Kit for sustaining luminescence of luminescent substrate and method for sustaining luminescence of luminescent substrate

Abstract

To provide a kit for sustaining luminescence of a luminescent substrate, capable of improving luminescence sustainability, and to provide a method for sustaining the luminescence of the luminescent substrate.SOLUTION: A kit for sustaining luminescence of a luminescent substrate, comprising a surfactant and an alcohol, wherein the luminescent substrate is a hydrophobic compound, and the molecular weight of the luminescent substrate is 300 or more and 500 or less.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a kit for sustaining the luminescence of a luminescent substrate and a method for sustaining the luminescence of a luminescent substrate. [Background technology]

[0002] In the fields of basic biology and diagnostic and testing technologies, a method for detecting a target substance (protein, antigen, etc.) by fusing a reporter substance to the target substance or a protein (antibody, etc.) that specifically binds to the target substance is known. Known reporter substances include luminescent enzymes, fluorescent proteins, fluorescent dyes, quantum dots, and peroxidase. For example, Japanese Patent Laid-Open Publication No. 2022-123828 (Patent Document 1) discloses a polypeptide having luminescent enzyme activity and containing a specific amino acid sequence. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2022-123828 Summary of the Invention [Problem to be solved by the invention]

[0004] Although the luminescence produced by luminescent enzymes is less intense than that produced when fluorescent proteins, fluorescent dyes, or quantum dots are used as reporter substances, it does not require excitation light, so it is not phototoxic to cells and is suitable for detecting trace amounts.

[0005] However, the reaction between a luminescent enzyme and a luminescent substrate (sometimes referred to as a "luminescence reaction") generally exhibits a luminescence pattern in which the luminescence intensity reaches a maximum immediately after the reaction, and then rapidly decays. Therefore, a method for improving the durability of luminescence is desired.

[0006] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a kit for sustaining the luminescence of a luminescent substrate, which is capable of improving the luminescence sustainability, and a method for sustaining the luminescence of a luminescent substrate. [Means for solving the problem]

[0007] As a result of extensive research, the present inventors have discovered that the duration of luminescence can be improved by reacting a luminescent enzyme with a luminescent substrate in the presence of a surfactant and an alcohol, and have thus completed the present invention.

[0008] A first aspect of the present invention is A kit for sustaining luminescence of a luminescent substrate, comprising: A surfactant and an alcohol are included, The luminescent substrate is a hydrophobic compound, The kit for sustained luminescence relates to the luminescent substrate, wherein the molecular weight of the luminescent substrate is 300 or more and 500 or less.

[0009] A second aspect of the present invention is A method for sustaining luminescence of a luminescent substrate, comprising: a preparation step of preparing a luminescent substrate and a luminescent enzyme; a reaction step of reacting the luminescent substrate with the luminescent enzyme; Including, The luminescent substrate is a hydrophobic compound, The molecular weight of the luminescent substrate is 300 or more and 500 or less, The reaction step is carried out in the presence of a surfactant and an alcohol. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a kit for sustaining the luminescence of a luminescent substrate and a method for sustaining the luminescence of a luminescent substrate, which are capable of improving the luminescence sustainability. [Brief explanation of the drawings]

