JPH10332594A - Detecting method for object to be inspected - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a detecting method in which a plurality of kinds of objects, to be inspected, in a sample are detected respectively in good S/N ratio. SOLUTION: A first detecting probe is composed of a donor probe 20a comprising a donor fluorescent molecule 21a and of an acceptor probe 30a comprising an acceptor fluorescent molecule 31a. A second detecting probe is composed of a donor probe 20b comprising a donor fluorescent molecule 21b and of an acceptor probe 30b comprising an acceptor fluorescent molecule 31b. At this time, the donor fluorescent molecule 21a and the donor fluorescent molecule 21a are identical, and the acceptor fluorescent molecule 31a and the acceptor molecule 31b are identical. The first and second detecting probes bond mutually different kinds of objects 10a, 10b, to be detected, so as to form a pair. At this time, the distance between the donor fluorescent molecules and the acceptor fluorescent molecules is different from each other. When they are irradiated with excitation light at an identical wavelength, they generate fluorescence at an identical wavelength. The objects 10a, 10b to be inspected are detected on the basis of the difference between their response characteristics.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the detection of a specimen sample by mixing a detection probe (fluorescent probe) with the specimen sample, irradiating the specimen sample with excitation light, and detecting the fluorescence generated thereby. The present invention relates to a technique for detecting an analyte to which a probe is bound.

[0002]

2. Description of the Related Art Conventionally, as a technique for detecting an object using a fluorescence method, one kind of fluorescent probe is mixed with a sample and allowed to bind to the object, and the sample is irradiated with excitation light and irradiated with excitation light. 2. Description of the Related Art There is known a technique for detecting a specimen in a specimen sample by measuring parameters derived from the intensity, spectrum, fluorescence lifetime, and the like of the fluorescence generated from a fluorescent probe bound to the specimen.

The detection and discrimination of a plurality of types of analytes in a sample sample using this analyte detection technique is performed as follows. That is, a plurality of types of fluorescent probes having different characteristics are mixed into a sample, and the plurality of types of fluorescent probes are respectively bound to the plurality of types of analytes. At this time, the type of the analyte to be bound and the type of the fluorescent probe correspond to each other. Then, the sample sample is irradiated with excitation light, fluorescence generated from the sample sample is identified based on the characteristics of each of the plurality of types of fluorescent probes, and each of the plurality of types of analytes in the sample sample is detected. Here, generally, a plurality of types of fluorescent probes having different fluorescence spectra and different fluorescence lifetimes are used.

[0004]

In order to detect a plurality of types of analytes by the above-mentioned conventional fluorescence detection method using an analyte, it is necessary to prepare a plurality of types of fluorescent molecules having different light emission phenomena. There is. In addition, since the emission phenomena of each of a plurality of types of analytes are distinguished from each other by using a parameter derived from the spectrum or the fluorescence lifetime that is a parameter other than the fluorescence intensity as a measurement target, selection of a fluorescent molecule is restricted.

On the other hand, it is conceivable to detect a plurality of types of analytes based on the fluorescence resonance energy transfer phenomenon between the donor fluorescent molecule and the acceptor fluorescent molecule contained in the donor probe and the acceptor probe, respectively. However, in this case, it is necessary to prepare a donor probe and an acceptor probe each composed of different types of donor fluorescent molecules and acceptor fluorescent molecules for each of a plurality of types of analytes. In the case where a plurality of types of analytes are detected by the fluorescence measurement, combinations of the types of the donor fluorescent molecule and the acceptor fluorescent molecule are limited. Therefore, it is difficult to select a donor fluorescent molecule and an acceptor fluorescent molecule, and the number of types of analytes that can be detected at one time is limited.

When the amount of each of a plurality of types of analytes present in a sample is different, the detection sensitivity is low for an analyte having a relatively small amount.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problem. By irradiating excitation light of the same wavelength, a plurality of types of specimens contained in a specimen can be reduced to a high S / E even if they are trace amounts. It is an object of the present invention to provide a method for detecting an analyte that can be detected at an N ratio.

[0008]

According to the present invention, there is provided a method for detecting an analyte, comprising the steps of: (1) providing a fluorescent resonance energy with respect to excitation light irradiation, A detection probe consisting of a donor probe and an acceptor probe, each having a donor fluorescent molecule and an acceptor fluorescent molecule that causes a migration phenomenon and generates fluorescence with predetermined response characteristics, and has the same donor fluorescent molecule and acceptor fluorescent molecule. A first step of mixing a plurality of types of detection probes, which are paired with different types of analytes and emit fluorescence with different response characteristics, into a sample, and (2) intensity at a constant frequency. The modulated excitation light irradiates the specimen sample, and each of multiple types of detection probes is coupled to the specimen in pairs. A second step of identifying fluorescence generated from the detection probe based on the response characteristics of the detection probe by frequency-domain time-resolved optical measurement, and detecting an analyte to which the detection probe is bound based on the identified fluorescence. And the step of:

