WO2018181796A1 - 生体試料中の測定対象物質を測定するためのキット及び方法 - Google Patents
生体試料中の測定対象物質を測定するためのキット及び方法 Download PDFInfo
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Images
Classifications
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
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- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
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- C07F5/02—Boron compounds
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
Definitions
- the present invention relates to a kit for measuring a substance to be measured in a biological sample and a method for measuring the substance to be measured in a biological sample.
- Fluorescence detection is widely used as a highly sensitive and easy measurement method for quantifying proteins, enzymes, inorganic compounds, and the like.
- the presence of a measurement target substance is detected by detecting the fluorescence emitted when the sample that is considered to contain the measurement target substance that is excited by light of a specific wavelength and emits fluorescence is irradiated with the excitation light of the specific wavelength. It is a method to confirm.
- the substance to be measured is not a fluorescent substance
- a substance that specifically binds to the substance to be measured is labeled with a fluorescent dye, contacted with the sample and then irradiated with excitation light in the same manner as described above
- excitation light By detecting the fluorescence emitted from the light, the presence of the substance to be measured can be confirmed.
- a method using the effect of electric field enhancement by plasmon resonance is known in order to improve the sensitivity for detecting a measurement target substance present in a minute amount.
- a sensor chip provided with a metal layer in a predetermined region on a transparent support is prepared, and a metal layer forming surface of the support is formed with respect to the interface between the support and the metal film.
- Excitation light is incident at a predetermined angle equal to or greater than the total reflection angle from the opposite surface side.
- the fluorescence detection method by surface plasmon excitation (hereinafter referred to as “SPF method”) has a signal enhancement of about 10 times that of the fluorescence detection method by epi-illumination excitation (also referred to as epi-illumination fluorescence method), and is measured with high sensitivity. can do.
- Patent Document 1 describes fluorescent microparticles produced by blending an initial donor dye having a preferable excitation peak and a final acceptor dye having a preferable emission peak into polymer microparticles. Patent Document 1 describes that a polyazaindacene dye is used as the dye.
- Non-Patent Document 1 the novel distyryl BODIPY R (R stands for boron-dipyrromethene) is described that is designed and synthesized dyes, the synthesized Jisuchiririru BODIPY R dye, absorbed in chloromethane solution And the emission spectrum is analyzed.
- R stands for boron-dipyrromethene
- the SPF method is known as a method capable of performing high-sensitivity measurement with a simple measurement method, but is not sufficiently satisfactory for measurement of a very small amount of a measurement target substance. It was.
- the detection methods in competitive methods that measure small molecules that cannot be sandwiched by antibodies, it is necessary to reduce the amount of particles in the reaction system in order to increase detection sensitivity, but in that case, the fluorescence intensity is insufficient. There was a problem that the high concentration range could not be measured accurately.
- the fluorescent microparticles described in Patent Document 1 have a preferable effective Stokes shift, but have a problem that the quantum yield is low.
- the absorption and emission spectra of the dye solution are analyzed, but there is no description about incorporating the dye into the particles.
- the present invention has an object to be solved by providing a kit and a method capable of realizing a highly accurate measurement of a measurement target substance in a biological sample in a wide concentration range from a low concentration to a high concentration.
- the present inventors have found that a labeled particle having a first binding substance having a binding property to a measurement target substance, and either the measurement target substance or the first binding substance In a kit including a substrate having a second binding substance having binding properties, by using a labeled particle having a maximum emission wavelength in a long wavelength region of 680 nm or more and exhibiting a high quantum yield as the labeled particle.
- the present inventors have found that the above problems can be solved.
- the present invention has been completed based on these findings. That is, according to the present invention, the following inventions are provided.
- R 11 to R 15 are each independently a hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group, alkylthio group.
- arylthio group which may have a substituent, and at least three of R 11 to R 15 represent an atom or group other than a hydrogen atom.
- X 1 and X 2 each independently represent a halogen atom, alkyl group, aryl group, heterocyclic group, hydroxy group, alkoxy group, aryloxy group, alkylthio group, arylthio group, ethenyl group, or ethynyl group, It may have a substituent, and X 1 and X 2 may be linked to each other to form a ring.
- Ar 1 and Ar 2 each independently represents an aryl group or a heterocyclic group, and these may have a substituent.
- L 1 and L 2 each independently represents any one of formulas (L-1) to (L-4).
- each of R 111 to R 116 is independently a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, an amino group, an acyl group, an alkoxy group, an aryloxy group, or an alkylthio group. Or an arylthio group, which may have a substituent.
- A represents —O—, —S—, or —NH—.
- ⁇ 2> The kit according to ⁇ 1>, wherein the labeled particles are labeled latex particles.
- ⁇ 3> The kit according to ⁇ 1> or ⁇ 2>, wherein the labeled particles have a carboxyl group.
- ⁇ 4> The kit according to any one of ⁇ 1> to ⁇ 3>, wherein the labeled particles have an average particle diameter of 70 to 500 nm.
- ⁇ 5> The kit according to any one of ⁇ 1> to ⁇ 4>, wherein the compound represented by the formula (1) is a compound represented by the following formula (3).
- R 11 , R 12 , R 14 , R 15 , X 1 , X 2 , Ar 1 , Ar 2 , L 1 and L 2 are as defined in formula (1), provided that R 11 , At least two of R 12 , R 14 and R 15 are atoms or groups other than hydrogen atoms.
- R 31 to R 35 are each independently a hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, cyano group, alkoxy group, aryloxy group, alkylthio group Or an arylthio group, which may have a substituent, and any one of R 31 , R 32 , R 34 and R 35 is a group consisting of two or more atoms.
- R 12 , R 13 , R 14 , X 1 , X 2 , Ar 1 , Ar 2 , L 1 and L 2 have the same definitions as in formula (1), provided that R 12 , R 13 and At least one of R 14 is an atom or group other than a hydrogen atom.
- R 41 and R 42 each independently represents an aryl group, a heterocyclic group, an ethenyl group, or an ethynyl group, and these may have a substituent.
- R 11 to R 15 , X 1 , X 2 , L 1 and L 2 have the same definitions as in formula (1).
- R 51 and R 52 each independently represents an alkyl group, aryl group, heteroaryl group, amino group, acyl group, alkoxy group, aryloxy group, alkylthio group, or arylthio group, and these have a substituent. May be.
- Q 1 and Q 2 each independently represents an aromatic hydrocarbon ring or an aromatic hetero ring, which may have a substituent.
- the labeling particle is a luminescent particle containing at least one energy donor compound, at least one energy acceptor compound, and particles, and is at least one of the energy donor compound and the energy acceptor compound.
- the energy donor compound contains at least one compound represented by the formula (1), and the energy acceptor compound contains at least one compound represented by the formula (1).
- the kit according to ⁇ 8>. ⁇ 10> The kit according to ⁇ 8> or ⁇ 9>, wherein the molar ratio of the energy donor compound to the energy acceptor compound is 1:10 to 10: 1.
- ⁇ 11> The kit according to any one of ⁇ 8> to ⁇ 10>, wherein the Stokes shift between the energy donor compound and the energy acceptor compound is 40 nm or more.
- ⁇ 12> The kit according to any one of ⁇ 1> to ⁇ 11>, wherein the substrate has a detection region having the second binding substance.
- the detection region is a metal film containing gold.
- a method for measuring a substance to be measured in a biological sample A reaction step of reacting a biological sample with a labeled particle having a first binding substance having binding properties with a measurement target substance, and a second binding having binding ability with either the measurement target substance or the first binding substance
- the labeled particles are luminescent labeled particles containing at least one compound represented by the following formula (1) and particles.
- R 11 to R 15 are each independently a hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group, alkylthio group. Or an arylthio group, which may have a substituent, and at least three of R 11 to R 15 represent an atom or group other than a hydrogen atom.
- X 1 and X 2 each independently represent a halogen atom, alkyl group, aryl group, heterocyclic group, hydroxy group, alkoxy group, aryloxy group, alkylthio group, arylthio group, ethenyl group, or ethynyl group, It may have a substituent, and X 1 and X 2 may be linked to each other to form a ring.
- Ar 1 and Ar 2 each independently represents an aryl group or a heterocyclic group, and these may have a substituent.
- L 1 and L 2 each independently represents any one of formulas (L-1) to (L-4).
- each of R 111 to R 116 is independently a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, an amino group, an acyl group, an alkoxy group, an aryloxy group, or an alkylthio group. Or an arylthio group, which may have a substituent.
- A represents —O—, —S—, or —NH—.
- ⁇ 15> The method according to ⁇ 14>, wherein the particles are latex particles.
- ⁇ 16> The method according to ⁇ 14> or ⁇ 15>, wherein the particles have a carboxyl group.
- ⁇ 17> The method according to any one of ⁇ 14> to ⁇ 16>, wherein the labeled particles have an average particle diameter of 70 to 500 nm.
- ⁇ 18> The method according to any one of ⁇ 14> to ⁇ 17>, wherein the compound represented by the formula (1) is a compound represented by the following formula (3).
- R 11 , R 12 , R 14 , R 15 , X 1 , X 2 , Ar 1 , Ar 2 , L 1 and L 2 are as defined in formula (1), provided that R 11 , At least two of R 12 , R 14 and R 15 are atoms or groups other than hydrogen atoms.
- R 31 to R 35 are each independently a hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, cyano group, alkoxy group, aryloxy group, alkylthio group Or an arylthio group, which may have a substituent, and any one of R 31 , R 32 , R 34 and R 35 is a group consisting of two or more atoms.
- R 12 , R 13 , R 14 , X 1 , X 2 , Ar 1 , Ar 2 , L 1 and L 2 have the same definitions as in formula (1), provided that R 12 , R 13 and At least one of R 14 is an atom or group other than a hydrogen atom.
- R 41 and R 42 each independently represents an aryl group, a heterocyclic group, an ethenyl group, or an ethynyl group, and these may have a substituent.
- ⁇ 20> The method according to any one of ⁇ 14> to ⁇ 17>, wherein the compound represented by the formula (1) is a compound represented by the following formula (5).
- R 11 to R 15 , X 1 , X 2 , L 1 and L 2 have the same definitions as in formula (1).
- R 51 and R 52 each independently represents an alkyl group, aryl group, heteroaryl group, amino group, acyl group, alkoxy group, aryloxy group, alkylthio group, or arylthio group, and these have a substituent. May be.
- Q 1 and Q 2 each independently represents an aromatic hydrocarbon ring or an aromatic hetero ring, which may have a substituent.
- the luminescent particles, wherein the labeling particles include at least one energy donor compound, at least one energy acceptor compound, and particles, wherein the energy donor compound and the energy acceptor compound are at least one kind.
- the energy donor compound contains at least one compound represented by the formula (1), and the energy acceptor compound contains at least one compound represented by the formula (1).
- ⁇ 23> The method according to ⁇ 21> or ⁇ 22>, wherein the molar ratio of the energy donor compound and the energy acceptor compound is 1:10 to 10: 1.
- ⁇ 24> The method according to any one of ⁇ 21> to ⁇ 23>, wherein the Stokes shift between the energy donor compound and the energy acceptor compound is 40 nm or more.
- ⁇ 25> The method according to any one of ⁇ 14> to ⁇ 24>, wherein the substrate has a detection region having the second binding substance.
- the detection region is a metal film containing gold.
- ⁇ 27> The method according to ⁇ 26>, wherein label information related to the measurement target substance is obtained by fluorescence detection by surface plasmon excitation.
- kit and method of the present invention it is possible to realize a highly accurate measurement of a substance to be measured in a biological sample in a wide concentration range from a low concentration to a high concentration.
- FIG. 1 shows a 400 MHz 1 H NMR spectrum of compound (4).
- FIG. 2 shows a 400 MHz 1 H NMR spectrum of compound (7).
- FIG. 3 shows a schematic diagram of the sensor chip.
- FIG. 4 shows an exploded view of the sensor chip.
- a numerical range indicated by using “to” means a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
- a kit for measuring a substance to be measured in a biological sample includes a labeled particle having a first binding substance having binding properties with a substance to be measured in a biological sample, and the substance to be measured or the first binding substance. And a substrate having a second binding substance having binding properties, wherein the labeling particles include at least one compound represented by the formula (1) and particles described later and a luminescent label Particles.
- the biological sample is not particularly limited as long as it is a sample that may contain a substance to be measured.
- a biological sample particularly an animal (eg, human, dog, cat, horse, etc.) Examples include body fluids (for example, blood, serum, plasma, spinal fluid, tears, sweat, urine, pus, runny nose, or sputum) or excretions (for example, feces), organs, tissues, mucous membranes, and skin.
- the substance to be measured is not particularly limited.
- thyroxine (T4) triiodothyronine (T3), estradiol (E2), aldosterone, symmetric dimethylarginine (SDMA), bile acid, cortisol, cholesterol, corticostero , Progesterone, testosterone, estrogen, vitamins, creatinine, amino acids, ⁇ -carotene, creatinine, digoxin, theophylline, folic acid, proteins such as inflammatory markers and sepsis markers.
- Progesterone is a sex hormone that is secreted from the ovary and placenta and is related to luteal function and pregnancy. Used to diagnose menstrual cycle abnormalities and infertility. It is also used for checking the mating time of dogs and the remains of cat ovaries.
- the first binding substance used in the present invention is a substance having binding properties with the measurement target substance.
- an antigen, an antibody, or a complex thereof can be used, but is not limited thereto.
- the first binding substance is an antibody.
- the first binding substance is an antibody, for example, an antiserum prepared from the serum of an animal immunized with the measurement target substance or an immunity purified from the antiserum as an antibody having a binding property to the measurement target substance Globulin fraction, monoclonal antibodies obtained by cell fusion using spleen cells of animals immunized with the substance to be measured, or fragments thereof [eg, F (ab ′) 2 , Fab, Fab ′, or Fv], etc. Can be used. These antibodies can be prepared by a conventional method. Furthermore, the antibody may be modified as in the case of a chimeric antibody, or may be a commercially available antibody or an antibody prepared from animal serum or culture supernatant by a known method.
- an anti-progesterone antibody having a binding property to progesterone preferably specifically recognizing progesterone is used as the first binding substance.
- a progesterone-BSA conjugate can be prepared by mixing progesterone, bovine serum albumin (hereinafter abbreviated as BSA), and a condensing agent.
- BSA bovine serum albumin
- the conjugate is used as a mouse immunization antigen and immunized subcutaneously on the back of the mouse several times.
- complete adjuvant Complete Fres' Adjuvant: CFA
- IFA incomplete adjuvant
- a complete adjuvant is a substance that stimulates immunity and is a mixture of paraffin and aracel.
- the incomplete adjuvant is obtained by adding dead mycobacteria or Mycobacterium tuberculosis killed to the complete adjuvant to further enhance the antigenicity.
- blood is collected from mice and the antibody titer is measured.
- an antigen is administered intraperitoneally and the spleen is removed several days later.
- myeloma myeloma
- a fused cell having an antibody-producing ability can be produced. From these fused cells, only antibody-producing cells against the target antigen are selected, and further limiting dilution is performed to grow only the cell lines.
- the diluted cells can be cultured (cloned).
- the fusion cell line thus obtained into the peritoneal cavity of a mouse and proliferating ascites-type antibody-producing cells, it becomes possible to produce monoclonal antibodies in ascites, and recover these antibodies.
- the target antibody can be obtained.
- label particles are luminescent labeling particles containing at least one compound represented by the following formula (1) and particles, and are also called fluorescent labeling particles.
- the meaning of each symbol in formula (1) is as defined in this specification.
- the normal dye compound is affected by the association when the amount of incorporation into the particle is increased, and the quantum yield decreases (this is also called concentration quenching).
- concentration quenching a fluorescent dye compound having an absorption wavelength of 650 nm or longer is easy to quench the concentration when it is incorporated into particles, and it is difficult to maintain the quantum yield.
- the compound represented by the formula (1) used in the present invention can emit light at a long wavelength by having a conjugated substituent, and in a polymer particle by having a plurality of substituents on the dipyrromethene skeleton. It is possible to suppress a decrease in the quantum yield. As a factor for suppressing the decrease in quantum yield, suppression of intermolecular interaction (for example, ⁇ - ⁇ interaction) by a plurality of substituents extending in a direction perpendicular to the dipyrromethene skeleton can be considered. According to the compound represented by the formula (1), it is possible to produce luminescent label particles (preferably fluorescent particles, more preferably fluorescent nanoparticles) having a high luminance particularly in a long wavelength region.
- luminescent label particles preferably fluorescent particles, more preferably fluorescent nanoparticles
- the luminance is the fluorescence intensity.
- the emission quantum yield is high in the biological window region (near infrared wavelength range of 650 to 900 nm, which is easily transmitted through the living organism), it is possible to improve the sensitivity of sensing using emission. is there.
- the alkyl group may be linear, branched, cyclic, or a combination thereof, and the linear or branched alkyl group preferably has 1 to 36 carbon atoms, more preferably 1 to 36 carbon atoms. 18, more preferably 1 to 12, and particularly preferably 1 to 6.
- the cyclic alkyl group include cycloalkyl having 3 to 8 carbon atoms.
- Specific examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, and n-hexyl.
- the aryl group is preferably an aryl group having 6 to 48 carbon atoms, more preferably an aryl group having 6 to 24 carbon atoms, still more preferably an aryl group having 6 to 14 carbon atoms, Examples thereof include a phenyl group, a naphthyl group, an anthryl group, a pyrenyl group, a phenanthrenyl group, a biphenyl group, and a fluorenyl group.
