Method for detecting target substances and test kit for target substances

Abstract

This invention provides a method for detecting target substances in a sample with high sensitivity, and a test kit for detecting target substances. [Solution] 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; 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.

Description

Technical Field

[0006] , ,

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

Background Art

[0002] In the fields of medicine and clinical examination, it is necessary to detect or quantify trace amounts of biological components from blood, a part of a collected organ, etc. with high sensitivity in order to investigate the cause and presence or absence of diseases. In recent years, among the methods for examining biological components, immunoassay is widely used. In many immunoassays, a washing step called B / F (Bound / Free) separation is required. One of the immunoassays that do not require B / F separation is the latex agglutination method and the fluorescence polarization method. In particular, the fluorescence polarization method is a method capable of highly sensitive detection based on the measurement principle.

[0003] Patent Document 1 discloses a method for quantifying a high molecular weight substance using a material in which a latex particle adsorbs a dye having long-lived luminescence characteristics as a luminescent material.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, in the field of clinical examination, a method for detecting a target substance and a test kit for the target substance that can detect with higher sensitivity are desired.

Means for Solving the Problems

[0006] One embodiment of the present disclosure is a detection method for detecting at least one of the presence and concentration of a target substance in a sample solution, comprising A step of obtaining a first liquid comprising 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; A step of obtaining a second liquid comprising a second complex having the first complex and an aggregate in which a plurality of the third sites of a second compound are bonded via the third sites, A step of measuring a value relating to the fluorescence polarization anisotropy of the second liquid, This is a method for detecting a target substance that has [certain properties].

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

[0008] Another embodiment of this disclosure is: A test kit that detects at least one of the presence or concentration of a target substance in a sample solution by obtaining a value related to fluorescence polarization anisotropy, A first reagent comprising: 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 specifically reacts with the luminescent reagent; and a first ion having a site that has repeating units including an ionic functional group; This is a target substance testing kit comprising a second reagent containing a second ion having a charge opposite to that of the aforementioned ionic functional group.

[0009] Another embodiment of the present disclosure is a test kit that detects at least one of the presence or concentration of a target substance in a sample solution by obtaining a value relating to fluorescence polarization anisotropy, 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 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 containing an ionic functional group, A second reagent containing a second ion having a charge opposite to that of the aforementioned ionic functional group, This is a test kit for target substances that have [specific characteristics / features].

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

[0011] Another embodiment of this disclosure is: A target substance test kit that detects at least one of the presence or concentration of a target substance in a sample solution by obtaining values ​​related to polarization anisotropy, A target substance testing kit characterized by comprising: a luminescent reagent having a first site that specifically reacts with the target substance; 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 different from the second site; a second compound having a plurality of fourth sites capable of forming 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 this disclosure is A method for detecting a target substance, which detects at least one of the presence or absence and concentration of the target substance in a sample solution, The target substance and, A step of obtaining a first liquid comprising a first complex having 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 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 step of 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, a third compound having a plurality of fifth sites that bind to the fourth site, and the first complex, A step of obtaining a value relating to the fluorescence polarization of the second liquid, This is a method for detecting a target substance that has [certain properties].

[0013] Another embodiment of this disclosure is: A target substance test kit that detects at least one of the presence or concentration of a target substance in a sample solution by obtaining a value related to fluorescence polarization, 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 different from the site that specifically reacts with the luminescent reagent among the target substances, and a third site different from the second site; A second compound having a plurality of fourth sites that bind to the third site; A third compound having a plurality of fifth sites that bind to the fourth site; It is a test kit for a target substance containing the above.

[0014] One embodiment of the present disclosure is A detection method for detecting at least one of the presence and concentration of a target substance in a sample solution, comprising: 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 among the target substances and a third site having a temperature-responsive polymer; By changing the temperature of a second liquid containing the first complex and a plurality of second compounds having the third site, obtaining a third liquid containing a second complex having the first complex and an aggregate of the second compounds; Obtaining a value related to the fluorescence polarization of the third liquid; It is a detection method for a target substance having the above.

[0015] Another embodiment of the present disclosure is A test kit for a target substance that detects at least one of the presence and concentration of a target substance in a sample solution by obtaining a value related to fluorescence polarization, comprising: A first reagent containing 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 among the target substances and a third site having a temperature-responsive polymer;

[0016] Another embodiment of the present disclosure is A target substance test kit that detects at least one of the presence or concentration of a target substance in a sample solution by obtaining a value related to fluorescence polarization, The test kit for a target substance comprises: 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. [Effects of the Invention]

[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. [Brief explanation of the drawing]

[0018] [Figure 1A] This is a schematic diagram illustrating an example of luminescent particles and a target material in one embodiment of the present disclosure. [Figure 1B] This is a schematic diagram illustrating an example of a first ion in one embodiment of the present disclosure. [Figure 1C] This is a schematic diagram illustrating an example of composite A in one embodiment of the present disclosure. [Figure 1D] This is a schematic diagram illustrating an example of a third ion in one embodiment of the present disclosure. [Figure 1E] This is a schematic diagram illustrating another example of composite A in one embodiment of the present disclosure. [Figure 2] This is a schematic diagram illustrating an example of composite B in one embodiment of the present disclosure. [Figure 3] This is a diagram illustrating the steps of a method for detecting a target substance according to one aspect of the present disclosure. [Figure 4A] This is a diagram illustrating luminescent particles and target material. [Figure 4B] This is a diagram illustrating compound 1a. [Figure 4C]This diagram illustrates the process of obtaining the mixed solution in step 1a. [Figure 4D] This is a diagram illustrating compound 2a. [Figure 4E] This diagram illustrates the case where the 1a process for obtaining the mixed solution includes sub-processes. [Figure 5A] This diagram illustrates the process of obtaining the mixed solution in step 2a. [Figure 5B] This figure illustrates an example in which compound 4a is used in the mixture acquisition process of 2a. [Figure 6A] This diagram illustrates the case where the reaction used to form the cross-linked structure is an oxidation reaction. [Figure 6B] This diagram illustrates the second a step in obtaining the mixed solution when the reaction used to form the cross-linked structure is an oxidation reaction. [Figure 7A] This diagram illustrates the case where the reaction used to form the cross-linked structure is an aminoacyltransferase reaction. [Figure 7B] This diagram illustrates the second a step in obtaining the mixed solution when the reaction used to form the cross-linked structure is an aminoacyltransferase reaction. [Figure 8] This diagram illustrates the inspection kit related to this disclosure. [Figure 9A] This is a diagram illustrating the steps of a method for detecting a target substance according to one aspect of the present disclosure. [Figure 9B] This is a diagram illustrating the steps of a method for detecting a target substance according to one aspect of the present disclosure. [Figure 10A] This diagram illustrates the process of obtaining the liquid in step 1b. [Figure 10B] This diagram illustrates the process of obtaining liquid 2b. [Figure 11] This diagram illustrates the flow from detection to measurement of a target substance according to the embodiment of this disclosure. [Figure 12A] This diagram illustrates a target substance testing kit related to this disclosure, specifically one composed of three types of solutions. [Figure 12B] This diagram illustrates a case where the target substance testing kit related to this disclosure is composed of two types of solutions. [Figure 13A] This is a diagram illustrating the steps of a method for detecting a target substance according to one aspect of the present disclosure. [Figure 13B] This is a diagram illustrating the steps of a method for detecting a target substance according to one aspect of the present disclosure. [Figure 14A] This is a diagram illustrating luminescent particles and target material. [Figure 14B] This is a diagram illustrating compound 1c. [Figure 14C] This diagram illustrates the process of obtaining the liquid in the first case (c). [Figure 14D] This diagram illustrates the case where the process of obtaining the liquid in step 1c includes sub-steps. [Figure 15] This diagram illustrates the process of obtaining the liquid in the third case. [Figure 16A] This is a diagram illustrating the test kit related to this disclosure. [Figure 16B] This diagram illustrates the case in which the test kit related to this disclosure includes reagent 2c and reagent 3c. [Modes for carrying out the invention]

[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, 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, This is a method for detecting a target substance that has [certain properties].

[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 following describes in detail a first embodiment, which is an example of one embodiment of the present disclosure, but this does 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. This is a method for detecting a target substance that has [certain properties].

[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 through bonding via the third site may be by at least one of the following types of bonds: 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 (an ionic site, a thiol group, an amino group, a group having an antibody or antigen, a group having biotin or avidin, or a hydrophobic functional group).

[0027] Furthermore, the "multiple second compounds having a third site" that form the aggregate may be the first compound that has not reacted with the target substance, a compound that was added to the first liquid between the step of obtaining the first liquid and the step of obtaining the second liquid, or both. In other words, the second compound may be the first compound, a compound different from the first compound, or both.

[0028] In the method for detecting a target substance of this embodiment, after a first complex is formed in the step of obtaining a first liquid, an aggregate (which may also be called a crosslinked structure, crosslinked body, or aggregate) is formed via a third site in the first complex and the third sites of a plurality of second compounds, thereby forming a second complex. Between the step of obtaining the first liquid and the step of obtaining the second liquid, there may be a step of adding a third compound to form the aggregate. For example, if the bond via the third site is an ionic bond, the third site may be an anionic site and the third compound may be a cation. For example, the third site may contain an ion derived from at least one of alginic acid, carboxymethylcellulose, pectin, carrageenan, cellulose sulfate, chondroitin sulfate, DNA, and polyvinylphosphonic acid. Furthermore, for example, the cation may include a cationic polymer containing at least one ion derived from Ca2+, Ba2+, Pb2+, Cu2+, Cd2+, Sr2+, Co2+, Ni2+, Zn2+, Mn2+, Al3+, and 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 allows for a significant change (increase) in the value related to fluorescence polarization anisotropy.

[0030] The size of the first complex is a value related to fluorescence polarization anisotropy. <r>There are no particular limitations as long as the value does not saturate. If the luminescent reagent has luminescent particles, if it is of a similar magnitude to the luminescent particles, the value related to fluorescence polarization anisotropy will be significantly reduced when the process of obtaining the second liquid is carried out. <r>This can be changed. As a result, the value related to fluorescence polarization anisotropy can be changed. <r>This allows for a greater increase in the amount of change before and after the reaction, thereby improving measurement sensitivity.

[0031] Therefore, specifically, the size of the first complex is preferably about the same as the size of the luminescent particles if 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] Note: Values ​​related to fluorescence polarization anisotropy <r>The size 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 more, the value related to the fluorescence polarization anisotropy of the second complex <r>The theoretical value far exceeds the maximum value.

