Composition and method for stabilizing coelenterazine and analog thereof

Abstract

Provided is a composition for stabilizing coelenterazine and an analog thereof, which composition contains coelenterazine and / or an analog thereof, and an aqueous-phase protective solution or an organic-phase protective solution. The coelenterazine and / or analog thereof is dissolved in the aqueous phase or organic phase, wherein the aqueous-phase protective solution has a pH≤5.5, and the organic-phase protective solution contains an organic acid or an inorganic acid. The composition can improve the stability of the substrates of coelenterazine and an analog thereof, can be directly used in an aqueous-phase substrate mother liquor without requiring pretreatment, has high substrate stability when used in an organic-phase substrate mother liquor, and can reduce inter-batch variation in experimental results and improve the stability of experimental results.

Description

Compositions and methods for stabilizing coelenterazine and analogs thereof TECHNICAL FIELD

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a composition and method for stabilizing coelenterazine and analogs thereof. BACKGROUND

[0002] Coelenterazine is a luciferin that emits light and is a substrate for many luciferases and photoproteins. Coelenterazine and its analogs are extremely unstable, and a prepared solution will lose efficacy within a few hours without a protective agent. There are reports that coelenterazine can be prepared just before use, but even if it is prepared just before use, improper storage will increase the batch-to-batch error of experimental results, reduce the reliability, and reduce the repeatability.

[0003] Some water-phase protective agents for coelenterazine analogs are mentioned in the prior art, and the pH range is 5.5-8.0. However, it has been found through research that coelenterazine analogs have poor stability in a water phase with a pH of 5.5-8.0, and the half-life at 37°C is not more than one day. Inouye et al. (The Use of Renilla Luciferase, Oplophorus Luciferase, and Apoaequorin as Bioluminescent Reporter Protein in the Presence of Coelenterazine Analogues as Substrate. Satoshi Inouye, and Osamu Shimomura. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 233, 349-353 (1997)) mentioned adding 2% by volume of 1M HCl to ethanol to prepare a coelenterazine stock solution (90 μM), but there is no experimental data to show that the stability of coelenterazine is improved after adding hydrochloric acid, and the ethanol solution is an organic solution that is volatile, and the concentration of the substrate will change during storage. In addition, ethanol will inhibit the activity of many luciferases.

[0004] The prior art also discloses a class of protectants (6-azido-2-thiothymine derivatives) which have good protection effect at high concentration (such as 225 mM). However, it is found through testing that its half-life at 37°C is about 5 days, which still does not meet the requirements of clinical diagnostic reagents. Moreover, the substance is difficult to dissolve in water and can only be stored in a mother liquor of an organic solvent with a coelenterazine substrate. Before use, the substrate needs to be diluted at a ratio of 20:1 or higher, otherwise the high concentration of organic solution will interfere with the enzyme reaction and reduce the luminescence value of the whole enzyme, especially the complementary enzyme. In addition, 6-azido-2-thiothymine also has a strong inhibitory effect on luciferase. At the same time, the stability of the diluted substrate is also poor, with a half-life of only about 12 hours, which cannot meet the needs of clinical use.

[0005] Therefore, it is necessary to develop a ready-to-use coelenterazine and analog thereof protection solution to meet the stability requirements of detection and reduce the configuration of the automatic dilution function of the instrument, thereby providing convenience for the clinical sample diagnosis of the luciferase detection system for automatic instruments. SUMMARY

[0006] To solve one of the above technical problems in the prior art, the present application provides a composition, kit and method for stabilizing coelenterazine and analogs thereof.

[0007] In a first aspect, the present application provides a composition comprising:

[0008] coelenterazine and / or analogs thereof; and

[0009] an aqueous phase protection solution,

[0010] wherein the coelenterazine and / or analogs thereof are dissolved in the aqueous phase,

[0011] wherein the pH of the aqueous phase protection solution is ≤5.5, preferably 1-5, more preferably 2-5.

[0012] In some embodiments, the aqueous phase protection solution comprises a buffer solution composed of an organic acid and / or an inorganic acid, a non-buffer solution.

[0013] In some embodiments, the coelenterazine analogs include, but are not limited to, one or more of Furimazine, coelenterazine-h, coelenterazine-h-h, coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, coelenterazine-I, coelenterazine-icp, coelenterazine-v, 2-methyl coelenterazine, JRW-0238, JRW-1743 and JRW-1744.

[0014] In some embodiments, the pH of the composition is any value in the range of < 5.5, preferably < 5.5, more preferably 2-5; for example, the pH can be 0.5, 1.0, 1.5, 1.72, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or 5.5.

[0015] The above composition of the present application can be used for the protection of coelenterazine and its analogs substrates in aqueous solution, the key is that the pH range is < 5.5. When the pH is in this range, the substrate can be guaranteed to be stable, not to be degraded, not to produce spontaneous light, and the stability of coelenterazine and its analogs substrates can be greatly improved. When the pH is greater than 5.5, the substrate stability is significantly decreased.

[0016] The concentration of coelenterazine and / or its analogs (substrate) in the aqueous protective solution ranges from 1 μM to 10000 μM, preferably 5 μM to 5000 μM, more preferably 15 μM to 1500 μM, further more preferably 30 μM to 600 μM.

[0017] In some embodiments, the organic acid includes, but is not limited to, one or more of carboxylic acid (R-COOH), sulfonic acid (R-SO3H), thiocarboxylic acid (R-COSH), squaric acid, wherein R is C 1-12 alkyl or C 6-30 aryl.

[0018] Preferably, the organic acid includes one or more of C 1-12 carboxylic acid, C 2-12 dicarboxylic acid, C 1-12 thiocarboxylic acid, C 1-12 amino carboxylic acid, C 1-12 alkyl sulfonic acid, C 6-30 aryl sulfonic acid.

[0019] Preferably, the C 1-12 carboxylic acid includes, but is not limited to, one or more of formic acid, acetic acid, propionic acid, isopropionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, 2-methylhexanoic acid, pivalic acid, 2-octynoic acid, trans-2-decenoic acid, n-decanoic acid, (2-benzimidazolylthio)acetic acid.

[0020] Preferably, the C 2-12 dicarboxylic acid includes, but is not limited to, one or more of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, dodecanedioic acid, phthalic acid, citric acid.

[0021] Preferably, the thio C 1-12 Carboxylic acids include, but are not limited to, one or more of thioacetic acid, acetylthioacetic acid, thiopropionic acid, thio butyric acid, thiovaleric acid, thiohexanoic acid, DL-thioctic acid, thio benzoic acid.

[0022] Preferably, the amino C 1-12 Carboxylic acids include, but are not limited to, one or more of glycine, glutamic acid, aspartic acid, and other acidic amino acids.

[0023] Preferably, the C 1-12 Alkyl sulfonic acids include, but are not limited to, one or more of methane sulfonic acid, ethane sulfonic acid, propane sulfonic acid, 4-morpholine ethanesulfonic acid (MES).

[0024] Preferably, the C 6-30 Aryl sulfonic acids include, but are not limited to, one or more of benzene sulfonic acid, xylene sulfonic acid.

[0025] In some embodiments, the organic acid further includes, but is not limited to, one or more of thio C 1-12 Alcohol acids, mercapto C 1-12 Carboxylic acids, cysteinyl amino C 1- 12 Carboxylic acids, C 6-30 Heteroaryl sulfonic acids.

[0026] In some embodiments, the thio C 1-12 The alcohol acid is thio glycolic acid.

[0027] In some embodiments, the mercapto C 1-12 The carboxylic acid is N-acetyl-L cysteine.

[0028] In some embodiments, the C 6-30 The heteroaryl sulfonic acid is 2-phenyl-5-benzimidazole sulfonic acid.

[0029] In some embodiments, the inorganic acid includes, but is not limited to, one or more of hydrochloric acid (HC1), sulfuric acid (H2SO4), sulfurous acid (H2SO3), hydrofluoric acid (HF), hydrobromic acid (HBr), hydroiodic acid (HI), nitric acid (HNO3), phosphoric acid (H3PO4), boric acid (H3BO3), preferably HC1.

[0030] In some embodiments, the buffer of organic acid and inorganic acid can be, for example, one or more of glycine-HC1, formic acid-HC1, acetic acid-HC1, malonic acid-HC1.

[0031] In some embodiments, the concentration of the organic acid and / or inorganic acid in the aqueous phase protection solution is 0.01% to 20%, preferably 0.1% to 10%, more preferably 0.1% to 5%, for example, it can be 0.01%, 0.05%, 0.1%, 0.11%, 0.15%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 15%, 20%.

[0032] In some embodiments, the concentration of the organic acid and / or inorganic acid in the aqueous phase protection solution is 0.1 mM to 5300 mM, preferably 5 mM to 1000 mM, more preferably 10 mM to 100 mM, for example, it can be 0.1 mM, 0.5 mM, 1 mM, 2.5 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, 2000 mM, 3000 mM, 4000 mM, 5000 mM, 5300 mM.

[0033] The organic acid that is liquid at room temperature is represented by volume percentage (v / v); the organic acid that is solid at room temperature is represented by mass percentage (m / v).

[0034] Since the optimal pH for luciferase catalyzed luminescence is around 6.0 to 6.5, if the acid concentration in the substrate protection solution is too high, it will cause subsequent neutralization to be difficult, and if the neutralization is not complete, the pH is low, and the activity is significantly reduced. If the acid concentration in the substrate protection solution is lower than the above range, the buffer capacity is not strong, the pH of the protection solution cannot be adjusted to the required range, and the protection effect on the substrate is reduced.

[0035] In some embodiments, the composition further comprises one or more of an antioxidant, a metal ion chelator, a surfactant, a biological preservative, and an anti-precipitation agent.

[0036] In some embodiments, the composition comprises one or more antioxidants. The antioxidant includes, but is not limited to, one or more of thiourea or thiourea analogues, DL thioctic acid, iodide (KI, NaI), thioacetic acid, dithiothreitol (DTT), ascorbic acid (sodium), methionine, thioglycolic acid, tris(2-carboxyethyl)phosphine hydrochloride, glucose, 6-aza-2-thiothymine (ATT), and Z-2(2-amino-4-thiazolyl)2-hydroxyiminoacetic acid.

[0037] In some embodiments, the thiourea or thiourea analog has a structure as shown in the following general formula 1:

[0038] wherein R1, R2, R3 are each independently selected from hydrogen, amino, substituted or unsubstituted C 1-12 alkyl, C 1-12 alkyl-C 6-30 aryl, C 1-12 alkyl-C 6-30 heteroaryl, C 3-12 cycloalkyl, C 6-30 aryl, C 6-30 heteroaryl, carboxylic acid group, imine group, hydroxyl group, oxo group, amidine group;

[0039] wherein R2 and R3 form a ring or do not form a ring with the adjacent atoms.

[0040] Preferably, the thiourea or thiourea analog includes one or more of thiourea, thiouracil, tetramethyl thiourea, amidine thiourea, N-ethyl thiourea, N,N'-dimethyl thiourea.

[0041] Preferably, the antioxidant is thiourea.

[0042] The addition of an antioxidant to the aqueous substrate protection solution has a synergistic effect on the protection of the substrate, and the protection effect of the addition of an antioxidant is better than that without the addition of an antioxidant. The effect of the substance with reducing effect under acidic conditions on the stability of the substrate is more significant, and can effectively prolong the half-life of the substrate.

[0043] In some embodiments, the composition further comprises one or more metal ion chelating agents. The metal ion chelating agent includes but is not limited to one or more of potassium gluconate, DTPA, EDDHA, HEDP. The metal ion chelating agent can further improve the stability.

[0044] In some embodiments, the composition further comprises one or more surfactants. The surfactant is an alkoxylated polyethylene hydroxyl ethanol, such as Tergitol 15S9.

[0045] In some embodiments, the composition further comprises one or more biological preservatives. The biological preservative is an isothiazolinone, such as ProClin300 (Sigma, 48912-U).

[0046] In some embodiments, the composition further comprises one or more anti-precipitation agents. The anti-precipitation agent includes but is not limited to one or more of DMSO, ethanol, DMF, cyclodextrin, methanol, propylene glycol.

