Background removal method for fluorescence lifetime measurement and quantitative determination method for target substance

Abstract

To improve accuracy of a quantitative determination on a target substance present in a solvent.SOLUTION: The present invention relates to a method which uses a fluorescent substance bonded to a target substance present in a solvent to quantitatively determine the target substance, and the method comprises: a first step of irradiating a first sample with excitation light exciting a fluorescent substance so as to acquire a first fluorescence attenuation curve, and then acquiring a first weight factor from the first fluorescence attenuation curve and a first fluorescence lifetime value, the first sample including a first solvent and the target substance or the fluorescent substance and not including the target substance bonded to the fluorescent substance; a second step of irradiating a second sample with the excitation light exciting the fluorescent substance so as to acquire a second fluorescence attenuation curve, and then acquiring a second weight factor from the second fluorescence attenuation curve, a second fluorescence lifetime value, the first fluorescence lifetime value, and the first weight factor, the second sample including a second solvent and the target substance bonded to the fluorescent substance; and a third step of quantitatively determining the target substance with the second weight factor.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for removing background in fluorescence lifetime measurement and a method for quantifying a target substance. [Background technology]

[0002] The amount of a target substance present in a test sample is quantified by a method in which a fluorescent substance is bound to the target substance and the fluorescence intensity is measured (e.g., Patent Documents 1 and 2). One of the factors that affects the results of measuring fluorescence intensity is the fluorescence lifetime, and accurate measurement of the fluorescence lifetime is important for correctly quantifying the target substance.

[0003] The fluorescence lifetime is determined by measuring the fluorescence decay curve and fitting the obtained fluorescence decay curve to a theoretical formula (generally an exponential function of multiple components). In measuring and analyzing the fluorescence decay curve, the presence of background light significantly affects the results and can cause incorrect analysis, so it is necessary to analyze the background light correctly. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2014-122846 A [Patent Document 2] International Publication No. 2004 / 090517 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to improve the accuracy of the quantification of a target substance present in a solvent. [Means for solving the problem]

[0006] The present invention relates to the following [1] to [9].

[0007] [1] A method for quantifying a target substance present in a solvent by using a fluorescent substance that binds to the target substance, comprising: a first step of irradiating a first sample containing a first solvent and the target substance or the fluorescent substance, but not the target substance bound to the fluorescent substance, with excitation light that excites the fluorescent substance to obtain a first fluorescence decay curve, and obtaining a first weighting factor from the first fluorescence decay curve and a first fluorescence lifetime value; a second step of irradiating a second sample containing a second solvent and the target substance bound to the fluorescent substance with excitation light that excites the fluorescent substance to obtain a second fluorescence decay curve, and obtaining a second weighting factor from the second fluorescence decay curve, the second fluorescence lifetime value, the first fluorescence lifetime value, and the first weighting factor; and a third step of quantifying the target substance from the second weighting factor. [2] The method according to [1], wherein the first fluorescence lifetime value is obtained from a first fluorescence decay curve. [3] The method according to [1] or [2], wherein the second fluorescence lifetime value is obtained from a second fluorescence decay curve. [4] The method according to any one of [1] to [3], wherein in the second step, the second fluorescence lifetime value and the second weighting factor are obtained for a plurality of fluorescent components or a plurality of background components. [5] The method according to any one of [1] to [4], wherein in the first step, the first fluorescence lifetime value and the first weighting factor are obtained for a plurality of fluorescent components or a plurality of background components. [6] The method according to any one of [1] to [5], wherein the first sample contains only the target substance out of the target substance and the fluorescent substance. [7] The method according to any one of [1] to [5], wherein the first sample contains only the fluorescent substance out of the target substance and the fluorescent substance. [8] The method according to any one of [1] to [7], wherein the target substance is a protein. [9] The method according to any of [1] to [8], wherein in the third step, a parameter having the second weighting factor is compared with a parameter having the second weighting factor obtained by carrying out the first step and the second step on a standard sample containing a known concentration of the target substance. Effect of the Invention

