A firefly-nanoluc-akaluc tri-luciferase reporter gene detection kit

Abstract

The application discloses a firefly-NanoLuc-AkaLuc three-luciferase reporter gene detection kit, which comprises a luciferase lysis solution, a firefly luciferase detection reagent, a NanoLuc luciferase detection buffer, a NanoLuc luciferase substrate concentrate, an AkaLuc luciferase detection buffer and an AkaLuc luciferase substrate concentrate. The firefly-NanoLuc-AkaLuc three-luciferase reporter gene detection kit prepared by the application has high detection sensitivity, a wider linear detection range, higher signals, a smaller signal attenuation degree, a slower light signal decline trend, and is more suitable for application in high-throughput detection.

Description

Technical Field

[0001] This invention relates to biological detection technology, specifically to a firefly-NanoLuc-AkaLuc three-luciferase reporter gene detection kit. Background Technology

[0002] A reporter gene is a group of genes encoding easily detectable proteins or enzymes. By fusing the coding sequence of a reporter gene with a gene expression regulatory sequence, or by fusing it with other target genes, the target gene is expressed under the control of the regulatory sequence. The expression regulation of the target gene is "reported" by detecting the expression product of the reporter gene. Commonly used reporter genes include luciferase, chloramphenicol acetyltransferase (CAT), β-galactosidase (β-gal), secretory human placental alkaline phosphatase (SEAP), and green fluorescent protein (GFP). Among these, luciferase reporter genes are widely used due to their simple analytical methods, wide linear range, and low cost.

[0003] The luciferase reporter gene assay (LCA) is a reporter system that uses luciferin as a substrate to detect luciferase activity in fireflies. The principle involves constructing a reporter gene plasmid by inserting a transcriptional regulatory element of the target gene into an expression vector containing luciferase. This sequence regulates the transcriptional expression of luciferase. The reporter gene plasmid is then transfected into cells, and after different treatments, the cells are lysed and luciferin is added. Luciferase catalyzes the emission of fluorescence from luciferin, and the fluorescence intensity indicates the effect of different treatments on the transcriptional regulatory element.

[0004] Firefly luciferase, NanoLuc luciferase, and AkaLuc luciferase can catalyze their respective substrates to emit fluorescence of different colors. Since the three types of light have different absorption wavelengths and do not interfere with each other, they can be used simultaneously in the same chemical reaction system. This has become an effective means of studying transcription factors' involvement in gene regulation. Summary of the Invention

[0005] To detect three simultaneously expressed independent reporter genes in the same system, this invention proposes a firefly-NanoLuc-AkaLuc triple luciferase reporter gene detection kit and innovatively develops formulations for NanoLuc luciferase detection buffer and AkaLuc luciferase detection buffer. The NanoLuc luciferase detection buffer effectively blocks the fluorescence signal of firefly luciferase, and the AkaLuc luciferase detection buffer effectively blocks the fluorescence signal of NanoLuc luciferase. Therefore, high-throughput screening and use with high light intensity, long duration, and high sensitivity can be achieved in the sequential detection of fluorescence signals of firefly luciferase, NanoLuc luciferase, and AkaLuc luciferase.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a firefly-NanoLuc-AkaLuc three-luciferase reporter gene detection kit, comprising luciferase lysis buffer, firefly luciferase detection reagent, NanoLuc luciferase detection buffer, NanoLuc luciferase substrate concentrate, AkaLuc luciferase detection buffer and AkaLuc luciferase substrate concentrate.

[0008] The luciferase lysis buffer comprises PBS buffer, nonionic surfactant, metal ion masking agent, reducing agent, glycerol, bovine serum albumin, trehalose, and sucrose. The luciferase lysis buffer used in the kit of this invention features gentle cell lysis, protection of the activity of the lysed luciferase, and ease of operation without requiring ice-based lysis. It also includes stabilizers and sugar protectants, further enhancing enzyme stability.

[0009] Furthermore, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4.

[0010] Furthermore, the nonionic surfactant includes one of Triton X-100 and NP-40, and its volume fraction in the luciferase lysis buffer is 0.1%-0.25%, preferably 0.1%. The nonionic surfactant can disrupt the cell lipid bilayer and dissolve the cytoplasm and cell membrane.

[0011] Furthermore, the metal ion masking agent includes ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, with a content of 3-5 mM in the luciferase lysis buffer, preferably 3 mM, which can mask metal ions under certain acidity conditions.

[0012] Furthermore, the reducing agent includes at least one of DTT, L-ascorbic acid, and reduced glutathione, and its content in the luciferase lysis buffer is 3-15 mM, preferably 3 mM. The reducing agent can protect the enzyme activity.

[0013] Furthermore, the volume fraction of glycerol in the luciferase lysis buffer is 10%-15%, preferably 10%, and glycerol can act as a stabilizer.

[0014] Furthermore, the mass-to-volume ratio of bovine serum albumin in the luciferase lysis buffer is 0.1%-0.2%, preferably 0.15%, and bovine serum albumin can act as a stabilizer.

[0015] Furthermore, the trehalose content in the luciferase lysis buffer is 10 mM, and trehalose can act as an enzyme protectant.

[0016] Furthermore, the sucrose content in the luciferase lysis buffer is 5 mM, and sucrose can act as an enzyme protectant.

[0017] The firefly luciferase detection reagent comprises PBS buffer, ascorbic acid, ATP, magnesium chloride hexahydrate, potassium D-luciferin, and a reducing agent. The firefly luciferase detection reagent used in this invention allows firefly luciferase to catalyze the oxidation of luciferin to generate oxidized luciferin in the simultaneous presence of the substrate, oxygen, ATP, and magnesium ions, producing yellow-green light. The fluorescence intensity is directly proportional to the amount of luciferase. It features high sensitivity, a wide linear range, and rapid and convenient operation, making it particularly suitable for detection and high-throughput screening applications in areas with low cell signal intensity, weak drug efficacy, and multi-cell interaction signals.

