JP2007033159A - Measuring object material labeled by fluorescent molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means capable of detecting, quantifying and identifying many measuring object materials simultaneously at low cost in a short time. <P>SOLUTION: The means is characterized by using the measuring object materials labeled by using a plurality of kinds of fluorescent molecules having different fluorescence life times, a test sample including them, and a reagent, a kit or the like using it. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a measurement target substance labeled with a plurality of types of fluorescent molecules, a test sample containing the same, a substance detection reagent using the same, a substance detection kit, and the like.

  In recent years, measurement of the concentration and mixing ratio of a fluorescently labeled substance present in a test sample has been performed mainly by measuring the fluorescence intensity of fluorescent molecules. That is, a fluorescent molecule with a known concentration (hereinafter referred to as “reference fluorescent molecule”) is placed in a test sample, and the concentration and mixing ratio are unknown by comparing the fluorescence intensity of the fluorescent molecule to be detected with the reference fluorescent molecule. The fluorescent molecules can be quantified. Applying such a method using fluorescence intensity, the fluorescent molecule is labeled on the probe or target molecule, and the intensity is compared, so that the target gene expression level or single nucleotide polymorphism (SNP: Single Nucleotide Polymorphism) ), It is industrially applied as a DNA microarray, TaqMan method, and invader method, and is now widely used. For example, a detection method for monitoring the amount of nucleic acid amplified by PCR by using two types of fluorescent molecules and comparing their fluorescence intensities is disclosed (Patent Document 1).

On the other hand, the fluorescence lifetime inherent to fluorescent molecules was thought to be less affected by the environment surrounding the fluorescent molecules and the degree of chemical reaction, and so has been used rarely. High-power lasers and laser diodes have been developed in the field, and the processing speed of electronic circuits has been increased and the measurement accuracy has been improved. Therefore, they have been actively used in the bio field. For example, a method for quantifying the abundance of a reaction product of an immune reaction using the fact that the transfer of photoluminescence energy appears in the apparent fluorescence lifetime is known (Patent Document 2). In addition, as a “fluorescence analysis method for biological systems”, the internal growth of the acceptor is determined by changing the fluorescence regulation lifetime and phase lifetime due to fluorescence resonance energy transfer between the donor and acceptor. A measurement method that can be monitored is disclosed (Patent Document 3).
Japanese National Patent Publication No. 8-510562 JP-A-6-66802 Japanese translation of PCT publication No. 2002-542453

  Currently quantified or identified test samples, especially nucleic acids, are performed by labeling samples amplified by PCR, LAMP, ICAN, etc. with fluorescent molecules and detecting their fluorescence intensity. . However, the types of test samples that can be identified have so far been limited to the number of fluorescent molecules whose fluorescence wavelengths can be separated, so simultaneous detection and quantification of several types of analytes labeled with about three types of fluorescent molecules・ Identification was the limit.

  Accordingly, an object of the present invention is to provide means for simultaneously detecting and quantifying and identifying a large number of substances to be measured at low cost and in a short time.

  The present inventor has intensively studied to solve the above problems. As a result, it has been found that the above-mentioned problems can be solved by using a measurement target substance labeled with a plurality of types of fluorescent molecules having different fluorescence lifetimes, a test sample containing the same, and a reagent, kit, or the like using the same. Was completed.

  That is, the present invention is as follows.

 (1) A substance to be measured, characterized by being labeled with a plurality of types of fluorescent molecules having different fluorescence lifetimes.

  In the measurement target substance of the present invention, for example, any one of the fluorescent molecules can be labeled at two or more locations. Further, at least two of the fluorescent molecules can be labeled at two or more locations.

  The fluorescence lifetime of the fluorescent molecule can be, for example, 100 ns or less.

  The plurality of types of fluorescent molecules include, for example, a group having an intrinsic fluorescence lifetime of 0.01 ns or more and less than 1.0 ns, a group having 1.0 ns or more and less than 2.0 ns, a group having 2.0 ns or more and less than 3.0 ns, 3.0 ns Groups with less than 4.0ns, groups with 4.0ns or more and less than 5.0ns, groups with 5.0ns or more and less than 6.0ns, groups with 6.0ns or more and less than 7.0ns, groups with 7.0ns or more and less than 10.0ns Groups with more than 10.0ns and less than 15.0ns, Groups with more than 15.0ns and less than 20.0ns, Groups with more than 20.0ns and less than 25.0ns, Groups with more than 25.0ns and less than 30.0ns, More than 30.0ns , Fluorescent molecules belonging to each group of two or more groups selected from the group consisting of a group having less than 40.0 ns and a group having 40.0 ns or more.

  Two or more of the fluorescent molecules may have a fluorescence lifetime that is different from each other by 0.1 ns or more, and may have a fluorescence lifetime that is 1.1 times or more different from each other. Here, the types of fluorescent molecules having different fluorescence lifetimes can be, for example, three or more.

  For example, at least two of the fluorescent molecules may have a difference in emission peak wavelength of 50 nm or less.

  In addition, the measurement target substance of the present invention can be composed of, for example, a plurality of types of measurement target substances having different combinations of fluorescent molecules. Here, the plurality of types of measurement target substances may include, for example, measurement target substances that have the same type of fluorescent molecule but have different labeling amounts for each type.

  In the measurement target substance of the present invention, for example, the fluorescent molecules can be three or more kinds.

  In addition, the measurement target substance of the present invention can be composed of, for example, a substance labeled with two selected from three or more fluorescent molecules having different fluorescence lifetimes.

  In addition, the measurement target substance of the present invention can further include, for example, a substance labeled with one kind of fluorescent molecule. Here, per substance to be labeled, a group of substances labeled with one fluorescent molecule, a group of substances labeled with two fluorescent molecules having different fluorescence lifetimes, and three groups with different fluorescence lifetimes A group consisting of a group of substances labeled with fluorescent molecules, a group of substances labeled with four fluorescent molecules with different fluorescence lifetimes, and a group of substances labeled with five fluorescent molecules with different fluorescence lifetimes Substances belonging to each group of two or more types of groups selected from the above can be included. Further, it can be composed of a substance labeled with one kind of fluorescent molecule and a substance labeled with two kinds of fluorescent molecules having different fluorescence lifetimes. Alternatively, it consists of a substance labeled with one kind of fluorescent molecule, a substance labeled with two kinds of fluorescent molecules having different fluorescence lifetimes, and a substance labeled with three kinds of fluorescent molecules having different fluorescence lifetimes. Can be.

