WO2015159580A1 - Biomolecule concentration measurement device and biomolecule concentration measurement method - Google Patents

Biomolecule concentration measurement device and biomolecule concentration measurement method Download PDF

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Publication number
WO2015159580A1
WO2015159580A1 PCT/JP2015/054223 JP2015054223W WO2015159580A1 WO 2015159580 A1 WO2015159580 A1 WO 2015159580A1 JP 2015054223 W JP2015054223 W JP 2015054223W WO 2015159580 A1 WO2015159580 A1 WO 2015159580A1
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biomolecule
site
concentration
fluorescent
fluorescence
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PCT/JP2015/054223
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French (fr)
Japanese (ja)
Inventor
悠策 中島
岸井　典之
拓哉 岸本
中尾　勇
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ソニー株式会社
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • 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
    • 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
    • G01N33/53—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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the present technology relates to a biomolecule concentration measuring apparatus and a biomolecule concentration measuring method. More specifically, the present invention relates to a technique for measuring density based on the number of light emitting points.
  • enzyme-linked immunosorbent assay is suitable for quantification of an antigen or antibody contained in a sample because it can quantitate a measurement object with relatively high sensitivity by using an antigen-antibody reaction. It is used for.
  • ELISA enzyme-linked immunosorbent assay
  • Patent Document 1 discloses that “a mixed liquid obtained by mixing the following competitive substance in a test sample solution containing a target substance is contacted in the order of the following competitive capture substance and the following determination substance.
  • An immune reaction step an enzyme reaction step of contacting a substrate of an enzyme included in the competitor with at least one of the competitive capture substance and the determination substance, and obtaining a reactant, and measuring the amount of the reactant
  • a calculation step of calculating the amount of the target substance is quantified by directly or indirectly measuring the amount of the competitive substance captured by the competitive capture substance by using the competitive substance.
  • the workability can be improved in the measurement of the biomolecule concentration by the measurement method described in Patent Document 1.
  • the method described in Patent Document 1 also requires a step of preparing a plurality of reagents including each of the competitive capture substance and the determination substance and mixing them in order. For this reason, a method for measuring the concentration of a biomolecule in a sample more simply is required.
  • the main object of the present disclosure is to provide a biomolecule concentration measuring device and the like that can easily quantify biomolecules.
  • the present disclosure provides a light irradiation unit that irradiates a sample including a labeled molecule that has a first fluorescent site and a second fluorescent site and binds to an arbitrary biomolecule, A detection unit that detects the emission of light, a counting unit that counts the number of emission points detected by the detection unit in the sample, the number of emission points derived from the first fluorescence site, and the second fluorescence
  • a biomolecule concentration measuring apparatus comprising: a concentration calculating unit that calculates the concentration of the arbitrary biomolecule contained in the sample based on the number of light emitting points derived from a region.
  • the labeling molecule irradiated with the excitation light emits a first fluorescence from the first fluorescent site when not bound to the arbitrary biomolecule, and binds to the arbitrary biomolecule Emits second fluorescence from the second fluorescent site, or emits second fluorescence from the second fluorescent site when not bound to the arbitrary biomolecule, and the arbitrary biomolecule
  • the first fluorescent site may emit the first fluorescence.
  • the concentration calculation unit emits fluorescence when it is bound to the arbitrary biomolecule with respect to the sum of the number of light emitting points derived from the first fluorescent site and the number of light emitting points derived from the second fluorescent site.
  • the counting unit can count the number of light emission points derived from the first fluorescent site and the number of light emission points derived from the second fluorescent site in the same region of an arbitrary size.
  • the second fluorescent site or the second fluorescent site is emitted from the first fluorescent site. Excitation energy may be applied from a site to the first fluorescent site.
  • the excitation light when the excitation light is irradiated to the labeled molecule that is not bonded to the arbitrary biomolecule, the first fluorescent site to the second fluorescent site, or the second fluorescent site to the first fluorescent site. Excitation energy may be given to the fluorescent site.
  • the excitation light may be evanescent light.
  • the sample may be held on a substrate.
  • the present disclosure also includes a light irradiation step of irradiating a sample containing a labeled molecule having a first fluorescent site and a second fluorescent site and binding to an arbitrary biomolecule, and detecting light emitted from the labeled molecule
  • a detecting step a counting step of counting the number of luminescent spots detected by the detection unit in the sample, a number of luminescent spots derived from the first fluorescent site, and a second fluorescent site
  • the concentration measurement method may include a concentration step of concentrating the sample by holding the sample on a substrate before the light irradiation step.
  • the present disclosure provides a biomolecule concentration measuring device and the like that can easily quantify biomolecules. Note that the effects described here are not necessarily limited, and may be any of the effects described in the present disclosure.
  • a and B are schematic diagrams for explaining switching of a fluorescent site that emits light in a labeled molecule. It is a schematic diagram which shows the outline
  • FIG. 1 is a schematic diagram showing an outline of a configuration of a biomolecule concentration measurement apparatus (hereinafter also simply referred to as a concentration measurement apparatus) according to the present embodiment.
  • the concentration measuring device D1 is detected by at least a light irradiation unit 1a that irradiates a sample with excitation light L11, a detection unit 2a that detects light emission L21 from the labeled molecule M, and a detection unit 2a.
  • a counting unit 3 that counts the number of emitted light points, and a concentration calculation unit 4 that calculates the concentration of an arbitrary biomolecule contained in the sample.
  • Each configuration of the concentration measuring device D1 will be described in order.
  • the light irradiation part 1a is a structure for irradiating the sample containing the labeled molecule M with the excitation light L11 in the concentration measuring device D1.
  • FIG. 2 schematically shows the structure of the labeled molecule M.
  • the labeled molecule M has a first fluorescent site F1 and a second fluorescent site F2.
  • the label molecule M has the binding site B with arbitrary biomolecules.
  • the binding site B has a structure in which one biomolecule T to be bound can be bound, and a plurality cannot be bound. Details of the labeling molecule M will be described later.
  • an appropriate light source 11 can be selected from a laser light source, an LED light source, a mercury lamp, a tungsten lamp, and the like according to the configuration of the labeling molecule M. Further, an excitation filter may be provided between the sample and the light source 11 in order to make the excitation light L11 light in a predetermined wavelength range (the excitation filter is not shown in FIG. 1).
  • the detection unit 2a is configured to detect the light emission L21 from the labeled molecule M in the sample in the concentration measurement device D1.
  • the configuration of the detection unit 2a is not particularly limited as long as the light emission from the labeled molecule M can be detected, and can be appropriately selected from known configurations.
  • the detector 2a can be provided with an area imaging device such as a CCD or CMOS device, a PMT (photomultiplier tube), a photodiode, or the like as the detector 21.
  • the detection unit 2a may be provided with a configuration such as an objective lens 22.
  • the labeling molecule M has a plurality of fluorescent sites F1 and F2 as described above. Regarding the first fluorescence and the second fluorescence emitted from these fluorescent sites F1 and F2, it is preferable that the wavelength ranges are different with respect to the excitation wavelength and the emission wavelength.
  • the detection unit 2a also detects light emission from any of the first fluorescent site F1 and the second fluorescent site F2.
  • the detection unit 2a is configured to detect the first fluorescence and the second fluorescence separately when detecting light emission from the labeled molecule M having the above-described characteristics. For example, by providing the fluorescent filter 23 between the labeled molecule M and the detector 21, only light in a desired wavelength region can be transmitted to the detector 21 side. In this case, the first fluorescence detection and the second fluorescence detection can be performed by switching the fluorescence filter 23 by providing a plurality of fluorescence filters 23 having different transmissive wavelength ranges.
  • the counting unit 3 is a configuration for counting the number of light emitting points in the sample detected by the detecting unit 2a in the concentration measuring device D1.
  • the concentration calculation unit 4 can select an arbitrary biomolecule included in the sample based on the number of emission points derived from the first fluorescence site F1 and the number of emission points derived from the second fluorescence site F2. It is the structure for calculating the density
  • the configuration of the counting unit 3 and the concentration calculation unit 4 is not particularly limited as long as the counting unit 3 and the concentration calculation unit 4 are configured to have the above-described functions, and can be appropriately adopted from known configurations.
  • the counting unit 3 and the concentration calculating unit 4 can be configured by general-purpose computers each including a CPU, a memory, a hard disk, and the like. Further, the counting unit 3 and the concentration calculating unit 4 may be configured by one general-purpose computer.
  • the concentration measuring device D1 has an input unit for inputting the concentration of a labeled molecule M in the sample, which will be described later, and a display unit for displaying the concentration of an arbitrary biomolecule in the sample.
  • a storage unit for storing the concentration of the labeled molecule M in the sample, the concentration of any measured biomolecule, and the like may be provided.
  • FIG. 3 is a flowchart illustrating an overview of the concentration measurement method according to the present disclosure.
  • the concentration measurement method includes a light irradiation step S1, a detection step S2, a counting step S3, and a concentration calculation step S4.
  • the light irradiation step S1 is a step of irradiating the sample containing the labeled molecule M with the excitation light L11 by the light irradiation unit 1a.
  • this step S1 light in a wavelength region corresponding to the excitation wavelength region of the fluorescent material employed in either the first fluorescent site F1 or the second fluorescent site F2 described above is irradiated.
  • the wavelength range of the excitation light L11 it can set suitably so that it may become a suitable wavelength in switching of the light emission site
  • the sample may be stored in any container as long as the sample can be irradiated with the excitation light L11.
  • the sample can be held on the substrate P (see FIG. 1 again). ).
  • the substrate P is configured to have affinity with the labeling molecule M. Since the substrate P has affinity with the label molecule M, the label molecule M is adsorbed to the substrate P, the Brownian motion of the label molecule M is suppressed, and the number of light emitting points is increased with higher accuracy in the counting step S3 described later. Can count.
  • a material having a high affinity with the label molecule M may be laminated on the surface of the substrate P in order to increase the affinity with the label molecule M.
  • centrifugation may be performed in order to bring the labeled molecule M closer to the surface of the substrate P.
  • a substance having a high affinity with the label molecule M may be applied to the surface of the substrate P.
  • the substrate P is preferably made of a material with little autofluorescence.
  • the sample only needs to contain a biomolecule whose concentration is to be measured, and its composition is not particularly limited.
  • the biomolecules whose concentration is to be measured include all molecules synthesized, metabolized or accumulated in vivo.
  • biomolecules include nucleic acids such as DNA and RNA, peptides, proteins, lipid-protein complexes, and the like.
  • the sample containing these biomolecules may be, for example, a liquid or gel-like biological sample.
  • the biological sample include whole blood, plasma, serum, cerebrospinal fluid, saliva, tear fluid, semen, synovial fluid, pleural effusion, and the like.
  • the method for measuring the concentration of the biomolecule according to the present disclosure includes a concentration step of concentrating the sample by holding it on the substrate before the light irradiation step S1. It may be. By concentrating the sample, the detection rate of luminescence derived from the labeled molecule M bound to the biomolecule can be increased.
  • the labeled molecule M is added to the sample.
  • concentration of the labeled molecule M in the sample is preferably known.
  • concentration of the labeled molecule M for example, a solution of the labeled molecule M prepared in advance to a predetermined concentration is prepared, and the concentration of the labeled molecule M in the sample is calculated from the amount added to the sample and the volume of the sample. You can also
  • the detection step S2 is a step in which the detection unit 2a detects the light emission L21 from the labeled molecule M irradiated with the excitation light L11 in the light irradiation step S1.
  • the label molecule M has two fluorescent sites, the first fluorescent site F1 and the second fluorescent site F2.
  • the labeling molecule M is configured so that the fluorescent site that emits light is switched depending on whether it is bound to a biomolecule or not.
  • the labeling molecule M emits the first fluorescence from the first fluorescent site F1 when not bound to any biomolecule, and the second fluorescent site when bound to any biomolecule. Those emitting second fluorescence from F2 are preferred. Further, the label molecule M emits the second fluorescence from the second fluorescent site F2 when it is not bound to any biomolecule, and the first fluorescence site when it is bound to any biomolecule. It is also possible to emit the first fluorescence from F1. Since the label molecule M has such a configuration, it is possible to determine whether or not the label molecule M is bound to a biomolecule to be bound by detecting light emission derived from the label molecule M. It becomes easy.