[0018] Furthermore, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4.

[0019] Furthermore, the ascorbic acid content in the firefly luciferase detection reagent is 2-4 mM, preferably 2 mM, as ascorbic acid can stabilize and protect the luciferin substrate.

[0020] Furthermore, the ATP content in the firefly luciferase detection reagent is 1-5 mM, preferably 5 mM, and the energy released during ATP hydrolysis serves as the energy source for the enzyme-catalyzed reaction in the PBS buffer.

[0021] Furthermore, the content of magnesium chloride hexahydrate in the firefly luciferase detection reagent is 1-5 mM, preferably 3 mM. The magnesium ions provided by magnesium chloride hexahydrate are essential ions for the reaction of firefly luciferase with the substrate.

[0022] Furthermore, the D-luciferin potassium salt is present in the firefly luciferase detection reagent at a concentration of 0.1-0.5 mg / mL, preferably 0.3 mg / mL, as a firefly luciferase substrate.

[0023] Furthermore, the reducing agent is reduced glutathione, and its content in the firefly luciferase detection reagent is 2-5 mM, preferably 2 mM, which plays a role in stabilizing and protecting the luciferin substrate.

[0024] The NanoLuc luciferase detection buffer contains PBS buffer, pyrophosphate, and EDTA. The NanoLuc luciferase detection buffer used in this kit effectively blocks the fluorescence signal of firefly luciferase, achieving an inhibition rate of 99%. This avoids the influence of the firefly luciferase fluorescence signal on subsequent NanoLuc luciferase detection. When prepared as a NanoLuc luciferase detection working solution with NanoLuc luciferase substrate concentrate, it can detect NanoLuc luciferase activity without affecting the activity itself.

[0025] Furthermore, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4.

[0026] Furthermore, the concentration of pyrophosphate in the NanoLuc luciferase detection buffer is 30-50 mg / mL, preferably 35 mg / mL.

[0027] Furthermore, the concentration of EDTA in the NanoLuc luciferase detection buffer is 8-12 mM, preferably 10 mM.

[0028] The NanoLuc luciferase substrate concentrate is HFFz, dissolved in DMSO to a concentration of 0.6-0.8 mg / mL, preferably 0.75 mg / mL. The NanoLuc luciferase substrate concentrate used in this kit contains the novel substrate HFFz (Hydroxylfluorofurimazine) for detecting NanoLuc luciferase, resulting in a high light signal intensity, long duration, and high sensitivity during detection. The NanoLuc luciferase signal can be detected by mixing the NanoLuc luciferase substrate concentrate with the NanoLuc luciferase detection buffer at a volume ratio of 1:50.

[0029] Furthermore, the preparation method of the substrate HFFz is described in Chinese invention patent application CN117924295A.

[0030] The AkaLuc luciferase detection buffer contains PBS buffer, sodium thiosulfate, and KSCN, which can effectively shield the fluorescence signal of NanoLuc luciferase with an inhibition rate of 99%, avoiding the influence of the NanoLuc luciferase fluorescence signal on subsequent AkaLuc luciferase detection. When prepared with AkaLuc luciferase substrate concentrate to form the AkaLuc luciferase detection working solution, it can detect the activity of AkaLuc luciferase without affecting the activity of AkaLuc luciferase.

[0031] Furthermore, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4.

[0032] Furthermore, the sodium thiosulfate content in the AkaLuc luciferase detection buffer is 40-100 mM, preferably 60 mM.

[0033] Furthermore, the KSCN content in the AkaLuc luciferase detection buffer is 20-50 mM, preferably 30 mM.

[0034] The AkaLuc luciferase substrate concentrate is Akalumine hydrochloride, dissolved in DMSO to a concentration of 0.5-1 mg / mL, preferably 0.6 mg / mL. The AkaLuc luciferase substrate concentrate is then mixed with the AkaLuc luciferase detection buffer at a volume ratio of 1:50 to detect the AkaLuc luciferase signal.

[0035] Furthermore, the method for preparing the substrate Akalumine hydrochloride is described in Chinese invention patent application CN117720477A.

[0036] In a second aspect, the present invention provides the application of the firefly-NanoLuc-AkaLuc triple luciferase reporter gene detection kit as described in the first aspect in improving the detection sensitivity of firefly luciferase reporter gene, NanoLuc luciferase reporter gene, and AkaLuc luciferase reporter gene.

[0037] The biofluorescence reaction catalyzed by the luciferase of fireflies is shown in the following process:

[0038]

[0039] The NanoLuc luciferase can catalyze a biofluorescent reaction using a novel substrate HFFz. The reaction process is shown below:

[0040]

[0041] The biofluorescence reaction catalyzed by AkaLuc luciferase on the substrate Akalumine hydrochloride is shown in the following process:

[0042]

[0043] This invention discloses a method for detecting bioactivity, the method comprising the following steps:

[0044] (1) Cell lysis

[0045] (1.1) After aspirating the cell culture medium from cells transfected with firefly luciferase, NanoLuc luciferase, and AkaLuc luciferase reporter genes, add 1 mL of 0.25% trypsin to digest the cells, then add 5 mL of culture medium to resuspend the cells and count them. Collect the required number of cells by centrifugation. After removing the supernatant, add luciferase lysis buffer to resuspend the cells to 1E7 cells / mL and incubate at room temperature for 5 minutes to fully lyse the cells.

[0046] (1.2) After complete lysis, centrifuge at 10,000-15,000×g for 3-5 minutes, and take the supernatant for determination.

[0047] (2) Detection of firefly luciferase

[0048] (2.1) Add 50 μL of supernatant to the 96-well plate to be tested, add 50 μL of firefly luciferase detection reagent, shake the plate to mix well, and detect the activity of firefly luciferase.