  In the measurement target substance of the present invention, for example, the number of labeling sites by the fluorescent molecule can be 3 to 100 per substance to be labeled. Further, for example, at least one of the fluorescent molecules may have a known fluorescence lifetime.

  Examples of the substance labeled with the fluorescent molecule include at least one selected from the group consisting of nucleic acids, amino acids, peptides, proteins, ligands, receptors, donors, hormones, sugar chains, cells, and embryos.

 (2) A test sample comprising the substance to be measured of the present invention.

  In the test sample of the present invention, for example, at least one of the measurement target substances may have a known concentration.

  In addition, the test sample of the present invention can further contain, for example, at least one fluorescent substance having a known concentration. Here, at least one of the fluorescent substances may have a known fluorescence lifetime, for example.

 (3) A substance detection reagent comprising the substance to be measured of the present invention or the test sample of the present invention.

 (4) A substance detection kit comprising the substance to be measured of the present invention or the test sample of the present invention.

 (5) A method for analyzing the structure and / or abundance ratio of the measurement target substance based on the fluorescence lifetime of the fluorescent molecule labeled on the measurement target substance of the present invention.

 (6) A method for identifying the type and / or abundance of the measurement target substance based on the fluorescence lifetime of the fluorescent molecule labeled on the measurement target substance of the present invention.

  According to the present invention, a measurement target substance labeled with a plurality of types of fluorescent molecules having different fluorescence lifetimes, a test sample containing the same, a substance detection reagent using the same, a substance detection kit, a structure of the measurement target substance, and It is possible to provide a method for analyzing the abundance ratio and a method for specifying the type and / or amount of the substance to be measured. The present invention can be said to be extremely useful in that detection or the like can be performed with high sensitivity even for a low-concentration measurement target substance.

Hereinafter, the present invention will be described in detail. However, the scope of the present invention is not limited to these descriptions, and modifications other than the following exemplifications can be made as appropriate without departing from the spirit of the present invention.
1. Outline The present inventor has focused on the fact that the fluorescence intensity emitted from a fluorescent molecule is attenuated over time, and has considered to measure the decay of the fluorescence intensity in a time-dependent manner. The fluorescence decay curve of the fluorescence intensity of the test sample containing fluorescent molecules with different fluorescence lifetimes (time dependence of the fluorescence intensity emitted from the sample) is calculated using the single photon counting method ("Fluorescence measurement" Kazuhiko Kinoshita and Satoshi Mihashi). Ed., Society Publishing Center).

  Note that the fluorescence lifetime measurement exemplified in the description of the present invention is not necessarily limited to using the single photon counting method, and any phase modulation method or pulse sampling method can be used as long as the fluorescence lifetime can be measured. Any other known method such as an excitation probe method may be used. Even when the fluorescence lifetime value and the intensity ratio are obtained using these other methods, the substance to be measured can be identified and quantified as in the case of using the single photon counting method described below.

In the present invention, the fluorescence lifetime means the time until the fluorescence intensity I ₀ by pulse excitation light reaches 1 / e (e represents the base of natural logarithm), and is a value inherent to the fluorescent molecule. . For example, FAM (made by Invitrogen, USA) is 4.0 nanoseconds (nsec), PyMPO (made by Invitrogen, USA) is 1.8 nsec, 5-carboxynaphthofluorescein is 0.65 nsec, and fluorescein is 4.04 nsec.

  The single-photon counting method is a method that detects each photon, which is the lowest unit of light, one by one, so it has the highest sensitivity among all photodetection methods, and is compared with the fluorescence intensity and absorbance that are generally used. It is suitable for stably measuring the fluorescence lifetime of a very small amount of sample without depending on the concentration. For example, as disclosed in the international application filed by the present inventor (WO2004 / 090517 (hereinafter referred to as “Patent Document 4”)), FIG. 1 shows a fluorescence lifetime measurement method using a single photon counting method, A method for quantification and identification of substances with minute differences in fluorescence lifetime is shown. Specifically, the fluorescence emitted from the test sample 1 by irradiating the pulsed light 3 from the pulsed light source 2 to the test sample 1 (the test sample 1 contains the substance to be measured) placed in a cell, tube, microplate or the like. 4 is detected by a photomultiplier tube (PMT) 5. The wavelength of the light source 2 and the wavelength of the light detected by the photomultiplier tube 5 are selected by a filter or a spectroscope. A signal from the pulse light source 2 is used as a start signal, and a signal from the photomultiplier tube 5 is used as a stop signal, via an amplifier 6 and a constant fraction discriminator (CFD) 7, a time-voltage converter (TAC). : When input to Time to Amplitude Converter (8), there is a difference in the output signal of TAC8 depending on the timing detected by photomultiplier tube 5. By taking this into Multichannel Analyzer (MCA) 9, It is possible to measure the fluorescence lifetime. The output signal is stored in a hard disk of a personal computer (PC) 10 or the like.

In the present invention, i types of fluorescent molecules having different fluorescence lifetimes “F ₁ (fluorescence lifetime τ ₁ ), F ₂ (fluorescence lifetime τ ₂ ), F ₃ (fluorescence lifetime τ ₃ ), F ₄ (fluorescence lifetime τ _4). ),..., F _i (fluorescence lifetime τ _i ) ”will be described as an example of measuring the concentration of a DNA probe (substance to be measured), but the measurement target substance is not limited to this. .

