Dye solution, kit for detecting reactive oxygen and application thereof

Abstract

The application provides a dye solution for detecting active oxygen, a kit and application thereof, and relates to the field of detection reagents. The dye solution for detecting active oxygen comprises a probe for detecting active oxygen and a heat stabilizer. The probe for detecting active oxygen comprises MitoSOX red mitochondrial superoxide fluorescence probe and / or 2,7-dichlorodihydrofluorescein diacetate. The heat stabilizer comprises one or a combination of several of alpha-cyclodextrin, sucrose, glucose, glycerol and beta-cyclodextrin. By adding the heat stabilizer to the dye solution, the heat stability of the probe is improved, and the problem of poor heat stability of the probe in the reagent for detecting active oxygen ROS is solved.

Description

Technical Field

[0001] This invention relates to the field of detection reagents, and in particular to a dye solution, kit, and application for detecting reactive oxygen species. Background Technology

[0002] During aerobic respiration, some oxygen cannot be completely reduced, generating reactive oxygen species (ROS) with strong oxidizing effects, including superanions, hydroxyl radicals, and hydrogen peroxide. Simultaneously, cells possess a set of antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase, which can eliminate the ROS continuously generated during metabolism, maintaining a relatively stable intracellular ROS level. When intracellular ROS levels are imbalanced, ROS can cause lipid peroxidation, protein denaturation, DNA breakage, and so on, participating in the formation of a series of inflammatory and degenerative diseases.

[0003] Reactive oxygen species (ROS) can be detected using fluorescent probes. For example, DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) itself is non-fluorescent and can freely cross the cell membrane. Once inside the cell, it is hydrolyzed by intracellular esterases to generate DCFH. Since DCFH is impermeable to the cell membrane, the probe can be easily loaded into the cell. Intracellular ROS can oxidize the non-fluorescent DCFH (2',7'-dichlorodihydrofluorescein) to generate fluorescent DCF (oxidized dichlorofluorescein). Detecting the fluorescence of DCF reveals the level of intracellular ROS.

[0004] The probes used for ROS detection are sensitive to ambient temperature and are easily oxidized and decomposed, reducing fluorescence signals. The preparation and preservation of reagents are also difficult, which brings difficulties to the preparation of detection products and detection operations. Therefore, how to improve the thermal stability of the probes used for ROS detection is a problem that needs to be solved.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the application of heat stabilizers in the preparation of dye solutions for detecting reactive oxygen species (ROS), and the resulting dye solutions for ROS detection. By adding heat stabilizers, primarily composed of sugars and / or alcohols, to the dye solution, the thermal stability of the probe is improved, facilitating storage and alleviating the problem of poor probe thermal stability in reagents containing probes for ROS detection in existing technologies. Another objective of this invention is to provide a ROS detection kit and its application.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] In a first aspect, a staining solution for detecting reactive oxygen species is provided, comprising a probe for detecting reactive oxygen species and a heat stabilizer; wherein the probe for detecting reactive oxygen species comprises MitoSOX red mitochondrial superoxide fluorescent probe and / or 2,7-dichlorodihydrofluorescein diacetate.

[0009] The heat stabilizer includes one or a combination of several of α-cyclodextrin, sucrose, glucose, glycerol, and β-cyclodextrin.

[0010] In an optional embodiment, the concentrations of α-cyclodextrin and β-cyclodextrin in the dye solution are each independently 10–50 mg / mL.

[0011] In an optional embodiment, the concentration of α-cyclodextrin in the dye solution is 12.5–40 mg / mL, more preferably 40 mg / mL.

[0012] In an optional embodiment, the concentrations of sucrose and glucose in the dye solution are each independently 10–200 mg / mL.

[0013] In an optional embodiment, the concentration of glycerol in the dye solution is 0.1 to 1 mL / mL.

[0014] In an optional embodiment, the dye solution further includes a buffer component and metal ions.

