WO2008053822A1 - Method of detecting specific bond reaction of molecule by single molecule fluorometry - Google Patents

Method of detecting specific bond reaction of molecule by single molecule fluorometry Download PDF

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Publication number
WO2008053822A1
WO2008053822A1 PCT/JP2007/071002 JP2007071002W WO2008053822A1 WO 2008053822 A1 WO2008053822 A1 WO 2008053822A1 JP 2007071002 W JP2007071002 W JP 2007071002W WO 2008053822 A1 WO2008053822 A1 WO 2008053822A1
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Prior art keywords
molecule
fluorescence
sample
immobilized
particle
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PCT/JP2007/071002
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French (fr)
Japanese (ja)
Inventor
Naoaki Okamoto
Sayoko Kobayashi
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Olympus Corporation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

  • the present invention relates to a protein, peptide, nucleic acid by measuring fluorescence from a single fluorescent molecule.
  • biomolecules Lipids, amino acids and other physiologically active substances or biomolecules (hereinafter referred to as “biomolecules”), and more particularly, between various biomolecules.
  • biomolecules Lipids, amino acids and other physiologically active substances or biomolecules
  • FCS fluorescence correlation spectroscopy
  • FCS fluorescence intensity distribution analysis
  • the force S can be used to detect various phenomena such as changes in the structure or size of molecules and molecular binding / dissociation or dispersion / aggregation.
  • Patent Document 1 Japanese Patent Laid-Open No. 2003-275000
  • Patent Document 2 JP 2005 337805 JP
  • Non-Patent Document 1 3 outside casks, PNAS 96, 24, 13756-13761, November 23, 1999
  • Non-Patent Document 2 Three people outside Kobayashi, Analytical Biochemistry, 2004 332 58-66
  • biological samples samples obtained from living organisms or samples derived from living organisms (serum, plasma, etc., hereinafter referred to as “biological samples”) that will be used for observation of interactions such as biomolecules and clinical diagnosis of diseases.
  • the influence of constituents or contaminants other than the molecule to be observed in the sample of single-molecule fluorescence analysis may remove those impurities from the sample or purify the molecule to be observed. It is possible to eliminate it by doing S.
  • extracting a specific molecule from a sample or removing other than a specific molecule requires time, labor and a large amount of sample, and can be measured in a short amount of time. The advantages of molecular fluorescence analysis technology will be lost.
  • the target substance may be denatured during the process of extracting / purifying or removing the substance, and eventually the target measurement may not be performed.
  • one of the main problems of the present invention is that detection and observation of molecular interactions, particularly specific binding of molecules, using single-molecule fluorescence analysis technology.
  • Another object of the present invention is to detect and observe the interaction of molecules using the single-molecule fluorescence analysis technique as described above.
  • a biological sample or a sample derived from a living body serum In the case of using plasma, etc., the effect of non-specific binding or adsorption in the sample is eliminated in the measurement result, and the accuracy and reliability of the measurement result are further improved.
  • specific binding of a specific molecule can be performed with high accuracy or reliability in a sample containing a large amount of contaminants, particularly in a biological sample solution, using the single-molecule fluorescence analysis technique as described above.
  • a method for detecting a specific binding reaction of a molecule that can be detected with high efficiency is provided.
  • a method for detecting specific binding of a specific molecule includes a first sample containing a first molecule that is fluorescently labeled, and the fluorescently labeled first molecule.
  • a process of preparing a mixed sample solution by mixing a second sample containing a second molecule to be determined whether or not it specifically binds to one molecule, and specifically binding to the second molecule The process of adding particles having an outer diameter of 1 11 m or less, in which a third molecule is immobilized on the surface, to the mixed sample solution, and the fluorescent labeling of the first molecule in the mixed sample solution to which the particles are added Measure fluorescence intensity And determining whether the first molecule is immobilized on the particle based on the fluorescence intensity, and determining whether the first molecule is immobilized on the particle. In this case, it is determined that the first molecule specifically binds to the second molecule. That is, in this embodiment, the force S is used to determine whether or not the first molecule specifically bind
  • a specific molecule for example, the above-mentioned first molecule
  • a certain other molecule the above-mentioned second molecule
  • a first sample containing a fluorescently labeled first molecule and a second sample containing a second molecule The sample was mixed and the fluorescence measurement of the mixed sample was performed and the result was analyzed.
  • the background light may be high and the S / N ratio may be poor, and even if there is nonspecific binding between the contaminants and the fluorescently labeled molecules. , It is indistinguishable from specific binding.
  • a third sample that specifically binds to the second molecule is mixed with the mixed sample solution containing the first and second molecules labeled with fluorescence as described above.
  • the process of the previous method is modified so that particles with an outer diameter of 1 11 m or less with molecules immobilized on the surface are added to the mixed sample solution, and then fluorescence measurement and analysis of the results are performed.
  • the third molecule only binds to the second molecule, resulting in the surface of the particle A second molecule will be collected on top.
  • the fluorescently labeled first molecule is also collected on the particle, so that the fluorescence intensity emitted from the fluorescent label is the fluorescence intensity. This reflects the state in which the label is fixed on particles with an outer diameter of 1 am or less.
  • the second molecule does not specifically bind to the first molecule, even if it is non-specifically bound to the contaminant, the fluorescence of the fluorescent labeling power on the first molecule The strength should be much different from the state in which the first molecule is free (bonded on the particle, the state!).
  • the Brownian motion of the fluorescent label is greatly limited as compared to the case where the fluorescent label is not, and the distribution of the fluorescent label within the measurement region is also reduced. Since the fluorescence label is collected on the particle, the fluorescence is emitted from a plurality or many of the fluorescence labels on the particle. The observed fluorescence intensity also increases and the S / N of the measurement is greatly improved.
  • the method is configured to determine whether the first molecule as described above is “fixed” on the particle through the specific binding of the second and third molecules. By correcting, it is possible to detect whether the first molecule specifically binds to the second molecule or not, at a higher S / N ratio.
  • a particle having an outer diameter of 1 ⁇ or less in which a third molecule that specifically binds to the second molecule is immobilized on the surface is added to a mixed sample solution, and fluorescence is measured and analyzed to obtain fluorescence.
  • a technique for determining whether a labeled first molecule is immobilized on a particle can also be used to detect whether a second molecule is present in a sample.
  • a method for detecting specific binding of a particular molecule of the present invention comprises a first sample comprising a first fluorescently labeled molecule and a fluorescently labeled first molecule.
  • a process of preparing a mixed sample solution by mixing a second sample to determine whether or not there is a second molecule that specifically binds to the first molecule, and specifically to the second molecule A process of adding particles having an outer diameter of 1 am or less, in which a third molecule to be bound is immobilized on the surface, to the mixed sample solution of the first and second samples, and in the mixed sample solution to which the particles are added
  • the above detection method in fluorescence measurement and analysis of the specific binding of the molecules of the present invention is, FCS, FIDA, fluorescence cross correlation spectroscopy (Fluorescence cross-correlation Sp e C tro SCO py: FCCS) or any other single-molecule fluorescence analysis method performed by a fluorescence measuring apparatus that combines an optical system of a laser confocal microscope with an ultrasensitive photodetection device.
  • any fluorescence analysis method that can observe changes in the structure, state, and motion of other molecules such as fluorescence depolarization spectroscopy (FDS), may be used! Depending on the configuration of The result of the fluorescence measurement / analysis will be explained in the explanation section of the embodiment. ).
  • FDS fluorescence depolarization spectroscopy
  • the first molecule, the second molecule and the third molecule to be fluorescently labeled need to be specified in advance.
  • the second sample containing molecules may contain contaminants, so serum used for research in the fields of biological science, medicine or pharmacology, clinical diagnosis of diseases or screening of bioactive substances, etc. Or a solution sample obtained from plasma or other living body.
  • the second sample as understood from the above description of the embodiment of the present invention is a solution sample obtained from a living body, whether or not the second molecule is contained in the biological sample! Whether or not it specifically binds to the first molecule can be detected even if the biological sample contains a certain amount of impurities.
  • the first molecule is DNA
  • the second molecule is a DNA-binding protein
  • the third molecule is an antibody against the second molecule
  • a certain DNA against a DNA having a certain base sequence can be obtained.
  • a binding protein specifically binds or is a DNA binding protein that specifically binds to DNA with a certain base sequence IJ included in a sample (eg, serum or plasma)? Whether or not is detected.
  • the particle on which the third molecule is immobilized on the surface is generally used in experiments in the field of biological science, medicine or pharmacy, and is in the nanometer order.
  • the method of immobilizing, ie immobilizing, the third molecule on the surface of the particle can be any method commonly known in the field of biological science, medicine or pharmacology. In this case, it may be appropriately selected according to the material of the beads and the type of the third molecule.
  • the surface of the particle is subjected to an anti-adsorption treatment for preventing the adsorption or binding of molecules present in the mixed sample solution excluding the specific binding between the second molecule and the third molecule.
  • an anti-adsorption treatment for preventing the adsorption or binding of molecules present in the mixed sample solution excluding the specific binding between the second molecule and the third molecule.
  • Such treatment can also be any method known in the field! / (For example, don't contribute to the reaction! /, Protein (bovine serum albumin, etc.) into a third molecule as a particle. Adsorb to the surface after fixing. ).
  • the mixed sample solution is mixed with the particles prior to addition. Measuring the fluorescence intensity of the fluorescent label of the first molecule in the solution and determining whether the first molecule is bound to the molecule in the second sample based on the measured fluorescence intensity. You can leave.
  • the method of fluorescence analysis performed before and after the addition of the particles may be the same or different. That is, before the addition of the particles, the fluorescence measurement for confirming the presence or absence of the binding of the first and second molecules is performed by FCS, and after adding the particles, the relationship between the particles and the first molecules is determined by FIDA. Measurements to confirm may be performed by FIDA.
  • the second molecule becomes the first molecule.
  • the fluorescent label is collected on the particles, so that the fluorescence obtained at one time is greater than when the first molecule is alone or only binds to another second molecule.
  • the amount of fluorescent light from the label increases, and this improves the ratio of fluorescence to be detected to fluorescence that is not detected, that is, the S / N ratio of the measurement.
  • the carrier of the first molecule becomes a nanometer-order particle, so that the state of movement of the fluorescent label is also regarded as a significant change. Can do.
  • the presence or absence of specific binding of molecules can be detected significantly more accurately than in the case of conventional single-molecule fluorescence analysis.
  • FIG. 1 schematically shows the state of molecules in a sample during the treatment process of the embodiment.
  • FIG. 2 is a flowchart showing the processing steps in a preferred embodiment of the method of the present invention.
  • Figure 1 shows the presence of DNA-binding protein P that specifically binds to DNA having a certain base sequence in biological sample solution A in order to detect whether or not fluorescently labeled DNA (No.
  • the method of the present invention is applied using a bead on which a monoclonal antibody (third molecule) is solid-phased on the surface using a single molecule) and DNA binding protein P (second molecule) as an epitope.
  • Fig. 2 (A) shows the measurement procedure in this case in the form of a flowchart.