First, the measurement target substance is a fluorescent molecule “F ₁ , F ₂ , F ₃ , F ₄ ,...” Having different fluorescence lifetimes, like the DNA probes of (1) to (8) in FIGS. Be labeled. Specifically, one or two or more fluorescent molecules are present at any one or two or more of the bases of the 5 ′ end, 3 ′ end and non-terminal portion of a DNA probe comprising an arbitrary base sequence. To label. For example, in the DNA probe (1), the fluorescent molecule F ₁ is bonded to the base at the 3 ′ end, and in the DNA probe (2), the fluorescent molecule F ₂ is bonded to the base at the non-terminal portion. In the DNA probe (6), fluorescent molecules F ₂ and F ₃ are bonded to _two bases of the non-terminal part, respectively. In the DNA probe (7), the bases of the 5 ′ end, 3 ′ end and non-terminal part F ₁ , F _3, and F ₂ are bonded to each in turn. Similarly to these, fluorescent molecules are bound to other DNA probes.

  The DNA probe as the measurement target substance is not limited to those having the base sequences shown in FIGS. 2A and 2B.

  Next, when the fluorescence intensity of the labeled DNA probes (1) to (8) is measured, attenuation curves (1) to (3) shown in FIGS. 3A and 3B can be obtained.

  Specifically, when the substance to be measured is labeled with only one type of fluorescent molecule, an attenuation curve (1) having the form shown in FIG. 3A is obtained. In this case, an attenuation curve based only on the intrinsic fluorescence lifetime of each fluorescent molecule is obtained.

  Similarly, when labeled with two kinds of fluorescent molecules, an attenuation curve (2) having the embodiment shown in FIG. 3A is obtained. In this case, first, up to a certain point, an attenuation curve based on the intrinsic fluorescence lifetime of the two fluorescent molecules having the shorter fluorescence lifetime is obtained, and thereafter, the intrinsic curve of the other (the longer fluorescence lifetime) is obtained. Thus, an attenuation curve based on the fluorescence lifetime is obtained. That is, as a whole, an attenuation curve is obtained by combining a part of the intrinsic attenuation curves of two kinds of fluorescent molecules, and unless the combination of the two kinds of fluorescent molecules is the same, They are different from each other.

  Similarly, when labeled with three kinds of fluorescent molecules, an attenuation curve (3) of the embodiment shown in FIG. 3B is obtained. In this case, it can be considered in the same manner as in the case of labeling with two kinds of fluorescent molecules. First, an attenuation curve based on the intrinsic fluorescence lifetime of the three fluorescent molecules having the shortest fluorescence lifetime is obtained until a certain point in time, and then the intrinsic fluorescence of the next shortest fluorescence lifetime is obtained until a certain point in time. An attenuation curve based on the lifetime is obtained, and thereafter an attenuation curve based on the intrinsic fluorescence lifetime of the longest fluorescence lifetime is obtained. That is, as a whole, an attenuation curve is obtained by combining a part of the intrinsic attenuation curves of the three types of fluorescent molecules, and unless the combination of the three types of fluorescent molecules is the same, the obtained attenuation curve is the relevant combination. They are different from each other.

  In addition, the above can be considered similarly also when it labels with 4 types, 5 types, ..., i type fluorescent molecules.

That is, when labeled with i types of fluorescent molecules, (2 ⁱ −1) types of different attenuation curves can be obtained except when the various fluorescent molecules are labeled in duplicate. Since this fluorescence decay curve is specific to the combination and type of fluorescent molecules once the excitation wavelength and fluorescence wavelength are determined, the substance to be measured labeled with the fluorescent molecule can be identified from the shape pattern of the decay curve.

  In addition, one of the fluorescent molecules contained in the test sample (fluorescent molecules contained without being involved in labeling) is used as a reference fluorescent molecule (internal standard) so that the concentration is always constant. In addition, by comparing the fluorescence intensities, the concentration of other fluorescent molecules whose concentration is unknown (that is, the concentration of the measurement target substance labeled with these fluorescent molecules) can be obtained.

In other words, this means that if a fluorescence decay curve is measured, (2 ⁱ −1) types of measurement target substances labeled with fluorescent molecules can be identified. Further, as disclosed in the above-mentioned “Patent Document 4”, the quantification can be obtained by comparing the fluorescence lifetime and coefficient A obtained by deconvolution of the fluorescence decay curve with a reference fluorescent molecule, Specifically, it is as follows.

  That is, generally, when obtaining a fluorescence decay curve when i types of fluorescent molecules having different intrinsic fluorescence lifetimes are used, the fluorescence lifetime function f (t) is expressed by the following equation (I).

[In formula (I), A _i represents a coefficient, t represents time, and τ _i represents fluorescence lifetime. ]
The fluorescence lifetime function f (t) can be expressed by the following formula (II) to which a background at the time of measurement is added as necessary.

[In formula (II), A _i is a coefficient, t is time, and τ _i is fluorescence lifetime. ]
The fluorescence lifetime and the intensity of the fluorescent molecule contained in the test sample are obtained by deconvolution of the measured fluorescence decay curve. Note that deconvolution is obvious to those skilled in the art. From the resulting coefficient A and fluorescence lifetime τ, the ratio of the fluorescence intensity of each fluorescent molecule is proportional to (A _i × τ _i ) (where i = 1, 2, 3, ...). The mixing ratio can be estimated by a method of (A ₁ × τ ₁ ) :( A ₂ × τ ₂ ) :( A ₃ × τ ₃ ):. If one of the fluorescent molecules contained in the test sample is used as a reference fluorescent molecule, the concentration of another fluorescent molecule whose concentration is unknown can be obtained by comparing the fluorescent intensities as described above.

For example, when two kinds of fluorescent molecules having different fluorescence lifetimes τ ₁ and τ ₂ are used, when the fluorescence decay curve of the test sample is measured, data as shown in FIG. The Then, deconvolution of the fluorescence intensity decay, which is the convolution integral of the response function g (t) of the device and the intrinsic fluorescence lifetime function f (t) of the fluorescent molecule, is included in the test sample. The fluorescence lifetime of the fluorescent molecule is obtained. From the coefficient A and the fluorescence lifetime τ obtained as a result, the mixing ratio is calculated by (A ₁ × τ ₁ ) :( A ₂ × τ ₂ ). If one of the fluorescent molecules contained in the test sample is used as a reference fluorescent molecule, the concentration of another fluorescent molecule whose concentration is unknown can be determined by comparing the fluorescence intensities.

