Polymers, test agents, and analyte concentration measurement methods

Abstract

A purpose of the present invention is to provide a polymer that makes it possible to suppress background fluorescence and to measure an analyte at a favorable detection sensitivity. Another purpose of the present invention is to provide a test agent containing said polymer, an analyte concentration measuring method using said polymer, and an analyte concentration measuring instrument used in said analyte concentration measuring method. The present invention is a polymer that has an aggregation-induced light-emitting group on the skeleton of the polymer and that has, on said polymer skeleton, a binding partner that can bind with an analyte.

Description

[Technical Field]

[0001] The present invention relates to a polymer capable of suppressing background fluorescence and measuring an analyte with good detection sensitivity, a test agent containing the polymer, a method for measuring an analyte concentration using the polymer, and an analyte concentration measuring device used in the method. [Background technology]

[0002] The method of measuring a substance to be measured in a sample by detecting fluorescence (fluorescence method) allows for simple and highly sensitive measurement, and can be automated using analytical equipment such as an immunoplate reader, so it is used in many fields, including clinical testing. The fluorescence method is extremely superior in terms of high efficiency and simplicity.

[0003] However, fluorescence methods can sometimes produce background fluorescence that is not attributable to the substance being measured. Background fluorescence can occur due to the autofluorescence of endogenous substances other than the substance being measured in the sample, from fluorescent dyes that nonspecifically adhere to proteins in the sample, or from the container (plate, etc.) into which the substance being measured is injected. In all cases, this affects sensitivity and specificity, making it a common problem for fluorescence methods. Therefore, there has been a demand for a measurement method that can suppress the effects of background fluorescence.

[0004] Patent Documents 1 and 2 disclose antibodies that use an analyte-dye complex containing a substantially non-fluorescent dye as an antigen. However, such antibodies only correspond to a specific antigen, and are affected by multiple proteins contained in the analyte, so that background fluorescence may not be reduced in some cases.

[0005] Patent Document 3 discloses particles containing an aggregated fluorescent material, which include a core particle, a binding partner that binds to an analyte, and an aggregated fluorescent material that aggregates and emits fluorescence when the analyte binds to the binding partner. The use of such particles containing an aggregated fluorescent material makes it possible to measure an analyte with a fairly good detection sensitivity while suppressing background fluorescence. However, there has been a demand for a measurement method that is even more effective in suppressing background fluorescence and has even better analyte detection sensitivity.

[0006] Furthermore, in nuclear power generation, when nuclear fission reactions of uranium or plutonium occur, radioactive isotopes such as cesium-137 (Cs) and cesium-134 (Cs) may be produced. Conventionally, measuring radioactive materials required a germanium semiconductor detector as disclosed in Patent Document 4, a NaI (Tl) scintillation spectrometer, or the like, but the equipment was expensive, and the operation was complicated, making measurement labor-intensive. Therefore, a low-cost, simple method for measuring radioactive materials was desired. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 9-5324 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-171213 [Patent Document 3] International Publication No. 2018 / 043688 [Patent Document 4] Japanese Patent Application Laid-Open No. 2013-246049 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a polymer that can suppress background fluorescence and enable analyte measurement with good detection sensitivity, a test agent containing the polymer, a method for measuring analyte concentration using the polymer, and an analyte concentration measurement device used in the method. [Means for solving the problem]

[0009] The present invention is a polymer having aggregation-induced luminescent groups on the polymer backbone and a binding partner on the polymer backbone that is capable of binding to an analyte. The present invention will be described in detail below.

[0010] The particles containing an aggregated fluorescent material as disclosed in Patent Document 3 have the problem that scattered light due to particle movement affects the background, i.e., background fluorescence tends to be large. In addition, the particles serving as carriers absorb or scatter the fluorescence generated by aggregation excitation, resulting in a decrease in detection sensitivity, and there is a problem that the analyte may not be detected, particularly when the analyte concentration is low. Therefore, the present inventors investigated the use of a polymer having an aggregation-induced luminescent group on the polymer backbone and a binding partner capable of binding to an analyte on the polymer backbone, and found that it is possible to obtain a polymer that can suppress background fluorescence and enable analyte measurement with good detection sensitivity, thereby completing the present invention. The polymer of the present invention hardly causes problems such as scattered light that occur when aggregated fluorescent material-containing particles are used, and therefore has an excellent effect of suppressing background fluorescence. Furthermore, since the aggregation-induced luminescent groups are brought into close proximity with each other as the polymer is entangled, which has a large spatial area that can participate in the reaction, and thus fluorescence is emitted, higher detection sensitivity can be achieved than when aggregated fluorescent material-containing particles are used.

[0011] The polymer of the present invention has aggregation-induced luminescent groups on the polymer backbone, and also has a binding partner on the polymer backbone that is capable of binding to an analyte. In this specification, "having an aggregation-induced luminescent group on a polymer backbone and having a binding partner on the polymer backbone that can bind to an analyte" includes not only the case where the aggregation-induced luminescent group and the binding partner are chemically bonded to the polymer backbone, but also the case where a compound having these is physically in contact with the polymer backbone. The case where they are chemically bonded is preferred. Furthermore, "having on the polymer backbone" may mean either having them in the main chain of the polymer or having them in the side chain of the polymer. Furthermore, "being in physical contact with the polymer backbone" refers to a state in which they are in close proximity due to hydrophobic interactions, π-π interactions, C-H interactions, etc.