[0033] In the following embodiments, luminescent particles are used as the luminescent reagent. Below, examples of each step (first liquid acquisition step to measurement step) of a method for detecting a target substance when the aggregate is formed by ionic bonding (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, A step of obtaining a liquid A comprising the target substance, a luminescent reagent having a site A that specifically reacts with the target substance, and a first ion having 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, A step of obtaining a liquid B containing the liquid A and a second ion having a charge opposite to that of the ionic functional group, A step of measuring a value relating to the fluorescence polarization anisotropy of the liquid B, This can be a method for detecting a target substance characterized by having the following features.

[0035] In this disclosure, "luminescent reagent having a first site that specifically binds to the target substance" corresponds to "luminescent reagent having site A that specifically reacts with the 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] <Process for obtaining the first liquid> Figure 1A is a schematic diagram showing an example of luminescent particles and target material, 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 of obtaining the first liquid (also referred to as liquid A) (hereinafter sometimes referred to as the first liquid acquisition step), the first liquid is obtained by mixing the sample solution containing the target material 3, the luminescent particles 2, and the first ion 4.

[0037] The complex A shown in 1 (hereinafter sometimes referred to as complex A1) is a complex 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 substrate 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-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 (indicated as 14 in Figures 1A-1E; hereafter sometimes referred to as site B14) that specifically reacts with site Y, which is different from site X that reacts with the luminescent particle 2 of the target substance 3, and an ionic site 12. Here, the ionic site 12 is a site having a repeating unit with 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 with 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 and a third site that specifically react with the fourth 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 set to be 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 site B14 and site C (shown as 15 in Figures 1D and 1E; hereafter sometimes referred to as site C15), which is different from site B14. The third ion 42 has site D (shown as 16 in Figures 1D and 1E; hereafter sometimes referred to as site D16), which specifically reacts with site C15, and site 12, which has ionic properties. 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 the first liquid (liquid A) has two sub-steps. In the first sub-step, target substance 3, luminescent particles 2, and compound A41 are mixed to form complex C (shown as 17 in Figure 1E; hereafter sometimes referred to as complex C17), which is a complex (subcomplex) of target substance 3, luminescent particles 2, and compound A41. Subsequently, in the second sub-step, the third liquid (also called liquid C) containing complex C17 formed in the first sub-step is mixed with a third ion 42 having repeating units containing ionic functional groups. This causes site C15 and site D16 to react, forming complex A1, which is a complex of complex C17 and the third ion 42 (a complex of target substance 3, luminescent particles 2, and the first ion 4).

[0044] In such cases, 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, thereby increasing the probability of reaction with the target substance 3 in the liquid. 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 for the 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 sites C15 and 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] <Process for obtaining the second liquid> In the step of obtaining the second liquid (hereinafter sometimes referred to as the second liquid acquisition step, or the second composite formation step), the second liquid is obtained by mixing the first liquid with a second ion having a charge opposite to that 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 repeating units of ionic functional groups that have a charge opposite to that of the ionic functional groups in the repeating units containing ionic functional groups 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, which has 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 complex B (shown as 13 in Figure 2; hereafter sometimes referred to as complex B13) which contains complex A1, the target substance and the unreacted first ion 4, and the second ion 6.

[0049] In this case, complex A1 is included in the compound having multiple ionic sites 12, 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 in the compound having other ionic sites 12 (for example, the first ion 4 that exists in a free state unreacted with the target substance), 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. In the measurement step described later, the value relating to the fluorescence polarization anisotropy of the second liquid is <r>By obtaining this data, it is possible to observe the change in the value related to fluorescence polarization anisotropy depending on the abundance of target substance 3.

[0051] The detection method according to this embodiment allows for the detection of high reactivity between the luminescent particles 2 and the target substance 3, and the change in the value of fluorescence polarization anisotropy obtained in the measurement step described later. <r>This makes it possible to increase the value of the target substance 3 even when it is present in trace amounts in the second liquid, and it is possible to detect the target substance 3 with high sensitivity. In other words, as complex B becomes a large structure, the value related to fluorescence polarization anisotropy becomes larger. <r>Because the amount of change is also large, the target substance can be detected with high sensitivity.

[0052] As mentioned 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, if alginate ions, which are obtained by the dissociation of sodium alginate in liquid, are used as the first ion 4, and a solution of alginate ions with a concentration of 1000 pM is used, it is preferable that the concentration of calcium ions be 5 mM or higher. In this case, the carboxylate group (carboxylate ion, COO-) in sodium alginate becomes approximately 0.2 mM, and calcium ions (Ca2+) are added at approximately 25 times the 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 likelihood 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 target substances such as site B. In such a case, for example, the fourth ion or an electrolyte that ionizes to generate the fourth ion may be added as an auxiliary in the second liquid acquisition step. 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 relating to the fluorescence polarization anisotropy of the second liquid. <r>The following is measured. For example, the temperature of the second liquid is preferably 0 to 50°C and the viscosity is preferably 0.5 to 50 mPa·s. Furthermore, it is preferable to measure 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 step may be performed simultaneously with the second liquid acquisition step. By performing the measurement step simultaneously with the second liquid acquisition step, the value related to the fluorescence polarization anisotropy immediately after the reaction can be measured. <r0>and the value relating to fluorescence polarization anisotropy after a certain reaction time. <r1>It can measure this. Details will be explained later, but this <r1>and <r0>By measuring the difference or the rate 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 solution.

[0058] In the detection method according to this embodiment, the formation of an aggregate containing luminescent particles via ionic bonding determines the value related to fluorescence polarization anisotropy. <r>The design is such that the amount of change is large, enabling highly sensitive measurements.

[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 although the following description is based on the premise of quantitative evaluation (measurement of the amount of target substance), 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 solution containing a luminescent reagent can be observed as changes in polarized emission properties. 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 observed as a change in the value related to fluorescence polarization anisotropy, as a result of the 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. 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.

[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) Here, Q: Rotational relaxation time of luminescent reagent V: Volume of the luminescent reagent η: viscosity of the solvent k: Boltzmann constant T: Absolute temperature That is the case.

[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) Here, p0: Polarization degree when the luminescent reagent is stopped (Q=∞) p: Polarization degree A: Constant τ: Luminescence lifetime of luminescent reagent Q: Rotation relaxation time That is the case.

[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 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, the ratio of I⊥ / I∥ measured with excitation light that has a vibration direction 90 degrees different from the excitation light used for sample measurement. That is the case.

[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, the luminescent reagent of this embodiment has a value related to fluorescence polarization anisotropy that can be determined by the following formula (1). <r>It is preferable that the polarization anisotropy is 0.01 or higher.

number

[0069] [Target substance testing kit] Next, a target substance testing kit using the above-described fluorescence polarization depolarization method will be described. The target substance testing kit according to this disclosure is a testing 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 containing 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 containing 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 to 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 A11 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. Below, using the example that the luminescent reagent is a luminescent particle as shown in the schematic diagram in Figure 1A, the various parts of the luminescent particle and the method for producing the luminescent particle will be described.

[0079] (base material) The substrate 9 of the base 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 preferable that the substrate is 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 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 mentioned above.

[0081] Examples of target substance 3 and ligand (site A11) combinations 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 DNA, RNA, cDNA, parts or fragments 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 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. In addition to the method of forming a hydrophilic layer 10 on the surface of the substrate 9 to suppress 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 luminescent 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 luminescent 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 molecule 8 contained in the substrate particle 7 of the luminescent particle 2 is preferably a rare earth complex, and more preferably a europium complex, considering the 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 a europium element and a ligand. 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 is obtained. The europium complex may also be a polynuclear complex.

[0087] Specific examples of europium complexes include tris(2-thenoyltrifluoroacetone)bis(triphenylphosphineoxide)europium(III), tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide)europium(III), and tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III).

[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 measurements, 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 between 25 nm and 500 nm, and more preferably between 50 nm and 300 nm. By setting the average particle diameter to between 50 nm and 300 nm, 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 where 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. Site B14, which reacts with target substance 3, has the same function as site A11 on the luminescent particle 2 that reacts with target substance 3, but it reacts with a different site (site Y) of target substance 3 than site A11 (site X) on the luminescent particle 2. In this embodiment, site B14 that reacts with target substance 3 can be an antibody or the like. When an antibody is used for site B14 that reacts with 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 reaction is specific via the target substance 3 because both site A11 on the luminescent particle 2 and site B14 on the first ion 4 specifically react with different sites on the target substance 3. Therefore, for example, if both site A11 that specifically reacts with the target substance 3 and site B14 that specifically reacts with the target substance 3 are antibodies, then different epitopes of the target substance 3 are recognized. Note that 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 ions derived from (or ionized in liquid) primary amines, secondary amines, tertiary amines, imidazolium salts, pyrrolidinium salts, pyridinyl groups (pyridinyl nitrogen), quaternary ammonium salts, and quaternary phosphonium salts, for example. 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] <The 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 ionic functional group charge of 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] When the first ion 4 has a negative charge, the second ion 6 can be a polyvalent ion such as Ca2+, Ba2+, Pb2+, Cu2+, Cd2+, Sr2+, Co2+, Ni2+, Zn2+, Mn2+, Al3+, a cationic polymer derived from primary amines, secondary amines, tertiary amines, structures containing nitrogen such as imidazolium groups, pyrrolidinium groups, pyridinyl groups, quaternary ammonium salts, or quaternary phosphonium salts, and amination polysaccharides. 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 detecting a target substance and a method for producing luminescent particles used in a 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 produce 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 monomer) The luminescent particles are produced by polymerizing a radically polymerizable monomer, which comprises at least styrene and a radically polymerizable organosilane. The radically 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 radically 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.

[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 radically polymerizable organic silanes 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 initiator) As radical polymerization initiators, a wide range of azo compounds and organic peroxides can be used. 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), and the like.

[0106] (Hydrophilic polymer) The luminescent particles may contain a hydrophilic polymer as a hydrophilic layer. The hydrophilic polymer preferably suppresses non-specific adsorption. Examples of hydrophilic polymers include those having repeating units such as ethers, betaines, and pyrrolidone rings. The hydrophilic layer is contained in the synthesized luminescent particles and preferably exists mainly on the surface of the substrate on which the luminescent particles are located. For example, by adding PVP during the synthesis of the substrate, it is possible to give the substrate surface non-specific 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. Furthermore, since PVP added during the synthesis of the substrate is more hydrophilic than radical polymerizable monomers, it exists at the interface between the solvent and the substrate during polymerization. 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, forming 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 an optical 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 nonspecific 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% to 100% by mass of water. The aqueous solvent is preferably water or a water-soluble organic solvent, and examples include solutions of methanol, ethanol, isopropyl alcohol, and acetone mixed with water. If the content of an organic solvent other than water exceeds 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 been pre-adjusted to a pH of 6 to 9. If the pH is less than 6 or greater than 9, the alkoxy 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. 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 manufacturing luminescent particles, it is preferable to first dissolve PVP in an aqueous medium whose pH has been adjusted to 6-9. 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. A mass ratio of 5:5 or higher suppresses significant aggregation of the generated luminescent particles. Furthermore, a mass ratio of 9.5:0.5 or lower ensures a sufficient amount of luminescent particles are 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% by mass and 10% by mass.

