JP4652868B2 - Fluorescence analysis method - Google Patents

Description

  The present invention relates to a fluorescence analysis method for detecting an object to be measured using a fluorescent label, wherein the fluorescence relaxation time constant of the fluorescent label is measured.

  Conventionally, when detecting an intracellular substance or cell surface, for example, an epitope, 1) a fluorescent dye is bound to a substance that binds to the detection target (for example, an antibody), and 2) a substance that binds to the detection target (for example, an antibody) Bind a fluorescent dye to a third substance (for example, an anti-antibody) that binds without inhibiting the binding of the substance to the detection target, or 3) bind to a substance (for example, an antibody) that competes with the detection target. A fluorescent dye was bound to the substance, and after B / F separation, the fluorescence emitted from the remaining fluorescent dye, that is, a fluorescent label, was measured.

  However, since there are various fluorescent substances (autofluorescent substances) inside and outside the cell, it is difficult to accurately detect only the fluorescence emitted by the fluorescent label.

  Further, when a carrier such as a microplate is used for the measurement, the carrier emits fluorescence, which may interfere with detection of fluorescence emitted by the fluorescent label.

  Under such circumstances, methods for detecting only fluorescence that is truly desired to be measured without being influenced by the so-called background fluorescence as described above have been studied. As an example of this, there is an attempt to detect only the fluorescence emitted by the fluorescent label by physically processing the measured fluorescence or calculating it with some parameters, instead of simply measuring the fluorescence intensity.

  More specifically, for example, in the method described in Patent Document 1, a fluorescently labeled antibody or the like is bound to two or more kinds of particles having different refractive indexes, irradiated with laser light, and forward scattered light and breaking scattered light are obtained. Each detection method is described.

Japanese Patent Laid-Open No. 6-271112

  The present invention is a method capable of measuring fluorescence emitted by a fluorescent label from background fluorescence including so-called autofluorescence and measuring it with higher accuracy, and is generated by modulated laser excitation light unlike the method described in Patent Document 1. An object is to provide a fluorescence analysis method for a measurement object by measuring and calculating a fluorescence relaxation time constant of fluorescence.

  In the present invention, a measurement object is directly or indirectly bound to a fluorescently labeled substance using a biological specific reaction, the fluorescent label is excited by irradiation with a modulated laser beam, and the emitted fluorescence is relaxed. The present invention relates to a fluorescence analysis method (1) characterized in that a constant is measured to detect a measurement object.

  Here, the direct binding between the measurement target and the fluorescently labeled substance, or the binding between the measurement target and the fluorescently labeled substance and the substance that binds both the measurement target and the fluorescently labeled substance is based on, for example, an antigen / antibody reaction. .

  In the present invention, the substance w that binds to the measurement target is either 1) competitively bound to the measurement target and the substance x that can be bound to the substance w and is fluorescently labeled, or 2) the measurement The target substance and the substance y that can bind to the substance w are competitively bound, and then the substance z that can be bound to the substance y and is not fluorescently labeled is bound to the substance y without binding to the measurement object. Fluorescence analysis method comprising detecting a measurement object by measuring a relaxation time constant of fluorescence emitted by exciting a fluorescent label of the substance x or the substance z by irradiation with a pulse-modulated laser beam. Regarding (2).

  In the analysis methods (1) and (2), the measurement object is, for example, a cell, an intracellular substance, a site existing on the cell surface, or a substance secreted by the cell.

  In the present invention, since the measurement object is detected using the relaxation time constant of the fluorescence emitted by the fluorescent label, it is possible to distinguish between the background fluorescence emitted from the autofluorescence and the measurement atmosphere and the fluorescence for the detection purpose. Accurate measurement is possible.

  In the present invention, modulated excitation light is irradiated onto a fluorescent label. In particular, depending on the purpose of detection, a measurement object such as an antigen or an antibody is labeled with a fluorescent dye having a different fluorescence relaxation time constant, and each fluorescence relaxation is performed. By measuring the time constant, it is possible to determine the origin of the labeled measurement object and to distinguish it from fluorescence emitted from the atmosphere. In addition, when a plurality of light sources are used, modulation can be performed under different conditions for each light source, and therefore it is possible to separately measure the measurement object using a plurality of fluorescent labels.

  First, the measurement object, the substance that binds to the measurement object, the substance to be fluorescently labeled, the fluorescent dye used for the fluorescent labeling, and the like in the method of the present invention will be described.

