carrier) is a carrier on which at least one of avidin, streptavidin, and neutraavidin (hereinafter sometimes abbreviated as avidins) is immobilized. Avidin, streptavidin, neutraavidin, and the avidin-immobilized carrier can bind to biotin and thus form affinity complex 6b. By having these avidins in compound 17b of 2b, compound 4b of 1b can be given biotin with a small molecular size, and as described above, the reactivity of compound 4b of 1b to target substance 3b can be increased. A particularly preferred example of compound 17b of 2b is streptavidin. Streptoavidin is inexpensive and readily available among avidins, and it causes fewer nonspecific reactions.

[0297] A more preferred example is compound 17b of the second b, which consists of a support on which many avidins are immobilized. Hereafter, this will be referred to as the avidin-immobilized support. Immobilizing many avidins not only improves the reaction efficiency with biotin, but also, because compound 17b of the second b has a certain size, a large affinity complex 6b can be rapidly obtained even when the biotin-avidin reaction efficiency is low.

[0298] In this case, the support A16b is preferably nanoparticles, which have a high specific surface area and thus high reactivity. The nanoparticles can be polystyrene nanoparticles, silica nanoparticles, gold nanoparticles, etc. From the viewpoint of the affinity bonding reaction rate, the size of the nanoparticles is preferably as small as possible, within a range that allows for multiple bonding sites (such as avidin and biotin). The average particle size is, for example, 5 nm to 200 nm, particularly 5 nm to 100 nm, and even more preferably 10 to 20 nm. This is because if the support A16b has a size of 10 to 40 nm, multiple avidins as the 4b site 14b can be immobilized, and the affinity bonding reaction rate (i.e., the diffusion rate of the nanoparticles) is sufficiently high. If it is 5 nm or less, it will be the same size as streptavidin, and there will be little advantage in using nanoparticles as a support. On the other hand, if it exceeds 40 nm, the affinity bonding reaction rate decreases, which is undesirable. Gold is preferred as the material for the nanoparticles because its aggregation properties can be controlled relatively easily, its particle surface is easily modified, and uniform particles can be obtained. It is known that the aggregation of gold nanoparticles can be controlled in solution by salts or pH. That is, the aggregation properties of gold nanoparticles can be adjusted with salts or acids / alkalis, and the size of the affinity complex 6b can be adjusted. Furthermore, gold nanoparticles immobilized with streptavidin are widely used and readily available commercially. For example, by increasing the salt concentration in solution to 1 M or higher, the gold nanoparticles become unstable, and by performing the affinity reaction under these conditions, a larger aggregate can be formed more rapidly. Therefore, gold nanoparticles are suitably used in the measurement method disclosed herein.

[0299] Compound 19b of the third b may have multiple sites of site 13b of the third b or site 15b of the fifth b, and may also contain carrier B18b. If a molecule has multiple sites of site 13b of the third b or site 15b of the fifth b, carrier B18b is not necessarily required. Antibodies, particularly polyclonal antibodies, are preferred as molecules having multiple sites of site 13b of the third b or site 15b of the fifth b. Furthermore, if site 14b of the fourth b, which reacts with site 13b of the third b or site 15b of the fifth b, is biotin, then compound 19b of the third b may be avidin. These have two or more binding sites within a single molecule. Therefore, when these are used as compound 19b of the third b, carrier B18b is not necessary.

[0300] Compound 19b of the third b only needs to have two or more binding sites to compound 17b of the second b; for example, a protein having multiple biotin molecules can be used. In Figure 10B, biotin is site 15b of the fifth b, and the protein is carrier B18b. Examples of carrier B18b include stable and highly water-soluble proteins, such as albumin, gelatin, casein, and globulin, with albumin being particularly suitable due to its high stability and availability.

[0301] Albumin containing multiple biotin molecules is, for example, biotinylated bovine serum albumin. It can be produced by chemically binding biotin to bovine serum albumin. If there are at least two biotin molecules, they can become components of affinity complex 6b. The number of biotin molecules on the albumin is preferably increased as much as possible, within a range that does not affect the binding affinity with compound 17b of the second b. Typically, it is preferable to label albumin with 2 to 30 biotin molecules, and in particular, 2 to 20 is preferred as it is within a range that does not affect the affinity reaction.

[0302] In the step of obtaining liquid 5b of the second b, an affinity bonding reaction between compound 17b of the second b and compound 19b of the third b yields liquid 5b having an affinity complex 6b. In this disclosure, a third compound may be added to form an aggregate (corresponding to the affinity complex 6b in this embodiment), which in this embodiment corresponds to compound 17b of the second b and compound 19b of the third b. The affinity bonding reaction between compound 17b of the second b and compound 19b of the third b is thought to be determined by the balance of the number of bonded molecules of both in the liquid. In order to efficiently form a large affinity complex 6b, the reaction should be carried out in an amount ratio such that the number of sites 13b of the third b or sites 15b of the fifth b and the number of sites 14b of the fourth b that react with them are equal. If either is added in a large excess, the size of the affinity complex 6b may not increase. The reaction concentrations of compound 17b of the second b and compound 19b of the third b are not particularly limited, as long as fluorescence polarization measurement is possible, but a range of 1 μM to 1 mM is preferred. Within this range, the antigen-antibody reaction and fluorescence measurement of the luminescent particles 2b are not inhibited, and affinity complex 6b with a size of 1 μm or larger can be obtained, which is effective in increasing the sensitivity of fluorescence polarization measurement.

[0303] The affinity complex 6b has a larger average particle size than the luminescent particles 2b, does not directly bind to the target substance 3b, but can bind to the compound 4b of the first b. The term "having affinity" is used synonymously with "specifically binding." The size (average particle size) of the affinity complex 6b is at least larger than the luminescent particles 2b, preferably 40 μm or less. Considering the magnitude of the change in values ​​related to the sedimentation and fluorescence polarization of the affinity complex 6b, it is preferably between 200 nm and 10,000 nm, and more preferably between 1,000 nm and 6,000 nm.

[0304] Furthermore, it is preferable that the affinity complex 6b does not interfere with the emission measurement of the luminescent particles 2b. For example, the excitation wavelength of the luminescent particles 2b and the excitation wavelength of the affinity complex 6b may be different, or the emission wavelength of the luminescent particles 2b and the emission wavelength of the affinity complex 6b may be different. Alternatively, the excitation wavelength of the luminescent particles 2b and the excitation wavelength of the affinity complex 6b may be different, and the emission wavelength of the luminescent particles 2b and the emission wavelength of the affinity complex 6b may be different. In other words, the affinity complex 6b can produce emission at an excitation wavelength different from that of the luminescent particles 2b, and can produce emission at an emission wavelength different from that of the luminescent particles 2b. On the other hand, it is preferable that the affinity complex 6b is not excited at wavelengths suitable for exciting the luminescent particles 2b. Specifically, it is preferable that the affinity complex 6b is not excited by excitation light with a wavelength of 300 nm to 450 nm. It is also preferable that the affinity complex 6b does not produce emission that overlaps with that of the luminescent particles 2b. Specifically, it is preferable that the affinity composite 6b does not have an emission maximum in the wavelength range of 550 nm to 650 nm. However, to simplify measurement, it is preferable that the affinity composite 6b is substantially non-emitting. Furthermore, the affinity composite 6b may scatter light as long as the emission intensity of the polarization can be measured. Since the emission intensity is preferably as high as possible from the viewpoint of detection accuracy of the emission particles 2b, it is desirable that the affinity composite 6b has minimal light scattering.

[0305] If the size of the composite 7b of 2b obtained in the step of acquiring the liquid 5b of 2b is large, the value related to the fluorescence polarization of the luminescent particles 2b contained in the composite 7b of 2b (polarization anisotropy <r> or fluorescence polarization degree mP) will also be large. It is important that the luminescent particles 2b in Figure 10B form a large reactant via the affinity composite 6b through the target substance 3b. Considering the stable fluorescence polarization measurement of the composite 7b of 2b, and taking into account the magnitude of sedimentation and changes in the value related to fluorescence polarization, the size of the composite 7b of 2b is 200 nm to 40000 nm, preferably 200 nm to 10000 nm, and more preferably 1000 nm to 6000 nm. The magnitude of the value related to fluorescence polarization depends on the rotational relaxation time and luminescence lifetime of the material, so in the case of luminescent particles 2b using a europium complex, if the size of the composite 7b of 2b is 1 μm or more, the value related to fluorescence polarization will theoretically reach its maximum value.

[0306] (Measurement step to obtain values ​​related to the fluorescence polarization of the liquid in step 2b) In the measurement step, values ​​related to the fluorescence polarization of the reaction solution (polarization anisotropy <r> or fluorescence polarization degree mP) are measured. When measuring to determine the values ​​related to fluorescence polarization, a polarizer such as a polarizing filter can be placed on the incident light side to irradiate with polarized excitation light (first polarization). If the polarizing filter is placed in a direction perpendicular to this, a second polarization whose vibration direction is perpendicular to the first polarization can be irradiated. If a polarizer is placed on the detection side in a direction parallel to the incident polarizer, the emission intensity of the emission component whose vibration direction is parallel to the excitation side can be measured. If the polarizer is placed on the detection side in a direction perpendicular to the incident polarizer, the emission intensity of the emission component whose vibration direction is perpendicular to the excitation light can be measured. The emission intensity can be measured by a spectrophotometer or the like.

