JP2010019553A - Specific bonding riaction detecting method of molecule by monomolecular fluorometric analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect and observe the specific bonding of a molecule using a monomolecular fluorometric analyzing technique without being affected by the background light or nonspecific reaction caused by the presence of impurities even in the presence of impurities other than a detection target molecule. <P>SOLUTION: Beads containing the third molecule specifically bonded to a first molecule in a solid-phase state are added to a mixed sample solution of a first sample containing a first molecule DNA labelled by fluorescence and a second sample containing a second molecule P determined whether to be specifically bonded to the first molecule or the second sample wherein it is determined whether the second molecule specifically bonded to the first molecule is present and it is determined whether the first molecule is fixed on the beads on the basis of the fluorescent intensity of the fluorescent label of the first molecule in the mixed sample solution. In the case where the first molecule is fixed on the beads, the first molecule is determined to be specifically bonded to the second molecule (or the second molecule is determined to be present in the second sample). <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  In the present invention, specific binding of proteins, peptides, nucleic acids, lipids, amino acids and other physiologically active substances or biomolecules (hereinafter referred to as “biomolecules”) is measured by measuring fluorescence from a single fluorescent molecule. The present invention relates to a method for detecting a reaction, and more particularly, to a detection method capable of distinguishing a specific binding reaction and a non-specific binding reaction between various biomolecules.

  With the development of optical measurement technology such as recent technology, at present, fluorescence correlation spectroscopy (FCS) for measuring and analyzing fluorescence from a single fluorescent molecule, and fluorescence-intensity distribution analysis (FIDA) ) Fluorescence analysis methods can be used. In these fluorescence analysis methods (single molecule fluorescence analysis technology) that measure fluorescence at the single molecule level, the optical system of a laser confocal microscope and photon counting (one-photon detection) are possible. The fluorescence intensity from a single fluorescent molecule is measured using a device, and fluctuations in the intensity are analyzed by various methods to determine the speed of movement of the fluorescent molecule or fluorescently labeled molecule or the size of the molecule (FCS ), Number density of molecules (or particles) or fluorescence intensity per molecule (in the case of FIDA). Therefore, based on such information, various phenomena such as a change in the structure or size of the molecule, a binding / dissociation reaction or dispersion / aggregation of the molecule can be detected.

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 at the molecular level. For example, at least one of a pair of molecules that interact specifically with each other (DNA and DNA-binding protein, antigen and antibody, etc.) is fluorescently labeled with a fluorescent molecule, and then these molecules are reacted. Then, the movement or state change of the fluorescent molecule on one molecule is reflected in the fluorescence intensity from the fluorescent molecule or the fluctuation thereof, whereby the interaction between molecules such as protein or DNA can be detected. Examples in which detection of binding reaction between protein and nucleic acid or antigen-antibody reaction at the molecular level using such single molecule fluorescence analysis technology has already been made (for example, Patent Document 1-2, Non-Patent Document). Literature 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, so in various fields such as medicine and pharmacology, Applications in clinical diagnosis and schooling of bioactive substances are also expected.
JP 2003-275000 A JP 2005-337805 A 3 outside casks, PNAS 96, 24, 13756-13761, November 23, 1999 Three people outside Kobayashi, Analytical Biochemistry, 2004, 332, 58-66

  The performance of the optical system and the photodetection device used in the above-described single molecule fluorescence analysis technology is certainly improved so that it is possible to detect and measure the amount of fluorescence from a single fluorescent molecule or about one photon. However, the light to be detected is still weak, so if the background light is high, the light to be detected is buried in the background and cannot be identified. In particular, biological samples or biological samples (serum, plasma, etc.) that are used for observation of interactions such as biomolecules or clinical diagnosis of diseases generally have various configurations that are not related to the reaction to be detected. There are many objects or impurities, and the background light becomes high due to autofluorescence from many molecules in addition to the molecules to be observed, which reduces the accuracy or reliability of the measurement results. It will be. In addition, when a specific binding reaction such as a biomolecule is to be detected, a biological sample contains a molecule that undergoes a reaction other than the specific reaction to be detected or a nonspecific binding or adsorption. In many cases, if a reaction other than the reaction to be detected or nonspecific binding / adsorption affects the measurement result and the analysis result, an error occurs and the reliability of the result is lowered.