According to this method for detecting an analyte, in the first step, the above-described donor fluorescent molecule and acceptor fluorescent molecule which are the same as each other as described above are combined and bound to different types of analytes in pairs. A plurality of types of detection probes that generate fluorescence with different response characteristics from each other are mixed into the sample. Then, in the second step, the sample sample is irradiated with excitation light whose intensity is modulated at a constant frequency, and for each of the plurality of types of detection probes, the response characteristics of the detection probes coupled to the subject in pairs. , The fluorescence generated from the detection probe is identified by frequency domain time-resolved optical measurement, and the analyte to which the detection probe is bound is detected based on the identified fluorescence.

In the second step, fluorescence is identified by synchronously detecting a light beam from the specimen sample based on a first synchronization signal obtained by adding a first phase delay to the intensity modulation of the excitation light. It is characterized by the following. In this case, the luminous flux from the sample is synchronously detected based on a first synchronization signal obtained by adding a first phase delay to the intensity modulation of the excitation light, and the fluorescence is converted to S based on the synchronization detection result. / N ratio.

In the second step, the light beam is synchronously detected based on a second synchronization signal obtained by adding a second phase delay to the intensity modulation of the excitation light, and the first and second light beams are detected. It is characterized in that fluorescence is identified based on each light beam synchronously detected based on each synchronization signal. In this case, the luminous flux from the sample is synchronously detected based on the second synchronization signal obtained by adding the second phase delay to the intensity modulation of the excitation light, and the first and second synchronization signals are respectively detected. The fluorescence is identified with a better S / N ratio on the basis of the synchronous detection result.

[0012]

Embodiments of the present invention will be described below in detail with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

First, a detection probe suitable for use in the subject detection method according to the present invention will be described.

This detection probe is composed of a donor probe and an acceptor probe. The donor probe is labeled with an energy donor donor fluorescent molecule. On the other hand, the acceptor probe is labeled with an acceptor fluorescent molecule having an energy acceptor property. Further, when the donor probe and the acceptor probe bind to, for example, a specific polynucleotide sequence, the distance between the donor fluorescent molecule and the acceptor fluorescent molecule is fixed to be constant, and the donor fluorescent molecule and the acceptor fluorescent molecule are bound. Has a molecular structure that takes a spatial position where a fluorescence resonance energy transfer phenomenon can occur efficiently.

Therefore, when the donor probe and the acceptor probe each having a pair bound to a specific polynucleotide sequence are irradiated with excitation light, the excitation light excites the donor fluorescent molecule,
A fluorescence resonance energy transfer phenomenon occurs from the donor fluorescent molecule to the acceptor fluorescent molecule, and fluorescence is generated from each of the donor fluorescent molecule and the acceptor fluorescent molecule.

As described above, when the donor fluorescent molecule and the acceptor fluorescent molecule are bound to a specific polynucleotide sequence at a certain distance from each other in a pair, the pulsed excitation light is applied to the donor fluorescent molecule and the acceptor fluorescent molecule. The above-described fluorescence resonance energy transfer phenomenon occurs by comparing and comparing the decay curve of the fluorescence generated from each of them with the decay curve of the fluorescence generated from each of the donor fluorescent molecule and the acceptor fluorescent molecule which exist independently in a free state. Can be determined.

The detection probe comprising the donor probe and the acceptor probe is configured to be spatially arranged so that the above-described fluorescence resonance energy transfer phenomenon occurs efficiently. Since this fluorescence resonance energy transfer phenomenon has occurred, it can be known that the detection probe is bound to a specific nucleotide sequence in a pair, that is, the presence of the analyte. Therefore, the use of this detection probe makes it possible to detect a subject with extremely high sensitivity.

This detection probe is used as follows in a fluorescence method based on a time-domain time-resolved optical measurement method. FIG. 1 is an explanatory diagram of a detection probe suitably used in the present invention.

First, a specific polynucleotide sequence possessed by the subject 10 is selected, and base sequences 11 and 12 of two types of oligonucleotide probes that complementarily bind to the selected sequence are selected. In this case, it is preferable that they have complete complementarity, but any sequence that substantially binds may be used. On the other hand, an appropriate donor fluorescent molecule 21 is bound at an appropriate position with a linker portion 22 of an appropriate length to prepare a donor probe 20 and an appropriate acceptor fluorescent molecule 31
At an appropriate position with a linker portion 32 of an appropriate length,
An acceptor probe 30 is prepared (FIG. 1A). These donor probe 20 and acceptor probe 3
0 is mixed with a specimen sample for which it is unknown whether or not the specimen 10 is contained. If the specimen 10 contains the specimen 10, some of the donor probes 20 and the acceptor probes 30 respectively bind to the base sequences 11 and 12 of the specimen 10 (FIG. 1 (b)).