[0011] [Figure 1]Figure 1 shows (A) a graph showing the change in luminescence intensity over time, (B) a graph showing the correlation between the maximum normalized luminescence intensity (vertical axis) and the concentrations of the luciferase and surfactant (horizontal axis), and (C) a graph showing the correlation between the half-life of the luminescence reaction (horizontal axis) and the concentrations of the luciferase and surfactant (vertical axis) in Experiment 1. In Figure 1A, the horizontal axis shows the time elapsed from immediately after mixing the enzyme solution and the substrate solution, and the vertical axis shows the relative amount of luminescence per second. [Figure 2] Figure 2 shows (A) a graph showing the correlation between the maximum normalized luminescence intensity (vertical axis) and the concentrations of the luminescent enzyme and surfactant (horizontal axis) in Experiment 1, and (B) a graph showing the correlation between the half-life of the luminescent reaction (horizontal axis) and the concentrations of the luminescent enzyme and surfactant (vertical axis). [Figure 3] Figure 3 shows (A) a graph showing the change in luminescence intensity over time, (B) a graph showing the correlation between the maximum normalized luminescence intensity (vertical axis) and the concentrations of the luciferase and surfactant (horizontal axis), and (C) a graph showing the correlation between the half-life of the luminescence reaction (horizontal axis) and the concentrations of the luciferase and surfactant (vertical axis) in Experiment 2. In Figure 3A, the horizontal axis shows the time elapsed from immediately after mixing the enzyme solution and the substrate solution, and the vertical axis shows the relative amount of luminescence per second. [Figure 4] Figure 4 is a graph showing the change in luminescence intensity over time in Experiment 3. In Figure 4, the horizontal axis shows the time elapsed from immediately after mixing the enzyme solution and the substrate solution, and the vertical axis shows the relative amount of luminescence per second. [Figure 5] FIG. 5 is a schematic diagram illustrating a general detection system using biotin and streptavidin. [Figure 6] Figure 6 shows (A) a schematic diagram of the simulated detection system, (B) a graph showing the change in luminescence intensity over time, (C) a graph showing the correlation between the maximum relative luminescence intensity (vertical axis) and the surfactant concentration (horizontal axis), and (D) a graph showing the correlation between the integrated value of the relative luminescence intensity (vertical axis) and the surfactant concentration (horizontal axis) in Experiment 4. In Figure 6B, the horizontal axis shows the elapsed time from immediately after mixing the enzyme solution and the substrate solution, and the vertical axis shows the relative luminescence intensity per second. [Figure 7] Fig. 7 is a graph showing the measurement results obtained by dynamic light scattering in Experiment 5. In Fig. 7, the horizontal axis indicates the size of the detected particles, and the vertical axis indicates the distribution rate. [Figure 8] FIG. 8 is a photograph of the particles in Experiment 6 observed under a microscope. [Figure 9] Figure 9 is a graph showing the change in luminescence intensity over time in Experiment 6. In Figure 9, the horizontal axis shows the time elapsed from immediately after mixing the enzyme solution and the substrate solution, and the vertical axis shows the relative amount of luminescence per second. [Figure 10] Figure 10 shows (A) a graph showing the correlation between the relative luminescence intensity (vertical axis) and the presence or absence of surfactants and substrate metabolites (horizontal axis) in Experiment 7, and (B) a graph showing the relationship between the rate of decrease in luminescence intensity due to the addition of substrate metabolites (vertical axis) and the presence or absence of surfactants (horizontal axis). [Figure 11] 11 is a graph showing the time course of the luminescent substrate and its metabolites in the reaction solution in Experiment 8. The vertical axis shows the concentration of the analyte compound, and the horizontal axis shows the presence or absence of the luminescent enzyme and surfactant, as well as the time elapsed since the start of the reaction. [Figure 12] 12 is a graph showing the time course of the luminescent substrate and its metabolites in the reaction solution in Experiment 8. The vertical axis shows the relative change in concentration of the analyte compound, and the horizontal axis shows the presence or absence of a surfactant and the time elapsed since the start of the reaction. DETAILED DESCRIPTION OF THE INVENTION

[0012] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited to this. In this specification, the notation in the format "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0013] <Kit for sustaining luminescence of luminescent substrates> A first aspect of the present invention is a kit for sustaining luminescence of a luminescent substrate, comprising: A surfactant and an alcohol are included, The luminescent substrate is a hydrophobic compound, The molecular weight of the luminescent substrate is 300 or more and 500 or less in this kit for sustained luminescence.

[0014] <Surfactant> In this embodiment, the term "surfactant" refers to a compound having both a portion with affinity for water (hydrophilicity) and a portion with affinity for nonpolar solvents in the molecule. Examples of the surfactant include anionic surfactants (anionic surfactants), cationic surfactants (cationic surfactants), amphoteric surfactants (zwitterionic surfactants), and nonionic surfactants (nonionic surfactants). In this embodiment, the surfactant preferably includes a nonionic surfactant or an amphoteric surfactant.

[0015] Examples of the nonionic surfactants include NP-40 (poly(oxyethylene) octylphenyl ether), Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Triton X-100 (polyoxyethylene (10) octylphenyl ether), n-octyl-β-D-thioglucoside, and MEGA-8 (n-octanoyl-N-methyl-D-glucamine). Examples of the amphoteric surfactants include CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate).

[0016] In one aspect of this embodiment, the surfactant preferably includes at least one selected from the group consisting of NP-40, CHAPS, Tween 20, Triton X-100, n-octyl-β-D-thioglucoside, and MEGA-8.

[0017] In this embodiment, the surfactant may be contained alone in a container, or may be contained in a container in the form of an aqueous solution. The surfactant may also be contained in the same container together with other components, such as alcohol, which will be described later. In one aspect of this embodiment, when the surfactant is in the form of an aqueous solution, the concentration of the surfactant may be, for example, 0.0001% by mass or more and 20% by mass or less, or 0.001% by mass or more and 5% by mass or less.

[0018] <Alcohol> In this embodiment, "alcohol" refers to a compound in which a hydrogen atom of an aliphatic hydrocarbon is replaced with a hydroxy group (-OH). Examples of the alcohol include saturated aliphatic alcohols having 1 to 4 carbon atoms. The alcohol may be a primary alcohol, a secondary alcohol, or a tertiary alcohol, but is preferably a primary alcohol. In this embodiment, the alcohol preferably includes at least one selected from the group consisting of ethanol, methanol, propanol, isopropanol, and butanol.