[0049] (3) NanoLuc luciferase assay

[0050] (3.1) Based on the requirement of 50 μL of NanoLuc luciferase detection working solution for each sample, take an appropriate amount of NanoLuc luciferase detection buffer and NanoLuc luciferase substrate concentrate and prepare NanoLuc luciferase detection working solution at a volume ratio of 50:1.

[0051] (3.2) Add 50 μL of NanoLuc luciferase detection working solution to the 96-well plate containing the firefly luciferase detection reagent added in step (2.1), shake the plate to mix, and detect the activity of NanoLuc luciferase.

[0052] (4) AkaLuc luciferase detection

[0053] (4.1) Based on the requirement of 50 μL of AkaLuc luciferase detection working solution for each sample, take an appropriate amount of AkaLuc luciferase detection buffer and AkaLuc luciferase substrate concentrate and prepare AkaLuc luciferase detection working solution at a volume ratio of 50:1.

[0054] (4.2) Add 50 μL of AkaLuc luciferase detection working solution to the 96-well plate containing the NanoLuc luciferase detection working solution added in step (3.2), shake the plate to mix, and detect the activity of AkaLuc luciferase.

[0055] Compared with the prior art, the beneficial effects of this application are:

[0056] The NanoLuc luciferase detection buffer of this invention can effectively block the fluorescence signal of firefly luciferase with an inhibition rate of 99%, avoiding the influence of the firefly luciferase fluorescence signal on subsequent NanoLuc luciferase detection without affecting the activity of NanoLuc luciferase. The AkaLuc luciferase detection buffer of this invention can also effectively block the fluorescence signal of NanoLuc luciferase with an inhibition rate of 99%, avoiding the influence of the NanoLuc luciferase fluorescence signal on subsequent AkaLuc luciferase detection without affecting the activity of AkaLuc luciferase. This invention uses D-luciferin potassium salt as a substrate to detect firefly luciferase, introduces a novel substrate HFFz (Hydroxylfluorofurimazine) for detecting NanoLuc luciferase, and introduces self-synthesized Akalumine hydrochloride for detecting AkaLuc luciferase. This results in a detection process with high light signal intensity, long duration, and high sensitivity, which is beneficial for high-throughput screening and use of three luciferases. Attached Figure Description

[0057] Figure 1 The graph shows the linearity test results of the kit in Example 3 for detecting firefly luciferase.

[0058] Figure 2 The graph shows the linearity test results of the kit in Example 3 for detecting NanoLuc luciferase.

[0059] Figure 3 The graph shows the linearity test results of the kit in Example 3 for detecting AkaLuc luciferase.

[0060] Figure 4 The image shows the signal stability detection results of the kit in Example 4 for detecting firefly luciferase.

[0061] Figure 5The image shows the signal stability detection results of the kit in Example 4 for detecting NanoLuc luciferase.

[0062] Figure 6 The image shows the signal stability detection results of the kit in Example 4 for AkaLuc luciferase.

[0063] Figure 7 This is a comparison graph showing the effects of different inhibitors on the inhibition rate of firefly luciferase in Example 5.

[0064] Figure 8 The bar chart shows the signal detection results of NanoLuc luciferase in Example 7.

[0065] Figure 9 The bar chart shows the signal detection results of AkaLuc luciferase in Example 9. Detailed Implementation

[0066] The technical solutions in the embodiments will be clearly and completely described below. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0067] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0068] It should also be understood that the terminology used in this specification of embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used in this specification of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0069] The method for preparing the HFFz substrate involved in this invention is described in Chinese invention patent application CN117924295A, and the preparation steps are as follows:

[0070] (1) Using p-toluenesulfonyl azide and sodium hydride as raw materials, dissolve them in ultra-dry tetrahydrofuran. After stirring under ice bath conditions, add diethylphosphonic acid tert-butyl ester and react under ice bath conditions for 20-40 min. Then transfer the mixture to room temperature and stir for 1.5-2.5 hours. After terminating the reaction, add ethyl acetate for extraction, dry, filter and evaporate to dryness to obtain intermediate 1, wherein the molar ratio of p-toluenesulfonyl azide, sodium hydride and diethylphosphonic acid tert-butyl ester is 1.2:1:1;

[0071] (2) Zinc powder and iodine were suspended in anhydrous tetrahydrofuran and dry DMF and stirred at room temperature until the iodine was decolorized. 2-fluorobenzyl bromide was added to react. After the reaction was completed, the reaction mixture was cooled to room temperature. 2-amino-3,5-dibromopyrazine, PdCl2(PPh3)2, anhydrous tetrahydrofuran and dry DMF were added and stirred to react. The mixture was then filtered, the filtrate was extracted, dried, concentrated and purified to obtain intermediate 2. The molar ratio of zinc powder, iodine, 2-fluorobenzyl bromide, 2-amino-3,5-dibromopyrazine and PdCl2(PPh3)2 was 3.8:0.38:1.5:1:0.05.

[0072] (3) Under an inert atmosphere, intermediate 1, intermediate 2 and rhodium dimer acetate were dissolved in toluene and reacted. After cooling, the mixture was extracted, washed, dried, concentrated and purified to obtain intermediate 3. The molar ratio of intermediate 2 to intermediate 1 and rhodium dimer acetate was 1:1 to 2:0.1. The reaction temperature was 100 to 110°C and the reaction time was 22 to 26 h.

[0073] (4) Intermediate 3 and 2-furfural were dissolved in methanol, and tetramethylguanidine was added to react. After extraction, drying, concentration and purification, intermediate 4 was obtained. The molar ratio of intermediate 3, 2-furfural and tetramethylguanidine was 1:1 to 2:2 to 3. The reaction temperature was room temperature and the reaction time was 1.5 to 3.5 h.