[0116] The step of heating the emulsion 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.

[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 around 3 to 14 hours at or below room temperature of around 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, one can select the optimal method for attaching the desired antibody or other substance by utilizing the functional groups present in the hydrophilic layer.

[0119] (Introduction of part A) The chemical reaction for chemically bonding a functional group capable of forming site A to 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 the electrolyte that generates the first ion used in this embodiment will be described. The method for producing the electrolyte that generates the first ion can be any method that imparts an ionic site, that is, a site having repeating units containing an ionic functional group (charged site), to a compound having a site B that reacts with a target substance, as long as the ligand activity 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 imparted 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> The following describes in detail an example of one embodiment of this disclosure, but this is not intended to limit the scope of this disclosure.

[0122] 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 liquid 1a containing a complex 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 complex of 2a having the complex of 1a and an aggregate in which a plurality of compounds 2a having the 3a sites are bound together 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 firsta / seconda.” “First compound” corresponds to “compound firsta.” “Second compound having a fourth site” corresponds to “compound seconda having a plurality of fourtha sites capable of forming covalent bonds with the thirda site” and “compound thirda for forming the covalent bond between the thirda site and the fourtha site.”

[0124] The formation of aggregates via bonding through site 3a may also 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 formation of the 1a complex in the step of obtaining the 1a liquid, an aggregate (which can also be called a crosslinked body or aggregate) is formed via the 3a portion of the 1a complex and the 3a portions of the multiple 2a compounds, thereby forming the 2a complex. Here, since the 2a complex has the structure of an aggregate containing the 1a complex, it is larger than the 1a complex. Therefore, in the step of obtaining the 1a liquid, it is possible to react the target substance with the 1a compound, which has a relatively small molecular weight, with high reactivity, and in the step of obtaining the 2a liquid, it is also possible to obtain the 2a 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 a value related to fluorescence polarization anisotropy. <r>There are no particular limitations as long as the value does not saturate. If the luminescent reagent has luminescent particles, if it is of a similar magnitude to the luminescent particles, the value related to fluorescence polarization anisotropy will be significantly reduced when the liquid in step 2a is obtained. <r>This can be changed. As a result, the value related to fluorescence polarization anisotropy can be changed. <r>This allows for a greater increase in the amount of change before and after the reaction, thereby improving measurement sensitivity.

[0127] Therefore, specifically, the size of the 1a complex is preferably about the same size as the luminescent particles if 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 2a complex is preferably at least twice the size of the luminescent particles (it may also be at least twice the size of the 1a complex). Considering the magnitude of sedimentation and anisotropy changes of the 2a complex, the 2a complex is preferably 200 nm to 6 μm, and more preferably 1 μm to 6 μm.

[0128] Note: Values ​​related to fluorescence polarization anisotropy <r>The size 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 more, the value relating to the fluorescence polarization anisotropy of the 2a complex <r>The theoretical value far exceeds 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 substances] The method for detecting a target substance in 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 to obtain a mixture of 1a (a step to obtain a mixture of 1a shown in S1001a), comprising: a target substance; a luminescent reagent having a site 1a that specifically reacts with the target substance; a compound 1a having a site 2a that specifically reacts with a site different from the site that reacts with the luminescent reagent; and a site 3a that is different from the site 2a. A step to obtain a mixture of 1a, a compound 2a having multiple 4a sites capable of forming covalent bonds with 3a sites, and a compound 3a for forming covalent bonds between 3a sites and 4a sites (a step to obtain a mixture of 2a shown in S1002a), A measurement step (shown in S1003a) for measuring the value related to the polarization anisotropy of the mixture of 2a.

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

[0132] As shown in Figure 4A, the target substance 3a in the sample solution binds to the luminescent particle 2a via the 1a site 11a. The luminescent particle 2a is not particularly limited as long as it has the 1a site 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 1a compound 4a via the 2a site 12a. In the figure, the site of the target substance 3a that binds to the 1a site 11a is shown as Xa, and the site that binds to the 2a site 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, 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 a mixture. The luminescent particles 2a and the first a compound 4a sandwich the target substance 3a, forming a so-called sandwich structure, and a mixture is prepared in which the first a composite 1a is formed.

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

[0136] <Step to obtain the mixture in 2a> Figures 5A and 5B illustrate the process of obtaining the mixture of 2a, which includes the composite 5a of 2a. While referred to as the mixture of 2a here, it can also be called the liquid of 2a. Furthermore, the process of obtaining the mixture of 2a can also be described as the process of forming 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 site 14a of the second a compound 6a is added to the first a mixture to form a covalent bond 30a with the third a site 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 site 13a and the fourth a site 14a, and the covalent bond 30a between the fourth a sites 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 1a, and the other types of compounds may be added as compound 3a 16a to prepare the mixture 2a, or multiple types of compounds may be added sequentially to prepare the mixture 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 complex 2a 5a for detecting the target substance 3a is prepared.

[0139] The size of composite 5a of 2a is preferably at least twice the size of composite 1a of 1a. Specifically, the size of composite 1a of 1a is preferably 5 nm to 500 nm, and the size of composite 5a of 2a is preferably 200 nm to 6000 nm.

[0140] In the method for detecting a target substance according to the above embodiment, (A) The 3a site 13a and the 4a site 14a have thiol groups, and the covalent bond 30a is a disulfide bond. Compound 6a of 2a contains any of the compounds selected from the group consisting of peroxidases, oxidizing agents, and compounds having a phenol group, or (B) Site 13a of 3a has a glutamine residue or a lysine residue, site 14a of 4a has a glutamine residue or a lysine residue, and the covalent bond 30a is an amide bond formed between the glutamine residue and the lysine residue. Compound 6a of 2a may contain 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] Site 11a and site 12a of site 2a 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, resulting in polarization anisotropy. <r>The design incorporates features that increase the size of the sensor, enabling highly sensitive measurements. The target substance detection method of this embodiment will be further explained using Figures 4A to 4E, and Figures 5A and 5B.

[0145] <Luminescent reagent> The luminescent reagent has a 1a site that reacts with the target substance. The luminescent reagent may be a luminescent molecule having a 1a 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 1a 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 1a 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 manufacturing method of 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] (base material) 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 site 11a described later can be imparted to the surface of the substrate 9a.

[0147] (The 1a site 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, and membrane proteins that specifically bind to them. Furthermore, examples include parts or fragments of DNA, RNA, or cDNA derived from bacteria, viruses, or cells, 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 onto 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 proteins 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 prone to 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. Among more specific examples of hydrophilic polymers, polymers having a pyrrolidone ring may be abbreviated as "PVP" in this specification.

[0151] The hydrophilic layer 10a preferably covers at least a portion of the surface of the substrate 9a, and more preferably 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 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 10a is less likely to become unstable due to hydration caused by ions in the solvent.

[0152] (Luminescent molecules) The luminescent molecule 8a contained in the luminescent particle 2a is preferably a rare-earth complex, and more preferably a europium complex, 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 equation (2a), which will be described later.

[0153] If 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 is obtained. The europium complex may also 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(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, be 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 more abundantly into the substrate 9a, 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 can 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> As shown in the schematic diagram of Figure 4B, compound 4a of the first a has a second a site 12a that specifically reacts with a site Y different from site Xa which specifically reacts with the luminescent particle 2a of the target substance 3a, and a third a site 13a which is different from site 12a of the second a. 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 which reacts with the target substance 3a, but it reacts with a site (site Ya) different from the site (site Xa) to which site 11a of the luminescent particle 2a reacts.

[0161] The 1a site 11a is, for example, a site where affinity bonding occurs with the target substance 3a. The 1a site 11a on the luminescent particle 2a and the 2a site 12a on the compound 4a of the 1a site both react specifically with different sites on the target substance 3a, thus reacting specifically via the target substance 3a. The 2a site 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 compound 1a is a site that forms a covalent bond 30a with the 4a site 14a of compound 6a of compound 2a, which will be described later, upon specific stimuli or chemical reactions, thereby forming an aggregate 17a (crosslinked structure).

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

[0164] When site 13a of 3a and site 14a of 4a are bonding functional groups, their chemical structure can be selected depending on the reaction used to form the crosslinked structure. For example, if the reaction used to form the crosslinked structure is an oxidation reaction, it is preferable that site 13a of 3a and site 14a of 4a (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] 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 6a of 2a has multiple 4a sites 14a that can form covalent bonds 30a with site 13a of 3a, as shown in Figure 4D. Because compound 6a of 2a has multiple 4a sites 14a, a crosslinked structure is formed by the covalent bond 30a between compound 4a of 1a and compound 6a of 2a, based on the bond between site 13a of 3a and site 14a of 4a. The hydrophilic polymer 15a of compound 6a of 2a shows a portion that connects to the multiple 4a sites 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 the hydrophilic polymer 15a has multiple 4a moieties 14a. However, a higher proportion 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 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 3a 16a forms a crosslinked structure via disulfide bonds between compound 1a 4a and compound 2a 6a, and also enables the formation of a crosslinked structure via disulfide bonds between the compounds 2a 6a. Compound 3a 16a preferably contains a peroxidase (HRP is exemplified in the example in Figure 6B), an oxidizing agent (H2O2 is exemplified in the example in Figure 6B), and a compound having a phenol group (thiamine is exemplified in the example in Figure 6B). The peroxidase is oxidized by hydrogen peroxide and functions as an oxidation catalyst. By utilizing this property, the thiol group is oxidized to form a crosslinked structure via disulfide bonds between site 3a 13a and site 4a 14a. This allows compound 1a 4a to be crosslinked to a part of the large aggregate 17a made up of compound 2a 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 that 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 site 14a) and a hydrophilic polymer 15a. In Figure 4D, compound 6a of 2a has multiple binding functional groups, the 4a site 14a, and a hydrophilic polymer 15a. In the step of obtaining the mixed solution of 2a, as shown in Figure 7B, compound 6a of 2a is bonded by the binding functional groups to form a composite 7a of compound 6a of 2a, and further bonded to compound 6a of 2a via the 4a site 14a to form an aggregate 17a, which is a larger measurement material. The bonding of compound 6a of 2a and the crosslinking reaction between the 4a site 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 16a of 3a, 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 (EC2.3.2.13; protein-glutamine:gamma-glutamyltransferase; protein-glutamine:amineγ-glutamyltransferase, CAS80146-85-6) can be any enzyme that functions as an isopeptide crosslinking catalyst for substrates having glutamine and lysine residues, but its origin is not limited. Examples include species of the genera Streptoperticillium, Streptomyces, Actinomadura, Bacillus (e.g., Bacillus circulans), and 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 is particularly suitable as compound 6a of the present disclosure because, when casein, gelatin, collagen, and keratin, which are hydrophilic proteins exemplified as having multiple sites 14a of 4a and multiple sites 13a of 3a, 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.