  • FIG. 4A and FIG. 4B show an example of switching the light emitting site of the labeled molecule M. 4A and 4B can be performed using, for example, the principle of fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the wavelength range of the excitation light L11 is the wavelength range corresponding to the excitation light of the first fluorescent site. Therefore, the first fluorescence L211 is emitted from the first fluorescence site F1.
  • the biomolecule T is bound to the binding region B, the three-dimensional structure of the label molecule M1 is changed, and the first fluorescent portion F1 and the second fluorescent portion F2 are close to each other.
  • the change in the three-dimensional structure of the labeled molecule M due to the binding between the binding region B and the biomolecule T is not limited to the case where the first fluorescent part F1 and the second fluorescent part F2 are close to each other.
  • the first fluorescent site F1 and the second fluorescent site F2 are close to each other. Therefore, when the excitation light L11 is irradiated to the labeled molecule M2 that is not bonded to the biomolecule T, excitation energy from the second fluorescent site F2 to the first fluorescent site F1 is given. As a result, the first fluorescence L211 is emitted from the first fluorescence site F1.
  • the biomolecule T is bound to the binding region B, the three-dimensional structure of the label molecule M2 is changed, and the first fluorescent site F1 and the second fluorescent site F2 are dissociated. Then, the second fluorescence L212 is emitted from the second fluorescence site F2 irradiated with the excitation light L11.
  • the light emitting site in a state where it is not bound to the biomolecule T is not limited to the first fluorescent site F1.
  • the light emitting site in the state of being bound to the biomolecule T is not limited to the second fluorescent site F2. That is, when the excitation light L11 is irradiated to the labeling molecule M bonded to the biomolecule T, the excitation energy is given from the second fluorescent part F2 to the first fluorescent part F1 in the labeling molecule M, and the light emitting part. However, the second fluorescent part F2 may be switched to the first fluorescent part F1.
  • the light emitting part may be switched.
  • Examples of combinations of fluorescent substances in the labeled molecule M using the FRET principle include CFP (excitation wavelength: 452 nm, emission wavelength: 505 nm) and YFP (excitation wavelength: 514 nm, emission wavelength: 527 nm), BFP ( Excitation wavelength: 380 nm, emission wavelength: 440 nm) and CFP (excitation wavelength: 452 nm, emission wavelength: 505 nm), GFP (excitation wavelength: 488 nm, emission wavelength: 509 nm), YFP (excitation wavelength: 514 nm, emission wavelength: 527 nm), etc. Is mentioned.
  • the combination of photoluminescence and fluorescent proteins such as Bioluminescence resonance energy transfer (BRET), combining two types of fluorescent molecules with different excitation and fluorescence wavelengths, excitation wavelength and fluorescence Using two types of Qdot (registered trademark) with different wavelengths, combining the molecules having the optical characteristics described above (fluorescent protein and fluorescent molecule, fluorescent molecule and Qdot (registered trademark), fluorescent protein and Qdot (registered trademark) ) And the like.
  • BRET Bioluminescence resonance energy transfer
  • binding site B of the labeled molecule M it is possible to adopt a configuration such as an antibody or an aptamer having a structure that specifically binds to an arbitrary biomolecule T.
  • the counting step S3 is a step of counting the number of light emitting points in the sample detected by the detecting unit 2a by the counting unit 3.
  • the counting unit 3 treats a point exceeding a predetermined reference from the distribution of the intensity of the light detected by the detection unit 2a as a light emitting point derived from the fluorescence L21 emitted from the labeled molecule M, and the number thereof. Can count.
  • the counting unit 3 switches the fluorescence filter 23 of the detection unit 2a to the first fluorescence L211 emitted from the first fluorescence site F1 with respect to the distribution of the detected light intensity.
  • Count the number of derived points S1 (FIG. 5A).
  • the counting unit 3 counts the number of points S2 (FIG. 5B) derived from the second fluorescence L212 emitted from the second fluorescence site F2.
  • the counting unit 3 counts the number of light emitting points S1 derived from the first fluorescent site F1 and the number of light emitting points S2 derived from the second fluorescent site F2 in the same region (region R) having an arbitrary size. It is preferable. By counting the number of light emitting points S1 and S2 in the same region R, the concentration can be calculated with higher accuracy in the concentration calculation step S4 described later.
  • concentration calculation step S4 an arbitrary biomolecule T contained in the sample is calculated based on the number S1 of light emitting points derived from the first fluorescent site F1 and the number of light emitting points S2 derived from the second fluorescent site F2. This is a step of calculating the concentration.
  • the labeled molecule M has the property that the fluorescent site that emits light can be switched depending on the presence or absence of binding to the biomolecule T that is the binding target. For this reason, one of the number of emission points S1 derived from the first fluorescence site F1 and the number of emission points S2 derived from the second fluorescence site F2 is bound to the biomolecule T in the sample.
  • the concentration calculation unit 4 selects an arbitrary biomolecule T with respect to the sum of the number of light emitting points S1 derived from the first fluorescent site F1 and the number of light emitting points S2 derived from the second fluorescent site F2.
  • the concentration of the biomolecule in the sample can be calculated based on the ratio of the number of emission points derived from the fluorescent sites that emit fluorescence when they are bound to each other.
  • the first fluorescence L211 is emitted from the first fluorescence site F1 when the labeled molecule M is not bound to the biomolecule T, and the second fluorescence is emitted when bound to the biomolecule T.
  • the second fluorescence L212 is emitted from the site F2 will be described as an example (see FIG. 4A, labeled molecule M1, again).
  • the number of emission points S2 derived from the second fluorescent site F2 corresponds to the number of labeled molecules M1 bound to the biomolecule T.
  • the concentration calculation unit 4 uses the one sample as a reference to calculate the light emission points S2. Depending on the number, the concentration of biomolecule T in a plurality of samples can be relatively quantified.
  • the ratio of the number of points S2 reflects the concentration of the biomolecule T in the sample.
  • the concentration of the labeled molecule M in the sample can be adjusted to a desired concentration when added to the sample in advance. For this reason, the concentration of the biomolecule T in the sample can be calculated based on the concentration of the labeled molecule M in the sample and the ratio.
  • the concentration of the biomolecule in the sample can be calculated using only the labeled molecule. For this reason, it is not necessary to use a plurality of reagents. Further, since no color reaction is required, it can be carried out easily in a short time compared with the conventional concentration calculation method.
  • a labeled molecule that is bound to a biomolecule and a labeled molecule that is not bound to a biomolecule can be distinguished based on the emission wavelength. Is unnecessary. For this reason, the concentration of the biomolecule can be measured more easily.
  • the concentration of the biomolecule in the sample can be calculated based on the ratio of the luminescent point derived from the labeled molecule bound to the biomolecule and the concentration of the labeled molecule in the sample. For this reason, the density
  • FIG. 6 is a schematic diagram illustrating an outline of a configuration of a concentration measuring device D2 according to the second embodiment of the present disclosure.
  • the light irradiation unit 1b of the concentration measuring device D2 has a configuration for irradiating the sample with evanescent light as the excitation light L12.
  • symbol is attached
  • the light irradiation unit 1b only needs to be configured so as to be able to irradiate the labeled molecules M in the sample with evanescent light, and the configuration can be appropriately adopted from the configuration of a known microscope or the like.
  • the mirror 14 may be provided in the light irradiation unit 1b.
  • the mirror 14 is provided at an angle at which the excitation light L11 emitted from the light source 11 is totally reflected on the sample contact surface P1 of the substrate P.
  • the excitation light L12 applied to the sample containing the labeling molecule M can be used as evanescent light. Since the evanescent light is irradiated only to a limited area of the sample, the detection unit 2a can detect the light emission L22 in a state with little background light.
  • evanescent light can be used as excitation light. For this reason, light emission derived from the labeled molecule can be detected with higher resolution, and counting of the light emission points can be performed with higher accuracy. Accordingly, it is possible to further increase the accuracy of concentration measurement in the concentration measuring apparatus.
  • the concentration measuring apparatus according to the second embodiment are the same as those of the concentration measuring apparatus according to the first embodiment described above.
  • FIG. 7 is a schematic diagram illustrating an outline of a configuration of a concentration measuring device D3 according to the third embodiment of the present disclosure.
  • the detector 2c of the concentration measuring device D3 has two light receivers 27a and 27b that receive photons of the light emission L21.
  • the detection unit 2c includes a dichroic mirror 24, a band pass filter 25, and a half mirror 26 as a configuration for guiding light to the two light receivers 27a and 27b.
  • the half mirror 26 has a transmittance and a reflectance of 50%.
  • the two light receivers 27 a and 27 b are installed at the same distance from the half mirror 26.
  • the density measuring device D3 may be provided with a polarizing beam splitter 28 or the like as necessary.
  • the light irradiation unit 1c is provided with a laser light source for irradiating the sample with the excitation light L11 as a light source 11c.
  • symbol is attached
  • the luminescence L21 emitted from the labeling molecule M by the excitation light L11 is guided to the half mirror 26 by removing the excitation light L11 by the dichroic mirror 24 and the band pass filter 25. If the photons contained in the light emission L21 are derived from one of the fluorescent sites F1 and F2 of the labeling molecule M, they are single photons and cannot be separated by the half mirror 26. Therefore, one of the two light receivers 27a and 27b detects a photon.
  • the photons contained in the light emission L21 are derived from the fluorescent sites of each of the plurality of labeled molecules M, for example, the photons are detected by both the two light receivers 27a and 27b because they are not single photons.
  • the excitation light L11 is pulse-irradiated, if it is not a single photon, the two light receivers 27a and 27b can detect the photon at the same timing. For this reason, it is possible to determine whether or not the light emission L12 is derived from one label molecule M by detecting the photons simultaneously or alternately in the two light receivers 27a and 27b. .
  • the concentration of a biomolecule is measured based on the number of luminescent spots. For this reason, depending on the density
  • the concentration measurement apparatus of the present embodiment it is possible to determine whether or not the light emission point is derived from one label molecule M by using two light receivers. Therefore, the light emitting points counted by the counting unit can be limited to only light emitting points derived from single photons in advance. As a result, the number of light emitting points can be counted more accurately, and the concentration can be measured with higher accuracy.
  • the present disclosure can have the following configurations.
  • a light irradiation unit that irradiates a sample including a labeled molecule that has a first fluorescent site and a second fluorescent site and binds to an arbitrary biomolecule, and a detection that detects luminescence from the labeled molecule ,
  • a counting unit for counting the number of light emitting points detected by the detection unit in the sample, the number of light emitting points derived from the first fluorescent site, and a light emitting point derived from the second fluorescent site
  • a concentration calculation unit that calculates the concentration of the arbitrary biomolecule contained in the sample based on the number of the biomolecules.
  • the labeling molecule irradiated with the excitation light does not bind to the arbitrary biomolecule, the labeling molecule emits a first fluorescence from the first fluorescent site and binds to the arbitrary biomolecule.
  • the second fluorescent site emits a second fluorescence, or the second fluorescent site emits a second fluorescence when not bound to the arbitrary biomolecule, and the optional fluorescent
  • the biomolecule concentration measuring apparatus according to (1) above, wherein the first fluorescent portion emits a first fluorescence when it is bound to a biomolecule.
  • the concentration calculation unit binds to the arbitrary biomolecule with respect to the sum of the number of emission points derived from the first fluorescence site and the number of emission points derived from the second fluorescence site.
  • the biomolecule concentration measuring apparatus wherein the concentration is calculated based on a ratio of the number of light emitting points derived from fluorescent sites that emit fluorescence.
  • the counting unit is configured to count the number of luminescent spots derived from the first fluorescent site and the number of luminescent spots derived from the second fluorescent site in the same area of an arbitrary size.
  • the biomolecule concentration measuring apparatus according to any one of 3).
  • (5) When the excitation light is irradiated to the labeled molecule bonded to the arbitrary biomolecule, the first fluorescent site to the second fluorescent site or the second fluorescent site to the first
  • the biomolecule concentration measurement apparatus according to any one of the above (1) to (4), wherein excitation energy is given to a fluorescent site.
  • the first fluorescent site to the second fluorescent site or the second fluorescent site to the first The biomolecule concentration measuring apparatus according to any one of (1) to (4), wherein excitation energy is applied to one fluorescent site.
