WO2012014778A1 - 発光プローブを用いて溶液中の希薄粒子を検出する方法 - Google Patents
発光プローブを用いて溶液中の希薄粒子を検出する方法 Download PDFInfo
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- WO2012014778A1 WO2012014778A1 PCT/JP2011/066576 JP2011066576W WO2012014778A1 WO 2012014778 A1 WO2012014778 A1 WO 2012014778A1 JP 2011066576 W JP2011066576 W JP 2011066576W WO 2012014778 A1 WO2012014778 A1 WO 2012014778A1
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- light
- probe
- particle
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- luminescent probe
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- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
- G01N5/04—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder
- G01N5/045—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder for determining moisture content
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- G—PHYSICS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0036—Scanning details, e.g. scanning stages
- G02B21/0048—Scanning details, e.g. scanning stages scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G01N2015/1486—Counting the particles
Definitions
- the present invention uses an optical system capable of detecting light from a minute region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope, and uses atoms, molecules or aggregates thereof dispersed or dissolved in a solution ( These are hereinafter referred to as “particles”), for example, biomolecules such as proteins, peptides, nucleic acids, lipids, sugar chains, amino acids or aggregates thereof, particulate objects such as viruses and cells, or non-
- the present invention relates to an optical analysis method capable of obtaining useful information in analysis or analysis of biological particle states (interaction, binding / dissociation state, etc.), and more specifically, using a luminescent probe.
- the present invention relates to a method for detecting a particulate object in a solution, measuring a concentration or a number density, and the like.
- the term “luminescent probe” refers to a substance that emits light by fluorescence, phosphorescence, chemiluminescence, bioluminescence, light scattering, etc., and is bound to particles to be observed. It is a substance that makes it possible to observe.
- the average residence time (translational diffusion time) of the fluorescent molecules and the like in the minute region determined from the value of the autocorrelation function of the measured fluorescence intensity, and the average number of staying molecules Based on this, acquisition of information such as the speed or size of movement of fluorescent molecules, concentration, concentration, molecular structure or size change, molecular binding / dissociation reaction, dispersion / aggregation, etc.
- the detection of various phenomena is achieved.
- the fluorescence intensity analysis (Fluorescence-Intensity Distribution Analysis: FIDA, for example, Patent Document 3) and the Photon Counting Histogram (PCH, for example, Patent Document 4) are measured in the same manner as FCS.
- a histogram of the fluorescence intensity of the fluorescent molecules entering and exiting the volume is generated, and by fitting a statistical model formula to the distribution of the histogram, the average value of the intrinsic brightness of the fluorescent molecules and the confocal
- An average value of the number of molecules staying in the volume is calculated, and based on such information, a change in the structure or size of the molecule, a bound / dissociated state, a dispersed / aggregated state, and the like are estimated.
- Patent Documents 5 and 6 propose a method of detecting a fluorescent substance based on the time lapse of a fluorescence signal of a sample solution measured using an optical system of a confocal microscope.
- Patent Document 7 describes the presence of fluorescent fine particles in a flow or on a substrate by measuring weak light from fluorescent fine particles circulated in a flow cytometer or fluorescent fine particles fixed on a substrate using a photon counting technique. A signal arithmetic processing technique for detecting the signal is proposed.
- the sample required for measurement has an extremely low concentration compared with the conventional method. It can be very small (the amount used in one measurement is about several tens of ⁇ L), and the measurement time is greatly shortened (measurement of time on the order of seconds is repeated several times in one measurement). . Therefore, these technologies are particularly useful for analyzing rare or expensive samples often used in the field of medical and biological research and development, for clinical diagnosis of diseases, screening for physiologically active substances, etc. When the number is large, it is expected to be a powerful tool capable of performing experiments or inspections at a lower cost or faster than conventional biochemical methods.
- the magnitude of the temporal fluctuation of the measured fluorescence intensity is calculated by statistical processing, and the sample solution is based on the magnitude of the fluctuation.
- Various characteristics such as fluorescent molecules entering and exiting the minute region are determined. Therefore, in order to obtain a significant result in the above optical analysis technique, the concentration or number density of the fluorescent molecules to be observed in the sample solution must be as long as one second in the equilibrium state. In order for the number of fluorescent molecules that can be statistically processed within the measurement time to enter and exit the micro area, it is preferable that there is always approximately one fluorescent molecule in the micro area.
- the volume of the confocal volume is typically about 1 fL, so that the concentration of the fluorescent molecule or the like is preferably about 1 nM or more.
- the concentration or number density of the particles to be observed in the sample solution is significantly lower than the level capable of statistical processing (for example, significantly lower than 1 nM)
- the observation target is measured in a minute region. A state that rarely enters in time occurs, and in the measurement result of fluorescence intensity, a state where the observation object is not present in the minute region is included for a long period of time, and is significant. Therefore, the optical analysis technique based on the statistical fluctuation of the fluorescence intensity as described above cannot expect a significant or accurate analysis result.
- Patent Documents 5 and 6 the statistical processing relating to the fluctuation of the fluorescence intensity as described above is not performed, and the measurement time over several seconds is used.
- the presence or absence of fluorescent molecules to be observed in the sample can be specified by the presence or absence of generation of a fluorescent signal with a significant intensity.
- the frequency of the fluorescent signal with a significant intensity and the number of fluorescent molecules or the like in the sample It is disclosed that a correlation can be obtained.
- Patent Document 6 suggests that detection sensitivity is improved by generating a random flow that stirs the sample solution.
- these methods are also limited to detecting the presence of fluorescent molecules or the like that enter the microregion stochastically by diffusion or random flow.
- Patent Document 7 The behavior cannot be grasped, and for example, particle counting and particle concentration or number density are not quantitatively calculated.
- the technique described in Patent Document 7 is to individually detect the presence of fluorescent fine particles in a flow in a flow cytometer or fluorescent fine particles fixed on a substrate, and in a normal state in a sample solution. This is not a technique for detecting molecules or colloid particles dissolved or dispersed in the sample solution, that is, particles moving randomly in the sample solution. Quantitative calculation of concentration or number density has not been achieved.
- the technique of Patent Document 7 includes a process such as measurement in a flow cytometer or immobilization of fluorescent particles on a substrate, the amount of sample required for inspection is optical analysis such as FCS, FIDA, PCH, etc. It is considered to require a complicated and sophisticated operation technique for the practitioner as well as the number of techniques.
- the applicant of the present application does not include statistical processing as performed in optical analysis techniques such as FCS, FIDA, and PCH in Japanese Patent Application No. 2010-044714 and PCT / JP2011 / 53481.
- optical analysis techniques such as FCS, FIDA, and PCH in Japanese Patent Application No. 2010-044714 and PCT / JP2011 / 53481.
- the particles to be observed are based on a novel principle.
- An optical analysis technique to observe In this new optical analysis technology, in short, an optical system capable of detecting light from a minute region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope, like FCS, FIDA and the like.
- the light which is dispersed and moving randomly in the sample solution is used.
- emitted particles hereinafter referred to as “luminescent particles”
- light emitted from the luminescent particles in the minute region is detected, whereby each of the luminescent particles in the sample solution is detected.
- a small amount of sample (for example, about several tens of ⁇ L) is required for measurement in the same manner as optical analysis techniques such as FCS, FIDA, and PCH.
- the measurement time is short, and the concentration or number density of the luminescent particles having a lower concentration or number density than those of optical analysis techniques such as FCS, FIDA, PCH, etc. It is possible to quantitatively detect the characteristics.
- the main object of the present invention is to further develop the scanning molecule counting method proposed in the above Japanese Patent Application No. 2010-044714, in particular, particles that are dispersed in a sample solution and randomly move using a luminescent probe. It is to propose a method that advantageously uses the scanning molecule counting method for observation.
- a method for detecting particles by detecting light from a luminescent probe that is dispersed in a sample solution and bonded to randomly moving particles using an optical system of a confocal microscope or a multiphoton microscope A process of preparing a sample solution containing particles and a luminescent probe, a process of moving the position of the light detection region of the optical system in the sample solution by changing the optical path of the optical system, and the sample solution. The process of detecting the light from the light detection region while moving the position of the light detection region inside, and detecting the light signal from the light emitting probe coupled to the individual particles from the detected light individually And individually detecting.
- a method is provided.
- particles dispersed in a sample solution and moving randomly are particles such as atoms, molecules or aggregates thereof dispersed or dissolved in the sample solution (either luminescent or non-luminescent). Any particle may be used as long as it is not fixed to a substrate or the like and is free to undergo Brownian motion in the solution.
- a “luminescent probe” is a substance (usually a molecule or an aggregate thereof) that has a property of binding or associating with a particle to be observed and emits light, and is typically a fluorescent particle. However, it may be a particle that emits light by phosphorescence, chemiluminescence, bioluminescence, light scattering, or the like.
- the “light detection area” of the optical system of a confocal microscope or multiphoton microscope is a minute area in which light is detected in those microscopes.
- the illumination light is Corresponds to the focused region.
- the confocal microscope such a region is determined particularly by the positional relationship between the objective lens and the pinhole.
- the luminescent probe emits light without illumination light, for example, in the case of a molecule or aggregate thereof that emits light by chemiluminescence or bioluminescence, illumination light is not required in the microscope.
- the term “optical signal” refers to a signal representing light from a light-emitting probe coupled to a particle unless otherwise specified.
- the basic configuration of the present invention first, after preparing a sample solution in which a particle to be detected and a luminescent probe bonded to the particle are mixed by any technique, Light is sequentially detected while moving the position of the photodetection region in the sample solution, that is, while scanning the sample solution with the photodetection region. Then, when the moving photodetection region includes a luminescent probe that is bound or associated with a randomly moving particle, the light from the luminescent probe is detected, thereby detecting the presence of one particle. (Depending on the mode of the experiment, the luminescent probe may be dissociated from the particle upon detection of light after binding to the particle to be detected once).
- the light signals from the light emitting probes are individually detected in the sequentially detected light, thereby detecting the presence of particles (coupled with the light emitting probes) one by one individually,
- Various information regarding the state of the particles in the solution will be acquired.
- the number of particles detected during the movement of the position of the light detection region may be counted by counting the individually detected particles (particles). Counting).
- information on the number density or concentration of particles in the sample solution can be obtained by combining the number of particles and the amount of movement of the position of the light detection region.
- the number density or concentration of the particles is concrete. Can be calculated automatically.
- the absolute number density value or concentration value is not directly determined, but the relative number density or concentration ratio with respect to a plurality of sample solutions or a standard sample solution serving as a reference for the concentration or number density is calculated. It may be like this.
- the optical detection area is moved to change the position of the photodetection area by changing the optical path of the optical system. In this case, no mechanical vibration or hydrodynamic action is generated. Therefore, it is possible to measure light in a stable state without being affected by the mechanical action.
- a luminescent probe coupled to one particle from sequentially detected optical signals.
- the determination of whether or not has been made may be made based on the shape of the optical signal detected in time series. In an embodiment, typically, when an optical signal having an intensity greater than a predetermined threshold is detected, it is detected that a luminescent probe bound to one particle has entered the light detection region. It may be.
- the moving speed of the position of the light detection region in the sample solution depends on the characteristics of the luminescent probe bound to the particles or the number density or concentration in the sample solution. It may be changed appropriately based on the above. As will be appreciated by those skilled in the art, the manner of light detected from a luminescent probe bound to a particle can vary depending on its properties or number density or concentration in the sample solution. In particular, when the moving speed of the light detection region is increased, the amount of light obtained from the light emitting probe bonded to one particle is reduced, so that light from the light emitting probe bonded to one particle is measured with high accuracy or sensitivity. It is preferable that the moving speed of the light detection region is appropriately changed so that it can be performed.
- the moving speed of the position of the light detection region in the sample solution is preferably a luminescent probe (that is, a particle coupled to the particle to be detected). And, depending on the form of the luminescent probe, or depending on the mode of the experiment, it is set to be higher than the diffusion movement speed (average movement speed of the particles due to Brownian motion) .
- the diffusion movement speed average movement speed of the particles due to Brownian motion
- the moving speed of the light detection region is set to be higher than the diffusion moving speed of the light emitting probe bonded to the particle, and thereby the light emitting probe bonded to one particle is represented by one (representing the presence of the particle).
- the diffusion movement speed varies depending on the luminescent probe bonded to the particle, as described above, the movement speed of the light detection region is appropriately changed according to the characteristics (particularly the diffusion constant) of the luminescent probe bonded to the particle. It is preferable.
- the change of the optical path of the optical system for moving the position of the light detection area may be performed by an arbitrary method.
- the position of the light detection region may be changed by changing the optical path using a galvanometer mirror employed in a laser scanning optical microscope.
- the movement trajectory of the position of the light detection region may be arbitrarily set, and may be selected from, for example, a circle, an ellipse, a rectangle, a straight line, and a curve.
- the presence of a particle to be detected is confirmed by detecting light from a luminescent probe bound or associated with the particle. Therefore, if there is a luminescent probe that is not bonded to the particles in the sample solution, the accuracy of the particle detection result is lowered. Therefore, in the embodiment of the method of the present invention, a configuration for preventing detection of light from a luminescent probe that is not bound to particles in the sample solution may be included.
- a process of separating the luminescent probe from within the sample solution may be performed.
- the specific method of separating a luminescent probe that is not bound to a particle is the difference in characteristics between a single luminescent probe or a luminescent probe that is not bound to a particle and a luminescent probe that is bound to a particle and a bound luminescent probe or a particle.
- an arbitrary method for physically separating a plurality of substances using a difference in size or molecular weight, affinity for an arbitrary substance, charged state, or the like may be selected.
- Examples of methods for separating a luminescent probe alone or a luminescent probe that is not bound to particles include chromatography (hydrophilic / hydrophobic chromatography, affinity chromatography, ion exchange chromatography, etc.), ultrafiltration, electrophoresis Fluorescent probes that are not bound to a single luminescent probe or particles can be combined with a luminescent probe by any method such as phase separation, centrifugation, solvent extraction or operation including adsorption / extraction or washing by filter adsorption, etc.