  The influence of the constituents or contaminants other than the molecule to be observed in the sample of single molecule fluorescence analysis as described above can be eliminated by removing those impurities from the sample or purifying the molecule to be observed. it can. 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 single molecule fluorescence that can be measured in a short amount of time. The advantage of 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.

  Thus, one main subject of the present invention is the accuracy and reliability of detection and observation results in the detection and observation of molecular interactions, particularly the specific binding of molecules, using single molecule fluorescence analysis technology. Without removing contaminants other than the molecules to be detected contained in the sample that cause damage to the sample, the effects of background light and non-specific reactions due to the presence of these contaminants can be eliminated or relative It is to be able to reduce it.

  Another object of the present invention is to detect and observe the molecular interaction using the single-molecule fluorescence analysis technique as described above. In particular, the sample is a biological sample or a biological sample (serum, plasma, etc.). ) Is used, the influence 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.

  According to the present invention, specific binding of a specific molecule is detected with high accuracy or reliability in a sample containing a large amount of contaminants, particularly 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 is provided.

  According to one aspect of the present invention, a method for detecting specific binding of a particular molecule comprises a first sample comprising a first fluorescently labeled molecule, the first fluorescently labeled molecule, A process of preparing a mixed sample solution by mixing a second sample containing a second molecule to be specifically determined whether or not to bind, and a third molecule that specifically binds to the second molecule A process of adding particles having an outer diameter of 1 μm or less solid-phased on the surface to the mixed sample solution, a process of measuring the fluorescence intensity of the fluorescent label of the first molecule in the mixed sample solution to which the particles are added, and Determining whether the first molecule is immobilized on the particle based on the fluorescence intensity, and if it is determined that the first molecule is immobilized on the particle, It is determined that one molecule is specifically bound to a second molecule. That is, in this embodiment, it can be determined whether or not the first molecule specifically binds to the second molecule.

  When determining whether or not a specific molecule (for example, the first molecule) and a certain other molecule (the second molecule) have specific binding using single molecule fluorescence analysis technology, In the method proposed so far (in the prior art), a first sample containing a fluorescently labeled first molecule and a second sample containing a second molecule are mixed together, The mixed sample was subjected to fluorescence measurement and analysis of the results. 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 / adsorption between the contaminants and the fluorescently labeled molecules, It is indistinguishable from specific binding.

  Therefore, in the present invention, as described above, the third molecule that specifically binds to the second molecule is on the surface of the mixed sample solution containing the first molecule and the second molecule that are fluorescently labeled. The process of the previous method is modified so that particles having an outer diameter of 1 μm or less that are solid-phased 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, and as a result, on the surface of the particle. The second molecule will be collected. If the second molecule specifically binds 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 Is reflected on the state of being fixed on particles having an outer diameter of 1 μm or less. On the other hand, if the second molecule does not specifically bind to the first molecule, the fluorescence intensity from the fluorescent label on the first molecule, even if it is non-specifically bound to a contaminant. Should not be significantly different from the free state of the first molecule (not bound on the particle). In addition, when the fluorescent label is fixed on a particle having an outer diameter of 1 μm 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 in the measurement region is also limited. Since the fluorescence label is collected on the particle, fluorescence is emitted from a plurality or many of the fluorescence labels on the particle, so that the fluorescence intensity observed at one time also increases, S / N is greatly improved. Thus, as described above, the configuration of the method is modified to determine whether the first molecule is “fixed” on the particle through the specific binding of the second and third molecules. Thus, the case where the first molecule specifically binds to the second molecule and the case where it does not can be detected at a higher S / N ratio.