[0015] In an optional embodiment, the staining solution comprises 0.05–1 μg / mL MitoSOX red mitochondrial superoxide fluorescent probe, 0.1–1.5 μg / mL 2,7-dichlorodihydrofluorescein diacetate, and 10–50 mg / mL α-cyclodextrin.

[0016] Secondly, the application of heat stabilizers in the preparation of dye solutions for detecting reactive oxygen species is also provided, wherein the dye solutions for detecting reactive oxygen species include MitoSOX red mitochondrial superoxide fluorescent probe and / or 2,7-dichlorodihydrofluorescein diacetate.

[0017] The heat stabilizer includes one or a combination of several of α-cyclodextrin, sucrose, glucose, glycerol, and β-cyclodextrin.

[0018] In an optional implementation, the dye solution is used to detect samples based on flow cytometry.

[0019] Thirdly, a reactive oxygen species detection kit is also provided, which includes the aforementioned dye solution and buffer reagent for detecting reactive oxygen species, packaged separately.

[0020] In an optional implementation, the buffer reagent includes a phosphate buffer.

[0021] Fourthly, the application of the above-mentioned staining solution for detecting reactive oxygen species, or the above-mentioned kit, in any of the following:

[0022] (a) Flow cytometry-based samples not intended for diagnostic or therapeutic purposes;

[0023] (b) Used to prepare in vitro diagnostic reagents; said in vitro diagnostic reagents are used to detect diseases caused by abnormal levels of reactive oxygen species.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention discovers that certain sugars and alcohols, acting as heat stabilizers, can enhance the thermal stability of the MitoSOX red mitochondrial superoxide fluorescent probe and 2,7-dichlorodihydrofluorescein diacetate. Based on this concept, this invention provides the application of sugars and alcohols in the preparation of staining solutions for detecting reactive oxygen species (ROS), and the staining solutions for ROS detection prepared based on this application. The sugars and alcohols include one or more combinations of α-cyclodextrin, sucrose, glucose, glycerol, and β-cyclodextrin.

[0026] By adding the aforementioned heat stabilizer to the staining solution, the influence of ambient temperature on the fluorescence signal of the MitoSOX red mitochondrial superoxide fluorescent probe and 2,7-dichlorodihydrofluorescein diacetate was reduced, improving the thermal stability of the probe in liquid reagents. This eliminates the need for lyophilization, facilitating storage, and has no impact on the fluorescence signal. Comparative experiments showed that after 7 days of storage at 37°C, the probe in the heat-stabilized solution exhibited significantly better thermal stability compared to the solutions without the added heat stabilizer. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is a graph showing the flow cytometry results of the control group in Example 1.

[0029] Figure 2 The image shows the flow cytometry results of the kit in Example 1 of Example 1.

[0030] Figure 3 The graph shows the flow cytometry results of the kit in Example 2 of Example 1.

[0031] Figure 4The image shows the flow cytometry results of the kit in Example 3 of Example 1.

[0032] Figure 5 The image shows the flow cytometry results of the kit in Example 4 of Example 1.

[0033] Figure 6 The graph shows the flow cytometry results of the kit in Example 5 of Example 1.

[0034] Figure 7 The image shows the flow cytometry results of the kit in Example 6 of Example 1.

[0035] Figure 8 The image shows the flow cytometry results of the kit in Comparative Example 1, which is the effect example. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The MitoSOX Red Mitochondrial Superoxide Indicator is a cationic derivative of dihydroethidum (DHE) and is a specially designed, highly specific detector for superoxide in live cell mitochondria. The MitoSOX Red Mitochondrial Superoxide Indicator specifically targets mitochondria, thus selectively detecting superoxide within them. This probe can penetrate the living cell membrane and selectively enter the mitochondria. Once inside the mitochondria, the probe is oxidized by superoxide, emitting red fluorescence.

[0038] 2,7-Dichlorodihydrofluorescein diacetate is a cell-permeable non-fluorescent probe. 2,7-Dichlorodihydrofluorescein diacetate is deesterified and oxidized in cells to convert into 2,7-dichlorofluorescein, which has strong fluorescence.