- a single luminescent probe or a luminescent probe that is not bonded to particles may be removed from the sample solution by separating from the luminescent probe bonded to the body or particles.
- a luminescent probe alone or a luminescent probe that is not bonded to particles in a sample solution and particles and luminescent probes
- the luminescent probe or the luminescent probe or the luminescent probe bound to the particle, or the luminescent probe or the luminescent probe so that light from the luminescent probe alone or the luminescent probe not bound to the particle is not emitted (from the sample solution).
- Other components in the sample solution may be selected so that the luminescent probe alone or the luminescent probe not bound to the particle and the luminescent probe bound to the particle and the luminescent probe conjugate or particle may be distinguished.
- Such a configuration is advantageous in that it does not require a processing operation for physically separating the luminescent probe alone or the luminescent probe not bonded to the particles from the luminescent probe bonded to the particles and the combined luminescent probes or the particles.
- a substance that changes its luminescent property when it is bound to a particle to be detected is selected, and in the process of detecting light.
- the light from the luminescent probe bound to the particle to be detected may be selectively detected.
- an intercalator fluorescent dye that causes an increase in fluorescence intensity or a change in fluorescence wavelength when selected as a luminescent probe when bound to the nucleic acid or nucleic acid analogue is selected.
- a dye for example, a fluorescent dye such as a hydrophobic probe ANS, MANS, or TNS
- a substance that is composed of at least two constituent elements and emits fluorescence when the position of the at least two constituent elements changes when bound to a particle to be detected may be employed. Examples of such substances are fluorescent proteins that change structure when bound to a certain particle and emit strong fluorescence, or aggregate when bound to a certain particle to form a fluorescent metal complex.
- Molecule ligand of the complex
- the light emitting probe alone or the light emitting probe that is not bonded to the particle emits little light or has a wavelength different from that of the particle and the combined body of the light emitting probe even when emitting light. It becomes possible to selectively detect light from the conjugate of the luminescent probe.
- the luminescent property is different between a luminescent probe which is not bonded to a single luminescent probe or a particle in a sample solution and a combined luminescent probe or a luminescent probe bonded to a particle.
- the fluorescence energy transfer phenomenon may be advantageously used.
- an acceptor that absorbs light emitted from the luminescent probe to a luminescent probe that is not bonded to particles
- an acceptor is a substance that binds or associates only with a luminescent probe alone or a luminescent probe that is not bound to a particle, and is an arbitrary substance that absorbs light emitted from the luminescent probe (immediately) by fluorescence energy transfer.
- Substance such as a quencher or an energy acceptor).
- the luminescent probe is bonded to the acceptor and does not emit light, or the particle and the luminescent probe Since only light having a wavelength different from the light emitted from the luminescent probe bound to the conjugate or the particle is emitted, it is possible to detect only the light of the particle and the luminescent probe bound to the conjugate of the luminescent probe or the particle.
- the particle to be detected is a nucleic acid or a nucleic acid analogue and the luminescent probe is a fluorescently labeled nucleic acid or nucleic acid analogue
- the particle to be detected is reacted with the luminescent probe in the sample solution.
- a nucleic acid or a nucleic acid analog to which an acceptor for the fluorescent label of the luminescent probe is added is added to the sample solution.
- the nucleic acid or nucleic acid analog to which the acceptor is added binds, and thus it is possible to selectively detect light only from the conjugate of the particle and the luminescent probe.
- Another example using the fluorescence energy transfer phenomenon is a substance having an energy donor site and an energy acceptor site that cause a fluorescence energy transfer phenomenon when they are close to each other, and binds to a particle to be detected.
- a substance configured to change the distance between the energy donor site and the energy acceptor site may be employed as the luminescent probe (molecular beacon method, scorpion method, etc.).
- the degree of occurrence of the fluorescence energy transfer phenomenon differs depending on whether or not the luminescent probe is bound to the particle to be detected. Therefore, the luminescent probe alone does not emit light, or its emission wavelength and the particle and luminescent probe Since the emission wavelength of the conjugate is different, it becomes possible to selectively detect light from the conjugate of the particles and the luminescent probe.
- a first probe that is an energy donor in the fluorescence energy transfer phenomenon, an energy acceptor in the fluorescence energy transfer phenomenon, and A second probe is prepared and mixed with the particles to be detected. Then, from the conjugate formed by binding both the first and second probes to the particle, the light of the second probe is emitted through the fluorescence energy transfer phenomenon, distinguishing it from the light from the first probe, Only the light from the conjugate can be selectively detected (in this case, the second probe that is not bound to the particles hardly emits light).
- the particle to be detected has a site that serves as an energy acceptor for light emitted by the luminescent probe
- the fluorescent energy generated by selecting the luminescent probe as the donor and binding the luminescent probe to the particle.
- the particle to be detected has a light emitting site
- a substance having a site that becomes an energy acceptor of light emitted from the light emitting site of the particle is selected as the light emitting probe, and the light emitting probe is bonded to the particle.
- the particles detected by the method of the present invention are typically biological particles such as proteins, peptides, nucleic acids, lipids, sugar chains, amino acids or aggregates thereof, and particulate biological such as viruses and cells. It can be an object or a non-biological particle (eg, atom, molecule, micelle, metal colloid, etc.) and the luminescent probe is any substance that binds or adsorbs specifically or non-specifically to the particle. It may be.
- the particle to be detected is a nucleic acid
- it may be a dye molecule that binds to the nucleic acid, a nucleic acid binding protein, or the like.
- the method of the present invention can be used in experiments for detecting arbitrary particles using various luminescent probes.
- the method of the present invention has, as a luminescent probe, an energy donor site and an energy acceptor site where a fluorescence energy transfer phenomenon occurs, and when it is bound to a certain particle, a predetermined decomposition reaction is performed. Adopting a substance to be decomposed, adding such a luminescent probe to the sample solution to be inspected, detecting the light of the sample solution, and based on the presence or absence of the fluorescence energy transfer phenomenon, the luminescent probe It can be used for an experiment such as inspecting whether or not the particles are present in the sample solution.
- the luminescent probe has an energy donor site and an energy acceptor site that cause fluorescence energy transfer, and the process of preparing the sample solution binds to the particle to be detected.
- a method is provided that includes performing a reaction that decomposes the probe, wherein the detected light is light emitted from a luminescent probe that has been decomposed by the reaction.
- optical analysis technique realized by the above-described method of the present invention is the same as in the case of optical analysis techniques such as FCS, FIDA, PCH, etc., with respect to the optical detection mechanism itself.
- the amount of the sample solution may be small as well.
- the optical analysis technique of the present invention is an optical analysis technique in which the number density or concentration of particles is FCS, FIDA, PCH, etc. It is applicable to sample solutions that are significantly lower than the levels required for
- each of the particles dispersed or dissolved in the solution is individually detected. Therefore, the information is used to quantitatively count the particle concentration or the concentration of the particles in the sample solution. It is possible to calculate the number density or obtain information on the density or number density. For example, in the techniques described in Patent Documents 5 and 6, the correlation between the total value of the frequency of fluorescent signals having an intensity equal to or higher than a predetermined threshold within a predetermined time and the number of particles such as fluorescent molecules in the sample solution is obtained. However, it is not possible to grasp the dynamic behavior of particles passing through the measurement region (whether the particles have passed straight through the measurement region or whether the particles have stayed in the measurement region).
- the particles that pass through the light detection region and the detected optical signal are detected one by one so that the particles are detected one by one. Counting, and the concentration or number density of particles in the sample solution can be determined with higher accuracy than before.
- the sample solution is not affected by mechanical vibration or hydrodynamic action.
- the sample solution is observed in a mechanically stable state, for example, when a flow is generated in the sample (when applying a flow, it is difficult to always give a uniform flow rate,
- the configuration of the apparatus is complicated, the amount of sample required is greatly increased, and particles, luminescent probes or conjugates or other substances in the solution may be altered or denatured due to the hydrodynamic action of the flow.
- the reliability of quantitative detection results is improved as compared to the above), and measurement can be performed without any influence or artifacts due to mechanical action on the particles to be detected in the sample solution. It made.
- FIG. 1A is a schematic diagram of the internal structure of an optical analyzer according to the present invention.
- FIG. 1B is a schematic diagram of a confocal volume (observation region of a confocal microscope).
- FIG. 1C is a schematic diagram of a mechanism for changing the direction of the mirror 7 to move the position of the light detection region in the sample solution.
- FIGS. 2A and 2B are a schematic diagram for explaining the principle of light detection by the optical analysis technique according to the present invention and a schematic diagram of a temporal change in measured light intensity, respectively.
- FIG. 3 is a diagram schematically illustrating an example of a configuration for preventing detection of light from a luminescent probe that is not bonded to particles.
- FIGS. 1A is a schematic diagram of the internal structure of an optical analyzer according to the present invention.
- FIG. 1B is a schematic diagram of a confocal volume (observation region of a confocal microscope).
- FIG. 1C is a schematic diagram of
- FIGS. 4A and 4B are a model diagram in the case where the observation target particle crosses the light detection region while performing the Brownian motion, and a diagram showing an example of the time change of the photon count (light intensity) in that case. It is.
- FIGS. 5A and 5B show the case where the observation target particle crosses the light detection region by moving the position of the light detection region in the sample solution at a speed faster than the diffusion movement speed of the observation target particle. It is a figure which shows the example of this, and the example of the time change of the photon count (light intensity) in that case.
- FIG. 6 is a flowchart showing a processing procedure for counting particles from the time change of photon count (light intensity) measured by the method of the present invention.
- FIG. 7 is a diagram for explaining an example of the signal processing process of the detection signal in the processing procedure for counting particles from the time change of the photon count (light intensity) measured by the method of the present invention.
- FIG. 8 shows an actual measurement example (bar graph) of photon count data measured by the method of the present invention, a curve (dotted line) obtained by smoothing the data, and a Gaussian function (solid line) fitted in the pulse existence region. Show. In the figure, a signal labeled “noise” is ignored as it is a signal due to noise or foreign matter.
- FIG. 8 shows an actual measurement example (bar graph) of photon count data measured by the method of the present invention, a curve (dotted line) obtained by smoothing the data, and a Gaussian function (solid line) fitted in the pulse existence region. Show. In the figure, a signal labeled “noise” is ignored as it is a signal due to noise or foreign matter.
- FIG. 9 shows (A) the result of a nucleic acid concentration detection experiment according to the method of the present invention, (B) the result of a nucleic acid concentration detection experiment using a plate reader, and (C) the molecule in the nucleic acid concentration detection experiment. Schematic diagrams of these states are shown respectively.
- FIG. 10 shows (A) the result of an experiment for detecting a nucleic acid having a certain base sequence using a molecular beacon according to the method of the present invention, and (B) the nucleic acid detection experiment using a molecular beacon in a plate reader. The results are shown respectively.
- FIG. 10 shows (A) the result of an experiment for detecting a nucleic acid having a certain base sequence using a molecular beacon according to the method of the present invention, and (B) the nucleic acid detection experiment using a molecular beacon in a plate reader. The results are shown respectively.
- FIG. 10 shows (A) the result of an experiment for detecting a nucleic acid having a certain
- FIG. 11 shows the result of an experiment for detecting particles to be observed in a sample solution from which an unreacted fluorescently labeled probe has been removed by physical purification according to the method of the present invention.
- the bar graph is the average value
- the error bar is the standard deviation.
- FIG. 12 (A) is a diagram schematically showing the state of a molecule in a nucleic acid detection experiment using fluorescence energy transfer (FRET) by the method of the present invention, and FIG. The result of the experiment of the number of pulses performed is shown.
- the bar graph is the average value
- the error bar is the standard deviation.
- FIG. 13 (A) is a diagram schematically showing the state of molecules in a nucleic acid (observation target particle) detection experiment using the fluorescence quenching method according to the method of the present invention, and FIG. The results of experiments on the number of detected pulses are shown.
- the bar graph is the average value
- the error bar is the standard deviation.
- FIG. 14 (A) is a diagram schematically showing the state of molecules in a nucleic acid detection experiment using a QUAL reaction according to the method of the present invention, and FIG. 14 (B) shows the number of detected pulses. The result of the experiment is shown.
- the bar graph is the average value
- the error bar is the standard deviation.
- FIG. 15 is an example of the time change of the photon count (light intensity) obtained in the conventional optical analysis technique for calculating the fluctuation of the fluorescence intensity.
- FIG. 15A shows that the concentration of particles in the sample is sufficient. This is a case where measurement accuracy is given, and (B) is a case where the concentration of particles in the sample is significantly lower than in (A).
- Optical analyzer confocal microscope 2 ... Light source 3 ... Single mode optical fiber 4 ... Collimator lenses 5, 14a ... Dichroic mirrors 6, 7, 11 ... Reflection mirror 8 ... Objective lens 9 ... Microplate 10 ... Well (sample solution container) DESCRIPTION OF SYMBOLS 12 ... Condenser lens 13 ... Pinhole 14 ... Barrier filter 15 ... Multi-mode optical fiber 16 ... Photo detector 17 ... Mirror deflector 17a ... Stage position change device 18 ... Computer
- optical analysis apparatus 1 includes optical systems 2 to 17 and a computer 18 for controlling the operation of each part of the optical system and acquiring and analyzing data.
- the optical system of the optical analyzer 1 may be the same as the optical system of a normal confocal microscope, in which the laser light (Ex) emitted from the light source 2 and propagated through the single mode fiber 3 is a fiber.
- the light is emitted as a divergent light at an angle determined by a specific NA at the outgoing end of the light, becomes parallel light by the collimator 4, is reflected by the dichroic mirror 5, the reflection mirrors 6, 7, and the objective lens 8. Is incident on.
- a microplate 9 in which a sample container or well 10 into which a sample solution of 1 to several tens of ⁇ L is dispensed is typically arranged is emitted from the objective lens 8.