Then, the sample is irradiated with pulsed excitation light using a fluorescence measuring device capable of time-resolved measurement, and the decay curve of the fluorescence generated from the acceptor fluorescent molecules 31 is measured. The fluorescence generated from the acceptor fluorescent molecule 31 includes the fluorescent light based on the fluorescence resonance energy transfer phenomenon from the donor fluorescent molecule 21 to the acceptor fluorescent molecule 31 (FIG. 1C). In addition, the sample in which the acceptor probe 30 exists alone in a free state is irradiated with pulsed excitation light, and the decay curve of the fluorescence generated from the acceptor fluorescent molecule 31 is measured (FIG. 1D).

As shown in this figure, when the donor probe and the acceptor probe are arranged at a fixed distance from each other (FIG. 1B), pulse excitation light is irradiated to cause a fluorescence resonance energy transfer phenomenon, thereby causing the acceptor probe to undergo fluorescence resonance energy transfer. A decay curve of the fluorescence generated from the fluorescent molecule (FIG. 1 (c)) and a decay curve of the fluorescence generated from the acceptor fluorescent molecule when irradiated with the pulse excitation light when the acceptor probe is present freely (FIG. 1 (d)). ) Have different shapes from each other. That is, the decay curve of the fluorescence generated from the acceptor fluorescent molecule due to the fluorescence resonance energy transfer phenomenon (FIG. 1)
(C)) shows the decay curve of the fluorescence generated from the acceptor fluorescent molecule of the acceptor probe in the free state (FIG. 1).
As compared with (d)), the rise is gradual and the decay after reaching the maximum point is gradual. This difference is due to the fluorescence resonance energy transfer phenomenon.

Therefore, by comparing these two obtained attenuation curves (FIGS. 1 (c) and 1 (d)),
It can be determined whether the subject is present. That is, when a significant difference is recognized between the two fluorescence decay curves, it is determined that the subject 10 is present in the sample sample. When no significant difference is recognized between the two fluorescence decay curves, the sample is included in the sample sample. It is determined that the subject 10 does not exist.

The detection probe comprising the donor probe and the acceptor probe has high sensitivity, and can sufficiently detect the analyte even under the condition that each probe is mixed in excess with the analyte. Having a specific structure. That is, according to the findings of the present inventor, usually, the spatial arrangement of each of the donor fluorescent molecule and the acceptor fluorescent molecule greatly changes due to the molecular motion of the subject, and the relative spatial positions of the donor fluorescent molecule and the acceptor fluorescent molecule are changed. A change in the fluorescence decay curve based on the expected fluorescence resonance energy transfer phenomenon when fixed may not be actually observed. Therefore, this detection probe
In order to substantially fix the donor fluorescent molecule and the acceptor fluorescent molecule at a preferable spatial position, the type of each fluorescent molecule, the binding mode of the probe (duplex or single chain) and the position (the number of bases between the fluorescent molecules) Etc. are optimized.

Therefore, the conjugate of the detection probe and the analyte is formed by the specific polynucleotide sequence even if the analyte changes its three-dimensional structure in the analyte sample based on molecular motion or the like. The portion of the duplex structure does not change significantly, and the preferred relative spatial position between each fluorescent molecule is fixed, resulting in a change in the expected sufficient fluorescence decay curve. Therefore, it is possible to detect a change in the fluorescence decay curve even under a condition where each free probe is in a large excess (in this case, it cannot be observed by a change in the fluorescence spectrum).

The term "fixing the relative spatial position between the donor fluorescent molecule and the acceptor fluorescent molecule" as used herein means that:
The donor probe and the acceptor probe are formed by binding to a specific polynucleotide sequence portion in the subject.
This means that the heavy chain portion substantially retains its three-dimensional structure with respect to the time width of the fluorescence decay curve measurement, and as a result, the relative spatial position of each fluorescent molecule bound to each probe. Also substantially holds in the time width. The time width of the fluorescence decay measurement is several to
It is on the order of several hundred nanoseconds.

The number of base sequences of the donor probe and the acceptor probe is not particularly limited. It is preferable that the number of double strands be stable at the time of binding and avoid misrecognition. It is possible to select the number of bases of each probe in consideration of the known base sequence of the peptide sequence and the like. The number of bases of each probe that can be used needs to be such that the donor fluorescent molecule and the acceptor fluorescent molecule are fixed in a specific spatial arrangement by binding, and a sufficient fluorescence resonance energy transfer phenomenon is possible. Furthermore, the total number of base sequences of each probe is not particularly limited. Generally, it is necessary that the duplex at the time of binding is sufficiently stable and has a sufficient length without erroneous recognition.