[0047] In some embodiments, the cyclodextrin is one or more of a water-soluble cyclodextrin or analog, in particular sulfobutyl-β-cyclodextrin sodium salt, 2-hydroxypropyl β cyclodextrin, methyl β cyclodextrin, and the like.

[0048] In some embodiments, the composition further comprises: an antioxidant 0-40 mM, for example 10 mM, 20 mM, 30 mM, 40 mM; a metal ion chelator 0-1.5 mM, for example 0.5 mM, 1 mM, 1.5 mM; a surfactant 0.01%-0.05%, for example 0.01%, 0.02%, 0.03%, 0.04%, 0.05%; a biological preservative 0.05%-0.2%, for example 0.05%, 0.1%, 0.15%, 0.2%; an anti-precipitant 0%-90%, for example 10%, 30%, 50%, 70%, 90%.

[0049] In some embodiments, the plurality of additives in the composition can further embody a synergistic effect of more optimal stabilization of the substrate.

[0050] In a second aspect, the present application provides a composition comprising:

[0051] coelenterazine and / or an analog thereof; and

[0052] an organic phase protection solution,

[0053] wherein the coelenterazine and / or the analog thereof is dissolved in the organic phase,

[0054] wherein the organic phase protection solution comprises an organic acid or an inorganic acid.

[0055] In some embodiments, the coelenterazine analog includes, but is not limited to, one or more of Furimazine, coelenterazine-h, coelenterazine-h-h, coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, coelenterazine-I, coelenterazine-icp, coelenterazine-v, 2-methylcoelenterazine, JRW-0238, JRW-1743, and JRW-1744.

[0056] In some embodiments, the organic acid includes, but is not limited to, one or more of C 1-12 carboxylic acid, C 2-12 dicarboxylic acid, thio 1-12 carboxylic acid, amino 1-12 carboxylic acid, C 1-12 alkylsulfonic acid, C 6-30 arylsulfonic acid, 2-phenyl-5-benzimidazole sulfonic acid, N-acetyl L-cysteine.

[0057] Preferably, the C 1-12Carboxylic acids include, but are not limited to, one or more of formic acid, acetic acid, propionic acid, isopropionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, 2-methylhexanoic acid, valproic acid, 2-octynoic acid, elaidic acid, n-decanoic acid, (2-benzimidazolylthio)acetic acid.

[0058] Preferably, the C 2-12 Dicarboxylic acids include, but are not limited to, one or more of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, dodecanedioic acid, phthalic acid, citric acid.

[0059] Preferably, the thio C 1-12 Carboxylic acids include, but are not limited to, one or more of thioacetic acid, acetylthioacetic acid, thiopropionic acid, thiobutyric acid, thiopentanoic acid, thiohexanoic acid, DL-thioctic acid, thiobenzoic acid.

[0060] Preferably, the amino C 1-12 Carboxylic acids include, but are not limited to, one or more of glycine, glutamic acid, aspartic acid, and other acidic amino acids.

[0061] Preferably, the C 1-12 Alkyl sulfonic acids include, but are not limited to, one or more of methane sulfonic acid, ethane sulfonic acid, propane sulfonic acid, 4-morpholine ethanesulfonic acid.

[0062] Preferably, the C 6-30 Aryl sulfonic acids include, but are not limited to, one or more of benzene sulfonic acid, xylene sulfonic acid.

[0063] In some embodiments, the organic acid is an anhydrous organic acid. However, it is also within the scope of the present application to have a small amount of water in the organic acid. In practical applications, the organic acid can be made to be free of water by mixing a pure liquid organic acid with an organic solvent, or dissolving an organic acid solid powder directly with an organic solvent, etc.

[0064] The above composition of the present application can be used to protect the substrate of coelenterazine and its analogues in an organic phase solution, the key point of which is to use an organic acid free of water in the organic phase protection solution, which is conducive to improving the stability of coelenterazine and its analogues in the organic phase solution; if the organic acid contains a large amount of water, it will seriously affect the stability of coelenterazine and its analogues in the organic phase solution.

[0065] In some embodiments, the inorganic acid includes, but is not limited to, one or more of hydrochloric acid (HCl), sulfuric acid (H2SO4), sulfurous acid (H2SO3), hydrofluoric acid (HF), hydrobromic acid (HBr), hydroiodic acid (HI), nitric acid (HNO3), phosphoric acid (H3PO4), boric acid (H3BO3), preferably HCl.

[0066] In some embodiments, the buffer of organic and inorganic acids can be, for example, one or more of glycine-HCl, formic acid-HCl, acetic acid-HCl, malonic acid-HCl.

[0067] In some embodiments, the organic acid further includes, but is not limited to, one or more of thioC 1-12 alcohols, mercaptoC 1-12 carboxylic acids, cysteinyl aminoC 1- 12 carboxylic acids, C 6-30 heteroaryl sulfonic acids.

[0068] In some embodiments, the thioC 1-12 alcohols is thioethanol.

[0069] In some embodiments, the mercaptoC 1-12 carboxylic acids is N-acetyl-L cysteine.

[0070] In some embodiments, the C 6-30 heteroaryl sulfonic acids is 2-phenyl-5-benzimidazole sulfonic acid.

[0071] In some embodiments, the concentration of organic or inorganic acids in the organic phase protecting solution is 0.01% to 20%, preferably 0.1% to 10%, more preferably 1% to 5%, for example, it can be 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 15%, 20%.

[0072] The organic acid that is liquid at room temperature is represented by % (v / v); the organic acid that is solid at room temperature is represented by % (m / v).

[0073] Alternatively, the concentration of organic or inorganic acids in the organic phase protecting solution is 0.1 mM to 5300 mM, preferably 10 mM to 1000 mM, more preferably 20 mM to 200 mM, for example, it can be 0.1 mM, 0.5 mM, 1 mM, 2.5 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, 2000 mM, 3000 mM, 4000 mM, 5000 mM, 5300 mM.

[0074] mM concentration of liquid organic acid = volume of acid used x density / molar molecular weight / final volume containing solvent x 1000.

[0075] mM concentration of solid organic acid = mass of weighed acid / molar molecular weight / final volume after dissolution x 1000.

[0076] When the organic acid or inorganic acid is within the above concentration range, the acidic environment can be maintained, and the pH is stable, thereby stabilizing the substrate and avoiding substrate degradation and spontaneous light emission; if the concentration is too high, the organic acid can react with the substrate, and the subsequent enzymatic luminescence reaction can be inhibited by the high concentration of acid; if the concentration is too low, the acidic environment can not be stably maintained for a long time, resulting in degradation of the substrate in a neutral or alkaline environment.

[0077] The concentration of coelenterazine and / or its analogues (substrate) in the organic phase protection solution ranges from 5 μΜ to 50,000 μΜ. For example, it can be 5 μΜ, 10 μΜ, 50 μΜ, 100 μΜ, 200 μΜ, 500 μΜ, 1000 μΜ, 2000 μΜ, 5000 μΜ, 10,000 μΜ, 20,000 μΜ, or 50,000 μΜ.

[0078] In some embodiments, the organic phase protection solution further comprises DMSO 0-50%, methanol 0-50%, ethanol 0-50%, propylene glycol 0-90%, and / or glycerol 0-90%.

[0079] Preferably, the organic phase protection solution further comprises DMSO 5-25%, methanol 5-25%, ethanol 5-25%, propylene glycol 50-90%, and / or glycerol 50-90%.

[0080] In some embodiments, one or more of an antioxidant, a metal ion chelator, a surfactant, a biological preservative, and an anti-precipitation agent are further included in the composition.

[0081] In some embodiments, one or more antioxidants are included in the composition. The antioxidant includes, but is not limited to, one or more of thiourea or thiourea analogues, DL thioctic acid, iodide (KI, NaI), thioacetic acid, dithiothreitol (DTT), ascorbic acid (sodium), methionine, thioglycolic acid, tris(2-carboxyethyl)phosphine hydrochloride, glucose, 6-aza-2-thiothymine (ATT), and Z-2(2-amino-4-thiazolyl)2-hydroxyiminoacetic acid.

[0082] In some embodiments, the thiourea or thiourea analogues have a structure as shown in the following general formula 1:

[0083] wherein R1, R2, R3 are each independently selected from the group consisting of hydrogen, amino, substituted or unsubstituted C 1-12 alkyl, C 1-12 alkyl-C 6-30 aryl, C 1-12 alkyl-C 6-30 heteroaryl, C 3-12 cycloalkyl, C 6-30 aryl, C 6-30 heteroaryl, carboxylic acid group, imine group, hydroxyl group, oxo group, amidine group;

[0084] wherein R2 and R3 form a ring or do not form a ring with the adjacent atoms.

[0085] Preferably, the thiourea or thiourea analogues include one or more of thiourea, thiouracil, tetramethyl thiourea, amidine thiourea, N-ethyl thiourea, N,N'-dimethyl thiourea.

[0086] Preferably, the antioxidant is thiourea.

[0087] In some embodiments, the composition further comprises one or more metal ion chelators. The metal ion chelators include, but are not limited to, one or more of potassium gluconate, DTPA, EDDHA, HEDP. Metal ion chelators can further improve stability.

[0088] In some embodiments, the composition further comprises one or more surfactants. The surfactant is an alkoxylated polyethylene hydroxyl ethanol, such as Tergitol 15S9.

[0089] In some embodiments, the composition further comprises one or more biological preservatives. The biological preservative is an isothiazolinone, such as ProClin 300 (Sigma, 48912-U).

[0090] In some embodiments, the composition further comprises one or more anti-precipitation agents. The anti-precipitation agents include, but are not limited to, one or more of DMSO, ethanol, DMF, cyclodextrin, methanol, propylene glycol.

[0091] In some embodiments, the cyclodextrin is a water-soluble cyclodextrin or analog, in particular one or more of sulfobutyl-β-cyclodextrin sodium salt, 2-hydroxypropyl β cyclodextrin, methyl β cyclodextrin, etc.

[0092] In some embodiments, the composition further comprises: an antioxidant 0-40 mM, for example 10 mM, 20 mM, 30 mM, 40 mM; a metal ion chelator 0-1.5 mM, for example 0.5 mM, 1 mM, 1.5 mM; a surfactant 0.01%-0.05%, for example 0.01%, 0.02%, 0.03%, 0.04%, 0.05%; a biological preservative 0.05%-0.2%, for example 0.05%, 0.1%, 0.15%, 0.2%; an anti-precipitant 0%-90%, for example 10%, 30%, 50%, 70%, 90%.

[0093] In some embodiments, the plurality of additives in the composition can further embody a synergistic effect of more optimal stabilization of the substrate.

[0094] In a third aspect, the present application provides a kit comprising the composition of the present application.

[0095] In some embodiments, the composition is contained in one or more containers.

[0096] Preferably, the container is a light-protective bottle or tube.

[0097] Preferably, the container is a tube.

[0098] In some embodiments, the structure of the kit is: reagent 1: the acidic aqueous substrate solution (containing 1-10,000 μM of a coelenterazine substrate) provided by the present application; reagent 2: a neutralization buffer, containing MES and / or Tris and / or PBS and the like buffer salts, with or without luciferase. After mixing reagent 2 with reagent 1 in a ratio (for example, 30 μL of reagent 1 and 50 μL of reagent 2), the pH of the mixed solution is neutralized to between 5.5 and 7.5; if reagent 2 contains luciferase, the luminescence value (luminescence wavelength 200-800 nm, preferably 400-600 nm) can be detected immediately after mixing reagent 1 with reagent 2. The magnitude of the luminescence value reflects the amount of luciferase.

[0099] Preferably, the reagent bottle containing the reagent is a light-protective container.

[0100] In a fourth aspect, the present application provides a method for stabilizing coelenterazine and / or its analogs, the method comprising the step of mixing the coelenterazine or its analogs with an effective amount of the organic phase protection solution, the solubilization medium of the coelenterazine and / or its analogs being an organic phase.

[0101] In some embodiments, the coelenterazine analogs include, but are not limited to, one or more of Furimazine, coelenterazine-h, coelenterazine-h-h, coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, coelenterazine-I, coelenterazine-icp, coelenterazine-v, 2-methylcoelenterazine, JRW-0238, JRW-1743, and JRW-1744.

