Acetaldehyde dehydrogenase activity detection kit, detection method and application thereof

Abstract

The invention provides an acetaldehyde dehydrogenase activity detection kit, a detection method and application thereof, and belongs to the technical field of enzyme activity detection. The acetaldehyde dehydrogenase activity detection kit provided by the invention comprises azidoacetaldehyde, an alkynyl fluorescent probe and a click reaction catalyst, the alkynyl fluorescent probe comprises cations and anions, and the cations of the alkynyl fluorescent probe have a structure as shown in a formula I. When the acetaldehyde dehydrogenase activity detection kit provided by the invention is used for detecting the acetaldehyde dehydrogenase activity, a detection reagent is easy to obtain, and the acetaldehyde dehydrogenase activity detection kit is convenient to popularize and apply. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme activity detection, and particularly relates to a kit and method for detecting the activity of aldehyde dehydrogenase and its application. Background Art

[0002] Acetaldehyde is cytotoxic and its accumulation in the body will damage the functions of biological macromolecules (such as proteins, DNA, and RNA). Aldehyde dehydrogenase (ALDH2) is an enzyme present in human cells, responsible for catalyzing the reaction of oxidizing acetaldehyde to acetic acid, and plays an important role in the aldehyde group metabolism process in the body. Therefore, it is of great significance to detect ALDH2.

[0003] ALDH2 is a tetrameric protein with three structural domains, including a coenzyme-binding domain, a catalytic domain, and an oligomeric domain. Although ALDH2 can be quantitatively detected for transcription and translation by methods such as real-time quantitative polymerase chain reaction fluorescence detection method (qPCR) and Western blotting (WB), these traditional detection methods based on immune recognition can only determine the protein expression level but cannot reflect its functional state. Since it is ultimately the activity rather than the concentration that determines the enzyme function, it is crucial to detect the total enzyme activity of ALDH2. Measuring the characteristic ultraviolet absorbance of the metabolite NADH at 340 nm to quantify the ALDH2 enzyme activity is an indirect detection method, which has the disadvantages of long time consumption, poor sensitivity, and non-in-situ imaging. Moreover, the above methods are complex in operation, high in detection cost, prone to rejection reactions with the detected tissues, have high requirements for substrates, and inevitably face the situation of substrate loss during the detection process.

[0004] Fluorescent probes have the characteristics of high detection sensitivity, low cost, real-time detection, and simple methods. Most of the existing fluorescent probe molecular structures are composed of a single lumophore connected to the corresponding enzyme substrate group, and the fluorescence signal is regulated by the interaction between the substrate group and the enzyme, either by switching the fluorescence signal or changing the fluorescence signal, so as to detect the change in enzyme activity through the signal change. However, the preparation of traditional enzyme activity detection fluorescent probes is difficult, not easily obtained, and not conducive to popularization and application. Summary of the Invention

[0005] The purpose of the present invention is to provide a kit and method for detecting the activity of aldehyde dehydrogenase and its application. Using the kit for detecting the activity of aldehyde dehydrogenase provided by the present invention to detect the activity of aldehyde dehydrogenase, the detection reagents are easily obtained and convenient for popularization and application.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a kit for detecting the activity of aldehyde dehydrogenase, which comprises azidoacetaldehyde, an alkynyl fluorescent probe and a click reaction catalyst. The alkynyl fluorescent probe comprises a cation and an anion, and the cation of the alkynyl fluorescent probe has the structure shown in Formula I:

[0008]

[0009] Preferably, the click reaction catalyst comprises copper sulfate, a reducing agent and a ligand; the reducing agent comprises vitamin C or tris(2-carboxyethyl)phosphine; the ligand comprises tris(3-hydroxypropyltriazolylmethyl)amine or tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.

[0010] Preferably, the click reaction catalyst comprises copper sulfate, vitamin C and tris(3-hydroxypropyltriazolylmethyl)amine; or the click reaction catalyst comprises copper sulfate, tris(2-carboxyethyl)phosphine and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.