[0307] The measurement conditions are preferably such that the liquid 5 of the second b is in a liquid at a temperature of 0 to 50°C, and the viscosity of the second b liquid 5 is preferably 0.5 to 50 mPa·s. The concentration of the luminescent particles 2b is preferably measured at 0.0001 mg / mL (0.00001 mass%) to 10 mg / mL (1 mass%), and the measurement wavelength is preferably 500 to 700 nm. The measurement process may be performed simultaneously with the process of obtaining the second b liquid 5b. By measuring simultaneously with the process of obtaining the second b liquid 5b, for example, when polarization anisotropy is used as a value related to fluorescence polarizability, it is possible to measure the polarization anisotropy <r0> immediately after the reaction and the polarization anisotropy <r1> after a certain reaction time. By measuring the difference between <r1> and <r0> or the amount of change with respect to time (dr / dt) and comparing it with a standard sample, it is possible to measure the concentration of the target substance 3b in the sample. The solution is preferably an aqueous solvent, such as a buffer solution, physiological saline, or water. The measurement conditions can be set as appropriate by those skilled in the art, and in doing so, they may refer to, but are not limited to, the examples described later in this specification.

[0308] (Method for producing luminescent particles) Next, an example of a method for producing luminescent particles 2b used in this embodiment will be described. The method for producing luminescent particles 2b comprises the following steps: A first manufacturing step of preparing an emulsion by mixing a radical polymerizable monomer containing at least styrene and a radical polymerizable organosilane, a radical polymerization initiator, a polarizing luminescent europium complex, and a hydrophilic polymer with an aqueous medium; a second manufacturing step of heating the emulsion to polymerize the radical polymerizable monomer; and a third manufacturing step of imparting a functional group to the surface of the luminescent particle for bonding the 1b portion 11b, which will be described later.

[0309] In this case, the functional group for attaching the first b portion 11b is a functional group that can attach the first b portion 11b to the substrate particle 20b, and specifically, any of the following can be used: a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, or an alkoxysilyl group (silicon alkoxide structure).

[0310] (Radical Polymerizable Monomers) The luminescent particles 2b are produced by polymerizing a radical polymerizable monomer, which comprises at least styrene and a radical polymerizable organosilane. The radical polymerizable monomer may further include monomers selected from the group consisting of acrylate monomers and methacrylate monomers. Examples of monomers include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, or mixtures thereof. That is, one or more of these monomers can be used in addition to styrene and the radical polymerizable organosilane. A monomer having two or more double bonds in a single molecule, such as divinylbenzene, may also be used as a crosslinking agent.

[0311] By including radically polymerizable organic silanes in the radically polymerizable monomer, siloxane bonds are imparted to the substrate 9b. Examples of radically polymerizable organic silanes include vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, or combinations thereof. By using radically polymerizable organic silanes, an inorganic oxide skeleton is formed within the substrate 9, which improves the physical and chemical stability of the luminescent particles 2b. Furthermore, by using radically polymerizable organic silanes, the affinity between the substrate 9b and the hydrophilic layer 10b and functional groups is increased. In addition, by including radically polymerizable organic silanes in the radically polymerizable monomer, silanol groups are imparted to the surface of the substrate 9b. As a result, the silanol groups and hydrophilic polymer are more firmly adsorbed onto the surface of the substrate 9b.

[0312] (Radical polymerization initiators) Radical polymerization initiators can be widely used from azo compounds, organic peroxides, etc. Specifically, examples include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionic acid) dimethyl, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), potassium persulfate (KPS), etc.

[0313] (Hydrophilic Polymer) The luminescent particle 2b may include a hydrophilic polymer as the hydrophilic layer 10b. The hydrophilic polymer preferably suppresses nonspecific adsorption. Examples of hydrophilic polymers include hydrophilic polymers containing units having ether, betaine, pyrrolidone rings, etc. The hydrophilic layer 10b is contained in the synthesized luminescent particle 2b and preferably exists mainly on the surface of the luminescent particle outside the substrate 9b. In this specification, polymers having pyrrolidone rings may be abbreviated as "PVP". By adding PVP during the synthesis of the luminescent particle 2b, it is possible to simultaneously impart nonspecific adsorption suppression ability and functional groups for binding the first b site 11b to the luminescent particle 2b. Since the PVP added during synthesis is more hydrophilic than the radical polymerizable monomer, it exists at the interface between the solvent and the substrate 9 during polymerization. The substrate 9b adsorbs PVP to its outside by partially incorporating PVP during polymerization or by physical and chemical adsorption such as the interaction between the pyrrolidone ring and styrene (radical polymerizable monomer).

[0314] The molecular weight of PVP is preferably between 10,000 and 100,000, and more preferably between 40,000 and 70,000. If the molecular weight is less than 10,000, the hydrophilicity of the surface of the luminescent particles 2 is weak, making nonspecific adsorption more likely. If the molecular weight is greater than 100,000, the hydrophilic layer 10b becomes too thick, causing gelation and making it difficult to handle. In addition to PVP, a hydrophilic polymer may be added as a protective colloid during the synthesis of the substrate 9b.

[0315] Furthermore, the luminescent particles 2b preferably satisfy A2 - A1 ≤ 0.1. In this case, A1 and A2 are defined as follows: A1 is the absorbance of a mixture obtained by adding 30 μL of a dispersion of 0.1% by mass of luminescent particles 2b to 60 μL of buffer mixed with 16 μL of 15-fold diluted human serum, immediately after addition, and A2 is the absorbance after being left at 37°C for 5 minutes after the addition. The absorbance is measured with an optical path of 10 mm and a wavelength of 572 nm. Substrate particles 20 in which A2 - A1 is 0.1 or less are preferred because they exhibit less nonspecific adsorption of impurities in the serum.

[0316] (Aqueous medium) The aqueous medium (aqueous solution) used in the above-described method for producing the luminescent particles 2b preferably contains 80% by mass or more and 100% by mass or less of water. The aqueous solvent is preferably water or an organic solvent soluble in water, and examples include a solution of methanol, ethanol, isopropyl alcohol, or acetone mixed with water. If an organic solvent other than water is included in an amount greater than 20% by mass, dissolution of polymerizable monomers may occur during the production of the luminescent particles 2b.

[0317] Furthermore, it is preferable that the above-mentioned aqueous medium has a pH of 6 to 9 beforehand. If the pH is less than 6 or greater than 9, the alkoxide or silanol groups of the radical polymerizable organic silane may undergo condensation polymerization or react with other functional groups before polymer formation, which may cause the resulting luminescent particles 2 to aggregate. In this embodiment, condensation polymerization of the alkoxide is not intentionally performed before polymerization.

[0318] The pH adjustment described above is preferably done using a pH buffer, but it may also be done with an acid or a base. In addition, surfactants, defoamers, salts, thickeners, etc. may be added to the aqueous medium in a proportion of 10% by mass or less.

[0319] When producing the luminescent particles 2b, it is preferable to first dissolve the PVP in an aqueous medium whose pH has been adjusted to 6 to 9. The PVP content is preferably 0.01% to 10% by mass relative to the aqueous medium, and more preferably 0.03% to 5% by mass. If it is less than 0.01% by mass, the amount adsorbed to the substrate 9b will be small and the effect will not be exhibited. If it is more than 10% by mass, the viscosity of the aqueous medium will increase, and sufficient stirring may not be possible.

[0320] Next, a radical polymerizable monomer containing styrene (A) and a radical polymerizable organic silane (B) is added to the aqueous medium to form an emulsion. The mass ratio of styrene (A) to radical polymerizable organic silane (B) is 6:4 to 100:1. Furthermore, a europium complex is mixed into the prepared emulsion. At this time, if the solubility of the europium complex is low, a water-insoluble organic solvent may be added. The mass ratio of the europium complex to the radical polymerizable monomer is 1:1000 to 1:10.

[0321] If the mass ratio of styrene (A) to radically polymerizable organic silane (B) is less than 6:4, the specific gravity of the entire luminescent particle 2b increases, which may lead to significant sedimentation of the luminescent particle 2b. Furthermore, in order to improve the adhesion of the luminescent particle 2 to the PVP, it is desirable to set the mass ratio of styrene (A) to radically polymerizable organic silane (B) to 100:1 or higher.

[0322] The mass ratio of the aqueous medium to the total amount of radically polymerizable monomers is preferably between 5:5 and 9.5:0.5. If the mass ratio of the aqueous medium to the total amount of radically polymerizable monomers is less than 5:5, aggregation of the generated luminescent particles 2b may become significant. If the mass ratio of the aqueous medium to the total amount of radically polymerizable monomers is greater than 9.5:0.5, there will be no problem in generating luminescent particles 2b, but the amount generated may be reduced.

[0323] The radical polymerization initiator is used after being dissolved in water, a buffer, etc. The amount of radical polymerization initiator relative to the total mass of styrene (A) and radically polymerizable organosilane (B) in the emulsion can be used between 0.5% by mass and 10% by mass. In the step of heating the emulsion, it is sufficient to heat the entire emulsion uniformly. The heating temperature can be arbitrarily set between 50°C and 80°C, and the heating time between 2 hours and 24 hours. By heating the emulsion, the radically polymerizable monomer is polymerized.

[0324] The substrate particle 20b may have a functional group on its surface for binding to the site 11b of the first b. The functional group is not particularly limited as long as it is a functional group to which the site 11b of the first b, such as an antibody, antigen, or enzyme, can be bound. For example, it may be a carboxyl group, amino group, thiol group, epoxy group, maleimide group, succinimidyl group, silicon alkoxide group, or contain these functional groups. For example, it is possible to impart a functional group to the surface of the substrate particle 20b by mixing a silane coupling agent having a functional group for binding to the site 11b of the first b with the synthesized particles. Specifically, a carboxyl group can be imparted to the surface of the substrate particle 20b by preparing an aqueous solution of a silane coupling agent having a carboxyl group and mixing it with a dispersion of the synthesized substrate particle 20b. At this time, a dispersant such as polysorbate 20 may be added to the reaction solution. The reaction temperature can be arbitrarily set between 0°C and 80°C, and the reaction time between 1 hour and 24 hours. To suppress the rapid condensation reaction of the silane coupling agent, it is preferable to set the reaction time to 3 to 14 hours at or below room temperature of about 25°C. Depending on the functional group used to bond the 1b site 11b, an acid or alkali catalyst may be added to accelerate the reaction on the surface of the substrate particle 20b.