  • a light irradiation step of irradiating a sample containing a labeled molecule having a first fluorescent site and a second fluorescent site and binding to an arbitrary biomolecule, and detection for detecting light emission from the labeled molecule A step, a counting step of counting the number of light emitting points detected by the detection unit in the sample, the number of light emitting points derived from the first fluorescent site, and a light emitting point derived from the second fluorescent site And a concentration calculating step of calculating a concentration of the arbitrary biomolecule contained in the sample based on the number of biomolecules.
  • D1, D2, D3 Biomolecule concentration measuring device (concentration measuring device)
  • B Binding site
  • F1 First fluorescent site
  • F2 Second fluorescent site
  • L11, L12 Excitation light
  • L21, L211, L212, L22 Luminescence M
  • M1, M2 Labeled molecule
  • R Region P: Substrate P1: Sample Contact surfaces S1, S2: Luminescent point T: Biomolecules 1a, 1b, 1c: Light irradiation unit 11, 11c: Light source 12: Condensing lens 13: Aperture 14: Mirror 2a, 2c: Detection unit 21: Detector 22: Objective Lens 23: Fluorescent filter 24: Dichroic mirror 25: Band pass filter 26: Half mirror 27a, 27b: Light receiver 28: Polarizing beam splitter 3: Counting unit 4: Density calculating unit

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Abstract

Provided is a biomolecule concentration measurement device capable of simply quantifying biomolecules. A biomolecule concentration measurement device is provided that has a light irradiation unit for irradiating excitation light onto a sample including a label molecule that has a first fluorescent moiety and a second fluorescent moiety and binds to an arbitrary biomolecule, a detection unit for detecting luminescence from the label molecule, a counting unit for counting the number of luminescent points detected in the sample by the detection unit, and a concentration calculation unit for calculating the concentration of the arbitrary biomolecule included in the sample on the basis of the number of luminescent points caused by the first fluorescent moiety and the number of luminescent points caused by the second fluorescent moiety.

Description

生体分子の濃度測定装置及び生体分子の濃度測定方法Biomolecule concentration measuring apparatus and biomolecule concentration measuring method
　本技術は、生体分子の濃度測定装置及び生体分子の濃度測定方法に関する。より詳しくは、発光点の数に基づき濃度を測定する技術に関する。 The present technology relates to a biomolecule concentration measuring apparatus and a biomolecule concentration measuring method. More specifically, the present invention relates to a technique for measuring density based on the number of light emitting points.
　生体由来の試料に含まれる物質の検出及び定量には、各種方法が用いられている。特に、酵素結合免疫吸着検定法（ＥＬＩＳＡ法）は、抗原抗体反応を利用することにより、比較的高感度に測定対象の定量を行うことができるため、試料に含まれる抗原又は抗体の定量に好適に用いられている。しかし、ＥＬＩＳＡ法では、複数の試薬や洗浄工程を必要とするため、その作業は煩雑である。 Various methods are used for detection and quantification of substances contained in biological samples. In particular, enzyme-linked immunosorbent assay (ELISA) is suitable for quantification of an antigen or antibody contained in a sample because it can quantitate a measurement object with relatively high sensitivity by using an antigen-antibody reaction. It is used for. However, since the ELISA method requires a plurality of reagents and washing steps, the operation is complicated.
　このような作業性の問題に対して、例えば、特許文献１には、「標的物質を含む被験試料液に下記競合物質を混合した混合液を、下記競合的捕捉物質、下記判定物質の順に接触させる免疫反応工程と、前記競合的捕捉物質及び前記判定物質のうちの少なくとも一方に前記競合物質が備える酵素の基質を接触させて反応物を得る酵素反応工程と、前記反応物の量を測定して前記標的物質の量を算出する算出工程と、を有することを特徴とする標的物質の測定方法」が開示されている。当該測定方法では、競合物質を利用することにより、競合物質が競合的捕捉物質に捕捉された量を直接的乃至間接的に測定することで、競合する標的物質の定量を行っている。 For such workability problem, for example, Patent Document 1 discloses that “a mixed liquid obtained by mixing the following competitive substance in a test sample solution containing a target substance is contacted in the order of the following competitive capture substance and the following determination substance. An immune reaction step, an enzyme reaction step of contacting a substrate of an enzyme included in the competitor with at least one of the competitive capture substance and the determination substance, and obtaining a reactant, and measuring the amount of the reactant And a calculation step of calculating the amount of the target substance. In this measurement method, the competitive target substance is quantified by directly or indirectly measuring the amount of the competitive substance captured by the competitive capture substance by using the competitive substance.
特開２００８－３２４９４号公報JP 2008-32494 A
　上記特許文献１に記載されている測定方法によって、生体分子の濃度の測定において、作業性を向上させることができる。しかし、上記特許文献１に記載の方法でも、競合的捕捉物質及び判定物質の各々の物質を含む複数の試薬を用意して、順に混合する工程が必須である。このため、より簡便に試料中の生体分子の濃度を測定する方法が求められている。 The workability can be improved in the measurement of the biomolecule concentration by the measurement method described in Patent Document 1. However, the method described in Patent Document 1 also requires a step of preparing a plurality of reagents including each of the competitive capture substance and the determination substance and mixing them in order. For this reason, a method for measuring the concentration of a biomolecule in a sample more simply is required.
　そこで、本開示は、簡便に生体分子を定量することができる、生体分子の濃度測定装置等を提供することを主な目的とする。 Therefore, the main object of the present disclosure is to provide a biomolecule concentration measuring device and the like that can easily quantify biomolecules.
　上記課題解決のため、本開示は第１の蛍光部位及び第２の蛍光部位を有し任意の生体分子に結合する標識分子を含む試料に励起光を照射する光照射部と、前記標識分子からの発光を検出する検出部と、前記試料中の前記検出部によって検出された発光点の数を数える計数部と、前記第１の蛍光部位に由来する発光点の数と、前記第２の蛍光部位に由来する発光点の数と、に基づき前記試料に含まれる前記任意の生体分子の濃度を算出する濃度算出部と、を有する生体分子の濃度測定装置を提供する。
　前記励起光を照射された前記標識分子は、前記任意の生体分子と結合していない場合には前記第１の蛍光部位から第１の蛍光を発し、前記任意の生体分子と結合している場合には前記第２の蛍光部位から第２の蛍光を発するもの、又は前記任意の生体分子と結合していない場合には前記第２の蛍光部位から第２の蛍光を発し、前記任意の生体分子と結合している場合には前記第１の蛍光部位から第１の蛍光を発するもの、であってもよい。
　前記濃度算出部は前記第１の蛍光部位に由来する発光点の数と前記第２の蛍光部位に由来する発光点の数の和に対する前記任意の生体分子と結合している場合に蛍光を発する蛍光部位に由来する発光点の数の割合に基づき前記濃度を算出してもよい。
　前記計数部は、任意の大きさの同一領域における前記第１の蛍光部位に由来する発光点の数及び前記第２の蛍光部位に由来する発光点の数を数えることができる。
　前記生体分子の濃度測定装置では、前記任意の生体分子と結合した前記標識分子へ前記励起光が照射されると、前記第１の蛍光部位から前記第２の蛍光部位、又は前記第２の蛍光部位から前記第１の蛍光部位へ励起エネルギーが与えられてもよい。
　また、前記任意の生体分子と結合していない前記標識分子へ前記励起光が照射されると、前記第１の蛍光部位から前記第２の蛍光部位、又は前記第２の蛍光部位から前記第１の蛍光部位へ励起エネルギーが与えられてもよい。
　また、前記励起光をエバネッセント光とすることもできる。
　さらに、前記試料は基板上に保持されていてもよい。 In order to solve the above problem, the present disclosure provides a light irradiation unit that irradiates a sample including a labeled molecule that has a first fluorescent site and a second fluorescent site and binds to an arbitrary biomolecule, A detection unit that detects the emission of light, a counting unit that counts the number of emission points detected by the detection unit in the sample, the number of emission points derived from the first fluorescence site, and the second fluorescence Provided is a biomolecule concentration measuring apparatus comprising: a concentration calculating unit that calculates the concentration of the arbitrary biomolecule contained in the sample based on the number of light emitting points derived from a region.
When the labeling molecule irradiated with the excitation light emits a first fluorescence from the first fluorescent site when not bound to the arbitrary biomolecule, and binds to the arbitrary biomolecule Emits second fluorescence from the second fluorescent site, or emits second fluorescence from the second fluorescent site when not bound to the arbitrary biomolecule, and the arbitrary biomolecule In the case where the first fluorescent site is bound to the first fluorescent site, the first fluorescent site may emit the first fluorescence.
The concentration calculation unit emits fluorescence when it is bound to the arbitrary biomolecule with respect to the sum of the number of light emitting points derived from the first fluorescent site and the number of light emitting points derived from the second fluorescent site. You may calculate the said density | concentration based on the ratio of the number of the light emission points originating in a fluorescence site | part.
The counting unit can count the number of light emission points derived from the first fluorescent site and the number of light emission points derived from the second fluorescent site in the same region of an arbitrary size.
In the biomolecule concentration measuring apparatus, when the excitation light is irradiated to the labeled molecule bound to the arbitrary biomolecule, the second fluorescent site or the second fluorescent site is emitted from the first fluorescent site. Excitation energy may be applied from a site to the first fluorescent site.
Further, when the excitation light is irradiated to the labeled molecule that is not bonded to the arbitrary biomolecule, the first fluorescent site to the second fluorescent site, or the second fluorescent site to the first fluorescent site. Excitation energy may be given to the fluorescent site.
The excitation light may be evanescent light.
Furthermore, the sample may be held on a substrate.
　本開示はまた、第１の蛍光部位及び第２の蛍光部位を有し任意の生体分子に結合する標識分子を含む試料に励起光を照射する光照射工程と、前記標識分子からの発光を検出する検出工程と、前記試料中の前記検出部によって検出された発光点の数を数える計数工程と、前記第１の蛍光部位に由来する発光点の数と、前記第２の蛍光部位に由来する発光点の数と、に基づき前記試料に含まれる前記任意の生体分子の濃度を算出する濃度算出工程と、を有する生体分子の濃度測定方法を提供する。
　前記濃度測定方法は、前記光照射工程の前に前記試料を基板上に保持させて濃縮する濃縮工程を有していてもよい。 The present disclosure also includes a light irradiation step of irradiating a sample containing a labeled molecule having a first fluorescent site and a second fluorescent site and binding to an arbitrary biomolecule, and detecting light emitted from the labeled molecule A detecting step, a counting step of counting the number of luminescent spots detected by the detection unit in the sample, a number of luminescent spots derived from the first fluorescent site, and a second fluorescent site And a concentration calculating step of calculating a concentration of the arbitrary biomolecule contained in the sample based on the number of light emitting points.
The concentration measurement method may include a concentration step of concentrating the sample by holding the sample on a substrate before the light irradiation step.
　本開示により、簡便に生体分子を定量することができる生体分子の濃度測定装置等が提供される。なお、ここに記載された効果は必ずしも限定されるものではなく、本開示中に記載されたいずれかの効果であってもよい。 The present disclosure provides a biomolecule concentration measuring device and the like that can easily quantify biomolecules. Note that the effects described here are not necessarily limited, and may be any of the effects described in the present disclosure.
本開示の第１実施形態に係る生体分子の濃度測定装置の構成の概要を示す模式図である。It is a mimetic diagram showing an outline of composition of a concentration measuring device of a biomolecule concerning a 1st embodiment of this indication. 標識分子の構造を示す模式図である。It is a schematic diagram which shows the structure of a labeled molecule. 本開示に係る生体分子の濃度測定方法の概要を示すフローチャートである。It is a flowchart which shows the outline | summary of the biomolecule density | concentration measuring method which concerns on this indication. Ａ及びＢは、標識分子における発光する蛍光部位の切り替えを説明するための模式図である。A and B are schematic diagrams for explaining switching of a fluorescent site that emits light in a labeled molecule. 計数工程の概要を示す模式図である。It is a schematic diagram which shows the outline | summary of a counting process. 本開示の第２実施形態に係る生体分子の濃度測定装置の構成の概要を示す模式図である。It is a schematic diagram which shows the outline | summary of a structure of the concentration measuring apparatus of the biomolecule which concerns on 2nd Embodiment of this indication. 本開示の第３実施形態に係る生体分子の濃度測定装置の構成の概要を示す模式図である。It is a schematic diagram which shows the outline | summary of a structure of the concentration measuring apparatus of the biomolecule which concerns on 3rd Embodiment of this indication.