[0008] According to the present invention, it is possible to improve the accuracy of the quantitative determination of a target substance present in a solvent. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a flow chart used to analyze the fluorescence lifetime for use in quantifying a target substance. [Diagram 2] FIG. 2 is a graph of the instrument response function E(t) of the measurement instrument. [Diagram 3] FIG. 3 shows the fluorescence decay curve of the background component (for verification). [Figure 4] FIG. 4 is a fluorescence decay curve (for verification) including the background component and the fluorescent component of the sample. [Diagram 5] FIG. 5 shows the fluorescence decay curve of a background sample containing Aβ(1-42) aggregates. [Figure 6] FIG. 6 shows the fluorescence decay curves of Thioflavin T bound to Aβ(1-42) aggregates. [Figure 7] FIG. 7 shows the fluorescence decay curve of a background sample containing Thioflavin T. [Figure 8] FIG. 8 shows the fluorescence decay curves of Thioflavin T bound to Aβ(1-42) aggregates. [Figure 9] FIG. 9(a) shows the fluorescence decay curve of a background sample containing biotin-modified ATTO390, and FIG. 9(b) shows the fluorescence decay curves of samples containing biotin-modified ATTO425 and biotin-modified ATTO390. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0011] The method according to this embodiment is a method for quantifying a target substance present in a solvent using a fluorescent substance that binds to the target substance, and includes the following steps: a first step of irradiating a first sample containing a first solvent and the target substance or the fluorescent substance, but not the target substance bound to the fluorescent substance, with excitation light that excites the fluorescent substance to obtain a first fluorescence decay curve, and acquiring a first weighting factor from the first fluorescence decay curve and the first fluorescence lifetime value; a second step of irradiating a second sample containing a second solvent and the target substance bound to the fluorescent substance with excitation light that excites the fluorescent substance to obtain a second fluorescence decay curve, and acquiring a second weighting factor from the second fluorescence decay curve, the second fluorescence lifetime value, the first fluorescence lifetime value, and the first weighting factor; and a third step of quantifying the target substance from the second weighting factor.

[0012] According to the method of this embodiment, it is possible to increase the accuracy of measuring the fluorescence lifetime, and thereby improve the accuracy of quantifying the target substance.

[0013] Examples of target substances include proteins, nucleic acids, viruses, bacteria, pollutants, etc. More specifically, examples of target substances include Aβ(1-42) aggregates, etc.

[0014] Examples of fluorescent substances include fluorescent dyes, fluorescent stains, quantum dots, fluorescent beads, etc. More specifically, examples include thioflavin S, 6-(2-fluoroethoxy)-2-(4-methylaminostyryl)benzoxazole (BF-168), 8-anilino-1-naphthalenesulfonic acid (ANS), thioflavin T, 2-(4'-aminophenyl)-6-methylbenzoxazole (MBPA), 2-(4'-methylaminophenyl)-6-methylbenzoxazole ((6-Me-)BTA-1), 2-(4'-dimethylaminophenyl)-6-iodobenzothiazole (TZDM), 2-(4'-methylaminophenyl)-6-hydroxybenzothiazole (PIB), 2-(4'-dimethylaminophenyl)-6-iodobenzoxazole (IBOX), ATTO425, ATTO390, etc.

[0015] Examples of the solvent include water, a buffer solution, a medium, etc. More specifically, examples of the solvent include purified water, ion-exchanged water, ultrapure water, phosphate-buffered saline (PBS), Tris-buffered saline (TBS), a glycine buffer solution (glycine-sodium hydroxide solution), a DMSO-containing medium, etc. The solvent may contain a first solvent and a second solvent, and the first solvent and the second solvent may be the same or different.

[0016] (First Step) In the first step, first, the first sample is irradiated with excitation light that excites the fluorescent substance to obtain a first fluorescence decay curve (background fluorescence decay curve B(t)). This operation corresponds to "(2) Measurement of background fluorescence decay curve" in FIG. 1. In the first step, the background fluorescence decay curve B(t) can be analyzed with a multi-component exponential function. This operation corresponds to "(3) Analysis of background decay curve" in FIG. 1. The function G shown in Equation 3 b (t) is convoluted according to Equation 4 to obtain I b (t) and calculate this I b (t) and the background fluorescence decay curve B(t), χ 2The value can be calculated. b χ so that (t) and B(t) match best. 2 Search for a combination of variables that minimizes τ in Equation 3. b1 ~τ bn , A b1 ~A bn The best combination of τ b1 ~τ bn (first fluorescence lifetime value) and A b1 ~A bn (First weighting factor) may be a parameter of the background component.

[0017] In Equation 3, bn can be the number of components of the exponential function used in background analysis. The value of the number of components bn can be any value, but χ 2 It is preferable to use a number sufficient to obtain good fitting, with <1.2 as a guideline. In practice, it is common to use about 2 to 3 components, but this does not prevent the acquisition of a first fluorescence lifetime value and a first weighting factor for a single component. In Equation 4, E(t) can be the instrument response function of the measuring device measured in advance. In Equation 5, c1 can be the start time of the analysis, and c2 can be the end time of the analysis.