[0325] By attaching the first b site 11b of various antibodies, etc., to the substrate particle 20b, it can be used as a particle for sample testing. By utilizing the functional groups present in the hydrophilic layer 10b for attaching the first b site 11b, the optimal method for attaching the target antibody, etc., can be selected.

[0326] (Introduction of the first b portion) The chemical reaction for chemically bonding the functional group for attaching the first b portion 11b to the first b portion 11b can be carried out using conventionally known methods to the extent that the objectives of this disclosure can be achieved. Furthermore, when amide bonding the first b portion 11b to the hydrophilic layer 10b, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate. In addition, in the bonding of the functional group to the first b portion 11b, the first b portion 11b may be introduced to the luminescent particle 2b by physical adsorption.

[0327] (Method for producing the compound of the first b) The compound of the first b used in this embodiment has a second b site 12b that specifically reacts with a site (site Yb) different from the site of the target substance 3b that reacts with the luminescent particle 2b, and a third b site 13b that is different from the second b site 12b. Below, an example of a method for producing the compound of the first b used in this embodiment will be described using a biotinylated antibody as an example. Here, the second b site 12b is an antibody, and the third b site 13b, which is different from the second b site 12b, is biotin.

[0328] The method for producing biotinylated antibodies involves an antibody molecule and at least one biotin molecule. Biotinylation of the antibody can be carried out by known methods, and any method is acceptable as long as the binding affinity of the antibody to the target substance 3 is maintained. The modification of the antibody with biotin can utilize the amino groups present in the antibody. By reacting the N-hydroxysuccinimide active ester of biotin with the amino groups present in the antibody, the amino groups of the antibody and the N-hydroxysuccinimide active ester group form a covalent bond (amide bond). As such a compound, N-[6-(biotinamide)hexanoyl]-6-aminohexanoate N-succinimidyl can be used.

[0329] (Method for producing compound 2b) Compound 2b 17b used in embodiment 1b is a compound having multiple 4b sites 14b that react with a 3b site 13b that is different from the 2b site 12b. Compound 2b 17b can be selected from avidin, streptavidin, and neutraavidin, and commercially available products thereof can be used, but it can also be produced as a recombinant protein by known methods. In addition, an avidin-immobilized carrier can be obtained or synthesized as appropriate and used.

[0330] An example of a method for producing streptavidin-immobilized carriers is described. Gold nanoparticles are used as the carrier. Gold nanoparticles can be produced by reducing gold compounds such as chloroauric acid. The dispersion of the obtained gold nanoparticles can be stabilized by modifying them with polymers or proteins as appropriate. Proteins can also be chemically bonded to the surface of the gold nanoparticles by presenting functional groups such as carboxyl groups. Gold nanoparticles with streptavidin immobilized on them are commercially available and easily obtainable.

[0331] If the 5b site 15b of compound 19b of the third b is a site that binds to biotin in avidin, then compound 17b of the second b can be any molecule having at least two biotin molecules as the 4b site 14b. Biotinylated albumin, which will be described later, is one example (the manufacturing method will be described later).

[0332] (Method for producing compound 3b) Compound 3b 19b used in this embodiment is a compound having multiple sites 15b of site 5b. An example of a method for producing the compound will be explained using biotinylated albumin as an example.

[0333] The method for producing biotinylated albumin is the same as the method for producing compound 4b in 1b, and known methods for biotinylation of proteins can be applied. Any method or configuration is acceptable as long as at least two biotin molecules are included. The modification of albumin with biotin can utilize the amino groups present in bovine serum albumin. By reacting the N-hydroxysuccinimide active ester of biotin with the amino groups present in bovine serum albumin, a covalent bond (amide bond) is formed between the amino group of bovine serum albumin and the -hydroxysuccinimide active ester group. As such a compound, N-[6-(biotinamide)hexanoyl]-6-aminohexanoic acid N-succinimidyl can be used. Commercially available biotinylated albumin may also be used. If the 4th b site 14b that reacts with the 3b site 13b of compound 2b 17b is biotin, then the 3b compound 19b can be any commercially available avidin or a carrier containing avidin (as described above for the manufacturing method).

[0334] (Method for measuring target substance by fluorescence polarization measurement) As a preferred embodiment of the present disclosure, a method for measuring target substance 3b by measuring values ​​related to fluorescence polarization will be described in detail with reference to Figure 11.

[0335] In this disclosure, the step of obtaining the liquid 1b of the first b is to prepare luminescent particles 2b, which are an example of a luminescent reagent containing a rare earth complex and specifically bind to the target substance 3b, and the compound 4b of the first b, which specifically reacts with the target substance 3b and has at least one or more 3b sites 13b, and to react with the target substance 3b. In the step of obtaining the liquid 1b of the first b, the liquid 1b of the first b is obtained, and this liquid 1b of the first b contains a reaction product of luminescent particles 2b (complex 21b of the first b) obtained by the specific reaction of the target substance 3b and the compound 4b of the first b.

[0336] In the next step of obtaining the liquid 5b of the second b, compound 17b of the second b having multiple sites 14b of the fourth b and compound 19b of the third b having multiple sites 15b of the fifth b are mixed to obtain affinity complex 6b. The driving force for the formation of affinity complex 6b is the affinity bonding reaction between site 15b of the fifth b and site 14b of the fourth b. Therefore, site 15b of the fifth b and site 14b of the fourth b can specifically bond, and affinity bonding is possible. At this time, the dissociation constant (K) of the bond between site 15b of the fifth b and site 14b of the fourth b is Ｄ The strength is 10 nM or less.

[0337] Since the obtained affinity complex 6b contains a fourth b site 14b that reacts with the third b site 13b, affinity bonding is possible between the first b complex 21b obtained in the step of obtaining the first b liquid 1b and the third b site 13b. As a result, the affinity complex 6b bonds with the first b complex 21b, which is composed of the luminescent particles 2b, the target substance 3b, and the first b compound 4b. Therefore, a second b complex 7b, which is larger than the first b complex 21b, is formed, and a second b liquid 5b containing the second b complex 7b is obtained.

[0338] Finally, the polarization anisotropy <r> or degree of fluorescence polarization, which is a value relating to the fluorescence polarization of the liquid 5b obtained in the step of acquiring the liquid 5b of the second b, is measured (measurement step). By measuring the value relating to fluorescence polarization, the target substance 3b can be measured. At this time, the measurement may be qualitative or quantitative.

[0339] In both the step of obtaining the first liquid 1b and the step of obtaining the second liquid 5b, the reaction is preferably carried out in the pH range of 3.0 to 11.0. The reaction temperature is in the range of 0°C to 100°C, or 4°C to 50°C, or even 20°C to 50°C, and the reaction time is in the range of 1 second to 2 hours, or 1 minute to 60 minutes. In the measurement method according to this embodiment, the concentration of the luminescent particles 2b is preferably as follows: that is, 0.00001% to 1% by mass in the reaction system is preferred, and more preferably 0.0001% to 0.1% by mass.

[0340] In this embodiment, the measurement method involves measuring the complex (second b complex 7b) of the luminescent particle 2b and affinity complex 6b via the target substance 3b using fluorescence polarization. Specifically, the measurement method includes the steps of: mixing the sample with a reagent to obtain a mixture; irradiating the reaction solution with polarized light; and separating and measuring the polarization component derived from the luminescent reagent in the reaction solution. At this time, a value related to fluorescence polarization (polarization anisotropy or fluorescence polarization degree) may be obtained after the step of obtaining the first b liquid 1b. This measurement allows confirmation of the blank level of the value related to fluorescence polarization. Furthermore, during the step of obtaining the second b liquid 5b, the value related to fluorescence polarization may be measured over time. This measurement allows confirmation of the time course of the value related to fluorescence polarization. In the final measurement step, the value related to fluorescence polarization obtained in the measurement can be used to determine at least one of the presence or absence of the target substance 3b and its concentration. This is because the value related to fluorescence polarization of the luminescent particle 2b changes depending on the amount of the target substance 3b.

[0341] The target substance 3b in the sample can be measured by measuring the fluorescence polarization value of the above reaction. For example, by obtaining a calibration curve (a graph showing the relationship between polarization anisotropy or fluorescence polarization degree and the concentration of target substance 3b) using the fluorescence polarization value (polarization anisotropy or fluorescence polarization degree) of a standard solution of target substance 3b of known concentration, it becomes possible to quantify the target substance 3b contained in the sample solution from the fluorescence polarization value.

[0342] (Target Substance Test Kit) A target substance test kit can also be made using the target substance detection method of this disclosure. The test kit for measuring the target substance in a sample in this embodiment is a target substance test kit that detects at least one of the presence or absence and concentration of the target substance in a sample solution by obtaining a value relating to fluorescence polarization, and comprises: a luminescent reagent having a first b site that specifically reacts with the target substance; a first b compound having a second b site that specifically reacts with a site in the target substance different from the site that specifically reacts with the luminescent reagent, and a third b site different from the second b site; a second b compound having a plurality of fourth b sites that bind to the third b site; and a third b compound having a plurality of fifth b sites that bind to the fourth b site. Here, the third b site and the fifth b site may be the same site.

[0343] In the target substance testing kit of this disclosure, "specifically binds" corresponds to "specifically reacts" in this embodiment. "First compound" corresponds to "compound 1b". "Second compound having a fourth site" corresponds to "compound 2b having multiple sites 4b that bind to site 3b" and "compound 3b having multiple sites 5b that bind to site 4b".