　以下、本開示を実施するための好適な形態について説明する。なお、以下に説明する実施形態は、本開示の代表的な実施形態を示したものであり、これにより本開示の範囲が狭く解釈されることはない。 Hereinafter, a preferred embodiment for carrying out the present disclosure will be described. In addition, embodiment described below shows typical embodiment of this indication, and, thereby, the range of this indication is not interpreted narrowly.
１．第１実施形態に係る生体分子の濃度測定装置
　先ず、本開示の第１実施形態に係る生体分子の濃度測定装置について説明する。図１は、本実施形態の生体分子の濃度測定装置（以下、単に濃度測定装置とも称する。）の構成の概要を示す模式図である。図１に示すように、濃度測定装置Ｄ１は、少なくとも、試料に励起光Ｌ１１を照射する光照射部１ａと、標識分子Ｍからの発光Ｌ２１を検出する検出部２ａと、検出部２ａによって検出された発光点の数を数える計数部３と、試料に含まれる任意の生体分子の濃度を算出する濃度算出部４と、を有する。濃度測定装置Ｄ１の各構成について順に説明する。 1. Biomolecule Concentration Measuring Device According to First Embodiment First, a biomolecule concentration measuring device according to a first embodiment of the present disclosure will be described. FIG. 1 is a schematic diagram showing an outline of a configuration of a biomolecule concentration measurement apparatus (hereinafter also simply referred to as a concentration measurement apparatus) according to the present embodiment. As shown in FIG. 1, the concentration measuring device D1 is detected by at least a light irradiation unit 1a that irradiates a sample with excitation light L11, a detection unit 2a that detects light emission L21 from the labeled molecule M, and a detection unit 2a. A counting unit 3 that counts the number of emitted light points, and a concentration calculation unit 4 that calculates the concentration of an arbitrary biomolecule contained in the sample. Each configuration of the concentration measuring device D1 will be described in order.
＜光照射部＞
　光照射部１ａは、濃度測定装置Ｄ１において、標識分子Ｍを含む試料に励起光Ｌ１１を照射するための構成である。図２に標識分子Ｍの構造を模式的に示す。標識分子Ｍとは、第１の蛍光部位Ｆ１及び第２の蛍光部位Ｆ２を有するものである。また、任意の生体分子に結合するために、標識分子Ｍは、任意の生体分子との結合部位Ｂを有する。結合部位Ｂは、結合対象とする生体分子Ｔを１つ結合でき、複数結合させることはできない構造を有する。標識分子Ｍの詳細は、後述する。 <Light irradiation part>
The light irradiation part 1a is a structure for irradiating the sample containing the labeled molecule M with the excitation light L11 in the concentration measuring device D1. FIG. 2 schematically shows the structure of the labeled molecule M. The labeled molecule M has a first fluorescent site F1 and a second fluorescent site F2. Moreover, in order to couple | bond with arbitrary biomolecules, the label molecule M has the binding site B with arbitrary biomolecules. The binding site B has a structure in which one biomolecule T to be bound can be bound, and a plurality cannot be bound. Details of the labeling molecule M will be described later.
光照射部１ａは、標識分子Ｍに所定の波長の励起光Ｌ１１を照射できれば、その構成は特に限定されず、公知の構成の中から適宜採用することができる。例えば、光照射部１ａには、励起光Ｌ１１を出射する光源１１や集光レンズ１２、絞り１３等を備えることができる。また、光照射部１ａは、走査しながら試料に対して励起光Ｌ１１を照射できるように構成されていてもよい。 If the light irradiation part 1a can irradiate the labeled molecule M with the excitation light L11 of a predetermined wavelength, the structure will not be specifically limited, It can employ | adopt suitably from well-known structures. For example, the light irradiation unit 1a can include a light source 11 that emits the excitation light L11, a condenser lens 12, a diaphragm 13, and the like. Moreover, the light irradiation part 1a may be comprised so that excitation light L11 can be irradiated with respect to a sample, scanning.
　光源１１としては、例えば、レーザー光源、ＬＥＤ光源、水銀ランプ、タングステンランプ等の中から、標識分子Ｍの構成等に合わせて適切な光源１１を選択することができる。また、励起光Ｌ１１を所定の波長域の光とするために、試料と光源１１との間に励起フィルタを設けることもできる（図１において、励起フィルタは不図示）。 As the light source 11, for example, an appropriate light source 11 can be selected from a laser light source, an LED light source, a mercury lamp, a tungsten lamp, and the like according to the configuration of the labeling molecule M. Further, an excitation filter may be provided between the sample and the light source 11 in order to make the excitation light L11 light in a predetermined wavelength range (the excitation filter is not shown in FIG. 1).
＜検出部＞
　検出部２ａは、濃度測定装置Ｄ１において、試料中の標識分子Ｍからの発光Ｌ２１を検出するための構成である。検出部２ａについても、標識分子Ｍからの発光を検出できれば、その構成は特に限定されず、公知の構成の中から適宜採用することができる。例えば、検出部２ａには、ＣＣＤやＣＭＯＳ素子等のエリア撮像素子、ＰＭＴ（光電子倍増管）、フォトダイオード等を検出器２１として備えることができる。また、検出部２ａには、対物レンズ２２等の構成が備えられていてもよい。 <Detector>
The detection unit 2a is configured to detect the light emission L21 from the labeled molecule M in the sample in the concentration measurement device D1. The configuration of the detection unit 2a is not particularly limited as long as the light emission from the labeled molecule M can be detected, and can be appropriately selected from known configurations. For example, the detector 2a can be provided with an area imaging device such as a CCD or CMOS device, a PMT (photomultiplier tube), a photodiode, or the like as the detector 21. Further, the detection unit 2a may be provided with a configuration such as an objective lens 22.
　標識分子Ｍは、上述したように複数の蛍光部位Ｆ１，Ｆ２を有しているものである。これらの蛍光部位Ｆ１，Ｆ２から発せられる第１の蛍光と第２の蛍光については、励起波長及び発光波長に関して、各々、波長域が異なることが好ましい。また、検出部２ａは、第１の蛍光部位Ｆ１と第２の蛍光部位Ｆ２の、いずれの蛍光部位からの発光についても検出する。 The labeling molecule M has a plurality of fluorescent sites F1 and F2 as described above. Regarding the first fluorescence and the second fluorescence emitted from these fluorescent sites F1 and F2, it is preferable that the wavelength ranges are different with respect to the excitation wavelength and the emission wavelength. The detection unit 2a also detects light emission from any of the first fluorescent site F1 and the second fluorescent site F2.
　検出部２ａは、上記の特徴を有する標識分子Ｍからの発光を検出する場合には、第１の蛍光と第２の蛍光とを、区別して検出できるように構成されていることが好ましい。例えば、標識分子Ｍと検出器２１との間に、蛍光フィルタ２３を設けることにより、所望の波長域の光のみを検出器２１側へ透過させることができる。この場合、透過可能な波長域が互いに異なる複数の蛍光フィルタ２３を設けることで、第１の蛍光の検出と、第２の蛍光の検出と、を、蛍光フィルタ２３の切り替えにより行うことができる。 It is preferable that the detection unit 2a is configured to detect the first fluorescence and the second fluorescence separately when detecting light emission from the labeled molecule M having the above-described characteristics. For example, by providing the fluorescent filter 23 between the labeled molecule M and the detector 21, only light in a desired wavelength region can be transmitted to the detector 21 side. In this case, the first fluorescence detection and the second fluorescence detection can be performed by switching the fluorescence filter 23 by providing a plurality of fluorescence filters 23 having different transmissive wavelength ranges.
＜計数部及び濃度算出部４＞
　計数部３は、濃度測定装置Ｄ１において、検出部２ａによって検出された試料中の発光点の数を数えるための構成である。また、濃度算出部４は、第１の蛍光部位Ｆ１に由来する発光点の数と、第２の蛍光部位Ｆ２に由来する発光点の数と、に基づいて、試料に含まれる任意の生体分子の濃度を算出するための構成である。これらの計数部３及び濃度算出部４については、上記の機能を有するように構成されていれば、その構成は特に限定されず、公知の構成の中から適宜採用することができる。例えば、計数部３及び濃度算出部４は、各々、ＣＰＵ、メモリ及びハードディスクなどを備える汎用のコンピュータによって構成することができる。また、計数部３と濃度算出部４とを、１台の汎用コンピュータから構成してもよい。 <Counter and concentration calculator 4>
The counting unit 3 is a configuration for counting the number of light emitting points in the sample detected by the detecting unit 2a in the concentration measuring device D1. In addition, the concentration calculation unit 4 can select an arbitrary biomolecule included in the sample based on the number of emission points derived from the first fluorescence site F1 and the number of emission points derived from the second fluorescence site F2. It is the structure for calculating the density | concentration of. The configuration of the counting unit 3 and the concentration calculation unit 4 is not particularly limited as long as the counting unit 3 and the concentration calculation unit 4 are configured to have the above-described functions, and can be appropriately adopted from known configurations. For example, the counting unit 3 and the concentration calculating unit 4 can be configured by general-purpose computers each including a CPU, a memory, a hard disk, and the like. Further, the counting unit 3 and the concentration calculating unit 4 may be configured by one general-purpose computer.
　上述した構成の他、濃度測定装置Ｄ１には、後述する試料中の標識分子Ｍの濃度等を入力するための入力部や、試料中の任意の生体分子の濃度等を表示するための表示部、試料における標識分子Ｍの濃度や測定された任意の生体分子の濃度等を記憶しておくための記憶部等が備えられていてもよい。 In addition to the configuration described above, the concentration measuring device D1 has an input unit for inputting the concentration of a labeled molecule M in the sample, which will be described later, and a display unit for displaying the concentration of an arbitrary biomolecule in the sample. In addition, a storage unit for storing the concentration of the labeled molecule M in the sample, the concentration of any measured biomolecule, and the like may be provided.
２．第１実施形態に係る濃度測定装置による生体分子の濃度の測定
　次に、本開示に係る生体分子の濃度測定方法について説明する。即ち、上述した第１実施形態に係る濃度測定装置Ｄ１の動作について説明する。図３は、本開示に係る濃度測定方法の概要を示すフローチャートである。図３に示すように、濃度測定方法は、光照射工程Ｓ１、検出工程Ｓ２、計数工程Ｓ３及び濃度算出工程Ｓ４の各工程を有する。 2. Measurement of Biomolecule Concentration by Concentration Measurement Device According to First Embodiment Next, a biomolecule concentration measurement method according to the present disclosure will be described. That is, the operation of the concentration measuring device D1 according to the first embodiment described above will be described. FIG. 3 is a flowchart illustrating an overview of the concentration measurement method according to the present disclosure. As shown in FIG. 3, the concentration measurement method includes a light irradiation step S1, a detection step S2, a counting step S3, and a concentration calculation step S4.
＜光照射工程＞
　光照射工程Ｓ１は、光照射部１ａによって、標識分子Ｍを含む試料に励起光Ｌ１１を照射する工程である。本工程Ｓ１では、上述した第１の蛍光部位Ｆ１又は第２の蛍光部位Ｆ２のいずれかに採用された蛍光物質の励起波長域に相当する波長域の光を照射する。励起光Ｌ１１の波長域については、後述する、標識分子Ｍにおける発光部位の切り替えにおいて適切な波長となるように適宜設定することができる。 <Light irradiation process>
The light irradiation step S1 is a step of irradiating the sample containing the labeled molecule M with the excitation light L11 by the light irradiation unit 1a. In this step S1, light in a wavelength region corresponding to the excitation wavelength region of the fluorescent material employed in either the first fluorescent site F1 or the second fluorescent site F2 described above is irradiated. About the wavelength range of the excitation light L11, it can set suitably so that it may become a suitable wavelength in switching of the light emission site | part in the labeling molecule M mentioned later.