[0011] The present invention provides a method for detecting the activity of aldehyde dehydrogenase by using the kit for detecting the activity of aldehyde dehydrogenase according to the above technical solution, comprising the following steps:

[0012] Mixing a sample to be tested, azidoacetaldehyde and a first reaction medium to obtain a first mixture solution, and performing a first incubation to obtain a first incubated sample;

[0013] Mixing the first incubated sample, the alkynyl fluorescent probe, the click reaction catalyst and a second reaction medium to obtain a second mixture solution, and performing a second incubation to obtain a second incubated sample;

[0014] Detecting the activity of aldehyde dehydrogenase in the sample to be tested according to the fluorescence intensity of the second incubated sample.

[0015] Preferably, the concentration of azidoacetaldehyde in the first mixture solution is 0.2-1.5 mM; the time of the first incubation is 4-12 h.

[0016] Preferably, the concentration of the alkynyl fluorescent probe in the second mixture solution is 1-5 μM, the concentration of copper sulfate is 0.05-5 mM, the concentration of the reducing agent is 1-10 mM, and the concentration of the ligand is 100-1000 μM; the time of the second incubation is 30-60 min.

[0017] Preferably, the sample to be tested includes living cells or tissues; when the sample to be tested is living cells or tissues, after obtaining the first incubation sample, it further includes: performing fixation-permeabilization treatment on the first incubation sample to obtain a fixation-permeabilization sample, and mixing the fixation-permeabilization sample with an alkynyl fluorescent probe, a click reaction catalyst, and a second reaction medium for the second incubation; the reagent used for the fixation-permeabilization treatment includes methanol.

[0018] The present invention provides an application of the method according to the above technical solution in screening ALDH2 activity regulators.

[0019] Preferably, the ALDH2 activity regulator includes an ALDH2 agonist or an ALDH2 inhibitor.

[0020] Preferably, the ALDH2 agonist includes sennoside A.

[0021] Beneficial effects: Using the acetaldehyde dehydrogenase activity detection kit provided by the present invention to detect the acetaldehyde dehydrogenase activity, the method is novel, the detection reagents are easily obtained, and it is convenient for popularization and application. The principle of the method of the present invention is as follows: First, incubate the metabolic substrate azidoacetaldehyde with a biological sample, which has an azide group, and through the catalytic reaction of ALDH2, catalyze azidoacetaldehyde to generate azidoacetic acid. Azidoacetic acid introduces an azide group into biological macromolecules during the subsequent biochemical process. Subsequently, utilize the click chemistry between the azide group and the alkynyl group to perform a bioorthogonal reaction to connect the fluorescent group and the substrate, and detect the corresponding ALDH2 activity using the fluorescence signal intensity in the imaging map. The feasibility of this method in detecting ALDH2 activity was verified in the examples of the present invention, providing a new approach for enzyme activity detection. Description of the Drawings

[0022] Figure 1 It is a schematic diagram of the operation process of the ALDH2 activity detection method in the present invention;

[0023] Figure 2 It is a cell staining diagram after incubation with azidoacetaldehyde for different times;

[0024] Figure 3 It is a cell staining diagram after incubation with azidoacetaldehyde at different concentrations;

[0025] Figure 4 It is a fluorescence quantitative statistical result diagram after incubation with azidoacetaldehyde under different conditions;

[0026] Figure 5 It is a cell staining diagram after incubation under different conditions using different click reaction catalysts and the alkynyl fluorescent probe RHO;

[0027] Figure 6It is a fluorescence quantitative statistical result graph after incubation with different click reaction catalysts and alkynyl fluorescent probe RHO under different conditions;

[0028] Figure 7 It is a schematic flow chart for screening ALDH2 activity regulators by using the ALDH2 activity detection method in the present invention;

[0029] Figure 8 It is a quantitative graph of cell fluorescence intensity after the action of different enzyme activity regulating drugs;

[0030] Figure 9 It is a dose-responsive cell staining graph of sennoside A and its fluorescence quantitative statistical result graph. Specific embodiments

[0031] The present invention provides a kit for detecting the activity of aldehyde dehydrogenase, which includes azidoacetaldehyde, alkynyl fluorescent probe and click reaction catalyst. The alkynyl fluorescent probe includes a cation and an anion, and the cation of the alkynyl fluorescent probe has the structure shown in Formula I:

[0032]

[0033] In the present invention, unless otherwise specified, the raw materials used are commercially available products well-known to those skilled in the art or prepared by methods well-known to those skilled in the art.