The “measurement object” in the method of the present invention is a substance that can be an antigen (for example, a cell, an intracellular substance, a substance secreted by a cell, a low molecular compound, a high molecular compound, a peptide, a protein, a polysaccharide, a bacterium, Microorganisms), antibodies (for example, immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), immunoglobulin E (IgE)), cells, etc. Orphans, which are ligands whose epitope sites and receptors are unknown. The cell type is not particularly limited, and may be any of cultured cells, dead cells, blood immune cells, microorganisms, and the like.
The “substance that binds to the measurement target” is an antibody when the measurement target is a substance or epitope site that can be an antigen, and is an antigen when the measurement target is an antibody. The reaction in which the anti-antibody binds to the antibody is interpreted in the same manner as when the antibody is an antigen and the anti-antibody is an antibody.
Since the “substance to be fluorescently labeled” is a substance that binds directly or indirectly to the measurement target in the fluorescence analysis method (1), it is a substance or epitope site where the measurement target can become an antigen. In some cases, it is an antibody (direct binding) or an anti-antibody (indirect binding with an antibody in between). Alternatively, if digoxigenin (DIG) is bound to the measurement object or antibody, the anti-DIG antibody can be used as a fluorescently labeled substance, and if biotin is bound to the measurement object or antibody, avidin Or streptavidin can be used as a fluorescently labeled substance.

  In the fluorescence analysis method (1), when the measurement target is an antibody, the fluorescently labeled substance is an antigen (direct binding), a substance having an epitope (direct binding), or the substance having the antigen or epitope. An antibody (indirect binding with an antigen in between). Alternatively, if digoxigenin (DIG) is bound to a measurement object, an antigen, a substance having an epitope, or an antibody against the substance having the antigen or epitope, the anti-DIG antibody can be used as a fluorescently labeled substance. If biotin is bound to a measurement object, an antigen, a substance having an epitope, or an antibody against the antigen or epitope-containing substance, avidin or streptavidin can be used as a fluorescently labeled substance.

In the fluorescence analysis method (2), when the measurement target is a substance or epitope site that can be an antigen, the substance w that binds to the measurement target is an antibody. The substance x is a substance that binds to the substance w competitively with the measurement target, such as an antigen. This substance x is fluorescently labeled.
Alternatively, a substance y that is an antigen or the like and is not fluorescently labeled can also be used as a substance that binds competitively to the substance w, that is, an antibody. In this case, the substance y that can be bound to the substance y and is fluorescently labeled is bound to the substance y without binding to the measurement target. Examples of substance z include fluorescent labeling of substance y, which is an antibody against an epitope different from the epitope possessed by the object to be measured, and a substance to which DIG is bound as substance y And anti-DIG antibody prepared, and fluorescently labeled avidin and streptavidin when a substance to which biotin is bound is used as the substance y.

In the said fluorescence analysis method (2), when a measuring object is an antibody, the substance w couple | bonded with a measuring object is a substance which has an antigen or an epitope. The substance x is a substance that binds to the substance w competitively with the measurement object, such as an antibody. This substance x is fluorescently labeled.
Alternatively, a substance y that is an antibody or the like and is not fluorescently labeled can also be used as a substance that binds competitively with the substance w, that is, a substance having an antigen or an epitope. In this case, the substance y that can be bound to the substance y and is fluorescently labeled is bound to the substance y without binding to the measurement target. Examples of the substance z include an anti-antibody against the substance y that is fluorescently labeled, a fluorescently labeled anti-DIG antibody when a substance to which DIG is bound is used as the substance y, and biotin as the substance y. Examples thereof include fluorescently labeled avidin and streptavidin when a bound substance is used.

Fluorescent dyes used for fluorescent labeling are not particularly limited. Examples thereof include FITC (manufactured by Dojin Chemical Co., Ltd.), PE (manufactured by Wako Pure Chemical Industries, Ltd.), Alexa, and the like. Fluor (Molecular Probes), Cy Dye (Amersham Biosciense), Rhodamine (Wako Pure Chemical Industries, etc.), Cascade Blue (Molecular Probes), Cascade Yellow (Molecular Probes) and Naphtofluorescein (Sigma) And Q-Dot (manufactured by Quantum Dot) and Evic-Tag (manufactured by Ocean Optics), which are semiconductor quantum fluorescent dyes having relatively long fluorescence relaxation time constants.
Fluorescent dyes can be bound to antigens, antibodies, etc. by, for example, a method in which the amino group of the antigen or antibody is chemically bonded to the carboxyl group of the fluorescent dye, or the fluorescent dye previously prepared on the antibody It can be performed by a method of binding a complex with an antibody.
In the fluorescence analysis methods (1) and (2), the binding between an antigen and an antibody, the binding between biotin and avidin or streptavidin, and the like can be performed under known conditions and methods.

In the fluorescence analysis method of the present invention, a plurality of antigen-antibody reactions and the like may be performed sequentially or simultaneously. The order of the reactions and the types of reactions that can be performed simultaneously are known to those skilled in the art. In addition, after each reaction, there is a case where B / F separation is performed or not, or a case where measurement is performed after removing an excessive fluorescent label by centrifugation or the like. Depending on the configuration of the system for carrying out the antigen-antibody reaction or the like in the method.
In the method of the present invention, the system for carrying out the antigen-antibody reaction or the like is not particularly limited, but examples are as follows.