　光照射工程Ｓ１において、試料は励起光Ｌ１１を照射可能であれば、どのような容器に収容されていてもよいが、例えば、試料を基板Ｐ上に保持することもできる（図１、再度参照）。試料を基板Ｐ上に保持する場合、基板Ｐは標識分子Ｍとの親和性を有するように構成されていることが好ましい。基板Ｐが標識分子Ｍとの親和性を有することにより、標識分子Ｍを基板Ｐに吸着させ、標識分子Ｍのブラウン運動を抑えて、後述する計数工程Ｓ３において、発光点の数をより精度高く数えることができる。 In the light irradiation step S1, the sample may be stored in any container as long as the sample can be irradiated with the excitation light L11. For example, the sample can be held on the substrate P (see FIG. 1 again). ). When the sample is held on the substrate P, it is preferable that the substrate P is configured to have affinity with the labeling molecule M. Since the substrate P has affinity with the label molecule M, the label molecule M is adsorbed to the substrate P, the Brownian motion of the label molecule M is suppressed, and the number of light emitting points is increased with higher accuracy in the counting step S3 described later. Can count.
　例えば、基板Ｐの表面に、標識分子Ｍとの親和性を高めるために、標識分子Ｍとの親和性が高い素材が積層されていてもよい。あるいは、基板Ｐの表面に標識分子Ｍを接近させるために、遠心処理を行ってもよい。また、基板Ｐの表面に、標識分子Ｍと親和性の高い物質が塗布されていてもよい。さらに、基板Ｐは、自家蛍光の少ない材質で構成されていることが好ましい。 For example, a material having a high affinity with the label molecule M may be laminated on the surface of the substrate P in order to increase the affinity with the label molecule M. Alternatively, centrifugation may be performed in order to bring the labeled molecule M closer to the surface of the substrate P. Further, a substance having a high affinity with the label molecule M may be applied to the surface of the substrate P. Furthermore, the substrate P is preferably made of a material with little autofluorescence.
　試料とは、濃度の測定対象となる生体分子が含まれていればよく、その組成は、特に限定されない。また、濃度の測定対象となる生体分子には、生体内で合成、代謝又は蓄積される分子全般が含まれる。例えば、生体分子としては、ＤＮＡ、ＲＮＡ等の核酸、ペプチド、タンパク質、脂質－タンパク質複合体等、が挙げられる。また、これらの生体分子を含む試料は、例えば、液状又はゲル状の生体試料であってもよい。生体試料としては、例えば、全血、血漿、血清、脳脊髄液、唾液、涙液、精液、滑液、胸水等が挙げられる。 The sample only needs to contain a biomolecule whose concentration is to be measured, and its composition is not particularly limited. In addition, the biomolecules whose concentration is to be measured include all molecules synthesized, metabolized or accumulated in vivo. Examples of biomolecules include nucleic acids such as DNA and RNA, peptides, proteins, lipid-protein complexes, and the like. The sample containing these biomolecules may be, for example, a liquid or gel-like biological sample. Examples of the biological sample include whole blood, plasma, serum, cerebrospinal fluid, saliva, tear fluid, semen, synovial fluid, pleural effusion, and the like.
　また、試料に含まれる生体分子の濃度が低い場合には、本開示に係る生体分子の濃度測定方法は、光照射工程Ｓ１の前に試料を基板上に保持させて濃縮する濃縮工程を有していてもよい。試料を濃縮することによって、生体分子と結合した標識分子Ｍに由来する発光の検出率を高めることができる。 In addition, when the concentration of the biomolecule contained in the sample is low, the method for measuring the concentration of the biomolecule according to the present disclosure includes a concentration step of concentrating the sample by holding it on the substrate before the light irradiation step S1. It may be. By concentrating the sample, the detection rate of luminescence derived from the labeled molecule M bound to the biomolecule can be increased.
　また、本工程Ｓ１の時点で、試料には標識分子Ｍが添加されている。試料中の標識分子Ｍの濃度は、既知であることが好ましい。標識分子Ｍの濃度については、例えば、予め所定の濃度に調製された標識分子Ｍの溶解液を用意して、試料への添加量と試料の容量から、試料中の標識分子Ｍの濃度を算出することもできる。 Further, at the time of this step S1, the labeled molecule M is added to the sample. The concentration of the labeled molecule M in the sample is preferably known. Regarding the concentration of the labeled molecule M, for example, a solution of the labeled molecule M prepared in advance to a predetermined concentration is prepared, and the concentration of the labeled molecule M in the sample is calculated from the amount added to the sample and the volume of the sample. You can also
＜検出工程＞
　検出工程Ｓ２は、光照射工程Ｓ１によって励起光Ｌ１１を照射された標識分子Ｍからの発光Ｌ２１を、上述した検出部２ａによって検出する工程である。上述したように、標識分子Ｍは、第１の蛍光部位Ｆ１と第２の蛍光部位Ｆ２の、２つの蛍光部位を有している。また、標識分子Ｍは、生体分子と結合している場合と結合していない場合とで、発光する蛍光部位が切り替わるように構成されていることが好ましい。 <Detection process>
The detection step S2 is a step in which the detection unit 2a detects the light emission L21 from the labeled molecule M irradiated with the excitation light L11 in the light irradiation step S1. As described above, the label molecule M has two fluorescent sites, the first fluorescent site F1 and the second fluorescent site F2. Moreover, it is preferable that the labeling molecule M is configured so that the fluorescent site that emits light is switched depending on whether it is bound to a biomolecule or not.
　例えば、標識分子Ｍは、任意の生体分子と結合していない場合には第１の蛍光部位Ｆ１から第１の蛍光を発し、任意の生体分子と結合している場合には第２の蛍光部位Ｆ２から第２の蛍光を発するものが好ましい。また、標識分子Ｍは、任意の生体分子と結合していない場合には第２の蛍光部位Ｆ２から第２の蛍光を発し、任意の生体分子と結合している場合には第１の蛍光部位Ｆ１から第１の蛍光を発するものとすることもできる。標識分子Ｍが、このような構成を有することにより、標識分子Ｍに由来する発光を検出することで、その標識分子Ｍが、結合対象である生体分子と結合しているか否か判定することが容易となる。 For example, the labeling molecule M emits the first fluorescence from the first fluorescent site F1 when not bound to any biomolecule, and the second fluorescent site when bound to any biomolecule. Those emitting second fluorescence from F2 are preferred. Further, the label molecule M emits the second fluorescence from the second fluorescent site F2 when it is not bound to any biomolecule, and the first fluorescence site when it is bound to any biomolecule. It is also possible to emit the first fluorescence from F1. Since the label molecule M has such a configuration, it is possible to determine whether or not the label molecule M is bound to a biomolecule to be bound by detecting light emission derived from the label molecule M. It becomes easy.
　図４Ａ及び図４Ｂに、標識分子Ｍの発光部位の切り替えについて、例を示す。また、図４Ａ及び図４Ｂに示すような、発光部位の切り替えは、例えば、蛍光共鳴エネルギー移動（Fluorescence Resonance Energy Transfer、ＦＲＥＴ）の原理を利用して行うこともできる。 FIG. 4A and FIG. 4B show an example of switching the light emitting site of the labeled molecule M. 4A and 4B can be performed using, for example, the principle of fluorescence resonance energy transfer (FRET).
　図４Ａに示す標識分子Ｍ１では、生体分子Ｔと結合していない標識分子Ｍ１へ励起光が照射されると、励起光Ｌ１１の波長域は、第１の蛍光部位の励起光に相当する波長域であるため、第１の蛍光部位Ｆ１から第１の蛍光Ｌ２１１が発せられる。一方、結合領域Ｂに生体分子Ｔが結合すると、標識分子Ｍ１の立体構造が変化して、第１の蛍光部位Ｆ１と第２の蛍光部位Ｆ２が近接する。そして、生体分子Ｔと結合した標識分子Ｍ１へ励起光Ｌ１１が照射されると、励起光Ｌ１１を照射された第１の蛍光部位Ｆ１から第２の蛍光部位Ｆ２へ励起エネルギーが与えられる。この結果、第２の蛍光部位Ｆ２から第２の蛍光Ｌ２１２が発せられる。 In the labeled molecule M1 shown in FIG. 4A, when excitation light is irradiated to the labeled molecule M1 that is not bonded to the biomolecule T, the wavelength range of the excitation light L11 is the wavelength range corresponding to the excitation light of the first fluorescent site. Therefore, the first fluorescence L211 is emitted from the first fluorescence site F1. On the other hand, when the biomolecule T is bound to the binding region B, the three-dimensional structure of the label molecule M1 is changed, and the first fluorescent portion F1 and the second fluorescent portion F2 are close to each other. When the labeling molecule M1 bonded to the biomolecule T is irradiated with the excitation light L11, excitation energy is given from the first fluorescent part F1 irradiated with the excitation light L11 to the second fluorescent part F2. As a result, the second fluorescence L212 is emitted from the second fluorescence site F2.
　結合領域Ｂと生体分子Ｔとの結合による標識分子Ｍの立体構造の変化は、第１の蛍光部位Ｆ１と第２の蛍光部位Ｆ２が近接する場合には限定されない。例えば、図４Ｂに示す標識分子Ｍ２は、結合部位Ｂに生体分子Ｔが結合していない場合、第１の蛍光部位Ｆ１と第２の蛍光部位Ｆ２は、近接している。そのため、生体分子Ｔと結合していない標識分子Ｍ２へ励起光Ｌ１１が照射されると、第２の蛍光部位Ｆ２から第１の蛍光部位Ｆ１への励起エネルギーが与えられる。この結果、第１の蛍光部位Ｆ１から第１の蛍光Ｌ２１１が発せられる。一方、結合領域Ｂに生体分子Ｔが結合すると、標識分子Ｍ２の立体構造が変化して、第１の蛍光部位Ｆ１と第２の蛍光部位Ｆ２が解離する。そして、励起光Ｌ１１を照射された第２の蛍光部位Ｆ２からは、第２の蛍光Ｌ２１２が発せられる。 The change in the three-dimensional structure of the labeled molecule M due to the binding between the binding region B and the biomolecule T is not limited to the case where the first fluorescent part F1 and the second fluorescent part F2 are close to each other. For example, in the labeled molecule M2 shown in FIG. 4B, when the biomolecule T is not bound to the binding site B, the first fluorescent site F1 and the second fluorescent site F2 are close to each other. Therefore, when the excitation light L11 is irradiated to the labeled molecule M2 that is not bonded to the biomolecule T, excitation energy from the second fluorescent site F2 to the first fluorescent site F1 is given. As a result, the first fluorescence L211 is emitted from the first fluorescence site F1. On the other hand, when the biomolecule T is bound to the binding region B, the three-dimensional structure of the label molecule M2 is changed, and the first fluorescent site F1 and the second fluorescent site F2 are dissociated. Then, the second fluorescence L212 is emitted from the second fluorescence site F2 irradiated with the excitation light L11.
　上述した標識分子Ｍにおいて、生体分子Ｔと結合していない状態における発光部位は、第１の蛍光部位Ｆ１に限定されるものではない。同様に、生体分子Ｔと結合した状態における発光部位についても、第２の蛍光部位Ｆ２に限定されるものではない。即ち、生体分子Ｔと結合した標識分子Ｍへ励起光Ｌ１１が照射されると、標識分子Ｍにおいては、第２の蛍光部位Ｆ２から第１の蛍光部位Ｆ１へ励起エネルギーが与えられて、発光部位が、第２の蛍光部位Ｆ２から第１の蛍光部位Ｆ１へ切り替えられてもよい。また、同様に、標識分子Ｍにおいては、生体分子Ｔと結合していない標識分子Ｍへ励起光Ｌ１１が照射されると、第２の蛍光部位Ｆ２から第１の蛍光部位Ｆ１へ励起エネルギーが与えられて、発光部位が切り替えられてもよい。 In the labeling molecule M described above, the light emitting site in a state where it is not bound to the biomolecule T is not limited to the first fluorescent site F1. Similarly, the light emitting site in the state of being bound to the biomolecule T is not limited to the second fluorescent site F2. That is, when the excitation light L11 is irradiated to the labeling molecule M bonded to the biomolecule T, the excitation energy is given from the second fluorescent part F2 to the first fluorescent part F1 in the labeling molecule M, and the light emitting part. However, the second fluorescent part F2 may be switched to the first fluorescent part F1. Similarly, in the labeled molecule M, when the excitation light L11 is irradiated to the labeled molecule M that is not bonded to the biomolecule T, excitation energy is given from the second fluorescent site F2 to the first fluorescent site F1. Then, the light emitting part may be switched.