- the heterocyclic group is preferably a 5- to 7-membered substituted or unsubstituted, saturated or unsaturated, aromatic or non-aromatic, monocyclic or condensed heterocyclic group.
- the heterocyclic group is preferably a heterocyclic group in which the ring-constituting atoms are selected from a carbon atom, a nitrogen atom, an oxygen atom and a sulfur atom, and have at least one heteroatom of any one of a nitrogen atom, an oxygen atom and a sulfur atom.
- a 5- or 6-membered aromatic heterocyclic group having 3 to 30 carbon atoms.
- heterocyclic group examples include a furyl group, a benzofuryl group, a dibenzofuryl group, a thienyl group, a benzothienyl group, a dibenzothienyl group, a pyridyl group, a pyrimidinyl group, a quinolyl group, an isoquinolyl group, an acridinyl group, a phenanthridinyl group, Pteridinyl group, pyrazinyl group, quinoxalinyl group, pyrimidinyl group, quinazolyl group, pyridazinyl group, cinnolinyl group, phthalazinyl group, triazinyl group, oxazolyl group, benzoxazolyl group, thiazolyl group, benzothiazolyl group, imidazolyl group, benzimidazolyl group, pyrazolyl group , Indazolyl group, is
- the acyl group is preferably a linear or branched alkanoyl group having 2 to 15 carbon atoms, such as an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a valeryl group, an isovaleryl group, and a pivaloyl group. , Hexanoyl group, heptanoyl group, benzoyl group and the like.
- the alkoxy group is preferably an alkoxy group having 1 to 20 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, an n-butoxy group, a pentyloxy group, a hexyloxy group, a heptyloxy group. Group and the like.
- the aryloxy group is preferably an aryloxy group having 6 to 14 carbon atoms, and examples thereof include a phenoxy group, a naphthoxy group, and an anthryloxy group.
- the alkylthio group is preferably an alkylthio group having 1 to 30 carbon atoms, and examples thereof include a methylthio group, an ethylthio group, and an n-hexadecylthio group.
- the arylthio group is preferably an arylthio group having 6 to 30 carbon atoms, and examples thereof include a phenylthio group, a p-chlorophenylthio group, and an m-methoxyphenylthio group.
- examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
- the aromatic ring is an aromatic hydrocarbon ring such as a benzene ring, naphthalene ring, anthracene ring, phenanthrene ring, pyrene ring, perylene ring and terylene ring; an indene ring, an azulene ring, a pyridine ring, a pyrazine ring, Pyrimidine ring, pyrazole ring, pyrazolidine ring, thiazolidine ring, oxazolidine ring, pyran ring, chromene ring, pyrrole ring, pyrrolidine ring, benzimidazole ring, imidazoline ring, imidazolidine ring, imidazole ring, pyrazole ring, triazole ring, triazine ring, Diazole ring, indoline ring, thiophene ring, thienothiophene ring, furan
- aromatic rings an aromatic ring and a condensed ring including an aromatic ring
- the aromatic ring may have a substituent
- aromatic ring means both an aromatic ring having a substituent and an aromatic ring having no substituent.
- substituents described in Substituent group A described later include substituents described in Substituent group A described later.
- an amino group includes an amino group; an alkyl-substituted amino group such as a mono or dimethylamino group, a mono or diethylamino group, and a mono or di (n-propyl) amino group; a mono or diphenylamino group and a mono or di
- An amino group substituted with an aromatic residue such as a naphthylamino group; an amino group substituted with one alkyl group such as a monoalkylmonophenylamino group and an aromatic residue; benzylamino group, acetylamino group, phenylacetyl An amino group etc. are mentioned.
- the aromatic residue means a group obtained by removing one hydrogen atom from an aromatic ring, and the aromatic ring is as described above in the present specification.
- the alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group, alkylthio group, or arylthio group represented by R 11 to R 15 has a substituent.
- the substituent may be the substituent described in Substituent Group A below.
- Substituent group A Sulfamoyl group, cyano group, isocyano group, thiocyanato group, isothiocyanato group, nitro group, nitrosyl group, halogen atom, hydroxy group, amino group, mercapto group, amide group, alkoxy group, aryloxy group, alkylthio group, arylthio group, carbamoyl Groups, acyl groups, aldehyde groups, carbonyl groups, aryl groups, alkyl groups, alkyl groups substituted with halogen atoms, ethenyl groups, ethynyl groups, silyl groups, and trialkylsilyl groups (such as trimethylsilyl groups).
- the alkyl group, aryl group, heterocyclic group, hydroxy group, alkoxy group, aryloxy group, alkylthio group, arylthio group, ethenyl group, or ethynyl group represented by X 1 and X 2 may have a substituent.
- substituents include those described in Substituent Group A.
- the aryl group or heterocyclic group represented by Ar 1 and Ar 2 may have a substituent, and examples of the substituent include the substituents described in Substituent Group A.
- the alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group, alkylthio group, or arylthio group represented by R 111 to R 116 has a substituent.
- examples of the substituent include the substituents described in the substituent group A.
- R 11 to R 15 are each independently a hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group Represents an alkylthio group or an arylthio group, and these may have a substituent.
- At least 3 of R 11 ⁇ R 15 represents an atom or group other than a hydrogen atom, preferably an at least four atoms or groups other than hydrogen atom of R 11 ⁇ R 15, more preferably R 11 ⁇ All of R 15 represent atoms or groups other than hydrogen atoms.
- R 11 and R 15 may be the same or different atoms or groups, but are preferably the same atoms or groups.
- R 12 and R 14 may be the same or different atoms or groups, but are preferably the same atoms or groups.
- R 11 and R 15 preferably represent a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, or an ethynyl group, and these may have a substituent.
- R 12 and R 14 preferably represent an alkyl group, and these may have a substituent.
- R 13 preferably represents an aryl group, which may have a substituent.
- X 1 and X 2 are each independently a halogen atom, alkyl group, aryl group, heterocyclic group, hydroxy group, alkoxy group, aryloxy group, alkylthio group, arylthio group, ethenyl group, or ethynyl. Represents a group, and these may have a substituent, and X 1 and X 2 may be linked to each other to form a ring.
- X 1 and X 2 preferably represent a halogen atom or an alkoxy group.
- X 1 and X 2 are more preferably a fluorine atom, a methoxy group, an ethoxy group, an isopropyloxy group, or a t-butyloxy group, and these are also preferably substituted with a fluorine atom or an alkoxy group.
- Ar 1 and Ar 2 each independently represent an aryl group or a heterocyclic group, and these may have a substituent.
- L 1 and L 2 each independently represent any of formula (L-1) to formula (L-4).
- each of R 111 to R 116 is independently a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, an amino group, an acyl group, an alkoxy group, an aryloxy group, or an alkylthio group. Or an arylthio group, which may have a substituent.
- A represents —O—, —S—, or —NH—.
- L 1 and L 2 preferably represents any one of formulas (L-1) or formula (L-2).
- R 111 to R 116 are preferably hydrogen atoms.
- Preferable examples of the compound represented by the formula (1) include a compound represented by the following formula (2).
- R 11 to R 15 , X 1 , X 2 , Ar 1 and Ar 2 have the same definition as in formula (1), and the preferred range is also the same as the preferred range in formula (1).
- L 21 and L 22 each independently represent a group represented by formula (L-1) or formula (L-2).
- R 11 , R 12 , R 14 , R 15 , X 1 , X 2 , Ar 1 , Ar 2 , L 1 and L 2 have the same definitions as in formula (1), and preferred ranges Is the same as the preferable range in Formula (1).
- at least two of R 11 , R 12 , R 14 and R 15 are atoms or groups other than hydrogen atoms, and preferably at least three of R 11 , R 12 , R 14 and R 15 are other than hydrogen atoms. More preferably, R 11 , R 12 , R 14, and R 15 are atoms or groups other than hydrogen atoms.
- R 31 to R 35 are each independently a hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, cyano group, acyl group, alkoxy group, Represents an aryloxy group, an alkylthio group, or an arylthio group, and these may have a substituent (the substituents described in Substituent Group A may be mentioned as the above-mentioned substituents), and R 31 , R 32 , R 34 and R 35 are each a group consisting of two or more atoms.
- the group consisting of two or more atoms is preferably an alkyl group, an aryl group, an ethenyl group, an ethynyl group, an amino group, a cyano group, or an alkoxy group, and more preferably an alkyl group.
- alkyl groups an alkyl group composed of only carbon atoms and hydrogen atoms, and an alkyl group substituted with a halogen atom are preferred, and an alkyl group composed of only carbon atoms having 1 to 6 carbon atoms and a hydrogen atom, a fluorine atom
- An alkyl group substituted with is more preferable, a methyl group, an isopropyl group, a t-butyl group, and a trifluoromethyl group are more preferable, and a methyl group is particularly preferable.
- Preferable examples of the compound represented by the formula (1) include a compound represented by the following formula (4).
- R 12 , R 13 , R 14 , X 1 , X 2 , Ar 1 , Ar 2 , L 1 and L 2 have the same definitions as those in the formula (1). This is the same as the preferred range in (1).
- at least one of R 12 , R 13 and R 14 is an atom or group other than a hydrogen atom, and preferably at least two of R 12 , R 13 and R 14 are atoms or groups other than a hydrogen atom. More preferably, R 12 , R 13 and R 14 are atoms or groups other than hydrogen atoms.
- R 41 and R 42 each independently represents an aryl group, a heterocyclic group, an ethenyl group, or an ethynyl group, and these may have a substituent.
- the substituent of the substituent group A is mentioned.
- R 41 and R 42 are each independently preferably an aryl group, an ethenyl group, or an ethynyl group. From the viewpoint of improving quantum yield, an aryl group is preferable. From the viewpoint of increasing the wavelength, an ethenyl group, An ethynyl group is preferred.
- the aryl group preferably has at least one substituent at the ortho position or the meta position, and more preferably has at least one substituent at the ortho position.
- the number of substituents substituted on the aryl group is preferably 1 to 3, more preferably 2 or 3.
- the substituent substituted on the aryl group is preferably an alkyl group, more preferably a methyl group, an isopropyl group, or a t-butyl group, and even more preferably a methyl group.
- R 11 to R 15 , X 1 , X 2 , L 1 and L 2 have the same definition as in the formula (1), and the preferred range is also the same as the preferred range in the formula (1). is there.
- R 51 and R 52 each independently represents an alkyl group, an aryl group, a heteroaryl group, an amino group, an acyl group, an alkoxy group, an aryloxy group, an alkylthio group, or an arylthio group. It may have a substituent.
- the substituent of the substituent group A is mentioned.
- R 51 and R 52 are each independently preferably an alkyl group or an alkoxy group, more preferably an alkyl group from the viewpoint of improving the quantum yield, a methyl group, an ethyl group, an isopropyl group, t-butyl. It is more preferably a group, and particularly preferably a methyl group.
- an alkoxy group is more preferable, a methoxy group, an ethoxy group, an isopropyloxy group, and a t-butyloxy group are more preferable, and a methoxy group is particularly preferable.
- Q 1 and Q 2 each independently represents an aromatic hydrocarbon ring or an aromatic hetero ring, which may have a substituent.
- the substituent of the substituent group A is mentioned.
- Q 1 and Q 2 are preferably aromatic hydrocarbon rings, more preferably benzene rings, naphthalene rings, anthracene rings, phenanthrene rings, and pyrene rings, and even more preferably benzene rings and naphthalene rings. Particularly preferred is a benzene ring.
- a tolyl group, a xylyl group, and a mesityl group are preferable, and a xylyl group and a mesityl group are more preferable.
- 1 or xylyl group having a methyl group at both ortho position with respect to the binding position with the L 2 with a mesityl group having a methyl group at both ortho and para position with respect to the binding position with the L 1 or L 2 More preferably, it is particularly preferably a mesityl group having a methyl group at both the ortho position and the para position with respect to the bonding position with L 1 or L 2 .
- the compound represented by the formula (5) is more preferably a compound represented by the following formula (6).
- R 11 , R 12 , R 14 and R 15 are each independently a hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, Represents an aryloxy group, an alkylthio group, or an arylthio group, which may have a substituent, and at least two of R 11 , R 12 , R 14, and R 15 are atoms or groups other than a hydrogen atom; .
- X 1 and X 2 each independently represent a halogen atom, alkyl group, aryl group, heterocyclic group, hydroxy group, alkoxy group, aryloxy group, alkylthio group, arylthio group, ethenyl group, or ethynyl group, It may have a substituent, and X 1 and X 2 may be linked to each other to form a ring.
- R 31 to R 35 are each independently a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, an amino group, an acyl group, a cyano group, an alkoxy group, an aryloxy group, or an alkylthio group. Or an arylthio group, which may have a substituent, and any one of R 31 to R 35 is a hydrogen atom.
- R 51 and R 52 each independently represents an alkyl group, aryl group, heteroaryl group, amino group, acyl group, alkoxy group, aryloxy group, alkylthio group, or arylthio group, and these have a substituent. May be.
- Q 1 and Q 2 each independently represents an aromatic hydrocarbon ring or an aromatic hetero ring, which may have a substituent.
- L 1 and L 2 each independently represents any one of formulas (L-1) to (L-4).
- each of R 111 to R 116 is independently a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, an amino group, an acyl group, an alkoxy group, an aryloxy group, or an alkylthio group. Or an arylthio group, which may have a substituent.
- A represents —O—, —S—, or —NH—.
- R 11 and R 15 are each independently preferably an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, or an amino group, and have the same meaning as R 41 and R 42 described above, that is, an aryl group , A heterocyclic group, an ethenyl group, or an ethynyl group, more preferably an aryl group, an ethenyl group, or an ethynyl group. From the viewpoint of improving the quantum yield, an aryl group is more preferable, and from the viewpoint of increasing the wavelength, an ethenyl group and an ethynyl group are more preferable.
- the aryl group preferably has at least one substituent at the ortho position or the meta position, and more preferably has at least one substituent at the ortho position.
- the number of substituents substituted on the aryl group is preferably 1 to 3, more preferably 2 or 3.
- the substituent substituted on the aryl group is preferably an alkyl group, more preferably a methyl group, an isopropyl group, or a t-butyl group, and even more preferably a methyl group.
- the labeling particle may be a labeling particle containing at least one energy donor compound, at least one energy acceptor compound, and particles.
- at least one of the energy donor compound and the energy acceptor compound is used. May be any compound represented by the above formula (1).
- the luminescent labeling particle contains the compound represented by the formula (1) as either one of the energy donor compound and the energy acceptor compound, and the energy donor compound and the energy acceptor.
- a compound represented by the following formula (10) is contained. That is, as the luminescent label particles, the luminescent label particles containing the compound represented by the formula (1) as the energy donor compound and the compound represented by the formula (10) as the energy acceptor compound may be used. Alternatively, it may be a luminescent label particle containing a compound represented by the formula (1) as an energy acceptor compound and a compound represented by the formula (10) as an energy donor compound.
- m1 and m2 each independently represents an integer of 0 to 4, and either m1 or m2 is at least 1 or more.
- M represents a metalloid atom or a metal atom.
- R 1 , R 2 and R 3 each independently represent a hydrogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, an acyl group, an alkoxy group, an aryloxy group, an alkylthio group, or an arylthio group, These may have a substituent.
- Y 1 and Y 2 each independently represent a halogen atom, an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an ethenyl group, or an ethynyl group, which are substituted It may have a group, and Y 1 and Y 2 may be linked to each other to form a ring.
- Ar 11 and Ar 12 each independently represents an aromatic ring which may have a substituent.
- Z 1 and Z 2 each independently represents an aryl group, a heterocyclic group or an amino group, and these may have a substituent.
- m1 and m2 each independently represent an integer of 0 to 4, and preferably both m1 and m2 are 1 or more.
- m1 and m2 may be the same or different integers, but are preferably the same integers.
- m1 and m2 are each independently 1 or 2, more preferably m1 and m2 are both 1 or both 2, and particularly preferably m1 and m2 are both 1.
- M represents a metalloid atom or a metal atom, preferably a metalloid atom, and particularly preferably a boron atom.
- R 1 , R 2 and R 3 are each independently a hydrogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, acyl group, alkoxy group, aryloxy group, alkylthio group, Or it represents an arylthio group, and these may have a substituent.
- R 1 and R 2 are each independently an aryl group or a heterocyclic group, and these may have a substituent.
- R 1 and R 2 may be the same or different, but are preferably the same.
- R 1 and R 2 are not linked to form a ring.
- R 3 is a hydrogen atom, an alkyl group, an aryl group, or a heterocyclic group, and these may have a substituent. More preferably, R 3 is a hydrogen atom.
- Y 1 and Y 2 are each independently a halogen atom, alkyl group, aryl group, heterocyclic group, hydroxy group, alkoxy group, aryloxy group, alkylthio group, arylthio group, ethenyl group, or ethynyl group. These may have a substituent, and Y 1 and Y 2 may be linked to each other to form a ring.
- Y 1 and Y 2 each independently represent a halogen atom, an alkyl group, an aryl group, hydroxy group, alkoxy group, or an aryloxy group, which may have a substituent, Y 1 and Y 2 may be linked to each other to form a ring.
- Y 1 and Y 2 are each independently a halogen atom. More preferably, Y 1 and Y 2 are fluorine atoms. Y 1 and Y 2 may be the same or different, but are preferably the same.
- Ar 1 and Ar 2 each independently represents an aromatic ring which may have a substituent.
- Ar 1 and Ar 2 represent a benzene ring.
- Z 1 and Z 2 are each independently a halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, acyl group, alkoxy group, aryloxy group, alkylthio group, arylthio group Or an amino group, which may have a substituent.