  • fluorescently labeled DNA (D) having a base sequence that specifically binds to DNA-binding protein P is preliminarily obtained.
  • Preparation (Step 10, Figure 1 (a)).
  • the length of the DNA is 10 to 10 from the cost of synthesis and ease of detection;! OOmer force S is preferable, 20 force, and 40mer force S is more preferable.
  • Fluorescent dye F added to DNA for fluorescent labeling can be any fluorescent dye commonly used in this field, such as T AMRA carboxymethylrhoaamine, i'MR (tetramethylrhodamine), Aiexa Ri 47, Rnodamine green, Alexa488.
  • FCCS Fluorescence depolarization
  • FIDA fluorescence depolarization
  • FCCS fluorescence depolarization
  • DNA derived from organisms that meet the above conditions may be used, but artificially synthesized DNA is more stable than organisms and can be mass-produced at low cost. Suitable for detecting DNA-binding protein P in many types of test samples!
  • Preparation of DNA having a desired base sequence and addition of a fluorescent label may be performed by any method for those skilled in the art. DNA is mass-produced all at once rather than being prepared each time the method of the present embodiment is performed, and then stored in an undenatured form so that it can be used in an amount necessary for performing the method of the present embodiment. It may be done.
  • the biological sample solution A may be any solution used in the art as long as it does not significantly reduce the transmission of excitation light and fluorescence, for example, serum, plasma, etc. Good (step 20, figure l (b)).
  • Preparation of a biological sample solution such as serum or plasma may be performed by a person skilled in the art by any method such as centrifugation, filtration, or ultrasonic treatment. If the transmittance of the sample to light is remarkably low, dilute with saline or phosphate buffer as appropriate (so as not to impair the activity of the internal molecules)!
  • the prepared biological sample solution A and the solution containing DNA (D) are mixed (step 30).
  • DNA and protein P bind (Fig. 1 (c)).
  • any biomolecule (Q) may bind or adsorb nonspecifically to DNA (D) or fluorescent label F (Fig. 1 (d)). Therefore, even if the fluorescence measurement described later is performed at this stage, the fluorescent dye F on the complex of DNA (D) and protein P and the complex of DNA (D) and biomolecule (Q) With the fluorescent dye F, its movement state or distribution state is almost distinguishable. There may not be.
  • the antibody X that specifically binds to protein P is further immobilized on the surface of the mixed sample solution of the biological sample solution A and the solution containing DNA (D). Beads are added (step 40, Fig. 1 (e), (f)).
  • the antibody X that specifically binds to protein P may be a monoclonal antibody having a part of protein P as an epitope.
  • the antibody X epitope does not inhibit the binding of the produced antibody X force S protein P to DNA.
  • Such antibodies may be prepared by any method known to those skilled in the art.
  • the beads may be any of plastics, latex, gold colloids, magnetic particles, glass, etc., commonly used in this field, preferably in the order of a few nm in diameter and less than lOOOnm.
  • antibody X may be fixed on the bead surface by using a functional group that is covalently bonded to the antibody on the bead surface.
  • any protein that does not participate in the binding reaction is adsorbed on the bead surface (blocking), and biomolecules are adsorbed. It ’s better to avoid it! /
  • Fluorescence analysis In the embodiment of the present invention described above, an optical system of a laser confocal microscope is used to detect the presence or absence of specific binding between DNA and protein P in the mixed sample solution prepared as described above.
  • the fluorescence intensity of the fluorescent dye F in the mixed sample solution is measured and analyzed by single molecule fluorescence analysis using a fluorescence measurement device combined with a sensitive light detection device.
  • a single molecule fluorescence analysis system MF20 (Olympus) may be used for fluorescence measurement.
  • FCS, FIDA, FDS, FCCS force S is executed to determine whether the fluorescent dye F is immobilized on the beads. The following explains how the measurement results reflect whether or not the fluorescent dye F is fixed on the beads in each analysis method.
  • FCS fluorescence correlation spectroscopy
  • the speed of movement of a molecule passing through a minute fluorescence observation region by Brownian motion is observed.
  • the speed of the translational movement of the molecule is reflected in the shape of the autocorrelation function with the time of the measured fluorescence intensity as a variable.
  • the length of time (translational diffusion time) from the start of measurement until the autocorrelation function value is halved is used. Since the movement of molecules becomes slower as the size of the molecules increases, the translational diffusion time becomes longer.
  • DNA bound to protein P as shown in FIG.
  • FDS fluorescence depolarization method
  • the speed of rotational movement of the molecule is reflected in the ratio of the intensity of the longitudinal and transverse polarization of the measured fluorescence or the degree of polarization (if performed in a single molecule fluorescence analysis system, the fluorescence is measured in FIDA. Detection is performed separately for longitudinally polarized light and laterally polarized light, and the degree of polarization is calculated (in this case, called FIDA-pol).
  • Molecular rotation is the size of the molecule The larger the thickness is, the slower it is, and the degree of polarization increases.
  • FIDA fluorescence intensity distribution analysis method
  • photons emitted from a minute fluorescence observation region are detected (photon counting), and the frequency of detection of photons per unit time is statistically processed.
  • the number density of fluorescent particles in the minute fluorescent observation region and the fluorescent intensity per fluorescent particle are calculated.
  • the fluorescent dye F is bound to the beads and moves as one fluorescent particle, so that the number density of the fluorescent particles is reduced.
  • the presence of a plurality of fluorescent dye molecules F on the beads increases the fluorescence intensity per fluorescent particle.
  • the DNA bound to the protein P is immobilized on the beads from the decrease in the number density of the fluorescent particles and the increase in the fluorescence intensity per fluorescent particle.
  • the fluorescence intensity per fluorescent particle increases, the fluorescence intensity (number of detected photons) measured at a time increases and the S / N ratio of the measurement improves.
  • FCCS fluorescence cross-correlation spectroscopy
  • DNAs labeled with different fluorescent dyes are used, DNAs carrying different fluorescent dyes are immobilized on the beads via the protein P, so that the fluorescence from the two dyes can be obtained.
  • the DN A bound to the protein P is present on the beads. It is detected whether it is fixed to.
  • one bead force Fluorescence is emitted by a plurality of dyes, thereby increasing the fluorescence intensity measured at a time and improving the S / N ratio of the measurement.
  • the amount of sample required to detect one result is about several tens of thousands, and the measurement time is Therefore, the sample amount and time can be greatly reduced compared to the conventional biochemical method.
  • the measured fluorescence intensity is several times larger, so the S / N ratio is improved, and even if there are contaminants in the sample, it is specific. Only a good binding reaction can be detected satisfactorily.
  • the fluorescence measurement methods executed in step 35 and step 50 may be different.
  • FCS may be executed in step 30, and FIDA may be executed in step 50 for the purpose of supporting the change in FCS in step 35.
  • FCS if the added bead diameter is too large, the translational Brownian motion will be small and may hardly move during the measurement time.
  • FIDA if no beads are added, (DNA with a fluorescent dye only binds one-to-one with the DNA-binding protein, so the number of fluorescent particles does not change, and the FIDA results do not change unless the fluorescence intensity of the dye changes for some reason.) . If separate fluorescence analysis methods are used in step 35 and step 50, it is recommended that measurements be made by both methods in step 15 for comparison. Yes.
  • DNA binding protein P In order to detect the base sequence of DNA that specifically binds to DNA binding protein P contained in a biological sample, in other words, whether DNA binding protein P binds to DNA of a certain base sequence. It can also be used to detect whether or not. In this case, a fluorescently labeled DNA having a base sequence that is thought to bind to DNA binding protein P is used. If it is detected that DNA is immobilized on the beads, it is identified that there is a site that specifically binds to DNA binding protein P in the DNA base sequence.
  • the first molecule and the second molecule to be fluorescently labeled may be any DNA, protein, and other biomolecules.
  • the first molecule to be fluorescently labeled may be an antibody of a molecule that is detected whether it is contained in a biological sample
  • the third molecule may be an antibody to an epitope of another part of the molecule to be detected. Good.
  • the fluorescently labeled antibody is immobilized on the beads, the presence of an antigen against the antibody as the first and third molecules in the biological sample is detected.
  • the method of the present invention can detect a specific binding of a specific molecule in a manner that distinguishes it from a non-specific binding, even if contaminants are present in the sample. It can be used as a screening tool for biologically active substances. If any molecule in a biological sample can be chemically or genetically engineered (by the expression of a fluorescent protein such as GFP), the first sample containing the first molecule is the biological sample. It should be understood that the second sample may be prepared or material in any manner and such cases are also within the scope of the present invention. .

Abstract

It is intended to provide a method of detecting a specific binding of a definite molecule with the use of the single molecule fluorometry technique by which the specific binding of the molecule can be detected and monitored even in the presence of contaminants other than the subject molecule to be detected while avoiding the effects of background light caused by the existence of the contaminants and a non-specific reaction. This method comprises adding, to a mixed sample solution, which consists of a first sample containing a fluorescent labeled first molecule and a second sample containing a second molecule that is to be determined whether or not binding specifically to the first molecule or another second sample that is to be determined whether or not containing a second molecule specifically binding to the first molecule, a bead on which a third molecule specifically binding to the second molecule has been immobilized. When it is determined that the first molecule has been immobilized on the bead based on the fluorescent intensity of the fluorescent label of the first molecule in the mixed sample solution, it is determined that the first molecule has specifically bound to the second molecule or the first sample contains the second molecule.

Description

明 細 書  Specification
一分子蛍光分析による分子の特異的結合反応検出方法  Method for detecting specific binding reaction of molecules by single molecule fluorescence analysis
技術分野  Technical field
[0001] 本発明は、蛍光一分子からの蛍光を計測することによりタンパク質、ペプチド、核酸 [0001] The present invention relates to a protein, peptide, nucleic acid by measuring fluorescence from a single fluorescent molecule.
、脂質、アミノ酸及びその他の生理活性物質又は生体分子（以下、「生体分子等」と する。）の特異的な結合反応を検出する方法に係り、より詳細には、種々の生体分子 間に於ける特異的な結合反応と非特異的な結合反応を識別可能な検出方法に係る 背景技術 , Lipids, amino acids and other physiologically active substances or biomolecules (hereinafter referred to as “biomolecules”), and more particularly, between various biomolecules. Related to a detection method capable of discriminating between a specific binding reaction and a non-specific binding reaction
[0002] 近年の技術などの光計測技術の発展により、現在では、蛍光一分子からの蛍光を 測定 '解析する蛍光相関分光分析 (Fluorescence Correlation Spectroscopy : FCS) 、 光強度分布分析 (Fluorescence—Intensity Distribution Analysis： といつ た蛍光分析方法が利用できるようになつている。これらの蛍光一分子レベルの蛍光 測定を行う蛍光分析法 (一分子蛍光分析技術）に於いては、レーザー共焦点顕微鏡 の光学系とフォトンカウンティング（1光子検出）も可能な超高感度の光検出装置とを 用いて蛍光一分子からの蛍光強度が測定され、その強度の揺らぎを種々の方法によ り解析して、蛍光分子又は蛍光標識された分子の運動の速さ又は分子の大きさ（FC Sの場合）、分子（又は粒子）の数密度又は一分子当たりの蛍光強度（FIDAの場合） といった情報を得ることができる。従って、これらの情報に基づいて、分子の構造又は 大きさの変化や分子の結合 ·解離反応又は分散 ·凝集といった種々の現象を検出す ること力 Sでさる。  [0002] With the recent development of optical measurement technology such as technology, fluorescence correlation spectroscopy (FCS), fluorescence intensity distribution analysis (Fluorescence Correlation Spectroscopy: FCS) Analysis: When the fluorescence analysis method is available, the fluorescence analysis method (single molecule fluorescence analysis technology) that measures fluorescence at the single molecule level is the optical system of the laser confocal microscope. And an ultrasensitive photodetection device capable of photon counting (single-photon detection), the fluorescence intensity from a single fluorescent molecule is measured, and fluctuations in the intensity are analyzed by various methods. Or to obtain information such as the speed of movement or size of the fluorescently labeled molecule (in the case of FC S), number density of molecules (or particles) or fluorescence intensity per molecule (in the case of FIDA) Therefore, based on this information, the force S can be used to detect various phenomena such as changes in the structure or size of molecules and molecular binding / dissociation or dispersion / aggregation.