[0018]

number

[0019] In the first step, a first fluorescence lifetime value and a first weighting factor can be obtained for a plurality of fluorescent components or a plurality of background components. By obtaining the first fluorescence lifetime value and the first weighting factor, the fluorescence lifetime value of the target substance bound to the fluorescent substance can be measured more accurately, and as a result, the accuracy of the quantification of the target substance is improved.

[0020] The first fluorescence lifetime value may be a known value or may be a value obtained from a first fluorescence decay curve.

[0021] Of the target substance and the fluorescent substance, the first sample may contain only the target substance, or may contain only the fluorescent substance.

[0022] (Second step) In the second step, first, a second sample is irradiated with excitation light that excites the fluorescent substance to obtain a second fluorescence decay curve (fluorescence decay curve F(t) including the background). This operation corresponds to "(1) Measurement of the fluorescence decay curve" in Fig. 1. In the second step, the parameters of the background components obtained in the first step are used to analyze the fluorescence decay curve F(t) including the background with a multi-component exponential function. This operation corresponds to "(4) Analysis of the fluorescence decay curve including the background parameters" in Fig. 1. The function G(t) is calculated according to Equation 6, and I(t) is calculated by convolution integration according to Equation 7. χ is calculated from I(t) and the fluorescence decay curve F(t) according to Equation 8. 2 We can calculate the value of χ so that I(t) and F(t) match best. 2 The variable τ that minimizes l ~τ n , A1~A n The resulting combination of τ l ~τ n and A1~A n can be the analysis result of the second fluorescence lifetime and the second weighting factor to be obtained. In the formula 8, c1 may be the start time of the analysis, and c2 may be the end time of the analysis. The value of the number of components n in the formula 6 may be any value, but χ 2 It is preferable to use a number sufficient to obtain good fitting, with a guideline of <1.2. In practice, it is common to use about 2 to 3 components, but this does not prevent the acquisition of a second fluorescence lifetime value and a second weighting factor for a single component. In Formula 6, bn, A1 to A bn and τ1~τ bn are the number of components and parameters of the background determined in the first step. In Equation 7, E(t) can be the instrument response function of the measuring device measured in advance.

[0023]

number

[0024] The second fluorescence lifetime value may be a known value or may be a value obtained from a second fluorescence decay curve.

[0025] (Third Step) The product of the second fluorescence lifetime value and the second weighting factor can be regarded as the amount of fluorescence of the fluorescent substance bound to the target substance whose concentration is unknown. The first and second steps are carried out for a plurality of standard samples having various concentrations (or weights) of the target substance, the products of the second fluorescence lifetime value and the second weighting factor (i.e., the amount of fluorescence) are obtained, and a calibration curve can be created by plotting the amount of fluorescence and the concentration (or weight). The concentration (or weight) of the target substance can be quantified from this calibration curve and the amount of fluorescence obtained for the target substance whose concentration (or weight) is unknown. In cases where the concentration of the target substance does not need to be obtained accurately, it is not necessary to create a calibration curve, and it is sufficient to simply compare the amount of fluorescence with that of several standard samples. In addition, in cases where it is guaranteed that the second fluorescence lifetime value of the target substance is the same, it is not necessary to obtain the amount of fluorescence from the product of the second fluorescence lifetime value and the second weighting factor, and it is sufficient to only compare the second weighting factors. EXAMPLES

[0026] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples.

[0027] Example 1 An example of an analysis of a fluorescence decay curve according to the present invention will be described below in accordance with the flowchart in Fig. 1. In this example, in order to show that a more accurate answer can be obtained compared to the conventional method, a verification decay curve theoretically obtained from known parameters is used for explanation, rather than an experimentally obtained decay curve.

[0028] (1-1) Calculation of the verification decay curve (1-1-1) Background fluorescence decay curve The method for creating a background fluorescence decay curve is described below. In an actual experiment, this procedure corresponds to “(2) Measurement of background fluorescence decay curve” in Figure 1. The background lifetime parameters shown in Table 1 were substituted into Equation 1 to determine the fluorescence decay curve G(t). The subscript i in Equation 1 indicates the order of the background components. The decay curve I(t) was determined by convolution integration using Equation 2 from G(t) and the instrument response function E(t) of the measuring device, which was measured in advance and shown in Figure 2. Finally, Poisson noise corresponding to the intensity was added to calculate the background fluorescence decay curve B(t). The calculated background fluorescence decay curve B(t) is shown in Figure 3.