- the laser light is focused in the sample solution in the sample container or well 10 to form a region with high light intensity (excitation region).
- particles to be observed and luminescent probes that bind to the particles are dispersed or dissolved, and are bound to the luminescent probes.
- an associated particle a luminescent probe that is once bound to the particle and then dissociated from the particle depending on the mode of the experiment
- the luminescent probe is excited and light is emitted during that time.
- the emitted light (Em) passes through the objective lens 8 and the dichroic mirror 5, is reflected by the mirror 11, is collected by the condenser lens 12, passes through the pinhole 13, and passes through the barrier filter 14.
- the pinhole 13 is disposed at a position conjugate with the focal position of the objective lens 8, and as a result, as shown in FIG. Only the light emitted from the focal region of the laser beam, that is, the excitation region as schematically shown, passes through the pinhole 13, and the light from other than the excitation region is blocked.
- 1B is usually a light detection region in the present optical analyzer having an effective volume of about 1 to 10 fL (typically, the light intensity is in the region). Gaussian distribution or Lorentzian distribution with the vertex at the center.
- the effective volume is the volume of a substantially elliptical sphere bounded by the surface where the light intensity is 1 / e 2.
- Called confocal volume The Further, in the present invention, light from a combined particle or luminescent probe of one particle and a luminescent probe, for example, faint light from one or several fluorescent dye molecules, is detected. In this case, an ultrasensitive photodetector that can be used for photon counting is used.
- a stage position changing device 17a for moving the horizontal position of the microplate 9 may be provided on a microscope stage (not shown) in order to change the well 10 to be observed.
- the operation of the stage position changing device 17a may be controlled by the computer 18. With this configuration, it is possible to achieve quick measurement even when there are a plurality of specimens.
- the optical path of the optical system is changed and the inside of the sample solution is scanned by the light detection region, that is, the focal region (ie, the light detection region) in the sample solution.
- the light detection region that is, the focal region (ie, the light detection region) in the sample solution.
- a mirror deflector 17 that changes the direction of the reflection mirror 7 may be employed as schematically illustrated in FIG.
- Such a mirror deflector 17 may be the same as a galvanometer mirror device provided in a normal laser scanning microscope.
- the mirror deflector 17 is driven in cooperation with light detection by the light detector 16 under the control of the computer 18.
- the movement trajectory of the position of the light detection region may be arbitrarily selected from a circle, an ellipse, a rectangle, a straight line, a curve, or a combination thereof (so that various movement patterns can be selected by the program in the computer 18). It may be.)
- the position of the light detection region may be moved in the vertical direction by moving the objective lens 8 up and down.
- mechanical vibrations and hydrodynamic actions are substantially caused in the sample solution. It is possible to eliminate the influence of the dynamic action on the observation object and to achieve stable measurement.
- the above optical system is used as a multiphoton microscope. In that case, since there is light emission only in the focal region (light detection region) of the excitation light, the pinhole 13 may be removed.
- the optical systems 2 to 5 for generating the excitation light may be omitted.
- the optical system of the confocal microscope is used as it is.
- a plurality of excitation light sources 2 may be provided, and the wavelength of the excitation light is appropriately determined according to the wavelength of the light that excites the particle and luminescent probe combination or the luminescent probe. Can be selected.
- a plurality of photodetectors 16 may be provided, and when a sample includes a combination of a plurality of types of particles having different wavelengths and a combination of luminescent probes or a luminescent probe, the light from them is changed depending on the wavelength. It may be possible to detect them separately.
- the spectroscopic analysis technology such as FCS, FIDA and the like is superior to the conventional biochemical analysis technology in that the required amount of sample is extremely small and inspection can be performed quickly. ing.
- the concentration and characteristics of the observation target particles are calculated based on the fluctuation of the fluorescence intensity. Therefore, in order to obtain an accurate measurement result, the sample solution
- the concentration or number density of the observation target particles therein is always about one observation target particle in the light detection region CV during the measurement of the fluorescence intensity.
- it is required that a significant light intensity (photon count) is always detected during the measurement time.
- the concentration or number density of the observation target particles is lower than that, for example, as shown in FIG. 15B, the observation target particles are at a level that only occasionally enters the light detection region CV. As illustrated on the right side of the figure, a significant light intensity (photon count) appears only in a part of the measurement time, making it difficult to accurately calculate the fluctuation of the light intensity.
- the concentration of the observation target particle is much lower than the level at which about one observation target particle is always present in the light detection area during measurement, the influence of the background is considered in the calculation of the light intensity fluctuation.
- the measurement time is long in order to obtain significant light intensity data sufficient for calculation.
- the concentration of the observation target particles is lower than the level required by the spectral analysis technology such as FCS or FIDA as described above, the characteristics such as the number density or the concentration of the observation target particles.
- the optical path is changed by driving a mechanism (mirror deflector 17) for moving the position of the photodetection region, as described briefly.
- a mechanism for moving the position of the photodetection region, as described briefly.
- light detection is performed while moving the position of the light detection region CV in the sample solution, that is, while scanning the sample solution by the light detection region CV. .
- FIG. 2A while the photodetection region CV moves (time to to t2 in the figure), one particle (in the figure, the fluorescent dye is bound as a luminescent probe). ), A significant pulsed light intensity (Em) is detected as shown in FIG. 2B.
- the fluorescence intensity measured by a fluorescence spectrophotometer or a plate reader is used. It is possible to measure to a lower concentration than when measuring the concentration of fluorescently labeled particles.
- the fluorescence intensity is proportional to the concentration of the fluorescently labeled particles.
- the concentration of the fluorescently labeled particles when the concentration of the fluorescently labeled particles is sufficiently low, the amount of noise signal with respect to the amount of signal due to the light emitted from the fluorescently labeled particles increases (deterioration of the S / N ratio), and fluorescence The proportional relationship between the concentration of the labeled particles and the amount of optical signal is lost, and the accuracy of the determined concentration value is deteriorated.
- the noise signal in the process of detecting the signal corresponding to each particle from the detected optical signal, the noise signal is excluded from the detection result, and only the signal corresponding to each particle is counted to obtain the concentration. Since the calculation is performed, it is possible to detect even a lower concentration than when the concentration is detected on the assumption that the fluorescence intensity is proportional to the concentration of the fluorescently labeled particles.
- the fluorescence intensity of the fluorescently labeled particles is increased.
- the measurement accuracy of the particle concentration on the higher particle concentration side is also improved.
- the number of the fluorescence intensity per observation target particle since the fluorescence intensity per observation target particle is reduced, the proportional relationship between the concentration of the fluorescently labeled particles and the light amount is lost, and the accuracy of the determined concentration value is deteriorated.
- the method of the present invention in the process of detecting the signal corresponding to each particle from the detected optical signal, the influence of the reduction of the fluorescence intensity per particle is small, and the concentration is calculated from the number of particles. Assuming that the fluorescence intensity is proportional to the concentration of the fluorescently labeled particles, it is possible to detect even higher concentrations than when detecting the concentration.
- the observation target particles of the method of the present invention are any particles as long as they are particles that are dispersed in the sample solution and move randomly in the solution, such as dissolved molecules.
- it may be a biomolecule such as a protein, peptide, nucleic acid, lipid, sugar chain, amino acid or aggregate thereof, virus, cell, metal colloid, or other non-biological molecule.
- the particles to be observed are typically luminescent labels (fluorescent molecules) in a sample solution (typically an aqueous solution, but not limited thereto, and may be an organic solvent or any other liquid).
- Phosphorescent molecules are mixed with a luminescent probe added in an arbitrary manner, and the luminescent probe binds or associates with the observation target particle to form a conjugate, thereby emitting the luminescent probe.
- the light becomes a mark of the presence of the observation target particle, and the observation target particle is detected (as will be described later, depending on the mode of the experiment, once coupled to the observation target particle, it is released from the particle through a predetermined process)
- the target particle is detected by detecting the luminescent probe.
- the luminescent probe when the observation target particle is a nucleic acid, the luminescent probe is a nucleic acid or nucleic acid analog having a base sequence complementary to the base sequence of the nucleic acid that is the observation target particle, a nucleic acid Binding proteins, nucleic acid binding antibodies and the like are selected.
- a nucleic acid Binding proteins, nucleic acid binding antibodies and the like are selected.
- hybrid capture method there is an example (hybrid capture method) in which a fluorescence-labeled DNA-RNA hybrid identification antibody is used as a luminescent probe with respect to an observation target particle that is an association of DNA-RNA.
- the mixing of the observation target particle and the luminescent probe to form a conjugate of the particle and the luminescent probe it should be noted that substantially all the observation target particles in the sample solution emit light. It is necessary to bind or associate with the probe. That is, if there are particles to be observed that are not bound or associated with the luminescent probe, the number and / or concentration of the particles to be observed in the sample solution is observed to be as low as that number. Therefore, in order to reliably bind or associate substantially all the observation target particles with the luminescent probe, the number of the luminescent probes in the sample solution is determined in the formation of the combination of the particles and the luminescent probe.
- a luminescent probe it is necessary to add a luminescent probe to the sample solution so as to exceed the number of particles.
- a luminescent probe that is not bound or associated with the observation target particle in the sample solution, and light from the luminescent probe or the luminescent probe alone that is not bonded to the observation target particle is present. If it is detected without distinction from the light from the combination of the particle and the luminescent probe, the detection accuracy of the observation target particle is deteriorated. Therefore, in the method of the present invention based on an optical analysis technique for individually detecting observation target particles using a luminescent probe, it is preferable that light from a luminescent probe or a luminescent probe alone not bound to the observation target particle be used.
- a configuration for preventing detection that is, a configuration for excluding light from a luminescent probe or a single luminescent probe that is not bound to an observation target particle from the detection result may be used.
- a configuration for preventing detection that is, a configuration for excluding light from a luminescent probe or a single luminescent probe that is not bound to an observation target particle from the detection result.
- the following configuration may be adopted.
- FIG. As schematically shown in (a), after formation of a combined particle and luminescent probe, a luminescent probe or a single luminescent probe not bonded to the observation target particle, and a combined particle or luminescent probe or target
- a luminescent probe or a single luminescent probe not bonded to the observation target particle and a combined particle or luminescent probe or target
- the luminescent probe or the luminescent probe alone that is not bound to the observation target particle may be separated from the sample solution and removed.
- adsorption by chromatography hydrophilic / hydrophobic chromatography, affinity chromatography, ion exchange chromatography, etc.
- ultrafiltration electrophoresis
- phase separation centrifugation
- solvent extraction solvent extraction
- filter adsorption etc.
- another probe that binds to the observation target particle is further mixed with the sample solution in which the observation target particle and the luminescent probe are mixed, and then combined with the observation target particle.
- the solution is exposed to a carrier that binds to the separation probe, and only the conjugate consisting of the observation target particle, the luminescent probe, and the separation probe is held on the carrier, while the luminescent probe alone in the sample solution is separated and removed (for example, A method of excluding the luminescent probe alone from the sample solution may be used.
- Such a fluorescent dye may be one that changes the excitation and / or emission wavelength when bound to the observation target particle, or one that significantly increases the fluorescence intensity when bound to the observation target particle.
- an observation target particle that is a nucleic acid or a nucleic acid analog
- a nucleic acid intercalator fluorescent dye ethidium bromide, acridine orange, SYTOX Orange, SYTOX Red, SYBR Green I, SYBR
- Green II ethidium bromide, acridine orange, SYTOX Orange, SYTOX Red, SYBR Green I, SYBR
- Green II ethidium bromide, acridine orange, SYTOX Orange, SYTOX Red, SYBR Green I, SYBR
- Green II ethidium bromide, acridine orange, SYTOX Orange, SYTOX Red, SYBR Green I, SYBR
- SYBR Gold Picogreen
- OllGreen Gel Red
- Gel Green Ribo Green
- EvaGreen a dye having a cyanine skeleton, etc.
- dyes examples include 1-anilinonaphthalene-8-sulfonic acid (ANS), N-methyl-2-anilinonaphthalene-6-sulfonic acid (MANS), 2-p-, which are hydrophobic probes.
- Naphthalenesulfonic acids such as toluidinylnaphthalene-6-sulfonic acid (TNS), dimethylaminonaphthalene (dansyl), TAMRA, fluorescein, 6-joe, BODIPY, TMR, which are susceptible to local pH and dielectric constant BODIPY TR, Alexa 488, Alexa 532, BODIPY FL, BODIPY FL / C3, BODIPY FL / C6, FITC, EDANS, Rhodamine 6G, TMR, TMRITC, x-Rhodamine, Texas Red, BODIPY 5-FAM, BODIPY R6G, BODIPY 581
- TSS toluidinylnaphthalene-6-sulf
- a substance composed of at least two constituents which, when bound to a particle to be detected, changes the position of the at least two constituents and emits fluorescence. It can be employed as a luminescent probe whose wavelength characteristics change when bound to a target particle. Examples of such substances are fluorescent proteins that change structure when bound to a certain particle and emit strong fluorescence, or aggregate when bound to a certain particle to form a fluorescent metal complex. Molecule (ligand of the complex).
- the effect of the fluorescence energy transfer phenomenon is achieved using at least two types of fluorescent dyes.
- the light wavelength from the light emitting probe which is single or not bonded to the observation target particle is different from each other, and only the light from the observation target particle and the combination of the light emission probe or the light emission probe bonded to the observation target particle is used. 1 may be employed.
- the aspect using the fluorescence energy transfer phenomenon may be as follows, for example.
- a fluorescence quenching method may be used. Specifically, as schematically shown in FIG. 3B, after formation of a conjugate of the observation target particle and the luminescent probe, it binds to a single or non-observed luminescent probe. However, a substance that does not bind to the conjugate of the particle and the luminescent probe and absorbs the light emitted by the luminescent probe by fluorescence energy transfer (photo acceptor) is added to the sample solution, It can be bonded only to a luminescent probe that is not bonded to the observation target particle or a luminescent probe alone.