  In the above example, the case where only one i-type fluorescent molecule is simultaneously labeled per measurement substance (that is, when various fluorescent molecules are not labeled in duplicate) is shown. In consideration of labeling the same type of fluorescent molecules so that they can be overlapped (ie, two or more), for example, more labeling patterns can be formed as in the DNA probes shown in (9) to (11) of FIG. 2B. . In addition, the DNA probes shown in (9) to (11) are also the 5 ′ end, 3 ′ end and non-end of a DNA probe consisting of an arbitrary base sequence, like the DNA probes of (1) to (8) described above. One or two or more fluorescent molecules can be bound and labeled at any one or two or more of the partial bases, and a DNA probe as a measurement target substance is shown in FIG. 2B. However, the present invention is not limited to those having the base sequence shown below.

Specifically, the DNA probes shown in (9) to (11) of FIG. 2B are the same in that there are two types of fluorescent molecules used as labels, F ₁ and F _2. When labeling is performed with a difference in one or both of the labeling amounts (for example, the number of F ₁ is different in the DNA probe shown in (9) and (10) of FIG. 2B), and the fluorescence intensity is measured. The attenuation curve (4) as shown in FIG. 3B is obtained. Since any of the above DNA probes are labeled with two types of fluorescent molecules, as described above, the obtained attenuation curves are partly each of the intrinsic attenuation curves of the two types of fluorescent molecules. It becomes a combined form. However, the time required for switching from the decay curve inherent to one fluorescent molecule to that of the other varies depending on the label amount (abundance) and the labeling ratio (abundance ratio) of the two types. As shown in the attenuation curve (4), the attenuation curve patterns are different from each other inherent in the labeling amount and the labeling ratio. Such an aspect of the present invention is very effective in terms of cost including a measuring apparatus and the like because many substances can be individually labeled with a few kinds of labels and measurement becomes easy. In addition, the above can be considered similarly when labeled with three, four, five,..., I types of fluorescent molecules.

2. Substance to be measured The substance to be measured of the present invention is characterized in that it is labeled with a plurality of types of fluorescent molecules having different fluorescence lifetimes as described above. The above-mentioned “labeled with a plurality of types (two or more types) of fluorescent molecules” means a plurality of types of fluorescent molecules having different fluorescence lifetimes, such as the DNA probes of (4) to (11) in FIGS. The labeling is performed using two or more of F ₁ , F ₂ , F ₃ , F ₄ ,..., But the present invention is not limited to these.

  Here, it is preferable that the substance to be measured is “there are three or more kinds of fluorescent molecules”, and means, for example, cases (7) to (8) in FIG. 2B. In addition, “any one of the fluorescent molecules is labeled at two or more locations” or “at least two of the labeled fluorescent molecules are each at two locations. “Labeled above” is also a preferred embodiment, which means, for example, the cases (9) to (11) in FIG. 2B and the case (11) in FIG. 2B, respectively.

  It is also preferable that the measurement target substance of the present invention is a group of so-called measurement target substances (measurement target substance group) composed of a plurality of types of measurement target substances having different combinations of a plurality of types of fluorescent molecules as labels. As mentioned. The above “consisting of a plurality of types of substances to be measured with different combinations of fluorescent molecules” means, for example, from two or more of the labeled DNA probes (4) to (11) in FIGS. 2A and 2B. It means the case where it is configured.

  Here, “a plurality of types of measurement target substances” in the measurement target substance (measurement target substance group) includes the measurement target substances having the same type of fluorescent molecule used as a label but different label amounts for each type. This means that it may be “thing”, which is one of the preferred embodiments. For example, when two or more of (4) and (9) to (11) in FIGS. 2A and 2B are included, this is an example of this aspect. In this case, it is further preferable that “the number of types of fluorescent molecules is 3 or more”, which means a case including, for example, (7) to (8) in FIG. 2B.

  In addition, the above-mentioned measurement target substance (measurement target substance group) is also a preferable embodiment in which the measurement target substance (measurement target substance group) is composed of a substance labeled with two selected from three or more fluorescent molecules having different fluorescence lifetimes. For example, it means a case including two or more of (4) to (6) in FIG. 2A.

  In addition, the above-described measurement target substance (measurement target substance group) is also preferably a "further containing substance labeled with one type of fluorescent molecule". For example, (4) to (4) in FIGS. In addition to at least one of (11), it means that at least one of (1) to (3) in FIG. In this case, it is also a preferable aspect that “comprising a substance labeled with one kind of fluorescent molecule and a substance labeled with two kinds of fluorescent molecules having different fluorescence lifetimes” is also a preferable embodiment. 2A including at least one of (1) to (3) and at least one of (4) to (6) of FIG. 2A and (9) to (11) of FIG. 2B Means the case. Similarly, “a substance labeled with one fluorescent molecule, a substance labeled with two fluorescent molecules with different fluorescence lifetimes, and a substance labeled with three fluorescent molecules with different fluorescence lifetimes” It is also a preferred embodiment, but for example, at least one of (1) to (3) in FIG. 2A and (4) to (6) in FIG. 2A and (9) to (9) in FIG. It means a case in which at least one of (11) and at least one of (7) to (8) in FIG. 2B are included.

  Furthermore, as the measurement target substance (measurement target substance group), the following can be exemplified as a preferred embodiment. That is, a group of substances that are labeled with only one fluorescent molecule per substance to be labeled (for example, (1) to (3) in FIG. 2A), labeled with only two fluorescent molecules having different fluorescence lifetimes. Group of substances (for example, (4) to (6) in FIG. 2A), group of substances labeled with only three fluorescent molecules having different fluorescence lifetimes (for example, (7) to (9) in FIG. 2B) ), Two or more kinds selected from the group consisting of a group of substances labeled only with four fluorescent molecules having different fluorescence lifetimes and a group of substances labeled only with five fluorescent molecules having different fluorescence lifetimes Preferred examples include those containing substances belonging to each group.

  In addition, the DNA probes of (1) to (11) in FIGS. 2A and 2B in the above description are shown in SEQ ID NOs: 1 to 11, respectively.