[0029]

number

[0030] [Table 1]

[0031]

number

[0032] (1-1-2) Fluorescence decay curve including background The method for creating a fluorescence decay curve including the background is described below. In an actual experiment, this operation corresponds to "(1) Measurement of the fluorescence decay curve" in Figure 1. The fluorescence lifetime parameters and background lifetime parameters shown in Table 2 were substituted into Equation 1 to obtain the fluorescence decay curve G(t). The subscript i in Equation 1 indicates the order of the background component. The decay curve I(t) was obtained by convolution integration using Equation 2 from G(t) and the instrument response function E(t) of the measuring device, which was measured in advance and shown in Figure 2, and finally the fluorescence decay curve F(t) including the background was calculated by adding Poisson noise corresponding to the intensity. In this parameter, the ratio of the intensity of the fluorescence component to the background component, i.e., the signal-to-noise ratio, was 5. The calculated fluorescence decay curve F(t) including the background is shown in Figure 4.

[0033] [Table 2]

[0034] (1-2) Analysis of Fluorescence Decay Curve Including Background According to the Present Invention The procedure and an example of analyzing the fluorescence decay curve including background parameters of the fluorescence decay curve F(t) including the background calculated in (1-1-2) according to the present invention will be described below.

[0035] (1-2-1) Analysis of background fluorescence decay curves The background fluorescence decay curve B(t) calculated in (1-1-1) was analyzed using a multi-component exponential function. This operation corresponds to "(3) Analysis of background fluorescence decay curve" in Figure 1. This analysis was performed in the following procedure. First, the function G b (t) is convoluted according to Equation 4 to obtain I b (t) and calculate this I b From (t) and the background fluorescence decay curve B(t) calculated in (1-1-1), χ 2 The value was calculated. b χ so that (t) and B(t) match best.2 Search for a combination of variables that minimizes τ in Equation 3. b1 ~τ bn , A b1 ~A bn The best combination of τ was obtained as a result of this analysis. b1 ~τ bn and A b1 ~A bn are the parameters of the background component.

[0036] In Equation 3, bn is the number of components of the exponential function used in background analysis. The value of the number of components bn is arbitrary, but χ 2 <1.2 is a sufficient number to obtain good fitting. In reality, there are about 2 to 3 components, but in this embodiment, two components (bn=2) were used, which is the number of background components assumed when calculating the background fluorescence decay curve. In Equation 4, E(t) is the instrument response function of the measuring device measured in advance. In Equation 5, c1 indicates the start time of the analysis, c2 indicates the end time of the analysis, and in this analysis, c1 was set to 101 channel, where the rise of the decay curve is 90% of the peak value, and c2 was set to 1024 channel, which is the end time of the measurement.

[0037] The results of the analysis are shown in Table 3. The error between each parameter obtained by the analysis and each parameter used in (1-1-2) was 2% or less.

[0038]

number

[0039] [Table 3]

[0040] (1-2-2) Analysis of fluorescence decay curves including background Using the background parameters obtained in (1-2-1), the fluorescence decay curve F(t) including the background was analyzed with a multi-component exponential function. This operation corresponds to "(4) Analysis of the fluorescence decay curve including the background parameters" in Figure 1. The function G(t) was calculated according to Equation 6, and I(t) was calculated by convolution integration according to Equation 7. From I(t) and the fluorescence decay curve F(t) calculated in (1-1-2), χ was calculated according to Equation 8. 2 We calculated the value of χ to obtain the best agreement between I(t) and F(t). 2 The variable τ that minimizes l ~τ n , A1~A n The combination of τ l ~τ n and A1~A n can be used as the analysis results of the second fluorescence lifetime and the second weighting factor to be obtained. In Equation 8, c1 indicates the start time of the analysis, and c2 indicates the end time of the analysis. In this analysis, c1 was set to 114 channel, where the rise of the decay curve is 90% of the peak value, and c2 was set to 1024 channel, which is the end time of the measurement. The value of the number of components n in Equation 6 is arbitrary, but a sufficient number is required to obtain good fitting. In practice, χ 2 <1.2 is a guideline. In this example, the number of components used in creating the fluorescence decay curve was n = 3. In addition, in Equation 6, bn, τ b1 ~τ bn and A b1 ~A bn are the number of components and parameters of the background determined in (1-2-1). In Equation 7, E(t) is the instrument response function of the measuring device measured in advance.