- the optical analyzer 1 when the sample solution mixed with the light acceptor is observed by the optical analyzer 1, light is emitted from the light emitting probe and detected by the light detector in the combination of the particles and the light emitting probe.
- the light emitted from a light emitting probe to which a light acceptor is bound or not bound to a particle to be observed is absorbed by the light acceptor, and thus light from a light emitting probe that is alone or not bound to a particle to be observed is absorbed. It will be extinguished and will not be reflected in the detection result.
- a fluorescently labeled nucleic acid or nucleic acid analog is used as a luminescent probe for a target particle that is a nucleic acid or a nucleic acid analog
- the luminescent probe is bound to the target particle.
- a light acceptor is added to the luminescent probe by adding a nucleic acid or nucleic acid analog having a light acceptor site that absorbs the light of the fluorescent label of the luminescent probe and having a base sequence complementary to the luminescent probe as a light acceptor. This makes it possible to quench the fluorescence of the luminescent probe that is not bound to the observation target particle.
- Example 2 A substance having an energy donor site and an energy acceptor site as a luminescent probe, and the distance between the energy donor site and the energy acceptor site when the luminescent probe binds to the observation target particle A material configured to change may be employed.
- luminescent probes are, for example, molecular beacons, ie two energy donors and energy acceptors in the fluorescence energy transfer phenomenon, as schematically depicted in FIG. 3 (d).
- Examples include nucleic acid molecules to which a dye has been added. In such a molecular beacon (FIG.
- the added dye is close to the molecular beacon, and this causes a fluorescence energy transfer phenomenon when the energy donor is irradiated with excitation light, resulting in energy.
- the light emitted by the donor moves to the energy acceptor and is quenched, or the light of the emission wavelength of the energy acceptor dye (wavelength 2 light) is emitted, but the molecular beacon is the target particle (nucleic acid) Or a nucleic acid analog) (FIG. 3 (d) right), the distance between the two dyes is increased, and the fluorescence energy transfer phenomenon does not occur when the energy donor is irradiated with the excitation light.
- Light having the emission wavelength of the donor (light having wavelength 1) is emitted.
- a scorpion probe may be used as a similar principle. Contrary to the example shown in the figure, the fluorescence energy transfer phenomenon does not occur in the light emitting probe alone (or the light emitting probe that is not bonded to the observation target particle), and the fluorescence in the combination of the particle and the light emitting probe.
- a light-emitting probe configured to generate an energy transfer phenomenon may be used (in this case, light having an emission wavelength of the energy acceptor is selectively detected).
- a luminescent probe is not limited to a nucleic acid or a nucleic acid analog, and has at least two luminescent sites. When bound or associated with a particle to be observed, the distance between the at least two luminescent sites changes and is thereby released. It may be a substance that changes the wavelength of light.
- Example 3 As schematically shown in FIG. 2 (e), as a light emitting probe, a first probe (light emitting probe 1) serving as an energy donor and a second probe (light emitting) serving as an energy acceptor. Probes 2) are prepared and added to the sample solution. Then, when the observation target particle is irradiated with light that excites the light-emitting probe 1 in such a state, both the light-emitting probe 1 and the light-emitting probe 2 are combined to form a combined body. A movement phenomenon occurs, and light having the emission wavelength of the light-emitting probe 2 (wavelength 2) is emitted from the combined body.
- light of the emission wavelength (wavelength 1) is emitted from the light emitting probe 1 that is not bonded to the observation target particle, and the light emission probe 2 that is not bonded to the observation target particle is not substantially excited and emits light.
- wavelength 1 when the light emitting probe 1 is caused to emit light, light having the emission wavelength of the light emitting probe 2 is emitted only from the conjugate.
- Example 4 When the observation target particle has a light emission site, a substance that serves as an energy donor for the light emission site of the observation target particle may be selected as the light emission probe.
- the light emitting probe alone emits light having the emission wavelength (wavelength 1), while the observation target particle to which the light emitting probe is bonded is used.
- the light emitted from the light emitting probe is absorbed by the light emitting portion of the observation target particle, and then light having the emission wavelength of the light emitting portion of the observation target particle (wavelength 2) is emitted.
- the observation target can be observed with high accuracy without physically removing the emission probe from the sample solution. It becomes possible to detect particles.
- the observation target particle is a nucleic acid
- any fluorescent label that gives energy to guanine in the nucleic acid when bound to the nucleic acid as a luminescent probe (guanine becomes an energy acceptor) is labeled.
- a substance may be selected.
- a particle to be observed is a protein
- an arbitrary labeled substance that gives energy to tryptophan in the protein ie, tryptophan becomes an energy acceptor
- tryptophan becomes an energy acceptor
- a substance that becomes an energy acceptor for the light emitting site of the observation target particle may be selected as the light emitting probe.
- the luminescent probe absorbs light emitted from the luminescent site of the observation target particle, and While the light emission wavelength light (wavelength 2) of the light emission probe is emitted, the light emission probe alone does not emit light.
- the observation object can be observed with high accuracy without physically removing the emission probe from the sample solution. It becomes possible to detect particles.
- the probe binds to the nucleic acid (see the left figure in FIG. 3 (h)).
- a predetermined decomposition reaction is performed in this state, only the probe bound to the nucleic acid or both the probe and the nucleic acid are decomposed, and the energy donor site and the energy acceptor site on the probe are separated (FIG. 3).
- H Refer to the right figure), the fluorescence energy transfer phenomenon does not occur, and as a result, light (wavelength 1) from the energy donor site can be observed.
- the probe will not bind to the nucleic acid and the probe will not be decomposed even if a predetermined degradation reaction is performed.
- light from the energy donor site is absorbed by the energy acceptor site and remains unemitted.
- the method of the present invention is used in an experiment using a luminescent probe that generates a fluorescence energy transfer phenomenon as described above, and that is decomposed by a predetermined decomposition reaction when bound to an observation target particle (for example, nucleic acid). It is possible to use. In that case, the light detected by the apparatus becomes light emitted from the light-emitting probe which is coupled to the observation target particle and further decomposed.
- an experimental method for example, (A) A luminescent probe, which is a DNA having an energy donor site and an energy acceptor site, in which a fluorescence energy transfer phenomenon occurs, is added to a sample solution containing a nucleic acid or nucleic acid analogue (observation target particle) to be examined.
- a method for detecting whether or not a luminescent probe is degraded by a DNA polymerase having 5′-3 ′ exonuclease activity (Taqman method), (B) DNA containing an energy donor site and an energy acceptor site where a fluorescent energy transfer phenomenon occurs in a sample solution containing a nucleic acid or nucleic acid analogue (observation target particle) to be examined, and partially containing RNA
- a method of detecting whether or not the luminescent probe is decomposed by RNase H (Cycleave method)
- the sample solution containing the nucleic acid or nucleic acid analogue (observation target particle) to be examined has an energy donor site and an energy acceptor site where a fluorescence energy transfer phenomenon occurs, and a restriction enzyme identification region in part
- a method for detecting whether or not a luminescent probe is degraded by a restriction enzyme comprising adding a luminescent probe that is DNA containing (D) A luminescent probe, which is a DNA having an energy
- the sample solution may be prepared in the usual manner in each experiment.
- the wavelength of light to be detected in the light intensity measurement described in detail later is the wavelength of light emitted after the luminescent probe is decomposed.
- the number of detected particles is directly the number of decomposed luminescent probes, but the number is equal to the number of nucleic acid or nucleic acid analog molecules to which the luminescent probe is bound.
- the light intensity in the optical analysis of the present invention is measured by driving the mirror deflector 17 during the measurement and moving the position of the light detection region in the sample solution (sample Other than performing scanning within the solution, it may be performed in the same manner as the light intensity measurement process in FCS or FIDA.
- the operation process typically, after injecting the sample solution into the well 10 of the microplate 9 and placing it on the stage of the microscope, the user inputs an instruction to start measurement to the computer 18. Then, the computer 18 stores a program stored in a storage device (not shown) (a procedure for changing the optical path to move the position of the light detection region in the sample solution, and during the movement of the position of the light detection region).
- the mirror deflector 17 drives the mirror 7 (galvanomirror) to move the position of the light detection region within the well 10.
- the photodetector 16 converts the sequentially detected light into an electrical signal and transmits it to the computer 18, and in the computer 18, time-series light is transmitted from the transmitted optical signal in an arbitrary manner. Generate and save intensity data.
- the photodetector 16 is an ultra-sensitive photodetector that can detect the arrival of one photon.
- the light detection is performed sequentially for a predetermined unit time over a predetermined time.
- photon counting executed in a mode in which the number of photons arriving at the photodetector every 10 ⁇ sec is measured, and time-series light intensity data is time-series photon count data. It may be.
- the moving speed of the position of the light detection region during the measurement of the light intensity may be a predetermined speed that is arbitrarily set, for example, experimentally or so as to suit the purpose of analysis.
- the size or volume of the region through which the light detection region has passed is required, so that the moving distance is grasped.
- the movement of the position of the light detection region is executed in the manner.
- the movement speed is basically a constant speed.
- the present invention is not limited to this.
- the moving speed of the position of the light detection region in order to quantitatively and accurately perform individual detection of the observation target particles from the measured time-series light intensity data or counting of the number of observation target particles.
- the moving speed is determined by the random movement of the observation target particle (more precisely, the combination of the particle and the luminescent probe or the luminescent probe which is decomposed and released after the binding to the particle), that is, the moving speed due to Brownian movement It is preferable to set a faster value. Since the observation target particle of the optical analysis technique of the present invention is a particle that is dispersed or dissolved in a solution and moves freely and randomly, the position moves with time by Brownian motion.
- the particle moves randomly within the region as schematically illustrated in FIG.
- the light intensity changes randomly as shown in FIG. 4B (as already mentioned, the excitation light intensity of the light detection area decreases outward with the center of the area as the apex), and each. It is difficult to specify a significant change in light intensity corresponding to the observation target particle. Therefore, preferably, as shown in FIG. 5 (A), the particle crosses the light detection region in a substantially straight line, so that the time-series light intensity data is illustrated in FIG. 5 (B).
- the change profile of the light intensity corresponding to each particle becomes substantially uniform (when the particle passes through the light detection region substantially linearly, the profile of the light intensity change is substantially the same as the excitation light intensity distribution. The same is true.)
- the moving speed of the position of the light detection region is based on the average moving speed (diffusion moving speed) due to the Brownian motion of the particles. Is also set faster.
- an observation target particle having a diffusion coefficient D (more precisely, a conjugate of a particle and a luminescent probe, or a luminescent probe that is decomposed and released after binding to the particle) has a photodetection region having a radius Wo due to Brownian motion.
- the moving speed of the position of the light detection region may be set to a value sufficiently faster than that with reference to the Vdif.
- Wo is about 0.62 ⁇ m
- Vdif is 1.2 ⁇ Since 10 ⁇ 3 m / s
- the moving speed of the position of the light detection region may be set to 15 mm / s or more, which is 10 times or more.
- the profile of the change in the light intensity by setting the moving speed of the position of the light detection region in various ways is expected (typically, the excitation light intensity distribution).
- a preliminary experiment for finding a condition that is substantially the same as that described above may be repeatedly performed to determine a suitable moving speed of the position of the light detection region.
- the computer 18 performs processing according to the program stored in the storage device to obtain the following light intensity data. An analysis may be performed.
- the trajectory of one observation target particle passing through the light detection region is a substantially straight line as shown in FIG.
- the change in the light intensity corresponding to the particle is a profile reflecting the light intensity distribution in the light detection region (determined by the optical system) as schematically illustrated in FIG. (Usually substantially bell-shaped). Therefore, in one method of detecting the observation target particle, when the threshold Io is set for the light intensity and the time width ⁇ in which the light intensity exceeding the threshold continues is within a predetermined range, the light intensity The profile may be determined to correspond to the passage of one particle through the light detection region, and one observation target particle may be detected.
- the predetermined range for the threshold value Io and the time width ⁇ for the light intensity is a combination of the observation target particle and the luminescent probe that moves relative to the light detection region at a predetermined speed (or is decomposed after the combination with the particle).
- the specific value may be arbitrarily set experimentally, and the binding between the observation target particle and the luminescent probe It may be selectively determined by the properties of the body (or the luminescent probe that has been degraded and released after binding to the particle).
- I A ⁇ exp ( ⁇ 2t 2 / a 2 ) (4)
- the intensity A and the width a calculated by fitting the expression (4) to a significant light intensity profile (a profile that can be clearly determined not to be background) are within a predetermined range.
- the profile of the light intensity corresponds to that one observation target particle has passed through the light detection region, and one observation target particle may be detected.
- the intensity A and the width a are outside the predetermined ranges, they are ignored in the analysis as noise or foreign matter.
- Counting of observation target particles may be performed by counting the number of particles detected by the above-described detection target particle detection method using an arbitrary method. However, when the number of particles is large, for example, the processing illustrated in FIGS. 6 and 7B may be performed.
- the light intensity measurement described above ie, After the time series optical signal data (photon count data) is acquired by performing scanning and photon counting in the sample solution by the photodetection region (step 100), the time series optical signal data (FIG. 7B) Smoothing (smoothing) processing is performed on the upper “detection result (unprocessed)” (step 110, upper “smoothing” in FIG. 7B).
- the light emitted from the combination of the particle and the luminescent probe or the light emitted from the luminescent probe is probabilistically emitted, and data values may be lost in a minute time.
- the smoothing process may be performed by a moving average method, for example. It should be noted that the parameters for executing the smoothing process, such as the number of data points averaged at a time in the moving average method and the number of moving averages, are the moving speed (scanning of the position of the light detection region at the time of optical signal data acquisition). Speed) and BIN TIME may be set as appropriate.
- a first-order differential value with respect to the time of the time-series optical signal data after the smoothing process Is calculated (step 120).
- the time differential value of the time series optical signal data has a large change in the value at the time of change of the signal value as illustrated in the lower “time differential” in FIG.