The relative positions of the donor probe and the acceptor probe on the specific polynucleotide sequence in the subject are not particularly limited, and the ends of each probe do not necessarily have to be adjacent to each other, and the single-stranded target nucleic acid May be sandwiched between them. According to various embodiments, it is possible to select a relative position of each probe that can fix the donor fluorescent molecule and the acceptor fluorescent molecule at a specific spatial position.

The donor fluorescent molecule and acceptor fluorescent molecule usable as the detection probe are not particularly limited, but a combination satisfying the following conditions is preferable. That is,
(1) The fluorescence lifetime of the fluorescence generated from the donor fluorescent molecule is longer than the fluorescence lifetime of the fluorescence generated from the acceptor fluorescent molecule, and the difference is large. (2) The acceptor is excited by the excitation light having the wavelength that excites the donor fluorescent molecule. It is preferable that the conditions satisfy the following conditions: the probability that the fluorescent molecule is excited is low; and (3) the overlap between the spectrum of the fluorescence generated from the donor fluorescent molecule and the spectrum of the fluorescence generated from the acceptor fluorescent molecule is small.

As a fluorescent molecule satisfying the above general conditions, for example, as a donor fluorescent molecule, BODIPY
(4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene) A fluorescent dye system (Molecular Probes), a FITC (fluorescein) system, and a rhodamine system can be suitably used, and in particular, BODIPY Is preferred. As the acceptor fluorescent molecule, rhodamine-based dyes and indocyanine-based dyes can be suitably used. In particular, Cy5, Cy3, and Cy are used.
3.5 (Amersham LIFE SCIENCE, FluoroLink, Cat. No.
PA25001, PA23001, PA23501) are preferred.

Further, there is no particular limitation on the bonding group between the donor fluorescent molecule and the acceptor fluorescent molecule and the probe oligonucleotide, but an appropriate linker portion is provided so that the above two types of fluorescent molecules cause a desirable fluorescence resonance energy transfer phenomenon. It is preferable that they are bonded through a bond. If the linker is too short, the interaction between each fluorescent molecule and the skeleton or base of the nucleic acid may become strong, which is not preferable. If the linker is too long, 2
Undesirably, the kinds of fluorescent molecules move freely, and the steric configuration between the two fluorescent molecules is not sufficiently fixed.
Therefore, a preferable length of the linker portion is a tetramethylene chain to a decamethylene chain. For binding to each fluorescent molecule, a known covalent bond formation reaction can be used. For example, an amide bond, an ester bond, an ether bond and the like are preferable, and an amide bond is particularly preferable.

The preferred structure of the detection probe having the above characteristics is, for example, the following structure. An oligonucleotide probe (donor probe) labeled with a donor fluorescent molecule at a specific position via a linker, and an oligonucleotide probe (acceptor probe) labeled with an acceptor fluorescent molecule at a specific position via a linker. A set of probes. The base sequence of each probe is selected so as to be able to substantially bind to all or a part of a specific site (specific polypeptide sequence portion of a single chain) in the subject. Accordingly, the two types of probes are selected so as to be bound to any positions on the single chain of the specific polypeptide sequence portion of the subject, and the two types of probes are selected.
The spatial relative positions of the types of probes can be arbitrarily selected.

In the method for detecting an object according to the present invention, when the object is coupled to the object, the object is arranged at a predetermined distance from the object, causes a fluorescence resonance energy transfer phenomenon with respect to the excitation light irradiation, and has a predetermined response characteristic. A detection probe consisting of a donor probe and an acceptor probe each having a donor fluorescent molecule and an acceptor fluorescent molecule that generate fluorescence, and having the same donor fluorescent molecule and acceptor fluorescent molecule, and capable of detecting different types of analytes. Fluorescent method based on frequency domain time-resolved optical measurement method detects multiple types of analytes in a sample using multiple types of detection probes that combine and generate fluorescence with mutually different response characteristics Things. In the following, a case where two sets of detection probes are used will be described for simplicity of description, but the same applies to a case where a larger number of sets of detection probes are used.

FIG. 2 is an explanatory diagram of two sets of detection probes used in the subject detection method according to the present invention. In each of the first and second detection probes shown in FIGS. 2A and 2B, the donor fluorescent molecule 2
1a and 21b are identical to each other, and acceptor fluorescent molecules 31a and 31b are also identical to each other. An analyte 10a having base sequences 11a and 12a to which the donor probe 20a and the acceptor probe 30a respectively bind (FIG. 2A), and base sequences 11b and 12b to which the donor probe 20b and the acceptor probe 30b respectively bind The subject 10b (FIG. 2B) is of a different type.

When the first detection probe consisting of the donor probe 20a and the acceptor probe 30a binds to the subject 10a in a pair, the donor fluorescent molecule 2
1a and the acceptor fluorescent molecule 31a are at a distance La from each other.
(FIG. 2A). When the second detection probe composed of the donor probe 20b and the acceptor probe 30b is paired and binds to the subject 10b, the donor fluorescent molecule 21b and the acceptor fluorescent molecule 3
1b are spaced from each other by a distance Lb (FIG. 2).
(B)).