[0003] 生物科学、医学又は薬学の分野に於いては、上記の如き一分子蛍光分析技術を 生体分子等の状態及び運動の検出 '観測に応用し、種々の生体分子等の現象-反 応を細胞レベル又は分子レベルで解明する試みがなされている。例えば、互いに特 異的に相互作用する一対の分子（DNAと DNA結合タンパク質、抗原と抗体など）の うち、少なくとも一方の分子に蛍光分子等で蛍光標識を施した上で、それらの分子を 反応させると、一方の分子上の蛍光分子の運動や状態の変化が蛍光分子からの蛍 光強度又はその揺らぎに反映され、これにより、タンパク質又は DNA等の分子間の 相互作用が検出できることとなる。既に、そのような一分子蛍光分析技術を用いて分 子レベルにてタンパク質と核酸の結合反応や抗原抗体反応の検出が為された例が 報告されつつある（例えば、特許文献 1 2、非特許文献 1 2)。また、一分子蛍光 分析技術は、従前の生化学的な方法に比して極めて微量な試料にて且短時間にて 計測が可能であるので、医学'薬理学等の分野に於いて、種々の病気の臨床診断や 生理活性物質のスクーリングに於ける応用も期待される。 [0003] In the field of biological science, medicine or pharmacy, the above-described single-molecule fluorescence analysis technology is applied to the detection and observation of the state and movement of biomolecules, etc. Attempts have been made to elucidate these at the cellular or molecular level. For example, among a pair of molecules that interact specifically with each other (DNA and DNA-binding protein, antigen and antibody, etc.), at least one of the molecules is fluorescently labeled with a fluorescent molecule, and then these molecules are reacted. , The movement and state change of the fluorescent molecule on one molecule Reflected by the light intensity or its fluctuations, this makes it possible to detect interactions between molecules such as proteins or DNA. Examples have already been reported in which binding reactions between proteins and nucleic acids and antigen-antibody reactions are detected at the molecular level using such single-molecule fluorescence analysis technology (for example, Patent Documents 12 and 2, non-patent documents). Reference 1 2). In addition, single-molecule fluorescence analysis technology can measure in a very short amount of sample and in a short time compared to conventional biochemical methods. Applications in clinical diagnosis of illnesses and in the screening of bioactive substances are also expected.
特許文献 1 :特開 2003— 275000公報 Patent Document 1: Japanese Patent Laid-Open No. 2003-275000
特許文献 2 :特開 2005 337805公報 Patent Document 2: JP 2005 337805 JP
非特許文献 1 :カスク外 3名、 PNAS 96巻、 24号、 13756— 13761頁 1999年 11 月 23日 Non-Patent Document 1: 3 outside casks, PNAS 96, 24, 13756-13761, November 23, 1999
非特許文献 2 :コバヤシ外 3名、アナリティカノいバイオケミストリー、 2004年 332巻 58— 66頁 Non-Patent Document 2: Three people outside Kobayashi, Analytical Biochemistry, 2004 332 58-66
発明の開示 Disclosure of the invention
発明が解決しょうとする課題 Problems to be solved by the invention
上記の如き一分子蛍光分析技術に於いて用いられる光学系及び光検出装置の性 能は、確かに、蛍光一分子からの蛍光又は一光子程度の光量を検出'測定すること が可能なほど向上されているが、検出したい光が微弱であることに変わりはない。従 つて、背景の光が高ければ、検出すべき光が背景に埋もれてしまい、識別することが できなくなってしまう。特に、生体分子等の相互作用の観測や病気の臨床診断等に 使用されることとなる生体から得られた試料又は生体由来の試料 (血清、血漿など。 以下、「生体試料」と称する。）は、一般に、検出したい反応に関わりのない種々の構 成物又は夾雑物が多く含まれており、その中の観測対象の分子以外に多数の分子 からの自家蛍光によって背景光が高くなり、このことにより、測定結果の精度又は信 頼性が低下してしまうこととなる。また、生体分子等の特異的な結合反応を検出しょう とする場合、生体試料中には検出したい特異的な反応とは別の反応又は非特異的 な結合又は吸着をする分子が存在している場合が多ぐそのような場合、検出したい 反応以外の反応や非特異的な結合 '吸着が測定結果及び解析結果に影響すると、 誤差が生じ、結果の信頼性が低下する。 The performance of optical systems and photodetection devices used in single-molecule fluorescence analysis techniques as described above is improved so that it is possible to detect and measure the amount of fluorescence from a single fluorescent molecule or the amount of light of one photon. However, the light to be detected is still weak. Therefore, if the background light is high, the light to be detected is buried in the background and cannot be identified. In particular, samples obtained from living organisms or samples derived from living organisms (serum, plasma, etc., hereinafter referred to as “biological samples”) that will be used for observation of interactions such as biomolecules and clinical diagnosis of diseases. In general, there are many various components or contaminants that are not related to the reaction to be detected, and the background light becomes high due to autofluorescence from a number of molecules other than the molecules to be observed. As a result, the accuracy or reliability of the measurement results will be reduced. In addition, when a specific binding reaction such as a biomolecule is to be detected, there are molecules in the biological sample that are different from the specific reaction to be detected or have nonspecific binding or adsorption. In many cases, reactions other than the reaction to be detected or non-specific binding 'adsorption affects the measurement and analysis results. Errors occur and the reliability of the results is reduced.
[0005] 上記の如き一分子蛍光分析の試料中の観測対象の分子以外の構成物又は夾雑 物の影響は、試料からそれらの夾雑物を除去するか或!/、は観測対象の分子を精製 することにより排除すること力 Sできる。し力もながら、試料から特定の分子を抽出したり 、或いは、特定の分子以外を除去することは、時間、労力及び多量の試料を必要とし 、微量にて短時間に計測が可能であるという一分子蛍光分析技術の利点を損なうこ ととなる。また、生体試料の場合、物質の抽出 ·精製又は除去の処理操作中に目的 の物質が変性してしまうこともあり、結局、 目的の測定が実施できなくなってしまうこと も有り得る。 [0005] As described above, the influence of constituents or contaminants other than the molecule to be observed in the sample of single-molecule fluorescence analysis may remove those impurities from the sample or purify the molecule to be observed. It is possible to eliminate it by doing S. However, extracting a specific molecule from a sample or removing other than a specific molecule requires time, labor and a large amount of sample, and can be measured in a short amount of time. The advantages of molecular fluorescence analysis technology will be lost. In the case of a biological sample, the target substance may be denatured during the process of extracting / purifying or removing the substance, and eventually the target measurement may not be performed.
[0006] 力、くして、本発明の一つの主な課題は、一分子蛍光分析技術を用いた分子の相互 作用、特に、分子の特異的な結合、の検出及び観測に於いて、検出'観測結果の精 度及び信頼性を損なう原因となる試料中に含まれる検出対象の分子以外の夾雑物 を除去することなぐそれらの夾雑物の存在に起因する背景光や非特異的な反応の 影響を排除するか又は相対的に低減できるようにすることである。  [0006] Thus, one of the main problems of the present invention is that detection and observation of molecular interactions, particularly specific binding of molecules, using single-molecule fluorescence analysis technology. The influence of background light and non-specific reactions caused by the presence of contaminants other than the molecules to be detected contained in the sample that cause the accuracy and reliability of the observation results to be lost. Is to be able to eliminate or relatively reduce.
[0007] また、本発明のもう一つの課題は、上記の如き一分子蛍光分析技術を用いた分子 の相互作用の検出及び観測に於いて、特に、試料として生体試料又は生体由来の 試料 (血清、血漿など)を用いる場合に、計測結果に於いて、試料中の非特異的な結 合又は吸着の影響を排除し、計測結果の精度、信頼性をより向上させることである。 課題を解決するための手段  [0007] Another object of the present invention is to detect and observe the interaction of molecules using the single-molecule fluorescence analysis technique as described above. Particularly, a biological sample or a sample derived from a living body (serum In the case of using plasma, etc., the effect of non-specific binding or adsorption in the sample is eliminated in the measurement result, and the accuracy and reliability of the measurement result are further improved. Means for solving the problem
[0008] 本発明によれば、上記の如き一分子蛍光分析技術を用いて、特定の分子の特異 的結合を、夾雑物を多く含む試料、特に生体試料溶液中に於いて精度良く又は信 頼性高く検出することのできる分子の特異的結合反応検出方法が提供される。  [0008] According to the present invention, specific binding of a specific molecule can be performed with high accuracy or reliability in a sample containing a large amount of contaminants, particularly in a biological sample solution, using the single-molecule fluorescence analysis technique as described above. A method for detecting a specific binding reaction of a molecule that can be detected with high efficiency is provided.
[0009] 本発明の一つの態様によれば、特定の分子の特異的結合を検出する方法は、蛍 光標識された第一の分子を含む第一の試料と、前記の蛍光標識された第一の分子 と特異的に結合するか否かが判定される第二の分子を含む第二の試料とを混合し混 合試料溶液を調製する過程と、第二の分子に特異的に結合する第三の分子が表面 に固相化された外径 1 11 m以下の粒子を混合試料溶液へ添加する過程と、前記の 粒子が添加された混合試料溶液中の第一の分子の蛍光標識の蛍光強度を測定す る過程と、蛍光強度に基づいて、第一の分子が前記粒子上に固定されているか否か を判定する過程とを含み、第一の分子が粒子上に固定されて!/、る判定された場合に は、第一の分子が第二の分子に特異的に結合したと判定することを特徴とする。即ち 、この態様に於いては、第一の分子が第二の分子と特異的な結合するか否力、を判定 すること力 Sでさる。 [0009] According to one embodiment of the present invention, a method for detecting specific binding of a specific molecule includes a first sample containing a first molecule that is fluorescently labeled, and the fluorescently labeled first molecule. A process of preparing a mixed sample solution by mixing a second sample containing a second molecule to be determined whether or not it specifically binds to one molecule, and specifically binding to the second molecule The process of adding particles having an outer diameter of 1 11 m or less, in which a third molecule is immobilized on the surface, to the mixed sample solution, and the fluorescent labeling of the first molecule in the mixed sample solution to which the particles are added Measure fluorescence intensity And determining whether the first molecule is immobilized on the particle based on the fluorescence intensity, and determining whether the first molecule is immobilized on the particle. In this case, it is determined that the first molecule specifically binds to the second molecule. That is, in this embodiment, the force S is used to determine whether or not the first molecule specifically binds to the second molecule.