  Particles having an outer diameter of 1 μm or less in which a third molecule that specifically binds to the second molecule is immobilized on the surface are added to a mixed sample solution, and fluorescence-labeled The technique of determining whether one 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 fluorescently labeled first molecule and a fluorescently labeled first molecule. A process of preparing a mixed sample solution by mixing a second sample to determine whether there is a second molecule that specifically binds to one molecule, and specifically binding to the second molecule Adding a particle having an outer diameter of 1 μm or less having a third molecule immobilized on the surface thereof to the mixed sample solution of the first and second samples, and a first sample in the mixed sample solution to which the particles are added The process includes measuring the fluorescence intensity of the fluorescent label of the molecule and determining whether the first molecule is immobilized on the particle based on the fluorescence intensity, and the first molecule is immobilized on the particle. The second molecule is present in the second sample. To. According to this configuration, whether or not a certain molecule (a molecule that specifically binds to the first molecule and the third molecule) is contained in an arbitrary sample (second sample) Similarly, it becomes possible to detect at a higher S / N ratio than before.

  Fluorescence measurement and analysis (or analysis) in the above-described method for detecting specific binding of the molecule of the present invention is performed by laser confocal microscopy such as FCS, FIDA, or fluorescence cross-correlation spectroscopy (FCCS). Any one of the single-molecule fluorescence analysis methods performed by a fluorescence measuring device in which an ultrasensitive photodetection device is combined with the above optical system may be used. In addition, any fluorescence analysis method capable of observing changes in the structure, state, and motion of other molecules, such as fluorescence depolarization spectroscopy (FDS), may be used (each of which is fluorescent according to the configuration of the present invention). How the result of fluorescence measurement / analysis in the analysis method will be described in the description of the embodiment). It should be understood that any fluorescence analysis method that can detect whether or not the first molecule is immobilized on a particle may be used, and such a case is also within the scope of the present invention.

  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, but contain the second molecule. The second sample may contain contaminants, and thus serum or plasma used in research in the fields of biological science, medicine or pharmacology, clinical diagnosis of diseases or screening of bioactive substances, etc. It may be a solution sample obtained from another living body. As will be understood from the above description of the embodiments of the present invention, when the second sample is a solution sample obtained from a living body, whether or not the second molecule is contained in the biological sample or 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-binding protein against DNA having a certain base sequence Is detected in a sample (eg, serum or plasma) that contains a DNA binding protein that specifically binds to DNA having a certain base sequence. The

  In the above configuration, the particle on which the third molecule is immobilized on the surface may be a nanometer-order bead commonly used in experiments in the field of biological science, medicine, or pharmacy, It may be a bead made of at least one material selected from the group consisting of plastic, latex, colloidal gold, magnetic particles and glass. The method of immobilizing, ie immobilizing, the third molecule on the surface of the particle may be any method commonly known in the field of biological science, medicine or pharmacology, and will be understood by those skilled in the art. 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 particles is subjected to an anti-adsorption treatment for preventing adsorption or binding of molecules present in the mixed sample solution excluding specific binding between the second molecule and the third molecule. Such treatment may be any method known in this field (for example, a protein that does not contribute to the reaction (such as bovine serum albumin) is immobilized on the particle and then adsorbed to the surface). .

  In the above-described configuration of the present invention, in order to compare with the result of fluorescence measurement / analysis after adding particles to the mixed sample solution, the particles in the mixed sample solution are added prior to the addition of the particles to the mixed sample solution. Measuring the fluorescence intensity of the fluorescent label of the first molecule and determining whether the first molecule is bound to the molecule in the second sample based on the measured fluorescence intensity. . The method of fluorescence analysis performed before and after the addition of the particles may be the same or different. That is, before adding the particles, fluorescence measurement is performed by FCS to confirm the presence or absence of binding between the first and second molecules, and after adding the particles, the relationship between the particles and the first molecules is confirmed by FIDA. Measurement for this purpose 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 results, the first molecule was completely applied to the second sample. If there is no change in the fluorescence measurement result (when it is recognized that there is almost no possibility of the first molecule binding to the second molecule), the addition of particles may be omitted (sample) And time savings).