[0012] The polymer backbone is preferably a chain or a network, and is more preferably a chain because the polymers are easily entangled and higher detection sensitivity can be achieved. In this specification, "chain" means that the polymer main chain is not two-dimensionally crosslinked and has no branched structure. Examples of the polymerizable monomer from which the polymer skeleton is derived include polymerizable monomers having an ethylenically unsaturated group. Examples of the polymerizable monomer having an ethylenically unsaturated group include a carboxyl group-containing monofunctional monomer, a hydroxyl group-containing monofunctional monomer, a hydroxyl group-containing polyfunctional monomer, an amino group-containing monofunctional monomer, an amino group-containing polyfunctional monomer, an amide group-containing monofunctional monomer, an amide group-containing polyfunctional monomer, and a sulfonic acid group-containing monofunctional monomer. Examples of the carboxyl group-containing monofunctional monomer include (meth)acrylic acid, β-carboxyethyl (meth)acrylate, and 2-(meth)acryloyloxyethyl succinate. Examples of the hydroxyl group-containing monofunctional monomer include 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, and 4-hydroxybutyl (meth)acrylate. Examples of the hydroxyl group-containing polyfunctional monomer include glycerin di(meth)acrylate. Examples of the amino group-containing monofunctional monomer include dimethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, and diethylaminoethyl (meth)acrylate. Examples of the amino group-containing polyfunctional monomer include PEG-NH2 and PEG-NHS. Examples of the amide group-containing monofunctional monomer include (meth)acrylamide, N-methylol(meth)acrylamide, isopropyl(meth)acrylamide, and sulfobetaine monomer FAM-101 manufactured by Fujifilm Corporation. Examples of the amide group-containing polyfunctional monomer include N,N'-methylenebis(meth)acrylamide, and polyfunctional acrylamide monomers FAM-401, 301, 201, and 402 manufactured by Fujifilm Corporation. Examples of the sulfonic acid group-containing monofunctional monomer include 2-(meth)acrylamido-2-methylpropanesulfonic acid, 2-(meth)acryloyloxyethyl acid phosphate, and p-styrenesulfonate. These may be used alone or in combination of two or more. Furthermore, the crosslinked structure in the polymer of the present invention can be obtained by copolymerizing the polyfunctional monomers listed above as polymerizable monomers having an ethylenically unsaturated group, and can also be obtained by intramolecular crosslinking (dehydration condensation) using a carboxyl group or a hydroxyl group. Of these, (meth)acrylic acid, 2-hydroxyethyl (meth)acrylate, (meth)acrylamide, N-methylol (meth)acrylamide, and N,N'-methylenebis(meth)acrylamide are preferred. If necessary, other monomers such as styrene, methyl (meth)acrylate, glycidyl (meth)acrylate, etc. may be copolymerized. When the other monomers are copolymerized, the upper limit of the proportion of the other monomers used is preferably 50% by weight, more preferably 30% by weight, and even more preferably 10% by weight. In this specification, the term "(meth)acrylic" means acrylic or methacrylic, and the term "(meth)acrylate" means acrylate or methacrylate.

[0013] The polymer backbone preferably contains a hydrophilic group, which makes the resulting polymer highly soluble in water, reducing background fluorescence and increasing the frequency of collisions between the analyte and the binding partner, which facilitates aggregation of the polymer, resulting in higher fluorescence intensity due to the aggregation-induced luminescent group.

[0014] When the polymer backbone contains a hydrophilic group, the hydrophilic group is preferably at least one group selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, an amide group, and a sulfonic acid group, and more preferably at least one group selected from the group consisting of a hydroxyl group, a carboxyl group, and an amino group.

[0015] The aggregation-induced luminescent group is a group that is non-luminescent when the molecular motion of the group is not suppressed, but emits fluorescence when the molecular motion of the group is significantly suppressed due to aggregation of the group moiety, spatial constraints, interactions with surrounding molecules, and the like, and the non-radiative deactivation pathway is inhibited.

[0016] The aggregation-induced luminescent group preferably contains a hydrophilic group. When the aggregation-induced luminescent group contains a hydrophilic group, the resulting polymer has excellent solubility in water, which reduces background fluorescence and increases the frequency of collisions between the analyte and the binding partner, making it easier for the polymer to aggregate, resulting in higher fluorescence intensity due to the aggregation-induced luminescent group.

[0017] When the aggregation-induced luminescent group contains a hydrophilic group, the hydrophilic group is preferably at least one group selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, an amide group, and a sulfonic acid group, and more preferably at least one group selected from the group consisting of a hydroxyl group, a carboxyl group, and an amino group.

[0018] Specifically, the aggregation-induced light-emitting group is preferably a group represented by the following formula (1) or a group represented by the following formula (2).

[0019] [ka]

[0020] In formula (1), E represents a silicon atom or a germanium atom, and R 1 and R 2 R may be the same or different and represent a saturated or unsaturated hydrocarbon group having 1 to 6 carbon atoms which may have a substituent, a phenyl group which may have a substituent, a hydrogen atom, a hydroxyl group, a halogen atom, an amino group, a carboxyl group, or a nitro group. 3 ~R 6 One of the bonds is R 3 ~R 6 Among these, those that are not bonds may be the same or different and represent an aromatic hydrocarbon group which may have a substituent, or an aromatic heterocyclic group which may have a substituent.