[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 1a: Acquisition of mixed solution] In the first a mixture acquisition step, a first a mixture is obtained which contains the first a complex 1a, which is a complex comprising 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 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 1a) to be formed between the luminescent particles 2a, the target substance 3a, and the compound 4a of the first a. For example, this could be a step in which a sample solution containing the target substance 3a is mixed with the luminescent particles 2a and compound 4a of the first a, or the sample solution and a reagent solution containing the luminescent particles 2a and compound 4a of the first a are mixed in two steps, and the complex is formed in the mixture.

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

[0178] As a sub-step of 1a, a 1a subcompound 20a having a 2a site 12a that reacts with the target substance 3a and a 5a site 18a that is different from the 2a site 12a, and not having a 3a site 13a that reacts with the 4a site 14a, is mixed with the target substance 3a and the luminescent particle 2a to form a subcompound 23a of the 1a subcompound 20a, the target substance 3a, and the luminescent particle 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 site 19a of 6a that reacts with site 18a of 5a and a site 13a of 3a (which reacts with site 14a of 4a). By reacting site 18a of 5a and site 19a of 6a to form compound 4a of 1a, a complex 1a of 1a is formed, which is a complex of the luminescent particle 2a, the 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 because the first a subcompound 20a, which has a smaller molecular weight than the first a compound 4a, reacts with the target substance 3a, and then the second a subcompound 21a reacts with it 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 site 18a of 5a and site 19a of 6a include a site containing avidin and a site containing biotin. Furthermore, it is preferable that the dissociation constant of site 18a of 5a and site 19a of 6a is smaller than the dissociation constant of site 12a of 2a and target substance 3a (the binding constant of site 18a of 5a and site 19a of 6a may be larger than the binding constant of site 12a of 2a and 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 approximately 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 with the luminescent reagent and target substance in the mixture acquisition step of 1a, regardless of whether it reacts with the luminescent reagent and target substance, in the mixture acquisition step of 2a due to the effect of site 13a of 3a and is incorporated into the composite 5a of 2a. On the other hand, in the mixture of 1a, it is possible to increase the probability of reaction with the target substance present in trace amounts by being present in large quantities in a free state in the liquid. As a result, even if the target substance is present in trace amounts, the polarization anisotropy of the mixture of 2a to be measured can be increased. <r>It can be made higher.

[0183] The concentration of compound 4a in the mixture of 1a should preferably be approximately 10 to 1,000,000 times the concentration of target substance 3a. If the concentration of 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 that reacts with the luminescent particle 2a, 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 below is the time for the site 11a of the first step and the site 12a of the second step 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 more than 90% of the reaction proceeds.

[0186] 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. The concentration of compound 4a of 1a in the mixture of 1a 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 2a, which has multiple 4a sites 14a capable of forming covalent bonds 30a with 3a sites 13a, in the mixture of 1a can be separated as a sub-step of the mixture acquisition step of 1a and performed immediately before or after the mixture acquisition step of 2a described later.

[0187] [Step 2a of obtaining mixed liquid] In the second a mixture acquisition step, 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, which forms a covalent bond 30a between the third site 13a and the fourth site 14a, are mixed, as shown in the schematic diagram in Figure 5B, a cross-linked 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 a single element) in the mixture and are capable of forming a covalent bond 30a with the third site 13a, and a large second complex 5a containing the cross-linked structure is formed.

[0189] The mixture of 2a may contain compound 16a of 3a to promote the covalent bond 30a between site 13a of 3a and site 14a of 4a. In such a case, compound 16a of 3a, which promotes the formation of the covalent bond 30a, may be present in the liquid, and the covalent bond between site 13a of 3a and site 14a may be promoted by mixing the liquid containing compound 16a of 3a with the mixture of 1a, or the covalent bond between site 13a of 3a and site 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 site 13a and site 14a. Furthermore, in the step of obtaining the mixture of 2a, compound 31a of 4a having site 13a may be included 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 enzyme-substrate reactions. A substance related to enzyme-substrate reactions is any substance related to enzyme-substrate reactions, and includes enzymes, substrates, or substances generated by the reaction of enzymes and substrates. Substances related to the reaction between peroxidase and substrates 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 in. 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 2a mixture acquisition step. In such an example, compound 16a of 3a may contain multiple types of substances related to the enzyme substrate reaction, and compound 16a of 3a may contain HRP and hydrogen peroxide, which is a compound that promotes the covalent bond between site 13a of 3a and site 14a of 4a when coexisting with HRP. In such a case, multiple types of substances related to the enzyme substrate reaction may be added to the 1a mixture separately for each type, and as a result, a 2a mixture containing compound 16a of 3a 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 compound 16a of the third a mixture is obtained to form a covalent bond 30a between site 13a of the third a mixture and site 14a of the fourth a mixture. The catalytic action of compound 16a of the third a mixture induces the formation of an isopeptide crosslinked structure between compound 4a of the first a mixture and compound 6a of the second a mixture, as well as the formation of a crosslinked structure by isopeptide bonds between the compounds 6a of the second a mixture, and compound 4a of the first a mixture is crosslinked to a large aggregate 17a made up of compounds 6a of the second a mixture.

[0193] The proportion of luminescent particles 2a contained in the 2a complex 5a in the 2a mixture depends on the proportion of the 1a complex 1a formed in the 1a mixture, i.e., the amount of target substance 3a. As a result, in the measurement process described later, the polarization anisotropy of the 2a mixture <r>By obtaining this data, it is possible to observe the change in the value related to polarization anisotropy depending on the amount of target substance 3a present.

[0194] This ensures high reactivity with target substance 3a and polarization anisotropy obtained in the measurement process described later, even when target substance 3a is present in trace amounts in the mixture of 2a. <r>It is possible to achieve both a large size and high sensitivity detection of the target substance 3a. In other words, the second a complex 5a becomes a large structure, <r>Since the amount of change is also large, the target substance 3a can be detected with high sensitivity.

[0195] Furthermore, the composite 5a of the second a is naturally larger than the luminescent particle 2a, and preferably more than twice the size of the luminescent particle 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, and more preferably 1 μm to 6 μm. <r>The size depends on the rotational relaxation time and luminescence lifetime of the luminescent particle 2a. For example, in the case of luminescent particle 2a using a europium complex, if the size of the 2a complex 5a is 1 μm or more, the polarization anisotropy of the 2a complex 5a <r>Theoretically, this significantly exceeds the maximum value. It is preferable that the 2a mixture acquisition step be performed after the 1a mixture acquisition step, but the 1a and 2a mixture acquisition steps may be performed simultaneously.

[0196] [Measurement process] In the measurement process, the polarization anisotropy of the 2a mixture <r>The following is measured. The measurement conditions are preferably, for example, in a liquid at a temperature of 0 to 50°C, with a viscosity of 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 mixed solution acquisition step 2a. By measuring simultaneously with the mixed solution acquisition step 2a, the polarization anisotropy immediately after the reaction can be measured. <r0>and polarization anisotropy after a certain reaction time <r1>This can be measured. <r1>and <r0>By measuring the difference or the rate of change over time (dr / dt) and comparing it with a standard sample, it becomes possible to measure the concentration of target substance 3 in the sample solution.

[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 goes without saying that although the following description is based on the premise of quantitative evaluation (measurement of the amount of target substance 3a), 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 of the luminescent reagent in a solution can be observed as changes in polarized emission properties. Specifically, when the site of the luminescent particle that reacts with the target substance 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 particle via the target substance can be observed as a change in the rotational Brownian motion of the luminescent particle, which is related to the 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. 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.

[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 light emission occurs. The rotational motion of the luminescent reagent can be expressed by the following equation (2a). Q = 3Vη / kT ···(2a) Here, Q: Rotational relaxation time of luminescent reagent V: Volume of the luminescent reagent η: viscosity of the solvent k: Boltzmann constant T: Absolute temperature That is the case.

[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 the 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) Here, p0: Polarization degree when the luminescent reagent is stopped (Q=∞) p: Polarization degree A: Constant τ: Luminescence lifetime of luminescent reagent Q: Rotation relaxation time That is the case.

[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 polarization degree 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 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) 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 that has a vibration direction 90 degrees different from the excitation light used for sample measurement. That is the case.

[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, the luminescent reagent of this embodiment has a value related to fluorescence polarization anisotropy that can be determined by the following formula (1a). <r>It is preferable that the polarization anisotropy is 0.01 or higher.

number

[0207] (Method for manufacturing 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 the luminescent particles 2a 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 1a moiety 11a described later to the surface of the luminescent particles 2a. Here, the functional group capable of forming the 1a moiety 11a is a functional group to which the 1a moiety 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 monomer) The luminescent particles 2a are produced by polymerizing a radically polymerizable monomer, the radically polymerizable monomer comprising at least styrene and a radically polymerizable organosilane. The radically 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 radically 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 conferred 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. 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 9a.

[0211] (Radical polymerization initiator) As radical polymerization initiators, azo compounds, organic peroxides, etc. can be widely used. Specifically, 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. can be mentioned.