[0074] (5) Under an inert atmosphere, intermediate 4 was dissolved in a 1,4-dioxane solution, and tetra-triphenylphosphine palladium, a phenylboronic acid compound, and a cesium carbonate aqueous solution were added. The mixture was reacted at 85°C for 3-4 hours. After cooling, the mixture was extracted, dried, concentrated, and purified to obtain intermediate 5. The molar ratio of intermediate 4, tetra-triphenylphosphine palladium, the phenylboronic acid compound, and the cesium carbonate aqueous solution was 1:0.1:1.3:3. The volume ratio of 1,4-dioxane to water in the 1,4-dioxane solution was 5:1. The phenylboronic acid compound was 3-(tert-butyldimethylsiloxy)-2-fluorophenylboronic acid.

[0075] (6) After dissolving intermediate 5 in dichloromethane, it was placed in an ice bath and then trifluoroacetic acid was added. The mixture was stirred at room temperature for 4 to 6 hours. The volatiles were removed under reduced pressure to obtain intermediate 6. The molar ratio of intermediate 5 to trifluoroacetic acid was 1:9 to 10.

[0076] (7) Intermediate 6 is dissolved in dichloromethane, and then CDI is added. The reaction is carried out in an ice bath for 0.5-2.5 hours. After extraction, drying, concentration and purification, intermediate 7 is obtained. The molar ratio of intermediate 6 to CDI is 1:1.5-2.5.

[0077] (8) Dissolve intermediate 7 in dichloromethane and methanol, cool, add sodium borohydride and react for 20-40 minutes, add hydrochloric acid to the reaction solution, adjust the pH, extract, dry, concentrate and purify to obtain intermediate 8, wherein the molar ratio of intermediate 7 to sodium borohydride is 1:2-4, and the concentration of hydrochloric acid is 0.1 mol / L.

[0078] (9) Intermediate 8 is dissolved in methanol, hydrochloric acid is added, and the reaction is carried out for 16 to 24 hours. After extraction, drying, concentration and purification, HFFz is obtained. The concentration of hydrochloric acid is 6 mol / L, and the molar ratio of intermediate 8 to hydrochloric acid is 1:8 to 20.

[0079] The method for preparing the Akalumine hydrochloride substrate involved in this invention is described in Chinese invention patent application CN117720477A, ​​and the preparation steps are as follows:

[0080] (1) D-cysteine ​​methyl ester hydrochloride was dissolved in an acid solution, triphenylmethanol was added, the reaction was stirred at room temperature, concentrated under reduced pressure, neutralized by anion exchange resin, washed with methanol, concentrated under reduced pressure, and purified to obtain intermediate 1.

[0081] (2) Dissolve 4-dimethylaminocinnamaldehyde in a solvent, add ethoxyformylmethylenetriphenylphosphine to react, evaporate under reduced pressure, extract, and purify to obtain intermediate 2;

[0082] (3) Intermediate 2 is dissolved in a solvent, and then sodium hydroxide aqueous solution is added and heated under reflux. After cooling to room temperature, the solid is concentrated under reduced pressure. The pH of the solid is adjusted to acidic, the precipitated solid is filtered, washed, and dried to obtain intermediate 3.

[0083] (4) Under a nitrogen atmosphere, intermediate 3 and intermediate 1 were dissolved in a solvent, EDC solution was added, then DMAP solution was added, the reaction was stirred at room temperature, extracted, evaporated to dryness, and purified to obtain intermediate 4.

[0084] (5) Under a nitrogen atmosphere, intermediate 4 was dissolved in a solvent, and triphenylphosphine oxide and trifluoromethanesulfonic anhydride were added to react. After quenching with water, the mixture was extracted, concentrated, and purified to obtain intermediate 5.

[0085] (6) Under a nitrogen atmosphere, intermediate 5 was reacted with a solvent, extracted, and passed through a column to obtain Akalumine;

[0086] (7) Sonicate Akalumine in a solvent, let it stand after sonication, centrifuge, collect the supernatant and freeze dry to obtain Akalumine hydrochloride.

[0087] All other raw materials involved in this invention can be purchased directly from the market. For process parameters not specifically specified, conventional techniques can be used as a reference.

[0088] Example 1

[0089] This embodiment provides a firefly-NanoLuc-AkaLuc triple luciferase reporter gene detection kit. The kit contains six reagents: luciferase lysis buffer, firefly luciferase detection reagent, NanoLuc luciferase detection buffer, NanoLuc luciferase substrate concentrate, AkaLuc luciferase detection buffer, and AkaLuc luciferase substrate concentrate. The raw material composition and preparation method of each reagent are as follows.

[0090] (1) Preparation of luciferase lysis buffer

[0091] In this embodiment, the luciferase lysis buffer includes PBS buffer, nonionic surfactant, metal ion masking agent, reducing agent, glycerol, bovine serum albumin, trehalose, and sucrose.

[0092] The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4; the nonionic surfactant is NP-40; the metal ion masking agent is ethylene glycol bis(2-aminoethyl ether)tetraacetic acid; and the reducing agents are DTT and reduced glutathione.

[0093] Measure 40 mL of PBS buffer into a container, then add 0.1% (v / v) NP-40, 3 mM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, 3 mM DTT, 3 mM reduced glutathione, 10% (v / v) glycerol, 0.15% (w / v) bovine serum albumin, 10 mM trehalose, and 5 mM sucrose. Finally, bring the volume to 50 mL with PBS buffer. All components can be dissolved using conventional stirring methods. The resulting clear, viscous liquid is the luciferase lysis buffer.

[0094] (2) Preparation of firefly luciferase detection reagent

[0095] In this embodiment, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, 1.84 mM KH2PO4, reduced glutathione as the reducing agent, as well as ascorbic acid, ATP, magnesium chloride hexahydrate, and potassium D-fluorescein.

[0096] Measure 40 mL of PBS buffer into a container, then add 3 mM reduced glutathione, 2 mM ascorbic acid, 5 mM ATP, 3 mM magnesium chloride hexahydrate, and 0.3 mg / mL D-luciferin potassium salt. Finally, bring the volume up to 50 mL with PBS buffer. The resulting pale yellow liquid is the firefly luciferase detection reagent.