[0344] In the target substance testing kit of this embodiment, there is no particular designation for the binding mode between site 3b and site 4b, and between site 4b and site 5b, however, the equilibrium dissociation constant (K) of the binding between site 3b and site 4b is specified. Ｄ ) and the equilibrium dissociation constant (K) of the bond between site 4b and site 5b. Ｄ The viscosity is preferably 10 nM or less.

[0345] Furthermore, the 3b site is a biotin-containing site, and the 2b compound may include at least one of avidin, streptavidin, neutraavidin, and an immobilization carrier for these avidins. In this case, the immobilization carrier included in the 2b compound may be a nanoparticle.

[0346] Site 5b is a biotin-containing site, and compound 3b may contain a protein. In this case, the immobilization carrier contained in compound 2b may be a nanoparticle. Furthermore, the protein contained in compound 3b may be any of albumin, gelatin, casein, or globulin.

[0347] A substance containing the target substance can be detected by obtaining a value related to its fluorescence polarization. In this case, the value related to fluorescence polarization can be a value related to the degree of fluorescence polarization or polarization anisotropy.

[0348] As shown in Figure 12A, the target substance testing kit 100b according to this embodiment may consist of, for example, a first b solution 101b containing two components, luminescent particles 2b and compound 4b of the first b, a second b solution 102b containing compound 17b of the second b, and a third b solution 103b containing compound 19b of the third b. This is a target substance testing kit composed of three types of solutions.

[0349] Furthermore, the target substance testing kit 100b according to this embodiment (Figure 12B) may consist of, for example, a first b solution 104b containing three components: luminescent particles 2b, compound 4b of the first b, and compound 19b of the third b, and a second b solution 105b containing compound 17b of the second b. This is a target substance testing kit composed of two types of solutions.

[0350] The amount of luminescent particles 2b according to this embodiment contained in the target substance testing kit 100b in this embodiment is preferably 0.00001% to 20% by mass, and more preferably 0.0001% to 2% by mass. The testing kit 100b according to this embodiment may also contain third substances such as solvents and blocking agents in addition to the configuration according to this embodiment, to the extent that the objectives of this disclosure can be achieved. Two or more types of third substances such as solvents and blocking agents may be included in combination. Examples of solvents used in this embodiment include various buffer solutions such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvents contained in the testing kit 100b in this embodiment are not limited to these. Furthermore, the target substance testing kit 100b according to this embodiment may also contain a dispersion stabilizer to stabilize dispersion and a sensitizer to promote reaction. Examples of dispersion stabilizers include surfactants, proteins, amino acids, hydrophilic polymers, and polysorbate 20b, albumin, glycine, arginine, and PVP. Examples of sensitizers include, but are not limited to, polyvinyl alcohol, polyvinylpyrrolidone, and polyalginic acid.

[0351] As an example of the target substance test kit 100b of this embodiment, it can also be an in vitro diagnostic kit. A kit refers to a set used for measuring a target substance in a sample, which includes the test kit 100b and other accessories. In addition to the test kit 100b, the kit may also include a standard solution, a positive control, a negative control, a serum diluent, etc. The standard solution is a solution of the target substance at a known concentration. As the medium for the positive control and negative control, serum, physiological saline, or a solvent may be used, in addition to serum that does not contain the target substance that can be measured. The kit according to this embodiment can be used in the method for measuring a target substance in a sample by fluorescence polarization according to this embodiment.

[0352] <Fourth Embodiment> An example of one embodiment of the present disclosure will be described in detail below, but this will not limit the scope of the present disclosure. In this embodiment, the target substance in the liquid is quantitatively evaluated by capturing the change in rotational Brownian motion of the luminescent reagent, which occurs as a result of the reaction of the luminescent reagent present in the liquid with the target substance, as a change in polarization anisotropy. The principle of fluorescence polarization method is described below. It should be noted that the following description is based on the premise of quantitative evaluation (measurement of the amount of target substance), but it goes without saying that each aspect of the present disclosure using fluorescence polarization method can be used not only for quantitative evaluation but also for qualitative evaluation (measurement of the presence or absence of target substance).

[0353] [Fluorescence Polarization Method] When a change occurs in the dispersion state of a luminescent reagent in a liquid, even a slight change can be detected as a change in polarized emission properties. When the first c site of the luminescent reagent that specifically reacts with the target substance (which can also be described as a site that specifically binds to the first c site of the target substance) specifically reacts with the target substance, the luminescent reagent aggregates due to the specific reaction between the target substance and the first c site. This then causes a change in the rotational Brownian motion of the particles. This change in rotational Brownian motion can be detected as a change in polarization anisotropy.

[0354] Polarization anisotropy means that there is anisotropy in the transition moment (transition dipole moment). Generally, if the luminescent molecule of a luminescent reagent is a luminescent dye with anisotropic transition moments, then if the excitation light is polarized along that transition moment, it means that the emitted light will also be polarized along that transition moment. For example, if a luminescent reagent has a europium complex as its luminescent molecule, it exhibits fluorescence emission based on energy transfer from the ligand to the central metal ion. Therefore, the transition moment of polarized emission becomes complex, but the red emission around 610 nm, which originates from the electron transition from the lowest excited state 5D0 to 7F2, exhibits polarization anisotropy.

[0355] The principle of fluorescence polarization is to measure the shift in the transition moment due to the rotational motion of the luminescent reagent during the time that polarized emission occurs. The rotational motion of the luminescent reagent can be expressed by equation (2c): Q = 3Vη / kT ... (2c) where, Q: rotational relaxation time of the luminescent reagent V: volume of the luminescent reagent η: viscosity of the solvent k: Boltzmann constant T: absolute temperature.

[0356] The rotational relaxation time of a luminescent reagent is the time required for the molecule to rotate by an angle θ (68.5°) such that cosθ = 1 / e.

[0357] From equation (2c), it can be seen that the rotational relaxation time of the luminescent reagent is proportional to the volume of the material, i.e., the cube of the radius. On the other hand, the relationship between the luminescence lifetime of the material and the degree of polarization in fluorescence depolarization can be expressed by equation (3c). p0 / p = 1 + A(τ / Q) ... (3c) Here, p0: degree of polarization when the luminescent reagent is stopped (Q = ∞) p: degree of polarization A: constant τ: luminescence lifetime of the luminescent reagent Q: rotational relaxation time.

[0358] From equations (2c) and (3c), it is clear that in order to measure a large change in polarization degree, the relationship between the luminescence lifetime of the luminescent reagent and the rotational relaxation time, i.e., the volume (particle size) of the luminescent reagent, is important, and the larger the particle size of the luminescent reagent, the longer the luminescence lifetime needs to be.

[0359] To determine the degree of polarization of the emission shown in equation (3c) experimentally, polarized light should be incident on the sample, and the emission should be detected at a 90-degree angle to the direction of propagation and vibration of the excitation light. At this time, the detected light should be separated into polarization components parallel and perpendicular to the polarization of the incident light and detected, and the degree of polarization should be evaluated using the equation shown in equation (4c). r(t) = (I∥(t) - GI⊥(t)) / (I∥(t) + 2GI⊥(t)) ... (4c) Here, r(t): Polarization anisotropy at time t I∥(t): Emission intensity of the emission component parallel to the excitation light at time t I⊥(t): Emission intensity of the emission component perpendicular to the excitation light at time t G: Correction value, the ratio of I⊥ / I∥ measured with excitation light whose vibration direction is 90 degrees different from the excitation light used for the sample measurement.

[0360] In other words, given the appropriate size and luminescence lifetime of the luminescent reagent, it is possible to sensitively read the change in the size of the luminescent reagent due to antigen-antibody reactions, etc., as a value of polarization anisotropy. Polarization anisotropy is the value of the degree of polarization corrected by G and 2G, and the degree of polarization is the value obtained by removing G and 2G from equation (4c). In actual measurements, a correction value for G is necessary, so polarization anisotropy is determined. Alternatively, even without determining the correction value G, it is possible to compare the relative magnitude of polarization anisotropy if the measurement conditions are the same.

[0361] Furthermore, it is preferable that the luminescent reagent of this embodiment has a polarization anisotropy <r> of 0.01 or higher, as determined by the following formula (1c). In equation (1c), <r>: polarization anisotropy I ＶＶ : The emission intensity of the emission component whose vibration direction is parallel to the first polarization when excited with the first polarization. ＶＨ : The emission intensity of the emission component whose vibration direction is perpendicular to the first polarization when excited with the first polarization. ＨＶ : The emission intensity of the emission component whose vibration direction is perpendicular to the second polarization when excited with a second polarization whose vibration direction is perpendicular to the first polarization. ＨＨ : The emission intensity of the emission component whose vibration direction is parallel to the second polarization when excited with a second polarization whose vibration direction is orthogonal to the first polarization. G: Correction value.

[0362] [Detection Method] The detection method of the present disclosure is a detection method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, and as shown in Figure 13A, comprises the following steps: (1) A step of obtaining a liquid of first c containing a complex of first c having a target substance, a luminescent reagent having a first c site that specifically reacts with the target substance, and a compound of first c having a second c site that specifically reacts with a site different from the site that specifically reacts with the luminescent reagent in the target substance, and a third c site different from the second c site (S1001c) (2) A step of obtaining a liquid of second c containing a complex of second c having a complex of first c and an aggregate in which a plurality of compounds of second c having third c sites are linked via the third c sites (S1002c) (3) A step of measuring a value relating to the fluorescence polarization of the liquid of second c (S1003c) At this time, the aggregate of compounds of second c may also be referred to as a crosslinked structure, a crosslinked body, or an aggregate of the compounds of second c.