[0102] In some embodiments, the organic acid includes, but is not limited to, one or more of a C 1-12 carboxylic acid, a C 1-12 dicarboxylic acid, a thio 1-12 carboxylic acid, an amino 1-12 carboxylic acid, a C 1-12 alkyl sulfonic acid, a C 6-30 aryl sulfonic acid, 2-phenyl-5-benzimidazole sulfonic acid, N-acetyl L cysteine.

[0103] Preferably, the C 1-12 carboxylic acid includes, but is not limited to, one or more of formic acid, acetic acid, propionic acid, isopropionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, 2-methylhexanoic acid, pivalic acid, 2-octynoic acid, elaidic acid, n-decanoic acid, (2-benzimidazolylthio)acetic acid.

[0104] Preferably, the C 2-12 dicarboxylic acid includes, but is not limited to, one or more of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, dodecanedioic acid, phthalic acid, citric acid.

[0105] Preferably, the thio 1-12 carboxylic acid includes, but is not limited to, one or more of thioacetic acid, acetylthioacetic acid, thiopropionic acid, thio butyric acid, thiovaleric acid, thiohexanoic acid, DL-thioctic acid, thiobenzoic acid.

[0106] Preferably, the amino 1-12 carboxylic acid includes, but is not limited to, one or more of glycine, glutamic acid, aspartic acid, and other acidic amino acids.

[0107] Preferably, the C 1-12 alkyl sulfonic acid includes, but is not limited to, one or more of methane sulfonic acid, ethane sulfonic acid, propane sulfonic acid, 4-morpholine ethanesulfonic acid.

[0108] Preferably, the C 6-30 aryl sulfonic acid includes, but is not limited to, one or more of benzene sulfonic acid, xylene sulfonic acid.

[0109] In some embodiments, the effective amount refers to the amount or concentration of the organic acid that is capable of stabilizing coelenterazine and its analogs in an organic phase solution.

[0110] For example, the concentration of the organic acid is 0.01% to 20%, preferably 0.1% to 10%, more preferably 1% to 5%, for example, it can be 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 15%, or 20%.

[0111] Alternatively, the concentration of the organic acid is 0.1 mM to 5300 mM, preferably 10 mM to 1000 mM, more preferably 20 mM to 200 mM, for example, it can be 0.1 mM, 0.5 mM, 1 mM, 2.5 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, 2000 mM, 3000 mM, 4000 mM, 5000 mM, or 5300 mM.

[0112] Fifthly, the present invention provides a method for stabilizing coelenterate and / or its analogues, the method comprising the step of mixing the coelenterate or its analogues with an effective amount of the aqueous protective solution, wherein the dissolution medium of the coelenterate and / or its analogues is an aqueous phase, and the pH of the aqueous protective solution is ≤5.5, preferably 1 to 5, more preferably 2 to 5.

[0113] In some embodiments, the aqueous phase solution includes aqueous solutions and heavy aqueous solutions.

[0114] In some embodiments, the coelenterin analogues include, but are not limited to, one or more of Furimazine, coelenterin-h, coelenterin-hh, coelenterin-n, coelenterin-f, coelenterin-hcp, coelenterin-cp, coelenterin-c, coelenterin-e, coelenterin-fcp, coelenterin-I, coelenterin-icp, coelenterin-v, 2-methylcoelenterin, JRW-0238, JRW-1743, and JRW-1744.

[0115] In some embodiments, the organic acid includes, but is not limited to, C 1-12 Carboxylic acid, C 2-12 Dicarboxylic acid, thioC 1-12 Carboxylic acid, amino C 1-12 Carboxylic acid, C 1-12 Alkyl sulfonic acid, C 6-30 One or more of the aryl sulfonic acids.

[0116] Preferably, the C 1-12Carboxylic acids include, but are not limited to, one or more of formic acid, acetic acid, propionic acid, isopropionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid.

[0117] Preferably, the C 1-12 Dicarboxylic acids include, but are not limited to, one or more of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid.

[0118] Preferably, the C 1-12 Carboxylic acids include, but are not limited to, one or more of thioacetic acid, acetylthioacetic acid, thiopropionic acid, thio butyric acid, thiovaleric acid, thiohexanoic acid, DL-thioctic acid.

[0119] Preferably, the C 1-12 Carboxylic acids include, but are not limited to, one or more of glycine, glutamic acid, aspartic acid, and other acidic amino acids.

[0120] Preferably, the C 1-12 Alkyl sulfonic acids include, but are not limited to, one or more of methane sulfonic acid, ethane sulfonic acid, propane sulfonic acid, 4-morpholine ethanesulfonic acid.

[0121] Preferably, the C 6-30 Aryl sulfonic acids include, but are not limited to, one or more of benzene sulfonic acid, xylene sulfonic acid.

[0122] In some embodiments, the inorganic acid includes, but is not limited to, one or more of hydrochloric acid (HC1), sulfuric acid (H2SO4), hydrofluoric acid (HF), hydrobromic acid (HBr), hydroiodic acid (HI), nitric acid (HNO3), phosphoric acid (H3PO4), boric acid (H3BO3).

[0123] In some embodiments, the effective amount refers to the amount or concentration of the organic acid and / or inorganic acid that is capable of stabilizing coelenterazine and its analogs in an aqueous solution.

[0124] For example, the concentration of the organic acid and / or inorganic acid can be 0.01% to 20%, preferably 0.1% to 10%, more preferably 0.1% to 5%, such as 0.01%, 0.05%, 0.1%, 0.11%, 0.15%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 15%, 20%.

[0125] Alternatively, the concentration of the organic acid and / or inorganic acid is 0.1 mM to 5300 mM, preferably 5 mM to 1000 mM, more preferably 10 mM to 100 mM, for example, it can be 0.1 mM, 0.5 mM, 1 mM, 2.5 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, 2000 mM, 3000 mM, 4000 mM, 5000 mM, 5300 mM.

[0126] In a sixth aspect, the present application provides use of the composition of the first aspect or the second aspect in stabilizing coelenterazine and / or its analogs.

[0127] In some embodiments, the coelenterazine and / or its analogs include one or more of coelenterazine, Furimazine, coelenterazine-h, coelenterazine-h-h, coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, coelenterazine-I, coelenterazine-icp, coelenterazine-v, 2-methyl coelenterazine, JRW-0238, JRW-1743 and JRW-1744.

[0128] In a seventh aspect, the present application provides use of a composition in performing detection, wherein a buffer is mixed with the aqueous phase protective solution of the first aspect or the oil phase protective solution of the second aspect, and the resulting mixture has a pH value in the range of 5.0 to 8.5, and the luminescent signal is detected.

[0129] The above-mentioned pH value range of the mixture is conducive to performing luminescent signal detection.

[0130] Preferably, the pH value of the mixture is in the range of 5.5 to 7.0.

[0131] In some embodiments, the buffer comprises a buffer salt component, and a luciferase.

[0132] In some embodiments, the buffer salt component includes, but is not limited to, one or more of MES, Tris, phosphate.

[0133] Since the activity of luciferase or the spontaneous luminescence of coelenterazine analogs is significantly affected by pH, low pH inhibits and high pH promotes, in order to maintain a relatively good signal-to-noise ratio, it is necessary to maintain the final pH of the enzymatic reaction of the substrate in the above-mentioned neutral to acidic range.

[0134] The successful protection of coelenterazine and its analogues by the protective solution of the present application provides great convenience for the luciferase detection system used in the clinical sample diagnosis of automatic instruments, and has obvious advantages in matching the automatic instrument of clinical diagnosis, which can reduce the automatic dilution function configuration of the instrument.

[0135] The protective solution provided in the present application can greatly improve the stability of coelenterazine and its analogues, and can be directly used without pretreatment in aqueous phase substrate mother liquor, and the substrate stability is obviously better than that of 6-azido-2-thiothymidine in organic phase substrate mother liquor.

[0136] In the protective solution of the present application, preferably, for example, thioglycolic acid and the like, the above substances have better protection effect on coelenterazine h and Furimazine in the aqueous solution of glycine-HCl buffer (10mM) at pH 2.5 (the protection effect of the aqueous protective solution added with the above substances is equivalent to that of glycine hydrochloride at pH 2.5, but the protection effect is better than that of 20mM HCl).

[0137] In the protective solution of the present application, preferably, for example, thioacetic acid, N-acetyl-L-cysteine, 2-phenyl-5-benzimidazole sulfonic acid and the like, the above substances have more significant protection effect on CTZ-h in organic phase.

[0138] Compared with the prior art, the present application has the following beneficial effects:

[0139] The present inventors have unexpectedly found that in the substrate mother liquor of organic phase, organic acids such as thioacetic acid, formic acid, acetic acid, oxalic acid and the like have good protection effect on coelenterazine and its analogues, especially thioacetic acid and DL-thioctic acid, which have obviously better protection effect than 6-azido-2-thiothymidine and ATCA; adding a certain amount of organic acids into the substrate mother liquor of organic phase can significantly prolong the half-life of the substrate, especially thioacetic acid can significantly prolong the half-life of the substrate to more than 35.6 days at 37℃. The stable substrate mother liquor provides guarantee for the reliability of test results.

[0140] The organic phase protective solution formula has the following advantages:

[0141] 1. Better protection effect, allowing to be stored for more than one year under refrigerated conditions (2-8℃), fully meeting the requirements of various use scenarios;

[0142] 2. Reduce batch difference of experimental results, improve stability of experimental results.

[0143] The inventors surprisingly found that the aqueous phase protection solution with a pH range of ≤5.5 has a good substrate protection effect on coelenterazine and its analogs, allowing the substrate to be stored in an aqueous solution without dilution and can be directly used for detection. When detecting, the aqueous phase protection solution with a pH range of ≤5.5 of the application is added to the neutralization buffer containing luciferase for neutralization, so that the pH value is adjusted to pH 5.5-7.5, which is beneficial to the activity of the enzyme and facilitates detection.

[0144] The aqueous phase protection solution formula has the following advantages:

[0145] 1. Better protection effect, under refrigeration conditions (2-8℃), the luminescent signal decreases by less than 10% after more than 3 months of storage, fully meeting the requirements of the use scene;

[0146] 2. Ready-to-use reagent, no pretreatment is required;

[0147] 3. It is aqueous, which is safer;

[0148] 4. It will not corrode the liquid path and connecting piece;

[0149] 5. It is not volatile. BRIEF DESCRIPTION OF DRAWINGS

[0150] Figure 1 shows the relative luminescent signal values of the substrate added in the protection solution with different pH values of Example 1 of the application, wherein A is the result of 37℃ treatment and B is the result of 60℃ treatment.

[0151] Figure 2 shows the relative luminescent signal values of the substrate added in the protection solution with different pH values of Example 2 of the application, wherein A is the result of substrate Furimazine and B is the result of substrate CTZ-h.

[0152] Figure 3 shows the relative luminescent signal values of the substrate added in the protection solution with different pH values of Example 3 of the application, wherein A is the result of substrate Furimazine and B is the result of substrate CTZ-h.

[0153] Figure 4 shows the relative luminescent signal values of the substrate added in the protection solution with different pH values of Example 4 of the application, wherein A is the result of substrate Furimazine and B is the result of substrate CTZ-h.

[0154] Figure 5 shows the relative luminescent signal values of the substrate added in the protection solution with different pH values of Example 5 of the application, wherein A is the result of substrate Furimazine and B is the result of substrate CTZ-h.

[0155] Figure 6 shows the relative luminescent signal values of the substrate added in the protection solution with different pH values of Example 6 of the application, wherein A is the result of substrate Furimazine and B is the result of substrate CTZ-h.

[0156] Figure 7 shows the relative luminescence signal values after adding substrates in different pH protective solutions of Example 7 of the present application, wherein A is the result of substrate Furimazine and B is the result of substrate CTZ-h.

[0157] Figure 8 shows the relative luminescence signal values after adding substrates in different pH protective solutions of Example 8 of the present application, wherein A is the result of substrate Furimazine and B is the result of substrate CTZ-h.

[0158] Figure 9 shows the relative luminescence signal values after adding substrates in different pH protective solutions of Example 9 of the present application, wherein A is the result of substrate Furimazine and B is the result of substrate CTZ-h.