[0010] 一分子蛍光分析技術を用いて或る分子（例えば、上記第一の分子）と或る別の分 子（上記第二の分子）とが特異的な結合をするか否力、を判定する場合、現在までに（ 従来の技術に於レ、て)提案されて!、る方法では、蛍光標識された第一の分子を含む 第一の試料と、第二の分子を含む第二の試料とを混合し、そのまま、その混合試料 の蛍光測定と結果の解析が行われていた。この場合、試料中に夾雑物が多いと、背 景光が高く S/N比が悪い場合があり、また、夾雑物と蛍光標識された分子との非特 異的な結合 '吸着があっても、それが特異的な結合とは区別がつかない。  [0010] Whether or not a specific molecule (for example, the above-mentioned first molecule) and a certain other molecule (the above-mentioned second molecule) specifically bind using a single-molecule fluorescence analysis technique is determined. In the method of determination, which has been proposed to date (in the prior art), a first sample containing a fluorescently labeled first molecule and a second sample containing a second molecule The sample was mixed and the fluorescence measurement of the mixed sample was performed and the result was analyzed. In this case, if there are many contaminants in the sample, the background light may be high and the S / N ratio may be poor, and even if there is nonspecific binding between the contaminants and the fluorescently labeled molecules. , It is indistinguishable from specific binding.
[0011] そこで、本発明に於いては、上記の如ぐ蛍光標識された第一の分子と第二の分子 とを含む混合試料溶液に、第二の分子に特異的に結合する第三の分子が表面に固 相化された外径 1 11 m以下の粒子を混合試料溶液へ添加し、その上で蛍光測定と結 果の解析が行われるよう従前の方法の過程が修正される。第二の分子に特異的に結 合する第三の分子を表面に担持する粒子が混合溶液に添加されると、第三の分子 が第二の分子にのみ結合し、その結果、粒子の表面上に第二の分子が収集されるこ ととなる。もし第二の分子が第一の分子と特異的に結合するものであれば、蛍光標識 された第一の分子も粒子上に集められ、その結果、蛍光標識から発せられる蛍光強 度は、蛍光標識が外径 1 a m以下の粒子上に固定された状態を反映されたものとな る。他方、もし第二の分子が第一の分子と特異的に結合しないものであれば、仮に夾 雑物と非特異的な結合をしていても、第一の分子上の蛍光標識力もの蛍光強度は、 第一の分子がフリーの状態 (粒子上に結合してレ、な!/、状態）と大きくは変わらなレ、は ずである。また、蛍光標識が外径 以下の粒子上に固定された状態となると、そ うでない状態に比して、蛍光標識のブラウン運動が大幅制限され、また、測定領域内 での蛍光標識の分布も大きく変化し、更に、蛍光標識が粒子上に収集されることによ り、粒子上の複数又は多数の蛍光標識より蛍光が発せられることになるので、一時に 観測される蛍光強度も大きくなり、測定の S/Nが大幅に改善される。力べして、上記 の如ぐ第一の分子が、第二の分子と第三の分子の特異的な結合を介して粒子上に 「固定」されるか否かを判定するよう方法の構成を修正することにより、第一の分子が 第二の分子と特異的に結合する場合とそうでない場合とが、より高い S/N比にて検 出すること力 Sできるようになる。 Therefore, in the present invention, a third sample that specifically binds to the second molecule is mixed with the mixed sample solution containing the first and second molecules labeled with fluorescence as described above. The process of the previous method is modified so that particles with an outer diameter of 1 11 m or less with molecules immobilized on the surface are added to the mixed sample solution, and then fluorescence measurement and analysis of the results are performed. When particles carrying a third molecule that specifically binds to the second molecule on the surface are added to the mixed solution, the third molecule only binds to the second molecule, resulting in the surface of the particle A second molecule will be collected on top. If the second molecule binds specifically to the first molecule, the fluorescently labeled first molecule is also collected on the particle, so that the fluorescence intensity emitted from the fluorescent label is the fluorescence intensity. This reflects the state in which the label is fixed on particles with an outer diameter of 1 am or less. On the other hand, if the second molecule does not specifically bind to the first molecule, even if it is non-specifically bound to the contaminant, the fluorescence of the fluorescent labeling power on the first molecule The strength should be much different from the state in which the first molecule is free (bonded on the particle, the state!). In addition, when the fluorescent label is fixed on a particle having an outer diameter or less, the Brownian motion of the fluorescent label is greatly limited as compared to the case where the fluorescent label is not, and the distribution of the fluorescent label within the measurement region is also reduced. Since the fluorescence label is collected on the particle, the fluorescence is emitted from a plurality or many of the fluorescence labels on the particle. The observed fluorescence intensity also increases and the S / N of the measurement is greatly improved. In force, the method is configured to determine whether the first molecule as described above is “fixed” on the particle through the specific binding of the second and third molecules. By correcting, it is possible to detect whether the first molecule specifically binds to the second molecule or not, at a higher S / N ratio.
[0012] 上記の第二の分子に特異的に結合する第三の分子が表面に固相化された外径 1 πι以下の粒子を混合試料溶液へ添加し、その蛍光測定及び分析により、蛍光標 識された第一の分子が粒子上に固定されているか否かを判定する手法は、或る試料 に第二の分子が存在するか否かを検出するために用いることもできる。従って、本発 明のもう一つの態様によれば、本発明の特定の分子の特異的結合を検出する方法 は、蛍光標識された第一の分子を含む第一の試料と、蛍光標識された第一の分子と 特異的に結合する第二の分子が存在するか否かが判定される第二の試料とを混合 して混合試料溶液を調製する過程と、第二の分子に特異的に結合する第三の分子 が表面に固相化された外径 1 a m以下の粒子を第一及び第二の試料の混合試料溶 液へ添加する過程と、粒子が添加された混合試料溶液中の第一の分子の蛍光標識 の蛍光強度を測定する過程と、蛍光強度に基づいて、第一の分子が粒子上に固定 されているか否かを判定する過程とを含み、第一の分子が粒子上に固定されている 判定された場合には、第二の分子が第二の試料に存在して!/、ると判定することを特 徴とする。力、かる構成によれば、或る分子（第一の分子と第三の分子に特異的に結 合する分子）が任意の試料 (第二の試料）に含まれているか否力、が、前記の態様と同 様に、従前に比して、より高い S/N比にて検出することができるようになる。  [0012] A particle having an outer diameter of 1 πι or less in which a third molecule that specifically binds to the second molecule is immobilized on the surface is added to a mixed sample solution, and fluorescence is measured and analyzed to obtain fluorescence. A technique for determining whether a labeled first molecule is immobilized on a particle can also be used to detect whether a second molecule is present in a sample. Thus, according to another aspect of the present invention, a method for detecting specific binding of a particular molecule of the present invention comprises a first sample comprising a first fluorescently labeled molecule and a fluorescently labeled first molecule. A process of preparing a mixed sample solution by mixing a second sample to determine whether or not there is a second molecule that specifically binds to the first molecule, and specifically to the second molecule A process of adding particles having an outer diameter of 1 am or less, in which a third molecule to be bound is immobilized on the surface, to the mixed sample solution of the first and second samples, and in the mixed sample solution to which the particles are added A process of measuring the fluorescence intensity of the fluorescent label of the first molecule, and a process of determining whether the first molecule is immobilized on the particle based on the fluorescence intensity. If it is determined that the second molecule is present in the second sample! /, It is referred to as Features. According to the force, such a configuration, whether a certain molecule (a molecule that specifically binds to the first molecule and the third molecule) is contained in an arbitrary sample (second sample), As in the above-described embodiment, detection can be performed at a higher S / N ratio than before.
[0013] 上記の本発明の分子の特異的結合の検出方法に於ける蛍光測定及び分析（又は 解析）は、 FCS、 FIDA、蛍光相互相関分光法（Fluorescence cross-correlation Sp eCtroSCOpy : FCCS)など、レーザー共焦点顕微鏡の光学系に超高感度光検出装置 を組み合わせた蛍光測定装置により実施される一分子蛍光分析法のいずれか任意 のものであってよい。また、その他の分子の構造 ·状態 ·運動の変化を観測できる任 意の蛍光分析法、例えば、蛍光偏光解消法（Fluorescence Depolarization Spectra scopy： FDS)などが用いられてもよ!/、（本発明の構成により各々蛍光分析方法に於 いて蛍光測定 ·分析の結果がどのようになるかは、実施の形態の説明の欄に於いて 説明される。）。理解されるべきことは、第一の分子が粒子に固定されているか否かを 検出できる蛍光分析方法であれば任意のものであってよぐそのような場合も本発明 の範囲に属する。 [0013] The above detection method in fluorescence measurement and analysis of the specific binding of the molecules of the present invention (or analysis) is, FCS, FIDA, fluorescence cross correlation spectroscopy (Fluorescence cross-correlation Sp e C tro SCO py: FCCS) or any other single-molecule fluorescence analysis method performed by a fluorescence measuring apparatus that combines an optical system of a laser confocal microscope with an ultrasensitive photodetection device. Also, any fluorescence analysis method that can observe changes in the structure, state, and motion of other molecules, such as fluorescence depolarization spectroscopy (FDS), may be used! Depending on the configuration of The result of the fluorescence measurement / analysis will be explained in the explanation section of the embodiment. ). It should be understood that any fluorescence analysis method capable of detecting whether or not the first molecule is immobilized on the particle may be any method, and such a case also belongs to the scope of the present invention.
[0014] 本発明の方法に於いて用いられる試料に於いて、蛍光標識される第一の分子、第 二の分子及び第三の分子は、予め特定されている必要があるが、第二の分子を含有 する第二の試料は、夾雑物を含んでいてもよぐ従って、生物科学、医学又は薬学の 分野の研究、病気の臨床診断又は生理活性物質のスクリーニング等に於いて使用さ れる血清又は血漿又はその他の生体から得られた溶液試料であってよレ、。上記の本 発明の態様の説明から理解される如ぐ第二の試料を生体から得られた溶液試料と する場合、第二の分子がその生体試料に含まれて!/、るか否か又は第一の分子と特 異的に結合するか否かが、生体試料に或る程度の量の夾雑物が含まれていても、検 出が可能となる。例えば、第一の分子を DNAとし、第二の分子を DNA結合タンパク 質とし、第三の分子が第二の分子に対する抗体とすれば、或る塩基配列を有する D NAに対して或る DNA結合タンパク質が特異的に結合するか否か又は或る塩基配 歹 IJを有する DNAに対して特異的に結合する DNA結合タンパク質が或る試料 (例え ば、血清や血漿）中に含まれているか否かが検出される。  [0014] In the sample used in the method of the present invention, the first molecule, the second molecule and the third molecule to be fluorescently labeled need to be specified in advance. The second sample containing molecules may contain contaminants, so serum used for research in the fields of biological science, medicine or pharmacology, clinical diagnosis of diseases or screening of bioactive substances, etc. Or a solution sample obtained from plasma or other living body. When the second sample as understood from the above description of the embodiment of the present invention is a solution sample obtained from a living body, whether or not the second molecule is contained in the biological sample! Whether or not it specifically binds to the first molecule can be detected even if the biological sample contains a certain amount of impurities. For example, if the first molecule is DNA, the second molecule is a DNA-binding protein, and the third molecule is an antibody against the second molecule, a certain DNA against a DNA having a certain base sequence can be obtained. Whether a binding protein specifically binds or is a DNA binding protein that specifically binds to DNA with a certain base sequence IJ included in a sample (eg, serum or plasma)? Whether or not is detected.