　このようなＦＲＥＴの原理を利用した標識分子Ｍにおける蛍光物質の組み合わせの例としては、ＣＦＰ（励起波長：４５２ｎｍ、発光波長：５０５ｎｍ）とＹＦＰ（励起波長：５１４ｎｍ、発光波長：５２７ｎｍ）、ＢＦＰ（励起波長：３８０ｎｍ、発光波長：４４０ｎｍ）とＣＦＰ（励起波長：４５２ｎｍ、発光波長：５０５ｎｍ）、ＧＦＰ（励起波長：４８８ｎｍ、発光波長：５０９ｎｍ）とＹＦＰ（励起波長：５１４ｎｍ、発光波長：５２７ｎｍ）等が挙げられる。 Examples of combinations of fluorescent substances in the labeled molecule M using the FRET principle include CFP (excitation wavelength: 452 nm, emission wavelength: 505 nm) and YFP (excitation wavelength: 514 nm, emission wavelength: 527 nm), BFP ( Excitation wavelength: 380 nm, emission wavelength: 440 nm) and CFP (excitation wavelength: 452 nm, emission wavelength: 505 nm), GFP (excitation wavelength: 488 nm, emission wavelength: 509 nm), YFP (excitation wavelength: 514 nm, emission wavelength: 527 nm), etc. Is mentioned.
　上述した蛍光タンパク質以外にも、発光タンパク質と蛍光タンパク質の組み合わせであるBioluminescence resonance energy transfer（ＢＲＥＴ）を用いることや、励起波長と蛍光波長の異なる２種類の蛍光分子等を組み合わせること、励起波長と蛍光波長の異なる２種類のＱｄｏｔ（登録商標）を使用すること、先述の光学特性を持つ分子を組み合わせること（蛍光タンパク質と蛍光分子、蛍光分子とＱｄｏｔ（登録商標）、蛍光タンパク質とＱｄｏｔ（登録商標））等が挙げられる。 In addition to the fluorescent proteins described above, the combination of photoluminescence and fluorescent proteins, such as Bioluminescence resonance energy transfer (BRET), combining two types of fluorescent molecules with different excitation and fluorescence wavelengths, excitation wavelength and fluorescence Using two types of Qdot (registered trademark) with different wavelengths, combining the molecules having the optical characteristics described above (fluorescent protein and fluorescent molecule, fluorescent molecule and Qdot (registered trademark), fluorescent protein and Qdot (registered trademark) ) And the like.
　また、標識分子Ｍの結合部位Ｂについては、任意の生体分子Ｔに特異的に結合する構造を有する、抗体やアプタマー等の構成を採用することができる。 Also, for the binding site B of the labeled molecule M, it is possible to adopt a configuration such as an antibody or an aptamer having a structure that specifically binds to an arbitrary biomolecule T.
＜計数工程＞
　計数工程Ｓ３は、計数部３によって、検出部２ａにおいて検出された試料中の発光点の数を数える工程である。計数部３は、検出部２ａにおいて検出された光の強度の分布から、予め定めた基準を超えている点を、標識分子Ｍから発せられた蛍光Ｌ２１に由来する発光点として扱い、その数を数えることができる。例えば、図５に示すように、計数部３は、検出部２ａの蛍光フィルタ２３を切り替えて検出された光の強度の分布について、第１の蛍光部位Ｆ１から発せられた第１の蛍光Ｌ２１１に由来する点Ｓ１（図５Ａ）の数を数える。同様に、計数部３は、第２の蛍光部位Ｆ２から発せられた第２の蛍光Ｌ２１２に由来する点Ｓ２（図５Ｂ）の数を数える。 <Counting process>
The counting step S3 is a step of counting the number of light emitting points in the sample detected by the detecting unit 2a by the counting unit 3. The counting unit 3 treats a point exceeding a predetermined reference from the distribution of the intensity of the light detected by the detection unit 2a as a light emitting point derived from the fluorescence L21 emitted from the labeled molecule M, and the number thereof. Can count. For example, as shown in FIG. 5, the counting unit 3 switches the fluorescence filter 23 of the detection unit 2a to the first fluorescence L211 emitted from the first fluorescence site F1 with respect to the distribution of the detected light intensity. Count the number of derived points S1 (FIG. 5A). Similarly, the counting unit 3 counts the number of points S2 (FIG. 5B) derived from the second fluorescence L212 emitted from the second fluorescence site F2.
　また、計数部３は、任意の大きさの同一領域（領域Ｒ）における第１の蛍光部位Ｆ１に由来する発光点Ｓ１の数及び第２の蛍光部位Ｆ２に由来する発光点Ｓ２の数を数えることが好ましい。同じ領域Ｒにおいて各々の発光点Ｓ１，Ｓ２の数を数えることにより、後述する濃度算出工程Ｓ４において、より精度高く濃度を算出することができる。 In addition, the counting unit 3 counts the number of light emitting points S1 derived from the first fluorescent site F1 and the number of light emitting points S2 derived from the second fluorescent site F2 in the same region (region R) having an arbitrary size. It is preferable. By counting the number of light emitting points S1 and S2 in the same region R, the concentration can be calculated with higher accuracy in the concentration calculation step S4 described later.
＜濃度算出工程＞
　濃度算出工程Ｓ４は、第１の蛍光部位Ｆ１に由来する発光点の数Ｓ１と、第２の蛍光部位Ｆ２に由来する発光点Ｓ２の数と、に基づき試料に含まれる任意の生体分子Ｔの濃度を算出する工程である。上述したように、標識分子Ｍは、結合対象である生体分子Ｔとの結合の有無によって、発光する蛍光部位を切り替えることができる性質を有する。このため、第１の蛍光部位Ｆ１に由来する発光点Ｓ１の数と、第２の蛍光部位Ｆ２に由来する発光点Ｓ２の数のうち、いずれか一方は、試料中の生体分子Ｔと結合していない標識分子Ｍの数に相当し、他方は、生体分子Ｔと結合した標識分子Ｍの数に相当する。このため、濃度算出部４は、第１の蛍光部位Ｆ１に由来する発光点Ｓ１の数と、第２の蛍光部位Ｆ２に由来する発光点Ｓ２の数と、の和に対する、任意の生体分子Ｔと結合している場合に蛍光を発する蛍光部位に由来する発光点の数の割合に基づき、試料中の生体分子の濃度を算出することができる。 <Concentration calculation process>
In the concentration calculation step S4, an arbitrary biomolecule T contained in the sample is calculated based on the number S1 of light emitting points derived from the first fluorescent site F1 and the number of light emitting points S2 derived from the second fluorescent site F2. This is a step of calculating the concentration. As described above, the labeled molecule M has the property that the fluorescent site that emits light can be switched depending on the presence or absence of binding to the biomolecule T that is the binding target. For this reason, one of the number of emission points S1 derived from the first fluorescence site F1 and the number of emission points S2 derived from the second fluorescence site F2 is bound to the biomolecule T in the sample. This corresponds to the number of label molecules M that are not present, and the other corresponds to the number of label molecules M bound to the biomolecule T. For this reason, the concentration calculation unit 4 selects an arbitrary biomolecule T with respect to the sum of the number of light emitting points S1 derived from the first fluorescent site F1 and the number of light emitting points S2 derived from the second fluorescent site F2. The concentration of the biomolecule in the sample can be calculated based on the ratio of the number of emission points derived from the fluorescent sites that emit fluorescence when they are bound to each other.
　本工程Ｓ４について、標識分子Ｍが、生体分子Ｔと結合していない状態では、第１の蛍光部位Ｆ１から第１の蛍光Ｌ２１１が発せられ、生体分子Ｔと結合した状態では、第２の蛍光部位Ｆ２から第２の蛍光Ｌ２１２が発せられる構成を有している場合を例に説明する（図４Ａ、標識分子Ｍ１、再度参照）。このような標識分子Ｍ１では、第２の蛍光部位Ｆ２に由来する発光点Ｓ２の数は、生体分子Ｔと結合した標識分子Ｍ１の数に相当する。従って、計数工程Ｓ３で、複数の試料について第２の蛍光部位Ｆ２に由来する発光点Ｓ２の数を数えている場合には、濃度算出部４は、一つの試料を基準として、発光点Ｓ２の数に応じて、複数の試料における生体分子Ｔの濃度を相対的に定量することもできる。 In this step S4, the first fluorescence L211 is emitted from the first fluorescence site F1 when the labeled molecule M is not bound to the biomolecule T, and the second fluorescence is emitted when bound to the biomolecule T. A case where the second fluorescence L212 is emitted from the site F2 will be described as an example (see FIG. 4A, labeled molecule M1, again). In such a labeled molecule M1, the number of emission points S2 derived from the second fluorescent site F2 corresponds to the number of labeled molecules M1 bound to the biomolecule T. Therefore, in the counting step S3, when the number of the light emission points S2 derived from the second fluorescent site F2 is counted for a plurality of samples, the concentration calculation unit 4 uses the one sample as a reference to calculate the light emission points S2. Depending on the number, the concentration of biomolecule T in a plurality of samples can be relatively quantified.
　さらに、下記に示す、第１の蛍光部位Ｆ１に由来する発光点Ｓ１の数と、第２の蛍光部位Ｆ２に由来する発光点Ｓ２の数の和に対する、第２の蛍光部位Ｆ２に由来する発光点Ｓ２の数の割合は、試料中の生体分子Ｔの濃度を反映したものである。 Furthermore, the light emission derived from the second fluorescent part F2 with respect to the sum of the number of light emitting points S1 derived from the first fluorescent part F1 and the number of light emitting points S2 derived from the second fluorescent part F2 shown below. The ratio of the number of points S2 reflects the concentration of the biomolecule T in the sample.
Figure JPOXMLDOC01-appb-M000001
Figure JPOXMLDOC01-appb-M000001
　試料中の標識分子Ｍの濃度は、予め試料へ添加する際に所望の濃度に調製することができる。このため、試料中の標識分子Ｍの濃度と、上記割合とに基づいて、試料中の生体分子Ｔの濃度を算出することができる。 The concentration of the labeled molecule M in the sample can be adjusted to a desired concentration when added to the sample in advance. For this reason, the concentration of the biomolecule T in the sample can be calculated based on the concentration of the labeled molecule M in the sample and the ratio.
　また、同じ領域Ｒから２種類の発光点Ｓ１，Ｓ２を数える場合、同一体積に含まれる、生体分子Ｔと結合していない標識分子Ｍの数と、生体分子Ｔと結合した標識分子Ｍの数と、を容易に数えることができる。従って、上記割合をより正確に求めることができる。 In addition, when counting two types of light emitting points S1 and S2 from the same region R, the number of label molecules M not bound to the biomolecule T and the number of label molecules M bound to the biomolecule T included in the same volume. And can be counted easily. Therefore, the ratio can be obtained more accurately.
　上述した本実施形態に係る濃度測定装置による生体分子の濃度測定方法では、標識分子のみを用いて、試料中の生体分子の濃度を算出することができる。このため、複数の試薬を用いる必要がない。また、呈色反応も不要であるため、従来の濃度の算出方法に比べて、短時間で簡便に行うことができる。 In the biomolecule concentration measurement method using the concentration measurement apparatus according to this embodiment described above, the concentration of the biomolecule in the sample can be calculated using only the labeled molecule. For this reason, it is not necessary to use a plurality of reagents. Further, since no color reaction is required, it can be carried out easily in a short time compared with the conventional concentration calculation method.
　また、上記濃度測定方法では、生体分子と結合した標識分子と、生体分子と結合していない標識分子と、を発光波長に基づいて区別できるため、従来のＥＬＩＳＡ法等で必要な試料の洗浄工程が不要である。このため、生体分子の濃度の測定をより簡便に行うことができる。 Further, in the above concentration measurement method, a labeled molecule that is bound to a biomolecule and a labeled molecule that is not bound to a biomolecule can be distinguished based on the emission wavelength. Is unnecessary. For this reason, the concentration of the biomolecule can be measured more easily.