  The first configuration example does not use a solid layer. For example, when a measurement target is a cell, the cell is suspended in an appropriate liquid, and a biological specific reaction such as a substance-specific agglutination reaction, antigen-antibody reaction, or ligand / receptor reaction is performed in the suspension system. Do. According to the fluorescence analysis method of the present invention, the reaction on the cell surface and inside can be detected. The fluorescence measurement described later can be performed using, for example, a flow cytometer. In this case, the B / F separation may or may not be performed. This is because the fluorescence due to the fluorescent dye indirectly bound to the cells and the fluorescence due to the fluorescent dye not bound to the cells can be measured separately.

The second configuration example uses particles such as microbeads as a carrier. Either an antigen or an antibody used in the reaction system (except for the measurement target) is bound to the particle, and the measurement target is bound thereto. Therefore, the measurement is performed on the fluorescence by the fluorescent dye bonded to the particles using, for example, a flow cytometer.
When carrying out the fluorescence analysis method (1), for example, the surface of the microbead is coated with an antibody against the antigen that is the measurement target, and after the antigen is bound to the antibody, the antigen is fluorescently labeled. The substance (antibody etc.) is bound (sandwich method).
When the fluorescence analysis method (2) is performed, for example, the surface of the microbead is coated with a substance w (antibody or the like) that binds to the measurement object, and the measurement object (antigen) Etc.) and substance x are competitively combined (competitive method).
When using microbeads, it is usually made of a synthetic high molecular polymer. Of the synthetic polymer, a water-insoluble water-dispersed synthetic polymer is preferred. Specific examples of water-insoluble water-dispersed synthetic polymer microbeads include polystyrene microbeads and (co) polymer microbeads of methacrylic acid fluoroalkyl ester derivatives such as trifluoroethyl methacrylate. it can.
The particle diameter of the microbeads used is not particularly limited as long as it has a size suitable for measurement. For example, when the fluorescence measurement is performed using a flow cytometer in the method of the present invention, usually, microbeads having a diameter of 1 to 20 μm are preferably used.

  The third configuration example uses a microplate such as a 96-well microtiter plate as a carrier. Any of the antigens and antibodies used in the reaction system (but not the measurement object) is bound to the microplate, and the measurement object is bound thereto. Therefore, the measurement is performed for fluorescence using a fluorescent dye bound to the plate, for example, using a fluorescence plate reader or a fluorescence imaging apparatus.

Next, the fluorescence measurement method in the present invention will be described.
In the present invention, the fluorescent label is excited by irradiation with modulated laser light, and the measurement target is detected by measuring the relaxation time constant of the emitted fluorescence.
Specific examples of laser light include semiconductor laser light and gas laser light. Further, the laser light may be monochromatic light, or may be obtained by converging laser light having a plurality of types of wavelengths at a predetermined position in the optical path by passing through a lens. The output of the laser beam is, for example, about 5 to 100 mW.
As a method for modulating the laser beam, the laser beam is modulated by any method selected from the group consisting of high-frequency superposition modulation, specific shape wave modulation, sine wave modulation, linear modulation, and pulse modulation. In the following present invention, description will be given by pulse modulation. The fluorescent label is irradiated with pulse-modulated laser light, the target fluorescence is identified from the fluorescence relaxation time constant of the generated fluorescence, and qualitative or quantitative is performed. This increases the accuracy of detection. By adopting this method, even if there are a plurality of detection or analysis objects, it is possible to measure each of them separately at the same time.

Hereinafter, measurement using a flow cytometer among various fluorescence measurement methods that can be employed in the method of the present invention will be described in detail.
FIG. 1 is a schematic configuration diagram of a flow cytometer 10 that can be used for a fluorescence measurement step in the method of the present invention.
The flow cytometer 10 modulates laser light by a pulse modulation method, and irradiates microbeads and cells (hereinafter, only described as “microbeads” unless otherwise required).
The flow cytometer 10 irradiates the microbeads 12 to be measured with laser light, and does not perform fluorescence light signals that are bound to the microbeads 12, that is, fluorescent light signals emitted by fluorescent labels, and B / F separation, for example. Etc., it is obtained by a signal processing device (fluorescence detection device) 20 for detecting and processing a fluorescent dye not bound to the microbeads 12, that is, a fluorescent light signal emitted from a fluorescent label, and the signal processing device 20. And an analyzer 50 for analyzing fluorescence derived from the fluorescent label bonded to the microbead 12 from the processing result.
Here, when there are two or more types of measurement objects, a plurality of types of fluorescent labels corresponding to the measurement objects one to one are used.