[0021] [ka]

[0022] In formula (2), R 7 ~R 10 One of the bonds is R 7 ~R 10 Among these, those that are not bonds may be the same or different and represent a saturated or unsaturated hydrocarbon group having 1 to 6 carbon atoms which may have a substituent, a hydrogen atom, a hydroxyl group, a halogen atom, an amino group, a carboxyl group, or a nitro group.

[0023] The aggregation-induced luminescent group can be introduced onto the polymer skeleton by a method of polymerization using a polymerizable monomer containing an aggregation-induced luminescent compound or an aggregation-induced luminescent compound having a polymerizable functional group, or by a method of introduction into the polymer skeleton by a side chain reaction. The aggregation-induced luminescent compound may be copolymerized on the polymer backbone, or may be chemically bonded via a functional group such as a carboxyl group, a hydroxyl group, an amino group, an amide group, or an epoxy group. The aggregation-induced luminescent group can also be introduced onto the polymer skeleton by bringing the aggregation-induced luminescent compound into physical contact with the polymer skeleton.

[0024] Examples of the aggregation-induced luminescent compound include tetraphenylethylene derivatives, hexaphenylbenzene derivatives, triphenylamine derivatives, ketoimine boron complex derivatives, diimine boron complex derivatives, aminomaleimide derivatives, aminobenzopyroxanthene derivatives, tetraphenylsilole derivatives, pentaphenylsilole derivatives, and hexaphenylsilole derivatives. Among these, from the viewpoint of availability, etc., tetraphenylethylene derivatives, hexaphenylbenzene derivatives, triphenylamine derivatives, tetraphenylsilole derivatives, pentaphenylsilole derivatives, and hexaphenylsilole derivatives are preferred, and tetraphenylethylene derivatives, tetraphenylsilole derivatives, pentaphenylsilole derivatives, and hexaphenylsilole derivatives are more preferred. Tetraphenylethylene derivatives are particularly preferred.

[0025] The tetraphenylethylene derivatives include tetraphenylethylenes whose phenyl groups may be substituted with functional groups. Specific examples include tetraphenylethylene, 4-(1,2,2-triphenylvinyl)phenyl(meth)acrylate, p-hydroxytetraphenylethylene(meth)acrylate, p-carboxytetraphenylethylene(meth)acrylate and its salts, 1-(4-bromophenyl)-1,2,2-triphenylethylene, tetrakis(4-hydroxyphenyl)ethylene, 1,2-Bis[4-(azidomethyl)phenyl]-1,2-diphenylethene, 1,2-Bis[4-(bromomethyl)phenyl]-1,2-diphenylethene, 1,2-Bis(4-methoxyphenyl)-1,2-diphenylethene, 4,4'-Bis(1,2,2-triphenylvinyl)-1,1'-biphenyl, [(1,2-Diphenylethene-1,2-diyl)bis(4,1-phenylene)]diboronic acid, 4,4'-(1,2-Diphenylethene-1,2-diyl)dibenzoic acid, 2,2'-[(1,2-Diphenyl-1,2-ethenediyl)di-4,1-phenylene]bis[4,4,5,5-tetramethyl-1,3,2-dioxaborolane], 1-{4-[ 1,2-Diphenyl-2-(p-tolyl)vinyl]phenyl}-1H-pyrrole-2,5-dione, 1-Ethynyl-4-(1,2,2-triphenylethenyl)benzene, Sodium Examples include 3,3'-{[(1,2-diphenylethene-1,2-diyl)bis(4,1-phenylene)]bis(oxy)}bis(propane-1-sulfonate), 4-(1,2,2-Triphenylethenyl)benzaldehyde, B-[4-(1,2,2-Triphenylethenyl)phenyl]boronic acid, and the like. An example of the above p-hydroxytetraphenylethylene(meth)acrylate having one hydroxyl group is 4-((4-hydroxyphenyl)diphenylvinyl)phenyl(meth)acrylate (the 4-hydroxyl group may be located at any of the four positions on the phenyl group of the compound). An example of the above p-hydroxytetraphenylethylene(meth)acrylate having two hydroxyl groups is 4-(bis(4-hydroxyphenyl)phenylvinyl)phenyl(meth)acrylate (the two 4-hydroxyl groups may be located at either of the four positions on the phenyl group of the compound). Among the above p-hydroxytetraphenylethylene(meth)acrylates, an example of one having three hydroxyl groups is 4-(1,2,2-tris(4-hydroxyphenyl)vinyl)phenyl(meth)acrylate. Among the above p-carboxytetraphenylethylene(meth)acrylates, an example having one carboxyl group is 4-((4-carboxyphenyl)diphenylvinyl)phenyl(meth)acrylate (the 4-carboxyl group may be located at any of the four positions on the phenyl group of the compound). An example of the p-carboxytetraphenylethylene(meth)acrylate having two carboxyl groups is 4-(bis(4-carboxyphenyl)phenylvinyl)phenyl(meth)acrylate (the two 4-carboxyl groups may be located at either of the four positions on the phenyl group of the compound). Among the above p-carboxytetraphenylethylene(meth)acrylates, an example of one having three carboxyl groups is 4-(1,2,2-tris(4-carboxyphenyl)vinyl)phenyl(meth)acrylate. Of these, tetraphenylethylene (meth)acrylate, p-hydroxytetraphenylethylene (meth)acrylate, p-carboxytetraphenylethylene (meth)acrylate and salts thereof, tetrakis(4-hydroxyphenyl)ethylene, 4,4'-(1,2-Diphenylethene-1,2-diyl)dibenzoic acid, and 4,4'-(1,2-Diphenylethene-1,2-diyl)diphenol are preferred.