- m1 is 2 or more
- the plurality of Z 1 may be the same group or different groups
- m2 is 2 or more
- the plurality of Z 2 may be the same group or different groups.
- Z 1 and Z 2 each independently represents an aryl group which may have a substituent.
- Z 1 and Z 2 each independently represent a phenyl group, a naphthyl group, or an anthryl group, and these may have a substituent.
- m1 2 or more
- the plurality of Z 1 are the same group.
- m2 2 or more
- the plurality of Z 2 are the same group.
- the compound represented by Formula (2) does not have acidic groups, such as a carboxylic acid group, a phosphoric acid group, and a sulfonic acid group, in a molecule
- Y 1 and Y 2 are each independently a halogen atom, alkyl group, aryl group, heterocyclic group, hydroxy group, alkoxy group, aryloxy group, alkylthio group, arylthio group, ethenyl group, or ethynyl group. These may have a substituent. As said substituent, the substituent of the substituent group A is mentioned.
- Y 1 and Y 2 each independently represent a halogen atom. Particularly preferably, Y 1 and Y 2 are fluorine atoms.
- R 3 represents a hydrogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, or an acyl group, and these may have a substituent.
- R 3 is a hydrogen atom, an alkyl group, an aryl group, or a heterocyclic group, and these may have a substituent. More preferably, R 3 is a hydrogen atom.
- Ar 3 and Ar 4 each independently represent an aryl group or a heterocyclic group, and these may have a substituent. As said substituent, the substituent of the substituent group A is mentioned.
- R 34 to R 41 are each independently a hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, acyl group, alkoxy group, aryloxy group, alkylthio group, An arylthio group or an amino group is represented, and these may have a substituent.
- substituent the substituent of the substituent group A is mentioned.
- R 34 to R 41 is an aryl group which may have a substituent. More preferably, at least one of R 34 to R 37 is an aryl group which may have a substituent, and at least one of R 38 to R 41 may have a substituent. A good aryl group.
- At least one of R 34 to R 41 is a group represented by the formula (11). More preferably, at least one of R 34 to R 37 is a group represented by the formula (11), and at least one of R 38 to R 41 is a group represented by the formula (11). is there.
- R 201 to R 205 are a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, an acyl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, Or an amino group, and at least one of R 201 and R 205 is an atom or group other than a hydrogen atom.
- R 201 and R 202 may be linked together to form a ring
- R 202 and R 203 may be linked together to form a ring
- R 203 and R 204 may be linked together to form a ring.
- R 204 and R 205 may be connected to each other to form a ring.
- At least one of R 34 to R 41 is a group represented by the formula (12). More preferably, at least one of R 34 to R 37 is a group represented by the formula (12), and at least one of R 38 to R 41 is a group represented by the formula (12). is there.
- R 101 represents a hydrogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, or an acyl group, and these may have a substituent. As said substituent, the substituent of the substituent group A is mentioned.
- Ar 101 represents an aryl group or a heterocyclic group, and these may have a substituent.
- Ar 101 and R 101 may be connected to each other to form a ring. It is preferable that the compound represented by Formula (10) does not have acidic groups such as a carboxylic acid group, a phosphoric acid group, and a sulfonic acid group in the molecule.
- the compound having the short wavelength absorption is the energy donor compound
- the compound having the long wavelength absorption is the energy acceptor compound
- the emission of the energy donor compound and the energy acceptor compound If there is any overlap in the absorption, there is a possibility that it can be used in luminescent label particles.
- the maximum wavelength of absorption of the energy acceptor compound is about 10 to 100 nm longer than the absorption wavelength of the energy donor compound. More preferably, the maximum wavelength of absorption of the energy acceptor compound is 10 to 70 nm longer than the absorption wavelength of the energy donor compound.
- the compound represented by the formula (1) has an absorption maximum wavelength of about +30 nm. There is a maximum of light emission, and an emission spectrum exists up to about +100 nm from there, and it is assumed that an energy transfer system can be realized by using an acceptor compound having absorption in the vicinity thereof.
- the absorption wavelength of each compound can be predicted not only by synthesizing and measuring the compound, but also by calculation by Gaussian et al. From the relationship between the calculated values, the combination of the energy donor compound and the energy acceptor compound Can also be estimated.
- the magnitude of the Stokes shift is preferably 25 nm or more, more preferably 30 nm or more, still more preferably 35 nm or more, still more preferably 40 nm or more, and even more preferably 45 nm or more. It is particularly preferably 50 nm or more, and most preferably 60 nm or more.
- the upper limit of the Stokes shift magnitude is not particularly limited, but is generally 150 nm or less.
- the content of the compound represented by the formula (1) with respect to the particles used in the present invention is not particularly limited as long as the effects of the present invention are not impaired. However, it is preferably 0.5 ⁇ mol / g to 400 ⁇ mol / g, more preferably 1 ⁇ mol / g to 300 ⁇ mol / g, still more preferably 2 ⁇ mol / g to 200 ⁇ mol / g, and particularly preferably 3 ⁇ mol / g to 100 ⁇ mol. / G.
- the content of the compounds represented by the formulas (1) to (6) relative to the particles used in the present invention is preferably 0.1% by mass to 30% by mass, more preferably 0.2% by mass to 20% by mass, and further preferably 0.3% by mass to It is 10% by mass, particularly preferably 0.4% by mass to 8% by mass.
- the luminescent labeling particles at least one compound represented by the formulas (1) to (6) is used, but two or more compounds represented by the formulas (1) to (6) are used. Also good. When two or more kinds of compounds represented by the formulas (1) to (6) are used, the total amount is preferably within the above range.
- the molar ratio of the energy donor compound and the energy acceptor compound is preferably 1:10 to 20: 1, and 1:10 to 10: 1. Is more preferably 1: 5 to 10: 1.
- the total amount of the compound represented by the formula (1) used is preferably within the above range.
- compound (3) is obtained using 3,5-bis (trifluoromethyl) benzaldehyde and 2,4-dimethylpyrrole as starting compounds. It can be produced via compound (3-A), compound (3-B), and compound (3-C).
- Compound (1) and Compound (3) are within the scope of the definition of the compound represented by Formula (1).
- a compound having a substituent corresponding to the target compound represented by the desired formula (1) is used as the compound represented by the formula (1) other than the compound (1) and the compound (3). It can manufacture by substituting.
- the compound represented by the formula (10) can be produced by, for example, the synthesis scheme shown below.
- R 1 and Z 1 in the synthesis scheme are the same as the definitions of R 1 and Z 1 in formula (10).
- Compound A-30 can be synthesized by reacting compound A-10 with compound A-20 according to the method described in Macromolecules 2010, 43, 193-200. Next, compound A-30, a compound represented by the formula: Z 1 -B (OH) 2 , and cesium fluoride (CsF) are added to a mixed solution of dimethoxyethane (DME) and water, and evacuated to replace with nitrogen. Repeat deaeration repeatedly.
- DME dimethoxyethane
- Compound D-10 is within the definition of the compound represented by formula (10).
- any one or more of the compound A-10, the compound A-20, and the compound represented by the formula: Z 1 -B (OH) 2 It can be prepared by replacing a compound with the corresponding compound.
- the label particles include particles.
- the material and form of the particles are not particularly limited.
- organic polymer particles such as polystyrene beads or inorganic particles such as glass beads can be used.
- Specific examples of the material of the particles include homopolymers obtained by polymerizing monomers such as styrene, methacrylic acid, glycidyl (meth) acrylate, butadiene, vinyl chloride, vinyl acetate acrylate, methyl methacrylate, ethyl methacrylate, phenyl methacrylate, or butyl methacrylate.
- a latex in which the above homopolymer or copolymer is uniformly suspended may be used.
- the particles include other organic polymer powders, inorganic substance powders, microorganisms, blood cells, cell membrane fragments, liposomes, and microcapsules.
- particles latex particles are preferred.
- latex materials include polystyrene, styrene-acrylic acid copolymer, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylate copolymer, and styrene-styrene sulfonic acid.
- examples thereof include a salt copolymer, a methacrylic acid polymer, an acrylic acid polymer, an acrylonitrile-butadiene-styrene copolymer, a vinyl chloride-acrylic acid ester copolymer, and a polyvinyl acetate acrylate.
- the latex a copolymer containing at least styrene as a monomer is preferable, and a copolymer of styrene and acrylic acid or methacrylic acid is particularly preferable.
- the method for producing the latex is not particularly limited, and the latex can be produced by any polymerization method. However, when an antibody is labeled on a luminescent labeling particle, it is difficult to immobilize the antibody if a surfactant is present.
- non-emulsifier emulsion polymerization that is, a surfactant, etc. Emulsion polymerization without using any emulsifier is preferred.
- the luminescent labeling particle in the present invention has a maximum emission wavelength in a long wavelength region of 680 nm or more and exhibits a high quantum yield.
- the light emission maximum wavelength represents a wavelength at which the absorbance is maximum in the absorption spectrum.
- the emission maximum wavelength of the luminescent labeling particles is preferably 680 nm or more, more preferably 700 nm or more, and particularly preferably 720 nm or more.
- the upper limit of the emission maximum wavelength of the luminescent labeling particles of the present invention is not particularly limited, but is preferably 900 nm or less, more preferably 800 nm or less.
- the emission maximum wavelength of the luminescent labeling particles can be measured using a commercially available fluorescence spectrophotometer, for example, using a fluorescence spectrophotometer RF-5300PC manufactured by Shimadzu Corporation.
- the quantum yield of luminescent label particles is the ratio of the number of photons emitted as fluorescence to the number of photons absorbed by the luminescent label particles.
- the quantum yield of the luminescent labeling particles is preferably 0.25 or more, more preferably 0.4 or more, still more preferably 0.5 or more, and even more preferably 0.6 or more. Yes, particularly preferably 0.7 or more.
- the upper limit of the quantum yield is not particularly limited, but is generally 1.0 or less.
- the quantum yield of the luminescent labeling particles can be measured using a commercially available quantum yield measuring device, for example, using an absolute PL quantum yield measuring device C9920-02 manufactured by Hamamatsu Photonics. can do.
- the average particle diameter of the luminescent labeling particles varies depending on the particle material, the concentration range for measuring the test substance, the measuring instrument, etc., but is preferably in the range of 0.001 to 10 ⁇ m (more preferably 0.01 to 1 ⁇ m).
- the range of 30 to 500 nm is more preferred, the range of 50 to 300 nm is more preferred, the range of 80 to 200 nm is particularly preferred, and the range of 100 to 150 nm is most preferred.
- the average particle diameter of the luminescent marker particles that can be used in the present invention can be measured with a commercially available particle size distribution meter or the like.
- Measurement methods of particle size distribution include optical microscopy, confocal laser microscopy, electron microscopy, atomic force microscopy, static light scattering, laser diffraction, dynamic light scattering, centrifugal sedimentation, electric pulse Measurement methods, chromatography methods, ultrasonic attenuation methods, and the like are known, and devices corresponding to the respective principles are commercially available.
- the method for producing the light-emitting label particles is not particularly limited, but can be produced by mixing at least one compound represented by the formula (1) and particles.
- a compound represented by the formula (1) for example, by adding a compound represented by the formula (1) to particles such as latex particles, luminescent label particles can be produced. More specifically, adding a solution containing the compound represented by formula (1) to a dispersion of particles containing at least one of water and a water-soluble organic solvent (tetrahydrofuran, methanol, etc.) and stirring.
- a water-soluble organic solvent tetrahydrofuran, methanol, etc.
- a dispersion containing the above-described luminescent labeling particles of the present invention may be prepared.
- the dispersion can be produced by dispersing the luminescent marker particles of the present invention in a dispersion medium.
- the dispersion medium include water, an organic solvent, or a mixture of water and an organic solvent.
- the organic solvent alcohols such as methanol, ethanol and isopropanol, and ether solvents such as tetrahydrofuran can be used.
- the solid content concentration of the luminescent label particles in the dispersion is not particularly limited, but is generally 0.1 to 20% by mass, preferably 0.5 to 10% by mass, more preferably 1 to 5%. % By mass.
- the method for immobilizing the first binding substance on the luminescent labeling particles is described in, for example, the protocol attached to JP-A-2000-206115 and Thermo Fisher's FluoSpheres (registered trademark) polystyrene microsphere F8813. Any known method for preparing a reagent for immunoaggregation reaction can be used. In addition, as a principle for immobilizing an antibody as a binding substance on a particle, any of physical adsorption and chemical bond by covalent bond can be adopted.
- the blocking agent ie, the first blocking agent
- the blocking agent that covers the surface of the particle that is not coated with the antibody after the antibody is immobilized on the particle
- examples of the blocking agent include, for example, albumin (such as BSA), skim milk, casein, soy-derived component, and fish-derived component
- albumin such as BSA
- blocking agents for immune reactions including components, polyethylene glycol, and the like, as well as the above substances or substances having the same properties as the above substances can be used.
- These blocking agents can be subjected to pretreatment such as partial modification with heat, acid, alkali, or the like, if necessary.
- an antibody that does not bind to the substance to be measured, or a protein (Protein A, Protein G) that is not used in the test area can also be used.
- a specific method for immobilizing the antibody on the particles is exemplified below.
- An antibody solution adjusted to a concentration of 0.01 to 20 mg / mL is added to and mixed with a solution in which the solid content concentration of the particles is 0.1 to 10% by mass. Stirring is continued at a temperature of 4 to 50 ° C. for 5 minutes to 48 hours. Subsequently, the particles and the solution are separated by centrifugation or other methods to sufficiently remove the antibody that is not bound to the particles contained in the solution. Thereafter, the operation of washing the particles with a buffer solution is repeated 0 to 10 times.
- Components that do not participate in antigen-antibody reaction preferably protein, more preferably globulin, albumin, Block Ace (registered trademark), skim milk, and casein after the operation of mixing particles and antibodies and binding the antibodies to the particles It is desirable to use a blocking agent such as to protect the part of the particle surface where the antibody is not bound.
- Stabilizers are not particularly limited as long as they stabilize antigens and antibodies, such as synthetic or natural polymers such as sucrose and polysaccharides, such as Immunoassay Stabilizer (Advanced Biotechnologies Inc (ABI)) Commercially available stabilizers can also be used.
- the labeled particles having the first binding substance are included in the kit of the present invention, and it is preferable that the labeled particles are included in a container, for example, a cup, which is a part of the kit.
- the measurement target substance in the biological sample and the first binding substance can be bound by injecting the biological sample into a container containing the labeled particles, mixing, and stirring.
- the present invention in order to achieve highly sensitive measurement, it is preferable to employ a measurement method that performs surface plasmon fluorescence (SPF) detection described later.
- SPF surface plasmon fluorescence
- a substrate having a metal film on the surface is not particularly limited as long as surface plasmon resonance can occur.
- a free electron metal such as gold, silver, copper, aluminum, or platinum is used, and gold is particularly preferable.
- gold is particularly preferable.
- the detection area described later is on the gold film.
- the above metals can be used alone or in combination.
- an intervening layer made of chromium or the like may be provided between the substrate and the layer made of metal.
- the thickness of the metal film is arbitrary, but is preferably, for example, 1 nm to 500 nm, and particularly preferably 10 nm to 200 nm. If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. Moreover, when providing the intervening layer which consists of chromium etc., it is preferable that the thickness of the intervening layer is 0.1 nm or more and 10 nm or less.
- the metal film may be formed by a conventional method, for example, sputtering, vapor deposition, ion plating, electroplating, or electroless plating.
- a sputtering method it is preferable to form the metal film by a sputtering method.
- the thickness of the mixed layer of the substrate material and the metal film is not particularly limited as long as sufficient adhesion can be secured, but is preferably 10 nm or less.
- the metal film is preferably disposed on the substrate.
- “arranged on the substrate” means that the metal film is arranged so as to be in direct contact with the substrate, and that the metal film is not directly in contact with the substrate, but through other layers. Including the case where it is arranged.
- optical glass such as BK7 (borosilicate glass) which is a kind of general optical glass, or synthetic resin, specifically polymethyl methacrylate, polyethylene terephthalate, What consists of a transparent material with respect to laser beams, such as a polycarbonate or a cycloolefin polymer, can be used.
- Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.
- a preferred embodiment of the substrate for SPF detection includes a substrate obtained by depositing a gold film on polymethyl methacrylate (PMMA).
- the substrate includes a detection region having a second binding substance having binding properties with either the measurement target substance or the first binding substance.
- the second binding substance is a substance having a binding property with the measurement target substance or a substance having a binding property with the first binding substance.
- a substance having binding properties with the substance to be measured can be used as the second binding substance.
- quantification is performed by a competitive method, a substance having binding properties with the first binding substance can be used as the second binding substance.
- quantification is preferably performed by a competitive method, and a substance having binding properties with the first binding substance is preferably used as the second binding substance.
- the second binding substance is not particularly limited, and preferred examples include an antigen, an antibody, or a complex thereof.
- the second binding substance is an antigen
- the second binding substance is a substance to be measured ( It is particularly preferable to use a substance having a binding property with the first binding substance.
- the second binding substance is preferably a conjugate of the measurement target substance and the carrier.
- a carrier means a substance to which a plurality of molecules of a substance to be measured can bind.
- An example of a preferred carrier is protein and the like, and specific examples include bovine serum albumin.
- the second binding substance is cholic acid and / or albumin conjugate of cholic acid, deoxycholic acid and / or deoxycholic acid / albumin conjugate, and chenodeoxycholic acid and / or chenodeoxycholic acid.
- -It is particularly preferred to include an albumin conjugate.
- the second binding substance is preferably a progesterone / albumin conjugate.