The signal processing device 20 includes a laser light source unit 22, light receiving units 24 and 26, a light source control unit that controls on / off of laser light emission from the laser light source unit 22, and fluorescence emitted by a fluorescent label coupled to the microbead 12. A control / processing unit 28 that includes a signal processing unit that discriminates an optical signal of the above and a fluorescent optical signal emitted from a fluorescent label that is not bonded to the microbead 12, and for excitation light irradiation and fluorescence detection, And a conduit 30 having a flow cell in which microbeads 12 are flowed in a sheath liquid that forms a high-speed flow. A recovery container 32 is provided at the outlet of the conduit 30.
The laser light source unit 22 is a part that emits three laser beams having different wavelengths, for example, laser beams having λ ₁ = 405 nm, λ ₂ = 533 nm, λ ₃ = 650 nm, and the like. A lens system is provided so that the laser beam is focused at a predetermined position in the pipe line 30, and a measurement point of the microbead 12 is formed at this focusing position.

FIG. 2 shows an example of the configuration of the laser light source unit 22.
The laser light source unit 22 emits visible pulsed laser light having a wavelength of 350 nm to 800 nm, and mainly emits a red laser light R intermittently as pulsed laser light with an extremely short pulse width. A G light source 22g that intermittently emits green laser light G as pulse laser light with an extremely short pulse width, and a B light source 22b that emits blue laser light B as pulse laser light with an extremely short pulse width; Dichroic mirrors 23a ₁ and 23a ₂ that transmit laser light in a specific wavelength band and reflect laser light in other wavelength bands, and laser light composed of laser light R, G, and B are used as measurement points in the pipe 30 And a lens system 23c for focusing.

The pulse width of the pulsed laser light is set so that the fluorescence emitted from the fluorescent label can be detected efficiently from the background noise, and is, for example, 0.5 nanoseconds to 4 nanoseconds.
The dichroic mirror 23a ₁ is a mirror that transmits the laser beam R and reflects the laser beam G, and the dichroic mirror 23a ₂ is a mirror that transmits the laser beams R and G and reflects the laser beam B.
With this configuration, the laser beams R, G, and B are combined to become irradiation light that irradiates the microbeads 12 at the measurement point.

The R light source 22r, the G light source 22g, and the B light source 22b are driven by laser drivers 34r, 34g, and 34b, respectively. The laser drivers 34r, 34g, and 34b are connected to the control / processing unit 28 and configured to control on / off of the emission of the laser beams R, G, and B. Here, each of the laser beams R, G, and B is modulated by controlling on / off of emission by a control signal as described later.
The R light source 22r, the G light source 22g, and the B light source 22b oscillate in a predetermined wavelength band so that the laser lights R, G, and B excite the fluorescent label to emit fluorescence in a specific wavelength band. There are fluorescent labels excited by the laser beams R, G, and B, and those that are bonded to the microbeads 12 and those that are not bonded to each other. Upon receiving the laser beams R, G, and B, fluorescence is emitted at a specific wavelength.

FIG. 3 schematically shows the oscillation wavelength of the laser beam and the spectral intensity distribution of the fluorescence emitted from the fluorescent label by this laser beam. For example, fluorescence having a center wavelength of λ ₁₂ is emitted by irradiation with a laser beam having a wavelength of λ ₁₁ emitted from a B light source. Similarly, fluorescence having a center wavelength of λ ₂₂ is emitted by irradiation with laser light of wavelength λ ₂₁ emitted from the G light source. In addition, fluorescence having a center wavelength of λ ₃₂ is emitted by irradiation with laser light of wavelength λ ₃₁ emitted from the B light source.
The light receiving unit 24 is disposed so as to face the laser light source unit 22 with the pipe line 30 interposed therebetween, and the microbead 12 passes through the measurement point by the forward scattering of the laser light by the microbead 12 passing through the measurement point. A photoelectric converter that outputs a detection signal indicating that the operation is to be performed. The signal output from the light receiving unit 24 is supplied to the control / processing unit 28, and is used as a trigger signal informing the timing at which the microbeads 12 pass the measurement point in the pipe line 30.
On the other hand, the light receiving unit 26 is arranged in a direction perpendicular to the emitting direction of the laser light emitted from the laser light source unit 22 and perpendicular to the moving direction of the microbeads 12 in the conduit 30. And a photoelectric converter that converts the fluorescence emitted from the fluorescent label bound to the microbeads 12 irradiated at the measurement point and the fluorescent label not bound to the microbeads 12 into an electrical signal.