[0041]

number

[0042] The above analysis procedure yields the fluorescence lifetime value τ lThe weighting factors A1 to A3 of the corresponding life values, as well as τ3, were calculated. The analysis results are shown in Table 4. The relative error with the assumed parameters is shown in Table 5. χ, which is a measure of the quality of the fitting, 2 was 1.11, and the fitting was good. Unlike the analysis results by the conventional method shown in Comparative Example 1 below, the lifetime values ​​and weighting factors used to create the decay curve for the fluorescent components were reproduced with an accuracy of 2% or less. This result demonstrated that the present invention can correctly separate the background and fluorescent components in the analysis of a fluorescence decay curve that includes a background, enabling highly accurate analysis.

[0043] [Table 4]

[0044] [Table 5]

[0045] (Comparative Example 1) Analysis of fluorescence decay curves including background by conventional method The results of the conventional method, in which the background component was treated as an additional component to the fluorescent component and analyzed simultaneously with the fluorescent component of interest, are described below. First, calculate the function G(t) according to Equation 9, and calculate I(t) by convolution integration according to Equation 10. Then, from this I(t) and the fluorescence decay curve F(t) calculated in (1-1-2), χ, which is a fitting guideline, is calculated according to Equation 11. 2 We calculated the value of χ to obtain the best agreement between I(t) and F(t). 2 The variable τ that minimizes l ~τ n , A1~A n The combination of τ l ~τ n and A1~A n are the analytical results of the desired fluorescence lifetime value and weighting factor.

[0046] In formula 9, n is the number of exponential functions used in the analysis. Here, n = 5, with 2 background components added to the 3 components of the fluorescence exponential function. In formula 11, c1 is the start time of the analysis, and c2 is the end time of the analysis. In this analysis, c1 was set to 114 channel, where the rise of the decay curve is 90% of the peak value, and c2 was set to 1024 channel, which is the end time of the measurement. The results of the analysis are shown in Table 6. Table 7 shows the error between each parameter obtained in the analysis and each parameter used in (1-1-2).

[0047] χ is a measure of the quality of the fitting. 2 was 1.09, and the fitting was good. However, the fluorescence lifetime values ​​of the fluorescent component (i=2) and the background component (i=4) were the same, and the fluorescent component and the background component could not be separated. As a result, there were large errors in the weighting factor of the fluorescent component (i=2) and the fluorescence lifetime values ​​and weighting factors of the fluorescent component (i=3) and the background components (i=4, i=5). This result shows that simply increasing the exponential function components to take the background components into account does not allow for a highly accurate analysis.

[0048]

number

[0049] [Table 6]

[0050] [Table 7]

[0051] Example 2 An example will be described in which the analytical method described in Example 1 is applied to the fluorescence decay curve of a sample of Aβ(1-42) aggregates bound to Thioflavin T. In this example, the fluorescent sample is Aβ(1-42) aggregates bound to Thioflavin T.

[0052] (2-1) Preparation of Aβ(1-42) aggregates Human Aβ(1-42) protein (Peptide Institute) was dissolved in dimethyl sulfoxide to a concentration of 5 mmol / L, and then further diluted to 100 μmol / L with 10 mmol / L hydrochloric acid. The resulting solution was left to stand in a 37°C incubator for 24 hours to prepare an Aβ(1-42) aggregate sample.

[0053] (2-2) Measurement of background fluorescence decay curve A background sample was prepared by mixing 10 μL of the Aβ(1-42) aggregate sample with 410 μL of glycine buffer solution (50 mmol / L glycine-sodium hydroxide solution pH 9.0) and 80 μL of distilled water. The background fluorescence decay curve of the background sample was measured using a fluorescence lifetime measurement device (Hamamatsu Photonics Quantaurus-Tau) at an excitation wavelength of 405 nm and an observation wavelength of 500 nm. The obtained background fluorescence decay curve B2(t) is shown in FIG. 5.

[0054] (2-3) Measurement of fluorescence decay curves of Aβ(1-42) aggregates stained with thioflavin T 10 μL of Aβ(1-42) aggregate sample was mixed with 410 μL of glycine buffer solution, 10 μL of 100 μmol / L thioflavin T aqueous solution and 70 μL of distilled water, or 20 μL of 100 μmol / L thioflavin T aqueous solution and 60 μL of distilled water to prepare a sample (hereinafter abbreviated as TA1) in which the molar ratio of thioflavin T to Aβ(1-42) is 1, and a sample (hereinafter abbreviated as TA2) in which the molar ratio of thioflavin T to Aβ(1-42) is 2. For each sample, a fluorescence decay curve was measured using a fluorescence lifetime measurement device (Hamamatsu Photonics Quantaurus-Tau) at an excitation wavelength of 405 nm and an observation wavelength of 500 nm. The two obtained fluorescence decay curves F TA1 (t) and F TA2 (t) is shown in Figure 6.