[0015] 上記の構成に於いて、第三の分子が表面に固相化される粒子は、生物科学、医学 又は薬学の分野の実験に於レ、て通常用いられてレ、るナノメートルオーダーのビーズ であってよく、プラスチック、ラテックス、金コロイド、磁性粒子及びガラスから成る群か ら選択された少なくとも一つの材料からなるビーズであってよ!/、。第三の分子を粒子 の表面上に固相化、即ち、固定する方法は、生物科学、医学又は薬学の実験の分 野に於いて通常知られている任意の方法であってよぐ当業者に於いて、ビーズの 材質、第三の分子の種類に応じて、適宜選択されてよい。好適には、粒子の表面は 、第二の分子と第三の分子との特異的結合を除く混合試料溶液中に存在する分子 の吸着又は結合を防止するための吸着防止処理が施される。かかる処理もこの分野 に於!/、て知られた任意の方法であってよ!/、（例えば、反応に寄与しな!/、タンパク質（ 牛血清アルブミンなど）を第三の分子を粒子に固定した後に表面に吸着させるなど。 )。 [0015] In the above configuration, the particle on which the third molecule is immobilized on the surface is generally used in experiments in the field of biological science, medicine or pharmacy, and is in the nanometer order. Bead of at least one material selected from the group consisting of plastic, latex, gold colloid, magnetic particles and glass! /. The method of immobilizing, ie immobilizing, the third molecule on the surface of the particle can be any method commonly known in the field of biological science, medicine or pharmacology. In this case, it may be appropriately selected according to the material of the beads and the type of the third molecule. Preferably, the surface of the particle is subjected to an anti-adsorption treatment for preventing the adsorption or binding of molecules present in the mixed sample solution excluding the specific binding between the second molecule and the third molecule. Such treatment can also be any method known in the field! / (For example, don't contribute to the reaction! /, Protein (bovine serum albumin, etc.) into a third molecule as a particle. Adsorb to the surface after fixing. ).
[0016] なお、上記の本発明の構成に於いて、混合試料溶液へ粒子を添加した後の蛍光 測定 ·分析の結果と比較するために、混合試料溶液への粒子を添加に先立って混合 試料溶液中の第一の分子の蛍光標識の蛍光強度を測定し、測定された蛍光強度に 基づいて、第一の分子が第二の試料中の分子と結合したか否かを判定する過程を 含んでいてよい。粒子を添加する前と後に於いて実行される蛍光分析の方法は、同 一であってもよいが、別のものであってよい。即ち、粒子の添加前は、第一及び第二 の分子の結合の有無を確認するための蛍光測定を FCSにより行い、粒子の添カロ後 は、 FIDAにより粒子と第一の分子との関係を確認するための測定を FIDAにより行う ようにしてよい。また、第一の分子の第二の分子に対する結合は、或る程度、蛍光強 度の測定'分析結果に反映されるはずであるので、第一の分子を第二の試料に作用 させた際に蛍光測定結果に全く変化が見られない場合 (第一の分子の第二の分子 に対する結合発生の可能性が殆どないと認められる場合）には、粒子の添加は省略 されてよ!/、 (試料及び時間の節約）。  In the configuration of the present invention described above, in order to compare the result of fluorescence measurement / analysis after adding particles to the mixed sample solution, the mixed sample solution is mixed with the particles prior to addition. Measuring the fluorescence intensity of the fluorescent label of the first molecule in the solution and determining whether the first molecule is bound to the molecule in the second sample based on the measured fluorescence intensity. You can leave. The method of fluorescence analysis performed before and after the addition of the particles may be the same or different. That is, before the addition of the particles, the fluorescence measurement for confirming the presence or absence of the binding of the first and second molecules is performed by FCS, and after adding the particles, the relationship between the particles and the first molecules is determined by FIDA. Measurements to confirm may be performed by FIDA. In addition, since the binding of the first molecule to the second molecule should be reflected to some extent in the fluorescence intensity measurement'analysis result, when the first molecule is allowed to act on the second sample. If there is no change in the fluorescence measurement results (when it is recognized that there is almost no possibility of binding of the first molecule to the second molecule), the addition of particles can be omitted! /, (Sample and time saving).
発明の効果  The invention's effect
[0017] 従来、多数の夾雑物を含む生体試料に於ける生体分子間の特異的な結合の検出 は、例えば、ゲルろ過クロマトグラフィー、タンパク質 'DNAの電気泳動法、ゥエスタ ンブロッテイング法、 ELISA法と!/、つた多量又は高濃度の試料量と時間を要する生 化学的な方法が必要であった。し力もながら、本発明によれば、高濃度若しくは多量 の試料を必要とせず、簡便に且迅速に、生体分子間の特異的な結合の検出を行うこ と力 Sできる。上記の説明から理解される如ぐ第三の分子を固相化した粒子を用いて 、第二の分子を特異的に収集することにより、或る程度の限界はあるが（例えば、励 起光が透過できない場合）、蛍光測定される試料中に夾雑物が存在していても、第 二の分子と (蛍光標識された)第一の分子との特異的な結合の有無を検出することが でき、従って、種々の病気の臨床診断や生理活性物質のスクーリングに於ける応用 も期待される  [0017] Conventionally, detection of specific binding between biomolecules in a biological sample containing a large number of contaminants, for example, gel filtration chromatography, protein 'DNA electrophoresis, Western blotting, ELISA There was a need for a method and a biochemical method that required a lot of sample volume and time. However, according to the present invention, it is possible to detect specific binding between biomolecules easily and quickly without requiring a high concentration or a large amount of sample. There are some limitations to the specific collection of the second molecule using particles solidified with the third molecule as understood from the above description (eg, excitation light). Can detect the presence or absence of specific binding between the second molecule and the first molecule (fluorescently labeled) even if contaminants are present in the sample to be measured for fluorescence. Therefore, application in clinical diagnosis of various diseases and schooling of bioactive substances is also expected.
[0018] 特記されるべきことは、第三の分子を固相化した粒子を用いて、第二の分子を特異 的に収集するという構成によれば、第二の分子が第一の分子に特異的に結合した場 合には、蛍光標識が粒子上に集められることにより、第一の分子が単独でいる場合 又は別の第二の分子とのみ結合してレ、る場合に比して、一時に得られる蛍光標識か らの蛍光光量が増大し、これにより、検出したい蛍光とそうでない蛍光との比、即ち、 測定の S/N比が向上される点である。また、第二の分子が第一の分子に特異的に 結合した場合には第一の分子の担体がナノメートルオーダーの粒子となるので、蛍 光標識の運動の状態も顕著な変化として捉えることができる。総じて、従前の一分子 蛍光分析による方法に比して、分子の特異的な結合の有無を顕著に正確に検出で さることとなる。 [0018] It should be noted that according to the configuration in which the second molecule is specifically collected using particles in which the third molecule is immobilized, the second molecule becomes the first molecule. Specific binding field In some cases, the fluorescent label is collected on the particles, so that the fluorescence obtained at one time is greater than when the first molecule is alone or only binds to another second molecule. The amount of fluorescent light from the label increases, and this improves the ratio of fluorescence to be detected to fluorescence that is not detected, that is, the S / N ratio of the measurement. In addition, when the second molecule is specifically bound to the first molecule, the carrier of the first molecule becomes a nanometer-order particle, so that the state of movement of the fluorescent label is also regarded as a significant change. Can do. In general, the presence or absence of specific binding of molecules can be detected significantly more accurately than in the case of conventional single-molecule fluorescence analysis.
[0019] 本発明のその他の目的及び利点は、以下の本発明の好ましい実施形態の説明に より明らかになるであろう。  [0019] Other objects and advantages of the present invention will become apparent from the following description of preferred embodiments of the present invention.
図面の簡単な説明  Brief Description of Drawings
[0020] [図 1]図 1は、実施形態の処理過程中に於ける試料中の分子の状態を模式的に表し たものである。  [0020] [FIG. 1] FIG. 1 schematically shows the state of molecules in a sample during the treatment process of the embodiment.
[図 2]図 2は、本発明の方法の好ましい実施形態に於ける処理過程をフローチャート の形式にて示したものである。  [FIG. 2] FIG. 2 is a flowchart showing the processing steps in a preferred embodiment of the method of the present invention.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0021] 以下に添付の図を参照しつつ、本発明を幾つかの好ましい実施形態について詳 細に説明する。  [0021] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.
[0022] 測定試料の調製極作の丰順  [0022] Procedure for preparation of measurement sample
図 1は、生体試料溶液 A中に、或る塩基配列を有する DNAに対して特異的に結合 する DNA結合タンパク質 Pが存在するか否かを検出するために、蛍光標識された D NA (第一の分子）と DNA結合タンパク質 P (第二の分子）の一部をェピトープとする モノクローナル抗体 (第三の分子)を表面に固相化したビーズとを用いて、本発明の方 法を適用した場合の試料の調製操作と分子の状態の変化を模式的に示したもので あり、図 2 (A)は、その場合の測定の手順をフローチャートの形式で示したものである  Figure 1 shows the presence of DNA-binding protein P that specifically binds to DNA having a certain base sequence in biological sample solution A in order to detect whether or not fluorescently labeled DNA (No. The method of the present invention is applied using a bead on which a monoclonal antibody (third molecule) is solid-phased on the surface using a single molecule) and DNA binding protein P (second molecule) as an epitope. Fig. 2 (A) shows the measurement procedure in this case in the form of a flowchart.