[0159] Figure 10 shows the relative luminescence signal values after adding substrates in different pH protective solutions of Example 10 of the present application, wherein A is the result of substrate Furimazine and B is the result of substrate CTZ-h.

[0160] Figure 11 shows the relative luminescence signal values after adding substrates in pH 6.08 protective solution of Example 11 of the present application, wherein A is the result of substrate Furimazine and B is the result of substrate CTZ-h.

[0161] Figure 12 shows the relative luminescence signal values after adding substrates in different pH protective solutions of Example 12 of the present application.

[0162] Figure 13 shows the relative luminescence signal values after adding substrates in different pH protective solutions of Example 13 of the present application, wherein A is the result of substrate Furimazine and B is the result of substrate CTZ-h.

[0163] Figure 14 shows the protective effect of the protective solution of Example 14 in the substrate mother liquor (organic phase). DETAILED DESCRIPTION

[0164] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application in combination with examples. The specific examples described herein are only used to explain the present application and do not constitute any limitation to the present application. In addition, in the following description, the description of the well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0165] Definitions

[0166] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will include the plural and vice versa.

[0167] In some embodiments, the coelenterazine referred to in the present disclosure has the following structure:

[0168] In some embodiments, exemplary coelenterazine analogs can include, but are not limited to, the following compounds:

[0169] Coelenterazine-h (CTZ-h): (2-deoxycoelenterazine or 2,8-dibenzyl-6-(4- hydroxyphenyl)imidazo[l,2-a]pyrazin-3(7H)-one);

[0170] Coelenterazine-h-h (CTZ-h-h): (dideoxycoelenterazine or 2,8-dibenzyl-6- phenylimidazo[l,2-a]pyrazin-3(7H)-one);

[0171] Furimazine (FZ): (8-benzyl-2-[(furan-2-yl)methyl]-6-phenylimidazo[l,2-a]pyrazin- 3(7H)-one);

[0172] JRW-0238: (8-benzyl-2-(furan-2-ylmethyl)-6-(3-hydroxyphenyl)imidazo[l,2- a]pyrazin-3(7H)-one);

[0173] JRW-1743: (6-(3-amino-2-fluorophenyl)-8-(2-fluorobenzyl)-2-(furan-2- ylmethyl)imidazo[l,2-a]pyrazin-3(7H)-one);

[0174] JRW-1744: (6-(3-amino-2-fluorophenyl)-8-benzyl-2-(furan-2-ylmethyl)imidazo[ 1,2-a]pyrazin-3(7H)-one;

[0175] The coelenterazine analogs have the following structures:

[0176] Other exemplary coelenterazine analogs include, for example, coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, coelenterazine-l, coelenterazine-icp, coelenterazine-v, 2-methylcoelenterazine, and the like.

[0177] In the context of the present application, C 1-12 Alkyl can include C 1-6 Alkyl, C 1-8 Alkyl, C 1-10 Alkyl, C 1-12 Alkyl, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, i-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl.

[0178] In the context of the present application, substituted C 1-12 Alkyl, the substituents can include hydroxyl, amino, cyano, oxo, C 1-6 Alkyl.

[0179] In the context of the present application, C 6-30 Aryl can include C 6-12 Aryl, C 6-18 Aryl, C 6-24 Aryl, C 6-30 Aryl, such as phenyl, naphthyl, phenanthryl, anthryl.

[0180] In the context of the present application, the heteroatom is selected from one or more of N, O, S.

[0181] As used herein, the terms "a" and "an" include plural referents unless the context clearly indicates otherwise. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0182] As used herein, the term "about" means ±20% of the numerical value of the number that it precedes. In some embodiments, the term "about" means ±10% of the numerical value of the number that it precedes. In some embodiments, the term "about" means ±5% of the numerical value of the number that it precedes.

[0183] Where numerical ranges are recited herein, any value within the range is included.

[0184] Abbreviations used in this document: DMSO: dimethylsulfoxide. DMF: N,N- Dimethylformamide. MES: 4-morpholineethanesulfonic acid (monohydrate). CDTA: cyclohexane-diamine-tetraacetic acid. EDTA: ethylene diamine tetraacetic acid. DTPA: diethylenetriamine pentaacetic acid. ATT: 6-AZA-2-THIOTHYMINE: chemical name 6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one. ATCA: chemical name 5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylic acid. DTT: Dithiothreitol. Tris: Tris(hydroxymethyl)aminomethane. PBS: Phosphate Buffer Saline. BSA: Bovine Serum Albumin. PEG: Polyethylene glycol. ProClin300: isothiazolinone, biological preservative. Triton X405: 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol solution.

[0185] The chemicals used in this document are commercially available.

[0186] Examples and figures are provided below to help understand the present invention. It should be understood, however, that these examples and figures are only intended to illustrate the present invention and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modification and change can be made without departing from the spirit of the present invention. The reagents used in the following examples are commercially available.

[0187] Example 1

[0188] Protective solution formula 1: Malonic acid 50 mM, 2-hydroxypropyl-β-cyclodextrin 60 g / L, Tergitol 15S9 200 μL / L, divided into 8 parts, adjusted to pH = 1, 2, 3, 4, 5, 6, 7 with HC1 and NaOH respectively. Add CTZ-h stock solution (methanol 10%, propylene glycol 75%, DMSO 15%, formic acid 0.2%, CTZ-h 15 mM) to the protective solution at different pH values at a ratio of 1:100 to make the final concentration 150 μM, and treat at 37℃ and 60℃ respectively for 0, 0.25, 1, 2, 3 days in the dark. The untreated substrate solution is stored at -60℃.

[0189] Residual activity test: Take 10 μL of the substrate solution treated at different pH, different temperature, and different time, and place it in a white flat-bottom 96-well luminescence plate well. Dilute luciferase (cytoplasmic expression in E. coli cells, nickel column purification, protein sequence see SEQ ID NO. 1) with buffer T43 (MES 13.67 g / L, Tris 4.24 g / L, BSA 20 g / L, 2-hydroxypropyl-β-cyclodextrin 10 g / L, PEG20000 25 g / L, TritonX405 300 μL / L, Mannitol 18 g / L, Antifoam 204 10 μL / L, pH 7.37) to 0.4 ng / ml, add 100 μL to each well, test RLU at 30s, 60s, 90s, take the RLU of 0-day treatment as 100%, calculate the residual RLU percentage after accelerated treatment at different times, and take the average of the residual RLU percentage at 30s, 60s, 90s. The experiment is repeated 1 time, and the average of the two times is taken.

[0190] Results: As shown in Table 1 and Figure 1 (A, B), the protective effect of substrate protective solution 1 at different pH values on the substrate is significantly different, and the pH value in the range of 1-5 has a significant substrate protection effect, with pH 3-4 being the best. In the case of pH 4, the 10% decay period is more than 3 days. The stability of the substrate decreases with the increase or decrease of pH value, and the effect of pH 7 is the worst, with the residual RLU being 18% at 37℃ for 6h and 0% at 60℃ for 6h.

[0191] Table 1. Protective effect of protective solution formula 1 at different pH values on the substrate

[0192] Example 2

[0193] Protective solution formula 2: Formic acid 265 mM, CDTA 1 mM, Tergitol 15S9 200 μL / L, KCl 150 mM, Thiourea 35 mM, ProClin300 1 ml / L, 5 parts each, adjusted to pH 2.12, 2.71, 3.26, 4.19, 5.25 respectively.

[0194] Substrate Furimazine or CTZ-h was dissolved in methanol to prepare a stock solution of 5 mM (methanol 10%, propylene glycol 75%, DMSO 15%), diluted to 25 μM with the above-mentioned protective solution formula 2 at 1:200, and placed at 37°C for 0 days, 0.5 days, 3 days, 5 days, and 7 days. After treatment, it was stored at -60°C for standby.

[0195] The above-mentioned frozen substrate solution was thawed, and the substrate solution was neutralized to pH 6.3 with 0.5M NaOH.

[0196] Luciferase (the sequence is shown in SEQ ID NO. 1, the same below) was diluted to 0.6 ng / mL with PBSA-9 (PBSA-9: anhydrous KH2PO4 0.2 g / L, anhydrous Na2HPO4 0.61 g / L, KCl 0.2 g / L, NaCl 8 g / L, BSA 1 g / L, PEG20000 20 g / L, TritonX405 100 μL / L, ProClin300 1 mL / L, salmon protamine (CAS: 53597-25-4, Aladdin) 300 μg / L, DTT 1 mM, pH 7.4), and 15 μL was added to each well. The luminescence signal value was measured on a bioluminescence detector, and the luminescence signal value data 40 s (CTZ-h) or 2 min (FZ) after the addition of the substrate were recorded, as shown in FIG. 2 (A, B).

[0197] Experimental results: the substrate protective solution 2 (265 mM formic acid) at pH 4.19 and lower pH has good substrate protection effect, and the remaining luminescence value is more than 40% after 7 days of accelerated treatment at 37°C.

[0198] Amino acid sequence of luciferase:

[0199] Example 3

[0200] Protective solution formula 3: acetic acid 115 mM, CDTA 1 mM, Tergitol 15S9 0.02%, KCl 150 mM, thiourea 35 mM, ProClin300 0.10%, in five parts, respectively adjusted to pH 3.2, 3.79, 4.0, 5.2, and 6.24 with NaOH.

[0201] Substrate Furimazine or CTZ-h was dissolved in methanol to prepare a stock solution of 5 mM (methanol 10%, propylene glycol 75%, DMSO 15%), diluted to 25 μM with the above-mentioned protective solution formula 3 at 1:200, and placed at 37°C for 0 days, 0.5 days, 3 days, 5 days, and 7 days. After treatment, it was stored at -60°C for standby.

[0202] The above frozen substrate solution was thawed and neutralized to pH 6.3 with 0.5M NaOH.

[0203] Luciferase was diluted with PBSA-9 to 0.6ng / mL, 15μL per well. The luminescent signal value was measured on a luminometer, and the luminescent signal value data at 40s (CTZ-h) or 2min (FZ) after substrate addition was recorded, as shown in Figure 3 (A, B).

[0204] Experimental results: Substrate protection solution 3 (115mM acetic acid) at pH 4.0 and lower pH has good substrate protection effect, and the residual luminescent value is above 25% (FZ) and 80% (CTZ-h) after 7 days of accelerated treatment at 37°C; the residual luminescent value of substrate protection solution 3 at pH 6.24 is close to 0%.

[0205] Example 4

[0206] Protection solution formula 4: propionic acid 267mM, CDTA 1mM, Tergitol 15S9 0.02%, KCl 150mM, Thiourea 35mM, ProClin300 0.10%, in five parts, respectively adjusted to pH 2.86, 3.54, 3.91, 4.24, 6.38 with NaOH.

[0207] Substrate Furimazine or CTZ-h was dissolved in methanol to prepare a 5mM stock solution (methanol 10%, propylene glycol 75%, DMSO 15%), diluted to 25μM with the above protection solution formula 4 at 1:200, and placed at 37°C for 0 days, 0.5 days, 3 days, 5 days, and 7 days. After treatment, it was stored at -60°C for standby.

[0208] The above frozen substrate solution was thawed and neutralized to pH 6.3 with 0.5M NaOH.

[0209] Luciferase was diluted with PBSA-9 to 0.6ng / mL, 15μL per well. The luminescent signal value was measured on a luminometer, and the luminescent signal value data at 40s (CTZ-h) or 2min (FZ) after substrate addition was recorded, as shown in Figure 4 (A, B).

[0210] Experimental results: Substrate protection solution 4 (267mM propionic acid) at pH 4.24 and lower pH has good substrate protection effect, and the residual luminescent value is above 10% (FZ) and 60% (CTZ-h) after 7 days of accelerated treatment at 37°C; the residual luminescent value of protection solution 4 at pH 6.38 is close to 0%.

[0211] Example 5

[0212] Protective solution formula 5: Malonic acid 50 mM, CDTA 1 mM, Tergitol 15S9 0.02%, KCl 150 mM, Thiourea 35 mM, ProClin300 0.10%, 5 replicates, respectively adjusted to pH 2.1, 2.5, 3.22, 4.92, 6.51 with NaOH.