[0026] Examples of the tetraphenylsilole derivative or the hexaphenylsilole derivative include 1,1,2,3,4,5-hexaphenylsilole, which may be substituted on the phenyl group with 1 to 5 functional groups; 2,3,4,5-tetraphenyl-1,1-dimethylsilole, which may be substituted on the phenyl group with 1 to 5 functional groups; 2,3,4,5-tetraphenyl-1,1-diallylsilole, which may be substituted on the phenyl group with 1 to 5 functional groups; and 1-methyl-1,2,3,4,5-pentaphenylsilole, which may be substituted on the phenyl group with 1 to 5 functional groups.

[0027] Examples of the hexaphenylbenzene derivative include benzene derivatives substituted with four or more phenyl groups or phenyl group derivatives, etc. Specific examples include hexaphenylsilole and hexaphenylbenzene.

[0028] Examples of the triphenylamine derivatives include 4-(di-p-triamino)benzaldehyde.

[0029] In particular, the aggregation-induced light-emitting compound is preferably a compound represented by the following formula (3) or a compound represented by the following formula (4).

[0030] [ka]

[0031] In formula (3), E represents a silicon atom or a germanium atom. 11 and R 12 R may be the same or different and represent a saturated or unsaturated hydrocarbon group having 1 to 6 carbon atoms which may have a substituent, a phenyl group which may have a substituent, a hydrogen atom, a hydroxyl group, a halogen atom, an amino group, a carboxyl group, or a nitro group. 13 ~R 16 may be the same or different and represent an aromatic hydrocarbon group which may have a substituent, or an aromatic heterocyclic group which may have a substituent.

[0032] [ka]

[0033] In formula (4), R 17 ~R 20 may be the same or different and represent a saturated or unsaturated hydrocarbon group having 1 to 6 carbon atoms, which may have a substituent, a hydrogen atom, a hydroxyl group, a halogen atom, an amino group, a carboxyl group, or a nitro group.

[0034] A binding partner capable of binding to the analyte can be introduced onto the polymer backbone by reacting a compound having the binding partner with a polymer into which aggregation-induced luminescent groups have been introduced. The binding partner may be copolymerized onto the polymer backbone or chemically bound via a functional group such as a carboxyl group, a hydroxyl group, an amino group, an amide group, an epoxy group, or a tosyl group. When chemically bonding via the functional group, a condensing agent may be used as a coupling agent. Examples of the condensing agent include carbodiimide-based condensing agents, imidazole-based condensing agents, triazine-based condensing agents, phosphonium-based condensing agents, uronium-based condensing agents, and haluronium-based condensing agents. Examples of the carbodiimide condensing agent include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), N,N′-dicyclohexylcarbodiimide (DCC), and N,N′-diisopropylcarbodiimide (DIC). The imidazole-based condensing agent includes, for example, N,N'-carbonyldiimidazole. Examples of the triazine-based condensing agent include 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM). Examples of the phosphonium-based condensing agent include 1H-benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), and the like. Examples of the uronium-based condensing agent include O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), {{[(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy}-4-morpholinomethylene}dimethylammonium hexafluorophosphate (COMU), and the like. Examples of the haluronium-based condensing agent include 1-(chloro-1-pyrrolidinylmethylene)pyrrolidinium hexafluorophosphate (PyCIU). In addition to the condensing agent, a reaction accelerator may be used. Examples of the reaction accelerator include 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), and N,N'-disuccinimidyl carbonate (DSC). The binding partner can also be introduced onto the polymer backbone by placing a compound having the binding partner in physical contact with the polymer backbone.

[0035] The analyte is not particularly limited, and examples thereof include molecules that can theoretically be measured by a measurement method, such as proteins, peptides, amino acids, lipids, sugars, nucleic acids, and haptens. Specific examples include CRP (C-reactive protein), Lp(a) (lipoprotein(a)), MMP3 (matrix metalloproteinase 3), anti-CCP (cyclic citrullinated peptide) antibody, antiphospholipid antibody, anti-syphilis antigen antibody, RPR, type IV collagen, PSA, AFP, CEA, BNP (brain natriuretic peptide), NT-proBNP, insulin, microalbumin, cystatin C, RF (rheumatoid factor), CA-RF, KL-6, PIVKA-II, FDP, D-dimer, SF (soluble fibrin), TAT (thrombin-antithrombin III complex), PIC, PAI, factor XIII, pepsinogen I, pepsinogen II, phenytoin, phenobarbital, carbamazepine, valproic acid, theophylline, and the like.