  Conventionally, detection of specific binding between biomolecules in a biological sample containing a large number of contaminants has been performed in a large amount or a high amount such as gel filtration chromatography, protein / DNA electrophoresis, Western blotting, and ELISA. A biochemical method requiring a sample amount of concentration and time was required. However, according to the present invention, specific binding between biomolecules can be detected easily and quickly without requiring a high concentration or a large amount of sample. As understood from the above description, there is a certain limit (for example, the excitation light is reduced) by specifically collecting the second molecule by using the particles in which the third molecule is immobilized. If there is a contaminant in the sample to be measured, the presence or absence of specific binding between the second molecule and the first molecule (fluorescently labeled) can be detected. Therefore, application in clinical diagnosis of various diseases and schooling of bioactive substances is also expected.

  It should be noted that according to the configuration in which the second molecule is specifically collected using the particles in which the third molecule is solid-phased, the second molecule is specific to the first molecule. When bound, the fluorescent label is collected on the particle, resulting in a fluorescence that is obtained at a time compared to when the first molecule is alone or bound only to another second molecule. The amount of fluorescent light from the label is increased, and this improves the ratio between the fluorescence desired to be detected and the fluorescence not desired, that is, the S / N ratio of the measurement. In addition, when the second molecule specifically binds 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 can also be regarded as a significant change. it can. In general, the presence or absence of specific binding of molecules can be detected remarkably accurately as compared with the conventional method using single-molecule fluorescence analysis.

  Other objects and advantages of the present invention will become apparent from the following description of preferred embodiments of the present invention.

  The present invention will now be described in detail with reference to a few preferred embodiments with reference to the accompanying drawings.

Procedure for Preparation of Measurement Sample FIG. 1 shows how to detect whether or not a DNA binding protein P that specifically binds to DNA having a certain base sequence exists in a biological sample solution A. Using labeled DNA (first molecule) and beads on which a monoclonal antibody (third molecule) having a part of DNA-binding protein P (second molecule) as an epitope is immobilized on the surface, FIG. 2A schematically shows a sample preparation operation and a change in molecular state when the method of the invention is applied, and FIG. 2A shows a measurement procedure in the form of a flowchart. is there.

Referring to FIGS. 1 and 2, before starting the method of the present invention, fluorescently labeled DNA (D) having a base sequence that specifically binds to DNA binding protein P is prepared (step). 10, FIG. 1 (a)). The length of the DNA is preferably 10 to 100 mer, more preferably 20 to 40 mer, from the cost of synthesis and ease of detection. As fluorescent dye F added to DNA for fluorescent labeling, any fluorescent dye usually used in this field, for example, TAMRA (carboxymethylrhodamine), TMR (tetramethylrhodamine), Alexa647, Rhodamine
It may be Green, Alexa488, etc., but is not limited thereto. As will be described later, when FCS, FIDA, or fluorescence depolarization is used as the fluorescence analysis method, only one type of fluorescent label may be used, but when two or more types of fluorescent labels are used (one for each DNA molecule). A kind of fluorescent molecule is added), and measurement by FCCS becomes possible. DNA derived from organisms that satisfy the above conditions may be used, but artificially synthesized DNA is more stable than organisms and can be mass-produced at low cost. It is suitable for detecting the DNA binding protein P in many kinds of test samples. Preparation of DNA having a desired base sequence and addition of a fluorescent label may be performed by a person skilled in the art by any method. Rather than preparing DNA each time the method of this embodiment is performed, the DNA is mass-produced at a time and then stored in a non-denaturing manner so that it can be used in the required amount when performing the method of this embodiment. May be.