- a method for immobilizing the second binding substance on the substrate is described in, for example, Tech Notes Vol. 2-12 and the like, and any known method for preparing a general ELISA (Enzyme-Linked ImmunoSorbent Assay) reagent can be used.
- surface modification may be performed by arranging a self-assembled monolayer (SAM) on the substrate.
- SAM self-assembled monolayer
- blocking agent As a blocking agent (second blocking agent) that covers the surface of the substrate not coated with the second binding substance after the second binding substance is fixed to the substrate, known substances such as BSA, globulin, skim milk, etc. , Casein, soybean-derived component, fish-derived component, polyethylene glycol, and the like, as well as commercially available blocking agents for immune reactions including the above-mentioned substances or substances having the same properties as the above-mentioned substances can be used. These blocking agents can be subjected to pretreatment such as partial modification with heat, acid, alkali, or the like, if necessary.
- a test area for detecting the presence or absence of a measurement target substance in a biological sample can be provided on the substrate.
- an antigen as a measurement target substance is captured, and the amount of the label bound to the antigen is detected and quantified, whereby the antigen can be quantified.
- the antigen can be quantified by a method in which only the label bound to the antigen cannot be bound, only the label not bound to the antigen is captured, and the amount of the label bound to the antigen is calculated.
- This detection method is called a competition method.
- a substrate related to the competition method will be described.
- the test area of the substrate has a site that reacts with a binding substance (for example, an antibody) present on the labeled particle.
- a binding substance for example, an antibody
- an embodiment having an antigen present in a biological sample on a test area of a substrate is preferable.
- the antigen and BSA are reacted in the presence of a condensing agent to produce an antigen / BSA conjugate, and the conjugate can be adsorbed onto the test area to produce a test area.
- the antigen-BSA conjugate which is the substance to be measured, is dissolved in a buffer solution, spotted on the substrate, allowed to stand for a certain period of time, and then the supernatant is aspirated and dried by a method such as drying. Can be combined.
- control area In the present invention, in order to suppress the influence of the measurement environment, particularly the measurement temperature as much as possible, a control area is provided on the substrate, and the information on the test area is normalized with the information on the control area, thereby greatly reducing the environmental dependency. It can be kept low.
- the control area is preferably designed so that it can bind to all the labels without depending on the amount of the substance to be measured present in the biological sample to be used. It is preferred to have an antibody that interacts with all antibodies present on the labeled particles.
- the information in the test area is normalized with the information in the control area.For example, even when the flow of the biological sample and the reaction rate are affected in a low temperature environment, It is possible to cancel the influence and obtain a result that is always accurate and unaffected by the measurement environment.
- a preferable antibody to be present in the control area is preferably an anti-mouse antibody if it has a function of recognizing a binding substance (for example, antibody) present on the labeled particle and the antibody is derived from a mouse. If the above antibody is derived from a goat, it is preferably an anti-goat antibody.
- These antibodies on the control area can be dissolved in a buffer solution, spotted on a substrate, allowed to stand for a certain period of time, and then the supernatant is sucked and dried to be bound to the substrate. .
- binding to both of the substances on the surface of the labeled particles it may be detected as negative even when a positive biological sample containing the measurement target substance is measured.
- blocking with BSA is used to suppress nonspecific adsorption to a solid phase surface (for example, the surface of a labeled particle, the surface of a gold film of a substrate).
- immunoglobulins other than immunoglobulins that have binding properties with the analyte include antisera prepared from the serum of animals immunized with an antigen different from the analyte, and immunoglobulin fraction purified from the antiserum.
- These antibodies can be prepared by a conventional method.
- the antibody may be modified as in the case of a chimeric antibody or the like, or a commercially available antibody prepared from an animal serum or culture supernatant by a known method can be used.
- antibodies can be used regardless of their animal species, subclasses, and the like.
- antibodies that can be used in the present invention are antibodies derived from organisms that can cause an immune reaction such as mice, rats, hamsters, goats, rabbits, sheep, cows, chickens, specifically mouse IgG, mouse IgM.
- a fragmented antibody is a molecule derived from a complete antibody having at least one antigen-binding site, and specifically, Fab, F (ab ′) 2 and the like. These fragmented antibodies are molecules obtained by enzyme or chemical treatment or using genetic engineering techniques.
- the kit of the present invention is used for a method of measuring a substance to be measured.
- the substance to be measured is bile acid
- it is a kit for diagnosing bile acid
- the substance to be measured is progesterone
- the measurement of the measurement target substance includes a sensor chip including a substrate on which a second binding substance is fixed and a member holding a label particle such as a fluorescent particle, and a surface plasmon excitation device
- You may include the various equipment or apparatus used for the measurement of a measuring object substance, such as a fluorescence measuring device.
- a sample including a known amount of a substance to be measured, an instruction manual, and the like may be included as elements of the kit.
- a method for measuring a substance to be measured in a biological sample comprises: A reaction step of reacting a biological sample with labeled particles having a first binding substance having binding properties with a measurement target substance; The reaction product obtained in the reaction step is brought into contact with a substrate having a second binding substance having binding properties with either the measurement target substance or the first binding substance, and the labeled particles are captured on the substrate. The capture process; A label information acquisition step of acquiring label information related to the measurement target substance; The labeled particles are luminescent labeled particles containing at least one compound represented by formula (1) and particles.
- the measurement target substance is measured by the measurement target substance related label information acquisition step of acquiring label information related to the amount of the measurement target substance.
- the measurement in the present invention is interpreted as the broadest concept as long as it is a measurement of the amount of the substance to be measured.
- Specific embodiments of the measurement method include a competitive method and a sandwich method, but the competitive method is preferred.
- the competitive method As an example of the competitive method, the case of quantifying progesterone will be described below. The same can be done when quantifying substances other than progesterone.
- a biological sample containing progesterone and anti-progesterone antibody-labeled fluorescent particles are brought into contact with a progesterone immunoassay substrate on which a progesterone-albumin conjugate is immobilized.
- progesterone is not present in the biological sample, an antigen-antibody reaction occurs on the substrate by the anti-progesterone antibody-labeled fluorescent particles and the progesterone on the substrate (that is, progesterone in the progesterone / albumin conjugate).
- an antigen-antibody reaction occurs between the progesterone in the biological sample and the anti-progesterone antibody-labeled fluorescent particles, and the anti-progesterone antibody-labeled fluorescent particles and the progesterone on the substrate (ie And the antigen-antibody reaction with the progesterone-albumin conjugate).
- anti-progesterone antibody-labeled fluorescent particles that have not bound to albumin on the substrate are removed.
- an immune complex on the substrate ie, a complex of anti-progesterone antibody-labeled fluorescent particles and progesterone in the progesterone / albumin conjugate on the substrate
- the concentration of progesterone can be measured.
- the measurement method of fluorescence in the competitive method can employ either plate reader measurement or flow measurement. For example, it can be measured by the following method.
- a plurality of samples with known progesterone amounts having different progesterone concentrations are prepared in advance, and this sample and anti-progesterone antibody-labeled fluorescent particles are mixed in advance.
- This mixed solution is brought into contact with the region where the progesterone / albumin conjugate is immobilized.
- the fluorescence signal from the region where the progesterone / albumin conjugate is immobilized is measured as a plurality of fluorescence signals while the mixed solution is in contact with the conjugate at a specific time interval.
- the temporal change (slope) of the fluorescence amount is obtained at each progesterone concentration.
- This time change is plotted on the Y axis and the progesterone concentration is plotted on the X axis, and a relational expression of the progesterone concentration with respect to the time change of the fluorescence amount is obtained using an appropriate fitting method such as a least square method.
- the amount of progesterone contained in the biological sample can be quantified using the result of temporal change in the fluorescence amount using the biological sample to be examined.
- Quantification of the amount of progesterone is preferably performed in a short time. Specifically, it is preferably performed within 10 minutes, more preferably within 8 minutes, and further preferably within 6 minutes.
- the sample and the anti-progesterone antibody-labeled fluorescent particles are added to the progesterone-and It includes the time to convert the amount of progesterone contained in the biological sample based on the results of time-dependent changes in the fluorescence amount using the biological sample to be tested after contacting the detection region where the albumin conjugate is immobilized. It is preferable.
- the measurement target substance can be measured by the following procedure.
- a biological sample that may contain a measurement target substance is brought into contact with a fluorescent particle having a first binding substance having binding properties with the measurement target substance on the substrate.
- a binding reaction antigen-antibody reaction or the like
- an immune complex composed of the second binding substance bound to the substrate, the measurement target substance, and fluorescent particles having the first binding substance is formed. Is done.
- the first binding substance that does not form the immune complex is included. Remove fluorescent particles and wash. Next, the concentration of the substance to be measured can be measured by detecting the degree of formation of the immune complex as the fluorescence intensity. The fluorescence intensity and the concentration of the measurement target substance have a positive correlation.
- a mixed liquid obtained by mixing a biological sample that may contain a substance to be measured and labeled particles having a first binding substance is applied on a substrate and developed in a flow path.
- the flow path is not particularly limited as long as it is a passage through which the biological sample and the labeled particles having the first binding substance flow down to the detection region.
- the sample has a structure that can pass over the metal film.
- a suction port can be provided on the side opposite to the spotting port with respect to the metal film.
- the method for detecting a label such as fluorescence in the present invention is not particularly limited.
- an apparatus capable of detecting fluorescence intensity specifically, a microplate reader or fluorescence detection (SPF) by surface plasmon excitation is used. It is preferable to detect the fluorescence intensity using a biosensor or the like.
- label information related to the amount of the substance to be measured can be acquired by fluorescence detection using surface plasmon resonance.
- fluorescence measurement may be plate reader measurement or flow measurement.
- the fluorescence detection method (SPF method) based on surface plasmon excitation can be measured with higher sensitivity than the fluorescence detection method (epifluorescence method) based on epi-illumination.
- the surface plasmon fluorescence (SPF) biosensor for example, an optical waveguide formed of a material that transmits excitation light of a predetermined wavelength, as described in Japanese Patent Application Laid-Open No. 2008-249361, and one of the optical waveguides are used.
- a fluorescence detection means for detecting fluorescence generated by excitation by the evanescent wave enhanced by the surface plasmon can be used.
- the fluorescence detection (SPF) system by surface plasmon excitation using the fluorescent particles of the present invention preferably detects fluorescence from a fluorescent substance depending on the amount of the measurement target substance immobilized on the metal film on the substrate.
- It is an assay method, for example, a method different from the so-called latex agglutination method in which a change in optical transparency is detected as turbidity by the progress of a reaction in a solution.
- the latex agglutination method an antibody-sensitized latex in a latex reagent and an antigen in a biological sample are combined and aggregated by an antibody reaction.
- This aggregate increases with time, and the antigen concentration is quantified from the change in absorbance per unit time obtained by irradiating the aggregate with near infrared light.
- the method of the present invention includes a label particle-related label information acquisition step for acquiring label information related to the amount of label particles; and a measurement target substance-related label information acquisition step for acquiring label information related to the amount of the measurement target substance
- a method including a normalization process in which the label information acquired in step 1 is normalized by the label information acquired in the label particle-related label information acquisition process may be used.
- the mixed liquid containing the biological sample and the labeled particles having the first binding substance having binding properties with the measurement target substance is brought into contact with the substrate having the detection area (test area) and the reference area (control area).
- the step of measuring the intensity of the fluorescence due to the surface plasmons generated on the detection region is It is a measurement target substance-related label information acquisition step for acquiring label information related to the quantity, and a step of measuring the intensity of fluorescence due to surface plasmons generated on the reference region is a label particle-related label information acquisition step.
- the step of obtaining the rate of increase of the fluorescence intensity obtained in these two steps per unit time as the rate of change of the fluorescence signal value and dividing the rate of change of the signal value of the detection region by the rate of change of the signal value of the reference region is a standardization step It is.
- the crude product obtained by concentrating the reaction solution under reduced pressure was purified by preparative TLC (developing solvent: hexane / ethyl acetate) and then recrystallized from methanol to obtain 71 mg of Compound (1).
- the compound was identified by 1 H-NMR and ESI-MS.
- This composition was purified by silica gel column chromatography (developing solvent: hexane / ethyl acetate) and then dissolved in 5 ml of dichloromethane. After further adding 15 ml of methanol, the dichloromethane was distilled off and reprecipitated. The precipitate was filtered to obtain 206 mg of compound (5-C).
- the crude product was purified by silica gel column chromatography (developing solvent: hexane / toluene), then dissolved in 3 ml of dichloromethane, 15 ml of methanol was added, and then dichloromethane was distilled off for reprecipitation. To give 16 mg of compound (5).
- the compound was identified by 1 H-NMR and ESI-MS.
- Synthesis of Compound (6) 181 mg of Compound (6-A), 237 mg of 2,4,6-trimethylbenzaldehyde, and 10 mL of dehydrated toluene were introduced into a 100 mL three-necked flask and stirred at room temperature. 2 mL of piperidine and 2 pieces of p-toluenesulfonic acid monohydrate (manufactured by Wako Pure Chemical Industries, Ltd., special grade reagent) were added, and the mixture was stirred at 140 ° C. for 1 hour while distilling off the solvent.
- the crude product obtained by concentrating the reaction solution under reduced pressure was purified by silica gel column chromatography (developing solvent: toluene) and then recrystallized from acetonitrile to obtain 194 mg of Compound (6).
- the compound was identified by 1 H-NMR and ESI-MS.
- the solvent was removed by evaporation under reduced pressure, 30 mL of dichloromethane was added thereto, washed with 20 mL of water and 20 mL of a saturated aqueous sodium chloride solution, and the organic layer was pre-dried with anhydrous sodium sulfate and then concentrated under reduced pressure.
- the crude product was purified by silica gel column chromatography (developing solvent: hexane / toluene) and then recrystallized from methanol to obtain 26 mg of compound (8).
- the compound was identified by 1 H-NMR and ESI-MS.
- Latex particles particles having an average particle diameter of 150 nm prepared by polymerizing a 9/1 (mass ratio) mixture of styrene and acrylic acid in a state of being dispersed in water were used. The average particle size was measured using a dynamic light scattering method.
- THF THF
- ⁇ mol / g of the amount of compound represents the number of moles of the compound used relative to 1 g of latex solid
- mass% represents the mass% of the compound used relative to 1 g of latex solid.
- High-intensity fluorescent latex particles (average particle size of 150 nm) labeled with an anti-progesterone antibody were prepared as follows.
- Tween 20 polyoxyethylene (20) sorbitan monolaurate, manufactured by Wako Pure Chemical Industries, Ltd.
- a PBS solution (pH 7.4) containing a concentration of mass% was prepared in advance, and washed with 300 ⁇ L of this solution repeatedly three times. After washing, in order to block the unadsorbed portion of the T4-BSA conjugate on the gold deposited film, 300 ⁇ L of PBS solution (pH 7.4) containing 1% by weight casein (Thermo Scientific) was added for 1 hour. And left at room temperature. After washing with the above washing solution, 300 ⁇ L of Immunoassay Stabilizer (manufactured by ABI) was added as a stabilizer, left at room temperature for 30 minutes, the solution was removed, and moisture was completely removed using a dryer.
- FIGS. FIG. 3 is a schematic view of the sensor chip 1
- FIG. 4 is an exploded view of the sensor chip 1.
- the sensor chip 1 includes an upper member 2, an intermediate member 3 and a substrate 4.
- the upper member 2 is provided with a first container 5 and a second container 6.
- the first container 5 and the second container 6 are collectively referred to as a container group 7.
- a flow path 10 is formed on the substrate 4, and a detection area 8 and a reference area 9 are formed on the flow path 10.
- concentrations of the antibody-labeled particles prepared with the anti-progesterone antibody prepared in ⁇ 2-1> and various samples are 0.080%, 0.040%, 0 Prepare multiple concentrations of each antibody-labeled particle so that the concentration is 0.020%, 0.010%, 0.005%, 0.002%, and 0.001%.
- Normalization was performed by calculating the rate of increase of the fluorescence intensity of each detection region and reference region obtained in each substrate per unit time as a fluorescence signal value and dividing the signal value of the detection region by the signal value of the reference region. .
- a sample having a progesterone concentration of 0 was prepared, and signal values from a sample not containing progesterone were normalized in the same manner.
- ⁇ 4> Preparation of calibration curve Correspondence between the fluorescence signal value normalized from the sample whose progesterone amount determined in ⁇ 3-2> is known and the measurement value obtained in the large machine determined in ⁇ 3-1> By obtaining the relationship, a calibration curve was prepared for the substrate using the progesterone-BSA conjugate prepared in ⁇ 2-2>.
- the literature “The Immunoassay Handbook Third Edition Edited by David Wild (2005)” describes that a four-parameter logistic curve model of a sigmoid function can be applied as a calibration curve for the competitive method. According to this method, an approximate line is obtained. Using a generally known least square method, 3-2. A 4-parameter logistic curve passing through the nearest point of each point of the fluorescence signal value at each progesterone concentration measured in (1) was obtained and used as a calibration curve.
- the performance was judged based on whether the calibration curve standard was satisfied.
- Calibration curves were defined at two locations. The first is the slope of the calibration curve in the low concentration range, and the reciprocal is taken to be within 2.0. The second is the deviation (deviation) from the calibration curve of the measurement points in the high concentration range, and the standard is within 4%. Within this range, it can be achieved that the variation coefficient of the measured value is within 10% and the accuracy is within 10%, and high-accuracy measurement is possible.