[0363] <Embodiment 4-1> The following embodiment 4-1, which is an example of the above embodiment, is a detection method for detecting at least one of the presence or absence and concentration of a target substance in a sample solution, and as shown in Figure 13B, comprises the following steps: A step of obtaining a liquid containing a first c complex having a target substance, a luminescent reagent having a first c site that specifically reacts with the target substance, and a first c compound having a second c site that specifically reacts with a site different from the site of the target substance that specifically reacts with the luminescent reagent, and a third c site having a temperature-responsive polymer (S2001c); A step of obtaining a liquid containing a liquid containing a second c complex having a first c complex and an aggregate of second c compounds bonded via the third c site by changing the temperature of a liquid containing a first c complex and a plurality of second c compounds having third c sites (S2002c); A step of obtaining a value relating to the fluorescence polarization of the liquid third c (S2003c).

[0364] In the method for detecting a target substance of this disclosure, “a sample solution that may contain a target substance” corresponds to “the sample solution” in this embodiment. “Specifically bind” corresponds to “specifically react.” “A first compound having a second site and a third site different from the second site” corresponds to “a firstc compound having a secondc site and a thirdc site having a temperature-responsive polymer.” “A second compound having a fourth site” corresponds to “(multiple) secondc compounds having a thirdc site.”

[0365] In the fluorescence polarization method according to this embodiment, the design is such that the polarization anisotropy <r> is increased by the aggregation of the luminescent reagent due to hydrophobic aggregation in the aqueous solution, thereby enabling highly sensitive measurements.

[0366] In the fourth-first embodiment, the portion of the third c portion of the third c portion that contributes to the formation of the aggregate of the second c compound in the method for detecting the target substance of the present disclosure is the portion made of a temperature-responsive polymer, and the aggregate of the second c compound is formed by changing the temperature of the second c liquid.

[0367] The method for detecting the target substance in the 4-1 embodiment will be further explained with reference to Figures 14A to 14D and Figure 15. Figures 14A to 14D illustrate the process of obtaining the first liquid c.

[0368] As shown in Figure 14A, the target substance 3c in the sample solution can bind to the luminescent particle 2c via the first c portion 11c. The luminescent particle 2c is not particularly limited as long as it has the first c portion 11c and is capable of emitting light, but in this example, the luminescent particle 2c has a substrate 9c containing a luminescent molecule 8c, a base particle having a hydrophilic layer 10c formed on its surface, and the first c portion 11c. As shown in Figure 14C, the target substance 3c can react specifically with the first c compound 4c via the second c portion 12c. A specific reaction is, for example, a specific binding. In the figure, the portion of the target substance 3c that binds to the first c portion 11c is shown as Xc, and the portion that binds to the second c portion 12c is shown as Yc.

[0369] As shown in Figure 14B, the compound 4c of the first c has a second c site 12c that specifically reacts with the target substance 3c, and a third c site 13c that, unlike the second c site 12c, has a temperature-responsive polymer 18c. Figure 14C shows an example of the process for obtaining the liquid 1c of the first c. A mixture is prepared in which the luminescent particle 2c and the compound 4c of the first c form a so-called sandwich structure, sandwiching the target substance 3c, thereby forming the complex 16c of the first c. At this time, for example, the target substance 3c can be an antigen and the second c site 12c can be an antibody, and the specific reaction between the target substance 3c and the compound 4c of the first c can be made into a binding by an antigen-antibody reaction.

[0370] As shown in Figure 14D, the step of obtaining the first liquid 1c may also include sub-steps. If sub-steps are included, the step of obtaining the first liquid 1c may include a first sub-step of obtaining a mixed liquid A22c having a subcomplex 21c comprising a target substance 3c, luminescent particles 2c, and a third compound 5c having a second portion 12c and a fourth portion 14c different from the second portion 12c; and a second sub-step of obtaining a mixed liquid B1c having a first complex 16c comprising the mixed liquid A22c and a fourth compound 6c having a fifth portion 15c that specifically reacts with the fourth portion 14c and a third portion 13c having a temperature-responsive polymer 18c.

[0371] In this case, there are no restrictions on the binding site and binding mode, as long as the dissociation constant between the site 14c of the fourth c and the site 15c of the fifth c is smaller than the dissociation constant between the target substance 3c and the site 12c of the second c. For example, one of the sites 14c of the fourth c and the site 15c of the fifth c may be a site containing avidin, and the other may be a site containing biotin. More specifically and preferably, the site 14c of the fourth c and the site 15c of the fifth c may be a site containing biotin, and the site 15c of the fifth c may be a site containing avidin.

[0372] Figure 15 illustrates the process of obtaining the liquid of 2c and the liquid of 3c 19c. The liquid of 2c refers to a mixture obtained by mixing a plurality of compounds of 2c 20c with the liquid of 1c 1c. In the process of obtaining the liquid of 3c 19c, by changing the temperature of the liquid of 2c, the temperature-responsive polymer 18c of the site 13c of 3c interacts, as shown in Figure 15, and a plurality of compounds of 2c 20c (Figure 15 shows an example where the compound of 2c 20c is the same as the compound of 1c 4c) form an aggregate 7c via the site 13c of 3c. That is, the liquid of 3c 19c is prepared, which contains the composite of 2c 17c including the composite of 1c 16c and the aggregate 7c. As mentioned above, Figure 15 shows an example where compound 4c of the first c is the same compound as compound 20c of the second c. However, compound 20c of the second c may be a different compound from compound 4c of the first c, as long as it has the third c moiety 13c.

[0373] The temperature-responsive polymer 18c contained in compound 20c of the second c is a stimulus-responsive polymer that changes its solubility in water when the temperature of the liquid of the second c changes, with a predetermined temperature as the boundary (for example, when the temperature rises above the lower critical solution temperature when the temperature is increased by heating, etc., or when the temperature falls below the upper critical solution temperature when the temperature is decreased by cooling, etc.). In this disclosure, the temperature-responsive polymer 18c can change from hydrophilic to hydrophobic to form an aggregate 7c of compound 20c of the second c. This makes it possible to form a large complex (complex 17c of the second c) containing the aggregate 7c of compound 20c of the second c, which is formed via the bond between the complex 16c of the first c and the site 13c of the third c (more specifically, the temperature-responsive polymer 18c of the site 3c). At this time, the size of complex 17c of the second c is at least twice the size of complex 16c of the first c, and specifically, the size of complex 17c of the second c is preferably 200 nm or more and 6000 nm or less.

[0374] As mentioned above, in the 4-1 embodiment, compound 4c of the first c and compound 20c of the second c were described as the same compound. However, as long as both have a third c portion 13c, if the temperature-responsive polymer 18c of the third c portion 13c of compound 4c of the first c and the temperature-responsive polymer 18c of the third c portion 13c of compound 20c of the second c interact to form a second c complex 17c containing an aggregate 7c of compound 2c 20c and a first c complex 16c, then compound 4c of the first c and compound 2c 20c may be different compounds.

[0375] [Luminescent Reagent] The luminescent reagent has luminescent properties and a first c site 11c that reacts with the target substance 3c. The luminescent reagent may be a luminescent molecule having a first c site 11c that reacts with a specific site (site Xc) of the target substance 3c and a site consisting of a luminescent molecule, or it may be a luminescent particle having a substrate containing a luminescent molecule (a molecule that is excited and emits light when irradiated with light) and a first c site 11c that specifically reacts with the target substance 3c. Among these, the luminescent reagent is preferably a luminescent particle because it can more sensitively detect polarization anisotropy when reacting with a trace amount of the target substance 3c. Preferably, the luminescent particle has a substrate 9c containing a luminescent molecule 8c, a first c site 11c that specifically reacts with the target substance 3c, and a hydrophilic layer 10c present on the surface of the substrate 9c, as shown in Figure 14A. Luminescence includes phosphorescence and fluorescence. Furthermore, it is preferable that the luminescent particles 2c have a small particle size distribution, and it is preferable that the surface of the luminescent particles 2c is covered with a hydrophilic layer 10c, as will be described later. Below, using the example that the luminescent reagent is luminescent particles 2c as shown in the schematic diagram in Figure 14A, we will describe the various parts of the luminescent particles 2c and the method for producing the luminescent particles 2c.

[0376] (Substrate) The substrate 9c of the luminescent particle 2c can be any material that can contain the luminescent molecule 8c. For example, if the luminescent molecule 8c is a europium complex, the substrate 9c is not particularly specified as long as it is a material that can stably incorporate the europium complex. The substrate 9c is preferably a polymer containing styrene units and organic silane units, and a polymer obtained by polymerizing a composition containing a radically polymerizable organic silane with styrene as the main component is particularly suitable. By including styrene as the main component in the composition, it is possible to produce particles with a very uniform particle size distribution by the emulsion polymerization method described later. Furthermore, by using a polymer containing organic silane units, silanol groups (Si-OH) are generated in the polymer in an aqueous solvent, and siloxane bonds (Si-O-Si) can be formed between them on the surface of the substrate 9c of the particles. Through such siloxane bonds, which are bonding functional groups, substances that specifically react with target substances 3c, such as the hydrophilic layer 10c and the site 11c of the first c, described later, can be imparted to the surface of the substrate 9c.

[0377] (First c portion that specifically reacts with the target substance) The first c portion 11c of the luminescent particle 2c that specifically reacts with the target substance 3c preferably includes a portion that specifically reacts with a specific target substance 3c. The portion that specifically reacts with the target substance 3c may include, for example, a substance that specifically reacts with a specific target substance 3c (this may be specific binding or specific capture), and any substance that shows affinity to a specific target substance 3c can be used. Furthermore, the concept that the first c portion 11c of the luminescent particle 2c includes a portion that specifically reacts with a specific target substance 3c includes, for example, a substrate 9c on which a substance that reacts with the target substance 3c is immobilized, as described above.