  The biological sample solution A may be any solution used by those skilled in the art as long as it does not significantly reduce the transmission of excitation light and fluorescence, and may be, for example, serum, plasma, etc. (step 20, FIG. 1 (b)). Preparation of biological sample solutions such as serum and plasma may be performed by any method for those skilled in the art, for example, through treatment such as centrifugation, filtration, and ultrasonic waves. When the transmittance of the sample with respect to light is remarkably low, it may be appropriately diluted with physiological saline or a phosphate buffer (so as not to impair the activity of the internal molecules).

  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 are bound (FIG. 1 (c)). However, even if the protein P is not contained, any biomolecule (Q) may bind or adsorb nonspecifically to the DNA (D) or the 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) In some cases, the movement state or distribution state of the fluorescent dye F is almost indistinguishable. Therefore, in the present invention, the antibody X that specifically binds to the 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, FIGS. 1 (e), (f)).

  The antibody X that specifically binds to the protein P may be a monoclonal antibody having a part of the 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 epitope of antibody X is such that the produced antibody X does not inhibit the binding between protein P and DNA. Selected to bind to protein P. Such an antibody may be prepared by any method known to those skilled in the art. The beads may be any of plastic, latex, colloidal gold, magnetic particles, glass and the like that are commonly used in this field, preferably having a diameter of about several nm to less than 1000 nm. In order to immobilize the prepared antibody X on the beads, a method of immobilizing the antibody on a carrier by ELISA, affinity chromatography or the like may be employed. Alternatively, the antibody X may be reliably fixed to the bead surface by using a functional group that is covalently bonded to the antibody on the bead surface. In addition, after the antibody X is added to the beads, any protein (skimmed milk, bovine serum albumin, etc.) that is not involved in the binding reaction can be adsorbed (blocking) on the bead surface, and further adsorption of biomolecules can be avoided. It is preferable that it is such.

  Thus, when a bead having antibody X that specifically binds to protein P immobilized on its surface is added to the mixed solution, if protein P is contained in the mixed solution, antibody X on the bead surface will Join. Therefore, in that case, DNA (D) carrying the fluorescent dye F bound to the protein P is also adsorbed on the beads (FIG. 1 (e)). On the other hand, if the protein P does not exist, even if the biomolecule (Q) is adsorbed nonspecifically to the DNA (D), the DNA is not adsorbed on the beads (FIG. 1 (f)). At this stage, when fluorescence measurement / analysis is executed by the fluorescence analysis method described later (step 50), the difference in the state of the DNA and the fluorescent dye F as shown in FIGS. It will appear prominently in the measurement and analysis results of the method, thereby detecting the presence or absence of specific binding between DNA and protein P and specifying whether protein P is present in the biological sample. It will be.

Fluorescence analysis In the above-described embodiment of the present invention, in order to detect the presence or absence of specific binding between DNA and protein P in the mixed sample solution prepared as described above, an optical system of a laser confocal microscope is used. 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 high-sensitivity photodetector. For the fluorescence measurement, typically, a single molecule fluorescence analysis system MF20 (Olympus) may be used. In such a system, FCS, FIDA, FDS, and FCCS are executed to determine whether or not the fluorescent dye F is immobilized on the beads. Hereinafter, it will be described how the measurement results reflect whether or not the fluorescent dye F is immobilized on the beads in each analysis method.

  In FCS (fluorescence correlation spectroscopy), the speed of movement (translational movement) of molecules 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 translational speed of the molecule, 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 the molecule becomes slower as the size of the molecule becomes larger, the translational diffusion time becomes longer. In the case of the present invention, as shown in FIG. 1 (e), when the DNA bound to the protein P is immobilized on the beads, the fluorescent dye F is bound to the beads. Therefore, compared with the case of FIG. 1 (f). The speed of the movement of the fluorescent dye F is remarkably reduced, the length of the translational diffusion time is remarkably increased, and it is detected whether or not the DNA bound to the protein P is immobilized on the beads. 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 the fluorescence intensity (signal) to the background light (noise) is improved compared to when the light passes, and the fluorescence intensity is measured with a good S / N ratio.