- Comparative Particles ⁇ 5-1> Preparation of Comparative Fluorescent Latex Particle Dispersion Add 100 mL of methanol to 100 mL of an aqueous dispersion having a solid content concentration of 2 mass% of the latex particle dispersion prepared in ⁇ 1-2>. Stir for 10 minutes at room temperature.
- a separately prepared fluorescent dye (Comparative compound: Compound 5 described in Japanese Patent No. 3442777) solution (1 mL of N, N-dimethylformamide, 9 mL of CHCl 3 and 16 mL of ethanol) is slowly added to the latex particle dispersion over 60 minutes. It was dripped.
- the organic solvent was distilled off under reduced pressure with an evaporator, and then centrifugal separation and redispersion in an aqueous PBS solution were repeated three times, and purification was performed to prepare a comparative fluorescent latex particle dispersion.
- the fluorescent latex dispersion liquid having a solid content concentration of 2% by mass was diluted 200 times with ultrapure water, and the excitation light of a fluorescence spectrophotometer RF-5300PC (manufactured by Shimadzu Corporation) was adjusted to 658 nm. Set and measured. When the fluorescence intensity of the fluorescent latex dispersion was high enough to exceed the measurement range, dilution was performed with ultrapure water until the maximum fluorescence intensity was measurable. 7-1.
- the integral value of the fluorescence intensity of the emission spectrum of the fluorescent latex dispersion with respect to the integral value of the fluorescence intensity of the fluorescence latex dispersion prepared in step 1 was defined as the particle fluorescence intensity (relative value).
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Abstract
Description
上記標識粒子は、下記式(1)で示される少なくとも一種の化合物と粒子とを含有する発光性の標識粒子である、上記キット。
<3> 上記標識粒子が、カルボキシル基を有する、<1>又は<2>に記載のキット。
<4> 上記標識粒子の平均粒子径が70~500nmである、<1>から<3>の何れか一に記載のキット。
<5> 上記式(1)で表される化合物が、下記式(3)で表される化合物である、<1>から<4>の何れか一に記載のキット。
<6> 上記式(1)で表される化合物が、下記式(4)で表される化合物である、<1>から<4>の何れか一に記載のキット。
<8> 上記標識粒子が、少なくとも一種のエネルギードナー化合物と、少なくとも一種のエネルギーアクセプター化合物と、粒子とを含有する発光性粒子であって、上記エネルギードナー化合物及び上記エネルギーアクセプター化合物の少なくとも一種が、上記式(1)で表される化合物である、<1>から<7>の何れか一に記載のキット。
<9> 上記エネルギードナー化合物として、少なくとも一種の上記式(1)で表される化合物を含有し、上記エネルギーアクセプター化合物として、少なくとも一種の上記式(1)で表される化合物を含有する、<8>に記載のキット。
<10> エネルギードナー化合物とエネルギーアクセプター化合物のモル比が1:10~10:1である、<8>又は<9>に記載のキット。
<11> エネルギードナー化合物とエネルギーアクセプター化合物のストークスシフトが40nm以上である、<8>から<10>の何れか一に記載のキット。
<12> 上記基板が、上記第二の結合物質を有する検出領域を有する、<1>から<11>の何れか一に記載のキット。
<13> 上記検出領域が金を含む金属膜である、<12>に記載のキット。
生体試料を、測定対象物質と結合性を有する第一の結合物質を有する標識粒子と反応させる反応工程と、上記測定対象物質又は上記第一の結合物質の何れかと結合性を有する第二の結合物質を有する基板に、上記反応工程で得られた反応産物を接触させて、標識粒子を基板上に捕捉させる捕捉工程と、
上記測定対象物質に関連した標識情報を取得する、標識情報取得工程と、
を含み、上記標識粒子は、下記式(1)で示される少なくとも一種の化合物と粒子とを含有する発光性の標識粒子である、方法。
<16> 上記粒子が、カルボキシル基を有する、<14>又は<15>に記載の方法。
<17> 上記標識粒子の平均粒子径が70~500nmである、<14>から<16>の何れか一に記載の方法。
<18> 上記式(1)で表される化合物が、下記式(3)で表される化合物である、<14>から<17>の何れか一に記載の方法。
<19> 上記式(1)で表される化合物が、下記式(4)で表される化合物である、<14>から<17>の何れか一に記載の方法。
<20> 上記式(1)で表される化合物が、下記式(5)で表される化合物である、<14>から<17>の何れか一に記載の方法。
<22> 上記エネルギードナー化合物として、少なくとも一種の上記式(1)で表される化合物を含有し、上記エネルギーアクセプター化合物として、少なくとも一種の上記式(1)で表される化合物を含有する、<21>に記載の方法。
<23> エネルギードナー化合物とエネルギーアクセプター化合物のモル比が1:10~10:1である、<21>又は<22>に記載の方法。
<24> エネルギードナー化合物とエネルギーアクセプター化合物のストークスシフトが40nm以上である、<21>から<23>の何れか一に記載の方法。
<25> 上記基板が、上記第二の結合物質を有する検出領域を有する、<14>から<24>の何れか一に記載の方法。
<26> 上記検出領域が金を含む金属膜である、<25>に記載の方法。
<27> 表面プラズモン励起による蛍光検出により、上記測定対象物質に関連した標識情報を取得する、<26>に記載の方法。
本明細書において「~」を用いて示された数値範囲は、「~」の前後に記載される数値をそれぞれ最小値及び最大値として含む範囲を意味する。
本発明による生体試料中の測定対象物質を測定するためのキットは、生体試料中の測定対象物質と結合性を有する第一の結合物質を有する標識粒子と、測定対象物質又は第一の結合物質の何れかと結合性を有する第二の結合物質を有する基板とを含むキットであって、標識粒子は、後述する式(1)で示される少なくとも一種の化合物と粒子とを含有する発光性の標識粒子である。
生体試料としては、測定対象物質を含む可能性のある試料である限り、特に限定されるものではなく、例えば、生物学的試料、特には動物(例えば、ヒト、イヌ、ネコ、ウマなど)の体液(例えば、血液、血清、血漿、髄液、涙液、汗、尿、膿、鼻水、又は喀痰)若しくは排泄物(例えば、糞便)、臓器、組織、粘膜や皮膚などを挙げることができる。
測定対象物質としては、特に限定されないが、例えば、サイロキシン(T4)、トリヨードサイロニン(T3)、エストラジオール(E2)、アルドステロン、対称性ジメチルアルギニン(SDMA)、胆汁酸、コルチゾール、コレステロール、コルチコステロン、プロゲステロン、テストステロン、エストロゲン、ビタミン類、クレアチニン、アミノ酸、βカロチン、クレアチニン、ジゴキシン、テオフィリン、葉酸、炎症マーカーや敗血症マーカーなどのタンパク質などが挙げられる。
本発明で用いる第一の結合物質は、測定対象物質と結合性を有する物質である。第一の結合物質としては、抗原、抗体、又はこれらの複合体を使用できるが、これらに限定されるものではない。好ましくは、第一の結合物質は抗体である。第一の結合物質が抗体である場合は、測定対象物質と結合性を有する抗体として、例えば、その測定対象物質によって免疫された動物の血清から調製する抗血清や、抗血清から精製された免疫グロブリン画分、その測定対象物質によって免疫された動物の脾臓細胞を用いる細胞融合によって得られるモノクローナル抗体、あるいは、それらの断片[例えば、F(ab’)2、Fab、Fab’、又はFv]などを用いることができる。これらの抗体の調製は、常法により行なうことができる。さらに、その抗体がキメラ抗体などの場合のように、修飾を加えられたものでもよいし、また市販の抗体でも、動物血清又は培養上清から公知の方法により調製した抗体でも使用可能である。
プロゲステロンと、牛血清アルブミン(Bovine Serum Albumin、以下BSAと略す)と、縮合剤を混合してプロゲステロン-BSA結合体を作製することができる。結合体をマウス免疫感作抗原として用いて、数回、マウス背部皮下に免疫する。この場合、完全アジュバント(Complete Freund‘s Adjuvant:CFA)、及び/又は不完全アジュバント(Incomplete Freund‘s Adjuvant:IFA)を適宜選択して免疫感作抗原と混合して使用することができる。完全アジュバントとは、免疫を刺激する物質であって、パラフィンとアラセルの混合物である。不完全アジュバントとは、完全アジュバントに死滅したミコバクテリア又は結核菌の死菌を加え、抗原性をさらに増強させたものである。数週間で数回、適宜免疫感作を行った後にマウスから採血し抗体価の測定を実施する。抗体価の十分な上昇が認められた場合に腹腔内に抗原を投与し、数日後に脾臓を摘出する。こうして免疫マウスより摘出した脾臓細胞を、変異株骨髄腫細胞(ミエローマ)と融合させることで、抗体産生能力を備えた融合細胞を作製することができる。この融合細胞の中から目的とする抗原に対する抗体産生細胞のみを選択し、さらにその細胞株だけを増殖するために限界希釈を行う。希釈後の細胞の培養(クローニング)を行うことができる。このようにして得られる融合細胞株を、マウスの腹腔内に注射して、腹水型の抗体産生細胞を増殖させることによってモノクローナル抗体を腹水中に産生することが可能となり、これらの抗体を回収することで、目的の抗体を入手することができる。
本発明で使用される標識粒子は、下記式(1)で表される少なくとも一種の化合物と粒子とを含有する発光性の標識粒子であり、蛍光標識粒子とも言う。
本明細書において、アリールオキシ基としては、好ましくは炭素数6~14のアリールオキシ基であり、例えば、フェノキシ基、ナフトキシ基、アントリルオキシ基などが挙げられる。
アリールチオ基としては、好ましくは、炭素数6から30のアリールチオ基であり、例えば、フェニルチオ基、p-クロロフェニルチオ基、m-メトキシフェニルチオ基等が挙げられる。
なお、芳香環は置換基を有していてもよく、「芳香環」との用語は、置換基を有する芳香環、及び置換基を有さない芳香環の両方を意味する。芳香環が有する置換基としては、後記する置換基群Aに記載の置換基が挙げられる。
スルファモイル基、シアノ基、イソシアノ基、チオシアナト基、イソチオシアナト基、ニトロ基、ニトロシル基、ハロゲン原子、ヒドロキシ基、アミノ基、メルカプト基、アミド基、アルコキシ基、アリールオキシ基、アルキルチオ基、アリールチオ基、カルバモイル基、アシル基、アルデヒド基、カルボニル基、アリール基、アルキル基、ハロゲン原子で置換されたアルキル基、エテニル基、エチニル基、シリル基、及びトリアルキルシリル基(トリメチルシリル基等)。
R111~R116が表すアルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基は、置換基を有していてもよく、上記置換基としては、置換基群Aに記載の置換基が挙げられる。
式(1)中、R11~R15はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよい。R11~R15のうち少なくとも3つは水素原子以外の原子又は基を表し、好ましくはR11~R15のうち少なくとも4つは水素原子以外の原子又は基を表し、より好ましくはR11~R15の全てが水素原子以外の原子又は基を表す。
R11及びR15は、好ましくは、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、又はエチニル基を表し、これらは置換基を有していてもよい。
R12及びR14は、好ましくは、アルキル基を表し、これらは置換基を有していてもよい。
R13は、好ましくは、アリール基を表し、これは置換基を有していてもよい。
X1及びX2は、好ましくは、ハロゲン原子、又はアルコキシ基を表す。X1及びX2は、フッ素原子、メトキシ基、エトキシ基、イソプロピルオキシ基、t-ブチルオキシ基であることがより好ましく、これらはフッ素原子、アルコキシ基によって置換されていることも好ましい。
L1及びL2は、好ましくは、式(L-1)又は式(L-2)の何れかを表す。
R111~R116は、好ましくは水素原子である。
式(1)で表される化合物の好ましい例としては、下記式(2)で表される化合物が挙げられる。
式(1)で表される化合物の好ましい例としては、下記式(4)で表される化合物が挙げられる。
式(4)中、R41及びR42はそれぞれ独立に、アリール基、ヘテロ環基、エテニル基、又はエチニル基を表し、これらは置換基を有していてもよい。上記置換基としては、置換基群Aに記載の置換基が挙げられる。R41及びR42はそれぞれ独立に、アリール基、エテニル基、又はエチニル基であることが好ましく、量子収率向上の観点からは、アリール基が好ましく、長波長化の観点からは、エテニル基、エチニル基であることが好ましい。アリール基である場合、アリール基のオルト位またはメタ位に少なくとも一つ置換基を有していることが好ましく、オルト位に少なくとも一つ置換基を有していることがより好ましい。アリール基に置換する置換基の数は、1~3つが好ましく、2つまたは3つがより好ましい。アリール基に置換する置換基としては、アルキル基であることが好ましく、メチル基、イソプロピル基、t-ブチル基であることがより好ましく、メチル基であることがさらに好ましい。
式(5)で表わされる化合物は、下記式(6)で表わされる化合物であることがより好ましい。
L1及びL2はそれぞれ独立に、式(L-1)~式(L-4)の何れかを表す。
式(1)~式(6)で表される化合物の具体例を以下に記載する。Meはメチル基を示し、Etはエチル基を示し、iPrはイソプロピル基を示す。
好ましくは、R1及びR2はそれぞれ独立に、アリール基又はヘテロ環基であり、これらは置換基を有していてもよい。
R1及びR2はそれぞれ同一でも異なっていてもよいが、好ましくは同一である。
R1及びR2は、連結して環を形成することはない。
好ましくは、R3は、水素原子、アルキル基、アリール基又はヘテロ環基であり、これらは置換基を有していてもよい。より好ましくは、R3は、水素原子である。
好ましくは、Y1及びY2はそれぞれ独立に、ハロゲン原子、アルキル基、アリール基、ヒドロキシ基、アルコキシ基、又はアリールオキシ基を表し、これらは置換基を有していてもよく、Y1及びY2は互いに連結して環を形成してもよい。
より好ましくは、Y1及びY2はそれぞれ独立に、ハロゲン原子である。
さらに好ましくは、Y1及びY2はフッ素原子である。
Y1及びY2はそれぞれ同一でも異なっていてもよいが、好ましくは同一である。
好ましくは、Ar1及びAr2はベンゼン環を表す。
好ましくは、Z1及びZ2は、それぞれ独立に、置換基を有していてもよいアリール基を表す。
より好ましくは、Z1及びZ2は、それぞれ独立に、フェニル基、ナフチル基、又はアントリル基を表し、これらは置換基を有していてもよい。
好ましくは、m1が2以上である場合、複数のZ1は同じ基である。
好ましくは、m2が2以上である場合、複数のZ2は同じ基である。
式(2)で表される化合物は、分子内に、カルボン酸基、リン酸基、スルホン酸基などの酸性基を有さないことが好ましい。
好ましくは、Y1及びY2はそれぞれ独立にハロゲン原子を表す。
特に好ましくは、Y1及びY2はフッ素原子である。
好ましくは、R3は、水素原子、アルキル基、アリール基又はヘテロ環基であり、これらは置換基を有していてもよい。
より好ましくは、R3は、水素原子である。
さらに好ましくは、R34~R37の少なくとも1つ以上が、置換基を有していてもよいアリール基であり、R38~R41の少なくとも1つ以上が、置換基を有していてもよいアリール基である。
式(10)で表される化合物は、分子内に、カルボン酸基、リン酸基、スルホン酸基などの酸性基を有さないことが好ましい。
式(10)又は式(10A)で表される化合物の具体例を以下に記載する。Meはメチル基を示し、Buはn-ブチル基を示し、Phはフェニル基を示す。
エネルギードナー化合物とエネルギーアクセプター化合物の組み合わせの具体例を以下に記載する。
本発明で用いる粒子(即ち、式(1)で表される化合物を添加する前の粒子)に対する式(1)で表される化合物の含有量は、本発明の効果を損なわない限り特に限定されないが、好ましくは0.5μmol/g~400μmol/gであり、より好ましくは1μmol/g~300μmol/gであり、さらに好ましくは2μmol/g~200μmol/gであり、特に好ましくは3μmol/g~100μmol/gである。
式(1)~式(6)で表される化合物は、例えば、後記する実施例に示す合成スキームにより製造することができる。