  In the measurement target substance of the present invention, if the number of labeling sites by a plurality of types of fluorescent molecules is too small, the number of identifiable measurement target substances is limited, but if it is too large, the cost of the fluorescent molecules increases. . Therefore, the number of labeling sites is preferably, for example, 3 to 100 sites, more preferably 4 to 50 sites, and further preferably 5 to 30 sites per single substance to be labeled.

  If the measurement target substance of the present invention, even if a plurality of fluorescent molecules for labeling that could not be used at the same time in the same solution due to the large overlap of the fluorescence spectrum, if the respective fluorescence lifetimes differ from each other by 1.1 times or more, There is an unprecedented advantage that detection and measurement can be multiplexed (multiplexed) and performed at a high throughput, regardless of the fluorescence spectrum. That is, it is possible to improve productivity by applying a combination of fluorescent molecules as shown in the present invention to many applications that have been analyzed by comparing fluorescence intensities so far. Specifically, when a band-pass filter is used as the fluorescence-side wavelength filter, not only the time dependency of the fluorescence intensity but also the wavelength dependency can be used, and the number of target substances to be detected has jumped. This means that the efficiency of the assay can be increased and the cost can be reduced.

  Here, the fluorescence lifetime of multiple types of fluorescent molecules to be labeled is stable for quantitative and qualitative analysis that two or more of them (and more than three of them) differ 1.1 times or more from each other as described above. However, it is more preferably 2 times or more, further preferably 3 times or more, and particularly preferably 5 times or more.

  In addition, the fluorescence lifetime of the multiple types of fluorescent molecules to be labeled may differ by 0.1 ns or more for each type (between all types), or for two or more of them (and more than three types). Although it is preferable in that the quantitative and qualitative analysis can be performed stably, it is more preferably 0.3 ns or more, further preferably 0.5 ns or more, and particularly preferably 1.0 ns or more. In addition, it is preferable that the fluorescence lifetimes of the fluorescent molecules to be labels are all 100 ns or less.

  Furthermore, it is possible to industrially realize quantification and qualification that the difference between the fluorescence peak wavelength and / or the absorption peak wavelength of at least two of the plurality of types of fluorescent molecules to be labeled is 50 nm or less. The difference is preferably 30 nm or less, more preferably 10 nm or less. Of course, you can use multiple types of fluorescent molecules even if the absorption and fluorescence peak wavelengths are far from each other, but if absorption and wavelength peaks are close, use the same light source or use the same fluorescence wavelength filter. Therefore, it is preferable in terms of effects such as high throughput and cost reduction. Here, the plurality of types of fluorescent molecules to be labeled have fluorescence lifetimes different from each other by 0.1 ns or more for each type, and at least two of them have a difference in emission peak wavelength of 50 nm or less, It can mention as a particularly preferable aspect at the point of the said effect.

  Moreover, the following can be mentioned as a preferable aspect of the measuring object substance of this invention. In other words, a plurality of types of fluorescent molecules to be labeled are groups having an intrinsic fluorescence lifetime of 0.01 ns or more and less than 1.0 ns, a group having 1.0 ns or more and less than 2.0 ns, a group having 2.0 ns or more and less than 3.0 ns, Groups with 3.0ns or more and less than 4.0ns, groups with 4.0ns or more and less than 5.0ns, groups with 5.0ns or more and less than 6.0ns, groups with 6.0ns or more and less than 7.0ns, groups with 7.0ns or more, 10.0ns Group with less than, 10.0ns or more, group with less than 15.0ns, Group with more than 15.0ns, less than 20.0ns, Group with more than 20.0ns, less than 25.0ns, Group with more than 25.0ns, less than 30.0ns, 30.0 Preferred examples include those containing fluorescent molecules belonging to each group of two or more groups selected from the group consisting of a group having ns or more and less than 40.0 ns, and a group having 40.0 ns or more (in a single substance to be measured). be able to.

  In the measurement target substance of the present invention, it is preferable that at least one of the fluorescent molecules used as a label has a known fluorescence lifetime, and it is naturally more preferable that all are known.

  The fluorescent molecule that can be used as a label in the measurement target substance of the present invention is not limited, and examples thereof include the following dyes: Cascade Yellow, Dapoxyl carboxylic acid, Pacific Blue, 7-Hydroxycourmarin-3-carboxylic acid , FAM (Invitrogen, USA), 5-carboxynaphthofluorescein, Dabcyl LysoSensor, Lucifer Yellow, Alexa Flour, PyMPO (Invitrogen, USA), NBD-X, DCCH, HEX, JOE, ROX, Texas Red, TET, TAMRA, AMC , Ant, Mant, DAN (Invitrogen), Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7 (Amersham Bioscience), Qdot (Quantum Dot, USA), FITC, GFP .

  Regarding the measurement target substance of the present invention, the substance labeled with a fluorescent molecule is preferably a probe or a target. As the probe or target, for example, at least one selected from the group consisting of nucleic acids, amino acids, peptides, proteins, ligands, receptors, donors, hormones, sugar chains, cells, embryos and the like can be preferably used. It is suitable for the purpose of identification and identification / detection of trace substances that emit fluorescence. It is also possible to discriminate between different types of measurement substances (for example, genes). Examples of means / methods include DNA microarray, TaqMan method, invader method, in situ hybridization, flow cytometry, various sequencers, fluorescence microscope, etc. By replacing the measurement target substance with one labeled with a plurality of fluorescent molecules having different fluorescence lifetimes as in the present invention, it becomes possible to further multiplex and analyze the measurement target substance such as a gene with high throughput. obtain.

3. Test Sample The test sample of the present invention is characterized by containing the above-described measurement target substance of the present invention.

  In the test sample, it is preferable that at least one of the substances to be measured contained has a known concentration.

  In addition to the measurement target substance, the test sample preferably further contains at least one fluorescent substance having a known concentration. Here, it is preferable that at least one of the fluorescent materials has a known fluorescence lifetime.

  The said test sample can contain suitably other components in the range which does not impair the effect of this invention. Examples of other components include buffer solutions and blocking solutions such as BSA, and RNase and DNase-free water.

4). Substance detection reagent, substance detection kit
(1) The reagent for detecting a substance of the present invention is characterized by containing the above-mentioned substance to be measured of the present invention or the test sample of the present invention. The above reagent can appropriately contain other components as long as the effects of the present invention are not impaired. Examples of other components include buffer solutions and blocking solutions such as BSA, and RNase and DNase-free water.