[0378] Examples of combinations of a target substance 3c and a site 11c of the first c that specifically reacts with the target substance 3c in the luminescent particle 2c include antigens and antibodies, small molecules and their receptors, enzymes and substrates, and complementary nucleic acids. More specific examples of antigens and antibodies include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and all kinds of small molecules, along with antibodies that specifically bind to them. Other examples include receptors and small molecules, hormones, neurotransmitters, signaling molecules, and membrane proteins that specifically bind to them. Furthermore, examples include parts or fragments of DNA, RNA, cDNA, synthetic nucleic acids, primers, probes, etc. derived from bacteria, viruses, cells, etc., and nucleic acids that are complementary to them. In addition to the above, any combination known to have affinity can be used as a combination of a target substance 3c and a site 11c of the first c that specifically reacts with the target substance 3c. In this embodiment, the site 11c of the first c that reacts with the target substance 3c is typically an antibody, an antigen, or a nucleic acid.

[0379] (Hydrophilic layer) From the viewpoint of maintaining particle uniformity and monodispersity, it is desirable that nothing be applied to the surface of the substrate 9c of the luminescent particles 2c. However, from the viewpoint of detecting the target substance 3c, it is preferable to suppress the nonspecific adsorption of substances other than the target substance 3c to the luminescent particles 2c. Therefore, it is preferable that a hydrophilic layer 10c is formed on the surface of the substrate 9c so that the surface of the luminescent particles 2c becomes hydrophilic. As a method for forming the hydrophilic layer 10c on the surface of the substrate 9c, there is also a method of supporting proteins such as BSA on the surface of the substrate 9c, but the method of forming a hydrophilic layer 10c containing hydrophilic polymers or hydrophilic molecules on the surface of the substrate 9c is preferable because it is less likely to cause variations between lots.

[0380] The hydrophilic layer 10c may include, for example, hydrophilic polymers or hydrophilic molecules. Hydrophilic polymers and hydrophilic molecules are polymers or molecules containing hydrophilic groups, and specific examples of hydrophilic groups include molecules and polymers having hydroxyl groups, ethers, pyrrolidones, betaine structures, etc. Specific examples of hydrophilic polymers include polyethylene glycol, polyvinylpyrrolidone, sulfobetaine polymers, phosphobetaine polymers, and polyglycidyl methacrylic acid, which has a glycidyl group opened and a hydroxyl group modified at the end of the molecule. These can be the main components of the hydrophilic layer 10c. Alternatively, the hydrophilic layer 10c may be formed by directly applying a single molecule having a hydrophilic group to the surface of the substrate 9c of the luminescent particle 2c using a silane coupling agent or the like. In this specification, a more specific example of a hydrophilic polymer, a polymer having a pyrrolidone ring, may be abbreviated as "PVP".

[0381] Preferably, the hydrophilic layer 10c covers at least a portion of the surface of the substrate 9c, and more preferably, it covers a large portion of the surface of the substrate 9c. This makes it possible to suppress the nonspecific adsorption of luminescent particles 2c, as described above. Also, as will be described later, if the hydrophilic layer 10c is formed during the synthesis of the substrate 9c, the hydrophilic layer 10c may be included in a portion of the substrate 9c, but it is preferable that the hydrophilic layer 10c mainly exists on the outer particle surface of the substrate 9c. There is no limit to the thickness of the hydrophilic layer 10c, but it does not need to be thicker than the thickness required to exhibit hydrophilicity. The thickness of the hydrophilic layer 10c is preferably between 1 nm and 15 nm. By keeping the thickness within this range, it is less likely to become like a hydrogel, and the thickness of the hydrophilic layer 10c is less likely to become unstable due to hydration caused by ions in the solvent.

[0382] (Luminescent Molecules) The luminescent molecules 8c contained in the luminescent particles 2c are preferably rare-earth complexes, and more preferably europium complexes, considering factors such as luminescence lifetime (i.e., long lifetime) and the visible emission wavelength range. Europium complexes have the characteristics of having a long luminescence lifetime, with the wavelength and intensity of the emission being less affected by the surroundings. Europium complexes are composed of europium elements and ligands. Europium complexes generally have a luminescence lifetime of 0.1 to 1.0 ms. It is necessary to appropriately adjust this luminescence lifetime and the rotational relaxation time obtained from the above formula (2c).

[0383] When the luminescent molecule 8c is a europium complex, at least one of the ligands constituting the europium complex is a ligand with a light-harvesting function. The light-harvesting function is the action of being excited at a specific wavelength and exciting the central metal of the complex by energy transfer. Furthermore, it is preferable that the ligands constituting the europium complex include ligands such as β-diketones to prevent coordination of water molecules. Ligands such as β-diketones that are coordinated to the europium ion suppress the deactivation process due to energy transfer to solvent molecules, etc., and strong luminescence can be obtained.

[0384] Europium complexes may be polynuclear complexes. Specific examples of europium complexes include tris(2-thenoyltrifluoroacetone)bis(triphenylphosphine oxide)europium(III), tris(2-thenoyltrifluoroacetone)(triphenylphosphine oxide)(dibenzyl sulfoxide)europium( Examples include tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide)europium(III) and tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III).

[0385] In addition, when it can be considered that the Brownian rotational motion of the europium complex has stopped in the medium, it is desirable that the polarization anisotropy <r> represented by the above formula (4c) is 0.10 or more. The state where the Brownian rotational motion can be considered to have stopped indicates a state where the rotational relaxation time of the luminescent particle 2c is sufficiently longer than the luminescence lifetime of the europium complex.

[0386] It is preferable that more luminescent molecules 8c are incorporated into the base material 9c because the luminescence intensity per particle becomes stronger. On the other hand, when the luminescent molecules 8c aggregate in the base material 9c, it may affect the excitation efficiency of the europium complex due to the interaction between the ligands, etc., and it may become difficult to measure the values related to fluorescence polarization with reproducibility. When the particle size of the luminescent particle 2c is a particle of 100 nm, the luminescent particle 2c containing about 1,000 to 3,000,000 luminescent molecules 8c per luminescent particle can be preferably used. Whether the europium complex shows a non-aggregated luminescence behavior in the base material 9c can be judged from the excitation spectrum of the sample. The luminescent particle 2c having strong luminescence not only enables highly sensitive measurement, but also maintains luminescence even when the particle size is reduced, making it possible to increase the biochemical reaction rate. Therefore, the smaller the particle size of the luminescent particle 2c, the larger the diffusion coefficient of the Brownian motion in the liquid, and it becomes possible to detect the reaction in a shorter time.

[0387] The diameter of the luminescent particle 2c can be determined by the dynamic light scattering method. When laser light is irradiated on the particles dispersed in the solution and the scattered light is observed with a photon detector, since the particles are constantly moving their positions due to Brownian motion, the intensity distribution due to the interference of the scattered light is constantly fluctuating. The dynamic light scattering method is a measurement method that observes the state of this Brownian motion as the fluctuation of the scattered light intensity. The fluctuation of the scattered light with respect to time is represented by an autocorrelation function, and the translational diffusion coefficient is determined. The Stokes diameter can be obtained from the determined diffusion coefficient, and the size of the luminescent particle 2 dispersed in the solution can be derived.

[0388] The diameter of the luminescent particles 2c preferably has an average particle diameter of 25 nm or more and 500 nm or less, more preferably 50 nm or more and 300 nm or less. By setting the average particle diameter to 50 nm or more and 300 nm or less, the polarization anisotropy after the aggregation reaction can be increased, and the amount of the luminescent molecules 8c such as europium complexes that can be contained per luminescent particle can be increased.

[0389] Also, the luminescent particles 2c preferably have a small particle size distribution. In FIG. 14A, an example in which both the luminescent particles 2c and the base material 9c are spherical is shown, but the shapes of the luminescent particles 2c and the base material 9c in the present embodiment are not limited.

[0390] [Compound of the first c] As shown in FIG. 14B, the compound 4c of the first c has a second c site 12c that reacts with the target substance 3c and a third c site 13c that has a temperature-responsive polymer 18c. The temperature-responsive polymer 18c exhibits hydrophilicity in the step of obtaining the liquid 1c of the first c and is in a state of being dissolved in the solution.

[0391] The compound 4c of the first c only needs to be in a state of being dissolved in an aqueous solution in the step of obtaining the liquid 1c of the first c, and may have a second c site 12c and a third c site 13c having a temperature-responsive polymer 18c. The compound 4c of the first c may be a ligand having a temperature-responsive polymer eighteen c. The second c site 12c that specifically reacts with the target substance 3c has the same function of specifically reacting with the target substance 3c as the first c site 11c of the luminescent particles 2c, but reacts with a site (site Yc) different from the site (site Xc) that reacts with the first c site of the target substance 3c. Examples of the second c site 12c in the present embodiment include an antibody or the like. When an antibody is used, either a monoclonal antibody or a polyclonal antibody can be used as the second c site 12c.

[0392] The compound 4c of the first c reacts specifically with (may bind to or capture) the luminescent particle 2c via the target substance 3c. The second c site 12c of the compound 4c of the first c is, for example, a site consisting of a ligand that specifically reacts with the target substance 3c, and both the first c site 11c of the luminescent particle 2c and the second c site 12c of the compound 4c of the first c react specifically with different sites of the target substance 3c, thereby reacting specifically via the target substance 3c. Although the second c site 12c has the function of reacting with the target substance 3c, it may include not only sites that contribute to the function of reacting with the target substance 3c but also sites that do not contribute.

[0393] The third c portion 13c of compound 4c of the first c, which contains the temperature-responsive polymer 18c, should be soluble in water at low temperatures but should become insoluble, turbid, and precipitate when heated to a predetermined temperature. The predetermined temperature is generally called the lower critical solution temperature and is unique to each temperature-responsive polymer 18c. In this specification, the lower critical solution temperature may be abbreviated as "LCST".