[0034] The kit for detecting the activity of aldehyde dehydrogenase in the present invention includes azidoacetaldehyde and alkynyl fluorescent probe (abbreviated as RHO). When detecting the activity of aldehyde dehydrogenase by using the kit for detecting the activity of aldehyde dehydrogenase provided by the present invention, first, the metabolic substrate azidoacetaldehyde with an azide group is incubated with a biological sample. Through the catalytic reaction of ALDH2, azidoacetaldehyde is catalyzed to generate azidoacetic acid. Azidoacetic acid introduces an azide group into biological macromolecules along with the subsequent biochemical process. Then, the click chemistry between the azide group and the alkynyl group is used for bioorthogonal reaction to connect the fluorescent group and the substrate, and the activity of the corresponding ALDH2 is detected by the fluorescence signal intensity in the imaging graph. As an embodiment of the present invention, the anion in the alkynyl fluorescent probe can be chloride ion.

[0035] The acetaldehyde dehydrogenase activity detection kit of the present invention includes a click reaction catalyst for catalyzing the click chemical reaction between an azide group and an alkyne group. As an embodiment of the present invention, the click reaction catalyst may include copper sulfate, a reducing agent and a ligand; the reducing agent may include vitamin C (Vc) or tris(2-carboxyethyl)phosphine (TCEP); the ligand may include tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) or tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA). As an embodiment of the present invention, the click reaction catalyst may specifically include copper sulfate, vitamin C and tris(3-hydroxypropyltriazolylmethyl)amine; or the click reaction catalyst may specifically include copper sulfate, tris(2-carboxyethyl)phosphine and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.

[0036] The present invention provides a method for detecting the activity of acetaldehyde dehydrogenase using the acetaldehyde dehydrogenase activity detection kit described in the above technical solution, including the following steps:

[0037] Mix the sample to be tested, azidoacetaldehyde and a first reaction medium to obtain a first mixed solution, and perform a first incubation to obtain a first incubated sample;

[0038] Mix the first incubated sample, an alkyne fluorescent probe, the click reaction catalyst and a second reaction medium to obtain a second mixed solution, and perform a second incubation to obtain a second incubated sample;

[0039] Detect the activity of acetaldehyde dehydrogenase in the sample to be tested according to the fluorescence intensity of the second incubated sample.

[0040] In the present invention, a sample to be measured, azidoacetaldehyde and a first reaction medium are mixed to obtain a first mixed liquid material, and a first incubation is carried out to obtain a first incubated sample. As an embodiment of the present invention, the sample to be measured may include living cells or tissues. In the embodiments of the present invention, the sample to be measured is specifically taken as an example of living cells for illustration. The present invention has no special limitation on the type of the first reaction medium, as long as the first incubation can proceed smoothly. For example, the first reaction medium may be a culture solution. In the embodiments of the present invention, cells can be specifically inoculated into a confocal dish and cultured overnight for adherent growth. After the culture is completed, azidoacetaldehyde is added to the confocal dish to obtain a first mixed liquid material, and a first incubation is carried out. As an embodiment of the present invention, the concentration of azidoacetaldehyde in the first mixed liquid material may be 0.2-1.5 mM, further may be 0.5-1.2 mM, and still further may be 0.8-1 mM; the time of the first incubation may be 4-12 h, further may be 6-10 h, and still further may be 8-9 h; the temperature of the first incubation is 37 °C. In the embodiments of the present invention, taking the sample to be measured as cells as an example, after the first incubation, it further includes: discarding the liquid material, washing the cells 3 times with PBS solution at intervals of 5 min between adjacent washings, and sucking dry the PBS solution in the confocal dish to obtain a first incubated sample.

[0041] As an embodiment of the present invention, when the sample to be measured is living cells or tissues, after obtaining the first incubated sample, it may further include: performing fixation-permeabilization treatment on the first incubated sample to obtain a fixed-permeabilized sample, and mixing the fixed-permeabilized sample with an alkynyl fluorescent probe, a click reaction catalyst and a second reaction medium for a second incubation; the reagent used for the fixation-permeabilization treatment includes methanol; the temperature of the fixation-permeabilization treatment may be 2-4 °C, and the time may be 10-15 min. In the embodiments of the present invention, taking the sample to be measured as cells as an example, after the fixation-permeabilization treatment, it further includes: discarding the liquid material, washing the cells 3 times with PBS solution at intervals of 5 min between adjacent washings, and sucking dry the PBS solution in the confocal dish to obtain a fixed-permeabilized sample.