 (2) The substance detection kit of the present invention is characterized by including the above-described measurement target substance of the present invention or the above-described test sample of the present invention. In addition to the above-mentioned measurement target substance or test sample, the kit can contain a buffer solution, instructions for use, various parts, and the like, and preferably contains the above-described substance detection reagent of the present invention. In addition, other components can be appropriately included as long as the effects of the present invention are not impaired. Examples of other components include a detection chip made of plastic and glass, a detection microtube, a detection microtiter plate, a column, and the like.

5. Various methods such as substance detection methods
(1) In the present invention, based on the fluorescence lifetime of the fluorescent molecule labeled on the measurement target substance of the present invention, the ratio of the amount of each fluorescent molecule in the measurement target substance and / or the fluorescence intensity ratio is analyzed. Methods can be included.

(2) Method for Analyzing Structure of Substance to be Measured The analysis method of the present invention is based on the fluorescence lifetime of the fluorescent molecule labeled on the substance to be measured of the present invention, and the structure and / or abundance ratio of the substance to be measured. Is a method of analyzing

  Specifically, for example, a plurality of fluorescent molecules having different fluorescence lifetimes are labeled on peptides having different amino acid sequences, and each peptide is labeled so as to be clearly distinguished by the fluorescence lifetime, and then a specific protein is labeled. After the binding and BF (Binding / Free) separation, which peptide is bound to the specific protein can be quantitatively estimated by measuring the fluorescence lifetime. If the amino acid sequence of a peptide bound to a specific protein is known, the types of amino acids constituting the active site of the protein, the three-dimensional structure, and the like can be known. As another example, a plurality of fluorescent molecules having different fluorescence lifetimes and one dark quencher (quenching molecule) are labeled at a plurality of sites of DNA or RNA. After the DNA or RNA forms a three-dimensional structure, the fluorescent molecules whose fluorescence intensity decreases with the fluorescence lifetime are identified from the fluorescence lifetime data, and the portion where the quencher is labeled and the fluorescence whose fluorescence intensity decreases It can be confirmed that the site where the molecule is labeled is close when a three-dimensional structure is formed.

(3) Identification method of the type of the measurement target substance The identification method of the present invention is based on the fluorescence lifetime of the fluorescent molecule labeled on the measurement target substance of the present invention, and the type and / or abundance of the measurement target substance. It is a method to specify. The type and / or abundance of the substance to be measured here refers to the type and / or abundance of a functional group that has reacted with the substance to be measured, and the type and / or presence of a substance that binds or associates with the substance to be measured. Including quantity.

  Regarding the specific method of the present invention, specifically, for example, proteins are known to have functional groups such as amino groups and thiol groups, but succinimidyl esters that can react with these functional groups, By labeling a fluorescent molecule in a state such as maleimide with a protein and analyzing the abundance ratio of the fluorescent molecule obtained from the fluorescence lifetime, what percentage of the amino group and thiol group are contained in the protein? Can be identified. In the analysis using a DNA microarray, a plurality of types of DNA probes labeled with a plurality of fluorescent molecules having different fluorescence lifetimes are prepared, and these are hybridized with a target DNA immobilized on a substrate, and BF (Binding / Free) After the separation, if the fluorescence lifetime of the probe left on the substrate is measured, it becomes possible to quantitatively confirm which probe is bound to or associated with which specific target.

 (4) In the present invention, the fluorescence lifetime of the fluorescent molecule labeled on the measurement target substance of the present invention is measured using a known fluorescence measurement method such as a single photon counting method, a phase modulation method, a pulse sampling method, or an excitation probe method. It can include a method for detecting a substance, characterized by measuring by a method.

  In the detection method, the fluorescence lifetime can be measured at a plurality of excitation wavelengths and / or a plurality of fluorescence wavelengths. Specifically, two or more types of fluorescence wavelengths can be detected by exciting at two or more types of wavelengths using a plurality of types of light sources or using various types of filters such as a plurality of types of bandpass filters.

 (5) The present invention can include a method for detecting a substance by flow cytometry, wherein the substance to be measured of the present invention is used as a detection substance in a test sample. In flow cytometry, cells and chromosomes to be measured are usually labeled with one type of fluorescent molecule for each type, and laser light is applied when flowing with water flow, and the spectrum and intensity of the emitted fluorescence. Depending on the type, the type of cell or the like is discriminated. That is, as the types of measurement objects to be discriminated increase, the types of fluorescent molecules are also required. On the other hand, if a plurality of fluorescent molecules having different fluorescence lifetimes are labeled by applying the present invention, the fluorescence to be labeled for each type is increased even when the types of measurement objects to be identified increase. By providing a difference in the combination of molecules and the amount of labeling, it is possible to discriminate and separate many types of measurement objects by using fewer types of fluorescent molecules.

 (6) The present invention can include a sequencing method characterized in that the measurement target substance of the present invention is used as a detection substance in a test sample. The present invention can also include a method for discriminating the type of gene, characterized in that the above-mentioned measurement target substance of the present invention is used as a detection substance in a test sample.

  In the sequencer, laser light is applied when DNA labeled with one type of fluorescent molecule flows on the gel, and the type of labeled DNA is determined according to the spectrum of emitted fluorescence and its intensity. Even in a sequencer that can read a sequence of only about 1000 bases with the conventional technology, the present invention is applied to label multiple types of fluorescent molecules with strong fluorescence intensity such as FAM and VIC by changing the ratio of their labeling amounts. Then, it becomes possible to distinguish a long array with high sensitivity. The same method can be applied to discrimination of gene type (detection of single nucleotide polymorphism, etc.) using a sequencer.

 (7) The present invention can include an in situ hybridization method characterized in that the above-mentioned measurement target substance of the present invention is used as a detection substance in a test sample. Further, the present invention can include a method for detecting a substance using a fluorescence microscope, wherein the substance to be measured of the present invention is used as a detection substance in a test sample.