[0039] Existing reactive oxygen species (ROS) detection technologies commonly use MitoSOX red mitochondrial superoxide fluorescent probes and 2,7-dichlorodihydrofluorescein diacetate as fluorescent probes. However, these two probes are sensitive to ambient temperature, easily decompose upon heating, reducing fluorescence signal, and are difficult to prepare and preserve in solutions. Certain sugars and alcohols can form special protective films or cavities on the cell surface under harsh conditions such as high temperature, high temperature, and dehydration, preventing the structure of the protected substance from being destroyed and thus improving the thermal stability of the protected substance. The inventors discovered that adding one or more of α-cyclodextrin, sucrose, glucose, glycerol, and β-cyclodextrin to the staining solution can improve the thermal stability of MitoSOX red mitochondrial superoxide fluorescent probes and 2,7-dichlorodihydrofluorescein diacetate, facilitating liquid preservation without the need for lyophilization.

[0040] Based on the above findings, in a first aspect, a staining solution for detecting reactive oxygen species (ROS) is provided, comprising a probe for detecting ROS and a heat stabilizer; wherein the probe for detecting ROS comprises MitoSOX red mitochondrial superoxide fluorescent probe and / or 2,7-dichlorodihydrofluorescein diacetate. The heat stabilizer comprises one or a combination of several of α-cyclodextrin, sucrose, glucose, glycerol, and β-cyclodextrin.

[0041] The experiments of this invention have shown that the above-mentioned heat stabilizer can improve the heat stability of both the MitoSOX red mitochondrial superoxide fluorescent probe and 2,7-dichlorodihydrofluorescein diacetate. Therefore, the staining solution for detecting reactive oxygen species can contain either of the above two probes, or it can contain both of the above probes simultaneously. In an optional embodiment, the staining solution for detecting reactive oxygen species is a combined staining solution, including the MitoSOX red mitochondrial superoxide fluorescent probe and 2,7-dichlorodihydrofluorescein diacetate.

[0042] The heat stabilizer in the dye solution for detecting reactive oxygen species provided by this invention has no effect on the fluorescence signal. It can be used in any method in the art that uses fluorescence signal detection to determine the sample state, and this invention does not limit the detection method for the fluorescence signal. In an optional embodiment, the dye solution for detecting reactive oxygen species is used in a flow cytometry-based detection method.

[0043] The concentrations of MitoSOX red mitochondrial superoxide fluorescent probe and 2,7-dichlorodihydrofluorescein diacetate in the dye solution for detecting reactive oxygen species provided by this invention are not limited, and those skilled in the art can select them according to the means and equipment for detecting fluorescence signals.

[0044] In an optional embodiment, when the dye solution used to detect reactive oxygen species is used for flow cytometry-based detection, the concentration of the MitoSOX red mitochondrial superoxide fluorescent probe is 0.05 to 1 μg / mL, for example, but not limited to 0.05, 0.1, 0.2, 0.5, 0.8 or 1 μg / mL, or a range between any two of the aforementioned points.

[0045] In an optional embodiment, when the dye solution used to detect reactive oxygen species is used for flow cytometry-based detection, the concentration of 2,7-dichlorodihydrofluorescein diacetate is 0.1 to 1.5 μg / mL, for example, but not limited to 0.1, 0.5, 1 or 1.5 μg / mL, or a range between any two of the aforementioned values.

[0046] In an optional embodiment, the heat stabilizer in the dye solution used for detecting reactive oxygen species includes α-cyclodextrin, wherein the concentration of α-cyclodextrin in the dye solution is independently 10–50 mg / mL, for example, but not limited to 10, 15, 20, 30, 35, 40, or 50 mg / mL, or a range between any two of the aforementioned points. Preferably, the concentration of α-cyclodextrin is 12.5–40 mg / mL, more preferably 30–40 mg / mL, and even more preferably 40 mg / mL.

[0047] In an optional embodiment, the heat stabilizer in the dye solution used to detect reactive oxygen species includes β-cyclodextrin, wherein the concentration of β-cyclodextrin in the dye solution is independently 10 to 50 mg / mL, for example, but not limited to 10, 15, 20, 30, 35, 40 or 50 mg / mL, or a range between any two of the aforementioned points.