FIG. 4 shows a schematic configuration of an example of the light receiving unit 26.
The light receiving unit 26 shown in FIG. 4 includes a lens system 26a that focuses a fluorescent light signal emitted from a fluorescent label bonded to the microbead 12 or a fluorescent label not bonded to the microbead 12, and dichroic mirrors 26b ₁ and 26b ₂ . , Band pass filters 26c _{1 to} 26c ₃ and photoelectric converters 27a to 27c such as photomultiplier tubes.
The lens system 26a is configured to focus the optical signal incident on the light receiving unit 26 onto the light receiving surfaces of the photoelectric converters 27a to 27c.
The dichroic mirrors 26b ₁ and 26b ₂ are mirrors that reflect fluorescence in a wavelength band within a predetermined range and transmit the other fluorescence. The reflection wavelength band and the transmission wavelength band of the dichroic mirrors 26b ₁ and 26b ₂ are set so as to be filtered by the band pass filters 26c _{1 to} 26c ₃ and captured by the photoelectric converters 27a to 27c. Has been.

The band-pass filters 26c _{1 to} 26c ₃ are provided in front of the light receiving surfaces of the photoelectric converters 27a to 27c, and transmit only fluorescence in a predetermined wavelength band. The wavelength band of the transmitted fluorescence corresponds to the wavelength band of the fluorescence emitted by the fluorescent label shown in FIG. 3, that is, the fluorescence emitted by the fluorescent label bound to the microbead 12 and the fluorescent label not bound to the microbead 12. The band is set in advance corresponding to the emitted fluorescence, for example, a band having a certain wavelength width centered on the wavelength λ ₁₂ emitted by the irradiation of the laser light having the wavelength λ ₁₁ emitted from the B light source.
The wavelength bands of the bandpass filters 26c _{1 to} 26c ₃ are, for example, that one of them transmits the fluorescence emitted by the fluorescent label bound to the microbead 12, and at least one of the other two is: The wavelength band is set so as to transmit the fluorescence emitted by the fluorescent label not bound to the microbeads 12.
The photoelectric converters 27a to 27c are sensors that include, for example, a sensor including a photomultiplier tube, and convert light received by the photoelectric surface into an electrical signal. The fluorescence received here is output as an electrical signal, amplified by an amplifier, and supplied to the control / processing unit 28.

As shown in FIG. 5, the control / processing unit 28 is coupled to the light source control unit 28 a that controls on / off of the laser light emission of the laser light source unit 22, and the fluorescent label or microbead 12 coupled to the microbead 12. The signal processing unit 28b for identifying the optical signal of the fluorescence emitted by the fluorescent label that has not been provided is configured.
When the detection signal output from the light receiving unit 24 is input as a trigger signal, the light source control unit 28a is configured to generate a pulse control signal that instantaneously controls on / off of laser light emission.
The light source controller 28a cyclically generates this pulse control signal and supplies it as control signals for the laser driver 34r, laser driver 34g, and laser driver 34b.
The control / processing unit 28 is driven by a constant clock signal in synchronization with the code generation timing in the light source control unit 28a, and an A / D converter 28c that performs A / D conversion of the received light signal and a constant clock signal. And an arithmetic processing unit 28d that performs predetermined arithmetic processing on the received light signal.
The A / D converter 28c samples the received light signals generated and amplified by the photoelectric converters 27a to 27c of the light receiving unit 26 by A / D conversion. The sampled received light signal is subjected to arithmetic processing by the arithmetic processing unit 28d.
The arithmetic processing unit 28d is configured to perform calculation processing of the received light signal in synchronization with the generation timing of the pulse control signal, and synchronizes the received light signal output from the light receiving unit 26 with the generation timing of the pulse control signal. Thus, the time-series data is used to obtain the fluorescence relaxation characteristics (see FIG. 7) of the fluorescent label bonded to the microbeads 12 with respect to laser light irradiation.

Here, the fluorescence relaxation time constant characterizing this fluorescence relaxation characteristic will be described. The fluorescence relaxation time constant is a parameter independent of the fluorescence intensity. The fluorescence relaxation time constant is a parameter indicating the extinction time estimated from the fluorescence decay curve. The larger the value, the longer the extinction time of the fluorescence. This fluorescence extinction time varies depending on the fluorescent reagent, that is, the fluorescent label. Therefore, it is possible to distinguish the fluorescence caused by the fluorescent label bound to the measurement object from the fluorescence emitted by the substance present in the measurement atmosphere. Further, when there are a plurality of measurement objects, a plurality of measurement objects can be simultaneously measured with high accuracy by using a plurality of types of fluorescent labels corresponding to each measurement object.
The fluorescence relaxation time constant in the present invention is measured by irradiating a fluorescent label with frequency-modulated excitation light, detecting the excitation light source and the generated fluorescence, and then demodulating.
In the fluorescence relaxation process, the fluorescence intensity of the emitted fluorescence is high at the time when the laser beam is started to be irradiated on the microbeads, but the fluorescence intensity decreases with time. The fluorescence relaxation time constant is the time when the fluorescence intensity decays to 37% (1 / e: e is the base of natural logarithm) with respect to the fluorescence intensity (maximum intensity) at the start of laser light irradiation at this time. Say.