[0212] (Hydrophilic polymer) The luminescent particle 2a can contain a hydrophilic polymer as the hydrophilic layer 10a. The hydrophilic polymer preferably suppresses non-specific adsorption. Examples of the hydrophilic polymer include hydrophilic polymers having an ether, betaine, pyrrolidone ring, etc. as repeating units (units). The hydrophilic layer 10a is preferably contained in the synthesized luminescent particle 2a and mainly exists on the surface of the substrate 9a that the luminescent particle 2a has. For example, by adding PVP when synthesizing the substrate 9a, it becomes possible to give the surface of the substrate 9a the ability to suppress non-specific adsorption and the ability to bind ligands, and by forming the first site 11a that specifically reacts with the target substance 3a on the luminescent particle 2a, a luminescent particle 2a having the ability to suppress specific adsorption can be obtained. Also, since PVP added during the synthesis of the substrate 9a is more hydrophilic than the radical-polymerizable monomer, it exists at the interface between the solvent and the substrate 9a during polymerization. By partially incorporating PVP during the polymerization of the substrate 9a or through physical and chemical adsorption such as the interaction between the pyrrolidone ring and styrene (radical-polymerizable monomer), PVP is adsorbed on the outside to form the hydrophilic layer 10a. In this specification, a polymer having a pyrrolidone ring may be abbreviated as "PVP".

[0213] 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 reagent is weak, and non-specific adsorption is likely to occur. If the molecular weight is greater than 100,000, the hydrophilic layer 10a 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 9a.

[0214] Also, A1 and A2, which are parameters related to the luminescent particles 2a, preferably satisfy A2 - A1 ≤ 0.1. A1 and A2 are defined as follows. That is, the absorbance immediately after adding 30 μL of a 0.1 mass% dispersion of the luminescent reagent to 60 μL of buffer 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 at an optical path of 10 mm and a wavelength of 572 nm. Luminescent particles 2a with A2 - A1 of 0.1 or less are preferred because the non-specific adsorption of impurities in the serum is small.

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

[0216] Also, the above aqueous medium is preferably adjusted in advance to have a pH of 6 or more and 9 or less. If the pH is less than 6 or greater than 9, the alkoxide group or silanol group of the radically polymerizable organosilane may react with other functional groups or undergo polycondensation before the formation of the polymer, and the resulting particles may aggregate. In this embodiment, intentional polycondensation of the alkoxide is not 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 sufficient stirring may not be possible.

[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, potentially leading to significant particle sedimentation. Furthermore, to improve the adhesion between the PVP and the 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 1a site 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., by utilizing the functional groups present in the hydrophilic layer 10a should be selected.

[0226] (Introduction of part 1a) The chemical reaction for chemically bonding the functional group for forming the 1a moiety 11a on the luminescent particle 2a to a substance containing the 1a moiety 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 moiety 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 the first a 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 target substance 3a, is maintained. When site 12a of 2a is an antibody and a thiol group is conferred as site 13a of 3a, the 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. N-Succinimidyl 3-(2-pyridyldithio)propionate (hereinafter abbreviated as SPDP) 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].

[0229] When using the aminoacyltransferase reaction to form a cross-linked structure, any method is acceptable for linking the peptide tag, which has 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 to which an N-hydroxysuccinimide active ester group has been introduced 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. However, click chemistry such as strain-enhanced azide-alkyne cycloaddition reactions and strain-enhanced inverse electron-demanded Diels-Alder reactions can be used. Additionally, 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 6a of 2a used in the embodiment will be described. Compound 6a of 2a can be produced by any method as long as the moiety 14a of 4a can be imparted to the hydrophilic polymer 15a. When the hydrophilic polymer 15a is sodium alginate and a thiol group is imparted as moiety 14a of 4a, compound 6a of 2a 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] (Diagnostic reagents for in vitro diagnostics) The in vitro diagnostic reagent in this embodiment, that is, the 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 reagent in this embodiment is preferably 0.000001% to 20% by mass, and more preferably 0.0001% to 1% by mass. The reagent in 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 reagent in this embodiment is not limited to these. When the 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 testing kit] This 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 (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 multiple 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 test kit for the target substance of the present disclosure, "specifically binds" corresponds to "specifically reacts" in the present embodiment. "The first compound" corresponds to "the first a compound". "The second compound having the fourth site" corresponds to "the second a compound having a plurality of fourth a sites capable of forming a covalent bond with the third a site" and "the third a compound for forming a covalent bond between the third a site and the fourth a site".

[0235] The test kit 100a according to the present embodiment can be used in the method for detecting a target substance according to the present embodiment, in the same manner as a kit for detecting a target substance in a specimen by ordinary in vitro diagnosis. Also, the concentration of the target substance can be measured by a conventionally known method, and in particular, it is preferably used for detecting the target substance in a specimen by the fluorescence polarization method.

[0236] The test kit 100a for the target substance of the present embodiment has a first a reagent 101a and a second a reagent 102a. The first a reagent 101a contains a luminescent reagent containing luminescent particles 2a and a first a compound 4a, and the second a reagent 102a can contain a second a compound 6a. At this time, the third a compound 16a contains a plurality of types of compounds for forming a covalent bond between the third a site 13a and the fourth a site 14a, and one of the first a reagent 101a or the second a reagent 102a and the other may each contain different types of compounds among the plurality of types of compounds. In addition to these reagents, it may have a housing that encloses these reagents, or when the target substance is an antigen or an antibody, it may have a viscosity modifier for use during the antigen-antibody reaction. Examples of the viscosity modifier include, but are not limited to, polyvinyl alcohol, polyvinylpyrrolidone, sodium alginate, and the like.

[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, 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. 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.

[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. Moreover, 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 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 is (A) Each of the 3a and 4a sites has a thiol group, The compound 3a includes any of the compounds selected from the group consisting of peroxidases, oxidizing agents, and compounds having a phenol group, or (B) Each of the 3a and 4a sites has at least one of two different glutamine residues and lysine residues, and the covalent bond is an amide bond formed between the glutamine residue and the lysine residue. Compound 3a may contain 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, then compound 2a may contain 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 luminescent particles present in the sample solution as a change in polarization anisotropy, resulting from the reaction of luminescent particles with the target substance. The principle of fluorescence polarization is described below. Although the following description assumes 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 exhibiting 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 larger aggregates 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 light emission occurs. The rotational motion of the light-emitting material can be expressed by equation (2b). Q = 3Vη / kT ···(2b) Here, Q: Material rotation relaxation time V: Volume of material η: viscosity of the solvent k: Boltzmann constant T: Absolute temperature That is the case.

[0246] The rotational relaxation time of a material is the time required for the numerator 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: Polarization degree when the material is stationary (Q=∞) p: Polarization degree A: Constant τ: Luminous lifetime of the material Q: Rotation relaxation time That is the case.

[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 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 that has a vibration direction 90 degrees different from the excitation light used for sample measurement. That is the case.

[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, the luminescent particles of this disclosure have polarization anisotropy determined by the following formula (1b) <r>It is preferable that it is 0.01 or higher.

number

[0252] In the fluorescence polarization method according to this embodiment, when particles aggregate via an antigen, polarization anisotropy <r>The design maximizes this potential, enabling highly sensitive measurements.

[0253] Furthermore, in this disclosure, polarization anisotropy <r>Instead, the degree of fluorescence polarization, which indicates a change in plane polarization, can be used as an indicator. The degree of fluorescence polarization can be expressed in units of polarization milli P (also called milli-polarization units; hereafter abbreviated as mP). The degree of fluorescence polarization (mP) can be calculated using the measured polarized fluorescence intensity as shown in the following formula (5b). mP=(I∥−I⊥) / (I∥+I⊥)···(5b) Here, 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 That is the case.

[0254] (Method for detecting target substances) The method for detecting a target substance in 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, it comprises the following steps. A step (S1001b) to obtain a liquid of the first b, which includes a target substance, a luminescent reagent having a first b site that specifically reacts with the target substance, and a first b compound having a second b site that specifically reacts with a site different from the site that specifically reacts with the luminescent reagent, and a third b site different from the second b site. A step (S1002b) to obtain a liquid of 2b containing a complex of 2b having a complex of 1b and an aggregate of compounds of 2b having a plurality of sites of 3b linked via sites of 3b, Step (S1003b) to measure the value related to the fluorescence polarization of the liquid in step 2b.

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

[0256] <Embodiment 3-1> The following third-first embodiment, which is an example of a target substance detection method in 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 has the following steps. A step (S2001b) to obtain a liquid of the first b, which includes a target substance, a luminescent reagent having a first b site that specifically reacts with the target substance, and a first b compound having a second b site that specifically reacts with a site different from the site that specifically reacts with the luminescent reagent, and a third b site different from the second b site. A step (S2002b) to obtain a liquid of 2b containing a 2b complex having an aggregate of a 2b compound having multiple 4b sites that bind to a 3b site, and a 3b compound having multiple 5b sites that bind to a 4b site, and a 1b complex, Step 2b: Obtaining values ​​related to the fluorescence polarization of the liquid (S2003b).

[0257] 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 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 sites of 4b that bind to sites of 3b, and a compound of 3b having multiple sites of 5b that bind to sites of 4b, 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 sites of 3b that are bound together via sites of 3b" 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 solution, 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 is an antibody.

[0261] In the step of obtaining liquid 5b of 2b in Figure 10B, compound 17b of 2b, which has multiple sites 14b of 4b that react with site 13b of 3b, and compound 19b of 3b, which has multiple sites 15b of 5b that react with site 14b of 4b, are mixed to form an affinity complex 6b (which can also be called an aggregate of compound 17b of 2b and compound 19b of 3b) consisting of compound 17b of 2b and compound 19b of 3b. At this time, site 13b of 3b and site 15b of 5b may be the same site. Next, by combining the affinity complex 6b with the complex 21b of 1b obtained in the step of obtaining liquid 1b of 1b, liquid 5b of 2b containing the measurement substance (also called complex 7b of 2b) can be obtained. Furthermore, when the 2b complex 7b is formed, the 3b compound 19b may bind to the 2b compound 17b after the 1b complex 21b and the 2b compound 17b have bound together, and then an aggregate of the 2b compound 17b and the 3b compound 19b may be formed, or the aggregate of the 2b compound 17b and the 3b compound 19b may bind to the compound formed by the binding of the 1b complex 21b, the 2b compound 17b and the 3b compound 19b.

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

[0263] While it is possible to perform the steps for obtaining liquid 1b (1b) and liquid 5b (2b) simultaneously, it is preferable to perform the step for obtaining liquid 1b (1b) before the step for obtaining liquid 5b (2b). This is because performing the steps for obtaining liquid 1b (1b) and liquid 5b (2b) simultaneously may reduce the reaction efficiency of the step for obtaining liquid 1b (1b).

[0264] The equilibrium dissociation constants (KD) of the binding 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 are preferably 10 nM or less. Furthermore, it is preferable that the equilibrium dissociation constants (KD) of the binding 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 are smaller than the equilibrium dissociation constants (KD) of the binding between target substance 3b and site 12b of the second b. This is because the affinity complex 6b is formed rapidly, and the efficiency of the formation of the second b complex 7b, which includes the luminescent particle 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 and no more than 400 times the size of the first complex 21b, i.e., between 200 nm and 40,000 nm.