[0048] In an optional embodiment, the heat stabilizer in the dye solution used to detect reactive oxygen species includes sucrose, wherein the concentration of sucrose in the dye solution is 10 to 200 mg / mL, for example, but not limited to 10, 50, 100, 150 or 200 mg / mL, or a range between any two of the aforementioned points, preferably 200 mg / mL sucrose.

[0049] In an optional embodiment, the heat stabilizer in the dye solution used to detect reactive oxygen species includes glucose, wherein the concentration of glucose in the dye solution is 10 to 200 mg / mL, for example, but not limited to 10, 50, 100, 150 or 200 mg / mL, or a range between any two of the aforementioned points, preferably 200 mg / mL glucose.

[0050] In an optional embodiment, the heat stabilizer in the dye solution used to detect reactive oxygen species includes glycerol, and the concentration of glycerol in the dye solution is 0.1 to 1 mL / mL, for example, but not limited to 0.1, 0.3, 0.5, 0.8 or 1 mL / mL, or a range between any two of the aforementioned points, preferably 0.5 mL / mL.

[0051] In an optional embodiment, the dye solution used to detect reactive oxygen species further includes a buffer component and metal ions.

[0052] In optional embodiments, the buffer salt includes, but is not limited to, buffer components constituting the following buffer solutions: phosphate buffer, Tris buffer, Bis-Tris buffer, Tris-HCl buffer, HEPES buffer, PIPES buffer, MOPS buffer, Tricine buffer, TEA buffer, glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, citrate-sodium citrate buffer, sodium bicarbonate-acetic acid buffer, acetic acid-sodium acetate buffer, glycine-sodium hydroxide buffer, potassium chloride-hydrochloric acid buffer, PBS, or PBST. In optional embodiments, the buffer component includes phosphates and carbonates.

[0053] In optional embodiments, the metal ions include magnesium ions and calcium ions. In optional embodiments, the magnesium ions are provided by a salt containing magnesium ions, such as, but not limited to, magnesium sulfate. In optional embodiments, the calcium ions are provided by a salt containing calcium ions, such as, but not limited to, calcium chloride.

[0054] In an optional embodiment, the staining solution used to detect reactive oxygen species is a combined staining solution containing two probes, namely MitoSOX red mitochondrial superoxide fluorescent probe 0.05-1 μg / mL, 2,7-dichlorodihydrofluorescein diacetate 0.1-1.5 μg / mL and α-cyclodextrin 10-50 mg / mL.

[0055] In an optional embodiment, the staining solution used for detecting reactive oxygen species is a combined staining solution containing two probes, wherein the staining solution comprises 0.152 μg / mL MitoSOX red mitochondrial superoxide fluorescent probe, 0.244 μg / mL 2,7-dichlorodihydrofluorescein diacetate, 0.04 g / mL α-cyclodextrin, 1 μg / mL glucose, 8 μg / mL NaCl, 0.126 μg / mL Na2HPO4·12H2O, 0.4 μg / mL KCl, 0.06 μg / mL KH2PO4, 0.098 μg / mL MgSO4, 0.14 μg / mL CaCl2, and 0.35 μg / mL NaHCO3.

[0056] Based on the aforementioned findings, the invention proposes that adding a heat stabilizer to the dye solution can improve the thermal stability of the MitoSOX red mitochondrial superoxide fluorescent probe and 2,7-dichlorodihydrofluorescein diacetate. Secondly, the invention also provides the application of a heat stabilizer in the preparation of a dye solution for detecting reactive oxygen species (ROS), wherein the dye solution comprises the MitoSOX red mitochondrial superoxide fluorescent probe and / or 2,7-dichlorodihydrofluorescein diacetate; and the heat stabilizer comprises one or a combination of several of α-cyclodextrin, sucrose, glucose, glycerol, and β-cyclodextrin.