[0055] (2-4) Analysis of background fluorescence decay curves The following describes "(3) Analysis of background fluorescence decay curve" in Figure 1. From the background fluorescence decay curve obtained in (2-2), the background parameters were determined according to the procedure in (1-2-1) of Example 1. 2 In calculating the value, we use the χ 2 Minimize the value of τ b1 ~τ bn and A b1 ~A bn Combinations of were explored. In determining the parameters, the decay curves were analyzed in three components. The obtained background parameters are shown in Table 8.

[0056] [Table 8]

[0057] (2-5) Analysis of fluorescence decay curves of Aβ(1-42) aggregates stained with thioflavin T The following describes "(4) Analysis of the fluorescence decay curve including background parameters" in Figure 1. The fluorescence decay curves of the Aβ(1-42) aggregates stained with thioflavin T obtained in (2-3) and the background parameters in Table 8 were analyzed according to the procedure in Example 1 (1-2-1). 2 In calculating the value, I(t) and F TA1 (t) or F TA2 (t) to best match χ 2 Minimize the value of τ1~τ n and A1~A n The combination of the above was explored. The fluorescent portion was analyzed with four components, i.e., n=4. The analysis results of the fluorescence lifetime of the TAl sample are shown in Table 9, and the analysis results of the TA2 sample are shown in Table 10. Note that the parameters of the background components are omitted in Tables 9 and 10.

[0058] [Table 9]

[0059] [Table 10]

[0060] χ 2 Both values ​​were below 1.2, and the fits were good. The fluorescence lifetime values ​​of TAl and TA2 were in good agreement, except for a slight difference in the second component.

[0061] Comparative Example 2 The results of directly analyzing the fluorescence decay curves of TAl and TA2 with four exponential components without relying on the present invention (using the same method as in Comparative Example 1) are shown in Tables 11 and 12, respectively.

[0062] [Table 11]

[0063] [Table 12]

[0064] Even when analyzed without relying on the present invention (using the same method as in Comparative Example 1), χ 2 Both values ​​were below 1.2, and the fitting was good. On the other hand, the fluorescence lifetime value showed different values ​​depending on the concentration. Considering that the fluorescence lifetime value does not change due to a change in concentration except when a special dynamic interaction acts on the fluorescent molecule, it is clear that the analysis results not based on the present invention are incorrect, and the analysis results based on the present invention are more accurate. From the above results, it was shown that the fluorescence lifetime value can be correctly analyzed by the present invention even in real samples, and it was suggested that the target substance can be correctly quantified.

[0065] Example 3 Here is another example in which the analytical method described in the Reference Example is applied to the fluorescence decay curve of an Aβ(1-42) aggregate sample bound to thioflavin T. In this example, unlike Example 2, the sample used to obtain the background fluorescence decay curve contains the fluorescent dye thioflavin T, and it is shown that the background effect due to fluorescent impurities in thioflavin T can be removed and the analysis can be performed correctly. For the sake of simplicity, the parts that overlap with Example 2 are omitted, and only the different parts are shown.

[0066] (3-1) Preparation of Aβ(1-42) aggregates An Aβ(1-42) aggregate sample was prepared according to the procedure in (2-1) of Example 2. The solvent used in the preparation (a liquid containing neither Aβ(1-42) protein nor Aβ(1-42) aggregates, and containing dimethyl sulfoxide and hydrochloric acid) is referred to as solvent A.

[0067] (3-2) Measurement of background fluorescence decay curve A background sample was prepared by mixing 800 μL of glycine buffer solution (50 mmol / L glycine-sodium hydroxide solution, pH 9.0), 100 μL of 100 μmol / L thioflavin T aqueous solution, and 100 μL of solvent A. The background fluorescence decay curve of the sample was measured using a fluorescence lifetime measurement device (Hamamatsu Photonics Quantaurus-Tau) at an excitation wavelength of 405 nm and an observation wavelength of 500 nm. The obtained background fluorescence decay curve B3(t) is shown in FIG. 7.