[0023] 図 1及び 2を参照して、本発明の方法の実施を開始する当たり、予め、 DNA結合タ ンパク質 Pに特異的に結合する塩基配列を有する蛍光標識された DNA(D)が準備 される（ステップ 10、図 1 (a) )。 DNAの長さは、合成のコスト及び検出の容易さから 1 0〜； !OOmer力 S好ましく、 20力、ら 40mer力 S更に好ましい。蛍光標識のために DNAに付 加される蛍光色素 Fとしては、この分野で通常使われる任意の蛍光色素、例えば、 T AMRA carboxymethylrhoaamine 、 i'MR(tetramethylrhodamine)、 Aiexaり 47、 Rnodami ne Green, Alexa488などであってよいが、これらに限定されない。後述の如ぐ蛍光 分析法として、 FCS、 FIDA、蛍光偏光解消法を用いる場合には、蛍光標識は、 1種 類でよいが、 2種類以上の蛍光標識を用いると（各 DNA分子には、一種類の蛍光分 子を付与する。）、 FCCSでの計測が可能となる。 DNAは、上記条件を満たす生物 由来の DNAでもよいが、人工的に合成した DNAは、生物由来のものより安定性が 高ぐ安価で大量生産ができるので、継続的に、病気の臨床診断や、多種類の被検 試料にて DNA結合タンパク質 Pの検出をする場合に適して!/、る。所望の塩基配列の DNAの調製と蛍光標識の付加は、当業者にとって任意の方法でなされてよい。 DN Aは、本実施形態の方法を実施する度に調製するのではなぐ一度に量産した後、 変性しない態様にて保存し、本実施形態の方法を実施する際に必要量ずつ使用す るようにされてよい。 [0023] Referring to Figs. 1 and 2, before starting the implementation of the method of the present invention, fluorescently labeled DNA (D) having a base sequence that specifically binds to DNA-binding protein P is preliminarily obtained. Preparation (Step 10, Figure 1 (a)). The length of the DNA is 10 to 10 from the cost of synthesis and ease of detection;! OOmer force S is preferable, 20 force, and 40mer force S is more preferable. Fluorescent dye F added to DNA for fluorescent labeling can be any fluorescent dye commonly used in this field, such as T AMRA carboxymethylrhoaamine, i'MR (tetramethylrhodamine), Aiexa Ri 47, Rnodamine green, Alexa488. However, it is not limited to these. When using FCS, FIDA, or fluorescence depolarization as a fluorescence analysis method as described later, only one type of fluorescent label may be used, but when two or more types of fluorescent labels are used (for each DNA molecule, One type of fluorescent molecule is added.) FCCS measurement is possible. DNA derived from organisms that meet the above conditions may be used, but artificially synthesized DNA is more stable than organisms and can be mass-produced at low cost. Suitable for detecting DNA-binding protein P in many types of test samples! Preparation of DNA having a desired base sequence and addition of a fluorescent label may be performed by any method for those skilled in the art. DNA is mass-produced all at once rather than being prepared each time the method of the present embodiment is performed, and then stored in an undenatured form so that it can be used in an amount necessary for performing the method of the present embodiment. It may be done.
[0024] 生体試料溶液 Aは、励起光及び蛍光の透過を著しく低下させないものであれば、 当業者に於いて使用されている任意の溶液であってよぐ例えば、血清、血漿等であ つてよい (ステップ 20、図 l (b) )。血清、血漿等の生体試料溶液の調製は、当業者に とって任意の方法にて、例えば、遠心分離、ろ過、超音波などの処理を経て為されて よい。光に対する試料の透過率が顕著に低い場合には、適宜、生理食塩水やリン酸 緩衝液により（内部の分子の活性を損なわなレ、ように）希釈されてよ!/、。  [0024] The biological sample solution A may be any solution used in the art as long as it does not significantly reduce the transmission of excitation light and fluorescence, for example, serum, plasma, etc. Good (step 20, figure l (b)). Preparation of a biological sample solution such as serum or plasma may be performed by a person skilled in the art by any method such as centrifugation, filtration, or ultrasonic treatment. If the transmittance of the sample to light is remarkably low, dilute with saline or phosphate buffer as appropriate (so as not to impair the activity of the internal molecules)!
[0025] 力、くして、調製された生体試料溶液 Aと DNA(D)を含む溶液とが混合される（ステ ップ 30)。この段階で、生体試料溶液 Aにタンパク質 Pが含まれていれば、 DNAとタ ンパク質 Pとが結合する（図 1 (c) )。しかしながら、タンパク質 Pが含まれていなくも、い ずれかの生体分子（Q)が DNA(D)又は蛍光標識 Fに非特異的に結合又は吸着す る場合も有り得る（図 1 (d) )。従って、もしこの段階で後述の蛍光測定を行ったとして も、 DNA(D)とタンパク質 Pとの複合体上の蛍光色素 Fと、 DNA (D)と生体分子（Q) とのの複合体上の蛍光色素 Fとで、その運動状態又は分布状態は、殆ど区別がつか ない場合がある。そこで、本発明に於いては、更に、生体試料溶液 Aと DNA(D)を 含む溶液との混合試料溶液へ、更に、タンパク質 Pに特異的に結合する抗体 Xが表 面に固相化されたビーズが添加される（ステップ 40、図 1 (e)、 （f) )。 [0025] Thus, the prepared biological sample solution A and the solution containing DNA (D) are mixed (step 30). At this stage, if protein P is contained in biological sample solution A, DNA and protein P bind (Fig. 1 (c)). However, even if protein P is not contained, any biomolecule (Q) may bind or adsorb nonspecifically to DNA (D) or fluorescent label F (Fig. 1 (d)). Therefore, even if the fluorescence measurement described later is performed at this stage, the fluorescent dye F on the complex of DNA (D) and protein P and the complex of DNA (D) and biomolecule (Q) With the fluorescent dye F, its movement state or distribution state is almost distinguishable. There may not be. Therefore, in the present invention, the antibody X that specifically binds to protein P is further immobilized on the surface of the mixed sample solution of the biological sample solution A and the solution containing DNA (D). Beads are added (step 40, Fig. 1 (e), (f)).
[0026] タンパク質 Pに特異的に結合する抗体 Xは、タンパク質 Pの一部をェピトープとする モノクローナル抗体であってよい。本発明の実験に於いては、タンパク質 Pは、 DNA に結合された状態となるので、好ましくは、抗体 Xのェピトープは、産生された抗体 X 力 Sタンパク質 Pと DNAとの結合を阻害せずにタンパク質 Pと結合するよう選択される。 そのような抗体の調製は、当業者にとって公知の任意の方法により調製されたもので あってよい。ビーズは、好ましくは、直径数 nm程度から lOOOnm未満のこの分野で 通常使用されているプラスチック、ラテックス、金コロイド、磁性粒子、ガラスなどからな る任意のものであってよい。調製された抗体 Xをビーズに固相化するために、 ELISA やァフィ二ティクロマトグラフィー等で担体に抗体を固定する手法が採用されてよい。 また、ビーズの表面に抗体と共有結合する官能基を修飾したものを用い、抗体 Xが確 実にビーズ表面へ固定されるようにされてもよい。なお、ビーズ上に抗体 Xが付加さ れた後、結合反応に関与しない任意のタンパク質 (スキムミルク、牛血清アルブミンな ど）をビーズ表面に吸着させ (ブロッキング）、更に生体分子等が吸着することを回避 できるようになって!/、ることが好まし!/、。 [0026] The antibody X that specifically binds to protein P may be a monoclonal antibody having a part of protein P as an epitope. In the experiment of the present invention, since protein P is in a state of being bound to DNA, preferably the antibody X epitope does not inhibit the binding of the produced antibody X force S protein P to DNA. Are selected to bind to protein P. Such antibodies may be prepared by any method known to those skilled in the art. The beads may be any of plastics, latex, gold colloids, magnetic particles, glass, etc., commonly used in this field, preferably in the order of a few nm in diameter and less than lOOOnm. In order to immobilize the prepared antibody X on the beads, a technique of immobilizing the antibody on a carrier by ELISA or affinity chromatography may be employed. Alternatively, antibody X may be fixed on the bead surface by using a functional group that is covalently bonded to the antibody on the bead surface. After antibody X is added to the beads, any protein that does not participate in the binding reaction (skimmed milk, bovine serum albumin, etc.) is adsorbed on the bead surface (blocking), and biomolecules are adsorbed. It ’s better to avoid it! /
[0027] 力、くして、タンパク質 Pに特異的に結合する抗体 Xが表面に固相化されたビーズが 混合溶液に添加されたとき、混合溶液中にタンパク質 Pが含まれていれば、ビーズ表 面上の抗体 Xに結合する。従って、その場合、タンパク質 Pに結合した蛍光色素 Fを 担持する DNA (D)もビーズ上に吸着することとなる（図 1 (e) )。他方、タンパク質 が 存在していなければ、仮に生体分子（Q)が DNA(D)に非特異的に吸着していたと しても、 DNAは、ビーズ上に吸着されない（図 l (f) )。この段階で、後述の蛍光分析 法により蛍光測定 ·解析を実行すると（ステップ 50)、図 1 (e)及び (f)の示されている 如き DNA及び蛍光色素 Fの状態の差が、蛍光分析法の測定'解析結果に於いて顕 著に表れることとなり、これにより、 DNAとタンパク質 Pとの特異的な結合の有無が検 出され、タンパク質 Pが生体試料中に存在するか否かが特定されることとなる。  [0027] When a bead having antibody X that specifically binds to protein P immobilized on its surface is added to the mixed solution, if the protein P is contained in the mixed solution, the beads Binds to antibody X on the surface. Therefore, in this case, DNA (D) carrying fluorescent dye F bound to protein P is also adsorbed on the beads (Fig. 1 (e)). On the other hand, if no protein is present, DNA will not be adsorbed on the beads even if biomolecules (Q) are adsorbed nonspecifically to DNA (D) (Fig. L (f)). At this stage, if fluorescence measurement / analysis is performed by the fluorescence analysis method described later (step 50), the difference in the state of DNA and fluorescent dye F as shown in Fig. 1 (e) and (f) is detected by fluorescence analysis. Method's analysis results, which are clearly shown in the analysis results. By this, the presence or absence of specific binding between DNA and protein P is detected, and whether or not protein P is present in the biological sample is identified. Will be.
[0028] 资光分析 上記の本発明の実施形態では、上記の如く調製された混合試料溶液に於ける DN Aとタンパク質 Pとの特異的な結合の有無の検出のために、レーザー共焦点顕微鏡 の光学系に超高感度光検出装置を組み合わせた蛍光測定装置を用いて、一分子 蛍光分析法により、混合試料溶液中の蛍光色素 Fの蛍光強度が計測され、解析され る。蛍光測定には、典型的には、 1分子蛍光分析システム MF20 (ォリンパス）が用 いられてよい。力、かるシステムに於いては、 FCS、 FIDA、 FDS、 FCCS力 S実行され、 蛍光色素 Fがビーズ上に固定されているか否かが判定される。以下、各分析方法に 於レ、て、蛍光色素 Fがビーズ上に固定されて!/、るか否かが計測結果に如何に反映さ れるかについて説明する。 [0028] Fluorescence analysis In the embodiment of the present invention described above, an optical system of a laser confocal microscope is used to detect the presence or absence of specific binding between DNA and protein P in the mixed sample solution prepared as described above. The fluorescence intensity of the fluorescent dye F in the mixed sample solution is measured and analyzed by single molecule fluorescence analysis using a fluorescence measurement device combined with a sensitive light detection device. Typically, a single molecule fluorescence analysis system MF20 (Olympus) may be used for fluorescence measurement. In the force system, FCS, FIDA, FDS, FCCS force S is executed to determine whether the fluorescent dye F is immobilized on the beads. The following explains how the measurement results reflect whether or not the fluorescent dye F is fixed on the beads in each analysis method.