[0213] Substrate Furimazine or CTZ-h was dissolved in methanol to prepare a stock solution of 5 mM (methanol 10%, propylene glycol 75%, DMSO 15%), diluted to 25 μM with the above protective solution formula 5 at 1:200, and placed at 37°C for 0 days, 0.5 days, 3 days, 5 days, and 7 days. After treatment, it was stored at -60°C for standby.

[0214] The above frozen substrate solution was thawed, and the substrate solution was neutralized to pH 6.3 with 0.5 M NaOH.

[0215] Luciferase was diluted to 0.6 ng / mL with PBSA-9, and 15 μL was added to each well. The luminescence signal value was measured on a bioluminescence detector, and the luminescence signal value data at 40 s (CTZ-h) or 2 min (FZ) after the addition of the substrate was recorded, as shown in FIG. 5 (A, B).

[0216] Experimental results: The substrate protective solution 5 (50 mM malonic acid) at pH 3.22 and lower pH has good substrate protection effect, and the remaining luminescence value is above 81% after 7 days of accelerated treatment at 37°C; the remaining luminescence value of the substrate protective solution 5 at pH 4.92 is above 31% (FZ) and 33% (CTZ-h), respectively, while the remaining luminescence value of the substrate protective solution 5 at pH 6.51 is close to 0%.

[0217] Example 6

[0218] Protective solution formula 6: Oxalic acid 55 mM, CDTA 1 mM, Tergitol 15S9 0.02%, KCl 150 mM, Thiourea 35 mM, ProClin300 0.10%, 5 replicates, respectively adjusted to pH 1.72, 2.27, 3.65, 4.68, 5.65 with NaOH.

[0219] Substrate Furimazine or CTZ-h was dissolved in methanol to prepare a stock solution of 5 mM (methanol 10%, propylene glycol 75%, DMSO 15%), diluted to 25 μM with the above protective solution formula 6 at 1:200, and placed at 37°C for 0 days, 0.5 days, 3 days, 5 days, and 7 days. After treatment, it was stored at -60°C for standby.

[0220] The above frozen substrate solution was thawed, and the substrate solution was neutralized to pH 6.3 with 0.5M NaOH, respectively.

[0221] Luciferase was diluted with PBSA-9 to 0.6ng / mL, 15μL per well. The luminescent signal value was measured on the bioluminescence detector, and the luminescent signal value data 40s (CTZ-h) or 2min (FZ) after the addition of the substrate was recorded, as shown in Figure 6 (A, B).

[0222] Experimental results: Substrate protection solution 6 (55mM oxalic acid) with pH 3.65 and lower pH has obvious substrate protection effect, and the residual luminescence value after 7 days of accelerated treatment at 37℃ is higher than 57%; the residual luminescence value of substrate protection solution 6 with pH 4.68 is 75% (FZ) and 15% (CTZ-h), respectively, and the residual luminescence value of substrate protection solution 6 with pH 5.65 is close to 0%.

[0223] Example 7

[0224] Protection solution formula 7: Thioacetic acid 70mM, CDTA 1mM, Tergitol 15S9 0.02%, KCl 150mM, Thiourea 35mM, ProClin300 0.10%, in quintuplicate, and adjusted to pH 2.74, 3.14, 4.04, 5.4, 7.2 with NaOH, respectively.

[0225] The substrate Furimazine or CTZ-h was dissolved in methanol to prepare a 5mM stock solution (methanol 10%, propylene glycol 75%, DMSO 15%), diluted with the above-mentioned protection solution formula 7 to 25μM at a dilution ratio of 1:200, and placed at 37℃ for 0 days, 0.5 days, 3 days, 5 days, and 7 days. After treatment, it was stored at -60℃ for standby use.

[0226] The above frozen substrate solution was thawed, and the substrate solution was neutralized to pH 6.3 with 0.5M NaOH, respectively.

[0227] Luciferase was diluted with PBSA-9 to 0.6ng / mL, 15μL per well. The luminescent signal value was measured on the bioluminescence detector, and the luminescent signal value data 40s (CTZ-h) or 2min (FZ) after the addition of the substrate was recorded, as shown in Figure 7 (A, B).

[0228] Experimental results: Substrate protection solution 7 (70mM thioacetic acid) with pH 5.4 and lower pH has obvious substrate protection effect, and the residual luminescence value after 7 days of accelerated treatment at 37℃ is higher than 48%; the residual luminescence value of substrate protection solution 7 with pH 3.14 is low in the CTZ-h test, which may be caused by the failure to neutralize to the right pH; the residual luminescence value of substrate protection solution 7 with pH 7.2 is close to 0% at this time.

[0229] Example 8

[0230] Protective solution formulation 8: Hydrochloric acid 15 mM, CDTA 1 mM, Tergitol 15S9 0.02%, KCl 150 mM, Thiourea 35 mM, ProClin300 0.10%, in 5 replicates, respectively adjusted to pH 2.18, 2.77, 3.3, 4.82, 8 with NaOH.

[0231] Substrate Furimazine or CTZ-h was dissolved in methanol to prepare a stock solution of 5 mM (methanol 10%, propylene glycol 75%, DMSO 15%), diluted to 25 μΜ with the above protective solution formulation 8 at 1:200, and placed at 37°C for 0 days, 0.5 days, 3 days, 5 days, and 7 days. After treatment, the samples were stored at -60°C for later use.

[0232] The above frozen substrate solution was thawed, and the substrate solution was neutralized to pH 6.3 with 0.5 M NaOH.

[0233] Luciferase was diluted to 0.6 ng / mL with PBSA-9, and 15 μΐ^was added to each well. The luminescence signal value was measured on a luminometer, and the luminescence signal value data at 40 s (CTZ-h) or 2 min (FZ) after the addition of the substrate was recorded, as shown in FIG. 8 (A, B).

[0234] Experimental results: The substrate protective solution 8 (15 mM hydrochloric acid) at pH 4.82 and lower pH had a significant substrate protection effect, and the remaining luminescence value after 7 days of accelerated treatment at 37°C was higher than 58%; the remaining luminescence value of the substrate protective solution 8 at pH 8.0 was close to 0% at this time.

[0235] Example 9

[0236] Protective solution formulation 9: Glycine-HCl 10 mM, CDTA 1 mM, Tergitol 15S9 0.02%, KCl 150 mM, Thiourea 35 mM, ProClin300 0.10%, in 4 replicates, respectively adjusted to pH 2.5, 2.87, 3.7, 7 with NaOH.

[0237] Substrate Furimazine or CTZ-h was dissolved in methanol to prepare a stock solution of 5 mM (methanol 10%, propylene glycol 75%, DMSO 15%), diluted to 25 μΜ with the above protective solution formulation 9 at 1:200, and placed at 37°C for 0 days, 0.5 days, 3 days, 5 days, and 7 days. After treatment, the samples were stored at -60°C for later use.

[0238] The above frozen substrate solution was thawed, and the substrate solution was neutralized to pH 6.3 with 0.5M NaOH, respectively.

[0239] Luciferase was diluted with PBSA-9 to 0.6ng / mL, 15μL per well. The luminescent signal value was measured on the bioluminescence detector, and the luminescent signal value data at 40s (CTZ-h) or 2min (FZ) after the addition of the substrate was recorded, as shown in Figure 9 (A, B).

[0240] Experimental results: Substrate protection solution 9 (10mM glycine-HCl) at pH 3.7 and lower pH has obvious substrate protection effect, and the remaining luminescence values after 7 days of accelerated treatment at 37℃ are all higher than 90%, and the remaining luminescence values of different treatment days have small fluctuation range; the remaining luminescence value of substrate protection solution 9 at pH 7.0 is close to 0%.

[0241] Example 10

[0242] Protection solution formula 10: acetylthioacetic acid 37.3mM, CDTA 1mM, Tergitol 15S9 0.02%, KCl 150mM, Thiourea 35mM, ProClin300 0.10%, in five parts, respectively adjusted to pH 2.38, 2.66, 3.23, 4.51, 11 with NaOH.

[0243] The substrate Furimazine or CTZ-h was dissolved in methanol to prepare a 5mM stock solution (methanol 10%, propylene glycol 75%, DMSO 15%), diluted with the above-mentioned protection solution formula 10 to 25μM at a dilution ratio of 1:200, and placed at 37℃ for 0 days, 0.5 days, 3 days, 5 days, and 7 days. After treatment, it was stored at -60℃ for standby use.

[0244] The above frozen substrate solution was thawed, and the substrate solution was neutralized to pH 6.3 with 0.5M NaOH, respectively.

[0245] Luciferase was diluted with PBSA-9 to 0.6ng / mL, 15μL per well. The luminescent signal value was measured on the bioluminescence detector, and the luminescent signal value data at 40s (CTZ-h) or 2min (FZ) after the addition of the substrate was recorded, as shown in Figure 10 (A, B).

[0246] Experimental results: Substrate protection solution 10 (37.3mM acetylthioacetic acid) at pH 4.51 and lower pH has obvious substrate protection effect, and the remaining luminescence values after 7 days of accelerated treatment at 37℃ are all between 10% and 71%, and have obvious pH dependence, and the remaining luminescence value is the highest in the appropriate pH range such as 2-4; the remaining luminescence value of substrate protection solution 10 at pH 11 is close to 0%.

[0247] Example 11

[0248] Protective solution formula 11: MES 10mM, CDTA 1mM, Tergitol 15S9 0.02%, KCl 150mM, Thiourea 35mM, ProClin300 0.10%, adjust pH to 6.08 with NaOH.

[0249] Substrate Furimazine or CTZ-h was dissolved in methanol to make a 5mM stock solution (methanol 10%, propylene glycol 75%, DMSO 15%), diluted to 25μM with the above protective solution formula 11 at 1:200, and placed at 37℃ for 0 days, 0.5 days, 3 days, 5 days, 7 days, and then stored at -60℃ after treatment. Nanoglo is the matched substrate of Nano-Glo® Luciferase Assay System (N1110, Promega), diluted with Assay buffer at 1:100 as control.

[0250] Luciferase was diluted with PBSA-9 to 0.6ng / mL, and 15μL was added to each well. The luminescence signal value was measured on a luminometer, and the data of luminescence signal value at 40s (CTZ-h) or 2min (FZ) after substrate addition was recorded, as shown in Figure 11 (A, B).

[0251] Experimental results: the protective effect of substrate protective solution 11 (10mM MES buffer) at pH 6.08 was poor.

[0252] Example 12

[0253] Protective solution formula 12: Glycine-HCl 10mM, CDTA 1mM, Tergitol 15S9 0.02%, KCl 150mM, Thiourea 35mM, ProClin300 0.10%, adjusted to pH 2.43, 2.79, 3.25, 3.71, 4.03, 4.45, 5.94, 6.5, 8.3 respectively with NaOH in 9 aliquots.

[0254] Substrate CTZ-h was dissolved in methanol to make a 5mM stock solution (methanol 10%, propylene glycol 75%, DMSO 15%), diluted to 25μM with the above protective solution formula 12 at 1:200, and placed at 37℃ for 0 days, 0.1 days, 2 days, 3 days, 5 days, 7 days, 10 days, 14 days, and then stored at -60℃ after treatment.

[0255] The above frozen substrate solution was thawed, and the substrate solution was neutralized to pH 6.3 with 0.5M NaOH.

[0256] Luciferase was diluted with PBSA-9 to 0.6 ng / mL, 15 μL per well. The luminescent signal value was measured on the bioluminescence detector, and the luminescent signal value data at 40 s (CTZ-h) after the substrate was added was recorded, as shown in Fig. 12.

[0257] Results: The substrate protection solution 12 (10 mM glycine-HCl) at pH 4.03 and lower pH can significantly maintain the stability of CTZ-h, and the luminescent value remained more than 40% after 14 days of treatment at 37°C; the substrate protection solution 12 at pH 4.45 also had obvious protective effect, and the remaining luminescent value was 15.2% after 14 days of treatment at 37°C, while the remaining luminescent value of the substrate protection solution 12 at pH 5.94, pH 6.5, and pH 8.3 was close to 0%. This indicates that low pH (pH≤5) is beneficial to the stability of coelenterazine analogs.