[0042] After obtaining the first incubation sample (or fixed-permeabilized sample), the present invention mixes the first incubation sample (or fixed-permeabilized sample), an alkynyl fluorescent probe, a click reaction catalyst, and a second reaction medium to obtain a second mixed solution, and performs a second incubation to obtain a second incubation sample. As an embodiment of the present invention, the concentration of the alkynyl fluorescent probe in the second mixed solution can be 1-5 μM, further 1.25-2.5 μM; the concentration of copper sulfate can be 0.05-5 mM, further 0.5-1 mM; the concentration of the reducing agent can be 1-10 mM, further 2.5-5 mM; the concentration of the ligand can be 100-1000 μM, further 600-800 μM. As an embodiment of the present invention, the time of the second incubation can be 30-90 min, further 60 min; the temperature of the second incubation can be 20-40 °C, further 25 °C. In the examples of the present invention, taking the test sample as cells, after the second incubation, it further includes: discarding the liquid material, washing the cells 3 times with PBS solution, with an interval of 5 min between adjacent washings, to obtain a second incubation sample.

[0043] After obtaining the second incubation sample, the present invention detects the activity of aldehyde dehydrogenase in the test sample according to the fluorescence intensity of the second incubation sample. In the examples of the present invention, taking the test sample as cells, after obtaining the second incubation sample, specifically, a laser confocal microscope can be used to take cell images, and the statistical software ImageJ can be used to calculate the cell fluorescence intensity as the basis for comparing the enzyme activities of cells in different groups.

[0044] The present invention provides a method for detecting the activity of aldehyde dehydrogenase based on metabolic labeling, using azidoacetaldehyde as an azide tool substrate and combining bioorthogonal reactions to detect the activity of ALDH2. The schematic diagram of the relevant operation process is as Figure 1 shown. This method has the following beneficial effects: (1) The method has chemical simplicity; (2) High signal-to-noise ratio and good sensitivity for fluorescence detection; (3) The azide substrate has excellent selectivity and can detect the activity of ALDH2 with high specificity; (4) It can be used for detecting the activity of ALDH2 in living cells; (5) Simple operation and fast detection speed; (6) This method can be combined with drug screening to obtain effective ALHD2 agonists.

[0045] The present invention provides an application of the method described in the above technical solution in screening ALDH2 activity regulators. As an embodiment of the present invention, cells are specifically inoculated into a confocal dish and cultured overnight; after the culture, the cells are divided into two groups, one group is added with an enzyme activity regulating drug (i.e., a candidate ALDH2 activity regulator), and the other group is added with an equal amount of blank culture medium, and then the two groups of cells are respectively incubated for the first time and the second time according to the method described in the above technical solution, and finally a laser confocal microscope is used to take cell images, and the fluorescence intensity of the two groups of cells is calculated using the statistical software ImageJ, and the regulatory effect of different enzyme activity regulating drugs on enzyme activity is compared, thereby screening to obtain ALDH2 activity regulators. As an embodiment of the present invention, the ALDH2 activity regulator may include an ALDH2 agonist or an ALDH2 inhibitor; the screening results in the embodiment of the present invention show that the ALDH2 agonist may include sennoside A. As an embodiment of the present invention, virtual screening can be performed from a natural drug library, and the drugability is ranked from high to low, and the compounds with the highest drugability ranking are used as candidate ALDH2 activity regulators as needed, and then further screened according to the above method to obtain ALDH2 activity regulators.

[0046] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Example 1 Experiment on optimization of incubation conditions of azidoacetaldehyde

[0048] (1) The tool cells (specifically HepG2) were inoculated into the confocal dish at a density of 70% and cultured overnight; after the culture, different concentrations of azidoacetaldehyde (final concentrations were 0.5 mM, 1 mM, and 1.5 mM, respectively) were added to the confocal dish and incubated at 37°C for different time periods (4 h or 8 h, respectively); after the incubation, the culture medium was discarded and the cells were washed 3 times with PBS solution, with an interval of 5 min between each wash;

[0049] (2) Aspirate the PBS solution in the confocal dish, add 4°C anhydrous methanol, and place the confocal dish in a 4°C refrigerator for 15 min. After the standing period, remove the confocal dish, discard the methanol, and wash the cells three times with PBS solution, with an interval of 5 min between each wash.