[0019] In this embodiment, the alcohol may be contained alone in a container, or may be contained in the same container together with the surfactant and other components such as a luminescent substrate, which will be described later. In one aspect of this embodiment, the surfactant and the alcohol may be contained in separate containers. In another aspect of this embodiment, the surfactant and the alcohol may be contained in the same container.

[0020] <Luminescent substrate> The luminescent substrate to be used in the luminescence-sustaining kit according to this embodiment is a hydrophobic compound. Because the luminescent substrate is a hydrophobic compound, the luminescent substrate accumulates in the micelle particles formed by the surfactant and the alcohol, thereby improving the sustainability of luminescence. In one aspect of this embodiment, the luminescent substrate may be fused with another compound such as a fluorescent dye.

[0021] The molecular weight of the luminescent substrate is 377 or more and 423 or less, and may be 300 or more and 376 or less, or 424 or more and 500 or less.

[0022] In this embodiment, the luminescent substrate may be, for example, a known luminescent substrate such as coelenterazine (Cypridina luciferin) furimazine, etc. Examples of coelenterazine substrates include natural coelenterazine (nCTZ), coelenterazine ip, coelenterazine i, coelenterazine hcp, coelenterazine 400A, coelenterazine, coelenterazine cp, coelenterazine f, and coelenterazine h (CTZh).

[0023] In one aspect of this embodiment, the luminescent substrate preferably includes at least one selected from the group consisting of coelenterazine and coelenterazine analogs.

[0024] In this embodiment, the luminescent substrate may or may not be included as a reagent constituting the luminescence-sustaining kit. In other words, the luminescence-sustaining kit may further include the luminescent substrate. The luminescent substrate may be contained alone in a container, or may be contained in a state of being dissolved in the alcohol. Furthermore, the luminescent substrate may be contained in the same container as other components.

[0025] <Other> In one aspect of this embodiment, the luminescence sustaining kit may further comprise one or more components selected from the group consisting of a luminescent enzyme, a buffer solution, a sample tube, a microplate, an instruction manual for the user of the kit, and a luminescent substrate. The instruction manual describes, for example, the procedure for a method for sustaining luminescence of a luminescent substrate according to the second aspect described below. <Luminescent enzyme> In this embodiment, the luminescent enzyme contained in the luminescence-sustaining kit is not particularly limited as long as it is a luminescent enzyme that catalyzes the luminescent reaction of the luminescent substrate. Examples of the luminescent enzyme include Renilla luciferase (RLuc), Caulerella luciferase (GLuc), NanoLuc (registered trademark) (a modified form of luciferase derived from Pseudomonas aeruginosa), ALuc (registered trademark) (a modified form of GLuc), and picALuc (registered trademark) (a modified form of GLuc). The luminescent enzymes described above may be produced by known methods or may be commercially available. For example, picALuc (registered trademark) can be produced by the method disclosed in Patent Document 1.

[0026] The luminescent enzyme may be contained in a container alone, or may be contained in a state of being dissolved in a buffer solution, or may be contained in the same container together with other components such as the surfactant.

[0027] <Method for sustaining the luminescence of luminescent substrates> A second aspect of the present invention is a method for sustaining luminescence of a luminescent substrate, comprising: a preparation step of preparing a luminescent substrate and a luminescent enzyme; a reaction step of reacting the luminescent substrate with the luminescent enzyme; Including, The luminescent substrate is a hydrophobic compound, The molecular weight of the luminescent substrate is 300 or more and 500 or less, The reaction step is carried out in the presence of a surfactant and an alcohol. In one aspect of this embodiment, the method for sustaining the luminescence of the luminescent substrate may be carried out using the luminescence sustaining kit according to the first aspect.

[0028] <Preparation process> In this step, a luminescent substrate and a luminescent enzyme are prepared. The luminescent substrate is a hydrophobic compound. The molecular weight of the luminescent substrate is 300 or more and 500 or less. Specific examples of the luminescent substrate and the luminescent enzyme are as described above. The luminescent substrate may be prepared alone or in a state of being dissolved in alcohol or the like. The luminescent enzyme may be prepared alone or in a state of being dissolved in a buffer solution or the like.

[0029] In one aspect of this embodiment, the preparing step may include preparing a substrate solution containing the luminescent substrate and the alcohol, and an enzyme solution containing the luminescent enzyme and the surfactant.

[0030] <Reaction process> In this step, the luminescent substrate and the luminescent enzyme are reacted. The reaction step is carried out in the presence of a surfactant and an alcohol. Specific examples of the surfactant and the alcohol are as described above.

[0031] In one aspect of this embodiment, the reaction step is preferably carried out in the presence of water, a surfactant, and an alcohol.