[0258] Example 13

[0259] The protection solution formula 13: glycine-HCl 10 mM, CDTA 1 mM, Tergitol 15S9 0.02%, KCl 150 mM, thiourea 35 mM, ProClin300 0.10%, prepared with heavy water (deuterium oxide), 5 replicates, respectively adjusted to pH 2.02, 2.59, 3.19, 3.59, and 9.0 with NaOH.

[0260] The substrate Furimazine or CTZ-h was dissolved in methanol to prepare a 5 mM stock solution (methanol 10%, propylene glycol 75%, DMSO 15%), diluted to 25 μM with the above-mentioned protection solution formula 13 at 1:200, and placed at 37°C for 0 days, 0.5 days, 3 days, 5 days, and 7 days, and then stored at -60°C after treatment.

[0261] The above-mentioned frozen substrate solution was thawed, and the substrate solution was neutralized to pH 6.3 with 0.5 M NaOH.

[0262] Luciferase was diluted with PBSA-9 to 0.6 ng / mL, 15 μL per well. The luminescent signal value was measured on the bioluminescence detector, and the luminescent signal value data at 40 s (CTZ-h) or 2 min (FZ) after the substrate was added was recorded, as shown in Fig. 13 (A, B).

[0263] The experimental results: the substrate protection solution 13 (10 mM glycine-HCL, heavy water preparation) with pH 3.59 and lower pH has obvious substrate protection effect, the remaining luminescence value after 7 days of accelerated treatment at 37°C is more than 75%, and the remaining luminescence value of the substrate protection solution 13 with pH 9 is close to 0% after 0.5 days. Similar to the results of Example 9, it shows that the heavy water prepared substrate protection solution also has good substrate protection effect in the appropriate pH range.

[0264] Example 14. Protection effect of organic acid in substrate mother liquor (organic phase)

[0265] Dissolve CTZ-h with a small amount of DMSO, add methanol to 10%, propylene glycol to 75%, and DMSO to 15% to make the final concentration of the substrate mother liquor 1.25 mM, and then add organic acids to the concentrations in Table 2 below, and set up a control without additives (methanol 10%, propylene glycol 75%, DMSO 15%, CTZ-h 1.25 mM). Mix well to ensure that the substrate protection solution is completely dissolved, and then continue to divide the substrate mother liquor with added protection solution into several aliquots, and treat them in a 60°C incubator for different times. Take out one aliquot on the 0th day (immediately after preparation and frozen at -20°C), 1st day, 3rd day, 5.5th day, and 7.5th day, and freeze them at -20°C for later use. Before use, dilute them with water phase substrate diluent (MES 100 mM, CDTA 1 mM, Tergitol 15S9 0.02%, KCl 150 mM, Thiourea 35 mM, ProClin300 0.10%, pH 6.37) at 1:100, and the final concentration of the substrate is 12.5 μM, and then add them to white flat-bottomed round-hole 96-well plates.

[0266] Dilute luciferase with PBSA-9 to 0.6 ng / mL, and add 15 μL per well. Place it in a bioluminescence detector to measure the luminescence signal value, and record the luminescence signal value data 40 s (CTZ-h) after the addition of the substrate.

[0267] Calculate the relative remaining luminescence value percentage based on the group with the highest remaining luminescence value after different days of treatment as 100%, and plot it to detect the remaining activity after different times of 60°C treatment, and verify the substrate stabilization effect of the protection solution, as shown in Figure 14.

[0268] Results: The tested organic acid protection solutions all have a certain stabilizing effect, and the residual activity is higher than that without protection solution (3 days). After 1 day of treatment at 60°C, the residual luminescence value of the organic acid protection solution group is still above 50%, and is better than 6-aza-2-thiothymidine, and most of them are above 70%. Among them, the protection effect of the high concentration group of thioacetic acid is particularly significant, which is significantly better than 6-aza-2-thiothymidine. After 7.5 days of treatment at 60°C, the residual luminescence value is still 63%, while the residual luminescence value of the 6-aza-2-thiothymidine treatment group is only 1.1% at this time. The control without protection solution has a residual luminescence value of only 1.6% after 3 days of treatment at 60°C. The results are shown in Table 2.

[0269] Table 2. Stabilizing effect of organic acid protection solution

[0270] Example 15. Protection effect of organic acids in substrate mother liquor (organic phase)

[0271] Experimental method:

[0272] Preparation of substrate mother liquor: Dissolve Furimazine with a small amount of DMSO, add methanol to 10%, propylene glycol to 65%, and DMSO to 25% to make the final concentration of Furimazine mother liquor 10 mM; Preparation of organic acid mother liquor: Dissolve or dilute organic acid with a small amount of DMSO, add methanol to 10%, propylene glycol to 65%, and DMSO to 25%; Preparation of organic acid substrate mother liquor: Mix the substrate mother liquor and the organic acid mother liquor according to the volume ratio of 1:4, so that the final concentration of Furimazine in the system is 2 mM, the solvent is methanol 10%, propylene glycol 65%, and DMSO 25%. The specific organic acid concentration is shown in Table 3 below.

[0273] Accelerated stability treatment: The above prepared organic acid substrate mother liquor is divided into 6 equal parts, and is placed in a 37°C incubator for 3 days, 7 days, 14 days, 21 days, 32 days, respectively, and the 0 day control is stored at -20°C.

[0274] Vitality test: The organic acid substrate stock solution was diluted with substrate working solution Oprop3.3 (malonic acid 12 mM, EDTA 1 mM, Tergitol 15S9 0.02%, KCl 150 mM, Thiourea 35 mM, ProClin300 0.10%, pH 3.0) at 1:100 before use, the final concentration of substrate was 20 μΜ, 50 μL was added to white flat-bottomed round hole 96-well plates. Luciferase was diluted with PBSA-16 to 0.6 ng / mL (PBSA-16: KH2PO4 anhydrous 1.2 g / L, Na2HPO4 anhydrous 3.66 g / L, MES 7.81 g / L, KCl 0.2 g / L, NaCl 8 g / L, BSA 10 g / L, PEG20000 20 g / L, TritonX405 100 μL / L, ProClin300 1 mL / L, DTT 1 mM, pH 8.0), 30 μL was added to each well, and it was mixed gently. The luminescence signal value was measured on a bioluminescence detector, and the luminescence signal value data 5 min after the enzyme was added was recorded.

[0275] The luminescence value of the substrate frozen at -20 °C immediately after preparation with the substrate stock solution was 100%, and the residual activity after treatment at 37 °C for different times was detected to verify the substrate stabilization effect of the protective solution (Table 3).

[0276] Results: Among the organic acid protective solutions, thioacetic acid has very good protective effect, and can stabilize Furimazine at 37 °C for at least 21 days (activity > 90%), with a half-life of about 35.6 days (37 °C); followed by DL-thioctic acid, with a half-life of about 20.1 days (37 °C); in addition, the half-lives of formic acid, acetic acid, n-butyric acid, n-valeric acid, n-hexanoic acid, 2-methylhexanoic acid, propylvaleric acid, trans-2-decenoic acid, n-decanoic acid, dodecanedioic acid, and phthalic acid all exceed 7 days (37 °C), and the protective effects of the above protective solutions are significantly better than those of 6-aza-2-thiothymine (half-life about 37 °C 4.9 days) and ATCA (half-life about 37 °C 6.4 days).

[0277] Table 3. Protective effect of organic acid protective solution

[0278] Example 16. Water-phase substrate protective solution additive-antioxidant 1

[0279] Formulation of aqueous phase protection solution formula, the basic formula is: malonic acid 12 mM, preservative ProClin300 1 mL / L, Tergitol 15S9 200 μL / L, methanol 37.5 mL / L, glycerol 100 mL / L, DMSO 24 mL / L, D-sorbitol 40 g / L, pH = 3.0, different formulations add different antioxidants (see Table 4). 15 mM CTZ-h stock solution (DMSO:methanol = 1:3) is diluted to 75 μM with the aqueous phase substrate protection solution in Table 4, each formula is divided into 4 tubes, respectively, at 37°C, avoid light treatment 0, 1, 3, 5 days, untreated at 2-4°C, avoid light storage.

[0280] Vitality test: add the aqueous phase protection solution containing the additives in Table 4 (37°C different treatment days) to the white flat bottom round hole 96 hole plate, add 15-71 μL per hole (the specific volume of aqueous phase substrate protection solution added is determined after the enzyme diluent and substrate solution are mixed and the pH is kept at 6.5±0.2, the volume of aqueous phase substrate protection solution with the same antioxidant additive is kept fixed); luciferase is diluted with P26 to 0.6 ng / mL (P26: Tris 2.42 g / L, MES 7.81 g / L, KCl 0.2 g / L, BSA 15 g / L, PEG20000 30 g / L, TritonX405 100 μL / L, ProClin300 1 mL / L, DTT 1 mM, pH 8.0), 40 μl per hole, mix gently. Immediately place on the bioluminescence detector to measure the luminescence signal value, record the RLU data at 30s, 60s, 90s after enzyme addition, take the average value. Then divide the average RLU of the substrate solution through 37°C different treatment days of the test hole by the RLU of the 0 day control hole respectively, to obtain the residual RLU percentage data. The results are shown in Table 4.

[0281] Results: The residual RLU of the aqueous phase protection solution of the thiourea, N,N'-dimethylthiourea, glucose added group is the highest, and the protection effect is the best; the half-life of the aqueous phase substrate solution formula of tris(2-carboxyethyl) phosphine hydrochloride, thio glycolic acid, DL-thioctic acid, 6-aza-2-thiothymine added group is also more than 5 days; the protection effect of the remaining antioxidant added groups such as Z-2-(2-amino-4-thiazolyl)-2-hydroxy imino acetic acid, thioacetic acid, N-acetyl L-cysteine is also better, and the protection effect is significantly stronger than that of the control group without antioxidant (half-life 1 day), see Table 4 for details.

[0282] Conclusion: In addition to the acidic pH in the aqueous phase substrate protection solution, the addition of antioxidants is also crucial, especially thiourea, N,N'-dimethylthiourea, glucose, etc. can significantly improve the stability of the substrate and prolong the half-life of the substrate.

[0283] Table 4. Water phase substrate protection solution formula with different antioxidants and its remaining RLU percentage

[0284] Example 17. Water phase substrate protection solution additives (antioxidants) 2

[0285] Water phase protection solution formula was prepared, the base formula was: malonic acid 12 mM; ProClin300 1 mL / L, Tergitol 15S9 200 μL / L, methanol 75 mL / L, glycerol 200 mL / L, DMSO 48 mL / L, D-sorbitol 80 g / L, pH 3.0, and different antioxidants were added to different formulas (see Table 5). The CTZ-h stock solution (DMSO:methanol = 1:3) was diluted to 75 μM with the water phase substrate protection solution in Table 5, and each formula was divided into 8 tubes, respectively, and treated at 37°C in the dark for 0, 1, 3, 5 days, and the untreated was stored at 2-4°C in the dark.

[0286] The water phase protection solution containing the additives in Table 5 (37°C for different treatment days) was added to a white flat-bottom round hole 96-well plate, 49-53 μL per well (the specific volume of water phase substrate protection solution added was determined after the enzyme diluent and substrate solution were mixed and the pH was maintained at 6.5±0.2, and the volume of water phase substrate protection solution with the same antioxidant additive was fixed); the luciferase was diluted to 0.6 ng / mL with P26, and 30 μL was added to each well, and mixed gently. Immediately place on the bioluminescence detector to measure the luminescence signal value, record the RLU data at 30s, 60s, 90s after the enzyme is added, and take the average value. Each test was repeated twice, and the average value was taken. Then the average RLU of the substrate solution through the test hole for different treatment days at 37°C was divided by the RLU of the 0-day control hole, respectively, to obtain the remaining RLU percentage data. The results are shown in Table 5.

[0287] Results: The protection effect of water phase substrate protection solution with thiourea, tetramethyl thiourea, N-ethyl thiourea added was significantly better than that without antioxidant, and the half-life was more than 5 days. The protection effect of DTT and ascorbic acid added groups was also significantly better than that without addition. The specific results are shown in Table 5.

[0288] Conclusion: In addition to the acidic pH in the water phase substrate protection solution, the addition of antioxidants is also crucial, especially thiourea, tetramethyl thiourea, N-ethyl thiourea, etc., which can significantly improve the stability of the substrate and prolong the half-life of the substrate.