[0057] In an optional embodiment, the application includes adding one or more of α-cyclodextrin, sucrose, glucose, glycerol and β-cyclodextrin to the staining solution when preparing a staining solution containing MitoSOX red mitochondrial superoxide fluorescent probe and / or 2,7-dichlorodihydrofluorescein diacetate.

[0058] In an optional embodiment, when the dye solution used to detect reactive oxygen species is used for flow cytometry-based detection, the concentration of the MitoSOX red mitochondrial superoxide fluorescent probe is 0.05–1 μg / mL.

[0059] In an optional embodiment, when the dye solution used to detect reactive oxygen species is used for flow cytometry-based detection, the concentration of 2,7-dichlorodihydrofluorescein diacetate is 0.1–1.5 μg / mL.

[0060] In an optional embodiment, the heat stabilizer in the dye solution used to detect reactive oxygen species includes α-cyclodextrin, wherein the concentration of α-cyclodextrin in the dye solution is independently 10–50 mg / mL.

[0061] In an optional embodiment, the heat stabilizer in the dye solution used to detect reactive oxygen species includes β-cyclodextrin, wherein the concentration of β-cyclodextrin in the dye solution is independently 10–50 mg / mL.

[0062] In an optional embodiment, the heat stabilizer in the dye solution used to detect reactive oxygen species includes sucrose, and the concentration of sucrose in the dye solution is 10-200 mg / mL.

[0063] In an optional embodiment, the heat stabilizer in the dye solution used to detect reactive oxygen species includes glucose, wherein the concentration of glucose in the dye solution is 10–200 mg / mL.

[0064] In an optional embodiment, the heat stabilizer in the dye solution used to detect reactive oxygen species includes glycerol, and the concentration of glycerol in the dye solution is 0.1 to 1 mL / mL.

[0065] In an optional embodiment, the staining solution used to detect reactive oxygen species is a combined staining solution containing two probes, namely MitoSOX red mitochondrial superoxide fluorescent probe and 2,7-dichlorodihydrofluorescein diacetate.

[0066] In an optional implementation, the dye solution used to detect reactive oxygen species is used to detect samples based on flow cytometry.

[0067] Thirdly, a reactive oxygen species (ROS) detection kit is also provided, comprising separately packaged dye solutions and buffer reagents for detecting ROS. In an optional embodiment, the buffer reagent comprises phosphate buffer.

[0068] It is understood that the kit may optionally include other detection reagents or consumables acceptable in the art, including but not limited to one or more of buffer reagents, negative controls, positive controls, blank controls, elution reagents, sample processing solutions, diluents, and solvents. Those skilled in the art can formulate other reagents or consumables according to the detection methods applicable to the kit and based on general knowledge in the art.

[0069] In an optional embodiment, the staining solution in the kit is a combined staining solution containing two probes: MitoSOX red mitochondrial superoxide fluorescent probe and 2,7-dichlorodihydrofluorescein diacetate. In an optional embodiment, the heat stabilizer in the combined staining solution includes α-cyclodextrin.

[0070] Fourthly, the above-mentioned dye solution for detecting reactive oxygen species, or the above-mentioned reactive oxygen species detection kit, may be used in any of the following:

[0071] (a) Flow cytometry-based samples not intended for diagnostic or therapeutic purposes;

[0072] (b) Used to prepare in vitro diagnostic reagents; said in vitro diagnostic reagents are used to detect diseases caused by abnormal levels of reactive oxygen species.

[0073] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0074] Example 1

[0075] Example 1 provides a kit for flow cytometry detection, comprising a dye solution and a buffer reagent.

[0076] (1) The buffer reagent is prepared as follows: Weigh 58g Na2HPO4·12H2O, 5.93g NaH2PO4·2H2O, and 9g NaCl, dissolve them completely in 800mL of purified water, and then bring the purified water to a final volume of 1000mL.