[0032] It has long been known that the reaction between a luminescent enzyme and a luminescent substrate generally exhibits a luminescence pattern in which the luminescence intensity reaches a maximum immediately after the reaction, followed by a rapid decay of the luminescence intensity. As a result of extensive research, the present inventors have discovered for the first time that the luminescence persistence is improved by reacting a luminescent enzyme with a luminescent substrate in the presence of a surfactant and an alcohol. The present inventors believe that the surfactant forms micellar particles in the reaction system, and that the luminescent substrate (a hydrophobic compound) accumulates on these particles, contributing to the persistence of luminescence. Furthermore, based on this mechanism, the present inventors believe that the luminescence persistence is improved regardless of the type of luminescent enzyme.

[0033] When the substrate solution and the enzyme solution are prepared in the preparation step, the reaction step preferably includes mixing the substrate solution and the enzyme solution. Hereinafter, the solution obtained by mixing the substrate solution and the enzyme solution will be referred to as a "reaction solution."

[0034] In this embodiment, the concentration of the luminescent substrate in the reaction solution is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include the concentrations described in the Examples.

[0035] In this embodiment, the concentration of the luminescent enzyme in the reaction solution is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include the concentrations described in the Examples.

[0036] In this embodiment, the concentration of the surfactant in the reaction solution is preferably 0.00001% by mass or more and 20% by mass or less, more preferably 0.0001% by mass or more and 10% by mass or less, and even more preferably 0.001% by mass or more and 5% by mass or less.

[0037] In this embodiment, the concentration of the alcohol in the reaction solution is preferably 0.001% by volume or more and 10% by volume or less, and more preferably 0.01% by volume or more and 1% by volume or less.

[0038] The buffer components, pH, and temperature of the reaction solution are not particularly limited as long as the luminescent reaction between the luminescent enzyme and the luminescent substrate proceeds appropriately, and can be set appropriately. Examples of the buffer components include sodium dihydrogen phosphate, disodium hydrogen phosphate, HEPES, and trishydroxymethylaminomethane. The pH of the reaction solution may be, for example, 5.0 to 8.0. The temperature of the reaction solution may be, for example, 4°C to 40°C, 4°C to 37°C, or 4°C to 30°C.

[0039] <Other processes> The method for sustaining the luminescence of a luminescent substrate according to this embodiment may further include other steps in addition to the preparation step and the reaction step, such as a detection step of detecting light emitted by the reaction of the luminescent substrate with the luminescent enzyme using a spectrophotometer, or a microscopic or visual observation step. [Example]

[0040] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0041] [Experiment 1: Effect of surfactants on luminescence properties] We investigated how the luminescence characteristics of the luminescent substrate change when the luminescent enzyme reacts with the luminescent substrate in the presence of a surfactant and alcohol. First, the luminescent enzyme and surfactant shown below were diluted in buffer (137 mM NaCl, 9.6 mM KH2PO4, 2.7 mM KCl, pH 7.0) to obtain enzyme solutions with different concentrations of the luminescent enzyme and surfactant. (luminescent enzyme) Luminescent enzyme name: picALuc (product name, manufactured by Shimadzu Corporation) Final concentration of luciferase: 1nM to 100nM (surfactant) Detergent name: NP-40, CHAPS, Tween 20, Triton X-100, n-octyl-β-D-thioglucoside, or MEGA-8 Final surfactant concentration: 0-5%

[0042] In addition, the luminescent substrate coelenterazine h (CTZh) was dissolved in ethanol (EtOH) to a final concentration of 2.4 mM to obtain an ethanol solution of CTZh, which was then further diluted 1000-fold with the above buffer to obtain a substrate solution (final concentration of CTZh: 2.4 μM).

[0043] 50 μL each of the enzyme solution and the substrate solution were mixed. Luminescence intensity was measured immediately after mixing. A GloMax® Navigator Microplate Luminometer (Promega) was used as the measuring device. The results are shown in FIGS. 1 and 2. FIG. 1A is a graph showing the change in luminescence intensity over time. In FIG. 1A, the horizontal axis indicates the time elapsed from immediately after mixing the enzyme solution and the substrate solution, and the vertical axis indicates the relative amount of light emitted per second. FIG. 1B is a graph showing the correlation between the maximum normalized luminescence intensity (vertical axis) and the concentrations of the luciferase and surfactant (horizontal axis). FIG. 1C is a graph showing the correlation between the half-life of the luminescence reaction (horizontal axis) and the concentrations of the luciferase and surfactant (vertical axis). FIG. 2A is a graph showing the correlation between the maximum normalized luminescence intensity (vertical axis) and the concentrations of the luciferase and surfactant (horizontal axis). FIG. 2B is a graph showing the correlation between the half-life of the luminescent reaction (horizontal axis) and the concentrations of the luminescent enzyme and surfactant (vertical axis).

[0044] The results in Figure 1C and Figure 2B show that the half-life of the luminescent reaction is longer depending on the concentration of the added surfactant. These results, along with the results of Experiment 5 described below, suggest that the luminescence of the luminescent substrate is sustained in the presence of surfactant and alcohol. Furthermore, the results in Figure 1B and Figure 2A show that, depending on the concentration of the luciferase (e.g., 1 nM) and the type of surfactant (e.g., NP-40, CHAPS, etc.), not only is luminescence sustained but also an increase in luminescence intensity is observed.