[0097] (3) Preparation of NanoLuc luciferase detection buffer

[0098] Measure 40 mL of PBS buffer into a container. The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4. Then add 35 mg / mL pyrophosphate and 10 mM EDTA. Finally, bring the volume up to 50 mL with PBS buffer. The resulting clear liquid is the NanoLuc luciferase detection buffer.

[0099] (4) Preparation of NanoLuc luciferase substrate (50×)

[0100] Measure 10 ml of DMSO into a container, weigh HFFz to make its concentration 0.65 mg / mL, which is the NanoLuc luciferase substrate concentrate.

[0101] (5) Preparation of AkaLuc luciferase detection buffer

[0102] Measure 40 ml of PBS buffer into a container. The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4. Then add 60 mM sodium thiosulfate and 30 mM KSCN. Finally, adjust the volume to 50 ml with PBS buffer. The resulting clear liquid is the AkaLuc luciferase detection buffer.

[0103] (6) Preparation of AkaLuc luciferase substrate concentrate

[0104] Measure 10 ml of DMSO into a container, weigh Akalumine hydrochloride to make its concentration 0.6 mg / mL, which is the AkaLuc luciferase substrate concentrate.

[0105] Example 2

[0106] This embodiment provides a firefly-NanoLuc-AkaLuc triple luciferase reporter gene detection kit. The kit contains six reagents: luciferase lysis buffer, firefly luciferase detection reagent, NanoLuc luciferase detection buffer, NanoLuc luciferase substrate concentrate, AkaLuc luciferase detection buffer, and AkaLuc luciferase substrate concentrate. The raw material composition and preparation method of each reagent are as follows.

[0107] (1) Preparation of luciferase lysis buffer

[0108] In this embodiment, the luciferase lysis buffer includes PBS buffer, nonionic surfactant, metal ion masking agent, reducing agent, glycerol, bovine serum albumin, trehalose, and sucrose.

[0109] The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, 1.84 mM KH2PO4, NP-40 as the nonionic surfactant, ethylene glycol bis(2-aminoethyl ether)tetraacetic acid as the metal ion masking agent, DTT as the reducing agent, and glycerol, bovine serum albumin, trehalose, and sucrose as the remainder.

[0110] Measure 40 mL of PBS buffer into a container, then add 0.2% (v / v) NP-40, 5 mM ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, 5 mM DTT, 15% (v / v) glycerol, 0.2% (w / v) bovine serum albumin, 10 mM trehalose, and 5 mM sucrose. Finally, bring the volume to 50 mL with PBS buffer. All components can be dissolved using conventional stirring methods. The resulting clear, viscous liquid is the luciferase lysis buffer.

[0111] (2) Preparation of firefly luciferase detection reagent

[0112] In this embodiment, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, 1.84 mM KH2PO4, reduced glutathione as the reducing agent, as well as ascorbic acid, ATP, magnesium chloride hexahydrate, and potassium D-fluorescein.

[0113] Measure 40 mL of PBS buffer into a container, then add 5 mM reduced glutathione, 4 mM ascorbic acid, 3 mM ATP, 5 mM magnesium chloride hexahydrate, and 0.5 mg / mL D-luciferin potassium salt. Finally, bring the volume up to 50 mL with PBS buffer. The resulting pale yellow liquid is the firefly luciferase detection reagent.

[0114] (3) Preparation of NanoLuc luciferase detection buffer

[0115] Measure 40 mL of PBS buffer into a container. The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4. Then add 50 mg / mL pyrophosphate and 12 mM EDTA. Finally, bring the volume up to 50 mL with PBS buffer. The resulting clear liquid is the NanoLuc luciferase detection buffer.

[0116] (4) Preparation of NanoLuc luciferase substrate concentrate

[0117] Measure 10 mL of DMSO into a container, weigh HFFz to make its concentration 0.75 mg / mL, which is the NanoLuc luciferase substrate concentrate.

[0118] (5) Preparation of AkaLuc luciferase detection buffer

[0119] Measure 40 ml of PBS buffer into a container. The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4. Then add 80 mM sodium thiosulfate and 45 mM KSCN. Finally, adjust the volume to 50 ml with PBS buffer. The resulting clear liquid is the AkaLuc luciferase detection buffer.

[0120] (6) Preparation of AkaLuc luciferase substrate concentrate

[0121] Measure 10 ml of DMSO into a container, weigh Akalumine hydrochloride to make its concentration 1 mg / mL, which is the AkaLuc luciferase substrate concentrate.

[0122] Comparative Example 1:

[0123] Measure 40 ml of PBS buffer into a container. The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4. Then add 80 mM sodium thiosulfate and finally adjust the volume to 50 ml with PBS buffer. The resulting clear liquid is the AkaLuc luciferase detection buffer contrast reagent.

[0124] Comparative Example 2

[0125] Measure 40 ml of PBS buffer into a container. The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4. Then add 45 mM KSCN and finally adjust the volume to 50 ml with PBS buffer. The resulting clear liquid is the AkaLuc luciferase detection buffer contrast reagent.

[0126] Comparative Example 3

[0127] Measure 40 ml of PBS buffer into a container containing 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4. Then add 50 mg / mL of pyrophosphate and finally bring the volume up to 50 ml with PBS buffer. The resulting clear liquid is the NanoLuc luciferase detection buffer contrast reagent.

[0128] Comparative Example 4

[0129] Measure 40 ml of PBS buffer into a container. The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4. Then add 12 mM EDTA and finally adjust the volume to 50 ml with PBS buffer. The resulting clear liquid is the NanoLuc luciferase detection buffer contrast reagent.

[0130] Comparative Example 5

[0131] The kit for this comparative example replaces the pyrophosphate component in the NanoLuc luciferase detection buffer of Example 1 with the firefly luciferase inhibitor KI at a concentration of 40 mM. The other components and preparation methods are the same as in Example 1.