[0050] (3) The PBS solution in the confocal dish was aspirated, and the alkyne fluorescent probe RHO (final concentration of 2.5 μM) and a click reaction catalyst were added, and incubated at 25° C. for 60 min, wherein the click reaction catalyst included CuSO 4 (final concentration of 0.5 mM), tris(2-carboxyethyl)phosphine (TCEP, final concentration of 1 mM) and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, final concentration of 100 μM); after the incubation, the click reaction solution was discarded and the cells were washed three times with PBS solution, with an interval of 5 min between each wash;

[0051] (4) Laser confocal microscopy was used to capture cell images, and statistical software ImageJ was used to calculate cell fluorescence intensity, which was used as the basis for analyzing cell enzyme activity.

[0052] Figure 2 The cell staining images after incubation with azidoacetaldehyde for different time periods (scale bar is 60 μm), wherein the concentration of azidoacetaldehyde is 0.5 mM, and the incubation time is 0 h, 4 h, and 8 h, respectively.

[0053] Figure 3 The cell staining images after incubation with different concentrations of azidoacetaldehyde (scale bar is 60 μm), where the incubation time is 8 h, and the azidoacetaldehyde concentrations are 0 mM, 0.5 mM, 1 mM, and 1.5 mM, respectively.

[0054] Figure 4 is the fluorescence quantitative statistical result diagram after azidoacetaldehyde was incubated under different conditions, that is Figure 2 and Figure 3 Quantitative results.

[0055] Depend on Figures 2 to 4 It can be seen that the brightness of the alkynyl fluorescent probe RHO staining of cells increases after the action of azidoacetaldehyde, and the brightness increases with the action time and concentration of azidoacetaldehyde.

[0056] Example 2 Orthogonal reaction condition optimization experiment

[0057] (1) The tool cells (specifically HepG2) were inoculated into the confocal dish at a density of 70% and cultured overnight; after the culture, azidoacetaldehyde (final concentration of 0.8 mM) was added to the confocal dish and incubated at 37°C for 8 h; after the incubation, the culture medium was discarded and the cells were washed 3 times with PBS solution, with an interval of 5 min between each wash;

[0058] (2) Aspirate the PBS solution in the confocal dish, add 4°C anhydrous methanol, and place the confocal dish in a 4°C refrigerator for 15 min. After the standing period, remove the confocal dish, discard the methanol, and wash the cells three times with PBS solution, with an interval of 5 min between each wash.

[0059] (3) The PBS solution in the confocal dish was aspirated, and the alkyne fluorescent probe RHO (final concentrations were 1.25 μM, 2.5 μM, and 5 μM, respectively) and a click reaction catalyst were added, and incubated at 25° C. for different time periods (20 min, 30 min, and 60 min, respectively), wherein the click reaction catalyst included CuSO 4 (final concentrations were 0.05mM, 0.5mM, 5mM, respectively), a reducing agent and a ligand, wherein the reducing agent was TCEP (final concentration was 1mM) or vitamin C (Vc, final concentrations were 1mM, 2.5mM, 5mM, 10mM, respectively), and the ligand was TBTA (final concentrations were 100μM, 600μM, respectively) or tris(3-hydroxypropyltriazolemethyl)amine (THPTA, final concentration was 600μM); after the incubation, the click reaction solution was discarded, and the cells were washed 3 times with PBS solution, with an interval of 5min between two adjacent times;

[0060] (4) Laser confocal microscopy was used to capture cell images, and statistical software ImageJ was used to calculate cell fluorescence intensity, which was used as the basis for analyzing cell enzyme activity.