[0036] The polymer of the present invention can also be suitably used for measuring radioactive substances as the analyte. Examples of the radioactive substance include cobalt 60 (60Co), strontium 90 (90Sr), radioactive zirconium, technetium 99 (99Tc), ruthenium 106 (106Ru), radioactive iodine, radioactive cesium, radioactive thorium, radioactive uranium, radioactive plutonium, radioactive americium, and radioactive curium. Examples of the radioactive zirconium include zirconium 93 (93Zr) and zirconium 95 (95Zr). Examples of the radioactive iodine include iodine 129 (129I) and iodine 131 (131I). Examples of the radioactive cesium include cesium 137 (137Cs) and cesium 134 (134Cs). The radioactive thorium includes, for example, thorium 230 (230Th). Examples of the radioactive uranium include uranium 235 (235U) and uranium 238 (238U). An example of the radioactive plutonium is plutonium 240 (240Pu). The radioactive americium includes, for example, americium 242 (242Am). The radioactive curium includes, for example, curium 244 (244Cm).

[0037] The binding partner is appropriately selected depending on the type of the analyte, and examples thereof include groups derived from proteins, peptides, amino acids, lipids, nucleic acids, haptens, and the like.

[0038] Furthermore, when the analyte is a radioactive substance, the binding partner is preferably a group derived from at least one compound selected from the group consisting of linear polyethers, cyclic ethers, calixarenes, macrocyclic heterocyclic compounds, cyclodextrins, tetraphenylboronic acids, and derivatives thereof, and more preferably a group derived from a compound represented by the following formula (5): In the group derived from a compound represented by the following formula (5), the bond is preferably located at any position on the benzene ring or at any position on the methyl group bonded to the carbonyl group.

[0039] [ka]

[0040] In particular, the binding partner is preferably an antibody or an antigen, since this allows the polymer of the present invention to be suitably used in clinical diagnostic reagents.

[0041] The polymer of the present invention preferably does not have a sugar chain in the polymer main chain, and more preferably does not have a sugar chain, since sugar chains may unexpectedly interact with substances in a sample.

[0042] The polymer of the present invention is preferably hydrophilic. Specifically, the polymer of the present invention has a solubility parameter of 12 to 25 (cal / cc). 1 / 2 It is preferable that the solubility parameter is 12 to 25 (cal / cc) and that the solubility parameter is 12 to 25 (cal / cc). 1 / 2 Dissolving the polymer in a liquid such as this leads to improved analyte detection sensitivity while suppressing precipitation and background fluorescence.

[0043] The polymer of the present invention may be an oligomer or a polymer. Furthermore, the polymer of the present invention preferably has a number-average degree of polymerization of at least 20 to 70,000 and a number-average molecular weight of at least 2,000 to 5,000,000. When at least one of the number-average degree of polymerization and the number-average molecular weight is within the above range, precipitation and background fluorescence can be suppressed, and the analyte detection sensitivity can be improved. A more preferred lower limit of the number average degree of polymerization is 125, and a more preferred upper limit is 14,000. A more preferred lower limit of the number average molecular weight is 10,000, and a more preferred upper limit is 1,000,000. In this specification, the number-average degree of polymerization and the number-average molecular weight are values ​​determined by size exclusion chromatography (SEC) using 150 mM phosphate buffer (pH 7.0) as a solvent and measuring calibration standards (ovalbumin, myoglobin, aprotinin, neurotensin, and angiotensin II). Columns used to measure the number-average molecular weight by SEC include, for example, AdvanceBio SEC (Agilent Technologies).

[0044] Examples of methods for producing the polymer of the present invention include the following methods. That is, first, the polymerizable monomer, the aggregation-induced luminescent compound, and the polymerization initiator are dissolved in a solvent. The resulting solution is then stirred while being heated to obtain a polymer having an aggregation-induced luminescent group introduced therein. Next, a compound having a binding partner corresponding to the type of analyte is added to the resulting aqueous dispersion of the polymer having the aggregation-induced luminescent group introduced therein and reacted to obtain the polymer of the present invention. The condensing agent and the reaction accelerator may be used when adding the compound having the binding partner to cause the reaction.

[0045] Examples of the solvent include N,N-dimethylformamide, ethyl acetate, tetrahydrofuran, acetonitrile, ethanol, methanol, and water.

[0046] Examples of the polymerization initiator include oil-soluble initiators such as azobisisobutyronitrile and benzoyl peroxide, and water-soluble initiators such as potassium persulfate and ammonium persulfate.

[0047] The polymer of the present invention is preferably used in a test agent. A test agent containing the polymer of the present invention also constitutes the present invention.

[0048] The diagnostic agent of the present invention can be suitably used as a clinical diagnostic agent in various methods that utilize biological reactions, such as enzyme immunoassay, fluorescent immunoassay, and immunochromatography, which utilize antigen-antibody reactions. The test agent of the present invention can also be suitably used for measuring radioactive substances. By using the test agent of the present invention, radioactive substances can be measured easily and at low cost.

[0049] The present invention also provides a method for measuring the concentration of an analyte, comprising the steps of: preparing a mixed solution by mixing a sample solution containing an analyte with a solution containing a polymer of the present invention; measuring the fluorescence intensity emitted from the polymer in the mixed solution; and correlating the fluorescence intensity emitted from the polymer with the analyte concentration in the mixed solution by comparing a calibration curve of fluorescence intensity versus analyte concentration with the fluorescence intensity emitted from the polymer.

[0050] The step of measuring the fluorescence intensity generated from the polymer in the mixed solution preferably includes a step of irradiating the mixed solution with excitation light and a step of measuring the amount of change in the intensity of light emission such as fluorescence or phosphorescence emitted from the mixed solution.