Since on / off of the laser light irradiation is intermittently controlled at a predetermined cycle by generating a pulse control signal, the fluorescent label bonded to the microbeads emits fluorescence when the laser light irradiation starts. And the fluorescence intensity attenuates over time. Therefore, the fluorescence relaxation time constant can be known by obtaining time series data of the fluorescence intensity.
When two types of fluorescent labels with different fluorescence relaxation time constants are used, the fluorescence relaxation time constants are different from the fluorescence relaxation time constants indicated by each of the two types of fluorescent labels. Indicates. This is because the two types of fluorescent labels interact with each other to create different fluorescence relaxation properties.
Fig. 8 shows how the fluorescence relaxation time constant changes when two types of fluorescent labeling dyes Q-dot525 (manufactured by Quantum Dot) and Cascade Blue (manufactured by Molecular Probes) coexist at different blending ratios. It is a graph which shows the result of having investigated whether to do.
Solution A with a Q-dot525 reference concentration of 20 nM and solution B with a Cascade Blue reference concentration of 20 μM are mixed in the following ratio, and the sample is stored in a spot sample storage part having a diameter of 1.5 m and a depth of 3 mm. Solutions 1 to 9 were prepared.

In common to sample solutions 1 to 9, 1 μL of 100 μM Cascade Blue is added. The laser beam was emitted by a semiconductor laser, and was pulse-modulated at 20 MHz using a laser beam having a wavelength of 405 nm and an output of 40 mW.
FIG. 8 is a graph showing the phase delay of the fluorescence emitted at this time. According to this graph, the phase delay changes according to the fluorescence relaxation time constant, and corresponds to the order of the ratio of the solution A and the solution B. This shows that the fluorescence relaxation time constant changes depending on the abundance ratio of the fluorescent label.
Thus, when two different types of fluorescent labels are present at different ratios, the fluorescence relaxation time constant changes.
The control / processing unit 28 supplies information on the identification result of the microbeads 12 to the analyzer 50.
The analysis device 50 is a device that identifies a fluorescent label that binds to the microbeads 12 using information on the identification result supplied from the control / processing unit 28. In the analyzer 50, the type of the fluorescent label used is known, and the wavelength band of the laser light excited by the fluorescent label is also known. Therefore, the microbeads can be obtained using information supplied from the control / processing unit 28. Fluorescent labels that bind to 12 can be identified.
Thus, the analyzer 50 can perform analysis in a short time.
The flow cytometer 10 is configured as described above.

In such a signal processing device 20 of the flow cytometer 10, first, the reaction mixture containing the microbeads 12 flows through the conduit 30, and irradiation with laser light is performed at the measurement point. At that time, a detection signal for detecting the passage of the microbeads 12 by the light receiving unit 24 is output to the control / processing device 28 as a trigger signal.
The control / processing device 28 uses a detection signal when the microbead 12 passes as a trigger signal, and generates a pulse modulation signal as shown in FIG. 6 in synchronization with the trigger signal. This pulse modulation signal is supplied to the laser drivers 34r, 34g, and 34b to be used as a control signal for controlling on / off of laser light emission from the laser light source section 22.
The laser light source unit 22 controls on / off of the emission of each laser beam according to this control signal, and emits the laser beam. This laser beam is used to excite a fluorescent label that is bonded to the microbead 12 that passes through the measurement point (and also a fluorescent label that is not bonded to the microbead 12 when B / F separation is not performed). The fluorescence emitted from the fluorescent label by light irradiation is received by the light receiving unit 26. At this time, the fluorescence from the fluorescent label is excited by the pulse-modulated laser beam, so that the fluorescence intensity is also modulated in response to the pulse modulation of the laser beam to emit fluorescence.

In this way, the time from the detection of the passage of the microbead 12 by the light receiving unit 24 to the irradiation of the laser beam is extremely short, and the pulse modulation is performed between several μ to several tens of μsec when the microbead 12 passes the measurement point. The laser beam modulated by the signal is cyclically applied to the microbeads 12.
Here, the pulse-modulated laser light has a series of pulse modulation control signals several times, with 0.510 microseconds as one cycle between several microseconds to several tens of microseconds when the microbead 12 passes the measurement point. It is circulated intermittently for several tens of times.
The received light signals received and output by the photoelectric converters 27a to 27c of the light receiving unit 26 are synchronized with the timing of the trigger signal output from the light receiving unit 24 in the A / D converter 28c, and generated pulse modulation. The received light signal is sampled with the same time resolution width as the signal time resolution width Δt. The sampling is, for example, 8-bit sampling (sampling with a gradation of 0 to 255).