[0267] (Luminous particles) An example of the luminescent particles of this embodiment will be described in detail with reference to Figures 10A and 10B. The luminescent particle 2b of 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 the interference of scattered light constantly fluctuates 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 the following: ether, betaine, or pyrrolidone ring. 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, making it more advantageous for measurements based on polarization anisotropy or fluorescence polarization degree.

[0274] (base material) 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 a polymer obtained by polymerizing a composition containing radically polymerizable organic silane with styrene as the main component is 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 luminescent 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 hydrophilically coated. 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 ring-opened glycidyl group 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) Europium complexes, exemplified as luminescent molecule 8b, are characterized by their low susceptibility to ambient influences in terms of emission wavelength and intensity, and their long emission lifetime. Europium complexes are composed of europium element and ligands. Considering the emission lifetime and the visible emission wavelength range, rare earth complexes, particularly europium complexes, are preferred as luminescent dyes. 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 equation (2b). In the case of europium in an aqueous dispersion, if the diameter of the luminescent particles 2b is about 50 to 300 nm, polarization anisotropy occurs before and after the process of obtaining liquid 5b of 2b. <r>It can change significantly.

[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(triphenylphosphineoxide)europium(III), 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 into the substrate 9b in large quantities, 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, allowing for reaction measurement in a shorter time.

[0282] (The 1b 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 (this can also be described as specifically capturing site Xb). 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, site 11b of 1b 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] (The compound of 1b having a site 2b and a site 3b that is different from site 2b) In this embodiment, the first compound 4b has a second site 12b that specifically reacts with a site (site Yb) different from the site 2b of the target substance 3b that specifically reacts with the luminescent particle 2b, and a third site 13b that is different from the second site 12b. A preferred example of the first compound 4b is a compound having an antibody that binds to site Yb via the second site 12b, and biotin as the third site 13b that is different from the second site 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 target substance 3b, the molecular weight of compound 4b of the first b can be reduced, so the preferred site 13b of the third b is a site containing biotin (for example, a group having biotin).

[0288] The key-to-dissociation (KD) of the bond between site 13b of the third b and site 14b of the fourth b 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 liquid 5b of the second b. The key-to-dissociation (KD) of the bond between biotin and avidin is 1 fM, and a stable bond can be formed very quickly, making this combination of binding sites particularly preferable.

[0289] Compound 4b of the first b preferably has at least one site 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 bound to the antibody molecule. This is because at least one biotin molecule allows 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 of the liquid 5b of the second b to be measured can be increased. <r>Alternatively, the fluorescence polarization degree mP can be increased.

[0291] The concentration of compound 4b in 1b should preferably be 10 to 1,000,000 times that of the target substance 3b. If the concentration of 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 the liquid 5b of the second b, the 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 the 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 in liquid to quickly form the affinity complex 6b.

[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 (KD) of the bond between the 4th b site 14b and the 5th b site 15b is 10 nM or less. As a result, affinity complex 6b is rapidly formed. The formed affinity complex 6b contains the 4th b site 14b of compound 2b 17b, which can undergo an affinity reaction with the 3b site 13b of compound 1b 4b. Therefore, in the step of obtaining liquid 2b 5b, the formation of luminescent particle 2b - target substance 3b - compound 1b 4b - affinity complex 6b becomes possible. Note that the 5th b site 15b may be the same site as the 3b site 13b.

[0294] A preferred combination of sites is one in which site 14b of 4b is the site that binds to biotin in avidin, and site 15b of 5b is the site that contains biotin. The dissociation constant (KD) for the binding of biotin to avidin is 1 fM, and a stable bond can be formed very quickly, making this combination of binding sites particularly preferable. Of course, site 14b of 4b may be the site that contains biotin, and site 15b of 5b may be the site that binds to biotin in avidin. Other examples include a site that contains avidin in site 14b and an anti-avidin antibody in site 15b. 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 2b has multiple sites 14b of 4b that react with site 13b of 3b, and contains carrier A16b. Carrier A16b is not necessarily required for any molecule that has multiple sites 14b of 4b that react with site 13b of 3b. Examples of compound 17b of 2b 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 2b only needs to have two or more binding sites with respect to compound 19b of 3b, which will be described later. If site 13b of 3b is a site containing biotin, compound 17b of 2b is preferably avidin, and site 14b of 4b is preferably a site within avidin that binds to biotin. On the other hand, if site 13b of 3b is a site containing avidin, then site 14b of 4b is preferably a site containing biotin.

[0296] Preferred examples of compound 17b of 2b include avidin, streptavidin, neutraavidin, and avidin-immobilized carriers (hereinafter sometimes referred to as avidin-immobilized carriers). 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, or neutraavidin (hereinafter sometimes abbreviated as avidins) is immobilized. Avidin, streptavidin, neutraavidin, and avidin-immobilized carriers 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 2b, 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 2b 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 nm to 20 nm. This is because if the support A16b has a size of 10 to 40 nm, multiple avidins as site 14b of site 4b 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 1M 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 containing 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 should be increased as much as possible, within a range that does not affect the binding affinity with compound 17b of 2b. Typically, it is preferable to label albumin with 2 to 30 biotin molecules, and in particular, 2 to 20 molecules is preferred as it does not affect the affinity reaction.

[0302] In the step of obtaining liquid 5b of 2b, an affinity bonding reaction between compound 17b of 2b and compound 19b of 3b yields liquid 5b of 2b 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 2b and compound 19b of 3b. The affinity bonding reaction between compound 17b of 2b and compound 19b of 3b 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 3b or site 15b of 5b and the number of sites 14b of 4b 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 particle 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] Affinity complex 6b has a larger average particle size than the luminescent particle 2b, does not directly bind to the target substance 3b, but can bind to compound 4b of the first b. Having affinity is used synonymously with having specific binding. The size (average particle size) of affinity complex 6b is at least larger than the luminescent particle 2b, preferably 40 μm or less. Considering the magnitude of the change in values ​​related to the sedimentation and fluorescence polarization of affinity complex 6b, it is between 200 nm and 10000 nm, more preferably between 1000 nm and 6000 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 process of acquiring the liquid 5b of 2b is large, the value relating to the fluorescence polarization of the luminescent particles 2b contained in the composite 7b of 2b (polarization anisotropy) <r>Alternatively, the fluorescence polarization degree (mP) also increases. It is important that the luminescent particle 2b in Figure 10B forms a large reactant via the affinity complex 6b through the target substance 3b. Considering the stable fluorescence polarization measurement of the complex 7b of the second b, and taking into account the magnitude of sedimentation and changes in the values ​​related to fluorescence polarization, the size of the complex 7b of the second b is between 200 nm and 40000 nm, preferably between 200 nm and 10000 nm, and more preferably between 1000 nm and 6000 nm. The magnitude of the value related to fluorescence polarization depends on the rotational relaxation time and emission lifetime of the material, so in the case of luminescent particle 2b using a europium complex, if the size of the complex 7b of the second b is 1 μm or more, the value related to fluorescence polarization will theoretically reach its maximum value.

[0306] (Measurement process to obtain values ​​related to the fluorescence polarization of liquid 2b) In the measurement process, the value related to the fluorescence polarization of the reaction solution (polarization anisotropy) is measured. <r>Alternatively, the degree of fluorescence polarization (mP) is measured. When measuring to determine the value 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 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 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 detection side is placed 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 using a spectrophotometer or the like.

[0307] The measurement conditions are preferably such that the viscosity of the liquid 5 of the second b is 0.5 to 50 mPa·s in a liquid at a temperature of 0 to 50°C. 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 liquid 5b of the second b. By measuring simultaneously with the process of obtaining the liquid 5b of the second b, for example, when using polarization anisotropy as a value related to fluorescence polarization, the polarization anisotropy immediately after the reaction can be measured. <r0>and polarization anisotropy after a certain reaction time <r1>This can be measured. <r1>and <r0>By measuring the difference or the change over time (dr / dt) and comparing it with a standard sample, the concentration of target substance 3b in the sample can be measured. 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 manufacturing luminescent particles) Next, an example of a method for manufacturing the luminescent particles 2b used in this embodiment will be described. The method for manufacturing the luminescent particles 2b has the following steps. A first manufacturing step involves 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. The second manufacturing step involves heating the emulsion to polymerize the radically polymerizable monomer. A third manufacturing step involves adding functional groups to the surface of the light-emitting particles to bond to the 1b portion 11b, which will be described later.

[0309] In this case, the functional group for attaching the 1b portion 11b is a functional group that can attach the 1b 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 monomer) The luminescent particles 2b are produced by polymerizing a radically polymerizable monomer, the radically polymerizable monomer comprising at least styrene and a radically polymerizable organosilane. The radically 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 radically 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] The radical polymerizable monomer, including radical polymerizable organic silanes, imparts siloxane bonds to the substrate 9b. Examples of radical polymerizable organic silanes include vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, or combinations thereof. The use of radical polymerizable organic silanes forms an inorganic oxide skeleton within the substrate 9, improving the physical and chemical stability of the luminescent particles 2b. Furthermore, the use of radical polymerizable organic silanes increases the affinity between the substrate 9b and the hydrophilic layer 10b and functional groups. In addition, the radical polymerizable monomer, including radical polymerizable organic silanes, imparts silanol groups 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 initiator) As radical polymerization initiators, a wide range of azo compounds and organic peroxides can be used. 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), and the like.

[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 ethers, betaines, 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 1b portion 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 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% to 100% by mass 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 the content of an organic solvent other than water exceeds 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 radically 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 is less than 5:5, aggregation of the generated luminescent particles 2b may become significant. If the mass ratio 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 1b site 11b of various antibodies, etc., to the substrate particle 20b, it can be used as a particle for sample testing. The optimal method for attaching the target antibody, etc., can be selected by utilizing the functional groups present in the hydrophilic layer 10b that are used to attach the 1b site 11b.

[0326] (Introduction of part 1b) The chemical reaction for chemically bonding the functional group for attaching the 11b portion of the 1b to the 11b portion of the 1b 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 11b portion of the 1b 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 11b portion of the 1b, the 11b portion of the 1b may be introduced to the luminescent particle 2b by physical adsorption.