[0132] Comparative Example 6

[0133] The kit for this comparative example replaces the pyrophosphate component in the NanoLuc luciferase detection buffer of Example 1 with resveratrol, a firefly luciferase inhibitor, at a concentration of 1 mM. Other components and preparation methods are the same as in Example 1.

[0134] Example 3

[0135] The linearity performance of the reagent kit was tested using the following steps:

[0136] (1) Cell lysis

[0137] (1.1) After aspirating the cell culture medium from cells transfected with firefly luciferase, NanoLuc luciferase, and AkaLuc luciferase reporter genes, add 1 mL of 0.25% trypsin to digest the cells, then add 5 mL of culture medium to resuspend the cells and count them. Collect the required number of cells by centrifugation. After removing the supernatant, add luciferase lysis buffer to resuspend the cells to 1E7 cells / mL and incubate at room temperature for 5 minutes to fully lyse the cells.

[0138] (1.2) After complete lysis, centrifuge at 10,000-15,000×g for 3-5 minutes and take the supernatant as the test sample.

[0139] (2) The supernatant from step (1.2) was diluted with luciferase lysis buffer at ratios of 1:2, 1:5, 1:10, 1:20, 1:50 and 1:100 respectively to obtain a total of 7 test samples.

[0140] (3) Firefly luciferase detection

[0141] (3.1) Take 50 μL of the supernatant of each sample from step (2) and add it to the 96-well plate to be tested. Add 50 μL of firefly luciferase detection reagent to each well, shake the plate to mix, and use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescent signal of firefly luciferase.

[0142] (4) NanoLuc luciferase detection

[0143] (4.1) Based on the requirement of 50 μL of NanoLuc luciferase detection working solution for each sample, take an appropriate amount of NanoLuc luciferase detection buffer and NanoLuc luciferase substrate concentrate and prepare NanoLuc luciferase detection working solution at a volume ratio of 50:1.

[0144] (4.2) Add 50 μL of NanoLuc luciferase detection working solution to the 96-well plate containing the firefly luciferase detection reagent added in step (2.1), shake the plate to mix, and use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescence signal of NanoLuc luciferase.

[0145] (5) AkaLuc luciferase assay

[0146] (5.1) Based on the requirement of 50 μL of AkaLuc luciferase detection working solution for each sample, take an appropriate amount of AkaLuc luciferase detection buffer and AkaLuc luciferase substrate concentrate and prepare AkaLuc luciferase detection working solution at a volume ratio of 50:1.

[0147] (5.2) Add 50 μL of AkaLuc luciferase detection working solution to the 96-well plate containing the NanoLuc luciferase detection working solution added in step (4.2), shake the plate to mix, and use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescent signal of AkaLuc luciferase.

[0148] The above tests were performed using the kits prepared in Examples 1 and 2. The test results were linearly fitted using a curve of dilution ratio and detection signal value (RLU).

[0149] The linearity test results for firefly luciferase are as follows: Figure 1 As shown, the linearity test results for NanoLuc luciferase are as follows: Figure 2 As shown, the linearity test results for AkaLuc luciferase are as follows: Figure 3 As shown, the kit of the present invention has a good linear fit and a wide linear range.

[0150] Example 4

[0151] The signal stability detection procedure for the reagent kit is as follows:

[0152] 1. Detection of signal stability of firefly luciferase

[0153] The supernatant from step (1.2) of Example 3 was used as the detection target. Three replicate wells were made. 50 μL of supernatant was added to each well of a 96-well plate, followed by 50 μL of firefly luciferase detection reagent. The plates were shaken to mix, and the chemiluminescence signal was detected using a multi-functional microplate reader (Berthokd LB941). The fluorescence value was measured every 5 minutes for a total of 30 minutes. The average fluorescence value at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min was read for subsequent analysis.

[0154] 2. Detection of signal stability of NanoLuc luciferase

[0155] (1) According to the amount of NanoLuc luciferase detection working solution required for each sample (50 μL), take an appropriate amount of NanoLuc luciferase detection buffer and NanoLuc luciferase substrate concentrate and prepare NanoLuc luciferase detection working solution at a volume ratio of 50:1.

[0156] (2) Take the supernatant from step (1.2) of Example 3 as the detection object, make 3 replicate wells, take 50 μL from each well and add it to a 96-well plate, add 50 μL of NanoLuc luciferase detection working solution, shake the plate to mix, use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescence signal, set the fluorescence value to be measured every 5 min, and measure the fluorescence value for a total of 30 min. Read the average fluorescence value at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30 min for subsequent analysis.

[0157] 3. Detection of signal stability of AkaLuc luciferase

[0158] (1) Based on the requirement of 50 μL of AkaLuc luciferase detection working solution for each sample, take an appropriate amount of AkaLuc luciferase detection buffer and AkaLuc luciferase substrate concentrate and prepare AkaLuc luciferase detection working solution at a volume ratio of 50:1.

[0159] (2) Take the supernatant from step (1.2) of Example 3 as the detection object, make 3 replicate wells, take 50 μL from each well and add it to a 96-well plate, add 50 μL of AkaLuc luciferase detection working solution, shake the plate to mix, use a multi-functional microplate reader (BerthokdLB941) to detect the chemiluminescence signal, set the fluorescence value to be measured every 5 min, and measure the fluorescence value for a total of 30 min. Read the average fluorescence value at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30 min for subsequent analysis.

[0160] The above tests were performed on Examples 1 and 2.

[0161] The luminescence stability results of firefly luciferase assay are as follows: Figure 4 As shown, the luminescence stability results of NanoLuc luciferase assay are as follows: Figure 5 As shown, the luminescence stability results of AkaLuc luciferase assay are as follows: Figure 6 As shown, the detection signal of the kit of the present invention decreases slowly within 30 minutes, indicating high signal stability.