[0051] The analyte concentration measuring device used in the analyte concentration measuring method of the present invention also constitutes the present invention. As the analyte concentration measuring device of the present invention, an automatic analyzer capable of performing measurements quickly and simply is suitable, and an automatic analyzer capable of measuring the intensity of luminescence such as fluorescence or phosphorescence is preferred.

[0052] The light source used in the step of irradiating the mixed liquid with excitation light is not particularly limited. Furthermore, the wavelength of the light irradiated in the step of irradiating the mixed liquid with excitation light is preferably in the ultraviolet region, and more preferably in the range of 10 nm to 400 nm. The above-described automatic analyzer can measure the change in fluorescence intensity at any two time points up to 1000 seconds after mixing the sample solution containing the analyte with the solution containing the polymer of the present invention. In particular, by measuring the change in fluorescence intensity at two time points within 300 seconds after mixing, the total measurement time per sample can be kept to 10 minutes or less, making it possible to take advantage of the maximum sample processing speed of various commercially available automatic analyzers.

[0053] The irradiation angle of light in the step of irradiating the mixed solution with excitation light is preferably 15 to 35 degrees. By setting the irradiation angle within this range, the light receiving unit for detecting fluorescence is not strongly affected by transmitted light, and this is also advantageous in terms of the ability to receive fluorescence. The irradiation angle is more preferably 20 to 30 degrees.

[0054] The amount of change in the fluorescence intensity is not particularly limited as long as it is calculated by an applicable method, such as the difference or ratio between two time points, or a converted value per unit time.

[0055] In the step of correlating the fluorescence intensity emitted from the polymer with the analyte concentration in the mixed solution, it is preferable to use a calibration curve of fluorescence intensity prepared using samples containing analyte of known concentration. For measurements of fluorescence intensity with a wide dynamic range, it is preferable to prepare a calibration curve over a wider concentration range. In the analyte concentration measurement method of the present invention, good accuracy and reproducibility of the measured values ​​of low concentrations of analyte are indicative of high sensitivity. The dynamic range of the analyte concentration measurement method of the present invention is the range in which a change in light intensity proportional to the analyte concentration can be detected. [Effects of the Invention]

[0056] The present invention provides a polymer capable of suppressing background fluorescence and enabling analyte measurement with good detection sensitivity, a test agent containing the polymer, a method for measuring analyte concentration using the polymer, and an analyte concentration measurement device used in the method for measuring analyte concentration. DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0058] (Synthesis Example 1) 1 part by weight of 4-(1,2,2-triphenylvinyl)phenyl acrylate, 99 parts by weight of acrylic acid, 5 parts by weight of azobisisobutyronitrile, and 100 parts by weight of N,N'-dimethylformamide were placed in a recovery flask and stirred uniformly. After that, heating was started and the reaction was carried out at 60°C for 18 hours, yielding a polymer into which aggregation-induced luminescent groups had been introduced. The resulting polymer having aggregation-induced luminescence groups introduced therein was dispersed in water, and 2 parts by weight of a PBS solution containing sialylated glycan antigen KL-6 (hereinafter simply referred to as "KL-6") antibody (KL-6 antibody concentration: 0.75 mg / mL) was added to the dispersion. The mixture was then stirred at 25°C for 24 hours to obtain Polymer A. 1 H-NMR and FT-IR measurements revealed that the polymer A contained a group (R 7 is a bond, R 8 ~R 10 is a hydrogen atom) and a group derived from the KL-6 antibody as a binding partner. The obtained polymer A was also measured by SEC using 150 mM phosphate buffer (pH 7.0) as a solvent, and the number average degree of polymerization and number average molecular weight calculated from the measurement results of the calibration standard were 350 and 25,000, respectively.

[0059] (Synthesis Example 2) Polymer B was obtained in the same manner as in Synthesis Example 1, except that the amount of azobisisobutyronitrile added was changed to 1 part by weight. 1 H-NMR and FT-IR measurements revealed that the polymer B contained a group (R 7 is a bond, R 8 ~R 10 is a hydrogen atom) and a group derived from the KL-6 antibody as a binding partner. The obtained polymer B was also measured by SEC using 150 mM phosphate buffer (pH 7.0) as a solvent, and the number average degree of polymerization calculated from the measurement results of the calibration standard was 1,400 and the number average molecular weight was 100,000.

[0060] (Synthesis Example 3) Polymer C was obtained in the same manner as in Synthesis Example 1, except that the amount of azobisisobutyronitrile added was changed to 0.001 parts by weight. 1H-NMR and FT-IR measurements revealed that the polymer C contained a group (R 7 is a bond, R 8 ~R 10 is a hydrogen atom) and a group derived from the KL-6 antibody as a binding partner. The obtained polymer C was also measured by SEC using 150 mM phosphate buffer (pH 7.0) as a solvent, and the number-average degree of polymerization and number-average molecular weight calculated from the measurement results of the calibration standard were 55,000 and 4,000,000, respectively.