[0061] Figure 5 The staining images of cells after incubation with different click reaction catalysts and alkyne fluorescent probe RHO under different conditions (the scale bar is 60 μm). Figure 6 This is the fluorescence quantitative statistical result diagram after incubation under different conditions using different click reaction catalysts and alkyne fluorescent probe RHO, that is, Figure 5 Quantitative results of Figure 5 and Figure 6 The types of click reaction catalysts corresponding to A, B, C, D and E and the incubation conditions of the alkyne fluorescent probe RHO are shown in Tables 1 to 5, respectively. 4 , Vc and TBTA as click reaction catalysts can significantly improve the brightness of the alkynyl fluorescent probe RHO after staining, and the appropriate alkynyl fluorescent probe RHO concentration and incubation time can increase the fluorescence growth multiple of the azidoacetaldehyde incubated group cells relative to the blank group cells.

[0062] Table 1 Types of click reaction catalysts and incubation conditions of alkyne fluorescent probe RHO (A)

[0063]

[0064]

[0065] Table 2 Types of click reaction catalysts and incubation conditions of alkyne fluorescent probe RHO (B)

[0066] Index B <![CDATA[CuSO 4 Concentration]]> Are 0.05 mM, 0.5 mM, and 5 mM respectively Type and concentration of reducing agent Vc 2.5 mM Type and concentration of ligand TBTA 100 μM Concentration of alkynyl fluorescent probe RHO 2.5 μM Incubation time 60 min

[0067] Table 3 Types of click reaction catalysts and incubation conditions of alkyne fluorescent probe RHO (C)

[0068] Index C <![CDATA[CuSO 4 Concentration]]> 0.5 mM Type and concentration of reducing agent Vc 2.5 mM Type and concentration of ligand Are TBTA 100 μM, TBTA 600 μM, and THPTA 600 μM respectively Concentration of alkynyl fluorescent probe RHO 2.5 μM Incubation time 60 min

[0069] Table 4 Types of click reaction catalysts and incubation conditions of alkyne fluorescent probe RHO (D)

[0070] Index D <![CDATA[CuSO 4 Concentration]]> 0.5 mM Type and concentration of reducing agent Vc 2.5 mM Type and concentration of ligand THPTA 600 μM Concentration of alkynyl fluorescent probe RHO 2.5 μM Incubation time Are 20 min, 30 min, and 60 min respectively

[0071] Table 5 Types of click reaction catalysts and incubation conditions of alkyne fluorescent probe RHO (E)

[0072] Index E <![CDATA[CuSO 4 Concentration]]> 0.5 mM Type and concentration of reducing agent Vc 2.5 mM Type and concentration of ligand THPTA 600 μM Concentration of alkynyl fluorescent probe RHO Are 1.25 μM, 2.5 μM, and 5 μM respectively Incubation time 60 min

[0073] Example 3 Screening for ALDH2 activity regulators

[0074] A total of 2784 compounds were virtual screened from the natural drug library, and the top 41 candidate compounds with the highest drugability were selected as enzyme activity regulating drugs (as shown in Table 6), and then ALDH2 activity regulators were further screened (the relevant process is shown in Figure 7 The specific steps are as follows:

[0075] (1) The tool cells (specifically HepG2) were inoculated into the confocal dish at a density of 70% and cultured overnight; after the culture, the cells were divided into two groups, one group was added with enzyme activity regulating drugs, and the other group was added with an equal amount of blank culture medium, and azidoacetaldehyde (final concentration of 0.8 mM) was added to the confocal dish and incubated at 37°C for 8 h; after the incubation, the culture medium was discarded and the cells were washed 3 times with PBS solution, with an interval of 5 min between each wash;

[0076] (2) Aspirate the PBS solution in the confocal dish, add 4°C anhydrous methanol, and place the confocal dish in a 4°C refrigerator for 15 min. After the standing period, remove the confocal dish, discard the methanol, and wash the cells three times with PBS solution, with an interval of 5 min between each wash.

[0077] (3) The PBS solution in the confocal dish was aspirated, and the alkyne fluorescent probe RHO (final concentration of 2.5 μM) and a click reaction catalyst were added, and incubated at 25° C. for 60 min, wherein the click reaction catalyst included CuSO 4 (final concentration of 0.5 mM), Vc (final concentration of 2.5 mM) and THPTA (final concentration of 600 μM); after the incubation, the click reaction solution was discarded, and the cells were washed three times with PBS solution, with an interval of 5 min between each wash;

[0078] (4) The cell images were taken by a laser confocal microscope, and the fluorescence intensities of the two groups of cells were calculated using the statistical software ImageJ as the basis for analyzing the cell enzyme activity. By comparing, the regulatory effects of different enzyme activity-regulating drugs on the enzyme activity were obtained, and thus an ALDH2 activity regulator was screened out.