[0162] Example 5: Effect of different inhibitors on the inhibition rate of firefly luciferase

[0163] The experimental steps are as follows:

[0164] (1) Detection of firefly luciferase

[0165] (1.1) Take 50 μL of the supernatant stock solution from step (1.2) of Example 3 and add it to a 96-well plate to make three replicate wells. Add 50 μL of firefly luciferase detection reagent, shake the plate to mix, and use a multifunctional microplate reader (Berthokd LB941) to detect the chemiluminescence signal of firefly luciferase. Take the average value of the fluorescence signal for final analysis.

[0166] (2) NanoLuc luciferase detection

[0167] (2.1) In the 96-well plate containing the firefly luciferase detection reagent added in step (1.1), add 49 μL of NanoLuc luciferase detection buffer to each well, shake the plate to mix, and detect the chemiluminescence signal. The obtained signal value reflects the inhibitory effect of the buffer on the firefly luciferase catalytic reaction in the previous step.

[0168] (2.2) Add 1 μL of NanoLuc luciferase substrate concentrate to the 96-well plate containing NanoLuc luciferase detection buffer from step (2.1), shake the plate to mix, and use a multi-functional microplate reader (Berthokd LB941) to detect the activity of NanoLuc luciferase.

[0169] The above tests were conducted using Examples 1, 2, 5, and 6.

[0170] The results are as follows Figure 7 As shown, pyrophosphate has a greater inhibitory effect on firefly luciferase than conventional KI and resveratrol, and has no inhibitory effect on NanoLuc luciferase, thus not affecting subsequent chemiluminescence signal measurement. In contrast, KI and resveratrol inhibit the activity of NanoLuc luciferase.

[0171] Example 6

[0172] The inhibitory effects of pyrophosphate and EDTA, inhibitors in NanoLuc luciferase assay buffer, on firefly luciferase were determined using the following steps:

[0173] 1. Firefly luciferase detection

[0174] Take 50 μL of each of the 7 samples from step (2) of Example 3 and add them to a 96-well plate. Add 50 μL of firefly luciferase detection reagent to each of the 96-well plates to be tested, shake the plates to mix, and use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescent signal of firefly luciferase.

[0175] 2. Inhibition rate detection

[0176] Add 49 μL of NanoLuc luciferase detection buffer to the 96-well plate from step 1, shake the plate to mix, and use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescence signal. The inhibition rate of the 7 samples can be obtained by using the formula: inhibition rate = 1 - (signal after adding NanoLuc luciferase detection buffer / firefly luciferase detection signal) × 100%.

[0177] The above tests were performed on Example 1, Example 2, Comparative Example 3, and Comparative Example 4.

[0178] The results are shown in the table below. The NanoLuc luciferase detection buffers in Examples 1 and 2 showed strong inhibitory effects on the fluorescence signal of firefly luciferase secreted by different cell numbers, with inhibition rates exceeding 99%. The inhibition rate in Comparative Example 3 was 76%-83%, indicating that pyrophosphate had a relatively good quenching effect on the fluorescence signal of firefly luciferase, but it did not meet the expected requirements. The inhibition rate in Comparative Example 4 was 25%-32%, indicating that EDTA, as a metal ion chelating agent, could chelate a certain amount of magnesium ions, thereby inhibiting the catalytic reaction of firefly luciferase and weakening the fluorescence signal, but the inhibitory effect was weak. Adding pyrophosphate or EDTA alone to the system failed to achieve the expected results; only when EDTA and pyrophosphate were in the same system could the fluorescence signal of firefly luciferase be quenched to a great extent without affecting subsequent tests.

[0179]

[0180]

[0181] Example 7

[0182] The following test steps were performed to determine whether the inhibitors pyrophosphate and EDTA in the NanoLuc luciferase assay buffer affected the detection of NanoLuc luciferase.

[0183] 1. Control Group 1 Test

[0184] Take the supernatant from step (1.2) of Example 3 into a 96-well plate, set up 3 replicate wells, add 49 μL of PBS buffer and 1 μL of NanoLuc luciferase substrate concentrate, shake the plate to mix well, and use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescence signal. The average fluorescence value is used for subsequent analysis.

[0185] 2. Experimental group testing

[0186] The test method is as follows: Take the supernatant of step (1.2) in Example 3 into a 96-well plate, set up 3 replicate wells, add 49 μL of NanoLuc luciferase detection buffer and 1 μL of NanoLuc luciferase substrate concentrate, shake the plate to mix well, and use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescence signal. The average fluorescence value is used for subsequent analysis.

[0187] The NanoLuc luciferase detection buffer and NanoLuc luciferase substrate concentrate from Examples 1 and 2 were used to conduct experimental group tests according to the above test method. The test results are as follows: Figure 8As shown, there was no significant difference in the signals between Examples 1 and 2 and the control group, and the detected fluorescence signals were similar, indicating that the inhibitors pyrophosphate and EDTA do not affect the activity of NanoLuc luciferase.

[0188] Example 8

[0189] The inhibitory effects of sodium thiosulfate and KSCN on NanoLuc luciferase in AkaLuc luciferase assay buffer were determined by the following steps:

[0190] 1. NanoLuc luciferase assay

[0191] (1.1) Take 50 μL of each of the 7 samples from step (2) of Example 3 and add them to a 96-well plate. According to the amount of 50 μL of NanoLuc luciferase detection working solution required for each sample, take an appropriate amount of NanoLuc luciferase detection buffer and NanoLuc luciferase substrate concentrate at a volume ratio of 50:1 to prepare NanoLuc luciferase detection working solution.

[0192] (1.2) Add 50 μL of NanoLuc luciferase detection working solution to each of the 96-well plates to be tested in step (1.1), shake the plates to mix well, and use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescence signal of NanoLuc luciferase.

[0193] 2. Inhibition rate detection

[0194] Add 49 μL of AkaLuc luciferase detection buffer to the 96-well plate from step (1.2), shake the plate to mix, and use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescence signal. The inhibition rate of the 7 samples can be obtained by using the formula: inhibition rate = 1 - (signal after adding AkaLuc luciferase detection buffer / NanoLuc luciferase detection signal) × 100%.