[0045] [Experiment 2: Dependence on the type of luciferase] The experiment was conducted in the same manner as in Experiment 1, except that NanoLuc (Promega) was used as the luciferase and NP-40 or CHAPS was used as the surfactant. The results are shown in Figure 3. Figure 3A is a graph showing the change in luminescence intensity over time. In Figure 3A, the horizontal axis represents the time elapsed from immediately after mixing the enzyme solution and the substrate solution, and the vertical axis represents the relative amount of light emitted per second. Figure 3B is a graph showing the correlation between the maximum normalized luminescence intensity (vertical axis) and the concentrations of the luciferase and surfactant (horizontal axis). Figure 3C is a graph showing the correlation between the half-life of the luminescence reaction (horizontal axis) and the concentrations of the luciferase and surfactant (vertical axis). The results in Figure 3C indicate that even when NanoLuc was used as the luciferase, the half-life of the luminescence reaction was extended depending on the concentration of the surfactant added. These results suggest that the duration of luminescence in the presence of surfactant and alcohol does not depend on the type of luciferase. Furthermore, the results in Figure 3B show that even when NanoLuc is used as the luminescent enzyme, not only is luminescence sustained but also an increase in luminescence intensity is observed depending on the luminescent enzyme concentration (e.g., 10 nM) and the type of surfactant (e.g., NP-40, CHAPS, etc.).

[0046] [Experiment 3: Alcohol and luminescent substrate dependence] The experiment was conducted in the same manner as in Experiment 1, except that methanol (MeOH) (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as the alcohol, NP-40 was used as the surfactant, and native coelenterazine (nCTZ, final concentration 8 μM) (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as the luminescent substrate. The final concentration of the luminescent enzyme (picALuc) was 100 nM. The results are shown in Figure 4. Figure 4 is a graph showing the change in luminescence intensity over time. In Figure 4, the horizontal axis represents the elapsed time from immediately after mixing the enzyme solution and the substrate solution, and the vertical axis represents the relative luminescence per second. The results in Figure 4 demonstrate that sustained luminescence and enhanced luminescence intensity were observed even when methanol was used as the alcohol and nCTZ was used as the luminescent substrate.

[0047] [Experiment 4: Examination of the usefulness for detecting target substances] We investigated whether the use of surfactants is useful for detecting target substances. In detection systems such as ELISA (Enzyme-linked Immunosorbent Assay) (including cases where target-binding proteins other than antibodies are used), a first target-binding protein is immobilized on a substrate, and a solution that may contain the target substance is added. Next, to detect the target substance bound to the first target-binding protein on the substrate, a biotinylated second target-binding protein is added, and a streptavidin-conjugated enzyme or fluorescent dye is bound to the biotin. In this manner, the target substance is detected by the enzyme or fluorescent dye (e.g., Figure 5).

[0048] In this experiment, biotinylated bovine serum albumin (BSA) was immobilized on a 96-well plate as a simulated detection system, and detection was performed using streptavidin-fused picALuc (Figure 6A). Specifically, 100 μL of biotinylated BSA solution (final concentration 10 μg / mL) was added to each well. After washing each well with washing buffer (PBST (0.1% Tween)), 200 μL of blocking buffer (20% Immnoblock (KAC) in PBS) was added to each well for blocking. After washing each well again with washing buffer, 100 μL of streptavidin-fused picALuc solution (final concentration 1 μg / mL, diluted with 5% Immunoblock in PBST) was added to each well. Each well was then washed with washing buffer.

[0049] Immediately before adding the luminescent substrate, NP-40 was diluted with buffer (PBS) to a final concentration of 0.002-0.2%, and 100 μL of the resulting diluted solution was added to each well. Next, 100 μL of the substrate solution prepared in the same manner as in Experiment 1 was added to each well. The luminescence intensity was measured immediately after the addition of the substrate solution. A GloMax® Navigator Microplate Luminometer (Promega) was used as the measuring device. The results are shown in Figure 6. Figure 6B is a graph showing the time course of luminescence intensity. In Figure 6B, the horizontal axis indicates the time elapsed from immediately after adding the substrate solution to the well, and the vertical axis indicates the relative luminescence per second. Figure 6C is a graph showing the correlation between the maximum relative luminescence (vertical axis) and the surfactant concentration (horizontal axis). Figure 6D is a graph showing the correlation between the integrated value of the relative luminescence (vertical axis) and the surfactant concentration (horizontal axis). The integrated value was the integrated value for 30 minutes after adding the substrate solution.