[0327] (Method for producing compound 1b) The compound 4b 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 that reacts with the luminescent particle 2b of the target substance 3b, 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 4b 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 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) The compound 17b of 2b used in the embodiment of 1b is a compound having multiple 4b sites 14b that react with a 3b site 13b that is different from the 2b site 12b. The compound 17b of 2b can be selected from avidin, streptavidin, and neutraavidin, and commercially available products of these 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 and used as appropriate.

[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 (3b) is the site that binds to biotin in avidin, then compound 17b (2b) can be any molecule that has at least two biotin molecules as the 4b site 14b. Biotinylated albumin, which will be described later, is one example (its manufacturing method will be described later).

[0332] (Method for producing compound 3b) The third compound 19b used in this embodiment is a compound having multiple fifth site 15b. 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 N-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, which reacts with the 3b site 13b of compound 17b (2b), is biotin, then the 3b compound 19b can be any commercially available avidin or a carrier containing avidin (as described above for manufacturing).

[0334] (Method for measuring target substances using fluorescence polarization measurement) As a preferred embodiment of this 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 sites 13b of the third b, 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 2b, compound 17b of 2b having multiple sites 14b of 4b and compound 19b of 3b having multiple sites 15b of 5b 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 5b and site 14b of 4b. Therefore, site 15b of 5b and site 14b of 4b can specifically bond, and affinity bonding is possible. At this time, the dissociation constant (KD) of the bond between site 15b of 5b and site 14b of 4b 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 particle 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 the second b liquid 5b containing the second b complex 7b is obtained.

[0338] Finally, the polarization anisotropy is a value relating to the fluorescence polarization of liquid 5b obtained in the process of acquiring liquid 5b 2b. <r>Alternatively, the degree of fluorescence polarization is measured (measurement step). By measuring the value related to fluorescence polarization, the target substance 3b can be measured. In this case, 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 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 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 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 from 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 testing kit) The method for detecting the target substance described herein can also be used to create a test kit for the target substance. The test kit used for measuring the target substance in a sample in this embodiment is a test kit for the target substance that detects at least one of the presence or absence and concentration of the target substance in the sample solution by acquiring a value related to fluorescence polarization, A luminescent reagent having a 1b site that specifically reacts with a target substance, A compound 1b having a site different from the site that specifically reacts with the luminescent reagent of the target substance, a site 2b that specifically reacts with the site 2b, and a site 3b that is different from the site 2b, A compound 2b having multiple sites 4b that bind to site 3b, A compound 3b having multiple sites 5b that bind to site 4b, This is a test kit for target substances containing [the specified substance]. Here, the sites 3b and 5b 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 modes between site 3b and site 4b, and between site 4b and site 5b, however, it is preferable that the equilibrium dissociation constants (KD) of the binding between site 3b and site 4b and the equilibrium dissociation constants (KD) of the binding between site 4b and site 5b are 10 nM or less.

[0345] Furthermore, the 3b site is a biotin-containing site, and the 2b compound may contain at least one of avidin, streptavidin, neutraavidin, and an immobilization carrier for these avidins. In this case, the immobilization carrier contained 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 test kit 100b in this embodiment is preferably 0.00001% to 20% by mass, and more preferably 0.0001% to 2% by mass. The test 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 test kit 100b in this embodiment are not limited to these. Furthermore, the target substance test kit 100b according to this embodiment may also contain a dispersion stabilizer to stabilize the dispersion and a sensitizer to promote the 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> The following describes in detail an example of one embodiment of this disclosure, but without limiting the scope of this 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. 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 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 observed as a change in polarized emission properties. When the 1c site of the luminescent reagent, which specifically reacts with the target substance (it can also be said to be a site that specifically binds to the 1c 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 1c site. This then causes a change in the rotational Brownian motion of the particles. This change in rotational Brownian motion can be observed 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) Here, Q: Rotational relaxation time of luminescent reagent V: Volume of the luminescent reagent η: viscosity of the solvent k: Boltzmann constant T: Absolute temperature That is the case.

[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: Polarization degree when the luminescent reagent is stopped (Q=∞) p: Polarization degree A: Constant τ: Luminescence lifetime of luminescent reagent Q: Rotation relaxation time That is the case.

[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 experimentally determine the degree of polarization of the emission shown in equation (3c), 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 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 that has a vibration direction 90 degrees different from the excitation light used for sample measurement. That is the case.

[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, the luminescent reagent of this embodiment has polarization anisotropy determined by the following formula (1c) <r>It is preferable that the value is 0.01 or higher.

number

[0362] [Detection method] The detection method disclosed herein 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 comprises the following steps as shown in Figure 13A. (1) A step of obtaining a liquid of the first c that includes a target substance, a luminescent reagent having a first c site that specifically reacts with the target substance, and a compound of the 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 the second c which includes the first c complex and an aggregate of the second c compound having a plurality of third c sites linked via the third c sites (S1002c) (3) Step of measuring the value related to the fluorescence polarization of the liquid in 2c (S1003c) In this case, the aggregate of compound 2c can also be referred to as a crosslinked structure, crosslinked material, or aggregate of compound 2c.

[0363] <Embodiment 4-1> The following 4-1 embodiment, 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, it has the following steps. A step (S2001c) to obtain a liquid of the first c, which includes a first c composite having a target substance, a luminescent reagent having a first c site that specifically reacts with the target substance, a first c compound 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 having a temperature-responsive polymer. A step (S2002c) to obtain a liquid 3c containing a complex 2c having a complex 1c and an aggregate of compounds 2c bonded via a site 3c, by changing the temperature of a liquid 2c containing a complex 1c and a plurality of compounds 2c having a site 3c, A step (S2003c) to obtain values ​​related to the fluorescence polarization of the liquid in step 3c.

[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 binds” corresponds to “specifically reacts.” “A first compound having a second site and a third site different from the second site” corresponds to “a firstc compound having a 2c site and a 3c site having a temperature-responsive polymer.” “A second compound having a fourth site” corresponds to “(multiple) secondc compounds having a 3c site.”

[0365] In the fluorescence polarization method according to this embodiment, polarization anisotropy is achieved by the aggregation of the luminescent reagent due to hydrophobic aggregation in an aqueous solution. <r>The design incorporates features that increase the size of the sensor, enabling highly sensitive measurements.

[0366] In the 4-1 embodiment, the portion of the 3c portion that contributes to the formation of the 2c compound aggregate in the method for detecting the target substance of the present disclosure is a portion made of a temperature-responsive polymer, and the 2c compound aggregate is formed by changing the temperature of the 2c 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 14C illustrate the process of obtaining the liquid 1c.

[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 substrate 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 specifically react with compound 4c of the first c via site 12c of the second c. A specific reaction is, for example, a specific bond. In the figure, the site of target substance 3c that binds to site 11c of the first c is denoted as Xc, and the site that binds to site 12c of the second c is denoted as Yc.

[0369] As shown in Figure 14B, compound 4c of the first c has a site 12c of the second c that specifically reacts with the target substance 3c, and a site 13c of the third c that, unlike site 12c of the second c, has a temperature-responsive polymer 18c. Figure 14C shows an example of the process for obtaining liquid 1c of the first c. A mixture is prepared in which the luminescent particle 2c and 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 used as an antigen and site 12c of the second c as an antibody, and the specific reaction between the target substance 3c and compound 4c of the first c can be described as binding by an antigen-antibody reaction.

[0370] As shown in Figure 14D, the process of obtaining the first liquid 1c may also include sub-steps. If sub-steps are included, the process of obtaining the first liquid 1c may include a first sub-step of obtaining a mixture A22c having a subcomplex 21c comprising a target substance 3c, luminescent particles 2c, and a third compound 5c having a second part 12c and a fourth part 14c different from the second part 12c; and a second sub-step of obtaining a mixture B1c having a first complex 16c comprising a mixture A22c and a fourth compound 6c having a fifth part 15c that specifically reacts with the fourth part 14c and 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 site 14c of 4c and site 15c of 5c is smaller than the dissociation constant between site 3c of target substance 3c and site 12c of 2c. For example, one of site 14c of 4c and site 15c of 5c may be a site containing avidin, and the other may be a site containing biotin. More specifically and preferably, site 14c of 4c and site 15c of 5c may be a site containing biotin, and site 15c of 5c 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 multiple 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 3c portion 13c interact, as shown in Figure 15, and multiple 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 3c portion 13c. That is, the liquid of 3c 19c is prepared, which contains the 1c complex 16c and the 2c complex 17c containing 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 site 13c.

[0373] The temperature-responsive polymer 18c contained in compound 2c 20c is a stimulus-responsive polymer that changes its solubility in water when the temperature of liquid 2c 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 2c 20c. This makes it possible to form a large complex (complex 2c 17c) containing the aggregate 7c of compound 2c 20c formed via the bond between the complex 16c of 1c and the site 13c of 3c (more specifically, the temperature-responsive polymer 18c of the site 3c). At this time, the size of complex 2c 17c is more than twice the size of complex 16c of 1c, and specifically, the size of complex 2c 17c is preferably 200 nm or more and 6000 nm or less.

[0374] As mentioned above, in the embodiment of 4-1, compound 4c of the first c and compound 20c of the second c were described as the same compound. However, as long as both compounds have a third c portion 13c, 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 complex 17c of the second c containing an aggregate 7c of compound 20c of the second c and a complex 16c of the first c. Compound 4c of the first c and compound 20c of the second c 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 trace amounts 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 particle 2c has a small particle size distribution, and it is preferable that the surface of the luminescent particle 2c is covered with the hydrophilic layer 10c, as will be described later. The following describes the various parts of the luminescent particle 2c and the method for producing the luminescent particle 2c, using the example that the luminescent reagent is a luminescent particle 2c as shown in the schematic diagram in Figure 14A.

[0376] (base material) 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 1st c site 11c described later, can be imparted to the surface of the substrate 9c.

[0377] (The 1c site 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 particular target substance 3c. The portion that specifically reacts with the target substance 3c may include, for example, any substance that specifically reacts with a particular target substance 3c (this may be through specific binding or specific capture), and any substance that shows affinity to a particular 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 particular 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 between 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, or cDNA derived from bacteria, viruses, or cells, 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 between 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 onto 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 lot-to-lot variability.

[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. Among more specific examples of hydrophilic polymers, polymers having a pyrrolidone ring may be abbreviated as "PVP" in this specification.

[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 molecule 8c contained in the luminescent particle 2c is preferably a rare-earth complex, and more preferably a europium complex, considering factors such as luminescence lifetime (i.e., long lifetime) and the visible emission wavelength range. Europium complexes have the characteristics of less susceptibility to ambient influence on the wavelength and intensity of emission, and long luminescence lifetime. Europium complexes are composed of europium element 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 aforementioned 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(triphenylphosphineoxide)europium(III), tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide)europium(III), and tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III).