- a significant signal (pulse signal) is sequentially detected on the time-series optical signal data, and it is determined whether or not the detected pulse signal is a signal corresponding to the observation target particle.
- pulse signal a significant signal
- the start point and the end point of one pulse signal are searched and determined, A pulse presence region is identified (step 130).
- the bell-shaped function fitting is performed on the smoothed time-series optical signal data in the pulse existence area (see FIG. 7B, “bell-shaped function fitting”).
- the peak intensity Imax of the bell-shaped function, the pulse width (full width at half maximum) w, and the correlation coefficient (of the least squares method) in the fitting are calculated (step 140).
- the bell-shaped function to be fitted is typically a Gaussian function, but may be a Lorentz-type function.
- the calculated bell-shaped function parameter is assumed as a bell-shaped profile parameter drawn by an optical signal detected when a combination of one particle and the light-emitting probe or the light-emitting probe passes through the light detection region. It is determined whether it is within the range, that is, whether the peak intensity, the pulse width, and the correlation coefficient are each within a predetermined range (step 150).
- the range that is, whether the peak intensity, the pulse width, and the correlation coefficient are each within a predetermined range
- the calculated bell-shaped function parameter is within an expected range in the optical signal corresponding to the combination of one particle and the luminescent probe or the luminescent probe.
- the signal is determined to be a signal corresponding to one observation target particle, whereby one observation target particle is detected and counted as one particle (the number of particles is counted up).
- Step 160 On the other hand, as shown in the right side of FIG. 8, a pulse signal whose calculated bell-shaped function parameter is not within the assumed range is ignored as noise.
- the search and determination of the pulse signal in the processing of steps 130 to 160 is repeatedly executed over the entire area of the time-series optical signal data, and is counted as a particle every time one observation target particle is detected. .
- the particle count value obtained so far is set as the number of observation target particles detected in the time-series optical signal data.
- (Iii) Determination of the number density or concentration of the observation target particles
- the total volume of the region through which the light detection region passes during acquisition of time-series optical signal data is used.
- the number density or concentration of the particles is determined.
- the effective volume of the light detection region varies depending on the wavelength of excitation light or detection light, the numerical aperture of the lens, and the adjustment state of the optical system, it is generally difficult to calculate from the design value. It is not easy to calculate the total volume of the region through which the light detection region passes. Therefore, typically, for a solution (reference solution) with a known particle concentration, the light intensity measurement, particle detection, and counting described above are performed under the same conditions as the measurement of the sample solution to be examined.
- the total volume of the region through which the light detection region has passed that is, the relationship between the number of detected particles and the concentration is determined from the number of detected particles and the concentration of the reference solution particles. It's okay.
- a plurality of solutions having different concentrations are prepared as reference solutions, and measurement is performed on each of the solutions.
- the calculated average value of Vt is adopted as the total volume Vt of the region through which the light detection region has passed. It may be.
- the volume of the light detection region and the total volume of the region through which the light detection region has passed are given by any method, for example, using FCS or FIDA, without depending on the above method. It's okay.
- the relationship between the concentration C and the number N of various standard particles (formula (5)) with respect to the assumed light detection region movement pattern may be stored in advance in a storage device of the computer 18 so that the information on the relationship stored as appropriate when the user of the device performs the optical analysis can be used.
- SYTOX Orange which is a DNA intercalator fluorescent dye (a dye whose fluorescence intensity increases remarkably when bound to DNA), is used in a sample solution according to the method of the present invention.
- the measurable range of the DNA concentration was verified (see FIG. 3C).
- the measurable range of the DNA concentration by the fluorescence intensity measured by a plate reader was also measured.
- DNA pbr322, Takara Bio
- SYTOX Orange is a fluorescent dye whose fluorescence intensity increases by about 500 times when bound to DNA (an example of a fluorescent dye whose wavelength characteristics change in FIG. 3C).
- a single molecule fluorescence measurement apparatus MF-20 (Olympus Corporation) equipped with an optical system of a confocal fluorescence microscope and a photon counting system is used as an optical analysis apparatus.
- Measurement of light intensity of sample solution time-series photon count data was obtained for each sample solution.
- a laser beam of 633 nm was used as excitation light
- a detection light wavelength was set to 660 to 710 nm using a bandpass filter.
- the moving speed of the position of the light detection region in the sample solution was 15 mm / second, the BIN TIME was 10 ⁇ sec, and the measurement time was 2 seconds.
- step 110 After measurement of light intensity, detection is performed in time-series data from time-series photon count data acquired for each sample solution according to the processing procedure described in “(3) (ii) Counting of observation target particles” above.
- the optical signal produced was counted.
- step 110 Nine data points were averaged at a time, and the moving average process was repeated five times.
- step 140 the Gaussian function was fitted to the time series data by the least square method, and the peak intensity (in the Gaussian function), the pulse width (full width at half maximum), and the correlation coefficient were determined. .
- step 150 The following conditions: 20 microseconds ⁇ pulse width ⁇ 400 microseconds peak intensity> 1 (photon / 10 microseconds) (A) Correlation coefficient> 0.95 While only the pulse signal that satisfies the above condition is determined to be an optical signal corresponding to the observation target particle, the pulse signal that does not satisfy the above condition is ignored as noise, and the signal is determined to be the optical signal corresponding to the observation target particle Was counted as “number of pulses”.
- the fluorescence intensity of each sample solution was measured using a plate reader SH-8000lab (Corona).
- the excitation light wavelength was 543 nm
- the detection light wavelength was 570 nm
- the bandwidth was set to 12 nm on both the excitation side and the detection side.
- the measurement was performed 3 times with the excitation light flushed 50 times, and the average was used as the final fluorescence intensity value.
- FIGS. 9 (A) and 9 (B) show the measurement results (number of pulses) and the measurement results (fluorescence intensity) obtained by the above-described method of the present invention detected for each concentration of the sample solution.
- the number of pulses measured by the method of the present invention increased approximately in proportion to the increase in nucleic acid concentration.
- the individual nucleic acid observed particle
- the concentration is quantified by counting the individual observed particle according to the method of the present invention. It became clear that it was possible to make a decision.
- each pulse signal in the time-series optical signal data corresponds to the observation target particle, and the optical signal determined as noise or foreign matter is determined. Since it is ignored (see FIG. 8), it is considered that the S / N ratio can be maintained relatively well even in a low concentration region where the contribution of noise or foreign matter to the fluorescence intensity is relatively large.
- the method of the present invention it is possible to determine the number density or concentration of particles to a concentration range lower than the number density or concentration limit that can be measured in the method using the conventional fluorescence intensity. It has been shown.
- the lower limit of the particle concentration that can be measured by optical analysis techniques such as FCS, FIDA, and PCH including statistical processing such as calculation of fluctuations in fluorescence intensity was about 1 nM, whereas the measurement of this example.
- the lower limit of the possible particle concentration is ⁇ 100 fM. Therefore, according to the present invention, it is shown that it is possible to measure particles in a concentration region that is significantly lower than in the case of optical analysis techniques such as FCS, FIDA, and PCH. It was done.
- the increase width of the number of pulses is about 10 times as expected with respect to the increase of the concentration 10 times from 10 pM to 100 pM.
- the increase in the fluorescence intensity was smaller than the expected 10 times as the concentration was increased from 10 pM to 100 pM.
- the present invention is configured to count the number of optical signals corresponding to the observation target particles, and thus has less influence due to a decrease in the number of dyes bound to one nucleic acid. It is considered that the number of pulses increased as expected up to the concentration range.
- the conventional fluorescence intensity is proportional to the number density or concentration of the luminescent particles. It shows that the number density or concentration of particles can be determined up to a concentration range higher than the upper limit of possible number density or concentration.
- a molecular beacon is a nucleic acid molecule to which a donor dye and an acceptor dye are added at both ends, as already mentioned. In a simple substance, the distance between the donor dye and the acceptor dye is close, and the fluorescence from the donor dye to the acceptor dye occurs. While energy transfer occurs, binding to a nucleic acid or nucleic acid analog having a base sequence complementary to its own base sequence increases the distance between the donor dye and the acceptor dye and prevents the fluorescence energy transfer phenomenon from occurring. It is a composed molecule (see FIG. 3 (d)).
- TAMRA donor dye
- BHQ-2 acceptor dye, but in this case, almost no fluorescence is emitted
- the nucleic acid having the following base sequence was used.
- grains used the nucleic acid which has the following base sequence. gtagttggagctgttggcgtaggcaagagtgccttgacgatacagctaattcag
- the above nucleic acids were synthesized upon request from Sigma Genosys.
- the molecular beacon and the observation target particle were dissolved in a phosphate buffer (containing 0.05% Tween 20) so as to be 500 pM and 100 nM, respectively, and used as a sample solution.
- a phosphate buffer containing 0.05% Tween 20
- grains (nucleic acid) but contains only a molecular beacon at 500 pM was prepared.
- the measurement of the sample solution and the control solution by the method of the present invention was performed under the same conditions as in Example 1.
- the fluorescence intensity of the sample solution and the control solution was measured in the same manner as in Example 1 using a plate reader.
- the excitation light wavelength was 550 nm and the detection light wavelength was 576 nm.
- FIGS. 10A and 10B show the measurement results (number of pulses) and the measurement results (fluorescence) according to the method of the present invention detected for the sample solution (MB + Target) and the control solution (only MB), respectively. Strength).
- the number of pulses of the sample solution containing the nucleic acid that is the observation target particle is remarkably increased as compared with the case of the control solution (difference of about 6 times). The variation was small.
- the control experiment of FIG. 10 (B) there was a difference in the average value of the fluorescence intensity of the sample solution and the fluorescence intensity of the control solution, but the variation was large.
- the concentration of the molecular beacon in this example is 500 pM as described above, and this concentration is the lower limit of the concentration measurable from the fluorescence intensity using a plate reader as understood from the result of Example 1.
- the result of FIG. 10 shows that according to the method of the present invention, detection of a nucleic acid having a specific base sequence using a molecular beacon is a conventional detection method based on fluorescence intensity. Compared to the above, it is shown that it can be achieved with higher accuracy even in a solution having a low concentration of the luminescent probe.
- Detection of nucleic acid in sample solution from which unreacted fluorescently labeled probe has been removed by physical purification As a luminescent probe, a short fluorescently labeled nucleic acid (fluorescently labeled probe) is bound to the target nucleic acid (target nucleic acid). Thereafter, it was verified that the target nucleic acid can be detected by the method of the present invention in the sample solution obtained by removing the unreacted fluorescently labeled probe by a physical purification method. (See Fig. 3 (a))
- annealing temperature annealing temperature
- 100 pM: 10 nM. 95 ° C, 90 ° C, 80 ° C, 70 ° C, 60 ° C, 50 ° C, 40 ° C, 30 ° C, 20 ° C, 10 minutes each, -0.1 ° C / second
- a sample (Probe) was prepared by similarly annealing a solution obtained by dissolving the fluorescently-labeled probe at 10 nM in STE buffer.
- Target nucleic acid 5'-gaaacagctatgaccatgattacgccaagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattcactggccgtcgttttac-3 '
- Fluorescently labeled probe ATTO647N-ggggatcctctagagtcgacc (ATTO647N is a fluorescent dye.)
- FIG. 11 shows the average value (bar graph) and standard deviation (error bar) of the number of pulses detected in each specimen.
- nucleic acids to be observed as two types of fluorescently labeled short nucleic acids (donor peptide nucleic acid and acceptor peptide nucleic acid) that cause fluorescence energy transfer when they are close to each other as luminescent probes In a state in which the light from the fluorescent label of the unreacted luminescent probe is quenched by binding to the (target nucleic acid) and further binding the light acceptor (quenching probe) that specifically binds to the unreacted luminescent probe.
- a fluorescence energy transfer phenomenon is caused between a donor peptide nucleic acid and an acceptor peptide nucleic acid on the target nucleic acid (see FIG.
- the acceptor peptide nucleic acid a luminescent probe serving as an energy acceptor
- the target nucleic acid was selectively detected, and it was verified that the concentration could be determined (see FIGS. 3B and 3E).
- nucleotide sequences of the donor peptide nucleic acid, acceptor peptide nucleic acid, donor peptide nucleic acid quenching nucleic acid, acceptor peptide nucleic acid quenching nucleic acid, and target nucleic acid are as follows.
- Donor peptide nucleic acid Alexa488-OO-cctacgccaccagctccaac Receptor peptide nucleic acid: agctgtatcgtcaaggcact-O-Lys-Alexa594 Quenching probe for donor peptide nucleic acid: ggagctggtggcg-BHQ1-dT-agg-BHQ1 Quenching probe for acceptor peptide nucleic acid: BHQ2-ag-BHQ2-dT-gccttgacgataca Target nucleic acid: atgactgaatataaacttgtggta gttggagctggtggcgtagg caa gagtgccttgacgatacagct aattcagaat In the target nucleic acid, the lower left line part is the binding sequence of the donor peptide nucleic acid, and the lower right line part is the binding sequence of the acceptor peptide nucle
- a single molecule fluorescence measurement device MF20 (Olympus Co., Ltd.) equipped with a confocal fluorescence microscope optical system and a photon counting system is used as an optical analysis device.
- Count data was acquired.
- 1 mW of 488 nm (corresponding to the excitation wavelength of the donor peptide nucleic acid fluorescent dye Alexa488) is used as the excitation light
- the detection light wavelength is 615 nm to (acceptor peptide nucleic acid) using a long-pass filter.
- the moving speed of the position of the light detection region in the sample solution is 67.5 mm / second, and BIN
- the TIME was 10 ⁇ s, and the measurement time was 2 seconds.
- the measurement was performed 5 times for each sample.
- the pulse signal was detected and counted in the time-series photon count data acquired for each sample solution, as in Example 1. Then, an average value and a standard deviation of the number of detected pulses were calculated.