[0050] The results in Figure 6 show that the maximum relative luminescence intensity increased and the luminescence persistence increased in an NP-40 concentration-dependent manner (Figure 6B). The addition of NP-40 increased the luminescence persistence, so that with the addition of 0.1% NP-40, the maximum relative luminescence intensity was 2.2 times higher than without NP-40 (Figure 6C), whereas the cumulative relative luminescence intensity was 3.2 times higher (Figure 6D). These results suggest that, even in the simulated detection system described above, the luminescence of the luminescent substrate persisted and the luminescence value increased in the presence of surfactants and alcohol.

[0051] [Experiment 5: Micelle formation of surfactants] The results of Experiment 1 show that the surfactant concentration at which sustained luminescence is observed tends to be higher as the critical micelle concentration (CMC) of the surfactant in question increases (Figures 1 and 2). Considering the possibility that particles such as micelles may have formed in the reaction system of Experiment 1, measurements were carried out using the dynamic light scattering method using the measurement samples and conditions shown below. (Composition of measurement sample) NP-40 (final concentration 0.01%) picALuc (final concentration 10nM) CTZh (final concentration 2.4μM) EtOH (final concentration 0.05%) Buffer (137 mM NaCl, 9.6 mM KH2PO4, 2.7 mM KCl, pH 7.0, balance) (Measurement conditions) Measuring device: ELSZ-2000 (Otsuka Electronics Co., Ltd.) Measurement temperature: room temperature

[0052] As a result, a signal indicating the presence of particles was observed in a mixture of CTZh dissolved in a buffer containing picALuc, NP-40, and EtOH (Figure 7A). To clarify the cause of particle formation, we performed the same measurements using the same sample but omitting other components. After removing picALuc from the sample, a signal was observed in a buffer containing NP-40 and EtOH (Figure 7B). This result indicated that picALuc is not involved in particle formation. Furthermore, when both picALuc and EtOH were further removed from the sample, a signal was not observed in a mixture of CTZh dissolved in a buffer containing only NP-40. Furthermore, a signal indicating the presence of particles was observed when a buffer containing only NP-40 and EtOH (a buffer in which CTZh was not dissolved) was measured (Figure 7C). These results suggest that surfactants and alcohol are necessary for the formation of these particles.

[0053] [Experiment 6: Microscopic observation of particles] When particles (NP-40 micelles) in a mixture of nCTZ and picALuc diluted with ethanol were observed using a microscope (product name IX83, Olympus) and an EM CCS camera (product name iXon Ultra888, Andor Technology), particles of various shapes and sizes were observed (Figure 8). The locations of the larger particles coincided with the fluorescence of nCTZ and the fluorescence due to the reaction between picALuc and nCTZ (Figure 8A). When the mixture was sonicated, small particles were observed, but the large particles were almost completely obscured. On the other hand, the fluorescence pattern remained unchanged before and after sonication (Figure 9). This raises two possible explanations: i) the signal localized in the particles accumulated within the larger particles, reaching a signal level detectable by microscope, and thus fluorescence also occurred on the small particles; or ii) particle formation is not related to the fluorescence pattern. However, considering that the surfactant concentration at which fluorescence persistence is observed tends to be higher with a higher CMC of the surfactant, this supports possibility i).

[0054] Furthermore, coelenteramine (CTM), a non-luminescent metabolite of nCTZ, 3-benzyl-5-(4-hydroxyphenyl)pyrazin-2(1H)-one (CTO), an oxidized form of nCTZ, and coelenteramide (CTMD), a luminescent metabolite of nCTZ, were diluted in MeOH, mixed with NP-40, and observed under a microscope (Keyence BZ-X810). The fluorescence of CTM, CTO, and CTMD coincided with the position of the large particles. Furthermore, in a mixture of mCherry-fused picALuc, MeOH, and NP-40, the position of mCherry fluorescence coincided with the position of the large particles (Figure 8B). These results suggest that i) the luminescence reaction may occur on the particle membrane, and ii) the hydrophobic nature of nCTZ, CTM, CTO, and CTMD may facilitate their capture by the particles.

[0055] [Experiment 7: Verification of the inhibitory effect of surfactants on the inhibition of luminescence reactions by substrate metabolites] Because CTM, CTO, and CTMD each have a similar structure to the luminescent substrate (nCTZ), it is thought that they may inhibit the luminescence reaction. For example, it has been reported that substrate metabolites inhibit the luminescence reaction of RLuc. Therefore, we investigated whether the addition of surfactants could suppress the inhibition of the luminescence reaction and maintain luminescence.