[0394] The third c portion 13c having the temperature-responsive polymer 18c includes, for example, poly(N-alkylacrylamide), poly(N-vinylalkylamide), polyvinyl alkyl ether, and polyoxazoline. Therefore, in the above example, the third c portion 13c having the temperature-responsive polymer 18c is a polymer containing the above. Accordingly, for example, poly(N-isopropylacrylamide), poly(N-2-isopropyl-2-oxazoline), etc. can be used for the temperature-responsive polymer 18c. In particular, poly(N-isopropylacrylamide) has an LCST near body temperature and can therefore be suitably used in specimen testing such as that described herein. The mechanism of temperature response is that in poly(N-isopropylacrylamide), the polymer chain is hydrated and stretched due to the strong interaction between the amide bond site below the LCST and water, and adopts a random coil conformation. On the other hand, at temperatures higher than the LCST, dehydration occurs, and the polymer chain aggregates into a globule state due to hydrophobic interactions.

[0395] Next, we will describe in more detail each step of the method for detecting the target substance 3c when using luminescent particles 2c as the luminescent reagent in this embodiment (from the step of obtaining the liquid 1c of the first c to the measurement step).

[0396] [Step to obtain the liquid of the first c] In the step to obtain the liquid of the first c, the liquid of the first c is obtained, which contains a target substance 3c in the sample liquid, a luminescent particle 2c having a site 11c of the first c that specifically reacts with the target substance 3c, and a compound 4c of the first c having a site 12c of the second c that specifically reacts with a site (site Yc) different from the site (site Xc) of the target substance 3c that reacts with the luminescent particle 2c, and a site 13c of the third c having a temperature-responsive polymer 18c.

[0397] In the step of obtaining the liquid 1c of the first c, as shown in Figure 14C, it is sufficient to form the first c complex 16c (a sandwich structure of luminescent particles - target substance - compound of the first c) which is a composite of luminescent particles 2c, target substance 3c, and compound of the first c 4c. That is, it may be a step of mixing a sample solution containing the target substance 3 with the luminescent particles 2c and compound of the first c 4c, or, for example, a sample solution containing the target substance 3c and a reagent solution containing luminescent particles 2c and compound of the first c 4c may be mixed in two steps to form the hydrophilic first c complex 16c in the liquid. Furthermore, as shown in Figure 14D, for example, the step of obtaining the first liquid 1c has two sub-steps. In the first sub-step, a compound 5c of the third c having a second c site 12c that reacts with the target substance 3c and a fourth c site 14c that is different from the second c site 12c, the target substance 3c, and luminescent particles 2c are mixed to prepare a mixed liquid A22c that forms a subcomplex 21c of the compound 5c of the third c, the target substance 3c, and luminescent particles 2c. Subsequently, in the second sub-step, the mixed liquid A22c is mixed with a compound 6c of the fourth c having a fifth c site 15c that reacts with the fourth c site 14c and a third c site 13c that has a temperature-responsive polymer 18c. This is the case when, for example, the 4th c site 14c and the 5th c site 15c are reacted to prepare a mixed solution B1c (liquid 1c of the 1st c site) which forms the 1st c complex 16c, a composite of the luminescent particle 2c, the target substance 3c, and the 1st c compound 4c. In such a case, the 3rd c compound 5c, which has a smaller molecular weight than the 1st c compound 4c, reacts with the target substance 3c, and then the 4th c compound 6c reacts with it to form the 1st c compound 4c, thereby increasing the probability of reaction with the target substance 3 in the liquid. This is because the smaller molecular weight leads to faster Brownian motion in the liquid, increasing the probability of reaction with the target substance 3c within a specific time before reaching equilibrium. Examples of combinations of the 4th c site 14c and the 5th c site 15c include one site having biotin and the other site having avidin.Furthermore, it is preferable that the dissociation constants of site 14c of the fourth c and site 15c of the fifth c are smaller than the dissociation constant of site 12c of the second c and target substance 3c (it is also preferable that the binding constant of site 14c of the fourth c and site 15c of the fifth c is larger than the binding constant of site 12c of the second c and target substance 3c).

[0398] The concentration of luminescent particles 2c in the liquid 1c of the first c is preferably 0.000001% to 1% by mass, more preferably 0.00001% to 0.01% by mass. Here, the concentration of compound 4c of the first c (including compound 4c of the first c contained in the complex 16c of the first c) in the liquid 1c of the first c is preferably about 10 to 10,000,000 times the concentration of the estimated target substance 3c. If the concentration of the estimated target substance 3c in the liquid 1c of the first c is low, it is desirable to use a higher concentration.

[0399] Next, a liquid of the second c is obtained, which contains the first c complex 16c and the second c compound 20c having the third c site 13c. Here, the liquid of the second c may be the same as the liquid of the first c 1c, or the second c liquid may be obtained by further adding the second c compound 20c to the liquid of the first c 1c. Furthermore, the second c compound 20c may be one of the first c compounds 4c present in the liquid of the first c that did not react with the target substance 3c, or it may be a different compound from the first c compound 4c as long as it has the third c site 13c.

[0400] [Step to obtain the liquid of the third c] In the step to obtain the liquid of the third c, 19c, the liquid of the third c is obtained by changing the temperature of the liquid of the second c, which contains the composite of the first c, 16c and the compound of the second c, 20c having the part of the third c, 13c (for example, by heating it to a temperature above the lower critical solution temperature), thereby changing the physical properties of the temperature-responsive polymer 18c from hydrophilic to hydrophobic.

[0401] As the temperature of the liquid in the second c changes, the properties of the temperature-responsive polymer 18c in the third c portion 13c change from hydrophilic to hydrophobic, as shown in Figure 15. Therefore, a composite 17c of the second c is formed, which includes an aggregate 7c of multiple compounds 20c of the second c formed in the liquid by hydrophobic bonds (which can also be called hydrophobic interactions) between the temperature-responsive polymers.

[0402] Furthermore, the third c portion 13c, which has a temperature-responsive polymer 18c that forms an aggregate 7c by hydrophobic interaction, includes the temperature-responsive polymer 18c of the third c portion 13c contained in compound 4c of the first c among the first c composite 16c (a composite of luminescent particles, target substance, and substance of the first c), and the temperature-responsive polymer 18c of the third c portion 13c possessed by compound 20 of the second c (as mentioned above, compound 20c of the second c may also be compound 4c of the first c) that exists in a free state (as a single element) in the liquid.

[0403] The proportion of luminescent particles 2c contained in the secondc complex 17c depends on the proportion of the firstc complex 16c formed in the process of obtaining the firstc liquid 1c, i.e., the amount of target substance 3c. As a result, when the polarization anisotropy <r> of the thirdc liquid 19c is obtained in the measurement process described later, a change in the value related to fluorescence polarization according to the amount of target substance 3c can be confirmed. This makes it possible to achieve both high reactivity with the target substance 3c and large values ​​related to fluorescence polarization, such as polarization anisotropy <r> and fluorescence polarization degree, obtained in the measurement process described later, even when the target substance 3c is present in trace amounts in the thirdc liquid 19c, and to detect the target substance 3c with high sensitivity. In other words, because the secondc complex 17c becomes a large reactant, the amount of change in the value related to fluorescence polarization also becomes large, so the target substance 3c can be detected with high sensitivity.

[0404] Furthermore, the second complex 17c is naturally larger than the luminescent particle 2c, and preferably at least twice the size of the luminescent particle 2c. Considering the sedimentation of the aggregate 7c and the magnitude of the change in polarization anisotropy, the second complex 17c is preferably 200 nm to 6000 nm, and more preferably 1 μm to 6 μm. When polarization anisotropy <r> is used as a value related to fluorescence polarizability, the magnitude of polarization anisotropy <r> depends on the rotational relaxation time and luminescence lifetime of the luminescent reagent. For example, in the case of luminescent particle 2c using a europium complex, if the size of the second complex 17c is 1 μm or more, the polarization anisotropy <r> of the second complex 17c will theoretically well exceed the maximum value.

[0405] To increase the size of the secondc complex 17c, a compound having a thirdc moiety 13c with a temperature-responsive polymer 18c can be used as the secondc compound 20c. In this case, the firstc compound 4c, which exists alone in the firstc liquid 1c without contributing to the formation of the firstc complex 16c, and the secondc compound 20c, which is added to the firstc liquid 1c, will aggregate as the thirdc moieties 13c combine with each other through a process of changing the temperature of the secondc liquid.

[0406] To increase the formation rate and size of the 2c composite 17c, salt can be added to the sample solution. There are no specific requirements for the salt to be added, but for example, if the temperature change of the 2c liquid is due to heating, adding a salt such as sodium chloride allows for efficient dehydration from the temperature-responsive polymer 18c during heating in the process of obtaining the 3c liquid 19c.

[0407] It is preferable to perform the step of obtaining the third liquid 19c after the step of obtaining the first liquid 1c, but the steps of obtaining the first liquid 1c and obtaining the third liquid 19c may be performed simultaneously in order to shorten the measurement time. However, if performed simultaneously, the temperature of the sample solution may reach the LCST before the first composite 16c is sufficiently formed, and if the second composite 17c is formed by the step of obtaining the third liquid 19c, the measurement sensitivity may decrease.

[0408] [Measurement Step] In the measurement step, a value relating to the fluorescence polarization of the liquid 19c of the third c is obtained. Here, the value relating to fluorescence polarization may be polarization anisotropy <r> or fluorescence polarization degree. There are no particular restrictions on the measurement conditions as long as the temperature of the sample liquid is LCST or higher, for example, it is preferable that the liquid is at a temperature of 0 to 50°C and the viscosity of the liquid is 0.5 to 50 mPa·s. It is preferable that the concentration of the luminescent particles 2c be measured at 0.0001 mg / mL to 0.1 mg / mL, and that the detection wavelength is 500 to 700 nm. The measurement step may be performed simultaneously with the step of obtaining the liquid 19c of the third c. By measuring simultaneously with the step of obtaining the liquid 19c of the third c, it is possible to measure the value relating to fluorescence polarization immediately after the reaction (e.g., polarization anisotropy <r0>) and the value relating to fluorescence polarization after a certain reaction time (e.g., polarization anisotropy <r1>). By measuring these differences (for example, the difference between <r1> and <r0>) and the rate of change over time (for example, dr / dt), and comparing them with a standard sample, it becomes possible to measure the concentration of the target substance 3c in the sample solution.