式(10)で表される化合物は、例えば、以下に示す合成スキームにより製造することができる。
化合物A-10と化合物A-20とをMacromolecules 2010、43、193-200に記載の方法に従って反応させることにより、化合物A-30を合成することができる。次いで、化合物A-30、式:Z1-B(OH)2で表される化合物、及びフッ化セシウム(CsF)をジメトキシエタン(DME)と水の混合溶液に加え、真空引き、窒素置換を繰り返して脱気を行う。酢酸パラジウム(Pd(OAc)2)、及び2-ジシクロヘキシルホスフィノ-2’、6’-ジメトキシビフェニル(SPhos)を加え、昇温し、還流下、所定の時間(例えば、2~24時間)反応させることにより、化合物D-10を製造することができる。
標識粒子は、粒子を含む。粒子の材質及び形態は特に限定されず、例えば、ポリスチレンビーズなどの有機高分子粒子、又はガラスビーズ等の無機粒子を用いることができる。粒子の材質の具体例としては、スチレン、メタクリル酸、グリシジル(メタ)アクリレート、ブタジエン、塩化ビニル、酢酸ビニルアクリレート、メチルメタクリレート、エチルメタクリレート、フェニルメタクリレート、又はブチルメタクリレートなどのモノマーを重合させたホモポリマー、並びに2種以上のモノマーを重合させたコポリマーなどが挙げられ、上記のホモポリマー又はコポリマーを均一に懸濁させたラテックスでもよい。また、粒子としては、その他の有機高分子粉末、無機物質粉末、微生物、血球、細胞膜片、リポソーム、マイクロカプセルなどが挙げられる。粒子としては、ラテックス粒子が好ましい。
本発明における発光性の標識粒子は、式(1)で表される化合物を含むことにより、680nm以上という長波長領域の発光極大波長を有し、高い量子収率を示す。
発光極大波長とは、吸収スペクトルにおいて吸光度が最も大きくなる波長のことを表す。
発光性の標識粒子の発光極大波長は、好ましくは680nm以上であり、より好ましくは700nm以上であり、特に好ましくは720nm以上である。本発明の発光性の標識粒子の発光極大波長の上限は特に限定されないが、好ましくは900nm以下であり、より好ましくは800nm以下である。
発光性の標識粒子が示す量子収率は、好ましくは0.25以上であり、より好ましくは0.4以上であり、さらに好ましくは0.5以上であり、さらに一層好ましくは0.6以上であり、特に好ましくは0.7以上である。量子収率の上限は特に限定されないが、一般的には、1.0以下である。
発光性の標識粒子の量子収率は、市販の量子収率測定装置を使用して測定することができ、例えば、浜松ホトニクス社製の絶対PL量子収率測定装置C9920-02を使用して測定することができる。
発光性の標識粒子の平均粒径は、粒子の材質や被検物質を測定する濃度範囲、測定機器などによって異なるが、0.001~10μm(より好ましくは0.01~1μm)の範囲が好ましく、30~500nmの範囲がより好ましく、50~300nmの範囲がさらに好ましく、80~200nmの範囲が特に好ましく、100~150nmの範囲が最も好ましい。本発明に用いることが可能な発光性の標識粒子の平均粒径は、市販の粒度分布計等で計測することができる。粒度分布の測定方法としては、光学顕微鏡法、共焦点レーザー顕微鏡法、電子顕微鏡法、原子間力顕微鏡法、静的光散乱法、レーザー回折法、動的光散乱法、遠心沈降法、電気パルス計測法、クロマトグラフィー法、超音波減衰法等が知られており、それぞれの原理に対応した装置が市販されている。これらの測定方法のうち、粒子径範囲及び測定の容易さから、動的光散乱法を用いて発光性の標識粒子の平均粒径を測定することが好ましい。動的光散乱を用いた市販の測定装置としては、ナノトラックUPA(日機装(株))、動的光散乱式粒径分布測定装置LB-550((株)堀場製作所)、濃厚系粒径アナライザーFPAR-1000(大塚電子(株))等が挙げられる。本発明では、平均粒径は、25℃にて、粘度0.8872CP、水の屈折率1.330の条件で測定したメジアン径(d=50)として求めるものとする。
発光性の標識粒子の製造方法は特に限定されないが、式(1)で表される少なくとも一種の化合物と粒子とを混合することによって製造することができる。例えば、ラテックス粒子などの粒子に、式(1)で表される化合物を添加することによって、発光性の標識粒子を作製することができる。より具体的には、水及び水溶性有機溶剤(テトラヒドロフラン、メタノール等)の何れか一種以上を含む粒子の分散液に、式(1)で表される化合物を含む溶液を添加して攪拌することにより、発光性の標識粒子を製造することができる。
分散液は、本発明の発光性の標識粒子を分散媒に分散することにより製造することができる。分散媒としては、水、有機溶媒、又は水と有機溶媒との混合物等が挙げられる。有機溶媒としては、メタノール、エタノール、イソプロパノール等のアルコール、テトラヒドロフラン等のエーテル系溶媒などを使用することができる。
分散液における発光性の標識粒子の固形分濃度は特に限定されないが、一般的には0.1~20質量%であり、好ましくは0.5~10質量%であり、より好ましくは1~5質量%である。
第一の結合物質を発光性の標識粒子に固定化する方法は、例えば、特開2000-206115号公報やThermo Fisher 社FluoSpheres(登録商標)ポリスチレンマイクロスフィアF8813に添付のプロトコールなどに記載されており、免疫凝集反応用試薬を調製する公知の方法がいずれも使用可能である。また、結合物質として抗体を粒子に固定化する原理として、物理吸着及び共有結合による化学結合のいずれの原理も採用可能である。抗体を粒子に固定させた後に抗体が被覆されていない粒子表面を覆うブロッキング剤(即ち、第一のブロッキング剤)としては、例えば、アルブミン(BSAなど)、スキムミルク、カゼイン、大豆由来成分、魚由来成分、又はポリエチレングリコールなど、並びに上記物質又は上記物質と性質が同じである物質を含む市販の免疫反応用ブロッキング剤などが使用可能である。これらのブロッキング剤は、必要に応じて熱や酸・アルカリ等により部分変性などの前処理を施すことも可能である。さらに、第一のブロッキング剤としては、測定対象物質と結合性を有しない抗体(グロブリン)、あるいはテストエリアに使用しないタンパク質(Protein A、Protein G)などを使用することもできる。
本発明では、高感度な測定を達成するために、後述する表面プラズモン蛍光(SPF)検出を行う測定法を採用することが好ましい。この場合における基板としては、表面に金属膜を有する基板を使用することが好ましい。金属膜を構成する金属としては、表面プラズモン共鳴が生じ得るようなものであれば特に限定されない。好ましくは金、銀、銅、アルミニウム、又は白金等の自由電子金属が挙げられ、特に金が好ましい。金を使用する場合、後記する検出領域は、金膜上にある。上記の金属は単独又は組み合わせて使用することができる。また、上記基板への付着性を考慮して、基板と金属からなる層との間にクロム等からなる介在層を設けてもよい。金属膜の膜厚は任意であるが、例えば、1nm以上500nm以下であるのが好ましく、特に10nm以上200nm以下であるのが好ましい。500nmを超えると、媒質の表面プラズモン現象を十分検出することができない。また、クロム等からなる介在層を設ける場合、その介在層の厚さは、0.1nm以上、10nm以下であることが好ましい。
第二の結合物質は、測定対象物質と結合性を有する物質であるか、又は第一の結合物質と結合性を有する物質である。定量をサンドイッチアッセイ法で行う場合には、第二の結合物質として、測定対象物質と結合性を有する物質を使用することができる。定量を競合法で行う場合には、第二の結合物質として、第一の結合物質と結合性を有する物質を使用することができる。本発明においては、定量を競合法で行うことが好ましく、第二の結合物質として、第一の結合物質と結合性を有する物質を使用することが好ましい。
第二の結合物質を基板に固定化する方法は、例えば、Nunc社の提供するTech Notes Vol.2-12などに記載されており、一般的なELISA(Enzyme-Linked ImmunoSorbent Assay:酵素結合免疫吸着法)試薬を調製する公知の方法がいずれも使用可能である。また、基板上に自己組織化単分子膜(SAM:Self-Assembled Monolayer)などを配することによる表面修飾を施しても良く、第二の結合物質を基板に固定化する方法としては、物理吸着を用いた方法、及び共有結合による化学結合を用いた方法のいずれの方法も採用可能である。第二の結合物質を基板に固定させた後に、第二の結合物質が被覆されていない基板表面を覆うブロッキング剤(第二のブロッキング剤)としては、公知の物質、例えば、BSA、グロブリン、スキムミルク、カゼイン、大豆由来成分、魚由来成分又はポリエチレングリコールなど、並びに上記物質又は上記物質と性質が同じである物質を含む市販の免疫反応用ブロッキング剤などが使用可能である。これらのブロッキング剤は、必要に応じて熱や酸・アルカリ等により部分変性などの前処理を施すことも可能である。
本発明においては、基板上に生体試料中の測定対象物質の有無を検出するテストエリアを設けることができる。このテストエリアでは、例えば測定対象物質である抗原を捕まえて、抗原に結合した標識の量を検出し定量することで、抗原を定量することが可能となる。あるいは、抗原に結合した標識のみを結合できないようにし、抗原に結合していない標識のみを捕獲して、抗原の結合した標識の量を算出する方法により、抗原を定量することが可能となる。この検出方法は競合法と呼ばれているが、ここでは、競合法に関する基板について説明する。
本発明では、測定環境、特に測定温度の影響を極力抑えるために、基板上にコントロールエリアを有し、テストエリアの情報を、コントロールエリアの情報で規格化することによって、環境依存性を非常に低く抑えることが可能となる。コントロールエリアとしては、使用する生体試料の中に存在する測定対象物質の量に依存せず、すべての標識と結合することが可能なように設計されていることが好ましい。標識粒子上に存在する抗体すべてに相互作用する抗体を有することが好ましい。このように設計することによって、テストエリアの情報をコントロールエリアの情報で規格化することにより、例えば、低温環境で、生体試料の流れや、反応速度が影響を受けた場合でも、規格化によってその影響をキャンセルして、常に精度よく、測定環境に影響されない結果を得ることが可能になる。
例えば、競合法において、測定対象物質を含まない陰性となる生体試料だけでなく、測定対象物質を含む陽性となる生体試料に対しても反応して陰性となる生体試料が存在しており、高値乖離の問題の解決が課題として認識されている。このような擬陰性を示す原因は明確にはなっていないが、抗体に覆われていない標識粒子表面と、検出領域(テストエリア)との非特異的な相互作用により、本来結合してほしくない標識粒子が存在することが原因の一つではないかと考えられている。また、テストエリア上に存在する物質と同じ物質が標識粒子表面上に存在する場合にも、遊離した抗体などが生体試料中に存在する場合には、その抗体が、テストエリア上に存在する物質と、標識粒子表面上の物質のどちらにも結合することで、測定対象物質を含む陽性となる生体試料を測定した場合においても陰性として検出される場合がある。
一般的に、固相表面(例えば標識粒子表面、基板の金膜表面)、への非特異吸着抑制のためにBSAでのブロッキングが用いられている。
本発明において、抗体は、その動物種やサブクラス等によらず使用できる。例えば、本発明に用いることが可能な抗体は、マウス、ラット、ハムスター、ヤギ、ウサギ、ヒツジ、ウシ、ニワトリなど免疫反応が起こり得る生物に由来する抗体、具体的には、マウスIgG、マウスIgM、ラットIgG、ラットIgM、ハムスターIgG、ハムスターIgM、ウサギIgG、ウサギIgM、ヤギIgG、ヤギIgM、ヒツジIgG、ヒツジIgM、ウシIgG、ウシIgM、トリIgY等であり、ポリクローナル又はモノクローナルのどちらも使用可能である。断片化抗体は、少なくとも1つの抗原結合部位を持つ、完全型抗体から導かれた分子であり、具体的にはFab、F(ab’)2等である。これらの断片化抗体は、酵素あるいは化学的処理によって、もしくは遺伝子工学的手法を用いて得られる分子である。
本発明のキットは、測定対象物質を測定する方法に用いられるものであり、測定対象物質が胆汁酸である場合には、胆汁酸測定診断用のキットであり、測定対象物質がプロゲステロンである場合には、プロゲステロン測定診断用のキットである。本発明において、測定対象物質の測定を実施するに当たり、第二の結合物質を固定した基板と、蛍光粒子などの標識粒子を保持した部材を含むセンサチップを含むものであるが、表面プラズモン励起装置、及び蛍光測定デバイスなどの、測定対象物質の測定に使用される各種の器材又は装置を含めてもよい。さらに、キットの要素として、既知量の測定対象物質を含む試料、取扱説明書などを含めてもよい。
本発明による生体試料中の測定対象物質を測定する方法は、
生体試料を、測定対象物質と結合性を有する第一の結合物質を有する標識粒子と反応させる反応工程と、
上記測定対象物質又は上記第一の結合物質の何れかと結合性を有する第二の結合物質を有する基板に、上記反応工程で得られた反応産物を接触させて、標識粒子を基板上に捕捉させる捕捉工程と、
上記測定対象物質に関連した標識情報を取得する、標識情報取得工程と、
を含む方法であり、上記標識粒子は、式(1)で示される少なくとも一種の化合物と粒子とを含有する発光性の標識粒子である。
本発明における測定は、測定対象物質の量の測定である限り、最も広い概念として解釈される。測定方法の具体的な実施態様としては、競合法及びサンドイッチ法が挙げられるが、競合法が好ましい。
競合法では、先ず、プロゲステロン・アルブミン結合体が固定化されているプロゲステロン免疫測定用基板に、プロゲステロンを含む生体試料及び抗プロゲステロン抗体標識蛍光粒子を接触させる。その生体試料中にプロゲステロンが存在しない場合には、抗プロゲステロン抗体標識蛍光粒子と、基板上のプロゲステロン(即ち、プロゲステロン・アルブミン結合体中のプロゲステロン)とにより、基板上で抗原抗体反応が起こる。一方、生体試料中にプロゲステロンが存在する場合には、生体試料中のプロゲステロンと抗プロゲステロン抗体標識蛍光粒子との間で抗原抗体反応が起こり、抗プロゲステロン抗体標識蛍光粒子と、基板上のプロゲステロン(即ち、プロゲステロン・アルブミン結合体中のプロゲステロン)との間の抗原抗体反応が阻害される。上記の反応が終了した後、基板上のアルブミンに結合しなかった抗プロゲステロン抗体標識蛍光粒子を除去する。次いで基板上の免疫複合体(即ち、抗プロゲステロン抗体標識蛍光粒子と、基板上のプロゲステロン・アルブミン結合体中のプロゲステロンとの複合体)の形成の度合いを蛍光強度として検出することにより、生体試料中のプロゲステロンの濃度などを測定することができる。
本発明の好ましい態様においては、測定対象物質を含む可能性のある生体試料と、第一の結合物質を有する標識粒子とを混合した混合液を、基板上に適用し、流路に展開することができる。流路とは、生体試料と、第一の結合物質を有する標識粒子とを、検出領域まで流下する通路であれば、特に制限はない。好ましい流路の態様としては、第一の結合物質を有する標識粒子を含む生体試料液を点着する点着口、検出領域としての金属膜、及び金属膜を超えて流路が存在し、生体試料が、金属膜上を通過できる構造を有するものである。好ましくは、金属膜に対して、点着口とは反対側に、吸引口を設けることができる。
本発明における蛍光などの標識の検出方法としては、特に限定されないが、例えば、蛍光強度を検出することができる機器、具体的には、マイクロプレートリーダー、又は表面プラズモン励起による蛍光検出(SPF)を行うためのバイオセンサーなどを用いて蛍光強度を検出することが好ましい。好ましくは、表面プラズモン共鳴による蛍光検出により、測定対象物質の量に関連した標識情報を取得することができる。
さらに本発明の方法は、標識粒子の量に関連した標識情報を取得する、標識粒子関連標識情報取得工程;及び測定対象物質の量に関連した標識情報を取得する測定対象物質関連標識情報取得工程で取得した標識情報を、標識粒子関連標識情報取得工程で取得した標識情報により規格化する、規格化工程を含む方法でもよい。
用語は以下の意味を示す。
MS:質量分析 (mass spectrometry)
ESI:エレクトロスプレーイオン化(electrospray ionization)
NMR:核磁気共鳴(nuclear magnetic resonance)
Me:メチル基
Et:エチル基
Bu:n-ブチル基
PL:フォトルミネッセンス
THF:テトラヒドロフラン
100mL三ツ口フラスコに、窒素雰囲気下、3,5-ビス(トリフルオロメチル)ベンズアルデヒド1.00g及びジクロロメタン20mLを導入し、室温で撹拌した。水冷しながら3-エチル-2,4-ジメチルピロール0.98gを滴下し、続いて、トリフルオロ酢酸を2滴加えた後、室温で30分間撹拌した。水冷しながらクロラニル1.0gを加え、室温で10分間撹拌した後、水冷しながらジイソプロピルエチルアミン(NiPr2Et)3.67gを滴下し、室温で15分間撹拌した。続いて、水冷しながら三フッ化ホウ素ジエチルエーテル錯体5.6mLを滴下し、室温で30分間撹拌した。飽和炭酸水素ナトリウムおよびトルエンを滴下し、抽出・分液して得られた有機層を無水硫酸ナトリウムで予備乾燥した後、減圧濃縮した。この粗生成物をシリカゲルカラムクロマトグラフィー(展開溶媒:ヘキサン/酢酸エチル)で精製した後、メタノールで再結晶することにより、化合物(1-A)を1.28g得た。
1H NMR(CDCl3,400MHz):δ 8.03(s,1H),7.83(s,2H),2.54(s,6H),2.31(q,J=7.6Hz,4H),1.21(s,6H),1.00(t,J=7.6Hz,6H).
100mL三ツ口フラスコに、化合物(1-A)100mg、2,4,6-トリメチルベンズアルデヒド115mg、及び脱水トルエン5mLを導入し、室温で撹拌した。ピペリジン1mLおよびp-トルエンスルホン酸1水和物(和光純薬工業(株)社製、試薬特級)1片を加えて140℃で溶媒を留去しながら1時間撹拌し、放冷後に脱水トルエン5mLを加えて140℃で溶媒を留去しながら1時間撹拌した。反応液を減圧濃縮して得られた粗生成物を分取TLC(展開溶媒:ヘキサン/酢酸エチル)で精製した後、メタノールで再結晶することにより、化合物(1)を71mg得た。化合物の同定は、1H-NMRとESI-MSにより行った。
1H NMR(CDCl3,400MHz):δ 8.06(s,1H),7.87(s,2H),7,38(d,J=17.2Hz,2H),7,32(d,J=17.2Hz,2H),6.93(s,4H),2.63(q,J=7.6Hz,4H),2.44(s,12H),2.30(s,6H),1.27(s,6H),1.17(t,J=7.6Hz,6H).