[0029] FCS (蛍光相関分光法）では、微小の蛍光観察領域をブラウン運動により通過する 分子の移動（並進運動）の速さが観測される。分子の並進運動の速さは、測定された 蛍光強度の時間を変数とした自己相関関数の形状に反映される。分子の並進運動 の速さの指標としては、測定開始時から自己相関関数の値が半分になるまでの時間 の長さ（並進拡散時間）が用いられる。分子の移動は、分子の大きさが大きいほど、 遅くなるので、並進拡散時間が長くなる。本発明の場合、図 1 (e)の如ぐタンパク質 P に結合した DNAがビーズ上に固定されると、蛍光色素 Fがビーズに拘束され、従つ て、図 1 (f)の場合に比して、蛍光色素 Fの運動の速さが顕著に低減し、並進拡散時 間の長さが顕著に長くなり、タンパク質 Pに結合した DNAがビーズ上に固定された否 かが検出される。更に特記すべきことは、ビーズ上に複数の蛍光色素が拘束されて いる場合には、それらの色素が一体的に蛍光観察領域を通過するので、蛍光強度 が増大し、従って、蛍光色素一分子が通過するときに比して、蛍光強度（シグナル） の背景光（ノイズ）に対する比がよくなり、良好な S/N比にて蛍光強度の測定がなさ れることとなる。 [0029] In FCS (fluorescence correlation spectroscopy), the speed of movement (translational movement) of a molecule passing through a minute fluorescence observation region by Brownian motion is observed. The speed of the translational movement of the molecule is reflected in the shape of the autocorrelation function with the time of the measured fluorescence intensity as a variable. As an index of the speed of translation of molecules, the length of time (translational diffusion time) from the start of measurement until the autocorrelation function value is halved is used. Since the movement of molecules becomes slower as the size of the molecules increases, the translational diffusion time becomes longer. In the case of the present invention, when DNA bound to protein P as shown in FIG. 1 (e) is immobilized on the beads, fluorescent dye F is bound to the beads, and therefore, compared with the case of FIG. 1 (f). Thus, the speed of movement of the fluorescent dye F is remarkably reduced, the length of the translational diffusion time is remarkably increased, and whether or not the DNA bound to the protein P is immobilized on the beads is detected. Further, it should be noted that when a plurality of fluorescent dyes are constrained on the beads, the dyes pass through the fluorescence observation region as a whole, so that the fluorescence intensity increases. The ratio of fluorescence intensity (signal) to background light (noise) is better than when light passes, and fluorescence intensity is measured with a good S / N ratio.
[0030] FDS (蛍光偏光解消法）では、この分野に於いて知られている如ぐ分子の回転ブ ラウン運動（自転）の速さが観測される。分子の回転運動の速さは、測定された蛍光 の縦偏光と横偏光の強度の割合又は偏光度に反映される（1分子蛍光分析システム で実行される場合には、 FIDAに於いて蛍光を縦偏光及び横偏光に分けて検出し偏 光度が算出される。その場合、 FIDA— polと称する。）。分子の回転は、分子の大き さが大きいほど、遅くなるので、偏光度が大きくなる。本発明の場合、前記の FCSと同 様に、図 1 (e)の如ぐタンパク質 Pに結合した DNAがビーズ上に固定されると、蛍光 色素 Fがビーズに拘束され、従って、図 1 (f)の場合に比して、蛍光色素 Fの回転運 動の速さが顕著に低減し、偏光度が長くなり、タンパク質 Pに結合した DNAがビーズ 上に固定された否かが検出される。また、 1分子蛍光分析システムにて測定する場合 には、ビーズ上に複数の蛍光色素が拘束されているときには、それらの色素が一体 的に蛍光観察領域を通過するので、蛍光強度が増大し、測定の S/N比が向上する [0030] In FDS (fluorescence depolarization method), the speed of rotational browning (rotation) of molecules as known in this field is observed. The speed of the rotational movement of the molecule is reflected in the ratio of the intensity of the longitudinal and transverse polarization of the measured fluorescence or the degree of polarization (if performed in a single molecule fluorescence analysis system, the fluorescence is measured in FIDA. Detection is performed separately for longitudinally polarized light and laterally polarized light, and the degree of polarization is calculated (in this case, called FIDA-pol). Molecular rotation is the size of the molecule The larger the thickness is, the slower it is, and the degree of polarization increases. In the case of the present invention, as in the FCS described above, when DNA bound to protein P as shown in FIG. 1 (e) is immobilized on the beads, the fluorescent dye F is bound to the beads, and accordingly, FIG. Compared to the case of f), the rotational speed of the fluorescent dye F is significantly reduced, the degree of polarization is increased, and whether or not the DNA bound to the protein P is immobilized on the beads is detected. . When measuring with a single-molecule fluorescence analysis system, when multiple fluorescent dyes are constrained on the beads, the dyes pass through the fluorescence observation region as a whole, and the fluorescence intensity increases. Improved S / N ratio of measurement
[0031] FIDA (蛍光強度分布解析法）では、微小の蛍光観察領域内から発せられる光子 の検出（フオトンカウンティング)を行い、単位時間当たりの光子が検出された頻度を 統計的に処理することによって、微小の蛍光観察領域内の蛍光粒子の数密度と、一 蛍光粒子当たりの蛍光強度が算出される。本発明の場合、タンパク質 Pに結合した D NAがビーズ上に固定されると、蛍光色素 Fがビーズに拘束され一つの蛍光粒子とし て運動することになるので、蛍光粒子の数密度が低減する一方、ビーズに複数の蛍 光色素分子 Fが存在することにより一蛍光粒子当たりの蛍光強度が増大する。従って 、蛍光粒子の数密度の低減と一蛍光粒子当たりの蛍光強度の増大からタンパク質 P に結合した DNAがビーズ上に固定されたことが検出される。また、一蛍光粒子当たり の蛍光強度が増大すると、一時に計測される蛍光強度 (検出光子数)が増大し、測定 の S/N比が向上する。 [0031] In FIDA (fluorescence intensity distribution analysis method), photons emitted from a minute fluorescence observation region are detected (photon counting), and the frequency of detection of photons per unit time is statistically processed. Thus, the number density of fluorescent particles in the minute fluorescent observation region and the fluorescent intensity per fluorescent particle are calculated. In the case of the present invention, when the DNA bound to protein P is immobilized on the beads, the fluorescent dye F is bound to the beads and moves as one fluorescent particle, so that the number density of the fluorescent particles is reduced. On the other hand, the presence of a plurality of fluorescent dye molecules F on the beads increases the fluorescence intensity per fluorescent particle. Therefore, it is detected that the DNA bound to the protein P is immobilized on the beads from the decrease in the number density of the fluorescent particles and the increase in the fluorescence intensity per fluorescent particle. In addition, when the fluorescence intensity per fluorescent particle increases, the fluorescence intensity (number of detected photons) measured at a time increases and the S / N ratio of the measurement improves.
[0032] FCCS (蛍光相互相関分光法）では、二つの発光波長の異なる蛍光標識が微小の 蛍光観察領域をブラウン運動により通過する際に、各々の標識の蛍光強度の変化か ら二つの蛍光標識の運動に相関があるか否かを判定することができる。もし蛍光標識 がーつの担体（分子）に存在する場合には、二つの蛍光強度の変化が一体的に変 化するが、蛍光標識が別々の担体に存在する場合には、二つの蛍光強度の変化は 、独立に変化することとなる。蛍光標識が一つの担体に乗っているか否かは、二つの 蛍光強度の相互相関関数から判定することができる。本発明の場合、互いに異なる 蛍光色素にて標識された DNAを用いれば、別々の蛍光色素を担持する DNAがタ ンパク質 Pを介してビーズ上に固定されることにより、二つの色素からの蛍光色素に 相関があることが検出され、各 DNAが自由に運動している場合（ビーズに固定され ていない場合）に比して、相互相関関数が高くなるので、タンパク質 Pに結合した DN Aがビーズ上に固定されたか否かが検出される。また、前記の蛍光分析法と同様に、 一つのビーズ力 複数の色素による蛍光が発せられることにより、一時に計測される 蛍光強度が増大し、測定の S/N比が向上する。 [0032] In FCCS (fluorescence cross-correlation spectroscopy), when two fluorescent labels with different emission wavelengths pass through a small fluorescence observation region by Brownian motion, two fluorescent labels are detected from the change in fluorescence intensity of each label. It can be determined whether or not there is a correlation between the movements. If the fluorescent label is present on one carrier (molecule), the change in the two fluorescent intensities changes together, but if the fluorescent label is present on a separate carrier, the two fluorescent intensities Change will change independently. Whether or not the fluorescent label is on one carrier can be determined from the cross-correlation function of the two fluorescent intensities. In the case of the present invention, if DNAs labeled with different fluorescent dyes are used, DNAs carrying different fluorescent dyes are immobilized on the beads via the protein P, so that the fluorescence from the two dyes can be obtained. To pigment Since the cross-correlation function is higher than when the correlation is detected and each DNA is moving freely (not immobilized on the beads), the DN A bound to the protein P is present on the beads. It is detected whether it is fixed to. In addition, as with the fluorescence analysis method described above, one bead force Fluorescence is emitted by a plurality of dyes, thereby increasing the fluorescence intensity measured at a time and improving the S / N ratio of the measurement.
[0033] 上記の一連の蛍光分析法に於!/、て、特記されるべきことは、一つの結果を検出す るために要する試料量は、数十 1程度でよぐまた、測定時間は、 5— 15秒程度の 測定を数回繰り返す程度よぐ従って、従前の生化学的な手法に比べ、試料量と時 間を大幅に低減することができる。しかも、従前の一分子蛍光分析の手法に比較す ると、測定される蛍光強度が数倍程度大きくなるので、 S/N比が向上し、試料に夾 雑物が在っても、特異的な結合反応のみを良好に検出できることとなる。  [0033] In the above-described series of fluorescence analysis methods, it should be noted that the amount of sample required to detect one result is about several tens of thousands, and the measurement time is Therefore, the sample amount and time can be greatly reduced compared to the conventional biochemical method. In addition, compared to the conventional single molecule fluorescence analysis method, the measured fluorescence intensity is several times larger, so the S / N ratio is improved, and even if there are contaminants in the sample, it is specific. Only a good binding reaction can be detected satisfactorily.