[0056] CTM, CTO, or CTMD was added to a mixture solution with the composition shown below to a final concentration of 0 μM or 1 μM, and the mixture was incubated at room temperature for 60 minutes. Luminescence was then measured using a GloMax® Navigator Microplate Luminometer (Promega). The time from substrate addition to maximum luminescence varied significantly depending on the conditions. Therefore, in this experiment, we compared luminescence immediately after substrate addition rather than maximum luminescence, so as not to require consideration of the amount of substrate consumed by the luminescence reaction or the amount of substrate oxidation over time. The results are shown in Figure 10. Figure 10A is a graph showing the correlation between relative luminescence (vertical axis) and the presence or absence of surfactants and substrate metabolites (horizontal axis). Figure 10B is a graph showing the relationship between the rate of decrease in luminescence due to the addition of substrate metabolites (vertical axis) and the presence or absence of surfactants (horizontal axis). (Composition of the mixed solution) NP-40 (final concentration 0% or 0.01%) picALuc (final concentration 100nM) nCTZ (final concentration 8μM) MeOH (final concentration 0.05%) Buffer (137 mM NaCl, 9.6 mM KH2PO4, 2.7 mM KCl, pH 7.0, balance)

[0057] When NP-40 was not added (NP-40(-)), a decrease in luminescence intensity was observed with the addition of CTM, CTO, or CTMD. The addition of CTM or CTMD significantly decreased luminescence intensity (Figure 10A). On the other hand, when NP-40 was added (NP-40(+)), a clear decrease in luminescence intensity was observed with the addition of CTM, but the decrease with the addition of CTO or CTM was small (Figure 10A). The percentage decrease in luminescence intensity with the addition of CTM, CTO, or CTMD was examined in the absence of surfactant (NP-40(-)) and with the addition of surfactant (NP-40(+)) (Figure 10B). The percentage decrease in luminescence intensity with the addition of CTM tended to be suppressed by the addition of surfactant, but not significantly. On the other hand, the percentage decrease in luminescence intensity with the addition of CTO or CTMD tended to be significantly suppressed by the addition of surfactant. These results suggest that, among the inhibition of the luminescence reaction by CTM, CTO, or CTMD, at least the inhibition by CTO or CTMD may be suppressed by the addition of a surfactant, allowing luminescence to persist.

[0058] [Experiment 8: Examination of the ratio of substrate and substrate metabolite in luminescence reaction] Using LC-MS analysis of reaction mixtures containing picALuc and nCTZ, we investigated the effect of surfactant (NP-40) on the consumption of substrate (nCTZ) and the production of substrate metabolites (CTM, CTO, CTMD).

[0059] A substrate solution (100 μL) having the following composition was added to an enzyme solution (100 μL) having the following composition to prepare a reaction solution. (enzyme solution) NP-40 (final concentration 0% or 0.01%) picALuc (final concentration 100nM) Buffer (PBS, pH 7.4, balance) (substrate solution) nCTZ (final concentration 8μM) MeOH (final concentration 0.05%) Buffer (PBS, pH 7.4, balance)

[0060] At 0 minutes (before adding the substrate solution), 10 minutes, or 60 minutes after the luminescence reaction, 1 / 1000 (volume ratio) of hydrochloric acid was added to the reaction solution to stop the luminescence reaction. Five volumes of methanol were added to the reaction solution, and the mixture was centrifuged. The supernatant was then collected to remove the luminescent enzyme. Each reaction solution was then analyzed by LC-MS to measure the amounts of nCTZ, CTM, CTO, and CTMD (Figure 11). The measurement conditions were as follows: (LC-MS measurement conditions) (MS measurement conditions) Device name: LCMS-8060 (Shimadzu Corporation) Measurement mode: multiple reaction monitoring (MRM) mode Interface temperature: 300℃ Heating gas flow rate: 10L / min Nebulizer gas flow rate: 3 L / min Drying gas flow rate: 10 L / min Desolvation line temperature: 250℃ Ionization method: Electrospray ionization (ESI) source in both positive and negative ion modes (LC measurement conditions) Instrument name: Nexera XS Inert UHPLC (Shimadzu Corporation) Column used: Wakosil 5C4 column (2.0 mm id × 250 mm) (Fujifilm Wako Pure Chemical Industries, Ltd.) Mobile phase: 0.1% formic acid (Mobile Phase A) · 0.1% formic acid in acetonitrile (Mobile Phase B) Flow rate: 0.2mL / min Mode: Gradient mode Column temperature: 25℃ Injection volume: 1μL

[0061] In the analysis of nCTZ, the amount of nCTZ differed between the NP-40-free (NP-40(-)) and NP-40-added (NP-40(+)) conditions at 0 min (before the addition of the substrate solution). The amount of nCTZ was thought to be higher than the actual amount when NP-40 was added due to ionic effects. Therefore, the amount of nCTZ was normalized to the amount without NP-40. Furthermore, the presence of CTM, CTO, and CTMD in the reaction solution at 0 min is likely due to the conversion of nCTZ to these compounds during storage. Therefore, the amounts present before the reaction were subtracted to calculate the amounts of nCTZ consumed and CTM, CTO, and CTMD produced (Figure 12). First, we found that the addition of surfactant increased nCTZ consumption. Meanwhile, the amount of CTO produced remained almost unchanged, but the amount of CTM produced decreased and the amount of CTMD produced increased with the addition of surfactant. These results suggest that the increased luminescence value may be due to the increased amount of CTMD produced when surfactant was added.