  In FDS (fluorescence depolarization), as is known in this field, the speed of rotational Brownian motion (rotation) of molecules 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 implemented in a single molecule fluorescence analysis system, the fluorescence is measured in FIDA. Detection is performed separately for longitudinally polarized light and transversely polarized light, and the degree of polarization is calculated (in this case, referred to as FIDA-pol). Since the rotation of the molecule becomes slower as the size of the molecule increases, the degree of polarization increases. In the case of the present invention, as in the FCS described above, as shown in FIG. 1 (e), when DNA bound to the protein P is immobilized on the beads, the fluorescent dye F is bound to the beads, and accordingly, FIG. ), The speed of the rotational movement of the fluorescent dye F is significantly reduced, the degree of polarization is increased, and it is detected whether the DNA bound to the protein P is immobilized on the beads. Also, when measuring with a single molecule fluorescence analysis system, if multiple fluorescent dyes are constrained on the beads, the fluorescence intensity increases because these dyes pass through the fluorescence observation region as a whole. In addition, the S / N ratio of the measurement is improved.

  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, thereby obtaining minute amounts. The number density of fluorescent particles in the fluorescent observation region and the fluorescent intensity per fluorescent particle are calculated. In the case of the present invention, when the DNA bound to the protein P is fixed 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. The presence of a plurality of fluorescent dye molecules F on the beads increases the fluorescence intensity per fluorescent particle. Therefore, it is detected from the decrease in the number density of the fluorescent particles and the increase in the fluorescence intensity per fluorescent particle whether the DNA bound to the protein P is immobilized on the beads. Further, 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 measurement improves.

  In FCCS (fluorescence cross-correlation spectroscopy), when two fluorescent labels with different emission wavelengths pass through a minute fluorescence observation region by Brownian motion, the change in fluorescence intensity of each label correlates with the movement of the two fluorescent labels. It can be determined whether or not there is. 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 intensity changes 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 fluorescent dyes from the two dyes are converted into the fluorescent dyes. Since the correlation is detected and the cross-correlation function is higher than when each DNA is freely moving (not fixed to the beads), the DNA bound to the protein P is present on the beads. Whether it is fixed or not is detected. Similarly to the fluorescence analysis method described above, fluorescence from a plurality of dyes is emitted from a single bead, thereby increasing the fluorescence intensity measured at a time and improving the S / N ratio of the measurement.

  In the above-mentioned series of fluorescence analysis methods, it should be noted that the sample amount required to detect one result may be about several tens of μl, and the measurement time is about 5-15 seconds. The measurement is repeated several times. Therefore, the sample amount and time can be greatly reduced as compared with the conventional biochemical method. Moreover, compared to the conventional single-molecule fluorescence analysis method, the measured fluorescence intensity is several times larger, so that the S / N ratio is improved and a specific binding reaction can be achieved even if there are impurities in the sample. Only can be detected satisfactorily.

  By the way, in the above method, even if the fluorescence measurement / analysis is executed only at the stage where the beads are added, it can be estimated from the numerical value of the result whether the fluorescent dye is on the beads. As illustrated in 2 (B), fluorescence measurement / analysis is performed for comparison in the stage where only the fluorescently labeled DNA is present (step 10) and the biological sample solution and the DNA solution are mixed (step 30). (Steps 15 and 35). At the stage of step 35, when it is considered that there is no significant difference between the result of fluorescence measurement / analysis in the case of step 15, that is, the occurrence of the binding reaction involving the first molecule irrespective of specific / non-specific. 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, fluorescence Since DNA having a dye only binds to a DNA binding protein on a one-to-one basis, the number of fluorescent particles does not change, and the result of FIDA does not change unless the fluorescence intensity of the dye changes due to binding for some reason. When separate fluorescence analysis methods are used in step 35 and step 50, it is desirable to perform measurement by both analysis methods in step 15 for comparison.