[0068] (3-3) Measurement of fluorescence decay curves of Aβ(1-42) aggregates stained with thioflavin T 12.5 μL of Aβ(1-42) aggregate sample and 87.5 μL of solvent A, and 6.25 μL of Aβ(1-42) aggregate sample and 93.75 μL of solvent A were mixed with 100 μL of 100 μmol / L thioflavin T aqueous solution and 800 μL of glycine buffer solution to prepare samples with a molar ratio of thioflavin T to Aβ(1-42) of 8 (hereinafter abbreviated as TA8) and a molar ratio of thioflavin T to Aβ(1-42) of 16 (hereinafter abbreviated as TA16). For each sample, a fluorescence decay curve was measured using a fluorescence lifetime measurement device (Hamamatsu Photonics Quantaurus-Tau) at an excitation wavelength of 405 nm and an observation wavelength of 500 nm. The two obtained fluorescence decay curves F TA8 (t) and F TA16 (t) is shown in Figure 8.

[0069] (3-4) Analysis of background fluorescence decay curves From the background fluorescence decay curve B3(t) obtained in (3-2), the background parameters were determined according to the procedure in (2-4) of Example 2. 2 In calculating the value, we use the χ 2 Minimize the value of τ b1 ~τ bn and A b1 ~A bn The combination of was explored. In the parameter determination, the decay curve was analyzed in three components. The obtained parameters are shown in Table 13. Here, the first component of the parameter τ b1 and A b1 is clearly the fluorescence from thioflavin T, and other components are the background. Therefore, τ b2 ~τ b3 , A b2 ~A b3 was adopted.

[0070] [Table 13]

[0071] (3-5) Analysis of fluorescence decay curves of Aβ(1-42) aggregates stained with thioflavin T Two fluorescence decay curves of Aβ(1-42) aggregates stained with thioflavin T obtained in (3-3) and the background parameter τ in Table 13 b2 ~τ b3 , A b2 ~A b3 According to the procedure of (2-4) of Example 2, the fluorescence decay curve F TA8 (t) and F TA16 (t) were analyzed respectively. 2 In calculating the value, I(t) and F TA8 (t) or F TA16 (t) to best match χ 2 Minimize the value of τ b1 ~τ bn and A b1 ~A bn The combination of the following was explored. In this analysis, both fluorescent components were analyzed with four components. The analysis results of the fluorescence lifetime in TA8 are shown in Table 14, and the analysis results of the fluorescence lifetime in TA16 are shown in Table 15.

[0072] [Table 14]

[0073] [Table 15]

[0074] χ 2 Both values ​​were below 1.2, and the fitting was good. There was almost no change in the lifetime value due to concentration, and it can be seen that the analysis was performed correctly.

[0075] Comparative Example 3 Two fluorescence decay curves F TA8 (t) and F TA16 The results of direct analysis of (t) with its four exponential components are shown in Tables 16 and 17.

[0076] [Table 16]

[0077] [Table 17]

[0078] χ 2 Both values ​​were below 1.2, and the fitting was good. The lifetime values ​​differed with changes in concentration, and could not be analyzed correctly.

[0079] Example 4 An example will be described in which the present invention is applied to the quantification of a fluorescent labeling reagent, ATTO 425. In this example, the fluorescent sample is ATTO 425, and ATTO 390 is mixed as a background fluorescent substance.

[0080] (4-1) Sample preparation The target fluorescent substance, biotin-modified ATTO425 (AD425-71, ATTO-TECGmbH), and the background fluorescent substance, biotin-modified ATTO390 (AD390-71, ATTO-TECGmbH), were dissolved in phosphate-buffered saline (pH 7.4) to concentrations of 5 nM and 10 nM, respectively. In this sample, the fluorescence intensity of ATTO425 is approximately 1.3-fold higher than background ATTO390.

[0081] (4-2) Measurement of background fluorescence decay curve Biotin-modified ATTO390 was dissolved in phosphate buffered saline (pH 7.4) to a concentration of 10 nM. This sample was placed in a fluorescence lifetime measurement device (Hamamatsu Photonics Quantaurus-Tau) and the fluorescence decay curve was measured at an excitation wavelength of 405 nm and an observation wavelength of 500 nm. The obtained background fluorescence decay curve is shown in Figure 9 (a).

[0082] (4-3) Measurement of the fluorescence decay curve of the sample The sample prepared in (4-1) was placed in a fluorescence lifetime measurement device (Hamamatsu Photonics Quantaurus-Tau) and the fluorescence decay curve was measured at an excitation wavelength of 405 nm and an observation wavelength of 500 nm. The fluorescence decay curve of the obtained sample is shown in Figure 9 (b).

[0083] (4-4) Analysis of background fluorescence decay curves The following describes "(3) Analysis of background fluorescence decay curve" in FIG. From the background fluorescence decay curve obtained in (4-3), the background parameters were determined according to the procedure in (1-2-1) of Example 1. In determining the parameters, the decay curve was analyzed in one component. The obtained background parameters are shown in Table 18.