[0062] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0063] (Item 1) A kit for sustaining luminescence of a luminescent substrate according to one embodiment comprises: A kit for sustaining luminescence of a luminescent substrate, comprising a surfactant and an alcohol, wherein the luminescent substrate is a hydrophobic compound and has a molecular weight of 300 or more and 500 or less. According to the kit for sustaining luminescence described in item 1, the luminescence sustainability of the luminescent substrate is improved.

[0064] (Item 2) In the luminescence-sustaining kit according to item 1, the surfactant includes a nonionic surfactant or an amphoteric surfactant. According to the luminescence-sustaining kit according to item 2, the luminescence sustainability of the luminescent substrate is further improved.

[0065] (Item 3) In the luminescence-sustaining kit according to item 1 or 2, the alcohol comprises at least one selected from the group consisting of ethanol, methanol, propanol, isopropanol, and butanol. The luminescence-sustaining kit according to item 3 further improves the luminescence sustainability of the luminescent substrate.

[0066] (Item 4) In the luminescence-sustaining kit according to any one of Items 1 to 3, the luminescent substrate comprises at least one selected from the group consisting of coelenterazine and coelenterazine analogs. The luminescence-sustaining kit according to Item 4 can suitably improve the luminescence durability of these luminescent substrates.

[0067] (Item 5) In the luminescence sustaining kits described in items 1 to 4, the surfactant and the alcohol are contained in separate containers. According to the luminescence sustaining kit described in item 5, the compounding ratio of the surfactant to the alcohol can be adjusted as appropriate.

[0068] (Item 6) In the luminescence-sustaining kit according to items 1 to 4, the surfactant and the alcohol are contained in the same container. The luminescence-sustaining kit according to item 6 allows for a simple and easy luminescence reaction.

[0069] (Item 7) The luminescence-sustaining kit according to any one of items 1 to 6, further comprising a luminescent enzyme. According to the luminescence-sustaining kit according to item 7, the preparation of the luminescent enzyme can be omitted.

[0070] (Item 8) A method for sustaining the luminescence of a luminescent substrate according to one embodiment includes a preparation step of preparing a luminescent substrate and a luminescent enzyme, and a reaction step of reacting the luminescent substrate with the luminescent enzyme, wherein the luminescent substrate is a hydrophobic compound and has a molecular weight of 300 or more and 500 or less, and the reaction step is carried out in the presence of a surfactant and an alcohol. According to the method described in Item 8, the luminescence sustainability of the luminescent substrate is improved.

[0071] (Item 9) In the method according to item 8, the preparing step includes preparing a substrate solution containing the luminescent substrate and the alcohol, and an enzyme solution containing the luminescent enzyme and the surfactant, and the reacting step includes mixing the substrate solution and the enzyme solution. According to the method according to item 9, the luminescence durability of the luminescent substrate is further improved.

[0072] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0073] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims rather than the above-described embodiments and examples, and it is intended to include any modifications within the scope of the claims that are equivalent to the claims.

Claims

1. A kit for sustaining luminescence of a luminescent substrate, comprising: A surfactant and an alcohol are included, the luminescent substrate is a hydrophobic compound, A kit for sustained luminescence, wherein the molecular weight of the luminescent substrate is 300 or more and 500 or less.

2. The kit for sustained luminescence according to claim 1 , wherein the surfactant comprises a nonionic surfactant or an amphoteric surfactant.

3. 3. The kit for sustaining luminescence according to claim 1, wherein the alcohol comprises at least one selected from the group consisting of ethanol, methanol, propanol, isopropanol, and butanol.

4. 3. The kit for sustained luminescence according to claim 1, wherein the luminescent substrate comprises at least one selected from the group consisting of coelenterazine and coelenterazine analogs.

5. 3. The luminescence sustaining kit according to claim 1, wherein the surfactant and the alcohol are contained in separate containers.

6. 3. The luminescence sustaining kit according to claim 1, wherein the surfactant and the alcohol are contained in the same container.

7. The kit for sustaining luminescence according to claim 1 or claim 2, further comprising a luminescent enzyme.

8. A method for sustaining luminescence of a luminescent substrate, comprising: a preparation step of preparing a luminescent substrate and a luminescent enzyme; a reaction step of reacting the luminescent substrate with the luminescent enzyme; Including, the luminescent substrate is a hydrophobic compound, the molecular weight of the luminescent substrate is 300 or more and 500 or less; The method wherein the reacting step is carried out in the presence of a surfactant and an alcohol.

9. the preparing step includes preparing a substrate solution containing the luminescent substrate and the alcohol, and an enzyme solution containing the luminescent enzyme and the surfactant; The method of claim 8 , wherein the reacting step comprises mixing the substrate solution and the enzyme solution.

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