ESI-MS:[M-H]-=775.8
1L三ツ口フラスコに、窒素雰囲気下、3,5-ビス(トリフルオロメチル)ベンズアルデヒド16.22g、及びジクロロメタン200mLを導入し、室温で撹拌した。水冷しながら2,4-ジメチルピロール15.75gを滴下し、続いて、トリフルオロ酢酸を5滴加えた後、室温で30分間撹拌した。水冷しながらクロラニル(Chloranil)19.45gを加え、室温で30分間撹拌した後、水冷しながらジイソプロピルエチルアミン(NiPr2Et)80mLを滴下し、室温で30分間撹拌した。続いて、水冷しながら三フッ化ホウ素ジエチルエーテル錯体(BF3・Et2O)85mLを滴下し、室温で30分間撹拌した。飽和炭酸水素ナトリウム400mLを滴下し、抽出・分液して得られた有機層を無水硫酸ナトリウムで予備乾燥した後、減圧濃縮した。この粗生成物をシリカゲルカラムクロマグラフィー(展開溶媒:ヘキサン/酢酸エチル)で精製した後、エタノールで再結晶することにより、化合物(3-A)を4.40g得た。
300mL三ツ口フラスコに、化合物(3-A)を3.05g、及び1,1,1,3,3,3-ヘキサフルオロ-2-プロパノール60mLを導入し、室温で撹拌した。N-ヨードスクシンイミド3.60gを導入し、室温で1時間半撹拌した。反応液を減圧濃縮した後、チオ硫酸ナトリウム水溶液50mL(チオ硫酸ナトリウム10g溶解)、及び塩化メチレン100mLを加え、抽出・分液して得られた有機層を無水硫酸ナトリウムで予備乾燥した後、減圧濃縮した。この粗生成物をエタノールで再結晶することにより、化合物(3-B)を3.90g得た。
100mL三ツ口フラスコに、化合物(3-B)を2.2g、2,4,6-トリメチルベンズアルデヒド2.6g及び脱水トルエン40mLを導入し、室温で撹拌した。ピペリジン4mLを導入して65℃で1時間撹拌した。反応液を減圧濃縮して得られた粗生成物をシリカゲルカラムクロマグラフィー(展開溶媒:ヘキサン/酢酸エチル)で精製した後、エタノールで再結晶することにより、化合物(3-C)を2.4g得た。
100mL三ツ口フラスコに、化合物(3-C)を96mg、2,4,6-トリメチルフェニルボロン酸64mg、フッ化セシウム130mg、及びメトキシシクロペンタン10mLを導入し、室温で撹拌しながら、減圧脱気後、窒素雰囲気にした。ここに、SPhos Pd G3(Aldrich製)63mgを加え、1時間加熱還流した。飽和塩化アンモニウム水溶液10mL、及び酢酸エチル10mLを加え、抽出・分液して得られた有機層を無水硫酸ナトリウムで予備乾燥した後、減圧濃縮した。この粗生成物を分取TLC(展開溶媒:ヘキサン/酢酸エチル)で精製した後、エタノールで再結晶することにより、化合物(3)を16mg得た。化合物の同定は、1H-NMRとESI-MSにより行った。
1H NMR(CDCl3,400MHz):δ 8.02(s,1H),8.00(s,2H),7,42(d,J=22.4Hz,2H),6.92(s,4H),6.80(s,4H),6.67(d,J=22.4Hz,2H),2.27(s,6H),2.17(s,6H),2.16(s,6H),2.11(s,12H),2.01(s,12H).
ESI-MS:[M-H]-=955.8
化合物(3)の合成において、3,5-ビス(トリフルオロメチル)ベンズアルデヒドを2,3,4,5,6-ペンタフルオロベンズアルデヒドに置き換え、さらに2,4-ジメチルピロールを2,4-ジメチル-3-エチルピロールに置き換えたこと以外は、同様にして合成を行い、粗生成物をシリカゲルカラムクロマトグラフィー(展開溶媒:ヘキサン/酢酸エチル)で精製した後、ジクロロメタン/メタノールで再結晶することにより、化合物(2)を8mg得た。化合物の同定は1H-NMR測定で行い、Org.Biomol.Chem.,2010,8,4546-4553と同様のNMRスペクトルであることを確認した。
化合物(2)の合成において、2,4,6-トリメチルベンズアルデヒドをo-トルアルデヒドに置き換えたこと以外は同様にして合成し、化合物(4)を合成した。化合物の同定は、1H-NMRとESI-MSにより行った。400MHz 1H-NMRスペクトルは図1に示す。
ESI-MS:[M-H]-=673.3
500mL三ツ口フラスコに、窒素雰囲気下、2,4-ジメチルピロール1.16ml及びジクロロメタン140mLを導入し、室温で撹拌した。2,3,5,6-テトラフルオロベンズアルデヒド1.0g、トリフルオロ酢酸1滴を加えた後、室温で15分間撹拌した。クロラニル(Chloranil)1.38gを加え、室温で15分間撹拌した後、水冷しながらジイソプロピルエチルアミン(NiPr2Et)6.8mLを滴下し、室温で20分間撹拌した。続いて、水冷しながら三フッ化ホウ素ジエチルエーテル錯体(BF3・Et2O)7.8mLを滴下し、室温で30分間撹拌した。飽和炭酸水素ナトリウム400mLを滴下し、ジクロロメタンで抽出・分液して得られた有機層を無水硫酸ナトリウムで予備乾燥した後、減圧濃縮した。この粗生成物をシリカゲルカラムクロマグラフィー(展開溶媒:ヘキサン/酢酸エチル)で精製した後、メタノールで再結晶することにより、化合物(5-A)を360mg得た。
1H NMR(CDCl3,400MHz):δ 7.43(s,1H),7.39(s,1H),7.29-7.21(m,1H),6.94(s,4H),6.80(s,4H),6.69(s,1H),6.65(s,1H),2.29(s,6H),2.23(s,6H),2.08(s,12H),2.03(s,12H),1.33(s,6H).
ESI-MS:[M-H]-=891.4
100mL三ツ口フラスコに、窒素雰囲気下、2,3,5,6-テトラフルオロベンズアルデヒド1.00g及びジクロロメタン20mLを導入し、室温で撹拌した。水冷しながら3-エチル-2,4-ジメチルピロール0.98gを滴下し、続いて、トリフルオロ酢酸を2滴加えた後、室温で15分間撹拌した。水冷しながらクロラニル1.0gを加え、室温で10分間撹拌した後、水冷しながらジイソプロピルエチルアミン3.67gを滴下し、室温で15分間撹拌した。続いて、水冷しながら三フッ化ホウ素ジエチルエーテル錯体5.6mLを滴下し、室温で60分間撹拌した。飽和炭酸水素ナトリウム及びトルエンを滴下し、抽出・分液して得られた有機層を無水硫酸ナトリウムで予備乾燥した後、減圧濃縮した。この粗生成物をシリカゲルカラムクロマトグラフィー(展開溶媒:トルエン)で精製した後、メタノールで再結晶することにより、化合物(6-A)を0.76g得た。
1H NMR(CDCl3,400MHz):δ 7.20-7.30(m,1H),2.54(s,6H),2.33(q,J=7.6Hz,4H),1.51(s,6H),1.01(t,J=7.6Hz,6H).
100mL三ツ口フラスコに、化合物(6-A)を181mg、2,4,6-トリメチルベンズアルデヒド237mg、及び脱水トルエン10mLを導入し、室温で撹拌した。ピペリジン2mLおよびp-トルエンスルホン酸1水和物(和光純薬工業(株)社製、試薬特級)2片を加えて140℃で溶媒を留去しながら1時間撹拌した。反応液を減圧濃縮して得られた粗生成物をシリカゲルカラムクロマトグラフィー(展開溶媒:トルエン)で精製した後、アセトニトリルで再結晶することにより、化合物(6)を194mg得た。化合物の同定は、1H-NMRとESI-MSにより行った。
1H NMR(CDCl3,400MHz):δ 7,40(d,J=17.2Hz,2H),7,32(d,J=17.2Hz,2H),7.20-7.30(m,1H),6.93(s,4H),2.66(q,J=7.6Hz,4H),2.44(s,12H),2.30(s,6H),1.55(s,6H),1.19(t,J=7.6Hz,6H).
ESI-MS:[M-H]-=711.7
化合物(2)の合成において、2,4,6-トリメチルベンズアルデヒドを2,4,6-トリメトキシベンズアルデヒドに置き換えたこと以外は同様にして合成し、化合物(7)を合成した。化合物の同定は、1H-NMRとESI-MSにより行った。400MHz 1H-NMRスペクトルを図2に示す。
ESI-MS:[M+H]+=825.3
50mL二口フラスコに、化合物(3-C)を97mg、2-エチニル-1,3,5-トリメチルベンゼン58mg、ヨウ化銅(I)3.8mg、THF4mL、及びトリエチルアミン1mLを導入し、室温で撹拌しながら、減圧脱気後、窒素雰囲気にした。ここにテトラキス(トリフェニルホスフィン)パラジウム(0)(Pd(PPh3)4)を加え、2時間加熱還流した。減圧留去で溶媒を除去し、そこにジクロロメタン30mLを加え、水20mLと飽和塩化ナトリウム水溶液20mLで洗浄し、有機層を無水硫酸ナトリウムで予備乾燥した後、減圧濃縮した。この粗生成物をシリカゲルカラムクロマトグラフィー(展開溶媒:ヘキサン/トルエン)で精製した後、メタノールで再結晶することにより、化合物(8)を26mg得た。化合物の同定は、1H-NMRとESI-MSにより行った。
1H NMR(CDCl3,400MHz):δ 8.60(s,1H),8.56(s,1H),8.09(s,1H),7.90(s,2H),7.41(s,1H),7.37(s,1H),6.88(s,4H),6.85(s,4H),2.36(s,12H),2.34(s,12H),2.28(s, 6H),2.27(s,6H).
ESI-MS:[M-H]-=1003.5
化合物(5-A)~化合物(5-C)を経由して化合物(5)を合成する方法において、化合物(5)の合成における2,4,6-トリメチルベンズアルデヒドをベンズアルデヒドに置き換え、その他は同様の方法で合成することにより、化合物(9)を合成した。
化合物(5-A)~化合物(5-C)を経由して化合物(5)を合成する方法において、化合物(5-A)の合成における2,3,5,6-テトラフルオロベンズアルデヒドを2,4,6-トリメチルベンズアルデヒドに置き換え、その他は同様の方法で合成することにより、化合物(10)を合成した。
化合物(5-A)~化合物(5-C)を経由して化合物(5)を合成する方法において、化合物(5)の合成における2,4,6-トリメチルベンズアルデヒドを2-ホルミルナフタレンに置き換え、その他は同様の方法で合成することにより、化合物(11)を合成した。
化合物(5-A)~化合物(5-C)を経由して化合物(5)を合成する方法において、化合物(5)の合成における2,4,6-トリメチルベンズアルデヒドを2,6-ジメトキシベンズアルデヒドに置き換え、その他は同様の方法で合成することにより、化合物(12)を合成した。
蛍光ラテックス粒子の作製を行った。ラテックス粒子としてはスチレンとアクリル酸の9/1(質量比)混合物を水中に分散させた状態で重合させて作製した、平均粒径150nmの粒子を用いた。平均粒径は動的光散乱法を用いて測定した。上記で作製した固形分2%のラテックス粒子分散液(ラテックス分散液)(25mL、固形500mg)に対してTHF(5mL)を滴下して10分攪拌した。そこに、化合物(1) 24μmol/gを含むTHF溶液(2.5mL)を15分間かけて滴下した。表中の化合物量のμmol/gはラテックスの固形1gに対する使用した化合物のモル数、質量%はラテックスの固形1gに対する使用した化合物の質量%を表す。化合物の滴下終了後、30分攪拌した後、減圧濃縮してTHFを除去した。その後、遠心分離して粒子を沈殿させた後、超純水を加えて再度分散させることで固形分濃度2%の高輝度蛍光ラテックス分散液を製造した。
また、化合物(1)の代わりに化合物(5)を用いて同様の操作を行い、化合物(5)を含む固形分濃度2%の高輝度蛍光ラテックス分散液を製造した。
さらに、化合物(1) 24μmol/gと化合物(5) 12μmol/gを混合したものを用いて同様の操作を行い、化合物(1)と化合物(5)を含む固形分濃度2%の高輝度蛍光ラテックス分散液を製造した。
抗プロゲステロン抗体で標識した高輝度蛍光ラテックス粒子(平均粒径150nm)を、以下の通り調製した。
<1-2>で作製した固形分濃度が2質量%の高輝度蛍光ラテックス分散液375μLに、50mMのMES(2-モルホリノエタノスルホン酸、同仁化学研究所社製)緩衝液(pH6.0)を117μL、10mg/mLのWSC(水溶性カルボジイミド)水溶液を5μL加え、室温で15分間攪拌した。その後、0.5mg/mLの抗プロゲステロンモノクローナル抗体(GeneTex社製)を182.4μL添加し、室温で1.5時間撹拌した。2mol/LのGlycine(和光純薬社製)水溶液を37.5μL添加し、15分間撹拌した後、遠心分離(15,000rpm、4℃、30分)にて、蛍光ラテックス粒子を沈降させた。上清液を取り除き、PBS(Phosphate Buffered Saline リン酸緩衝生理食塩水;和光純薬社製)溶液(pH7.4)を750μL加え、超音波洗浄機により蛍光ラテックス粒子を再分散させた。さらに遠心分離(15,000rpm、4℃、15分)を行い、上清液を除いた後、1質量%BSAを含むPBS (pH7.4)溶液750μLを加え、蛍光ラテックス粒子を再分散させることで、抗プロゲステロン抗体結合蛍光ラテックス粒子の1質量%溶液を得た。
プロゲステロン-BSA結合体(BIO-RAD社製)150μgを、50mM濃度のクエン酸緩衝液1mL(pH 5.2, 150 mM NaCl)に添加して溶解させ、クエン酸緩衝液の溶液を得た。
ポリメチルメタクリレート(PMMA)の基体(三菱レイヨン(株)社製、アクリペット(登録商標)VH)を準備し、蒸着法により、厚さ50nmの金膜を片面に蒸着し7mmの幅に裁断して、同じ基板を7枚作製した。この基板の金蒸着面上に、1.で調製したプロゲステロン-BSA結合体のクエン酸緩衝液による溶液を点着して乾燥させ、プロゲステロン-BSA結合体を固定化した基板を作製した。
このように調製した基板をセンサチップの流路に取り付ける前に、Tween20(ポリオキシエチレン(20)ソルビタンモノラウラート、和光純薬社製)を0.05質量%の濃度で含むPBS溶液(pH7.4))を予め調製し、この溶液300μLを用いて3回繰り返し洗浄した。洗浄終了後、金蒸着膜上のT4-BSA結合体の未吸着部分のブロッキングを行うため、1質量%カゼイン(Thermo Scientific社製)を含むPBS溶液(pH7.4)を300μL添加し、1時間、室温で静置した。上記の洗浄用溶液で洗浄後、安定化剤としてImmunoassay Stabilizer(ABI社製)300μLを添加し、室温で30分間放置し、溶液を除去して乾燥機を用いて水分を完全に取り除いた。
特開2010-190880号公報の第2の実施形態の構成となるように、流路型センサチップを作製した。その概略図を図3及び図4に示した。図3は、センサチップ1の概略図であり、図4は、センサチップ1の分解図である。センサチップ1は、上部部材2、中間部材3及び基板4から構成されている。上部部材2には、第一の容器5及び第二の容器6が設けられている。なお、第一の容器5及び第二の容器6を併せて、容器群7と称する。基板4には、流路10が形成されており、流路10の上には、検出領域8及び参照領域9が形成されている。
免疫測定で、当業者により広く使用されている大型機であるイムライズ1000(株式会社LSIメディエンス)により、取り扱い説明書に従い、プロゲステロンの量が既知である試料の測定を行い、プロゲステロンの測定値を得た。
各種濃度(0.00ng/mL、0.5ng/mL、2.0ng/mL、15.0ng/mL、30.0ng/mL、45.0ng/mL)のプロゲステロンを含む試料を用意した。次に、<2-1>で調製した抗プロゲステロン抗体で標識した抗体標識粒子と各種試料の混合液を作製した時点での抗体標識粒子の濃度が、0.080%、0.040%、0.020%、0.010%、0.005%、0.002%、0.001%となるように、各抗体標識粒子の濃度のカップを複数準備し、それぞれの濃度のカップに上記で用意した各種濃度の試料を添加し、抗体標識粒子と試料との混合液を10分間攪拌しながら調製した。次に、2-5.で作製した基板を封入した流路型センサチップに抗体標識粒子と試料との混合液をそれぞれ点着した。点着後、ポンプ吸引を行いながら混合液を10μL/minの速度で混合液を流下させ、プロゲステロン-BSA結合体を固定した金膜面上に接触させてから、蛍光強度を1.5分間継続して測定した。各基板において得られた検出領域と参照領域のそれぞれの蛍光強度の単位時間における増加速度を蛍光シグナル値として求め、検出領域のシグナル値を参照領域のシグナル値で除することで規格化を行った。また、プロゲステロン濃度0の試料を用意して同様にして、プロゲステロンを含まない試料からのシグナル値の規格化を行った。
<3-2>で求めたプロゲステロンの量が既知である試料から規格化した蛍光シグナル値と、<3-1>で求めた大型機での測定値、との対応関係を求めることで、<2-2>で調製した、プロゲステロン-BSA結合体を用いた基板に対して、検量線を作成した。文献「The Immunoassay Handbook Third Edition Edited by David Wild (2005)」に競合法の検量線として、シグモイド関数の4パラメータロジスティック曲線モデルが適用できることが記載されており、この方法に従って、近似線を得る方法として一般的に知られている最小二乗法を用いて、3-2.で測定した各プロゲステロン濃度における蛍光シグナナル値の各点の最近傍を通る4パラメータロジスティック曲線を求め、検量線とした。
<5-1>比較蛍光ラテックス粒子分散液の作製
<1-2>で作製したラテックス粒子分散液の固形分濃度2質量%の水分散液100mLにメタノール100mLを加え、10分間、室温で攪拌した。一方、別途用意した蛍光色素(比較化合物:特許3442777号公報記載の化合物5)溶液(N,N-ジメチルホルムアミド1mL、CHCl3 9mL、エタノール16mLに溶解)を60分間かけてゆっくりラテックス粒子分散液に滴下した。滴下完了後、エパポレーターで有機溶媒を減圧留去した後、遠心分離とPBS水溶液への再分散を3回繰り返し、精製を行うことで、比較用蛍光ラテックス粒子分散液を調製した。
<2-1>で調製した蛍光ラテックス粒子の調製において、蛍光ラテックス粒子の代わりに、<5-1>で調製した2質量%(固形分濃度)の比較用蛍光ラテックス粒子に置き換え、それ以外の作業は同様に行って、比較用抗体標識粒子を調製した。それ以外の作業は同様に<4>の測定まで実施した。
上記で固形分濃度2質量%の蛍光ラテックス分散液を超純水で200倍に希釈し、蛍光分光光度計RF-5300PC(島津製作所製)の励起光を658nmに設定し、測定を行った。蛍光ラテックス分散液の蛍光強度が測定範囲を超えるほど高い場合には、蛍光強度の極大値が測定可能な範囲まで超純水で希釈を行った。7-1.で作製した蛍光ラテックス分散液の発光スペクトルの蛍光強度の積分値に対する、蛍光ラテックス分散液の発光スペクトルの蛍光強度の積分値を粒子蛍光強度(相対値)とした。算出に用いた計算式を以下に示す。
蛍光強度(相対値)= (蛍光ラテックス分散液の発光スペクトルの蛍光強度の積分値)/(比較例で作製した蛍光ラテックス分散液の発光スペクトルの蛍光強度の積分値)
低濃度域の検量線傾きの逆数は、2.0以下の場合の判定をAとし、2.0より大きい場合の判定をBとした。
高濃度域での検量線からのズレは、4%以下の場合の判定をAとし、4%より大きい場合の判定をBとした。
2 上部部材
3 中間部材
4 基板
5 第一の容器
6 第二の容器
7 容器群
8 検出領域
9 参照領域