Thus, each of the first and second detection probes has the same donor fluorescent molecule and the same acceptor fluorescent molecule, and
It is adjusted so that the different types of analytes bind to each other in pairs and the distance between the donor fluorescent molecule and the acceptor fluorescent molecule is different from each other. Therefore, when the excitation light of a predetermined wavelength is irradiated,
The first fluorescence generated from the first detection probe paired with a and the second fluorescence generated from the second detection probe paired with the analyte 10b are as follows. Have the same wavelength but different response characteristics.

FIGS. 3 to 6 show the degree of modulation of fluorescence with respect to the excitation light and the distance between the donor fluorescent molecule and the acceptor fluorescent molecule when the donor probe and the acceptor probe respectively bind to the subject in pairs. 9 is a graph showing an example of a phase delay. FIG.
Indicates the degree of modulation of the fluorescence generated from the donor fluorescent molecule,
FIG. 4 shows the phase lag of the fluorescence generated from the donor fluorescent molecule, FIG. 5 shows the modulation degree of the fluorescent light generated from the acceptor fluorescent molecule, and FIG. 6 shows the phase lag of the fluorescent light generated from the acceptor fluorescent molecule. . In each of these figures, DA5, DA7, DA9 and DA13 represent detection probes with different distances between the donor fluorescent molecule and the acceptor fluorescent molecule, and the distances are shorter in this order. D represents a donor probe in a free state, and A represents
Indicates a free acceptor probe.

When measuring the degree of modulation and the phase delay, a wavelength 488n intensity-modulated to a frequency of 100 MHz is used.
m, and emits fluorescence generated from the donor fluorescent molecule to a band-pass filter (transmission wavelength band 500-550n).
m), and the fluorescence generated from the acceptor fluorescent molecule is detected by a color glass filter (transmission wavelength band 600n).
m or more).

As can be seen from these figures, the degree of modulation and the phase lag of the fluorescence generated from the donor fluorescent molecule and the acceptor fluorescent molecule of the donor probe and the acceptor probe respectively bound to the subject in a pair are:
It differs according to the distance between the donor fluorescent molecule and the acceptor fluorescent molecule, and also differs in the modulation degree and phase delay of the fluorescence generated from the free donor fluorescent molecule and the free acceptor fluorescent molecule, respectively. Furthermore, even if the distance between the donor fluorescent molecule and the acceptor fluorescent molecule is the same, the degree of modulation and the phase delay differ between the fluorescence generated from the donor fluorescent molecule and the fluorescent light generated from the acceptor fluorescent molecule.

Next, an object detecting apparatus suitable for applying the object detecting method according to the present embodiment will be described with reference to FIG.

The excitation light source 40 emits excitation light to be applied to the sample 70, and has an appropriate wavelength according to a donor probe or an acceptor probe constituting a detection probe mixed into the sample 70. A device that emits light is used. For example, as the excitation light source 40,
An argon ion laser light source that emits excitation light having a wavelength of 488 nm is preferably used.

The acousto-optic modulator 42 that receives the excitation light emitted from the excitation light source 40 converts the excitation light into a sine wave based on a sine wave signal of a predetermined frequency (for example, 100 MHz) output from the synthesizer 50. The intensity is modulated. The excitation light whose intensity has been modulated by the acousto-optic modulator 42 has its beam diameter adjusted by the beam expander 44, is reflected by the reflecting mirror 46, and is irradiated on the sample 70.

The sample 70 contains the donor probe and the acceptor probe as described above. When the sample 70 is irradiated with the excitation light, the donor probe and the acceptor probe emit donor fluorescent molecules and Fluorescence is generated from each of the acceptor fluorescent molecules. The intensity of the fluorescence changes sinusoidally at the same frequency as the intensity modulation frequency of the excitation light. The phase difference and the degree of modulation of the fluorescence with respect to the excitation light differ depending on the intensity modulation frequency of the excitation light, and whether each of the donor probe and the acceptor probe is paired and bound to a predetermined analyte in the analyte sample 70 is determined. It differs depending on the distance between the donor fluorescent molecule and the acceptor fluorescent molecule even when each probe is bound to the analyte in a pair. The fluorescence generated from the sample 70 passes through the filter 48 that blocks the wavelength component of the excitation light but transmits the wavelength component of the fluorescence, and is imaged on the imaging surface of the framing camera 56 by the macro lens 58.