[0289] Table 5. Water phase substrate protection solution formula with different antioxidants and its remaining RLU percentage

[0290] Example 18. Water phase substrate protection solution additives (antioxidants) 3

[0291] Formulate the aqueous phase substrate protection solution, the basic formula is: malonic acid 12 mM, 2-hydroxypropyl β-cyclodextrin 60 g / L, Tergitol 15s9 400 μL / L, add different antioxidants to the specified concentration (see Table 6), adjust the pH to 3.0, then add the substrate CTZ-h to a final concentration of 150 μM, and store after dispensing in the dark. Respectively, at 37°C, avoid light treatment for 0, 1, 3, 5 days, and untreated at 4°C in the dark.

[0292] Vitality test: first add 50 μL of the aqueous phase substrate solution treated at 37°C for different days to a white flat-bottomed round-hole 96-well plate; add 40 μL of P26 containing 0.6 ng / mL luciferase according to the test sequence, mix gently, immediately place in a bioluminescence detector to measure the luminescence signal value, record the RLU data at 30 s, 60 s, 90 s after enzyme addition, and take the average value; each test is repeated twice, and the average value is taken. Then the average RLU of the substrate solution treated in the test wells for different days at 37°C is divided by the RLU of the 0-day control well, and the residual RLU percentage data is obtained. The results are shown in Table 6.

[0293] Results: In the basic aqueous phase substrate protection solution described in this example, the antioxidants sodium iodide and thiourea analogs (thiourea, amidine thiourea) have good substrate protection effect, and the 10% decay period is more than 10 days, which is significantly better than the control without antioxidant (10% decay period 1.6 days), in addition, oxalic acid and acetaldehyde also have good protection effect on the substrate, and the 10% decay period is significantly longer than the control without antioxidant.

[0294] Compared with Example 18, there are differences in the basic aqueous phase protection solution and the difference in substrate concentration, the half-life of the control without antioxidant in Example 18 is more than 5 days, while the half-life of the control without antioxidant in Example 17 is only 0.1 day, which shows that the substrate concentration and the basic aqueous phase protection solution formula also significantly affect the substrate protection effect.

[0295] Table 6. Formulation of aqueous phase substrate protection solution with different antioxidants and residual RLU percentage

[0296] Example 19. Additives (antioxidants) for aqueous phase substrate protection solution

[0297] Formulation of thiocyanate-free aqueous substrate protection solution: malonic acid 23 mM, 2-hydroxypropyl-β-cyclodextrin 60 g / L, Tergitol 15S9 200 μL / L, divided into 17 parts, and added with thiocyanate and its analogues in Table 7 to 35 mM or saturation, and adjusted to pH 3.0, wherein the control is not added. Add CTZ-h stock solution (methanol 10%, propylene glycol 75%, DMSO 15%, formic acid 0.2%, CTZ-h 15 mM) to the aqueous substrate protection solution containing different thiocyanate analogues at a ratio of 1:100 to make the final concentration 150 μM, divided into 4 parts, and treated at 60°C in the dark for 0, 1, 3, and 5 days, and the untreated substrate solution is stored at 4°C in the dark.

[0298] Residual activity test: take 50 μL of the aqueous substrate protection solution containing different thiocyanate analogues and place it in a white flat-bottom 96-well luminescence plate well, dilute the luciferase with buffer T48A (MES 5.47 g / L, Tris 1.7 g / L, BSA 20 g / L, 2-hydroxypropyl-β-cyclodextrin 10 g / L, PEG2000 25 g / L, Triton X405 300 μL / L, mannitol 18 g / L, Antifoam 204 10 μL / L, ethanol 1.25 ml / L, pH 7.5) to 0.4 ng / ml, add 100 μL per well, test RLU at 30 s, 60 s, and 90 s, take the RLU at 0 day of treatment as 100%, calculate the residual RLU percentage after accelerated treatment for different times, and take the average of the residual RLU percentages at 30 s, 60 s, and 90 s. The experiment is repeated 1 time, and the average of the two times is taken.

[0299] Results: As shown in Table 7, compared with the control without addition, all thiocyanate analogues tested in this example can significantly improve the stability of the substrate, and the half-life at 60°C is more than 3 days, indicating that the addition of thiocyanate analogues as antioxidants can further improve the stability of the substrate in the aqueous phase under acidic pH conditions.

[0300] Table 7. Formulation of aqueous substrate protection solution with different antioxidants and its residual RLU percentage

[0301] Note: The concentration of the formulations lower than 35 mM is about the saturation concentration.

[0302] Example 20. Additives in aqueous substrate protection solution-metal ion chelating agent

[0303] The series of aqueous substrate protection solution formulations shown in Table 8 were prepared by adding 1.2 mg / L of FeCl3 to the base formulation, and different metal ion chelating agents were added to different formulations. The 15 mM CTZ-h stock solution (DMSO:methanol = 1:3) was diluted to 75 μM with the aqueous substrate protection solutions in Table 8, and each formulation was divided into 8 tubes and treated at 37°C in the dark for 0, 1, 2, 3, 5, 7 days, respectively, and the untreated samples were stored at 2-4°C in the dark.

[0304] Vitality test: R1 in Table 8 is the enzyme dilution P26, and R2 is the aqueous substrate solution. The luciferase was diluted to 0.6 ng / mL (P26: Tris 2.42 g / L, MES 7.81 g / L, KCl 0.2 g / L, BSA 15 g / L, PEG20000 30 g / L, TritonX405 100 μL / L, ProClin300 1 mL / L, DTT 1 mM, pH 8.0) with P26, 40 μL was added to each well, and the aqueous substrate solution R2 was added to the white flat-bottomed round-hole 96-well plate according to the R1:R2 ratio in Table 8, and mixed gently. Immediately place it in a bioluminescence detector to measure the luminescence signal value, record the RLU data at 30s, 60s, 90s after enzyme addition, and take the average value. Then divide the average RLU of the substrate solution treated for different days by the RLU of the 0-day control well to obtain the residual RLU percentage data, repeat the test once, and take the average value. The results are shown in Table 8.

[0305] Results: The aqueous protection solution formulations with metal ion chelating agents such as potassium gluconate, DTPA, EDDHA, HEDP, etc. have stronger stability than the control without chelating agents, and can significantly reverse the instability of the substrate caused by FeCl3.

[0306] Table 8. Aqueous protection solution formulations and residual RLU percentage

[0307] Example 21. Effect of substrate concentration on substrate stability

[0308] The substrate protection solution base formulation OB9-5A (malonic acid 12 mM, 2-hydroxypropyl β-cyclodextrin 60 g / L, Tergitol 15s9 400 μL / L, thiourea 35 mM, pH 3.0) was prepared, and the substrate CTZ-h was added to make its final concentration 5, 15, 30, 90, 180, 360 μM, and divided into 6 tubes, respectively, and placed at 37°C in the dark for 0, 1, 3, 5, 7, 9 days.

[0309] Activity test: luciferase was diluted with P26 to 0.6 ng / mL, 40 μL per well; different substrate concentrations, different treatment days of OB9-5A substrate solution 50 μL per well, using bioluminescence detector to detect the RLU of the mixture of substrate and enzyme after 30, 60, 90 seconds, taking the average value of RLU; repeated twice to take the average value. Taking 0 day RLU as 100%, calculate the relative RLU% of the substrate of different treatment days.

[0310] Results: As shown in Table 9, at a concentration of 5 μM or less, the substrate has poor stability, and the 10% decay period is less than 3 days, at a concentration of 15 μM and above, such as 15 μM to 360 μM, the substrate stability is significantly enhanced, and the 10% decay period is more than 3 days, the higher the substrate concentration, the better the stability.

[0311] Conclusion: The substrate stability is enhanced with the increase of substrate concentration; when the substrate concentration is ≥15 μM, the 10% decay period is more than 3 days under acceleration at 37°C.

[0312] Increasing the substrate concentration actually makes the substrate concentration reach or exceed the saturation concentration required for enzymatic reaction, even if the substrate concentration decreases with the extension of 37°C treatment time, which is not obvious in RLU value, so higher substrate concentration can show better substrate stability.

[0313] Table 9. Stability difference of different substrate concentrations after accelerated treatment (percentage of remaining RLU)

[0314] Example 22. Effect evaluation of substrate anti-precipitation agent

[0315] From the results of Example 21, substrate concentrations higher than 5 μM significantly improve the stability of the substrate, but the core skeleton of coelenterazine analogues is hydrophobic, and most of them are not water-soluble. In the aqueous substrate protection solution, increasing the substrate concentration will bring the risk of substrate precipitation, thereby reducing the reliability of the detection results. In order to solve the problem of substrate precipitation, the present application tries to use various organic solvents, anti-precipitation agents and inclusion agents to solve the problem of substrate precipitation.

[0316] Prepare the substrate protection solution basic formula OB9-5B (malonic acid 12 mM, Tergitol 15s9 400 μL / L, thiourea 35 mM, add different concentrations of different anti-precipitation agents (see Table 10), adjust pH to 3.0, add CTZ-h to 150 μM, treat at 37°C for 5 days, observe the precipitation after 5 days, take the precipitation level of the substrate protection solution without anti-precipitation agent as “-----”, and define “+++++” as completely no visible precipitation, and select the formula with better anti-precipitation level, detect the relative activity ratio before and after 37°C treatment for 5 days, and evaluate whether the anti-precipitation agent has adverse effects on the stability of the substrate.

[0317] Activity test method: 50 μL of the substrate to be tested was added to the wells of a 96-well white flat-bottom plate, luciferase was diluted to 0.6 ng / mL with P26, and 40 μL was added to each well; the RLUs at 30, 60, 90 seconds after mixing the substrate and enzyme were detected using a bioluminescence detector, and the average value of the RLUs was taken; the average value was taken twice. Taking the RLUs at day 0 as 100%, the relative RLU% of the substrate at different treatment days was calculated. The results are shown in Table 10.

[0318] Results:

[0319] 1. ≥30% ethanol, ≥30% DMSO, ≥30% methanol, ≥30% propylene glycol, ≥15% DMF, ≥15% (DMSO + glycerol + sorbitol), ≥15% (ethanol + propylene glycol + sorbitol), ≥7.5% (ethanol + DMSO + glycerol + sorbitol), ≥25 g / L sulfobutyl-β-cyclodextrin sodium salt, ≥25 g / L methyl β-cyclodextrin, ≥25 g / L 2-hydroxypropyl β-cyclodextrin, ≥50 g / L (2-hydroxyethyl)-β-cyclodextrin, ≥50 g / L carboxymethyl-β-cyclodextrin sodium salt, ≥50 g / L 2-hydroxypropyl α-cyclodextrin, ≥100 g / L 2-hydroxypropyl γ-cyclodextrin, all of which can make the substrate dissolution efficiency higher than 50%.

[0320] 2. α-cyclodextrin and γ-cyclodextrin have low water solubility and poor anti-precipitation effect, while cyclodextrins with higher water solubility can significantly improve the substrate dissolution efficiency, especially sulfobutyl-β-cyclodextrin sodium salt, methyl β-cyclodextrin, and 2-hydroxypropyl β-cyclodextrin.

[0321] 3. After 5 days of accelerated treatment at 37°C, the relative RLU of the substrate preservation solution with various substrate anti-precipitation agents was ≥62% (except for carboxymethyl-β-cyclodextrin sodium salt, which was 39%).

[0322] Conclusion: The addition of organic solvents at 15% or more (such as ethanol, DMSO, DMF) and / or cyclodextrin derivatives with better water solubility at a concentration of 12.5 g / L or more in the aqueous substrate protection solution can significantly improve the substrate anti-precipitation effect and maintain good accelerated stability at 37°C.

[0323] Table 10. Effect of different anti-precipitation agents and influence on stability

[0324] Note: 1, 2, and 3 are added in equal proportions, and the percentage concentration is the volume ratio of each substance. CK- is the supernatant after centrifugation without adding anti-precipitation agents, and CK+ is the non-centrifuged substrate suspension without adding anti-precipitation agents.