The above procedure for other embodiments can also be used to detect the base sequence of DNA that specifically binds to the DNA binding protein P contained in the biological sample. In that case, a fluorescently labeled DNA having a base sequence that is supposed to bind to the DNA binding protein P is used. If it is detected that the DNA is immobilized on the beads, it is specified that a site that specifically binds to the DNA binding protein P exists in the base sequence of the DNA.

  In the above method, it should be understood that the first molecule and the second molecule to be fluorescently labeled may be any DNA, protein, or other biomolecule. For example, the first molecule to be fluorescently labeled may be an antibody for a molecule to be detected whether it is contained in a biological sample, and the third molecule may be an antibody against an epitope of another part of the molecule to be detected. . When the fluorescently labeled antibody is immobilized on the beads, it is detected that an antigen against the antibody as the first and third molecules is present in the biological sample.

  In the method of the present invention, even when contaminants are present in the sample, it is possible to detect the specific binding of a specific molecule in a mode that is advantageously distinguished from non-specific binding. It can be used as a screening tool for substances. If any molecule in a biological sample can be fluorescently labeled specifically or chemically (by the expression of a fluorescent protein such as GFP), the first sample containing the first molecule is the biological sample. It should also be understood that the second sample may be prepared or material in any manner and such cases are within the scope of the present invention.

FIG. 1 schematically shows the state of molecules in a sample during the process of the embodiment. FIG. 2 is a flowchart showing the processing steps in a preferred embodiment of the method of the present invention.

Claims (11)

A method for detecting specific binding of a specific molecule, comprising:
A first sample containing a fluorescently labeled first molecule and 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;
Adding a particle having an outer diameter of 1 μm 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 the particles are added;
Determining whether the first molecule is immobilized on the particle based on the fluorescence intensity, and when determining that the first molecule is immobilized on the particle Determining that the first molecule is specifically bound to the second molecule. A method for detecting specific binding of a specific molecule, comprising:
A first sample containing a fluorescently labeled first molecule; a second sample to determine whether there is a second molecule that specifically binds to the fluorescently labeled first molecule; Mixing the sample solution to prepare a mixed sample solution,
Adding a particle having an outer diameter of 1 μm 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 the particles are added;
Determining whether the first molecule is immobilized on the particle based on the fluorescence intensity, and when determining that the first molecule is immobilized on the particle Determining that the second molecule is present in the second sample.   3. The method of claim 1 or 2 based on 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. A method comprising determining whether a first molecule has bound to a molecule in the second sample.   4. The method according to claim 1, wherein the measurement of the fluorescence intensity is performed by fluorescence correlation spectroscopy.   4. The method according to claim 1, wherein the measurement of the fluorescence intensity is performed by a fluorescence depolarization method.   4. The method according to claim 1, wherein the fluorescence intensity is measured by a fluorescence intensity distribution analysis method.   4. The method according to claim 1, wherein the fluorescence intensity is measured by fluorescence cross-correlation spectroscopy.   4. The method according to claim 1, wherein the second sample is serum or plasma or a solution sample obtained from a living body.   4. The method according to claim 1, wherein the first molecule is DNA, the second molecule is a DNA-binding protein, and the third molecule is an antibody against the second molecule. Feature method.   4. 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, gold colloid, magnetic particles and glass. 4. The method according to claim 1, wherein the surface of the particle prevents adsorption or binding of molecules present in the mixed solution excluding specific binding between the second molecule and the third molecule. A method characterized in that an anti-adsorption treatment is applied.

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