[0061] (Synthesis Example 4) Polymer D was obtained in the same manner as in Synthesis Example 1, except that 1 part by weight of 4-(1,2,2-tris(4-hydroxyphenyl)vinyl)phenyl acrylate was used in place of 1 part by weight of 4-(1,2,2-triphenylvinyl)phenyl acrylate. 1 H-NMR and FT-IR measurements revealed that the polymer D contained a group (R 7 is a bond, R 8 ~R 10 It was confirmed that the polymer had a hydroxyl group and a group derived from the KL-6 antibody as a binding partner. The obtained polymer D was also measured by SEC using 150 mM phosphate buffer (pH 7.0) as a solvent, and the number-average degree of polymerization and number-average molecular weight calculated from the measurement results of the calibration standard were 1,350 and 100,000, respectively.

[0062] (Synthesis Example 5) Polymer E was obtained in the same manner as in Synthesis Example 1, except that 1 part by weight of 4-(1,2-bis(4-carboxyphenyl)2-phenylvinyl)phenyl acrylate was used instead of 1 part by weight of 4-(1,2,2-triphenylvinyl)phenyl acrylate. 1H-NMR and FT-IR measurements revealed that the polymer E contained a group (R 7 is a bond, R 8 and R 9 is a carboxyl group, R 10 is a hydrogen atom) and a group derived from the KL-6 antibody as a binding partner. The obtained polymer E was also measured by SEC using 150 mM phosphate buffer (pH 7.0) as a solvent, and the number-average degree of polymerization and number-average molecular weight calculated from the measurement results of the calibration standard were 1,330 and 100,000, respectively.

[0063] (Synthesis Example 6) Polymer F was obtained in the same manner as in Synthesis Example 4, except that the amount of 4-(1,2,2-tris(4-hydroxyphenyl)vinyl)phenyl acrylate added was changed to 5 parts by weight and the amount of acrylic acid added was changed to 95 parts by weight. 1 H-NMR and FT-IR measurements revealed that the polymer F contained a group (R 7 is a bond, R 8 ~R 10 It was confirmed that the polymer had a hydroxyl group and a group derived from the KL-6 antibody as a binding partner. The obtained polymer F was also measured by SEC using 150 mM phosphate buffer (pH 7.0) as a solvent, and the number-average degree of polymerization calculated from the measurement results of the calibration standard was 1,300 and the number-average molecular weight was 100,000.

[0064] (Synthesis Example 7) Polymer G was obtained in the same manner as in Synthesis Example 6, except that 95 parts by weight of acrylic acid was changed to 94 parts by weight of acrylic acid and 1 part by weight of ethylene glycol diacrylate (EGDA). 1H-NMR and FT-IR measurements revealed that the polymer G contained a group (R 7 is a bond, R 8 ~R 10 It was confirmed that the polymer had a hydroxyl group and a group derived from the KL-6 antibody as a binding partner. The obtained polymer G was also measured by SEC using 150 mM phosphate buffer (pH 7.0) as a solvent, and the number-average degree of polymerization and number-average molecular weight calculated from the measurement results of the calibration standard were 1,350 and 100,000, respectively.

[0065] (Synthesis Example 8) The polymer having aggregation-induced luminescent groups introduced therein, obtained in the same manner as in Synthesis Example 6, was designated as Polymer H. 1 H-NMR and FT-IR measurements revealed that the polymer H contained a group (R 7 is a bond, R 8 ~R 10 It was confirmed that the polymer has a hydroxyl group and does not have a binding partner capable of binding to the KL-6 antigen. The obtained polymer H was also measured by SEC using 150 mM phosphate buffer (pH 7.0) as the solvent, and the number-average degree of polymerization and number-average molecular weight calculated from the measurement results of the calibration standard were 1,300 and 100,000, respectively.

[0066] (Synthesis Example 9) 100 parts by weight of water was mixed with 3.6 parts by weight of styrene and 0.136 parts by weight of V-50 (Fujifilm Wako Pure Chemical Industries, Ltd.) as a polymerization initiator. The resulting mixture was stirred at 60°C for 4 hours, after which 0.375 parts by weight of 2-chloropropionyloxyethyl methacrylate was added and further stirred at 60°C for 6 hours. The resulting solution was filtered and then purified by centrifugation to obtain core particles. The resulting core particles were dispersed in water to a content of 1.0 wt% to obtain a dispersion. To 30 parts by weight of the resulting dispersion, 0.517 parts by weight of methacrylic acid and 21.1 parts by weight of ascorbic acid as a reducing agent were added in the presence of copper(I) chloride / tris[2-(dimethylamino)ethyl]amine as a metal complex, and the mixture was stirred at 30°C for 2 hours. The resulting solution was purified by centrifugation, and organic graft chains were attached to the surfaces of the core particles. 1.0 part by weight of the obtained particles was dispersed in ethylene glycol, 0.578 part by weight of 4-(1,2,2-triphenylvinyl)phenol and 0.28 part by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and the mixture was stirred at room temperature for 6 hours. The obtained dispersion was purified by centrifugation, and aggregation-induced luminescent groups were introduced into the organic graft chains. The resulting particles with aggregation-induced luminescent groups introduced into the organic graft chains were dispersed in water to a content of 0.5 wt% to obtain a dispersion. 20 parts by weight of a PBS solution containing KL-6 antibody (KL-6 antibody concentration: 0.75 mg / mL) was added to 10 parts by weight of the resulting dispersion, and the mixture was stirred at room temperature for 24 hours. The resulting solution was purified by centrifugation to obtain aggregated luminescent material-containing particles with the aggregated luminescent material on their surfaces.