　従来のＥＬＩＳＡ法などでは、標準品を用いて希釈系列を作製して、検量線を求める工程が必要であった。しかし、上記濃度測定方法では、生体分子と結合した標識分子に由来する発光点の割合と、試料中の標識分子の濃度に基づき、試料中の生体分子の濃度を算出することができる。このため、より簡便に生体分子の濃度を測定することができる。 In the conventional ELISA method and the like, a process for obtaining a calibration curve by preparing a dilution series using a standard product is required. However, in the above concentration measurement method, the concentration of the biomolecule in the sample can be calculated based on the ratio of the luminescent point derived from the labeled molecule bound to the biomolecule and the concentration of the labeled molecule in the sample. For this reason, the density | concentration of a biomolecule can be measured more simply.
３．本開示の第２実施形態に係る濃度測定装置
　図６は、本開示の第２実施形態に係る濃度測定装置Ｄ２の構成の概要を示す模式図である。濃度測定装置Ｄ２の光照射部１ｂは、励起光Ｌ１２として、エバネッセント光を試料に照射するための構成を有する。なお、第１実施形態に係る濃度測定装置Ｄ１と同一の構成については同一の符号を付し、その説明は省略する。 3. Concentration Measuring Device According to Second Embodiment of Present Disclosure FIG. 6 is a schematic diagram illustrating an outline of a configuration of a concentration measuring device D2 according to the second embodiment of the present disclosure. The light irradiation unit 1b of the concentration measuring device D2 has a configuration for irradiating the sample with evanescent light as the excitation light L12. In addition, the same code | symbol is attached | subjected about the structure same as the density | concentration measuring apparatus D1 which concerns on 1st Embodiment, and the description is abbreviate | omitted.
　光照射部１ｂは、エバネッセント光を試料中の標識分子Ｍに照射できるように構成されていればよく、その構成は公知の顕微鏡等の構成から適宜採用できる。例えば、濃度測定装置Ｄ２において、光照射部１ｂにミラー１４が設けられていてもよい。 The light irradiation unit 1b only needs to be configured so as to be able to irradiate the labeled molecules M in the sample with evanescent light, and the configuration can be appropriately adopted from the configuration of a known microscope or the like. For example, in the concentration measurement device D2, the mirror 14 may be provided in the light irradiation unit 1b.
　ミラー１４は、光源１１から出射された励起光Ｌ１１を、基板Ｐの試料接触面Ｐ１において全反射させる角度に備えられている。励起光Ｌ１１を試料接触面Ｐ１において全反射させることにより、標識分子Ｍを含む試料に照射される励起光Ｌ１２をエバネッセント光とすることができる。エバネッセント光は、試料の限定された領域にのみ照射されるため、検出部２ａは、発光Ｌ２２を背景光の少ない状態で検出することができる。 The mirror 14 is provided at an angle at which the excitation light L11 emitted from the light source 11 is totally reflected on the sample contact surface P1 of the substrate P. By totally reflecting the excitation light L11 at the sample contact surface P1, the excitation light L12 applied to the sample containing the labeling molecule M can be used as evanescent light. Since the evanescent light is irradiated only to a limited area of the sample, the detection unit 2a can detect the light emission L22 in a state with little background light.
　第２実施形態に係る濃度測定装置においては、励起光としてエバネッセント光を利用することができる。このため、より高解像度に標識分子に由来する発光を検出することができ、発光点の計数をより精度高く行うことができる。従って、濃度測定装置における濃度の測定の精度をより高めることができる。 In the concentration measurement apparatus according to the second embodiment, evanescent light can be used as excitation light. For this reason, light emission derived from the labeled molecule can be detected with higher resolution, and counting of the light emission points can be performed with higher accuracy. Accordingly, it is possible to further increase the accuracy of concentration measurement in the concentration measuring apparatus.
　また、エバネッセント光を用いる場合は、上述した基板Ｐにおいては、エバネッセント光の到達が基板Ｐ近傍に限定されるため、基板Ｐの面積を決定すれば、体積を算出することができるため、単位体積当たりの分子数を算出することも可能である。この他、第２実施形態に係る濃度測定装置における効果は、上述した第１実施形態に係る濃度測定装置と同様である。 Further, when evanescent light is used, in the substrate P described above, the arrival of the evanescent light is limited to the vicinity of the substrate P. Therefore, if the area of the substrate P is determined, the volume can be calculated. It is also possible to calculate the number of molecules per hit. In addition, the effects of the concentration measuring apparatus according to the second embodiment are the same as those of the concentration measuring apparatus according to the first embodiment described above.
４．本開示の第３実施形態に係る濃度測定装置
　図７は、本開示の第３実施形態に係る濃度測定装置Ｄ３の構成の概要を示す模式図である。濃度測定装置Ｄ３の検出部２ｃには、発光Ｌ２１の光子を受光する２つの受光器２７ａ，２７ｂを有する。この他、検出部２ｃは、２つの受光器２７ａ，２７ｂへ導光するための構成として、ダイクロイックミラー２４、バンドパスフィルタ２５及びハーフミラー２６を有する。ハーフミラー２６は、透過率と反射率が各々５０％のものである。また、２つの受光器２７ａ，２７ｂは、ハーフミラー２６から同じ距離に設置されている。さらに、濃度測定装置Ｄ３には、必要に応じて、偏光ビームスプリッタ２８等が設けられていてもよい。 4). Concentration Measuring Device According to Third Embodiment of Present Disclosure FIG. 7 is a schematic diagram illustrating an outline of a configuration of a concentration measuring device D3 according to the third embodiment of the present disclosure. The detector 2c of the concentration measuring device D3 has two light receivers 27a and 27b that receive photons of the light emission L21. In addition, the detection unit 2c includes a dichroic mirror 24, a band pass filter 25, and a half mirror 26 as a configuration for guiding light to the two light receivers 27a and 27b. The half mirror 26 has a transmittance and a reflectance of 50%. The two light receivers 27 a and 27 b are installed at the same distance from the half mirror 26. Furthermore, the density measuring device D3 may be provided with a polarizing beam splitter 28 or the like as necessary.
　濃度測定装置Ｄ３において、光照射部１ｃには、光源１１ｃとして、励起光Ｌ１１を試料へパルス照射するためのレーザー光源が備えられている。この他、第１実施形態に係る濃度測定装置Ｄ１と同一の構成については同一の符号を付し、その説明は省略する。 In the concentration measuring apparatus D3, the light irradiation unit 1c is provided with a laser light source for irradiating the sample with the excitation light L11 as a light source 11c. In addition, the same code | symbol is attached | subjected about the structure same as the density | concentration measuring apparatus D1 which concerns on 1st Embodiment, and the description is abbreviate | omitted.
　本実施形態の濃度測定装置Ｄ３では、励起光Ｌ１１によって標識分子Ｍから発せられた発光Ｌ２１について、ダイクロイックミラー２４とバンドパスフィルタ２５によって励起光Ｌ１１を除いて、ハーフミラー２６へ導光する。発光Ｌ２１に含まれる光子が、標識分子Ｍの蛍光部位Ｆ１，Ｆ２のうち、一つの蛍光部位に由来していれば、単一光子であるため、ハーフミラー２６において分離することができない。このため、２台の受光器２７ａ，２７ｂのうち、いずれか一方が光子を検出する。 In the concentration measuring device D3 of the present embodiment, the luminescence L21 emitted from the labeling molecule M by the excitation light L11 is guided to the half mirror 26 by removing the excitation light L11 by the dichroic mirror 24 and the band pass filter 25. If the photons contained in the light emission L21 are derived from one of the fluorescent sites F1 and F2 of the labeling molecule M, they are single photons and cannot be separated by the half mirror 26. Therefore, one of the two light receivers 27a and 27b detects a photon.
　一方、発光Ｌ２１に含まれる光子が、例えば、複数の標識分子Ｍの各々の蛍光部位に由来していれば、単一光子ではないため、２つの受光器２７ａ，２７ｂの両方で光子が検出される。また、励起光Ｌ１１はパルス照射されているため、単一光子ではない場合、２台の受光器２７ａ，２７ｂは、同じタイミングで光子を検出することができる。このため、２台の受光器２７ａ，２７ｂにおいて、同時に光子を検出するか、又は交互に検出するかによって、発光Ｌ１２が一つの標識分子Ｍに由来するものであるか否か判定することができる。 On the other hand, if the photons contained in the light emission L21 are derived from the fluorescent sites of each of the plurality of labeled molecules M, for example, the photons are detected by both the two light receivers 27a and 27b because they are not single photons. The In addition, since the excitation light L11 is pulse-irradiated, if it is not a single photon, the two light receivers 27a and 27b can detect the photon at the same timing. For this reason, it is possible to determine whether or not the light emission L12 is derived from one label molecule M by detecting the photons simultaneously or alternately in the two light receivers 27a and 27b. .
　本開示に係る濃度測定方法においては、発光点の数に基づき生体分子の濃度を測定している。このため、試料に含まれる標識分子の濃度によっては、複数の標識分子Ｍが近接して、２つの標識分子Ｍに由来する発光Ｌ２１を一つの発光点として検出するおそれがある。上述したように、本実施形態の濃度測定装置では、２台の受光器によって、発光点が一つの標識分子Ｍに由来するものであるか否か判定することができる。そこで、計数部が数える発光点について、予め単一光子に由来する発光点のみに限定することができる。この結果、発光点の数をより正確に数えることができ、濃度の測定をより精度高く行うことができる。 In the concentration measurement method according to the present disclosure, the concentration of a biomolecule is measured based on the number of luminescent spots. For this reason, depending on the density | concentration of the label molecule contained in a sample, there exists a possibility that the some label molecule M may adjoin and may detect the light emission L21 originating in the two label molecules M as one light emission point. As described above, in the concentration measurement apparatus of the present embodiment, it is possible to determine whether or not the light emission point is derived from one label molecule M by using two light receivers. Therefore, the light emitting points counted by the counting unit can be limited to only light emitting points derived from single photons in advance. As a result, the number of light emitting points can be counted more accurately, and the concentration can be measured with higher accuracy.
　なお、上記に記載された効果はあくまで例示であって、限定されるものではなく、また他の効果があってもよい。 It should be noted that the effects described above are merely examples and are not limited, and may have other effects.
　本開示は、以下のような構成もとることができる。
　（１）第１の蛍光部位及び第２の蛍光部位を有し任意の生体分子に結合する標識分子を含む試料に励起光を照射する光照射部と、前記標識分子からの発光を検出する検出部と、前記試料中の前記検出部によって検出された発光点の数を数える計数部と、前記第１の蛍光部位に由来する発光点の数と、前記第２の蛍光部位に由来する発光点の数と、に基づき前記試料に含まれる前記任意の生体分子の濃度を算出する濃度算出部と、を有する生体分子の濃度測定装置。
　（２）前記励起光を照射された前記標識分子は、前記任意の生体分子と結合していない場合には前記第１の蛍光部位から第１の蛍光を発し、前記任意の生体分子と結合している場合には前記第２の蛍光部位から第２の蛍光を発するもの、又は前記任意の生体分子と結合していない場合には前記第２の蛍光部位から第２の蛍光を発し、前記任意の生体分子と結合している場合には前記第１の蛍光部位から第１の蛍光を発するもの、である上記（１）に記載の生体分子の濃度測定装置。
　（３）前記濃度算出部は前記第１の蛍光部位に由来する発光点の数と前記第２の蛍光部位に由来する発光点の数の和に対する前記任意の生体分子と結合している場合に蛍光を発する蛍光部位に由来する発光点の数の割合に基づき前記濃度を算出する上記（２）に記載の生体分子の濃度測定装置。
　（４）前記計数部は任意の大きさの同一領域における前記第１の蛍光部位に由来する発光点の数及び前記第２の蛍光部位に由来する発光点の数を数える上記（１）～（３）のいずれかに記載の生体分子の濃度測定装置。
　（５）前記任意の生体分子と結合した前記標識分子へ前記励起光が照射されると、前記第１の蛍光部位から前記第２の蛍光部位、又は前記第２の蛍光部位から前記第１の蛍光部位へ励起エネルギーが与えられる上記（１）～（４）のいずれかに記載の生体分子の濃度測定装置。
　（６）前記任意の生体分子と結合していない前記標識分子へ前記励起光が照射されると、前記第１の蛍光部位から前記第２の蛍光部位、又は前記第２の蛍光部位から前記第１の蛍光部位へ励起エネルギーが与えられる上記（１）～（４）のいずれかに記載の生体分子の濃度測定装置。
　（７）前記励起光がエバネッセント光である上記（１）～（６）のいずれかに記載の生体分子の濃度測定装置。
　（８）前記試料は基板上に保持されている上記（１）～（７）のいずれかに記載の生体分子の濃度測定装置。
　（９）第１の蛍光部位及び第２の蛍光部位を有し任意の生体分子に結合する標識分子を含む試料に励起光を照射する光照射工程と、前記標識分子からの発光を検出する検出工程と、前記試料中の前記検出部によって検出された発光点の数を数える計数工程と、前記第１の蛍光部位に由来する発光点の数と、前記第２の蛍光部位に由来する発光点の数と、に基づき前記試料に含まれる前記任意の生体分子の濃度を算出する濃度算出工程と、を有する生体分子の濃度測定方法。
　（１０）前記光照射工程の前に前記試料を基板上に保持させて濃縮する濃縮工程を有する上記（９）に記載の生体分子の濃度測定方法。 The present disclosure can have the following configurations.