10 流路
Claims (27)
- 生体試料中の測定対象物質と結合性を有する第一の結合物質を有する標識粒子と、前記測定対象物質又は前記第一の結合物質の何れかと結合性を有する第二の結合物質を有する基板とを含む、生体試料中の測定対象物質を測定するためのキットであって、
前記標識粒子は、下記式(1)で示される少なくとも一種の化合物と粒子とを含有する発光性の標識粒子である、前記キット。
- 前記標識粒子が標識ラテックス粒子である、請求項1に記載のキット。
- 前記標識粒子が、カルボキシル基を有する、請求項1又は2に記載のキット。
- 前記標識粒子の平均粒子径が70~500nmである、請求項1から3の何れか一項に記載のキット。
- 前記式(1)で表される化合物が、下記式(3)で表される化合物である、請求項1から4の何れか一項に記載のキット。
- 前記標識粒子が、少なくとも一種のエネルギードナー化合物と、少なくとも一種のエネルギーアクセプター化合物と、粒子とを含有する発光性粒子であって、前記エネルギードナー化合物及び前記エネルギーアクセプター化合物の少なくとも一種が、前記式(1)で表される化合物である、請求項1から7の何れか一項に記載のキット。
- 前記エネルギードナー化合物として、少なくとも一種の前記式(1)で表される化合物を含有し、前記エネルギーアクセプター化合物として、少なくとも一種の前記式(1)で表される化合物を含有する、請求項8に記載のキット。
- エネルギードナー化合物とエネルギーアクセプター化合物のモル比が1:10~10:1である、請求項8又は9に記載のキット。
- エネルギードナー化合物とエネルギーアクセプター化合物のストークスシフトが40nm以上である、請求項8から10の何れか一項に記載のキット。
- 前記基板が、前記第二の結合物質を有する検出領域を有する、請求項1から11の何れか一項に記載のキット。
- 前記検出領域が金を含む金属膜である、請求項12に記載のキット。
- 生体試料中の測定対象物質を測定する方法であって、
生体試料を、測定対象物質と結合性を有する第一の結合物質を有する標識粒子と反応させる反応工程と、前記測定対象物質又は前記第一の結合物質の何れかと結合性を有する第二の結合物質を有する基板に、前記反応工程で得られた反応産物を接触させて、標識粒子を基板上に捕捉させる捕捉工程と、
前記測定対象物質に関連した標識情報を取得する、標識情報取得工程と、
を含み、前記標識粒子は、下記式(1)で示される少なくとも一種の化合物と粒子とを含有する発光性の標識粒子である、方法。
- 前記粒子がラテックス粒子である、請求項14に記載の方法。
- 前記粒子が、カルボキシル基を有する、請求項14又は15に記載の方法。
- 前記標識粒子の平均粒子径が70~500nmである、請求項14から16の何れか一項に記載の方法。
- 前記式(1)で表される化合物が、下記式(3)で表される化合物である、請求項14から17の何れか一項に記載の方法。
- 前記標識粒子が、少なくとも一種のエネルギードナー化合物と、少なくとも一種のエネルギーアクセプター化合物と、粒子とを含有する発光性粒子であって、前記エネルギードナー化合物及び前記エネルギーアクセプター化合物の少なくとも一種が、前記式(1)で表される化合物である、請求項14から20の何れか一項に記載の方法。
- 前記エネルギードナー化合物として、少なくとも一種の前記式(1)で表される化合物を含有し、前記エネルギーアクセプター化合物として、少なくとも一種の前記式(1)で表される化合物を含有する、請求項21に記載の方法。
- エネルギードナー化合物とエネルギーアクセプター化合物のモル比が1:10~10:1である、請求項21又は22に記載の方法。
- エネルギードナー化合物とエネルギーアクセプター化合物のストークスシフトが40nm以上である、請求項21から23の何れか一項に記載の方法。
- 前記基板が、前記第二の結合物質を有する検出領域を有する、請求項14から24の何れか一項に記載の方法。
- 前記検出領域が金を含む金属膜である、請求項25に記載の方法。
- 表面プラズモン励起による蛍光検出により、前記測定対象物質に関連した標識情報を取得する、請求項26に記載の方法。
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Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH07508309A (ja) * | 1992-05-13 | 1995-09-14 | モレキュラー・プロウブズ・インコーポレーテッド | 制御可能な強化ストークスシフトを有する蛍光微小粒子 |
JP2000206115A (ja) | 1999-01-11 | 2000-07-28 | Eiken Chem Co Ltd | 抗体断片を用いた免疫凝集反応試薬 |
CN1944540A (zh) * | 2006-09-15 | 2007-04-11 | 大连理工大学 | 生物分析用近红外氟化硼络合二吡咯甲川类荧光染料 |
JP2008249361A (ja) | 2007-03-29 | 2008-10-16 | Fujifilm Corp | 表面プラズモンセンサーおよび免疫学的測定方法 |
JP2010190880A (ja) | 2008-04-18 | 2010-09-02 | Fujifilm Corp | 光信号検出方法、光信号検出装置、光信号検出用試料セルおよび光信号検出用キット |
US20110054187A1 (en) * | 2009-08-28 | 2011-03-03 | Bam Bundesanstalt Fuer Materialforschung Und -Pruefung | Difluoroboradiazaindacene dyes |
WO2015129361A1 (ja) * | 2014-02-26 | 2015-09-03 | コニカミノルタ株式会社 | 表面プラズモン励起増強蛍光分光測定用センサーチップ |
JP2016057145A (ja) * | 2014-09-09 | 2016-04-21 | 富士フイルム株式会社 | 試薬キット、測定キット及び被検物質の測定方法。 |
WO2018038138A1 (ja) * | 2016-08-23 | 2018-03-01 | 富士フイルム株式会社 | 発光性粒子及び化合物 |
WO2018038137A1 (ja) * | 2016-08-23 | 2018-03-01 | 富士フイルム株式会社 | 発光性粒子 |
Family Cites Families (30)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS60256057A (ja) | 1984-06-01 | 1985-12-17 | Dai Ichi Pure Chem Co Ltd | 免疫学的測定法 |
US5670381A (en) | 1988-01-29 | 1997-09-23 | Abbott Laboratories | Devices for performing ion-capture binding assays |
US5723218A (en) | 1990-04-16 | 1998-03-03 | Molecular Probes, Inc. | Dipyrrometheneboron difluoride labeled flourescent microparticles |
JP3185215B2 (ja) | 1990-07-13 | 2001-07-09 | 三菱化学株式会社 | 免疫測定法 |
EP0586590B1 (en) | 1991-05-30 | 1999-07-07 | Abbott Laboratories | Reagents containing a nonspecific binding blocker in ion-capture binding assays |
DE9216110U1 (de) | 1992-11-26 | 1993-01-28 | Biolab GmbH, 80995 München | Progesteron-Schnelltest für Mensch und Haustiere |
JPH10153599A (ja) | 1996-11-22 | 1998-06-09 | Nof Corp | 蛋白質非特異的吸着防止剤およびその用途 |
JP3623657B2 (ja) | 1998-05-22 | 2005-02-23 | 積水化学工業株式会社 | 非特異反応抑制剤、免疫測定試薬及び免疫測定方法 |
JP4086266B2 (ja) | 1998-11-26 | 2008-05-14 | 日本化薬株式会社 | 免疫測定方法 |
JP2001021563A (ja) | 1999-07-06 | 2001-01-26 | Nitto Denko Corp | 特異的結合体 |
CA2592254A1 (en) | 2004-12-31 | 2006-07-13 | Genentech, Inc. | Detecting human antibodies in non-human serum |
JP4668768B2 (ja) | 2005-11-01 | 2011-04-13 | 積水化学工業株式会社 | 免疫測定方法及び非特異反応抑制方法 |
JP2010019553A (ja) | 2006-11-02 | 2010-01-28 | Olympus Corp | 一分子蛍光分析による分子の特異的結合反応検出方法 |
JP2008190946A (ja) | 2007-02-02 | 2008-08-21 | Kyushu Institute Of Technology | タンパク質吸着阻害剤を用いた免疫測定法と免疫測定用の基板及びキット |
JP5152917B2 (ja) | 2008-11-04 | 2013-02-27 | 富士フイルム株式会社 | 検出方法、検出用試料セルおよび検出用キット |
JP5573499B2 (ja) | 2010-08-30 | 2014-08-20 | コニカミノルタ株式会社 | 蛍光測定方法 |
CN102174144B (zh) | 2011-01-31 | 2013-03-20 | 长沙三诺生物传感技术股份有限公司 | 一种荧光乳胶颗粒及其制备方法 |
JP6194882B2 (ja) | 2012-03-28 | 2017-09-13 | コニカミノルタ株式会社 | 生体物質の検出方法 |
FR2993565B1 (fr) | 2012-07-19 | 2016-01-08 | Centre Nat Rech Scient | Composes fluorescents de type thienyldipyrromethene bores et leurs utilisations. |
JP2015072249A (ja) | 2013-03-29 | 2015-04-16 | 富士フイルム株式会社 | 被検物質の測定方法、被検物質測定キット及び被検物質測定試薬 |
KR20140137676A (ko) | 2013-05-23 | 2014-12-03 | 도레이케미칼 주식회사 | 광학필름용 폴리머닷 및 이를 포함하는 광학필름 |
JP6236881B2 (ja) | 2013-06-03 | 2017-11-29 | コニカミノルタ株式会社 | 免疫染色方法、蛍光免疫染色用前処理液および免疫染色用キット |
CN105462576B (zh) | 2015-09-01 | 2018-03-30 | 南京林业大学 | 一种近红外bodipy类荧光染料及其制备方法 |
WO2017150516A1 (ja) | 2016-02-29 | 2017-09-08 | 富士フイルム株式会社 | 生体試料中の測定対象物質を定量するためのキット及び生体試料中の測定対象物質を定量する方法 |
CN106008581B (zh) | 2016-07-15 | 2017-10-20 | 福州大学 | 含六个三氟甲基基团的氟硼二吡咯衍生物及其制备和应用 |
EP3605092B1 (en) | 2017-03-30 | 2024-07-24 | FUJIFILM Corporation | Kit and method for measuring measurement target substance in biological sample |
CN110476062A (zh) * | 2017-03-30 | 2019-11-19 | 富士胶片株式会社 | 用于测定活体样品中的测定对象物质的试剂盒及方法 |
JP6808821B2 (ja) * | 2017-03-30 | 2021-01-06 | 富士フイルム株式会社 | 測定対象物質を測定するためのキット、方法及び試薬 |
WO2019163929A1 (ja) * | 2018-02-22 | 2019-08-29 | 富士フイルム株式会社 | プロゲステロン測定キット、プロゲステロンの測定方法およびプロゲステロン測定試薬 |
EP3922992A4 (en) * | 2019-02-06 | 2022-05-11 | FUJIFILM Corporation | KIT FOR MEASUREMENT OF SUBSTANCE OBJECT OF MEASUREMENT, AND METHOD FOR MEASURING SUBSTANCE OBJECT OF MEASUREMENT |
-
2018
- 2018-03-29 EP EP18774345.5A patent/EP3605092B1/en active Active
- 2018-03-29 WO PCT/JP2018/013406 patent/WO2018181796A1/ja unknown
- 2018-03-29 CN CN201880022541.0A patent/CN110506208A/zh active Pending
- 2018-03-29 JP JP2019510171A patent/JP6808819B2/ja active Active
- 2018-03-29 KR KR1020197028398A patent/KR20190120325A/ko not_active Application Discontinuation
-
2019
- 2019-09-27 US US16/585,306 patent/US11821896B2/en active Active
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH07508309A (ja) * | 1992-05-13 | 1995-09-14 | モレキュラー・プロウブズ・インコーポレーテッド | 制御可能な強化ストークスシフトを有する蛍光微小粒子 |
JP3442777B2 (ja) | 1992-05-13 | 2003-09-02 | モレキュラー・プロウブズ・インコーポレーテッド | 制御可能な強化ストークスシフトを有する蛍光微小粒子 |
JP2000206115A (ja) | 1999-01-11 | 2000-07-28 | Eiken Chem Co Ltd | 抗体断片を用いた免疫凝集反応試薬 |
CN1944540A (zh) * | 2006-09-15 | 2007-04-11 | 大连理工大学 | 生物分析用近红外氟化硼络合二吡咯甲川类荧光染料 |
JP2008249361A (ja) | 2007-03-29 | 2008-10-16 | Fujifilm Corp | 表面プラズモンセンサーおよび免疫学的測定方法 |
JP2010190880A (ja) | 2008-04-18 | 2010-09-02 | Fujifilm Corp | 光信号検出方法、光信号検出装置、光信号検出用試料セルおよび光信号検出用キット |
US20110054187A1 (en) * | 2009-08-28 | 2011-03-03 | Bam Bundesanstalt Fuer Materialforschung Und -Pruefung | Difluoroboradiazaindacene dyes |
WO2015129361A1 (ja) * | 2014-02-26 | 2015-09-03 | コニカミノルタ株式会社 | 表面プラズモン励起増強蛍光分光測定用センサーチップ |
JP2016057145A (ja) * | 2014-09-09 | 2016-04-21 | 富士フイルム株式会社 | 試薬キット、測定キット及び被検物質の測定方法。 |
WO2018038138A1 (ja) * | 2016-08-23 | 2018-03-01 | 富士フイルム株式会社 | 発光性粒子及び化合物 |
WO2018038137A1 (ja) * | 2016-08-23 | 2018-03-01 | 富士フイルム株式会社 | 発光性粒子 |
Non-Patent Citations (6)
Title |
---|
"The Immunoassay Handbook", 2005 |
HECHT, MANDY ET AL.: "Fluorinated Boron-Dipyrromethene (BODIPY) Dyes: Bright and Versatile Probes for Surface Analysis", CHEMISTRY0PEN, vol. 2, 2013, pages 25 - 38, XP055466454, DOI: doi:10.1002/open.201200039 * |
MACROMOLECULES, vol. 43, 2010, pages 193 - 200 |
OLIVIER GALANGAU ET AL., ORG. BIOMOL. CHEM., vol. 8, 2010, pages 4546 - 4553 |
See also references of EP3605092A4 |
XU, JUNCHAO ET AL.: "meso-C6F5 substituted BODIPYs with distinctive spectroscopic properties and their application for bioimaging in living cells", TETRAHEDRON, vol. 70, 2014, pages 5800 - 5805, XP029038855, DOI: doi:10.1016/j.tet.2014.06.040 * |
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US20200025748A1 (en) | 2020-01-23 |
CN110506208A (zh) | 2019-11-26 |
KR20190120325A (ko) | 2019-10-23 |
JPWO2018181796A1 (ja) | 2020-01-09 |
EP3605092B1 (en) | 2024-07-24 |
US11821896B2 (en) | 2023-11-21 |
JP6808819B2 (ja) | 2021-01-06 |
EP3605092A4 (en) | 2020-03-11 |
EP3605092A1 (en) | 2020-02-05 |
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