A fluorescence relaxation characteristic as shown in FIG. 7 is obtained by averaging the time-series data of the fluorescence intensity from the sampled received light signal in correspondence with the pulse modulation signal generated by circulation.
In the waveform of the fluorescence relaxation characteristic, the time when the fluorescence intensity becomes 1 / e% = 37% (e is the base of natural logarithm) is obtained as the fluorescence relaxation time constant with respect to the maximum fluorescence intensity. From the value of this fluorescence relaxation time constant, the type of fluorescent label bound to the microbead 12 is specified.
Note that the photoelectric converter 27a~27c Since then received by the band-pass filter 26c ₁ ~26c ₃ to each wavelength band to output a light reception signal, it is also possible to know a signal of the fluorescence of which wavelength band received signal. Therefore, the type of the fluorescent label can be more accurately identified using the fluorescence relaxation time constant of the fluorescence emitted from the fluorescent label bound to the microbead 12 and the wavelength band.

The flow cytometer 10 described above measures the fluorescence relaxation time constant by irradiating a fluorescent label that is pulse-modulated with laser light and bound to the microbeads 12, but the intensity of the laser light is measured at a predetermined frequency. The fluorescence relaxation time constant may be measured by modulating and irradiating the microbead. Hereinafter, this method will be described.
For example, the intensity of the laser beam having a wavelength of 405 nm is modulated at 20 MHz, and this laser beam is irradiated to the microbead that passes through the measurement point in the flow cell. Fluorescence emitted upon irradiation with the modulated pulsed light is received by a photoelectric converter or the like.
The fluorescence signal output from the photoelectric converter is amplified, mixed with the modulation signal used for modulation of the laser light, and detected through a low-pass filter. Thereby, the signal of the cos component of the fluorescence signal is extracted. Furthermore, the fluorescence signal is mixed with a signal whose phase is shifted by 90 degrees with respect to the modulation signal used for frequency modulation of the laser light, and detected through a low-pass filter. Thereby, the signal of the sine component of the fluorescence signal is extracted.

Using the extracted signals of the sin component and the cos component, the phase shift angle (phase delay angle) of the fluorescence signal with respect to the modulation signal can be obtained.
The obtained phase shift angle depends on the fluorescence relaxation time constant of the fluorescence emitted from the fluorescent label. For example, when expressed in the first order relaxation process, the cos component and the sin component are expressed by the following formulas (1) and (2). expressed.
cos (θ) = 1 / (1+ (ωτ) ² ) ^(1/2) (1)
sin (θ) = ωτ / (1+ (ωτ) ² ) ^(1/2) (2)
Here, θ is a phase shift angle, ω is a modulation frequency of laser light, and τ is a fluorescence relaxation time constant.

The fluorescence relaxation time constant τ can be obtained from the above equations (1) and (2) using the phase shift angle θ obtained from the ratio of the cos component and the sin component of the fluorescence signal.
As described above, this fluorescence relaxation time constant τ varies depending on the type of fluorescent label, and when the ratio of two types of fluorescent labels is changed and mixed, the fluorescence relaxation time constant τ also varies according to the ratio. For this reason, the ratio of two types of fluorescent labels can be specified by obtaining the fluorescence relaxation time constant τ.
In this way, the type of fluorescence emitted can be identified by irradiating the microbeads to which the fluorescent label is bound with laser light intensity-modulated at a predetermined frequency and measuring the fluorescence emitted at that time.
Such microbeads are used in the flow cytometer as in the above embodiment, but the use is not limited to the flow cytometer.
However, in the flow cytometer, it is possible to accurately identify the type of the fluorescent label by obtaining the fluorescence relaxation time constant of the fluorescent label bound to the microbead 12, so that the fluorescence measurement step in the method of the present invention is used. Can suitably use a flow cytometer.

The fluorescence detector used needs to be able to measure the fluorescence intensity and the fluorescence relaxation time constant of the fluorescence having a wavelength corresponding to the labeled fluorescent dye. As such a detector, an apparatus described in Japanese Patent Application No. 2005-37399 can be used. As a detection method, a detector of a method such as a flow cytometer, a fluorescence plate reader, or a fluorescence imaging device can be selected according to the state of a single substance or a cell.
Prior to the execution of the fluorescence measurement step in the method of the present invention, a blank test is performed without using a fluorescently labeled substance, and a fluorescence relaxation time constant of so-called background fluorescence emitted from cells, measurement atmosphere, etc. is measured. It becomes easy to distinguish from the fluorescence emitted by the fluorescent label bound to the measurement object.
As mentioned above, although the specific aspect was demonstrated in detail among the fluorescence analysis methods concerning this invention, this invention is not limited to the said embodiment, In the range which does not deviate from the meaning of this invention, various improvement and a change are carried out. Of course.