The synthesizer 52 connected to the synthesizer 50 outputs a synchronization signal synchronized with the synthesizer 50. The delay unit 54 inputs the synchronization signal output from the synthesizer 52 to the framing camera 56 after giving a predetermined phase delay, and the phase delay added to the synchronization signal is variable. In addition, the framing camera 56 has a PSD
(Phase Sensitive detector) is a two-dimensional photodetector based on the synchronization signal output from the delay unit 54, and synchronously detects the light intensity of the fluorescent image formed on the imaging surface for each pixel. Is what you do.

Therefore, by appropriately setting the phase delay in the delay unit 54, the specimen 70
It is possible to analyze the frequency response characteristics of the fluorescence generated from, and based on this, it is possible to detect the analyte in the specimen sample 70 in which the donor probe and the acceptor probe are coupled in pairs. That is, the fluorescence image intensity synchronously detected by the framing camera 56 based on the synchronization signal output from the delay unit 54 is:
The delay unit 54 changes according to a phase delay given to the synchronization signal. The dependence of the fluorescence image intensity on the phase delay differs depending on whether the donor probe and the acceptor probe are coupled to the analyte in the analyte sample 70 in pairs, and each probe is paired. When they are bonded together, they differ depending on the distance between the donor fluorescent molecule and the acceptor fluorescent molecule.
By utilizing this, the subject in the sample sample 70 can be detected.

The image processing unit 60 for inputting a fluorescent image picked up by the framing camera 56 performs image processing such as image integration on the fluorescent image. The display unit 62 displays the fluorescent image captured by the framing camera 56 as it is, or displays the image after the image processing is performed by the image processing unit 60.

Next, the method of detecting an object according to the present embodiment will be described together with the operation of the object detecting apparatus described above. Hereinafter, two types of detection probes will be described for simplicity.

The two types of detection probes described with reference to FIG. 2 are prepared. Among them, the first detection probe is the sample 7
0, which can be coupled to the first analyte in pairs, wherein the distance between the donor fluorescent molecule and the acceptor fluorescent molecule at that time is adjusted to be a predetermined distance La, while Is capable of binding to the second analyte in the analyte sample 70 in a pair, and the distance between the donor fluorescent molecule and the acceptor fluorescent molecule at that time is a predetermined distance Lb (≠ La ). Each of the first and second detection probes is mixed into a sample 70, and the sample 70 is placed at a predetermined position of the apparatus.

Then, the intensity of the excitation light emitted from the excitation light source 40 is modulated into a sinusoidal wave and irradiates the specimen 70.
That is, the excitation light emitted from the excitation light source 40 is intensity-modulated by the acousto-optic modulator 42 on the basis of the sine wave signal output from the synthesizer 50 to have a sine wave shape, and the beam expander 44 adjusts the beam diameter. Then, the light is reflected by the reflecting mirror 44 and irradiated onto the sample 70.

The fluorescence generated from the specimen 70 by irradiation with the sine-wave excitation light passes through the filter 48,
An image is formed on the imaging surface of the framing camera 56 by the macro lens 58. The fluorescence image is synchronously detected for each pixel based on the sinusoidal excitation light and a synchronization signal having a certain phase delay. That is, a synchronizing signal synchronized with the sine wave signal output from the synthesizer 50 is output to the synthesizer 5.
2 and the synchronization signal is generated by the delay unit 5
Framing camera 5
Input to 6. The fluorescent image formed on the imaging surface of the framing camera 56 is
The synchronization is detected for each pixel based on the synchronization signal output from the delay unit 54.

Here, the delay unit 54 adjusts the phase of the synchronization signal input to the framing camera 56 to the first
One of the first fluorescence generated from the first detection probe paired with the second analyte and the second fluorescence generated from the second detection probe paired with the second analyte On the other hand, a phase delay is given to the synchronization signal output from the synthesizer 52 so as to coincide with the phase of the intensity change of the fluorescent light imaged on the imaging surface of the framing camera 56. That is, the delay unit 54 is configured such that the donor probe and the acceptor probe each
An appropriate phase delay is given to the synchronization signal in accordance with one of the distances La and Lb between the donor fluorescent molecule and the acceptor fluorescent molecule when the pair is bound to the analyte in the pair. Further, the phase delay in the delay unit 54 is based on the fact that the fluorescence generated from either the donor fluorescent molecule or the acceptor fluorescent molecule
6 may be different depending on whether the detection is performed.

By doing so, the first fluorescence generated from the first detection probe paired with the first analyte and the second fluorescence paired with the second analyte are generated. Even if the second fluorescent light generated from the detection probe reaches the imaging surface of the framing camera 56, the respective response characteristics are different from each other, and therefore, based on the synchronization signal added with a predetermined phase delay by the delay unit 54, By performing synchronous detection by the framing camera 56 and performing image processing by the image processing unit 60, it is possible to select and detect only one fluorescent light. Further, even when background light arrives at the imaging surface of the framing camera 56 in addition to the fluorescence to be detected, the background light is similarly removed by utilizing the difference in response characteristics to the excitation light irradiation. can do. Therefore, even if each of the first and second analytes in the sample 70 is very small, excellent S
/ N ratio.