(1) A light irradiation unit that irradiates a sample including a labeled molecule that has a first fluorescent site and a second fluorescent site and binds to an arbitrary biomolecule, and a detection that detects luminescence from the labeled molecule , A counting unit for counting the number of light emitting points detected by the detection unit in the sample, the number of light emitting points derived from the first fluorescent site, and a light emitting point derived from the second fluorescent site And a concentration calculation unit that calculates the concentration of the arbitrary biomolecule contained in the sample based on the number of the biomolecules.
(2) When the labeling molecule irradiated with the excitation light does not bind to the arbitrary biomolecule, the labeling molecule emits a first fluorescence from the first fluorescent site and binds to the arbitrary biomolecule. The second fluorescent site emits a second fluorescence, or the second fluorescent site emits a second fluorescence when not bound to the arbitrary biomolecule, and the optional fluorescent The biomolecule concentration measuring apparatus according to (1) above, wherein the first fluorescent portion emits a first fluorescence when it is bound to a biomolecule.
(3) When the concentration calculation unit binds to the arbitrary biomolecule with respect to the sum of the number of emission points derived from the first fluorescence site and the number of emission points derived from the second fluorescence site. The biomolecule concentration measuring apparatus according to (2), wherein the concentration is calculated based on a ratio of the number of light emitting points derived from fluorescent sites that emit fluorescence.
(4) The counting unit is configured to count the number of luminescent spots derived from the first fluorescent site and the number of luminescent spots derived from the second fluorescent site in the same area of an arbitrary size. The biomolecule concentration measuring apparatus according to any one of 3).
(5) When the excitation light is irradiated to the labeled molecule bonded to the arbitrary biomolecule, the first fluorescent site to the second fluorescent site or the second fluorescent site to the first The biomolecule concentration measurement apparatus according to any one of the above (1) to (4), wherein excitation energy is given to a fluorescent site.
(6) When the excitation light is irradiated to the labeled molecule that is not bound to the arbitrary biomolecule, the first fluorescent site to the second fluorescent site or the second fluorescent site to the first The biomolecule concentration measuring apparatus according to any one of (1) to (4), wherein excitation energy is applied to one fluorescent site.
(7) The biomolecule concentration measuring apparatus according to any one of (1) to (6), wherein the excitation light is evanescent light.
(8) The biomolecule concentration measuring apparatus according to any one of (1) to (7), wherein the sample is held on a substrate.
(9) A light irradiation step of irradiating a sample containing a labeled molecule having a first fluorescent site and a second fluorescent site and binding to an arbitrary biomolecule, and detection for detecting light emission from the labeled molecule A step, a counting step of counting the number of light emitting points detected by the detection unit in the sample, the number of light emitting points derived from the first fluorescent site, and a light emitting point derived from the second fluorescent site And a concentration calculating step of calculating a concentration of the arbitrary biomolecule contained in the sample based on the number of biomolecules.
(10) The biomolecule concentration measurement method according to (9), further including a concentration step of concentrating the sample on a substrate before the light irradiation step.
Ｄ１，Ｄ２，Ｄ３：生体分子の濃度測定装置（濃度測定装置）
Ｂ：結合部位
Ｆ１：第１の蛍光部位
Ｆ２：第２の蛍光部位
Ｌ１１，Ｌ１２：励起光
Ｌ２１，Ｌ２１１，Ｌ２１２，Ｌ２２：発光
Ｍ，Ｍ１，Ｍ２：標識分子
Ｒ：領域
Ｐ：基板
Ｐ１：試料接触面
Ｓ１，Ｓ２：発光点
Ｔ：生体分子
１ａ，１ｂ，１ｃ：光照射部
１１，１１ｃ：光源
１２：集光レンズ
１３：絞り
１４：ミラー
２ａ，２ｃ：検出部
２１：検出器
２２：対物レンズ
２３：蛍光フィルタ
２４：ダイクロイックミラー
２５：バンドパスフィルタ
２６：ハーフミラー
２７ａ，２７ｂ：受光器
２８：偏光ビームスプリッタ
３：計数部
４：濃度算出部 D1, D2, D3: Biomolecule concentration measuring device (concentration measuring device)
B: Binding site F1: First fluorescent site F2: Second fluorescent site L11, L12: Excitation light L21, L211, L212, L22: Luminescence M, M1, M2: Labeled molecule R: Region P: Substrate P1: Sample Contact surfaces S1, S2: Luminescent point T: Biomolecules 1a, 1b, 1c: Light irradiation unit 11, 11c: Light source 12: Condensing lens 13: Aperture 14: Mirror 2a, 2c: Detection unit 21: Detector 22: Objective Lens 23: Fluorescent filter 24: Dichroic mirror 25: Band pass filter 26: Half mirror 27a, 27b: Light receiver 28: Polarizing beam splitter 3: Counting unit 4: Density calculating unit

Claims (10)

  1. 　第１の蛍光部位及び第２の蛍光部位を有し任意の生体分子に結合する標識分子を含む試料に励起光を照射する光照射部と、
    　前記標識分子からの発光を検出する検出部と、
    　前記試料中の前記検出部によって検出された発光点の数を数える計数部と、
    　前記第１の蛍光部位に由来する発光点の数と、前記第２の蛍光部位に由来する発光点の数と、に基づき前記試料に含まれる前記任意の生体分子の濃度を算出する濃度算出部と、
    を有する生体分子の濃度測定装置。     A light irradiation unit that irradiates a sample including a labeling molecule that has a first fluorescent part and a second fluorescent part and binds to an arbitrary biomolecule;
    A detection unit for detecting light emission from the labeled molecule;
    A counting unit for counting the number of light emitting points detected by the detection unit in the sample;
    A concentration calculation unit that calculates the concentration of the arbitrary biomolecule contained in the sample based on the number of emission points derived from the first fluorescence site and the number of emission points derived from the second fluorescence site. When,
    An apparatus for measuring the concentration of biomolecules.
  2. 　前記励起光を照射された前記標識分子は、前記任意の生体分子と結合していない場合には前記第１の蛍光部位から第１の蛍光を発し、前記任意の生体分子と結合している場合には前記第２の蛍光部位から第２の蛍光を発するもの、又は前記任意の生体分子と結合していない場合には前記第２の蛍光部位から第２の蛍光を発し、前記任意の生体分子と結合している場合には前記第１の蛍光部位から第１の蛍光を発するもの、である
    請求項１に記載の生体分子の濃度測定装置。     When the labeling molecule irradiated with the excitation light emits a first fluorescence from the first fluorescent site when not bound to the arbitrary biomolecule, and binds to the arbitrary biomolecule Emits second fluorescence from the second fluorescent site, or emits second fluorescence from the second fluorescent site when not bound to the arbitrary biomolecule, and the arbitrary biomolecule The biomolecule concentration measurement apparatus according to claim 1, wherein the first fluorescence site emits a first fluorescence when it is bound to a biomolecule.
  3. 　前記濃度算出部は前記第１の蛍光部位に由来する発光点の数と前記第２の蛍光部位に由来する発光点の数の和に対する前記任意の生体分子と結合している場合に蛍光を発する蛍光部位に由来する発光点の数の割合に基づき前記濃度を算出する
    請求項２に記載の生体分子の濃度測定装置。     The concentration calculation unit emits fluorescence when it is bound to the arbitrary biomolecule with respect to the sum of the number of light emitting points derived from the first fluorescent site and the number of light emitting points derived from the second fluorescent site. The biomolecule concentration measuring apparatus according to claim 2, wherein the concentration is calculated based on a ratio of the number of luminescent spots derived from a fluorescent site.
  4. 　前記計数部は任意の大きさの同一領域における前記第１の蛍光部位に由来する発光点の数及び前記第２の蛍光部位に由来する発光点の数を数える
    請求項３に記載の生体分子の濃度測定装置。     4. The biomolecule according to claim 3, wherein the counting unit counts the number of light emitting points derived from the first fluorescent site and the number of light emitting points derived from the second fluorescent site in the same region of an arbitrary size. Concentration measuring device.
  5. 　前記任意の生体分子と結合した前記標識分子へ前記励起光が照射されると、前記第１の蛍光部位から前記第２の蛍光部位、又は前記第２の蛍光部位から前記第１の蛍光部位へ励起エネルギーが与えられる
    請求項１に記載の生体分子の濃度測定装置。     When the excitation light is irradiated to the labeled molecule bound to the arbitrary biomolecule, the first fluorescent site is transferred to the second fluorescent site, or the second fluorescent site is transferred to the first fluorescent site. The biomolecule concentration measurement apparatus according to claim 1, wherein excitation energy is applied.
  6. 　前記任意の生体分子と結合していない前記標識分子へ前記励起光が照射されると、前記第１の蛍光部位から前記第２の蛍光部位、又は前記第２の蛍光部位から前記第１の蛍光部位へ励起エネルギーが与えられる
    請求項１に記載の生体分子の濃度測定装置。     When the excitation light is irradiated to the label molecule that is not bound to the arbitrary biomolecule, the first fluorescence site to the second fluorescence site or the second fluorescence site to the first fluorescence The biomolecule concentration measurement apparatus according to claim 1, wherein excitation energy is applied to the site.
  7. 　前記励起光がエバネッセント光である
    請求項１に記載の生体分子の濃度測定装置。     The biomolecule concentration measurement apparatus according to claim 1, wherein the excitation light is evanescent light.
  8. 　前記試料は基板上に保持されている
    請求項１に記載の生体分子の濃度測定装置。     The biomolecule concentration measurement apparatus according to claim 1, wherein the sample is held on a substrate.
  9. 　第１の蛍光部位及び第２の蛍光部位を有し任意の生体分子に結合する標識分子を含む試料に励起光を照射する光照射工程と、
    　前記標識分子からの発光を検出する検出工程と、
    　前記試料中の前記検出部によって検出された発光点の数を数える計数工程と、
    　前記第１の蛍光部位に由来する発光点の数と、前記第２の蛍光部位に由来する発光点の数と、に基づき前記試料に含まれる前記任意の生体分子の濃度を算出する濃度算出工程と、
    を有する生体分子の濃度測定方法。     A light irradiation step of irradiating a sample containing a labeling molecule having a first fluorescent site and a second fluorescent site and binding to an arbitrary biomolecule with excitation light;
    A detection step of detecting luminescence from the labeled molecule;
    A counting step of counting the number of luminescent spots detected by the detection unit in the sample;
    A concentration calculating step of calculating the concentration of the arbitrary biomolecule contained in the sample based on the number of light emitting points derived from the first fluorescent site and the number of light emitting points derived from the second fluorescent site. When,
    A method for measuring the concentration of a biomolecule having
  10. 　前記光照射工程の前に前記試料を基板上に保持させて濃縮する濃縮工程を有する
    請求項９に記載の生体分子の濃度測定方法。
    　     The biomolecule concentration measurement method according to claim 9, further comprising a concentration step in which the sample is held on a substrate and concentrated before the light irradiation step.
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WO1997047968A1 (en) * 1996-06-10 1997-12-18 Laboratory Of Molecular Biophotonics Highly sensitive fluorescent immunoassay
JP2012525582A (en) * 2009-04-27 2012-10-22 コリア リサーチ インスティテュート オブ バイオサイエンス アンド バイオテクノロジー Ligand detection method using FRET biosensor
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