[0079] Table 6 41 enzyme activity-regulating drugs

[0080]

[0081]

[0082] Figure 8 It is a quantitative graph of the cell fluorescence intensity after the action of different enzyme activity-regulating drugs. Among them, A is the quantitative result graph, and B is the docking result graph of sennoside A and ALDH2; the results show that the method of the present invention can be applied to the screening of high-throughput ALDH2 enzyme activity drugs, and the ALDH2 activity regulator sennoside A (Sennoside A, C3) was successfully screened out.

[0083] Test Example 1

[0084] Sennoside A was incubated with HepG2 cells and the ALDH2 enzyme activity was detected according to the method in Example 3 at different concentrations, and then cell imaging was performed to obtain the effect of different concentrations of sennoside A on the ALDH2 enzyme activity in the cells.

[0085] Figure 9 It is the dose-responsive cell staining graph of sennoside A and its fluorescence quantitative statistical result graph. Among them, A is the cell staining graph (scale bar is 60 μm), and B is the fluorescence quantitative statistical result graph. The results show that the sennoside A screened by the present invention has a typical dose-response curve of ALDH2 activity.

[0086] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An acetaldehyde dehydrogenase activity detection kit, comprising azidoacetaldehyde, an alkynyl fluorescent probe and a click reaction catalyst, wherein the alkynyl fluorescent probe comprises a cation and an anion, and the cation of the alkynyl fluorescent probe has a structure shown in Formula I:

2. The acetaldehyde dehydrogenase activity detection kit according to claim 1, characterized in that: The click reaction catalyst includes copper sulfate, a reducing agent and a ligand; the reducing agent includes vitamin C or tris(2-carboxyethyl)phosphine; the ligand includes tris(3-hydroxypropyltriazolemethyl)amine or tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.

3. The acetaldehyde dehydrogenase activity detection kit according to claim 1 or 2, characterized in that: The click reaction catalyst includes copper sulfate, vitamin C and tris(3-hydroxypropyltriazolylmethyl)amine; or the click reaction catalyst includes copper sulfate, tris(2-carboxyethyl)phosphine and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.

4. A method for detecting acetaldehyde dehydrogenase activity using the acetaldehyde dehydrogenase activity detection kit according to any one of claims 1 to 3, comprising the following steps: Mixing the sample to be tested, azidoacetaldehyde and a first reaction medium to obtain a first mixed liquid, and performing a first incubation to obtain a first incubation sample; The first incubation sample, the alkyne fluorescent probe, the click reaction catalyst and the second reaction medium are mixed to obtain a second mixed solution, and a second incubation is performed to obtain a second incubation sample; The activity of acetaldehyde dehydrogenase in the sample to be tested is detected according to the fluorescence intensity of the second incubated sample.

5. The method according to claim 4, characterized in that The concentration of azidoacetaldehyde in the first mixed liquid is 0.2-1.5 mM; the first incubation time is 4-12 hours.

6. The method according to claim 4, characterized in that The concentration of the alkynyl fluorescent probe in the second mixed liquid is 1-5 μM, the concentration of copper sulfate is 0.05-5 mM, the concentration of the reducing agent is 1-10 mM, and the concentration of the ligand is 100-1000 μM; the second incubation time is 30-60 min.

7. The method according to any one of claims 4 to 6, characterized in that: The sample to be tested includes living cells or tissues; when the sample to be tested is living cells or tissues, after obtaining the first incubation sample, the method further includes: fixing and permeabilizing the first incubation sample to obtain a fixed-permeabilized sample, mixing the fixed-permeabilized sample with an alkynyl fluorescent probe, a click reaction catalyst and a second reaction medium to perform the second incubation; the reagent used for the fixation-permeabilization treatment includes methanol.

8. Use of the method according to any one of claims 4 to 7 in screening ALDH2 activity regulators.

9. The use according to claim 8, characterized in that: The ALDH2 activity regulator includes an ALDH2 agonist or an ALDH2 inhibitor.

10. The use according to claim 9, characterized in that: The ALDH2 agonist includes sennoside A.

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