  In the in situ hybridization method (FISH method) using fluorescence, generally, fluorescent molecules are labeled on DNA, RNA, etc., and the measurement target substance labeled with a fluorescence microscope is observed. Even in this case, if the present invention is applied and a plurality of fluorescent molecules having different fluorescence lifetimes are labeled on DNA or RNA, more genes and RNAs can be simultaneously discriminated in situ based on the fluorescence lifetime and quantified. Can be realized.

  In the above methods (1) to (7), a laser diode (LD) or a laser may be used as a light source, but a flash lamp or a light emitting diode (LED) may also be used. Absent. In the present invention, the pulse frequency, pulse intensity, and pulse diameter can be appropriately selected depending on the measurement object. For example, the pulse frequency is 1 kHz to 1 GHz, and the pulse intensity is several μW to several hundred W. The pulse diameter is several tens of μm to several tens of mm.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

<Identification and quantitative measurement of DNA labeled with fluorescent molecules having different fluorescence lifetimes>
In this example, FAM having a fluorescence lifetime of 4.0 ns (τ ₁ ) is defined as F ₁ , PyMPO having a fluorescence lifetime of 1.8 ns (τ ₂ ) is defined as F _2, and a measurement target substance labeled with both fluorescent molecules is defined as The fluorescence decay curves of the test samples contained were measured and observed using the DNA probes (1), (2), and (4) of FIG. The peak wavelengths of the absorption spectrum and fluorescence spectrum of FAM are 415 nm and 570 nm, respectively. The peak wavelengths of the absorption spectrum and the fluorescence spectrum of PyMPO are 495 nm and 516 nm, respectively.

As a procedure, a solution containing the DNA probes (FIGS. 4 (1) to (3)) labeled with fluorescent molecules FAM and PyMPO (in order, SEQ ID NOs: 12, 13, and 14) was prepared, and the fluorescence decay curve was obtained. It was measured. When the test sample was excited with an LD light source having a wavelength of 408 nm and measured at a fluorescence wavelength of 570 nm, a result as shown in FIG. 5 was obtained. FIG. 5 shows data obtained by measuring different solutions (pH = 8.5, concentration 1.0 μM) containing the DNA probes (1) and (2) of FIG. The fluorescence lifetime of the solution containing the DNA probe shown in FIGS. 4 (1) and (2) obtained from FIG. 5 is the same as that of FAM alone or PyMPO alone not labeled with DNA, 4.0ns and 1.8ns respectively. Met. Next, the FAM and PyMPO-labeled DNA probe described in FIG. 4 (3) was excited at 408 nm, and fluorescence decay curves were measured at 515 nm and 570 nm. The result shown in FIG. 6 was obtained. It was. As is clear from FIG. 6, the fluorescence decay curve of the DNA probe labeled with FAM and PyMPO simultaneously is a combination of the fluorescence lifetime of PyMPO and the fluorescence lifetime of FAM. The decay curve obtained at 515 nm is The ratio of PyMPO is higher than the attenuation curve at 570 nm. This is probably because the wavelength of 515 nm is close to the fluorescence peak wavelength of PyMPO. The fluorescence intensity ratios (A ₁ × τ ₁ : A ₂ × τ ₂ ) of FAM and PyMPO at 515 nm and 570 nm were 42:58 and 21:79, respectively. Here, the coefficient Ai (where i = 1, 2) of the exponential function (described above) described in “Patent Document 4” was calculated by applying it to the fluorescence decay curve. As shown in FIG. 6, when the wavelength at which the attenuation curve is monitored is changed, the component ratio of the fluorescence obtained from FAM and PyMPO changes, so by measuring the attenuation curve at several wavelengths, It becomes possible to measure the abundance ratio with high accuracy.

FIG. 7 shows the results when DNAs labeled with FAM and PyMPO at the same time were measured at different concentrations (0.33 μM and 1.00 μM) with a constant measurement time. Here, parameters other than concentration (excitation wavelength, fluorescence wavelength, slit width, light source intensity, etc.) are exactly the same. As is clear from FIG. 7, when the concentration is reduced to 1/3, the count (fluorescence intensity) is also reduced to about 1/3, but the ratio of the FAM fluorescence lifetime component to the PyMPO component depends on the concentration. Is constant without. That is, the fluorescence intensity ratio (A ₁ × τ ₁ : A ₂ × τ ₂ ) of FAM and PyMPO was 21:79 at any concentration. From this, DNA labeled with two different fluorescent molecules at the same time can be easily distinguished by measuring the fluorescence lifetime, and easily quantified by comparing the fluorescence lifetime intensities. Indicates that it is possible.

  As is clear from this example, since the ratio of the fluorescence lifetime of FAM and PyMPO is more than twice, it is easy to separate the components of fluorescence lifetime derived from FAM and PyMPO, and FAM and PyMPO are labeled simultaneously. When the concentration of DNA is changed, the ratio of FAM and PyMPO does not change, but the intensity (count) of the fluorescence decay curve changes accordingly. At this time, if DNA labeled with FAM is used as a reference molecule with known concentration, the fluorescence decay curve of DNA labeled with PyMPO with unknown concentration and DNA labeled with FAM and PyMPO at the same time can be compared with the reference fluorescent molecule. It is possible to determine the concentration of each DNA. It is also possible to further expand the concentration detection range (dynamic range) by mixing two or more types of reference fluorescent molecules with known concentrations in the solution.