[0077] (2) The staining solution was prepared as follows: Weigh 0.244g of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), 0.152g of red mitochondrial superoxide fluorescent probe (MitoSOX Red Mitochondrial Superoxide Indicator), 40g of α-cyclodextrin, 8g of NaCl, 0.126g of Na2HPO4·12H2O, 0.4g of KCl, 0.06g of KH2PO4, 0.098g of MgSO4, 0.14g of CaCl2, 1g of D-glucose, and 0.35g of NaHCO3. After dissolving them completely in 800mL of purified water, bring the volume up to 1000mL.

[0078] (3) After dispensing each of the prepared components separately, the buffer solution and dye solution are assembled into a kit.

[0079] Example 2

[0080] Example 2 provides a kit for flow cytometry detection, comprising a dye solution and a buffer reagent, differing from Example 1 only in that the 40 mg / mL α-cyclodextrin in the dye solution is replaced with 200 mg / mL sucrose.

[0081] Example 3

[0082] Example 3 provides a kit for flow cytometry detection, comprising a dye solution and a buffer reagent, differing from Example 1 only in that the 40 mg / mL α-cyclodextrin in the dye solution is replaced with 200 mg / mL glucose.

[0083] Example 4

[0084] Example 4 provides a kit for flow cytometry detection, comprising a dye solution and a buffer reagent, differing from Example 1 only in that the 40 mg / mL α-cyclodextrin in the dye solution is replaced with 0.5 mL / mL glycerol.

[0085] Example 5

[0086] Example 5 provides a kit for flow cytometry detection, comprising a dye solution and a buffer reagent, which differs from Example 1 only in that the concentration of α-cyclodextrin in the dye solution is 30 mg / mL.

[0087] Example 6

[0088] Example 6 provides a kit for flow cytometry detection, comprising a dye solution and a buffer reagent, differing from Example 1 only in that the concentration of α-cyclodextrin in the dye solution is 12.5 mg / mL.

[0089] Comparative Example 1

[0090] Comparative Example 1 provides a kit for flow cytometry detection, comprising dye and buffer reagent, which differs from Example 1 only in that it does not contain α-cyclodextrin.

[0091] (1) The buffer reagent is prepared as follows: Weigh 58g Na2HPO4·12H2O, 5.93g NaH2PO4·2H2O, and 9g NaCl, dissolve them completely in 800mL of purified water, and then bring the purified water to a final volume of 1000mL.

[0092] (2) The staining solution was prepared as follows: Weigh 0.244g of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), 0.152g of red mitochondrial superoxide fluorescent probe (MitoSOX Red Mitochondrial Superoxide Indicator), 8g of NaCl, 0.126g of Na2HPO4·12H2O, 0.4g of KCl, 0.06g of KH2PO4, 0.098g of MgSO4, 0.14g of CaCl2, 1g of D-glucose, and 0.35g of NaHCO3. After dissolving them completely in 800mL of purified water, bring the purified water to a final volume of 1000mL.

[0093] (3) After dispensing each of the prepared components separately, the buffer solution and dye solution are assembled into a kit.

[0094] Example 1

[0095] The dye solutions in the kits of Examples 1 to 6 and Comparative Example 1 were placed at 37°C for 7 days; the dye solution in the kit of Example 1 was stored at 2 to 8°C as a control group.

[0096] The kits from Examples 1-6, Comparative Example 1, and the control group were subjected to flow cytometry detection according to the following steps:

[0097] (1) Sample dilution: Take 1×10 6 Add one sperm and an appropriate amount of buffer solution to make up to 0.2 mL.

[0098] (2) Staining: Take the above sperm suspension, add 2 μl of staining solution, and incubate at 37°C in the dark for 15 min.

[0099] (3) Detection: Under light-protected conditions, take the above sperm suspension and test it on the machine.

[0100] Experimental results: The flow cytometry results of the control group are as follows: Figure 1 As shown, the flow cytometry results of Examples 1-6 are as follows: Figures 2-7 As shown, the flow cytometry results of Comparative Example 1 are as follows: Figure 8 As shown. Figures 1-8In the diagram, a is a scatter plot of flow cytometry results, b is a density plot of flow cytometry results, c is a red histogram of flow cytometry results, and d is a green histogram of flow cytometry results.