[0067] (Synthesis Example 10) Polystyrene particles with an average particle size of 0.15 μm were prepared as seed particles. An emulsion was prepared by mixing 0.5 parts by weight of tetraphenylethylene as an aggregation-induced luminescent compound, 5 parts by weight of ethyl acetate, 0.1 parts by weight of sodium styrenesulfonate, and 10 parts by weight of ion-exchanged water. The emulsion was added to a dispersion of 1 part by weight of the seed particles, and the mixture was stirred at room temperature for 24 hours to obtain a dispersion of swollen particle droplets of seed particles that had absorbed tetraphenylethylene and ethyl acetate. The resulting dispersion of swollen particle droplets was stirred at 65° C. for 24 hours to dry the ethyl acetate, thereby obtaining aggregated particles containing a light-emitting material.

[0068] (Synthesis Example 11) A PBS solution containing sodium dodecyl sulfate (SDS) and KL-6 antibody (KL-6 antibody concentration: 0.75 mg / mL) was used to prepare a PBS solution containing 1.0 × 10 -2 A PBS solution (hereinafter also referred to as "SDS solution") with a TPS concentration of 1.0 × 10 mol / L was prepared using 2,3,4,5-tetraphenyl-1,1-dimethylsilole (TPS) and methanol. -3 A 100 mol / L methanol solution (hereinafter also referred to as "TPS solution") was prepared. 0.1 mL of the TPS solution was added to 10 mL of the SDS solution, and the mixture was stirred for 1 hour, and then allowed to stand overnight in a cool, dark place to obtain aggregated micelles encapsulating the light-emitting material.

[0069] (Reference example 1~ 4、6、7 , Example 5 , Comparative Examples 1 to 4) Aqueous solutions or aqueous dispersions containing polymers A to H obtained in Synthesis Examples 1 to 8, aggregated light-emitting material-containing particles obtained in Synthesis Examples 9 and 10, and aggregated light-emitting material-encapsulating micelles obtained in Synthesis Example 11 at the concentrations shown in Table 1 were obtained.

[0070] <Evaluation> Reference Examples, Examples, and 、 The aqueous solutions or dispersions obtained in the comparative examples were evaluated as follows, and the results are shown in Table 1.

[0071] (fluorescence intensity) The KL-6 antigen was added as an analyte to a buffer solution containing bovine serum albumin and stirred to prepare a sample solution containing the analyte (analyte concentration: 100 U / mL). Reference Examples, Examples, and 、 The resulting mixture was mixed with 10 parts by weight of each of the aqueous solutions or aqueous dispersions obtained in the comparative examples, and the resulting mixture was shaken for 1 minute using a wave rotor. The fluorescence intensity of the mixture before shaking (immediately after mixing) was defined as the fluorescence intensity before the antigen-antibody reaction, and the fluorescence intensity of the mixture after shaking was defined as the fluorescence intensity after the antigen-antibody reaction.The fluorescence intensities of each were measured using an F-2700 (Hitachi High-Tech Science Corporation).

[0072] [Table 1]

[0073] Examples 4 to 6 show that by converting the functional group on the tetraphenylethylene into a hydrophilic group, the solubility of the polymer in water can be improved while keeping the background low, and as a result, the frequency of interaction between the analyte and the binding partner increases, and more polymers aggregate, thereby increasing the fluorescence intensity after the antigen-antibody reaction. [Industrial Applicability]

[0074] The present invention provides a polymer capable of suppressing background fluorescence and enabling analyte measurement with good detection sensitivity, a test agent containing the polymer, a method for measuring analyte concentration using the polymer, and an analyte concentration measurement device used in the method for measuring analyte concentration.

Claims

1. a polymer having aggregation-induced luminescent groups on a polymer backbone and a binding partner on the polymer backbone capable of binding to an analyte; The aggregation-induced luminescent group is a group represented by the following formula (2) containing a carboxyl group as a hydrophilic group: A polymer characterized by: 【Chemistry 1】 In formula (2), any one of R 7 to R 10 is a bond, and those of R 7 to R 10 that are not bonds may be the same or different and represent a saturated or unsaturated hydrocarbon group having 1 to 6 carbon atoms, which may have a substituent, a hydrogen atom, a hydroxyl group, a halogen atom, an amino group, a carboxyl group, or a nitro group, and at least one of R 7 to R 10 that are not bonds is a carboxyl group.

2. The polymer of claim 1 , wherein the polymer backbone is linear.

3. 3. The polymer according to claim 1, which satisfies at least one of a number average degree of polymerization of 20 to 70,000 and a number average molecular weight of 2,000 to 5,000,000.

4. 4. The polymer according to claim 1, 2 or 3, which does not have a sugar chain in the polymer main chain.

5. The polymer according to claim 4, which does not have a sugar chain.

6. 6. The polymer of claim 1, 2, 3, 4 or 5, wherein the binding partner is an antibody or an antigen.

7. A test agent comprising the polymer according to claim 1, 2, 3, 4, 5 or 6.

8. a step of mixing a sample solution containing an analyte with a solution containing the polymer according to claim 1 to prepare a mixed solution; measuring the intensity of fluorescence emitted from the polymer in the mixed solution; comparing the fluorescence intensity emitted from the polymer with a calibration curve of fluorescence intensity versus analyte concentration to correlate the fluorescence intensity emitted from the polymer with the analyte concentration in the mixed solution; A method for measuring analyte concentration comprising:

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