The measurement beads were prepared as follows. Magnetic microbeads (Dynabeads M270 manufactured by Dynal) were washed three times with 100 μL of distilled water, 10 μL of CMC (0.005 M, manufactured by Sigma) was added, and the mixture was reacted at 4 ° C. for 10 minutes. The top was removed, 6 μL of CMC (0.005 M) and 4 μL of MES buffer (0.3 M) were added, and the mixture was reacted at 4 ° C. for 30 minutes. The beads were washed with 100 μL of MES buffer (0.3M), and the beads were diluted with 2 μL of MES buffer (0.3M) and 0.3M, MES buffer. 8 μL) was added and reacted at 4 ° C. for 20 minutes. 5 μL of BSA (10 mg / mL) was added to the beads and allowed to react at 4 ° C. overnight. The microbeads were washed twice with 200 μL of PBS buffer to obtain measurement beads.
10 μL of 1.1 μL BSA (10 mg / mL Sigma) and transferrin (1 mg / mL) are added to the measurement beads, reacted at room temperature for 1 hour, and then washed with 200 μL TBS-T buffer. In this way, beads to which transferrin to be measured was bound were prepared.

10 μL of anti-transferrin biotin-labeled antibody (manufactured by Rockland) was added to the transferrin-bound beads from which the supernatant had been removed, and reacted at room temperature for 1 hour. The microbeads were washed 3 times with 200 μL of TBS-T buffer, the supernatant was removed, 20 μL of 2 × B & W buffer was added, and 5 μL of Q-Dot655 (manufactured by Q-Dot) and 18. Q-Dot was labeled by adding 5 μL of TBS-T buffer and reacting at room temperature for 40 minutes.
After completion of the labeling reaction of the above reaction, the microbeads were washed three times with 200 μL of B & W buffer solution and suspended in 100 μL of TBS-T warm buffer solution, and used for measurement with a fluorescence relaxation time constant measurement flow cytometer.
In measurement using a flow cytometer, a 30 mW modulated excitation laser was used as a light source, and measurement was performed while flowing a sheath liquid at a flow rate of 6 m / sec.
The measured values at this time and the fluorescence relaxation time constants of the microbeads not labeled with Q-Dot are shown in Table 2 below.

It is a schematic block diagram of the flow cytometer which implements a fluorescence measurement process in the method of this invention. It is a schematic block diagram which shows an example of the laser light source part used for the flow cytometer shown in FIG. It is a figure which shows typically the spectrum intensity distribution of the laser beam radiate | emitted from the laser light source part shown in FIG. 2, and the light which a fluorescent label emits. It is a schematic block diagram which shows an example of the light-receiving part used for the flow cytometer shown in FIG. It is a schematic block diagram which shows an example of the light source control part and signal processing part which are used for the flow cytometer shown in FIG. It is a figure which shows the example of the pulse modulation signal produced | generated with the flow cytometer shown in FIG. It is a figure explaining the characteristic of the intensity | strength of the fluorescence which a fluorescent label emits. (A) shows a fluorescence decay curve, and (B) shows a phase difference caused by modulation of excitation light. It is a graph which shows the result of the phase delay of the fluorescence when irradiating the pulse-modulated laser beam when there are two types of fluorescent labels.

Explanation of symbols

10 flow cytometer 12 microbead 20 signal processor 22 laser light source unit 22r R light source 22 g G light 22b B light source _{23a 1, 23a 2, 23b 1} , 23b 2 dichroic mirrors 23c. 26a a lens system 24, 26 light receiving unit 26c _1, 26c _2, 26c ₃ band pass filters 27a~27c photoelectric sensor 28 control and processing unit 28a light source control section 28b the signal processing unit 28c A / D converter 28d arithmetic processing unit 30 pipeline 32 Collection container 34r, 34g, 34b Laser driver

Claims (5)

A measurement object is directly or indirectly bound to a fluorescently labeled substance using an antigen-antibody reaction, the fluorescent label is excited by irradiation with a modulated laser beam, and the relaxation time constant of the emitted fluorescence is measured to determine the background. A fluorescence analysis method characterized by detecting a measurement object by distinguishing from fluorescence.   2. The fluorescence analysis according to claim 1, wherein the modulation of the laser light is performed by any method selected from the group consisting of high-frequency superposition modulation, specific shape wave modulation, sine wave modulation, linear modulation, and pulse modulation. Method.   Either 1) competitively bind the substance to be measured with the substance to be measured and 1) the substance to be measured and the substance x that can be bound to the substance w and is fluorescently labeled, or 2) the substance to be measured and the substance to be measured The substance y that can bind to w is competitively bound, and then the substance z that can bind to the substance y and is fluorescently labeled without binding to the measurement target is bound to the substance y, A fluorescence analysis method comprising: exciting a fluorescent label of x or the substance z by irradiation with a pulse-modulated laser beam; and measuring a relaxation time constant of the emitted fluorescence to detect a measurement object. The fluorescence analysis method according to any one of claims 1 to 3, which is a method in which binding is performed using the antigen-antibody reaction and B / F separation is not performed.   The fluorescence analysis method according to any one of claims 1 to 4, wherein the measurement object is a cell, an intracellular substance, a site existing on the cell surface, or a substance secreted by the cell.

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