[0084] [Table 18]

[0085] (4-5) Analysis of the fluorescence decay curve of the sample In accordance with the present invention, the lifetime values ​​and weighting factors of the background fluorescence were previously determined and both were treated as constants when analyzing the decay curves of the samples. The results are shown in Table 19.

[0086] [Table 19]

[0087] In Table 19, the fluorescence lifetime value of ATTO425 was 3.51 ns and the fluorescence intensity ratio was 1.36, which are almost equivalent to the correct values ​​of 3.6 ns and 1.3. Therefore, it was shown that the analysis method according to the present invention can correctly determine the fluorescence lifetime value and weighting factor even in a sample in which background fluorescence is present (even in a sample in which the difference between the background component and the target fluorescent component is small).

[0088] Comparative Example 4 Table 20 shows the results of an analysis without relying on the present invention, in which the background fluorescence was explicitly included in the analysis components, that is, the analysis was performed using two components, the fluorescent component of the sample and the background component.

[0089] [Table 20]

[0090] In Table 20, the first component is thought to be the target fluorescence of ATTO425, but the value is different from the correct lifetime value of 3.6 ns for ATTO425, and the fluorescence intensity is weaker than the background ATTO390, although it should be 1.3 times stronger. Therefore, this sample could not be analyzed correctly by the method of explicitly including the background fluorescence in the analysis components.

[0091] Comparative Example 5 For example, Japanese Patent No. 6133053 discloses a method in which the background lifetime is calculated in advance, and then the background lifetime is used as a constant and the weighting factor is used as a variable when analyzing the decay curve of a sample. Table 21 shows the results of analysis based on this method using the lifetime of 5.14 ns of ATTO390 measured in advance.

[0092] [Table 21]

[0093] In Table 21, the fluorescence lifetime value of ATTO425 was almost correctly determined as 3.67 ns, but the fluorescence intensity ratio was 1.83, which is different from the correct value of 1.3, and the correct analysis result was not obtained. Therefore, the fluorescence intensity cannot be correctly determined by the method of Patent No. 6133053.

[0094] The above results demonstrate that the present invention can analyze fluorescence lifetime values ​​with higher accuracy than conventional methods. Accurate measurement of fluorescence lifetime (and weighting factor) leads to accurate quantification of target substances, so it is believed that the target substances can be accurately quantified by using the fluorescence lifetime values ​​and weighting factors obtained in Examples 1 to 4. This example shows that even when the fluorescent reagent contains impurities, it is possible to more accurately analyze the fluorescence decay curve by taking these into account.

Claims

1. A method for quantifying a target substance present in a solvent using a fluorescent substance that binds to the target substance, comprising the steps of: a first step of irradiating a first sample containing a first solvent and the target substance or the fluorescent substance, but not containing the target substance bound to the fluorescent substance, with excitation light that excites the fluorescent substance to obtain a first fluorescence decay curve, and obtaining a first weighting factor from the first fluorescence decay curve and a first fluorescence lifetime value; a second step of irradiating a second sample containing a second solvent and the target substance bound to the fluorescent substance with excitation light that excites the fluorescent substance to obtain a second fluorescence decay curve, and obtaining a second weighting factor from the second fluorescence decay curve, the second fluorescence lifetime value, the first fluorescence lifetime value, and the first weighting factor; and a third step of quantifying the target substance from the second weighting factor.

2. The method of claim 1 , wherein the first fluorescence lifetime value is obtained from a first fluorescence decay curve.

3. The method of claim 1 or 2, wherein the second fluorescence lifetime value is obtained from a second fluorescence decay curve.

4. The method according to any one of claims 1 to 3, wherein in the second step, the second fluorescence lifetime values ​​and the second weighting factors are obtained for a plurality of fluorescent components or a plurality of background components.

5. The method according to any one of claims 1 to 4, wherein in the first step, the first fluorescence lifetime values ​​and the first weighting factors are obtained for a plurality of fluorescent components or a plurality of background components.

6. The method according to any one of claims 1 to 5, wherein the first sample contains only the target substance out of the target substance and the fluorescent substance.

7. The method according to any one of claims 1 to 5, wherein the first sample contains only the fluorescent substance out of the target substance and the fluorescent substance.

8. The method according to any one of claims 1 to 7, wherein the target substance is a protein.

9. 9. The method according to claim 1, wherein in the third step, a parameter having the second weighting factor is compared with a parameter having the second weighting factor obtained by carrying out the first step and the second step on a standard sample containing the target substance of a known concentration.

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