[0385] Furthermore, when the Brownian rotation of the europium complex can be considered to have stopped in the medium, the polarization anisotropy represented by the aforementioned equation (4c) is <r>It is desirable that this value be 0.10 or higher. The state in which Brownian rotation can be considered to have stopped is when the rotational relaxation time of the luminescent particle 2c is sufficiently longer than the luminescence lifetime of the europium complex.

[0386] It is preferable for the luminescent molecules 8c to be incorporated in large quantities into the substrate 9c, as this increases the luminescence intensity per particle. On the other hand, if the luminescent molecules 8c aggregate in the substrate 9c, the interaction between ligands may affect the excitation efficiency of the europium complex, making it difficult to measure values ​​related to fluorescence polarization while maintaining reproducibility. If the particle size of the luminescent particles 2c is 100 nm, then luminescent particles 2c containing approximately 1,000 to 3,000,000 luminescent molecules 8c per particle are preferably used. Whether the europium complex exhibits non-aggregative luminescence behavior in the substrate 9c can be determined from the excitation spectrum of the sample. Luminescent particles 2c with strong luminescence not only enable high-sensitivity measurements, but also maintain luminescence even with small particle sizes, thus enabling faster biochemical reaction rates. Therefore, smaller particle sizes of luminescent particles 2c result in a larger diffusion coefficient of Brownian motion in the liquid, making it possible to detect the reaction in a shorter time.

[0387] The diameter of the luminescent particle 2c 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.

[0388] The diameter of the luminescent particles 2c is preferably such that the average particle diameter 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 diameter 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 8c, such as europium complexes, that can be contained in each luminescent particle can be increased.

[0389] Furthermore, it is preferable that the luminescent particles 2c have a small particle size distribution. Also, although Figure 14A shows an example where both the luminescent particles 2c and the substrate 9c are spherical, the shapes of the luminescent particles 2c and the substrate 9c in this embodiment are not limited.

[0390] [Compounds of type 1c] As shown in Figure 14B, 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 is hydrophilic and dissolved in the solution during the process of obtaining liquid 1c of the first c.

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

[0392] Compound 4c of the first c reacts specifically with (may bind to or capture) the luminescent particle 2c via the target substance 3c. The site 12c of the second c of 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 site 11c of the first c of the luminescent particle 2c and the site 12c of the second c of compound 4c of the first c react specifically with different sites of the target substance 3c, thereby reacting specifically via the target substance 3c. Note that the site 12c of the second c has the function of reacting with the target substance 3c, but 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, causing turbidity and precipitation when heated to a predetermined temperature. The predetermined temperature is generally called the lower critical solution temperature and is specific 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 liquid 1c] In the step of obtaining the liquid 1c of the first c, a liquid 1c of the first c is obtained, which includes 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 4c of the first c. That is, it may be a step of mixing a sample solution containing target substance 3 with luminescent particles 2c and compound 4c of the first c, or, for example, a sample solution containing target substance 3c and a reagent solution containing luminescent particles 2c and compound 4c of the first c 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 process 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, is mixed with the target substance 3c and luminescent particles 2c to prepare a mixed solution 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 solution 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 preparing a mixed solution B1c (liquid 1c of the first c) in which the luminescent particle 2c, target substance 3c, and compound 4c of the first c are reacted to the site 14c of the fourth c and the site 15c of the fifth c to form the complex 16c of the first c. In such cases, the compound 5c of the third c, which has a smaller molecular weight than compound 4c of the first c, reacts with the target substance 3c, and then the compound 6c of the fourth c reacts with it to form compound 4c of the first c, 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 site 14c of the fourth c and site 15c of the fifth c include one site having biotin and the other site having avidin.Furthermore, it is preferable that the dissociation constants of site 14c of the 4th c and site 15c of the 5th c are smaller than the dissociation constant of site 12c of the 2nd c and target substance 3c (it is also acceptable for the binding constant of site 14c of the 4th c and site 15c of the 5th c to be larger than the binding constant of site 12c of the 2nd 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 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 2c is obtained containing the complex 16c of 1c and compound 20c of 2c having the site 13c of 3c. Here, liquid 2c may be the same as liquid 1c of 1c, or liquid 2c may be obtained by further adding compound 20c of 2c to liquid 1c of 1c. Furthermore, compound 20c of 2c may be a compound of compound 4c of 1c that did not react with the target substance 3c present in liquid 1c, or it may be a different compound from compound 4c of 1c as long as it has the site 13c of 3c.

[0400] [Process for obtaining liquid 3c] In the step of obtaining the third liquid 19c, the temperature of the second liquid 19c, which contains the first composite 16c and the second compound 20c having the third portion 13c, is changed (for example, by heating it to a temperature above the lower critical solution temperature) to obtain the third liquid 19c in which the physical properties of the temperature-responsive polymer 18c have changed 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. This leads to the formation of a second c composite 17c containing an aggregate 7c of multiple second c compounds 20c formed in the liquid by hydrophobic bonds (also 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 through hydrophobic interactions, 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 material, and the first c material), 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 second complex 17c depends on the proportion of the first complex 16c formed in the process of obtaining the first liquid 1c, i.e., the amount of target substance 3c. As a result, in the measurement process described later, the polarization anisotropy of the third liquid 19c <r>By obtaining this data, it is possible to confirm the change in the value related to fluorescence polarization depending on the amount of target substance 3c present. This allows us to confirm the high reactivity with target substance 3c and the polarization anisotropy obtained in the measurement process described later, even when target substance 3c is present in trace amounts in liquid 19c of the third c. <r>It is possible to achieve both a large increase in fluorescence polarization values ​​such as fluorescence polarization degree and a large increase in fluorescence polarization values, enabling highly sensitive detection of the target substance 3c. In other words, because the complex 17c of the second c becomes a large reactant, the change in fluorescence polarization values ​​also becomes large, allowing for highly sensitive detection of the target substance 3c.

[0404] Furthermore, the composite 17c of the second c is naturally larger than the luminescent particle 2c, and preferably more than 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 composite 17c of the second c is preferably 200 nm to 6000 nm, and more preferably 1 μm to 6 μm. Note that the value related to fluorescence polarization is polarization anisotropy. <r>When using polarization anisotropy <r>The size depends on the rotational relaxation time and luminescence lifetime of the luminescent reagent. For example, in the case of luminescent particles 2c using a europium complex, if the size of the 2c complex 17c is 1 μm or more, the polarization anisotropy of the 2c complex 17c <r>The theoretical value far exceeds the maximum value.

[0405] To increase the size of the 2c complex 17c, a compound having a temperature-responsive polymer 18c and a 3c moiety 13c can be used as the 2c compound 20c. In this case, the 1c compound 4c, which exists alone in the 1c liquid 1c without contributing to the formation of the 1c complex 16c, and the 2c compound 20c, which is added to the 1c liquid 1c, will aggregate as the 3c moieties 13c combine through a process of changing the temperature of the 2c liquid.

[0406] To increase the formation rate and size of the 2c complex 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 can efficiently dehydrate 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 c liquid 19c after the step of obtaining the first c liquid 1c, but the steps of obtaining the first c liquid 1c and obtaining the third c 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 c composite 16c is sufficiently formed, and if the second c composite 17c is formed by the step of obtaining the third c liquid 19c, the measurement sensitivity may decrease.

[0408] [Measurement process] In the measurement process, a value related to the fluorescence polarization of liquid 19c of the third c is obtained. Here, the value related to fluorescence polarization is the polarization anisotropy. <r>It may be either the degree of fluorescence polarization or the degree of fluorescence polarization. There are no particular restrictions on the measurement conditions as long as the temperature of the sample solution is above the LCST, for example, it is preferable that the liquid viscosity is 0.5 to 50 mPa·s in a solution at a temperature of 0 to 50°C. 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 be 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 obtain a value related to the fluorescence polarization immediately after the reaction (for example, polarization anisotropy). <r0>) and the value relating to fluorescence polarization after a certain reaction time (e.g., polarization anisotropy) <r1>) can be measured. These differences (for example, <r1>and <r0>By measuring the difference between 0 and 1 / 2 (difference) and the rate of change over time (e.g., dr / dt), and comparing these 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 producing the 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 capturer of the target substance 3c is used to form the luminescent particles 2c of the first c moiety 11c that reacts with the target substance 3c.

[0410] A method for producing luminescent particles 2c comprises the steps 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 (step Ac); heating the emulsion to polymerize the radical polymerizable monomer (step Bc); and further comprising the step of imparting a functional group for introducing the first c moiety 11c, described later, to the surface of the luminescent particles 2c (step Cc).

[0411] Here, the functional group for introducing the 11c site is a functional group to which the 11c site 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 monomer) The luminescent particles 2c are produced by polymerizing a radically polymerizable monomer, the radically polymerizable monomer comprising at least styrene and a radically polymerizable organosilane. The radically 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 radically 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 conferred 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. 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 9c.

[0415] (Radical polymerization initiator) As radical polymerization initiators, a wide range of azo compounds and organic peroxides can be used. 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), and the like.

[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 such as ethers, betaines, and pyrrolidone rings. 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 to sites ...

Claims

1. 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.

2. The method for detecting a target substance according to claim 1, wherein the second complex is larger than the first complex.

3. The method for detecting a target substance according to claim 1, wherein the second complex is at least twice the size of the first complex.

4. A method for detecting a target substance according to claim 1, 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.

5. The method for detecting a target substance according to claim 4, wherein the third compound is the same compound as the second compound.

6. The method for detecting a target substance according to claim 1, wherein the third portion and the fourth portion are the same portion.

7. 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 claim 1, 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.

8. 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.

9. The method for detecting a target substance according to claim 7, wherein one of the fifth and sixth sites is a site containing avidin and the other is a site containing biotin.

10. 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.

11. The method for detecting a target substance according to claim 10, wherein the bond between the third and fourth portions is a hydrophobic bond.

12. 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.

13. 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.

14. 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.

15. The target substance testing kit according to claim 14, wherein the third compound is the same compound as the second compound.

16. The target substance testing kit according to claim 13, wherein the third portion and the fourth portion are the same portion.

17. A target substance testing kit according to claim 13, 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.

18. 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.

19. The target substance testing kit according to claim 13, wherein the bond via the third site is any of an ionic bond, a covalent bond, an affinity bond, or a hydrophobic bond.

20. The target substance testing kit according to claim 19, wherein the bond via the third site is a hydrophobic bond.

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