- FIG. 12B shows the average value (bar graph) and standard deviation (error bar) of the number of detected pulses in the sample solution of the target nucleic acid at each concentration described above.
- the number of pulses increased with increasing concentration when the target nucleic acid concentration was 1 pM or more.
- two types of luminescent probes that cause fluorescence energy transfer are coupled to the observation target particle, and the light of the luminescent probe that serves as an energy acceptor is detected to enable selective detection of the observation target particle and determination of its concentration. It was shown that
- nucleic acid observation target particle
- fluorescence quenching method After binding a short fluorescently labeled nucleic acid (fluorescence-labeled probe) to the nucleic acid (target nucleic acid) that becomes the observation target particle, unreacted fluorescence It was verified that the target nucleic acid can be detected by the method of the present invention in a sample solution in which a nucleic acid (quenching probe) labeled with a fluorescence quenching molecule is allowed to act on the labeled probe. (See Fig. 3 (b))
- a single-stranded nucleic acid probe fluorescent probe
- a single-stranded nucleic acid probe quenched probe
- Tris buffer 100 mM Tris-HCl (pH 8.0) so as to be 5 nM and 2 ⁇ M, respectively.
- double-stranded nucleic acid target nucleic acid was dissolved in Tris buffer so as to be 100 pM, 1 nM, and 10 nM.
- the temperature of these solutions was raised to 95 ° C. using a thermal cycler and then lowered to 20 ° C. over 90 minutes to hybridize the fluorescent probe to the target nucleic acid.
- the base sequences of the fluorescent probe, quenching probe, and target nucleic acid are as follows.
- Fluorescent probe aaacttgtggtagttggagctgttggcgtagg Quenching probe: aacagctccaactaccacaagttt Target nucleic acid: gaagtacagttcattacgatacacgtctgcagtcaactggaattttcatgattgaatttttgtaaggtattttgaaataatttttcatataaaggtgagtttgtattaaaaggtactggtggagtatttgatagtgtattaaccttatgtgtgacatgttctaatatagtcacattttttttattataaggcctgctgaaaatgactgaaaatgactgaaatataaacttgtggtagttggagctggcgtaggcaagagtgccttgac
- the photodetection area in the sample solution was rotated at a moving speed of 15 mm / sec, the BIN TIME was set to 10 ⁇ sec, and the measurement time was 5 seconds for 2 seconds. Then, similarly to Example 1, the time series light intensity data obtained by the measurement was smoothed, and then the peak was detected by differentiation. Among the regions regarded as pulse signals, pulses that can approximate a Gaussian function and have an intensity of 1 or more were counted.
- the number of pulses (average value of five measurements) obtained from each sample solution is shown in FIG. As can be seen from the figure, the number of pulses increased with increasing target nucleic acid concentration. In particular, since there is no overlap between the case where the standard deviation of the five measurements is 0M and the case of 100pM, it is suggested that the nucleic acid concentration of 100pM or more can be measured according to the method of this embodiment.
- Non-Patent Document 4 a single-stranded nucleic acid (E-probe-) in which the 5 ′ end is labeled with a fluorescent molecule (white circle) and a quenching molecule (black circle) in close proximity.
- N probe a single-stranded nucleic acid
- target nucleic acid-observed particle target nucleic acid-observed particle
- the fluorescent molecule on the 5 ′ end emits light, whereby the presence of a nucleic acid having a specific base sequence is detected. That is, this is an example of a reaction in which the luminous efficiency changes depending on the type of observation target particle. (See Fig. 3 (h))
- the quenching molecule Dabcyl at the 5 ′ end a single-stranded nucleic acid probe in which the fluorescent molecule Alexa488 is modified at the third base t from the 5 ′ end (E probe, Japan Bioservice), and the phosphosal at the 3 ′ end
- a fate-modified single-stranded nucleic acid probe (N probe, Nippon Bioservice) was dissolved in a solution of 100 mM Tris-HCl (pH 8.0) so as to be 10 nM and 100 nM, respectively.
- a single-stranded nucleic acid was dissolved in a solution of 100 mM Tris-HCl (pH 8.0) so as to be 10 pM, 100 pM, 1 nM, and 10 nM, respectively. Then, 3 ⁇ L of the E probe solution, 3 ⁇ L of the N probe solution, 3 ⁇ L of each target nucleic acid solution, and 21 ⁇ L of a solution of 100 mM Tris-HCl (pH 8.0) containing 400 mM NaCl are mixed, and a thermal cycler is used. After raising the temperature of these solutions to 95 ° C., they were reacted at 50 ° C. for 1 hour.
- the base sequences of the E probe, N probe, and target nucleic acid are as follows.
- E probe tcttgcctacgccaccagctccaac
- N probe ttctgaattagctgtatcgtcaaggcac
- Target nucleic acid gttggagctggtggcgtaggcaagagtgccttgacgatacagctaattcagaa Therefore, the light of Alexa488 on the E probe is detected only when the E probe and the N probe bind to the target nucleic acid and the quenching molecule Dabcyl on the E probe is released.
- a single-molecule fluorescence measuring device MF20 (Olympus Corporation) equipped with an optical system of a confocal fluorescence microscope and a photon counting system is used as an optical analysis device. Intensity data (photon count data) was obtained. At that time, laser light of 488 nm of 200 ⁇ W was used as excitation light, and light in a wavelength band of 510 to 560 nm was measured using a bandpass filter to generate time-series light intensity data.
- the photodetection area in the sample solution was rotated at a moving speed of 15 mm / sec, the BIN TIME was set to 10 ⁇ sec, and the measurement time was 5 seconds for 2 seconds. Then, similarly to Example 1, the time series light intensity data obtained by the measurement was smoothed, and then the peak was detected by differentiation. Among the regions regarded as pulses, pulses that can be approximated to a Gaussian function and have an intensity of 1 or more were counted.
- the number of pulses increased with increasing target nucleic acid concentration.
- the nucleic acid concentration of 10 pM or more can be measured according to the method of this embodiment. .
- the particles crossing the light detection region are moved while moving the position of the micro region which is the light detection region in the sample solution, that is, while scanning the sample solution.
- Calculation of fluctuations in fluorescence intensity executed in FCS, FIDA, etc. by detecting the presence of particles individually by detecting the bound luminescent probe, or by counting such particles, etc. Therefore, it is possible to detect the state or characteristics of the observation target particles in the sample solution in which the concentration or number density of the observation target particles is lower than the level handled by FCS, FIDA, or the like.
- the optical analysis technique of the present invention basically uses the same optical system as FCS, FIDA, etc., it may be executed in combination with FCS, FIDA, etc.
- FCS optical analysis technique
- the optical analysis technique of the present invention in the case of detecting an interaction between a plurality of types of substances in a solution containing a plurality of types of substances, and when the concentration difference between the substances is large, for example, the concentration of one of the substances is on the order of nM.
- the other substance is on the order of pM
- the high concentration substance is measured and analyzed by FCS or FIDA, and the low concentration substance is measured using the optical analysis technique of the present invention.
- an analysis aspect such as performing an analysis will be performed. In such a case, it is advantageous to prepare a plurality of photodetectors as illustrated in FIG.