Here, the fluorescence to be detected is:
A fluorescence generated from any of the donor fluorescent molecule and the acceptor fluorescent molecule of the first detection probe bound to the first analyte in the analyte sample 70 in a pair;
Is the fluorescence generated from either the donor fluorescent molecule or the acceptor fluorescent molecule of the second detection probe bound to the analyte in a pair. In addition, the background light is, in addition to the auto-fluorescence generated from any optical component in the optical system, the fluorescence generated from the donor fluorescent molecule of the donor probe in a free state,
Fluorescence generated from an acceptor fluorescent molecule of an acceptor probe in a free state, fluorescence generated from a donor fluorescent molecule of a donor probe bound to an analyte alone, and
It also includes fluorescence generated from an acceptor fluorescent molecule of an acceptor probe that is solely bound to an analyte.

The first fluorescence generated from the first detection probe paired with the first analyte in the sample 70 and the second detection probe coupled with the second analyte in the second sample In order to extract only the first fluorescent light and remove the others from the mixture of the second fluorescent light and background light generated from, the following is specifically performed. First, a first phase delay θ1 is added to the synchronization signal output from the synthesizer 52 by the delay unit 54, and an image is captured by the framing camera 56 based on the synchronization signal of the phase delay θ1, and the image M1 is subjected to image processing. Send to section 60. Next, the delay unit 54 applies a second phase delay θ2 (≠ θ) to the synchronization signal output from the synthesizer 52.
1) is added, the framing camera 56 takes an image based on the synchronization signal of the phase delay θ2, and sends the image M2 to the image processing unit 60. When the fluorescence intensity is low, image integration is performed.

Here, the phase delays θ1 and θ2 are set so that the influence of the second fluorescent light and the background light is as small as possible in the difference image obtained by calculating the difference between the images M1 and M2. Further, each of the phase delays θ1 and θ2 is set such that a component caused by the first fluorescence is sufficiently larger than a component caused by the second fluorescence and the background light in the difference image. The component caused by the first fluorescence after the difference calculation is set to be larger than that before the difference calculation. Each of the phase delays θ1 and θ2 can be appropriately set together with the distance between the donor fluorescent molecule and the acceptor fluorescent molecule when the donor probe and the acceptor probe bind to the subject in a pair.

The images M1 and M thus obtained
2, the image processing unit 60 obtains an image M by performing an image calculation as follows: M = (M1−M2) / (M1 + M2) (1)
The image M is processed so as to have 56 gradations and is displayed on the display unit 62. In the image M obtained by performing such an operation, while the influence of the second fluorescent light and the background light is almost eliminated, a sufficiently large value of the first fluorescent light component is obtained. As described above, even with a weak fluorescence emission phenomenon, fluorescence can be detected with a good S / N ratio, and a very small amount of the analyte in the sample 70 can be detected. Note that the second fluorescence can be detected in the same manner.

The present invention is not limited to the above embodiment, and various modifications are possible. For example, in the above-described embodiment, a case has been described in which each of two types of analytes is detected using each of two types of detection probes.
The number of types of the detection probes is not limited to two, and may be more types. In short, each detection probe is composed of a donor probe having the same donor fluorescent molecule and an acceptor probe having the same acceptor fluorescent molecule. The distance between the fluorescent molecule and the acceptor fluorescent molecule is different from each other, and the response characteristics to the excitation light irradiation are different from each other.

When the fluorescence emission phenomenon is extremely weak, the image may be taken by the photon counting method. When the photon noise is remarkable, the image processing unit 60 may perform filtering image processing or perform integration of adjacent images.

Also, in the method of observing the change over time of the specimen 70 by the heterodyne method (cross-correlation method), the fluorescence generated from the first detection probe paired with the first analyte and By utilizing the difference in the frequency response characteristics of each of the fluorescences generated from the second detection probe paired with the second analyte, only one of the fluorescences can be identified.

In place of the framing camera, an image intensifier with a gate function may be used as a photodetector. If point photometry is sufficient, a streak camera or a photomultiplier with a gate function may be used. May be used.

[0060]

As described above in detail, according to the present invention, each of which has the same donor fluorescent molecule and the same acceptor fluorescent molecule, binds to a different kind of analyte in a pair, and has a different response. A plurality of types of detection probes that emit fluorescence with their characteristics are mixed in the sample. Then, the sample sample is irradiated with excitation light whose intensity is modulated at a constant frequency, and the detection is performed for each of the plurality of types of detection probes based on the response characteristics of the detection probes coupled to the subject in pairs. Fluorescence generated from the probe is identified by frequency-domain time-resolved optical measurement, and an analyte to which the detection probe is bound is detected based on the identified fluorescence.