1 represents a measurement principle and measurement apparatus of a single photon counting method that can be used in the present invention. As an example of the present invention, it is a diagram showing a DNA sequence labeled with a fluorescent molecule. As an example of the present invention, it is a diagram showing a DNA sequence labeled with a fluorescent molecule. The schematic diagram of the fluorescence decay curve which shows that the decay curve of DNA which labeled the fluorescent molecule of this invention is intrinsic | native to a combination is shown. The schematic diagram of the fluorescence decay curve which shows that the decay curve of DNA which labeled the fluorescent molecule of this invention is intrinsic | native to a combination is shown. It is a figure which shows the arrangement | sequence of DNA labeled with the fluorescent molecule used in the present Example. The plot which shows that the decay curve of DNA which labeled the fluorescent molecule of a present Example is intrinsic | native to a fluorescent molecule. The plot which shows that the decay curve of DNA which labeled the fluorescent molecule of a present Example simultaneously changes shape with the fluorescence wavelength monitored. The plot which shows that the attenuation | damping curve of DNA which labeled the fluorescent molecule of a present Example simultaneously, changes the fluorescence intensity in proportion to a density | concentration, but the component ratio of a fluorescent molecule does not change.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Test sample 2 Pulse light source 3 Pulse light 4 Fluorescence 5 Photomultiplier tube 6 Amplifier 7 Constant fraction discriminator 8 Time-voltage converter 9 Multichannel analyzer 10 Personal computer 11 Excitation side wavelength filter 12 Fluorescence side wavelength filter

Sequence number 1: DNA probe sequence number 2: DNA probe sequence number 3: DNA probe sequence number 4: DNA probe sequence number 5: DNA probe sequence number 6: DNA probe sequence number 7: DNA probe sequence number 8: DNA probe sequence number 9: DNA probe sequence number 10: DNA probe sequence number 11: DNA probe sequence number 12: DNA probe sequence number 13: DNA probe sequence number 14: DNA probe

Claims (28)

  A substance to be measured, which is labeled with a plurality of types of fluorescent molecules having different fluorescence lifetimes.   The measurement target substance according to claim 1, wherein any one of the fluorescent molecules is labeled at two or more locations.   The measurement target substance according to claim 1, wherein at least two of the fluorescent molecules are labeled at two or more locations.   The measurement target substance according to any one of claims 1 to 3, wherein a fluorescence lifetime of the fluorescent molecule is 100 ns or less.   The plurality of types of fluorescent molecules have a specific fluorescence lifetime of 0.01 ns or more, a group having less than 1.0 ns, a group having 1.0 ns or more, less than 2.0 ns, a group having 2.0 ns or more, less than 3.0 ns, a group having 3.0 ns or more, Groups with less than 4.0ns, groups with more than 4.0ns, less than 5.0ns, groups with more than 5.0ns, less than 6.0ns, groups with more than 6.0ns, less than 7.0ns, groups with more than 7.0ns, less than 10.0ns , 10.0 ns or more, group with less than 15.0 ns, 15.0 ns or more, group with less than 20.0 ns, 20.0 ns or more, group with less than 25.0 ns, 25.0 ns or more, group with less than 30.0 ns, 30.0 ns or more, 40.0 The measurement target substance according to any one of claims 1 to 4, comprising a fluorescent molecule belonging to each of two or more kinds of groups selected from the group consisting of a group having less than ns and a group having 40.0ns or more. .   The measurement target substance according to any one of claims 1 to 5, wherein two or more of the fluorescent molecules have fluorescence lifetimes different from each other by 0.1 ns or more.   The measurement target substance according to any one of claims 1 to 6, wherein two or more of the fluorescent molecules have fluorescence lifetimes different from each other by 1.1 times or more.   The measurement target substance according to claim 6 or 7, wherein there are three or more types of fluorescent molecules having different fluorescence lifetimes.   The substance to be measured according to any one of claims 1 to 8, wherein at least two of the fluorescent molecules have a difference in emission peak wavelength of 50 nm or less.   The measurement target substance according to claim 1, which is composed of a plurality of types of measurement target substances having different combinations of fluorescent molecules.   The measurement target substance according to claim 10, wherein the plurality of types of measurement target substances include measurement target substances having the same type of fluorescent molecule but having different labeling amounts for the respective types.   The measurement target substance according to any one of claims 1 to 11, wherein the fluorescent molecule has three or more kinds.   The measurement object substance according to any one of claims 1 to 12, comprising a substance labeled with two selected from three or more fluorescent molecules having different fluorescence lifetimes.   Furthermore, the measuring object substance of any one of Claims 1-13 containing the substance labeled with 1 type of fluorescent molecule.   For each substance to be labeled, a group of substances labeled with one fluorescent molecule, a group of substances labeled with two fluorescent molecules with different fluorescence lifetimes, and three fluorescent molecules with different fluorescence lifetimes Selected from the group consisting of a group of substances labeled, a group of substances labeled with four fluorescent molecules with different fluorescence lifetimes, and a group of substances labeled with five fluorescent molecules with different fluorescence lifetimes The measurement target substance according to claim 14, comprising a substance belonging to each group of two or more types of groups.   The measurement target substance according to claim 14 or 15, comprising a substance labeled with one kind of fluorescent molecule and a substance labeled with two kinds of fluorescent molecules having different fluorescence lifetimes.   It is composed of a substance labeled with one kind of fluorescent molecule, a substance labeled with two kinds of fluorescent molecules having different fluorescence lifetimes, and a substance labeled with three kinds of fluorescent molecules having different fluorescence lifetimes. The measurement target substance according to claim 14 or 15.   The substance to be measured according to any one of claims 1 to 17, wherein the number of labeling sites by the fluorescent molecules is 3 to 100 per one substance to be labeled.   The measurement target substance according to claim 1, wherein at least one of the fluorescent molecules has a known fluorescence lifetime.   The substance labeled with the fluorescent molecule is at least one selected from the group consisting of nucleic acids, amino acids, peptides, proteins, ligands, receptors, donors, hormones, sugar chains, cells and embryos. The measurement target substance according to any one of the above.   A test sample comprising the substance to be measured according to any one of claims 1 to 20.   The test sample according to claim 21, wherein at least one of the measurement target substances has a known concentration.   The test sample according to claim 21 or 22, further comprising at least one fluorescent substance having a known concentration.   The test sample according to claim 23, wherein at least one of the fluorescent substances has a known fluorescence lifetime.   A substance detection reagent comprising the measurement target substance according to any one of claims 1 to 20 or the test sample according to any one of claims 21 to 24.   A substance detection kit comprising the measurement target substance according to any one of claims 1 to 20, or the test sample according to any one of claims 21 to 24.   A method for analyzing the structure and / or abundance ratio of a measurement target substance based on the fluorescence lifetime of a fluorescent molecule labeled on the measurement target substance according to any one of claims 1 to 20.   A method for identifying the type and / or abundance of the measurement target substance based on the fluorescence lifetime of the fluorescent molecule labeled on the measurement target substance according to any one of claims 1 to 20.