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Abstract
Description
2…光源
3…シングルモードオプティカルファイバー
4…コリメータレンズ
5、14a…ダイクロイックミラー
6、7、11…反射ミラー
8…対物レンズ
9…マイクロプレート
10…ウェル(試料溶液容器)
12…コンデンサーレンズ
13…ピンホール
14…バリアフィルター
15…マルチモードオプティカルファイバー
16…光検出器
17…ミラー偏向器
17a…ステージ位置変更装置
18…コンピュータ
本発明による方法は、基本的な構成に於いて、図1(A)に模式的に例示されている如き、FCS、FIDA等が実行可能な共焦点顕微鏡の光学系と光検出器とを組み合わせてなる光分析装置により実現可能である。同図を参照して、光分析装置1は、光学系2~17と、光学系の各部の作動を制御すると共にデータを取得し解析するためのコンピュータ18とから構成される。光分析装置1の光学系は、通常の共焦点顕微鏡の光学系と同様であってよく、そこに於いて、光源2から放射されシングルモードファイバー3内を伝播したレーザー光(Ex)が、ファイバーの出射端に於いて固有のNAにて決まった角度にて発散する光となって放射され、コリメーター4によって平行光となり、ダイクロイックミラー5、反射ミラー6、7にて反射され、対物レンズ8へ入射される。対物レンズ8の上方には、典型的には、1~数十μLの試料溶液が分注される試料容器又はウェル10が配列されたマイクロプレート9が配置されており、対物レンズ8から出射したレーザー光は、試料容器又はウェル10内の試料溶液中で焦点を結び、光強度の強い領域(励起領域)が形成される。試料溶液中には、観測対象物である粒子と、かかる粒子と結合する発光プローブ、典型的には、蛍光色素等の発光標識が付加された分子が分散又は溶解されており、発光プローブと結合又は会合した粒子(実験の態様によっては、粒子と一旦結合した後粒子から解離した発光プローブ)が励起領域に進入すると、その間、発光プローブが励起され光が放出される。放出された光(Em)は、対物レンズ8、ダイクロイックミラー5を通過し、ミラー11にて反射してコンデンサーレンズ12にて集光され、ピンホール13を通過し、バリアフィルター14を透過して(ここで、特定の波長帯域の光成分のみが選択される。)、マルチモードファイバー15に導入されて、光検出器16に到達し、時系列の電気信号に変換された後、コンピュータ18へ入力され、後に説明される態様にて光分析のための処理が為される。なお、当業者に於いて知られている如く、上記の構成に於いて、ピンホール13は、対物レンズ8の焦点位置と共役の位置に配置されており、これにより、図1(B)に模式的に示されている如きレーザー光の焦点領域、即ち、励起領域内から発せられた光のみがピンホール13を通過し、励起領域以外からの光は遮断される。図1(B)に例示されたレーザー光の焦点領域は、通常、1~10fL程度の実効体積を有する本光分析装置に於ける光検出領域であり(典型的には、光強度が領域の中心を頂点とするガウス型分布又はローレンツ型分布となる。実効体積は、光強度が1/e2となる面を境界とする略楕円球体の体積である。)、コンフォーカル・ボリュームと称される。また、本発明では、1つの粒子及び発光プローブの結合体又は発光プローブからの光、例えば、一個又は数個の蛍光色素分子からの微弱光が検出されるので、光検出器16としては、好適には、フォトンカウンティングに使用可能な超高感度の光検出器が用いられる。また、顕微鏡のステージ(図示せず)には、観察するべきウェル10を変更するべく、マイクロプレート9の水平方向位置を移動するためのステージ位置変更装置17aが設けられていてよい。ステージ位置変更装置17aの作動は、コンピュータ18により制御されてよい。かかる構成により、検体が複数在る場合にも、迅速な計測が達成可能となる。
FCS、FIDA等の分光分析技術は、従前の生化学的な分析技術に比して、必要な試料量が極めて少なく、且つ、迅速に検査が実行できる点で優れている。しかしながら、FCS、FIDA等の分光分析技術では、原理的に、観測対象粒子の濃度や特性は、蛍光強度のゆらぎに基づいて算定されるので、精度のよい測定結果を得るためには、試料溶液中の観測対象粒子の濃度又は数密度が、図15(A)に模式的に描かれているように、蛍光強度の計測中に常に一個程度の観測対象粒子が光検出領域CV内に存在するレベルであり、同図の右側に示されている如く、計測時間中に常に有意な光強度(フォトンカウント)が検出されることが要求される。もし観測対象粒子の濃度又は数密度がそれよりも低い場合、例えば、図15(B)に描かれているように、観測対象粒子がたまにしか光検出領域CV内へ進入しないレベルである場合には、同図の右側に例示されている如く、有意な光強度(フォトンカウント)が、計測時間の一部にしか現れないこととなり、精度のよい光強度のゆらぎの算定が困難となる。また、観測対象粒子の濃度が計測中に常に一個程度の観測対象粒子が光検出領域内に存在するレベルよりも大幅に低い場合には、光強度のゆらぎの演算に於いて、バックグラウンドの影響を受けやすく、演算に十分な量の有意な光強度データを得るために計測時間が長くなる。
図1(A)に例示の光分析装置1を用いた本発明による方法に於いては、具体的には、(1)発光プローブとそれが結合する観測対象粒子を含む試料溶液の調製、(2)試料溶液の光強度の測定処理過程と、(3)測定された光強度の分析処理過程とが実行される。
本発明の方法の観測対象粒子は、溶解された分子等の、試料溶液中にて分散し溶液中にてランダムに運動する粒子であれば、任意のものであってよく、例えば、タンパク質、ペプチド、核酸、脂質、糖鎖、アミノ酸若しくはこれらの凝集体などの生体分子、ウイルス、細胞、或いは、金属コロイド、その他の非生物学的分子などであってよい。観測対象粒子は、典型的には、試料溶液(典型的には水溶液であるが、これに限定されず、有機溶媒その他の任意の液体であってよい。)中にて、発光標識(蛍光分子、りん光分子、化学・生物発光分子)が任意の態様にて付加された発光プローブと混合され、発光プローブが観測対象粒子と結合又は会合して結合体を形成することにより、発光プローブの発する光が観測対象粒子の存在の目印となり、観測対象粒子が検出されることとなる(後で述べる如く、実験の態様によっては、観測対象粒子に一旦結合した後、所定の処理を経て粒子から遊離した発光プローブが検出されることにより観測対象粒子が検出される。)。観測対象粒子と発光プローブの組合せの例としては、観測対象粒子が核酸であるときには、発光プローブは、観測対象粒子である核酸の塩基配列に相補的な塩基配列を有する核酸又は核酸類似物、核酸結合性タンパク質、核酸結合性抗体などが選択される。具体的な例として、DNA-RNAの会合体である観測対象粒子に対して、発光プローブとして蛍光標識されたDNA-RNAハイブリッド識別抗体とする例(ハイブリッドキャプチャー法)が挙げられる。
観測対象粒子に結合していない発光プローブからの光を検出結果から排除する構成の一つに於いては、図3(a)に模式的に示されている如く、粒子及び発光プローブの結合体の形成後に、観測対象粒子に結合していない発光プローブ又は発光プローブ単体と、粒子及び発光プローブの結合体又は観測対象粒子に結合した発光プローブとの特性の違い、例えば、それらの大きさ又は分子量、任意の物質に対する親和性、帯電状態等の違いを利用して複数の物質を物理的に分離する任意の手法により、観測対象粒子に結合していない発光プローブ又は発光プローブ単体が試料溶液から分離され除去されてよい。具体的には、クロマトグラフィー(親水/疎水性クロマトグラフィー、アフィニティ・クロマトグラフィー、イオン交換クロマトグラフィーなど)、限外濾過、電気泳動、相分離、遠心分離、溶媒抽出、或いは、フィルター吸着等による吸着・抽出又は洗浄を含む操作等の、この分野で通常行われる物理的な物質の分離方法が採用されてよい。また、ELISA法の如く、観測対象粒子及び発光プローブを混合した試料溶液に、更に、観測対象粒子に結合する別のプローブ(分離用プローブ)を混合して観測対象粒子に結合させ、次いで、試料溶液を分離用プローブに結合する担体に曝して、観測対象粒子と発光プローブと分離用プローブとからなる結合体のみを担体に保持する一方、試料溶液中の発光プローブ単体を分離及び除去し(例えば、洗浄する)、試料溶液から発光プローブ単体を排除する方法が用いられてもよい。
この分野に於いて、或る物質に結合している場合と結合していない場合とで波長特性が変化する蛍光色素が種々知られている。そこで、観測対象粒子に結合していない発光プローブ又は発光プローブ単体からの光を検出結果から排除するために、図3(c)に模式的に描かれている如く、観測対象粒子に結合すると波長特性が変化する蛍光色素が、発光プローブとして採用されてよい。かかる発光プローブを採用する場合には、発光プローブが観測対象粒子に結合した場合に発光プローブから放出される光を選択的に検出することにより、単体の又は観測対象粒子に結合していない発光プローブを物理的に排除する操作を要することなく、観測対象粒子及び発光プローブの結合体又は観測対象粒子に結合した発光プローブからの光を選択的に検出できることとなる。そのような蛍光色素としては、観測対象粒子に結合すると、励起及び/又は発光波長が変化するもの、或いは、観測対象粒子に結合すると、蛍光強度を顕著に増大するものであってよい。そのような例としては、例えば、核酸又は核酸類似物である観測対象粒子に対しては、核酸のインターカレーター蛍光色素(エチジウム・ブロマイド、アクリジン・オレンジ、SYTOX Orange、SYTOX Red、SYBR Green I、SYBR Green II、SYBR Gold、Picogreen、OllGreen、Gel Red、Gel Green、Ribo Green、EvaGreen、シアニン骨格を有する色素など)が発光プローブとして採用可能である。また、観測対象粒子がタンパク質である場合には、タンパク質と結合して周囲環境が変化することにより蛍光強度や蛍光波長が変化する色素が、発光プローブとして採用可能である。そのような色素としては、例えば、疎水性プローブである1-アニリノナフタレン-8-スルホン酸(ANS)、N-メチル-2-アニリノナフタレン-6-スルホン酸(MANS)、2-p-トルイジニルナフタレン-6-スルホン酸(TNS)などのナフタレンスルホン酸類、ジメチルアミノナフタレン(ダンシル)類、局所的なpHや誘電率の影響を受けやすいTAMRA、フルオレセイン、6-joe、BODIPY、TMR、BODIPY TR、Alexa 488、Alexa 532、BODIPY FL、BODIPY FL/C3、BODIPY FL/C6、FITC、EDANS、ローダミン6G、TMR、TMRITC、x-ローダミン、Texas Red、BODIPY 5-FAM、BODIPY R6G、BODIPY 581といった蛍光色素が利用可能である。
観測対象粒子に結合していない発光プローブからの光を検出結果から排除するための別の構成として、少なくとも2種類の蛍光色素を用いて蛍光エネルギー移動現象の効果を利用して、観測対象粒子に結合していない発光プローブから光が発せられないようにするか、観測対象粒子及び発光プローブの結合体又は観測対象粒子に結合した発光プローブからの光の波長と、単体の又は観測対象粒子に結合していない発光プローブからの光の波長とが互いに異なるようにし、観測対象粒子及び発光プローブの結合体又は観測対象粒子に結合した発光プローブからの光のみを装置1で検出する手法が採用されてもよい。蛍光エネルギー移動現象を利用した態様は、例えば、以下の如くであってよい。
ところで、核酸の塩基配列、構造又は特性を研究する分野に於いて、蛍光エネルギー移動現象が発生するエネルギー・ドナー部位とエネルギー・アクセプター部位とを有する核酸分子であって、その塩基配列と相補的な核酸に結合した状態に於いて所定の分解反応で分解されるよう構成された核酸分子を、核酸の塩基配列を探索するためのプローブとして利用する実験方法が知られている。かかる実験方法に於いては、端的に述べれば、まず、検査したい試料へ上記のプローブが添加され、もしその試料中にプローブの塩基配列と相補的な塩基配列を含む核酸が存在している場合には、その核酸にプローブが結合することとなる(図3(h)左図参照)。その状態で所定の分解反応が実行されると、核酸に結合したプローブのみ又はプローブと核酸の両方が分解され、プローブ上のエネルギー・ドナー部位とエネルギー・アクセプター部位とがばらばらになるため(図3(h)右図参照)、蛍光エネルギー移動現象が発生しなくなり、その結果、エネルギー・ドナー部位からの光(波長1)が観測可能となる。一方、試料中にプローブの塩基配列と相補的な塩基配列を含む核酸が存在しない場合には、プローブは、核酸に結合せず、所定の分解反応を実行してもプローブは分解されず、従って、プローブ上で、エネルギー・ドナー部位からの光は、エネルギー・アクセプター部位に吸収され外部に放出されないままとなる。即ち、上記の実験では、エネルギー・ドナー部位からの光が検出されるか否かによって、試料中に検出したい核酸が試料中に存在するか否かが検出できることとなる。
(a)検査されるべき核酸又は核酸類似物(観測対象粒子)を含む試料溶液に、蛍光エネルギー移動現象が発生するエネルギー・ドナー部位とエネルギー・アクセプター部位とを有するDNAである発光プローブを添加し、5’-3’エキソヌクレアーゼ活性を有するDNAポリメラーゼにより発光プローブが分解されるか否かを検出する方法(Taqman法)、
(b)検査されるべき核酸又は核酸類似物(観測対象粒子)を含む試料溶液に、蛍光エネルギー移動現象が発生するエネルギー・ドナー部位とエネルギー・アクセプター部位とを有し一部にRNAを含むDNAである発光プローブを添加し、RNaseHにより発光プローブが分解されるか否かを検出する方法(Cycleave法)、
(c)検査されるべき核酸又は核酸類似物(観測対象粒子)を含む試料溶液に、蛍光エネルギー移動現象が発生するエネルギー・ドナー部位とエネルギー・アクセプター部位とを有し一部に制限酵素識別領域を含むDNAである発光プローブを添加し、制限酵素により発光プローブが分解されるか否かを検出する方法、
(d)検査されるべき核酸又は核酸類似物(観測対象粒子)を含む試料溶液に、蛍光エネルギー移動現象が発生するエネルギー・ドナー部位とエネルギー・アクセプター部位とを有するDNAである発光プローブを添加し、二重鎖核酸を特異的に分解するエキソヌクレアーゼにより発光プローブが分解されるか否かを検出する方法
などが挙げられる。これらの実験を本発明の方法を用いて実行する場合、試料溶液は、それぞれの実験に於ける通常の態様にて調製されてよい。後に詳細に述べる光強度の測定に於いて検出されるべき光の波長は、発光プローブが分解された後に放出される光の波長となる。また、検出される粒子数は、直接的には、分解した発光プローブの数であるが、その数は、発光プローブが結合した核酸又は核酸類似物の分子の数と等しいこととなる。
本発明の光分析に於ける光強度の測定は、測定中にミラー偏向器17を駆動して、試料溶液内での光検出領域の位置の移動(試料溶液内の走査)を行う他は、FCS又はFIDAに於ける光強度の測定過程と同様の態様にて実行されてよい。操作処理に於いて、典型的には、マイクロプレート9のウェル10に試料溶液を注入して顕微鏡のステージ上に載置した後、使用者がコンピュータ18に対して、測定の開始の指示を入力すると、コンピュータ18は、記憶装置(図示せず)に記憶されたプログラム(試料溶液内に於いて光検出領域の位置を移動するべく光路を変更する手順と、光検出領域の位置の移動中に光検出領域からの光を検出する手順)に従って、試料溶液内の光検出領域に於ける励起光の照射及び光強度の計測が開始される。かかる計測中、コンピュータ18のプログラムに従った処理動作の制御下、ミラー偏向器17は、ミラー7(ガルバノミラー)を駆動して、ウェル10内に於いて光検出領域の位置の移動を実行し、これと同時に光検出器16は、逐次的に検出された光を電気信号に変換してコンピュータ18へ送信し、コンピュータ18では、任意の態様にて、送信された光信号から時系列の光強度データを生成して保存する。なお、典型的には、光検出器16は、一光子の到来を検出できる超高感度光検出器であるので、光の検出は、所定時間に亘って、逐次的に、所定の単位時間毎(BIN TIME)に、例えば、10μ秒毎に光検出器に到来するフォトンの数を計測する態様にて実行されるフォトンカウンティングであり、時系列の光強度のデータは、時系列のフォトンカウントデータであってよい。
(2Wo)2=6D・Δt …(1)
から、
Δt=(2Wo)2/6D …(2)
となるので、観測対象粒子がブラウン運動により移動する速度(拡散移動速度)Vdifは、概ね、
Vdif=2Wo/Δt=3D/Wo …(3)
となる。そこで、光検出領域の位置の移動速度は、かかるVdifを参照して、それよりも十分に早い値に設定されてよい。例えば、観測対象粒子の拡散係数が、D=2.0×10-10m2/s程度であると予想される場合には、Woが、0.62μm程度だとすると、Vdifは、1.2×10-3m/sとなるので、光検出領域の位置の移動速度は、その10倍以上の15mm/sなどと設定されてよい。なお、観測対象粒子の拡散係数が未知の場合には、光検出領域の位置の移動速度を種々設定して光強度の変化のプロファイルが、予想されるプロファイル(典型的には、励起光強度分布と略同様)となる条件を見つけるための予備実験を繰り返し実行して、好適な光検出領域の位置の移動速度が決定されてよい。
上記の処理により試料溶液の時系列の光強度データが得られると、コンピュータ18に於いて、記憶装置に記憶されたプログラムに従った処理により、下記の如き光強度の分析が実行されてよい。