As described above, a plurality of types of detection probes which have the same donor fluorescent molecule and acceptor fluorescent molecule, but bind to a pair of different types of analytes and generate fluorescence with different response characteristics from each other. Because we use
Since the fluorescence of the same wavelength is generated without changing the wavelength of the excitation light, it is possible to detect each of a plurality of types of subjects without changing the filter for detecting fluorescence. Further, the fluorescence generated from the detection probe bound to each of a plurality of types of analytes in a pair is identified based on appropriately set response characteristics, and thus is detected with a good S / N ratio. Therefore, even if the amount of the analyte in the sample is very small, the analyte can be detected.

Further, a light beam is synchronously detected based on a first synchronization signal obtained by adding a first phase delay to the intensity modulation of the excitation light, and fluorescence is identified based on the synchronously detected light beam. In such a case, by appropriately setting the first phase delay, the fluorescence is identified with a good S / N ratio. Therefore, even if the analyte in the sample is extremely small, the analyte can be detected.

Further, a light beam is synchronously detected based on a second synchronization signal obtained by adding a second phase delay to the intensity modulation of the excitation light, and based on the first and second synchronization signals, respectively. When the fluorescence is identified based on each of the synchronously detected light beams, the first and second phase delays are appropriately set, and based on the first and second synchronization signals, respectively. Since each of the synchronously detected light beams is appropriately processed, the fluorescence is further increased by S.
/ N ratio. Therefore, even if the analyte in the sample is extremely small, the analyte can be detected more efficiently.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of a detection probe suitably used in the present invention.

FIG. 2 is an explanatory diagram of two sets of detection probes used in the subject detection method according to the present invention.

FIG. 3 is a graph showing a modulation degree of fluorescence generated from a donor fluorescent molecule for each distance between a donor fluorescent molecule and an acceptor fluorescent molecule.

FIG. 4 is a graph showing a phase delay of fluorescence generated from a donor fluorescent molecule for each distance between a donor fluorescent molecule and an acceptor fluorescent molecule.

FIG. 5 is a graph showing a modulation degree of fluorescence generated from an acceptor fluorescent molecule for each distance between a donor fluorescent molecule and an acceptor fluorescent molecule.

FIG. 6 is a graph showing the phase lag of fluorescence generated from an acceptor fluorescent molecule for each distance between a donor fluorescent molecule and an acceptor fluorescent molecule.

FIG. 7 is a configuration diagram of a subject detection apparatus suitable for applying the subject detection method according to the present invention.

[Explanation of symbols]

10: analyte, 11, 12: base sequence of oligonucleotide probe, 20: donor probe, 21: donor fluorescent molecule, 22: linker part, 30: acceptor probe,
DESCRIPTION OF SYMBOLS 31 ... Acceptor fluorescent molecule, 32 ... Linker part, 40 ... Excitation light source, 42 ... Acousto-optic modulator, 44 ... Beam expander, 46 ... Reflecting mirror, 48 ... Filter, 50, 52 ... Synthesizer, 54 ... Delay unit, 56 ... framing camera, 58 ... macro lens, 60 ... image processing unit,
62: display unit, 70: specimen sample.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Akihiko Tsuji 5000 Hiraguchi, Hamakita City, Shizuoka Prefecture Inside Molecular Biophotonics Research Institute Co., Ltd.

Claims (3)

[Claims] 1. A donor fluorescent molecule which is arranged at a predetermined distance when coupled to a subject in a pair, causes a fluorescence resonance energy transfer phenomenon with respect to excitation light irradiation, and generates fluorescence with predetermined response characteristics, and A detection probe consisting of a donor probe and an acceptor probe each having an acceptor fluorescent molecule, each having the same donor fluorescent molecule and the acceptor fluorescent molecule, and binding to a pair of different types of analytes in a pair, A first step of mixing a plurality of types of detection probes that generate fluorescence with mutually different response characteristics into the sample, and irradiating the sample with excitation light whose intensity is modulated at a constant frequency; For each of the detection probes, based on the response characteristics of the detection probe bound to the analyte in pairs. Fluorescence generated from the detection probe to identify the frequency domain time-resolved optical measurement method,
A second step of detecting a subject to which the detection probe is bound based on the identified fluorescence. 2. The method according to claim 1, wherein the second step includes synchronously detecting a light beam from the sample based on a first synchronization signal obtained by adding a first phase delay to the intensity modulation of the excitation light. 2. The method according to claim 1, wherein the fluorescence is identified. 3. The method according to claim 2, wherein the second step further includes adding a second phase delay to the intensity modulation of the excitation light.
Synchronously detects the light beam based on the synchronization signal of
3. The object detection method according to claim 2, wherein the fluorescence is identified based on each of the light beams synchronously detected based on each of the second synchronization signals.