[0409] [Method for Manufacturing Luminescent Particles] Next, an example of a method for manufacturing luminescent particles 2c used in the detection method and test kit of this embodiment will be described. In the following example, a europium complex is used as the luminescent molecule 8c, styrene and siloxane particles are used as the substrate 9c, and a capture agent of the target substance 3c is used to form the first c portion 11c that reacts with the target substance 3c to form the luminescent particles 2c.

[0410] A method for producing luminescent particles 2c includes a step (Step A) of mixing a radical polymerizable monomer containing at least styrene and a radical polymerizable organosilane, a radical polymerization initiator, a polarizing luminescent europium complex, and a hydrophilic polymer with an aqueous medium to prepare an emulsion; a step (Step Bc) of heating the emulsion to polymerize the radical polymerizable monomer; and a step (Step Cc) of introducing a functional group for the first c moiety 11c, which will be described later, onto the surface of the luminescent particles 2c.

[0411] Here, the functional group for introducing the first c moiety 11c is a functional group to which the first c moiety 11c can be attached, and specifically, any of the following can be used: a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, or an alkoxysilyl group (silicone alkoxide structure).

[0412] (Radical Polymerizable Monomers) The luminescent particles 2c are produced by polymerizing a radical polymerizable monomer, which comprises at least styrene and a radical polymerizable organosilane. The radical polymerizable monomer may further include monomers selected from the group consisting of acrylate monomers and methacrylate monomers. Examples of monomers include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, and mixtures thereof. That is, one or more of these monomers can be used in addition to styrene and the radical polymerizable organosilane. A monomer having two or more double bonds in a single molecule, such as divinylbenzene, may also be used as a crosslinking agent.

[0413] By including radically polymerizable organic silanes in the radically polymerizable monomer, siloxane bonds are imparted to the substrate 9c of the luminescent particle 2c. Examples of radically polymerizable organic silanes include vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, or combinations thereof. By using radically polymerizable organic silanes, an inorganic oxide skeleton is formed within the substrate 9c, which improves the physical and chemical stability of the luminescent particle 2c. Furthermore, by using radically polymerizable organic silanes, the affinity between the substrate 9c and the functional groups for introducing the hydrophilic layer 10c and the first c moiety 11c is increased.

[0414] Furthermore, the inclusion of a radically polymerizable organic silane in the radically polymerizable monomer imparts silanol groups to the surface of the substrate 9c. The silanol groups form hydrogen bonds with hydrophilic polymers, such as PVP. As a result, hydrophilic polymers such as PVP are more strongly adsorbed onto the surface of the substrate 9c.

[0415] (Radical polymerization initiators) Radical polymerization initiators can be widely used from azo compounds, organic peroxides, etc. Specifically, examples include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionic acid) dimethyl, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), potassium persulfate (KPS), etc.

[0416] (Hydrophilic Polymer) The luminescent particles 2c may include a hydrophilic polymer as the hydrophilic layer 10c. The hydrophilic polymer preferably suppresses nonspecific adsorption. Examples of hydrophilic polymers include hydrophilic polymers containing units having ethers, betaines, pyrrolidone rings, etc. The hydrophilic layer 10c is contained in the synthesized luminescent particles 2c and preferably exists mainly on the surface of the substrate 9c on which the luminescent particles 2c are located. Particles on which the hydrophilic layer 10c is formed on the surface of the substrate 9c may also be called substrate particles. By adding PVP during the synthesis of the luminescent particles 2c, it is possible to simultaneously impart nonspecific adsorption suppression ability and binding ability of sites that react with target substance 3c to the luminescent particles 2c. Since the PVP added during synthesis has higher hydrophilicity than radical polymerizable monomers, it is present at the interface between the solvent and the substrate 9c of the luminescent particles 2c during polymerization. The substrate 9c of the luminescent particle 2c adsorbs PVP to its outer surface through physical and chemical adsorption, such as partially incorporating PVP during polymerization and interactions between the pyrrolidone ring and styrene (a radically polymerizable monomer).

[0417] The molecular weight of PVP is preferably 10,000 or more and 100,000 or less, more preferably 40,000 or more and 70,000 or less. If the molecular weight is less than 10,000, the hydrophilicity on the surface of the luminescent particles is weak, and non-specific adsorption is likely to occur. If the molecular weight is greater than 100,000, the hydrophilic layer 10c becomes too thick and gels, making it difficult to handle. In addition to PVP, a hydrophilic polymer may be added as a protective colloid during the synthesis of the base material 9c of the luminescent particles 2c.

[0418] Also, the luminescent particles 2c preferably satisfy A2 - A1 ≤ 0.1. At this time, A1 and A2 are defined as follows. The absorbance immediately after the addition of 30 μL of a 0._{1}% by mass dispersion of the luminescent particles 2 to 60 μL of a buffer solution mixed with 16 μL of 15-fold diluted human serum is defined as A1, and the absorbance after leaving it at 37 °C for 5 minutes after the addition is defined as A2. The absorbance is measured with an optical path of 10 mm and a wavelength of 572 nm. The luminescent particles 2c for which A2 - A1 is 0.1 or less are preferable because non-specific adsorption of impurities in the serum is small.

[0419] (Aqueous medium) The aqueous medium (aqueous solution) used in the method for producing the above-described luminescent particles 2c preferably contains 80% by mass or more and 100% by mass or less of water in the medium. The aqueous solvent is preferably water or an ...

Claims

A method for detecting a target substance in a sample solution, A step of mixing a sample solution that may contain the target substance, a luminescent reagent having a first site that specifically binds to the target substance, and a first compound having a second site that specifically binds to a site in the target substance different from the site that specifically binds to the luminescent reagent, and a third site different from the second site, to form a first complex in a first liquid; A step of forming a second complex in a second liquid, comprising a second compound having a fourth site that binds to the third site, an aggregate formed by the binding of the third site and the fourth site together, and the first complex; A step of measuring a value relating to the fluorescence anisotropy of the second liquid, A method for detecting a target substance, characterized by having the following features. 　 The method for detecting a target substance according to claim 1, wherein the second complex is larger than the first complex. 　 The method for detecting a target substance according to claim 1 or 2, wherein the second complex is at least twice the size of the first complex. 　 A method for detecting a target substance according to any one of claims 1 to 3, further comprising the step of adding a third compound for forming the aggregate between the step of forming the first complex in the first liquid and the step of forming the second complex in the second liquid. 　 The method for detecting a target substance according to claim 4, wherein the third compound is the same compound as the second compound. 　 A method for detecting a target substance according to any one of claims 1 to 5, wherein the third portion and the fourth portion are the same portion. 　 The first compound is a compound formed by the bonding of a fourth compound having a second site and a fifth site different from the second site, and a fifth compound having a sixth site that specifically binds to the fifth site and the third site. The step of forming the first composite in the first liquid is A first sub-step for forming a subcomplex having the target substance, the luminescent reagent, and the fourth compound, A method for detecting a target substance according to any one of claims 1 to 6, comprising a second sub-step of mixing a liquid containing the sub-complex obtained in the first sub-step with the fifth compound to obtain the first complex having the sub-complex and the fifth compound. 　 The method for detecting a target substance according to claim 7, wherein the dissociation constant between the fifth and sixth regions is smaller than the dissociation constant between the second region and the target substance. 　 A method for detecting a target substance according to claim 7 or 8, wherein one of the fifth and sixth sites is a site containing avidin and the other is a site containing biotin. 　 A method for detecting a target substance according to any one of claims 1 to 9, wherein the bond between the third and fourth sites is an ionic bond, a covalent bond, an affinity bond, or a hydrophobic bond. 　 The method for detecting a target substance according to claim 10, wherein the bond between the third and fourth portions is a hydrophobic bond. 　 The method for detecting a target substance according to claim 11, wherein the third portion contains a temperature-responsive polymer, and the second composite is formed by heating the first liquid. 　 A target substance detection kit that detects a target substance in a sample solution by obtaining values ​​related to fluorescence polarization, A target substance testing kit comprising: a luminescent reagent having a first site that specifically binds to the target substance; a first reagent containing a first compound having a second site that specifically binds to a site in the target substance different from the site that specifically binds to the luminescent reagent, and a third site different from the second site; and a second reagent containing a second compound having a fourth site that binds to the third site. 　 The target substance testing kit according to claim 13, further comprising a third reagent containing a third compound for promoting binding via the third site. 　 The target substance testing kit according to claim 14, wherein the third compound is the same compound as the second compound. 　 A target substance testing kit according to any one of claims 13 to 15, wherein the third part and the fourth part are the same part. 　 A target substance testing kit according to any one of claims 13 to 16, wherein the first compound is a compound formed by the combination of a fourth compound having a second site and a fifth site different from the second site, and a fifth compound having a sixth site that specifically binds to the fifth site and the third site. 　 A target substance testing kit according to claim 17, wherein one of the fifth and sixth regions is a site containing avidin and the other is a site containing biotin. 　 A test kit for a target substance according to any one of claims 13 to 18, wherein the bond via the third site is an ionic bond, a covalent bond, an affinity bond, or a hydrophobic bond. 　 The target substance testing kit according to claim 19, wherein the bond via the third site is a hydrophobic bond.

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