[0325] Example 23. Long-term stability of CTZ-h in aqueous substrate protection solution

[0326] CTZ-h was diluted to 25 μΜ in Oproll 3 (malonic acid 20 mM, EDTA 1 mM, Tergitol 15S9 0.02%, KCI 150 mM, Thiourea 35 mM, ProClin300 0.10%, pH 3.0) and divided into 20 tubes of 150 μΐ^each, and stored in the dark at 4-8°C. One tube was taken out every other day and stored at -80°C. After all the tubes were processed, the stored tubes were thawed and tested.

[0327] Test method: Luciferase was diluted to 0.4 ng / mL with P26, 30 μΐ^was added to each well, and 50 μΐ^of the substrate solution treated for different times at 4-8°C was added to each well. The RLU was immediately tested, and the average of three measurements was taken. The RLU of the 0-day treatment was taken as 100%, and the remaining RLU of the different treatment days was calculated. The results are shown in Table 11.

[0328] Table 11. Long-term stability of 25 μΜ CTZ-h in Oproll 3 protection solution

[0329] Results: At 4-8°C, the remaining luminescence of CTZ-h in the pH 3.0 aqueous substrate protection solution Oproll 3 was still more than 90% after 3 months.

[0330] Conclusion: The acidic substrate protection solution Oproll 3 can stabilize the coelenterazine substrate CTZ-h, and meet most application scenarios. In combination with the results of Example 20, the substrate concentration used in this experiment was 25 μΜ, which was relatively low. When the substrate concentration is increased to 90-180 μΜ, the stability can be further improved.

[0331] Example 24. Long-term stability of CTZ-h in organic phase at 4°C

[0332] CTZ-h was dissolved in a small amount of DMSO and prepared into an organic phase stock solution of 2.5 mM. The solvent formula of the organic phase stock solution was: ethanol 25%, propylene glycol 62.5%, thioacetic acid 2% (-263 mM), DMSO 12.5%. It was divided into 10 tubes and stored in the dark at 4-8°C. One tube was taken out every other day and stored at -20°C. After all the tubes were taken out, the activity was tested.

[0333] Test method: The luciferase mutant was diluted to 0.4 ng / mL with P26, 40 μL was added to each well, and the substrate mother liquor treated for different times at 4-8 DEG C was diluted to 25 μM with OB9-5A, 50 μL was added to each well, and the RLU was tested for 30 s and 60 s, the average was taken, each treatment was measured twice and then the average was taken, the RLU of 0 days (prepared directly and then frozen at-20 DEG C) was taken as 100%, and the residual RLU of different treatment months was calculated. The results are shown in Table 12.

[0334] Results: In the organic phase substrate mother liquor containing thioacetic acid, the 10% decay period of CTZ-h is >12 months.

[0335] Conclusion: The long-term stability of the coelenterazine substrate in the thioacetic acid organic phase substrate mother liquor is more than 12 months, which meets the vast majority of application scenarios. The RLU of the substrate treated for different times fluctuates, which may be related to the container tightness, solvent volatility, light strictness and the like, but still has overall excellent stability.

[0336] Table 12. 4 DEG C long-term stability of CTZ-h in organic phase protection solution

[0337] The coelenterazine and analog protection solution provided by the present application greatly improves the stability of coelenterazine and analogs, and has easy usability.

[0338] The technical scheme of the present application is not limited to the limitation of the above specific embodiments, and any technical modification made according to the technical scheme of the present application falls within the protection scope of the present application.

Claims

1. A composition characterized in that, The composition comprises: coelenterazine and / or an analogue thereof; and an aqueous phase protective solution, wherein the coelenterazine and / or an analogue thereof is dissolved in the aqueous phase, wherein the aqueous phase protective solution has a pH of ≤ 5.5, preferably 1-5, more preferably 2-5.

2. The composition of claim 1, wherein, The aqueous phase protective solution comprises a buffer of organic acid and / or inorganic acid, non-buffer.

3. The composition of claim 1, wherein, The coelenterazine analogue includes one or more of Furimazine, coelenterazine-h, coelenterazine-h-h, coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, coelenterazine-I, coelenterazine-icp, coelenterazine-v, 2-methylcoelenterazine, JRW-0238, JRW-1743, and JRW-1744.

4. The composition of claim 1, wherein, The concentration of the coelenterazine and / or an analogue thereof is 1 µM-10000 µM, preferably 5 µM-5000 µM, more preferably 15 µM-1500 µM, further more preferably 30 µM-600 µM.

5. The composition of claim 2, wherein The organic acid includes one or more of carboxylic acid (R-COOH), sulfonic acid (R-SO3H), thioacid (R-COSH), squaric acid, wherein R is C 1-12 alkyl or C 6-30 aryl; Preferably, the organic acid comprises one or more of a C 1-12 carboxylic acid, C 2-12 dicarboxylic acid, a C 1-12 carboxylic acid, an amino C 1-12 carboxylic acid, C 1-12 alkylsulfonic acid, C 6-30 aryl sulfonic acid; and / or, The inorganic acid includes one or more of hydrochloric acid, sulfuric acid, sulfurous acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, boric acid.

6. The composition of claim 5, wherein, The C 1-12 carboxylic acids include one or more of formic acid, acetic acid, propionic acid, isopropionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, 2-methylcaproic acid, valproic acid, 2-octynoic acid, elaidic acid, n-decanoic acid, (2-benzimidazolylthio)acetic acid; and / or, The C 2-12 The dicarboxylic acid includes one or more of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, dodecanedioic acid, phthalic acid, citric acid; and / or, said thio C 1-12 carboxylic acids include one or more of thioacetic acid, acetylthioacetic acid, thiopropionic acid, thio butyric acid, thiovaleric acid, thiohexanoic acid, DL- thioctic acid, thiobenzoic acid; and / or, said amino C 1-12 carboxylic acids include one or more of glycine, glutamic acid, aspartic acid; and / or, The C 1-12 Alkylsulfonic acids include one or more of methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, 4-morpholineethanesulfonic acid; and / or, The C 6-30 Arylsulfonic acids include one or more of benzene sulfonic acid, xylene sulfonic acid.

7. The composition according to any one of claims 2-6, characterized in that, The concentration of the organic acid and / or inorganic acid in the aqueous phase protective solution is 0.01%-20% or 0.1 mM-5300 mM.

8. The composition of claim 1, wherein, The composition further comprises one or more of an antioxidant, a metal ion chelator, a surfactant, a biological preservative, an anti-precipitant.

9. The composition of claim 8, wherein, The composition comprises one or more of an antioxidant.

10. The composition of claim 9, wherein, The antioxidant includes one or more of thiourea or thiourea analogues, DL thioctic acid, iodide, thioacetic acid, dithiothreitol, ascorbic acid, methionine, thioglycolic acid, tris(2-carboxyethyl)phosphine hydrochloride, glucose, 6-aza-2-thiothymine, Z-2(2-amino-4-thiazolyl)2-hydroxyiminoacetic acid.

11. The composition of claim 10, wherein, The thiourea or thiourea analog has a structure as shown in the following general formula 1: wherein R1, R2, R3, R4are each independently selected from the group consisting of hydrogen, amino, substituted or unsubstituted C 1-12 alkyl, C 1-12 alkyl-C 6-30 aryl, C 1-12 alkyl-C 6-30 heteroaryl, C 3-12 cycloalkyl, C 6-30 aryl, C 6-30 heteroaryl, carboxylic, imino, hydroxyl, oxyl, amidino; wherein R2 and R3 form a ring or not with the adjacent atoms.

12. The composition of claim 9, wherein, The antioxidant is thiourea.

13. The composition of claim 8, wherein, The composition further comprises one or more of a metal ion chelator; preferably, the metal ion chelator includes one or more of potassium gluconate, DTPA, EDDHA, HEDP.

14. The composition of claim 8, wherein, The composition further comprises one or more of an anti-precipitant; preferably, the anti-precipitant includes one or more of DMSO, ethanol, DMF, cyclodextrin, methanol, propylene glycol.

15. A composition characterized in that, The composition comprises: coelenterazine and / or an analogue thereof; and an organic phase protective solution, wherein the coelenterazine and / or an analogue thereof is dissolved in the organic phase, wherein the organic phase protective solution comprises an organic acid or an inorganic acid.

16. The composition of claim 15, wherein, The coelenterazine analogue includes one or more of Furimazine, coelenterazine-h, coelenterazine-h-h, coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, coelenterazine-I, coelenterazine-icp, coelenterazine-v, 2-methylcoelenterazine, JRW-0238, JRW-1743, and JRW-1744.

17. The composition of claim 15, wherein, The organic acid includes one or more of C 1-12 carboxylic acid, C 2-12 dicarboxylic acid, thio C 1- 12 carboxylic acid, amino C 1-12 carboxylic acid, C 1-12 alkyl sulfonic acid, C 6-30 aryl sulfonic acid, 2-phenyl-5-benzimidazole sulfonic acid, N-acetyl L-cysteine; and / or, The inorganic acid comprises one or more of hydrochloric acid, sulfuric acid, sulfurous acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, boric acid.

18. The composition of claim 17, wherein, The C 1-12 carboxylic acids include one or more of formic acid, acetic acid, propionic acid, isopropionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, 2-methylcaproic acid, valproic acid, 2-octynoic acid, elaidic acid, n-decanoic acid, (2-benzimidazolylthio)acetic acid; and / or, The C 2-12 The dicarboxylic acid includes one or more of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, dodecanedioic acid, phthalic acid, citric acid; and / or, said thio C 1-12 carboxylic acids include thioacetic acid, acetylthioacetic acid, thiopropionic acid, thio butyric acid, thiovaleric acid, thiohexanoic acid, DL-thioctic acid, thio benzoic acid; and / or, said amino C 1-12 carboxylic acids include one or more of glycine, glutamic acid, aspartic acid; and / or, The C 1-12 Alkylsulfonic acids include one or more of methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, 4-morpholineethanesulfonic acid; and / or, The C 6-30 Arylsulfonic acids include one or more of benzene sulfonic acid, xylene sulfonic acid.

19. The composition according to any one of claims 15-18, characterized in that, The concentration of the organic acid or inorganic acid in the organic phase protective solution is 0.01% to 20% or 0.1 mM to 5300 mM.

20. The composition of any one of claims 15-18, wherein, The organic phase protective solution further comprises DMSO 0 to 50%, methanol 0 to 50%, ethanol 0 to 50%, propylene glycol 0 to 90%, and / or glycerol 0 to 90%; Preferably, the organic phase protective solution further comprises DMSO 5 to 25%, methanol 5 to 25%, ethanol 5 to 25%, propylene glycol 50 to 90%, and / or glycerol 50 to 90%.

21. The composition of claim 15, wherein, The composition further comprises one or more of an antioxidant, a metal ion chelator, a surfactant, a biological preservative, and an anti-precipitation agent; Preferably, the antioxidant, the metal ion chelator, or the anti-precipitation agent is the same as defined in claims 9-14.

22. A kit comprising the composition of any one of claims 1-21.

23. The kit of claim 22, wherein The composition is contained in one or more containers; preferably, the container is a light-protected bottle or tube.

24. A method of stabilizing coelenterazine and / or an analog thereof, comprising, The method comprises the step of mixing the coelenterazine or its analogues in claims 15-21 with an effective amount of the organic phase protective solution, the solubilization medium of the coelenterazine and / or its analogues being an organic phase.

25. A method of stabilizing coelenterazine and / or an analog thereof, comprising, The method comprises the step of mixing the coelenterazine or its analogues in claims 1-14 with an effective amount of the aqueous phase protective solution, the solubilization medium of the coelenterazine and / or its analogues being an aqueous phase, the pH of the aqueous phase protective solution being ≤5.5, preferably 1 to 5, more preferably 2 to 5.

26. Use of the composition of any one of claims 1-21 for stabilizing coelenterazine and / or its analogues.

27. Use of the composition of any one of claims 1-21 for performing an assay, wherein a buffer is mixed with the aqueous phase protective solution or the organic phase protective solution in any one of claims 1-21, the pH of the resulting mixture being in the range of 5.0 to 8.5, and the luminescent signal is detected; Preferably, the pH is in the range of 5.5 to 7.

0. Preferably, the buffer comprises a buffer salt component, and luciferase; Preferably, the buffer salt component comprises one or more of MES, Tris, phosphate.

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