時系列の光強度データに於いて、一つの観測対象粒子の光検出領域を通過する際の軌跡が、図5(A)に示されている如く略直線状である場合、その粒子に対応する光強度の変化は、図7(A)に模式的に描かれている如く、(光学系により決定される)光検出領域の光強度分布を反映したプロファイル(通常、略釣鐘状)を有する。そこで、観測対象粒子の検出の一つの手法に於いて、光強度に対して閾値Ioが設定され、その閾値を超える光強度が継続する時間幅Δτが所定の範囲にあるとき、その光強度のプロファイルが一つの粒子が光検出領域を通過したことに対応すると判定され、一つの観測対象粒子の検出が為されるようになっていてよい。光強度に対する閾値Io及び時間幅Δτに対する所定の範囲は、光検出領域に対して所定の速度にて相対的に移動する観測対象粒子と発光プローブとの結合体(又は粒子との結合後分解され遊離した発光プローブ)から発せられる光の強度として想定されるプロファイルに基づいて定められるところ、具体的な値は、実験的に任意に設定されてよく、また、観測対象粒子と発光プローブとの結合体(又は粒子との結合後分解され遊離した発光プローブ)の特性によって選択的に決定されてよい。
ガウス分布:
I=A・exp(-2t2/a2) …(4)
であると仮定できるときには、有意な光強度のプロファイル(バックグラウンドでないと明らかに判断できるプロファイル)に対して式(4)をフィッティングして算出された強度A及び幅aが所定の範囲内にあるとき、その光強度のプロファイルが一つの観測対象粒子が光検出領域を通過したことに対応すると判定され、一つの観測対象粒子の検出が為されてよい。しかしながら、強度A及び幅aが所定の範囲外にあるときには、ノイズ又は異物として分析に於いて無視される。
観測対象粒子のカウンティングは、上記の観測対象粒子の検出の手法により検出された粒子の数を、任意の手法により、計数することにより為されてよい。しかしながら、粒子の数が大きい場合には、例えば、図6及び図7(B)に例示された処理により為されてよい。
観測対象粒子のカウンティングが為されると、時系列光信号データの取得の間に光検出領域の通過した領域の総体積を用いて、観測対象粒子の数密度又は濃度が決定される。しかしながら、光検出領域の実効体積は、励起光又は検出光の波長、レンズの開口数、光学系の調整状態に依存して変動するため、設計値から算定することは、一般に困難であり、従って、光検出領域の通過した領域の総体積を算定することも簡単ではない。そこで、典型的には、粒子の濃度が既知の溶液(参照溶液)について、検査されるべき試料溶液の測定と同様の条件にて、上記に説明した光強度の測定、粒子の検出及びカウンティングを行い、検出された粒子の数と参照溶液の粒子の濃度とから、光検出領域の通過した領域の総体積、即ち、観測対象粒子の検出数と濃度との関係が決定されるようになっていてよい。参照溶液の粒子としては、好ましくは、観測対象粒子が形成する粒子及び発光プローブ結合体(又は観測対象粒子に結合後遊離した発光プローブ)と同様の波長特性を有する発光標識(蛍光色素等)であってよい。具体的には、例えば、粒子の濃度Cの参照溶液について、その粒子の検出数がNであったとすると、光検出領域の通過した領域の総体積Vtは、
Vt=N/C …(5)
により与えられる。また、参照溶液として、複数の異なる濃度の溶液が準備され、それぞれについて測定が実行されて、算出されたVtの平均値が光検出領域の通過した領域の総体積Vtとして採用されるようになっていてよい。そして、Vtが与えられると、粒子のカウンティング結果がnの試料溶液の粒子の数密度cは、
c=n/Vt …(6)
により与えられる。なお、光検出領域の体積、光検出領域の通過した領域の総体積は、上記の方法によらず、任意の方法にて、例えば、FCS、FIDAを利用するなどして与えられるようになっていてよい。また、本実施形態の光分析装置に於いては、想定される光検出領域の移動パターンについて、種々の標準的な粒子についての濃度Cと粒子の数Nとの関係(式(5))の情報をコンピュータ18の記憶装置に予め記憶しておき、装置の使用者が光分析を実施する際に適宜記憶された関係の情報を利用できるようになっていてよい。
発光プローブとして、DNAのインターカレーター蛍光色素(DNAに結合したときに蛍光強度が顕著に増大する色素)であるSYTOX Orangeを用い、本発明の方法による試料溶液中のDNAの濃度の測定可能範囲を検証した(図3(c)参照)。なお、対照実験として、プレートリーダーにより計測される蛍光強度によるDNAの濃度の測定可能範囲も測定した。
下記の条件:
20μ秒<パルス幅<400μ秒
ピーク強度>1(フォトン/10μ秒) …(A)
相関係数>0.95
を満たすパルス信号のみを観測対象粒子に対応する光信号であると判定する一方、上記の条件を満たさないパルス信号はノイズとして無視し、観測対象粒子に対応する光信号であると判定された信号の数を「パルス数」として計数した。
本発明の方法によって、モレキュラー・ビーコンを用いて特定の塩基配列の核酸分子が検出可能であることを検証した。モレキュラー・ビーコンとは、既に触れた如く、両端にドナー色素とアクセプター色素がそれぞれ付加された核酸分子であり、単体では、ドナー色素とアクセプター色素との距離が近接しドナー色素からアクセプター色素への蛍光エネルギー移動現象が発生する一方、自身の塩基配列に相補的な塩基配列を有する核酸又は核酸類似物に結合すると、ドナー色素とアクセプター色素との間の距離が離れ、蛍光エネルギー移動現象が発生しないよう構成された分子である(図3(d)参照)。
TAMRA-cctacgccaacagctccaactacgtagg-BHQ2
また、観測対象粒子は、下記の塩基配列を有する核酸を用いた。
gtagttggagctgttggcgtaggcaagagtgccttgacgatacagctaattcag
なお、上記の核酸は、シグマジェノシス株式会社に依頼して合成した。そして、上記のモレキュラー・ビーコン及び観測対象粒子(核酸)は、それぞれ、500pM、100nMとなるように、リン酸緩衝液(0.05% Tween20を含む)に溶解し、試料溶液とした。なお、対照溶液としては、観測対象粒子(核酸)を含まず、500pMにてモレキュラー・ビーコンのみを含む溶液を調製した。
発光プローブとして、蛍光標識された短い核酸(蛍光標識プローブ)を観測対象粒子となる核酸(標的核酸)に結合させた後、未反応の蛍光標識プローブを物理的な精製方法により除去して得られた試料溶液に於いて、本発明の方法により、標的核酸が検出できることを検証した。(図3(a)参照)
標的核酸:
5’-gaaacagctatgaccatgattacgccaagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattcactggccgtcgttttac-3’
蛍光標識プローブ:ATTO647N-ggggatcctctagagtcgacc
(ATTO647Nは、蛍光色素である。)
発光プローブとして互いに近接すると蛍光エネルギー移動を起こす2種類の蛍光標識された短い核酸(供与体ペプチド核酸、受容体ペプチド核酸)を観測対象粒子となる核酸(標的核酸)に結合させ、更に、未反応の発光プローブに特異的に結合する光アクセプター(消光プローブ)を結合させて未反応の発光プローブの蛍光標識からの光を消光させた状態にて、標的核酸上の供与体ペプチド核酸と受容体ペプチド核酸との間で蛍光エネルギー移動現象を起こさせ(図12(a)参照)、受容体ペプチド核酸(エネルギー・アクセプターとなる発光プローブ)からの光を検出することにより、標的核酸を選択的に検出し、その濃度が決定できることを検証した(図3(b)、(e)参照)。
供与体ペプチド核酸:Alexa488-OO-cctacgccaccagctccaac
受容体ペプチド核酸:agctgtatcgtcaaggcact-O-Lys-Alexa594
供与体ペプチド核酸用消光プローブ:ggagctggtggcg-BHQ1-dT-agg-BHQ1
受容体ペプチド核酸用消光プローブ:BHQ2-ag-BHQ2-dT-gccttgacgataca
標的核酸:
atgactgaatataaacttgtggtagttggagctggtggcgtaggcaagagtgccttgacgatacagctaattcagaat
標的核酸に於いて、左下線部は、供与体ペプチド核酸の結合配列であり、右下線部は、受容体ペプチド核酸の結合配列である。Alexa488、Alexa594は、蛍光色素であり、BHQ1、BHQ2は、それぞれ、Alexa488、Alexa594に対する消光分子である。
TIMEを10μ秒とし、測定時間は、2秒間とした。また、測定は各試料5回行った。光強度の測定後、実施例1の場合と同様に、各試料溶液について取得された時系列フォトンカウントデータに於いてパルス信号の検出及び計数を行った。そして、それらの検出パルス数の平均値と標準偏差を算出した。
発光プローブとして、蛍光標識された短い核酸(蛍光標識プローブ)を観測対象粒子となる核酸(標的核酸)に結合させた後、未反応の蛍光標識プローブに対して蛍光消光分子を標識した核酸(消光プローブ)を作用させた試料溶液に於いて、本発明の方法により、標的核酸が検出できることを検証した。(図3(b)参照)
蛍光プローブ:aaacttgtggtagttggagctgttggcgtagg
消光プローブ:aacagctccaactaccacaagttt
標的核酸:
gaagtacagttcattacgatacacgtctgcagtcaactggaattttcatgattgaattttgtaaggtattttgaaataatttttcatataaaggtgagtttgtattaaaaggtactggtggagtatttgatagtgtattaaccttatgtgtgacatgttctaatatagtcacattttcattatttttattataaggcctgctgaaaatgactgaatataaacttgtggtagttggagctggtggcgtaggcaagagtgccttgacgatacagctaattcagaatcattttgtggacgaatatgatccaacaatagaggtaaatcttgttttaatatgcatattactggtgcaggaccattctttgatacagataaaggtttctctgaccattttcatgagtacttattacaagataattatgctgaaagttaagttatctgaaatgtaccttgggtttcaagttatatgtaaccattaatatgggaactttact
上記の試料溶液に於いて、消光プローブは多量に存在するので、標的核酸に結合しなかった蛍光プローブは、全て消光プローブに結合し、消光プローブに結合した蛍光プローブからの蛍光は、実質的に消光される。(図13(A)参照)
本発明の方法によって、QUAL(Quenched auto-ligation)反応(非特許文献4)を用いて特定の塩基配列の核酸分子が検出可能であることを検証した。図14(A)に示されている如く、QUAL反応に於いては、5’末端が蛍光分子(白丸)と消光分子(黒丸)とが近接して標識された一本鎖核酸(Eプローブ-発光プローブ)と、3’末端がホスホサルフェイト修飾された一本鎖核酸(Nプローブ)とが、塩基配列が検査されるべき一本鎖核酸(標的核酸-観測対象粒子)上に、Eプローブの5’末端とNプローブの3’末端とが互いに近接して結合したとき(図14(A)上段)、EプローブとNプローブとが互いに結合すると共に(図中Xが付された個所)、5’末端上の消光分子が遊離することにより(図14(A)下段)、5’末端の蛍光分子が発光し、これにより特定の塩基配列を有する核酸の存在が検出される。即ち、観測対象粒子の種類に依存して発光効率が変化する反応の一例である。(図3(h)参照)
Eプローブ:tcttgcctacgccaccagctccaac
Nプローブ:ttctgaattagctgtatcgtcaaggcac
標的核酸:
gttggagctggtggcgtaggcaagagtgccttgacgatacagctaattcagaa
従って、標的核酸にEプローブとNプローブとが結合し、Eプローブ上の消光分子Dabcylが遊離した場合のみ、Eプローブ上のAlexa488の光が検出されることとなる。
Claims (15)
- 共焦点顕微鏡又は多光子顕微鏡の光学系を用いて試料溶液中にて分散しランダムに運動する粒子に結合する発光プローブからの光を検出して前記粒子を検出する方法であって、
前記粒子と前記発光プローブとを含む試料溶液を調製する過程と、
前記光学系の光路を変更することにより前記試料溶液内に於いて前記光学系の光検出領域の位置を移動する過程と、
前記試料溶液内に於いて前記光検出領域の位置を移動させながら前記光検出領域からの光を検出する過程と、
前記検出された光から個々の粒子に結合した発光プローブからの光信号を個別に検出して前記粒子を個別に検出する過程と
を含むことを特徴とする方法。 - 請求項1の方法であって、更に、前記個別に検出された粒子の数を計数して前記光検出領域の位置の移動中に検出された前記粒子の数を計数する過程を含むことを特徴とする方法。
- 請求項1又は2の方法であって、前記光検出領域の位置を移動する過程に於いて、前記光検出領域の位置が所定の速度にて移動されることを特徴とする方法。
- 請求項1乃至3のいずれかの方法であって、前記光検出領域の位置を移動する過程に於いて、前記光検出領域の位置が前記粒子に結合した前記発光プローブの拡散移動速度よりも速い速度にて移動されることを特徴とする方法。
- 請求項1乃至4のいずれかの方法であって、前記個々の粒子に結合した発光プローブからの光信号を個別に検出して前記粒子を個別に検出する過程に於いて、検出された時系列の光信号の形状に基づいて、1つの粒子が前記光検出領域に入ったことが検出されることを特徴とする方法。
- 請求項1乃至5のいずれかの方法であって、前記試料溶液を調製する過程に於いて、前記粒子に結合していない前記発光プローブを前記試料溶液内から分離する過程を含むことを特徴とする方法。
- 請求項1乃至5のいずれかの方法であって、前記試料溶液を調製する過程に於いて、前記粒子に結合していない前記発光プローブに該発光プローブの発する光を吸収するアクセプターを結合させる過程を含むことを特徴とする方法。
- 請求項1乃至5のいずれかの方法であって、前記発光プローブが、前記粒子に結合すると発光特性が変化する物質であり、前記検出される光が、前記粒子に結合した前記発光プローブから放出される光であることを特徴とする方法。
- 請求項1乃至5のいずれかの方法であって、前記発光プローブが、互いに近接しているときに蛍光エネルギー移動現象を発生するエネルギー・ドナー部位とエネルギー・アクセプター部位とを有し且つ前記粒子に結合した状態と前記粒子に結合していない状態との間で前記エネルギー・ドナー部位と前記エネルギー・アクセプター部位との距離が異なり、前記粒子に結合した状態と前記粒子に結合していない状態とで放出する光の波長特性が異なる物質であり、前記検出される光が前記粒子に結合した前記発光プローブから放出される光であることを特徴とする方法。
- 請求項1乃至5のいずれかの方法であって、前記発光プローブが蛍光エネルギー移動を生ずるエネルギー・ドナー部位とエネルギー・アクセプター部位とを有し、前記試料溶液を調製する過程が前記粒子と結合した前記発光プローブを分解する反応を実行する過程を含み、前記検出される光が前記反応により分解された前記発光プローブから放出される光であることを特徴とする方法。
- 請求項1乃至5のいずれかの方法であって、前記発光プローブが、蛍光エネルギー移動現象に於けるエネルギー・ドナーと成る第一のプローブと、前記蛍光エネルギー移動現象に於けるエネルギー・アクセプターと成る第二のプローブとを含み、前記検出される光が、前記第一及び第二のプローブの両方が前記粒子に結合した状態に於いて生ずる蛍光エネルギー移動現象を経て発せられる前記第二のプローブの光であることを特徴とする方法。
- 請求項1乃至5のいずれかの方法であって、前記粒子が前記発光プローブの発する光のエネルギー・アクセプターと成る部位を有し、前記検出される光が、前記発光プローブが前記粒子に結合することにより生ずる蛍光エネルギー移動現象を経て前記粒子の有する前記エネルギー・アクセプター部位から発せられる光であることを特徴とする方法。
- 請求項1乃至5のいずれかの方法であって、前記粒子が発光部位を有し、前記発光プローブが前記粒子の発光部位の発する光のエネルギー・アクセプターと成る部位を有し、前記検出される光が、前記発光プローブが前記粒子に結合することにより生ずる蛍光エネルギー移動現象を経て前記発光プローブから発せられる光であることを特徴とする方法。
- 請求項1乃至5のいずれかの方法であって、前記粒子が核酸であり、前記発光プローブが核酸結合性タンパク質であることを特徴とする方法。
- 請求項1乃至5のいずれかの方法であって、前記発光プローブが、少なくとも二つの構成要素から成る物質であって、前記粒子に結合すると前記少なくとも二つの構成要素の互いの位置が変化して蛍光を発する物質であることを特徴とする方法。
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Also Published As
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CN103026205B (zh) | 2016-03-23 |
EP2584343A1 (en) | 2013-04-24 |
JP5877155B2 (ja) | 2016-03-02 |
EP2584343A4 (en) | 2014-01-08 |
US9395357B2 (en) | 2016-07-19 |
US20130122488A1 (en) | 2013-05-16 |
JPWO2012014778A1 (ja) | 2013-09-12 |
CN103026205A (zh) | 2013-04-03 |
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