[0195] The above tests were performed on Example 1, Example 2, Comparative Example 1, and Comparative Example 2.

[0196] The results are shown in the table below. The AkaLuc luciferase detection buffers of Examples 1 and 2 showed strong inhibitory effects on the fluorescence signal of NanoLuc luciferase secreted by different cell numbers, with inhibition rates exceeding 99%. Comparative Examples 1 and 2 showed increased inhibition of NanoLuc luciferase as the cell number decreased, but the inhibition rate was only 50-70%. This indicates that neither sodium thiosulfate nor KSCN alone in the buffer can achieve a good inhibitory effect on NanoLuc luciferase. Only the simultaneous presence of sodium thiosulfate and KSCN in the buffer can quench the fluorescence signal of NanoLuc luciferase to a great extent without affecting subsequent tests.

[0197]

[0198]

[0199] Example 9

[0200] The following test steps were performed to determine whether the inhibitors sodium thiosulfate and KSCN in the AkaLuc luciferase assay buffer affected the detection of AkaLuc luciferase.

[0201] 1. Control Group 2 Test

[0202] Take the supernatant from step (1.2) of Example 3 into a 96-well plate, set up 3 replicate wells, add 49 μL of PBS buffer and 1 μL of AkaLuc luciferase substrate concentrate, shake the plate to mix well, and use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescence signal. The average fluorescence value is used for subsequent analysis.

[0203] 2. Experimental group testing

[0204] The test method is as follows: Take the supernatant of step (1.2) in Example 3 into a 96-well plate, set up 3 replicate wells, add 49 μL of AkaLuc luciferase detection buffer and 1 μL of AkaLuc luciferase substrate concentrate, shake the plate to mix well, and use a multi-functional microplate reader (Berthokd LB941) to detect the chemiluminescence signal. The average fluorescence value is used for subsequent analysis.

[0205] The AkaLuc luciferase detection buffer and AkaLuc luciferase substrate concentrate from Examples 1 and 2 were used to conduct experimental group tests according to the above test method. The test results are as follows: Figure 9 As shown, there was no significant difference in the signals between Examples 1 and 2 and the control group, and the detected fluorescence signals were similar, indicating that the inhibitors sodium thiosulfate and KSCN do not affect the activity of AkaLuc luciferase.

[0206] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A firefly-NanoLuc-AkaLuc three-luciferase reporter gene detection kit, characterized in that, Includes luciferase lysis buffer, firefly luciferase assay reagent, NanoLuc luciferase assay buffer, NanoLuc luciferase substrate concentrate, AkaLuc luciferase assay buffer, and AkaLuc luciferase substrate concentrate; The luciferase lysis buffer includes PBS buffer, nonionic surfactant, metal ion masking agent, reducing agent, glycerol, bovine serum albumin, trehalose and sucrose; The firefly luciferase detection reagent includes PBS buffer, ascorbic acid, ATP, magnesium chloride hexahydrate, D-luciferin potassium salt and reduced glutathione; The NanoLuc luciferase detection buffer contains PBS buffer, pyrophosphate, and EDTA. The NanoLuc luciferase substrate concentrate is HFFz; The AkaLuc luciferase detection buffer contains PBS buffer, sodium thiosulfate and KSCN; The AkaLuc luciferase substrate concentrate is Akalumine hydrochloride.

2. The reagent kit according to claim 1, characterized in that, In the luciferase lysis buffer, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4 and 1.84 mM KH2PO4; The nonionic surfactant is selected from Triton X-100 and NP-40, and its volume fraction in the luciferase lysis buffer is 0.1%-0.25%. The metal ion masking agent is ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, and its content in the luciferase lysis buffer is 3-5 mM. The reducing agent is selected from at least one of DTT, L-ascorbic acid, and reduced glutathione, and its content in the luciferase lysis buffer is 3-15 mM. The volume fraction of glycerol in the luciferase lysis buffer is 10%-15%; The bovine serum albumin content in the luciferase lysis buffer is 0.1%-0.2% by mass / volume. The trehalose content in the luciferase lysis buffer is 10 mM; The sucrose content in the luciferase lysis buffer is 5 mM.

3. The reagent kit according to claim 1, characterized in that, In the firefly luciferase assay reagent, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4 and 1.84 mM KH2PO4; The ascorbic acid content in the firefly luciferase detection reagent is 2-4 mM; The ATP content in the firefly luciferase assay reagent is 1-5 mM; The content of magnesium chloride hexahydrate in the firefly luciferase detection reagent is 1-5 mM; The concentration of the D-luciferin potassium salt in the firefly luciferase detection reagent is 0.1-0.5 mg / mL; The content of the reduced glutathione in the firefly luciferase detection reagent is 2-5 mM.

4. The reagent kit according to claim 1, characterized in that, In the NanoLuc luciferase assay buffer, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4 and 1.84 mM KH2PO4; The concentration of pyrophosphate in the NanoLuc luciferase detection buffer is 30-50 mg / mL; The concentration of EDTA in the NanoLuc luciferase detection buffer is 8-12 mM.

5. The reagent kit according to claim 1, characterized in that, The content of HFFz is 0.6-0.8 mg / mL.

6. The reagent kit according to claim 1, characterized in that, In the AkaLuc luciferase assay buffer, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4 and 1.84 mM KH2PO4; The sodium thiosulfate content in the AkaLuc luciferase detection buffer is 40-100 mM; The concentration of KSCN in the AkaLuc luciferase detection buffer is 20-50 mM.

7. The kit according to claim 1, characterized in that, The content of the Akalumine hydrochloride is 0.5-1 mg / mL.

8. The use of the firefly-NanoLuc-AkaLuc triple luciferase reporter gene assay kit according to any one of claims 1-7 in improving the detection sensitivity of firefly luciferase reporter gene, NanoLuc luciferase reporter gene, and AkaLuc luciferase reporter gene.

Images