[0034] ところで、上記の方法に於レ、て、ビーズを添加した段階でのみ蛍光測定'分析を実 行しても、その結果の数値から蛍光色素がビーズ上にあるか否かを推定することがで きる力 図 2 (B)に例示されている如ぐ蛍光標識された DNAのみの段階 (ステップ 1 0)、生体試料溶液と DNA溶液を混合した状態 (ステップ 30)で、比較のために蛍光 測定 ·解析を実行してもよい（ステップ 15、 35)。ステップ 35の段階で、ステップ 15の 場合と蛍光測定'解析の結果に有意な差、即ち、特異'非特異によらず、第一の分子 が関与する結合反応の発生が認められないと考える場合には、ビーズの添加及びそ の後の蛍光測定を実施しなくてもよいであろう。この点に関し、ステップ 35とステップ 5 0とで実行する蛍光測定の方法は、別々であってもよい。例えば、ステップ 30に於い ては、 FCSを実行し、ステップ 50では、ステップ 35の FCSの変化を裏付ける目的で FIDAを実行するようにしてもよい。 （FCSの場合、添加されるビーズ径が大き過ぎる 場合、並進ブラウン運動が小さくなり、測定時間中に殆ど移動しないといったことが起 き得る。他方、 FIDAの場合、ビーズを添加しない場合には、蛍光色素を有する DN Aは、 DNA結合タンパク質に一対一に結合するのみなので、蛍光粒子数は変化せ ず、結合により色素の蛍光強度が何らかの理由で変化しない限り、 FIDAの結果は 変化しない。）。ステップ 35とステップ 50とで別々の蛍光分析法を用いる場合、比較 のためには、ステップ 15に於いて両方の分析法による測定を行っておくことが望まし い。 [0034] By the way, in the above method, even if the fluorescence measurement 'analysis is performed only at the stage where the beads are added, it is estimated whether the fluorescent dye is on the beads from the numerical value of the result. Forces that can be usedFor comparison, in the stage of only fluorescently labeled DNA as shown in Figure 2 (B) (Step 10), the biological sample solution and the DNA solution are mixed (Step 30). Fluorescence measurement / analysis may be performed at steps 15 and 35. In step 35, if there is a significant difference between the results of fluorescence measurement and analysis in step 35, i.e., no specific or non-specific binding reaction involving the first molecule is observed. In some cases, the addition of beads and subsequent fluorescence measurements may not be performed. In this regard, the fluorescence measurement methods executed in step 35 and step 50 may be different. For example, FCS may be executed in step 30, and FIDA may be executed in step 50 for the purpose of supporting the change in FCS in step 35. (In the case of FCS, if the added bead diameter is too large, the translational Brownian motion will be small and may hardly move during the measurement time. On the other hand, in the case of FIDA, if no beads are added, (DNA with a fluorescent dye only binds one-to-one with the DNA-binding protein, so the number of fluorescent particles does not change, and the FIDA results do not change unless the fluorescence intensity of the dye changes for some reason.) . If separate fluorescence analysis methods are used in step 35 and step 50, it is recommended that measurements be made by both methods in step 15 for comparison. Yes.
[0035] その他の実施形態について  [0035] Other embodiments
上記の手順は、生体試料に含まれる DNA結合タンパク質 Pに特異的に結合する D NAの塩基配列を検出するために、換言すれば、 DNA結合タンパク質 Pが或る塩基 配列の DNAが結合するか否かを検出するために用いることもできる。その場合、 DN A結合タンパク質 Pに結合すると思われる塩基配列を有する DNAを蛍光標識したも のが用いられる。 DNAがビーズ上に固定されたことが検出されれば、その DNAの塩 基配列に DNA結合タンパク質 Pと特異的に結合する部位が存在することが特定され  In order to detect the base sequence of DNA that specifically binds to DNA binding protein P contained in a biological sample, in other words, whether DNA binding protein P binds to DNA of a certain base sequence. It can also be used to detect whether or not. In this case, a fluorescently labeled DNA having a base sequence that is thought to bind to DNA binding protein P is used. If it is detected that DNA is immobilized on the beads, it is identified that there is a site that specifically binds to DNA binding protein P in the DNA base sequence.
[0036] また、上記の方法に於!/、て、蛍光標識される第一の分子、第二の分子は、任意の D NA、タンパク質、その他の生体分子であってよいことは理解されるべきである。例え ば、蛍光標識される第一の分子を生体試料中に含まれるか否かが検出される分子の 抗体とし、第三の分子をその検出対象の分子の別の部分のェピトープに対する抗体 としてもよい。蛍光標識された抗体がビーズ上に固定されれば、生体試料中に第一 及び第三の分子である抗体に対する抗原が存在することが検出される。 [0036] In the above method, it is understood that the first molecule and the second molecule to be fluorescently labeled may be any DNA, protein, and other biomolecules. Should. For example, the first molecule to be fluorescently labeled may be an antibody of a molecule that is detected whether it is contained in a biological sample, and the third molecule may be an antibody to an epitope of another part of the molecule to be detected. Good. When the fluorescently labeled antibody is immobilized on the beads, the presence of an antigen against the antibody as the first and third molecules in the biological sample is detected.
[0037] 本発明の方法は、試料中に夾雑物が存在していても、有利に特定の分子の特異的 結合を非特異的な結合と見分けた態様にて検出可能なので、病気の臨床診断、生 理活性物質のスクリーニングのツールとして利用可能である。生体試料中の任意の 分子を特異的に化学的又は遺伝子工学的に (GFPなど蛍光タンパク質の発現により )蛍光標識することが可能であれば、第一の分子を含む第一の試料が生体試料であ つてもよく、また、第二の試料が、任意の態様にて調製された又は物質であってもよ いことは理解されるべきであり、そのような場合も本発明の範囲に属する。  [0037] The method of the present invention can detect a specific binding of a specific molecule in a manner that distinguishes it from a non-specific binding, even if contaminants are present in the sample. It can be used as a screening tool for biologically active substances. If any molecule in a biological sample can be chemically or genetically engineered (by the expression of a fluorescent protein such as GFP), the first sample containing the first molecule is the biological sample. It should be understood that the second sample may be prepared or material in any manner and such cases are also within the scope of the present invention. .

Claims

請求の範囲 The scope of the claims
[1] 特定の分子の特異的結合を検出する方法であって、  [1] A method for detecting specific binding of a specific molecule comprising:
蛍光標識された第一の分子を含む第一の試料と、前記蛍光標識された第一の分 子と特異的に結合するか否かが判定される第二の分子を含む第二の試料とを混合し 混合試料溶液を調製する過程と、  A first sample containing a fluorescently labeled first molecule; a second sample containing a second molecule to be determined whether or not it specifically binds to the fluorescently labeled first molecule; Mixing and preparing a mixed sample solution;
前記第二の分子に特異的に結合する第三の分子が表面に固相化された外径 1 m以下の粒子を前記第一及び第二の試料の混合試料溶液へ添加する過程と、 前記粒子が添加された前記混合試料溶液中の前記第一の分子の蛍光標識の蛍 光強度を測定する過程と、  Adding a particle having an outer diameter of 1 m or less in which a third molecule that specifically binds to the second molecule is immobilized on the surface thereof, to the mixed sample solution of the first and second samples; Measuring the fluorescence intensity of the fluorescent label of the first molecule in the mixed sample solution to which particles are added;
前記蛍光強度に基づいて、前記第一の分子が前記粒子上に固定されているか否 かを判定する過程と  Determining whether the first molecule is immobilized on the particle based on the fluorescence intensity;
を含み、前記第一の分子が前記粒子上に固定されていると判定された場合には、前 記第一の分子が前記第二の分子に特異的に結合したと判定することを特徴とする方 法。  And the first molecule is determined to be specifically bound to the second molecule when it is determined that the first molecule is immobilized on the particle. how to.
[2] 特定の分子の特異的結合を検出する方法であって、  [2] A method for detecting specific binding of a specific molecule,
蛍光標識された第一の分子を含む第一の試料と、前記蛍光標識された第一の分 子と特異的に結合する第二の分子が存在するか否かが判定される第二の試料とを 混合し混合試料溶液を調製する過程と、  A first sample containing a fluorescently labeled first molecule and a second sample to determine whether there is a second molecule that specifically binds to the fluorescently labeled first molecule A process of preparing a mixed sample solution by mixing
前記第二の分子に特異的に結合する第三の分子が表面に固相化された外径 1 m以下の粒子を前記第一及び第二の試料の混合試料溶液へ添加する過程と、 前記粒子が添加された前記混合試料溶液中の前記第一の分子の蛍光標識の蛍 光強度を測定する過程と、  Adding a particle having an outer diameter of 1 m or less in which a third molecule that specifically binds to the second molecule is immobilized on the surface thereof, to the mixed sample solution of the first and second samples; Measuring the fluorescence intensity of the fluorescent label of the first molecule in the mixed sample solution to which particles are added;
前記蛍光強度に基づいて、前記第一の分子が前記粒子上に固定されているか否 かを判定する過程と  Determining whether the first molecule is immobilized on the particle based on the fluorescence intensity;
を含み、前記第一の分子が前記粒子上に固定されていると判定された場合には、前 記第二の分子が前記第二の試料に存在していると判定することを特徴とする方法。  And it is determined that the second molecule is present in the second sample when it is determined that the first molecule is immobilized on the particle. Method.
[3] 請求項 1乃至 2の方法であって、前記蛍光強度の測定が蛍光相関分光法、蛍光偏 光解消法、蛍光強度分布解析法又は蛍光相互相関分光法により行われることを特 徴とする方法。 [3] The method according to claim 1 or 2, wherein the fluorescence intensity is measured by fluorescence correlation spectroscopy, fluorescence depolarization, fluorescence intensity distribution analysis, or fluorescence cross correlation spectroscopy. How to make a sign.
[4] 請求項 1乃至 3の方法であって、前記第二の試料が血清又は血漿又は生体から得 られた溶液試料であることを特徴とする方法。  4. The method according to claim 1, wherein the second sample is serum, plasma, or a solution sample obtained from a living body.
[5] 請求項 1乃至 4の方法であって、前記粒子の表面が前記第二の分子と前記第三の 分子との特異的結合を除く前記混合溶液中に存在する分子の吸着又は結合を防止 するための吸着防止処理が施されていることを特徴とする方法。 [5] The method according to any one of claims 1 to 4, wherein the particle surface adsorbs or binds molecules present in the mixed solution excluding specific binding between the second molecule and the third molecule. A method characterized by being provided with an anti-adsorption treatment to prevent it.
[6] 請求項 1乃至 5の方法であって、前記粒子が、プラスチック、ラテックス、金コロイド、 磁性粒子及びガラスから成る群から選択された少なくとも一つの材料からなるビーズ であることを特徴とする方法。 6. The method according to claim 1, wherein the particles are beads made of at least one material selected from the group consisting of plastic, latex, colloidal gold, magnetic particles, and glass. Method.
[7] 請求項 1乃至 6の方法であって、前記混合試料溶液へ前記粒子を添加するのに先 立って測定された前記混合試料溶液中の前記第一の分子の蛍光標識の蛍光強度 に基づいて、前記第一の分子が前記第二の試料中の分子と結合したか否力、を判定 する過程を含むことを特徴とする方法。 [7] The method according to any one of claims 1 to 6, wherein the fluorescence intensity of the fluorescent label of the first molecule in the mixed sample solution measured prior to adding the particles to the mixed sample solution. And determining whether or not the first molecule is bound to the molecule in the second sample.
[8] 請求項 1乃至 7の方法であって、前記第一の分子が DNAであり、前記第二の分子 力 ¾ΝΑ結合タンパク質であり、前記第三の分子が前記第二の分子に対する抗体で あることを特徴とする方法。 [8] The method according to any one of claims 1 to 7, wherein the first molecule is DNA, the second molecular force is a binding protein, and the third molecule is an antibody against the second molecule. A method characterized by being.
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