[0101] Compare Figures 1-8 It can be seen that:

[0102] (1) Comparison of the red light histograms shows that the fluorescence signal of the MitoSOX red mitochondrial superoxide fluorescent probe decreased significantly after 7 days of accelerated fluorescence at 37°C without the addition of α-cyclodextrin. α-cyclodextrin, glucose, sucrose, and glycerol can improve the thermal stability of the MitoSOX red mitochondrial superoxide fluorescent probe to some extent, with α-cyclodextrin showing the best effect.

[0103] (2) Comparison of the green light histogram shows that the fluorescence signal of the 2,7-dichlorodihydrofluorescein diacetate fluorescent probe shifted significantly and was extremely unstable when α-cyclodextrin was not added and the fluorescence was accelerated at 37°C for 7 days. α-cyclodextrin, glucose and glycerol can improve the thermal stability of the 2,7-dichlorodihydrofluorescein diacetate fluorescent probe to a certain extent, among which α-cyclodextrin has the best effect.

[0104] In summary, the MitoSOX red mitochondrial superoxide fluorescent probe and 2,7-dichlorodihydrofluorescein diacetate in the staining solution showed significantly higher thermal stability after the addition of α-cyclodextrin, glucose, sucrose and glycerol compared to the solution without the addition of α-cyclodextrin.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dye solution for detecting reactive oxygen species, characterized in that, This includes probes for detecting reactive oxygen species and heat stabilizers; The probes used to detect reactive oxygen species include MitoSOX red mitochondrial superoxide fluorescent probe and / or 2,7-dichlorodihydrofluorescein diacetate. The heat stabilizer includes one or a combination of several of α-cyclodextrin, sucrose, glucose, glycerol, and β-cyclodextrin; In the dye solution, the concentrations of α-cyclodextrin and β-cyclodextrin are independently 10-50 mg / mL, the concentrations of sucrose and glucose are independently 10-200 mg / mL, and the concentration of glycerol is 0.1-1 mL / mL.

2. The dyeing solution according to claim 1, characterized in that, The concentration of α-cyclodextrin in the dye solution is 12.5~40 mg / mL.

3. The dyeing solution according to claim 2, characterized in that, The concentration of α-cyclodextrin in the dye solution is 40 mg / mL.

4. The dyeing solution according to claim 1, characterized in that, It also includes buffer components and metal ions.

5. The dyeing solution according to claim 4, characterized in that, The buffer components include phosphates and carbonates.

6. The dyeing solution according to claim 5, characterized in that, The metal ions include magnesium ions and calcium ions.

7. The dyeing solution according to any one of claims 1 to 6, characterized in that, It includes MitoSOX red mitochondrial superoxide fluorescent probe 0.05~1 μg / mL, 2,7-dichlorodihydrofluorescein diacetate 0.1~1.5 μg / mL and α-cyclodextrin 10~50 mg / mL.

8. The dyeing solution according to claim 7, characterized in that, The staining solution includes 0.152 μg / mL MitoSOX red mitochondrial superoxide fluorescent probe, 0.244 μg / mL 2,7-dichlorodihydrofluorescein diacetate, 40 mg / mL α-cyclodextrin, 1 μg / mL glucose, 8 μg / mL NaCl, 0.126 μg / mL Na2HPO4·12H2O, 0.4 μg / mL KCl, 0.06 μg / mL KH2PO4, 0.098 μg / mL MgSO4, 0.14 μg / mL CaCl2, and 0.35 μg / mL NaHCO3.

9. The use of heat stabilizers in the preparation of dye solutions for detecting reactive oxygen species, wherein the dye solutions for detecting reactive oxygen species include MitoSOX red mitochondrial superoxide fluorescent probe and / or 2,7-dichlorodihydrofluorescein diacetate; The heat stabilizer includes one or a combination of several of α-cyclodextrin, sucrose, glucose, glycerol, and β-cyclodextrin; In the dye solution, the concentrations of α-cyclodextrin and β-cyclodextrin are independently 10-50 mg / mL, the concentrations of sucrose and glucose are independently 10-200 mg / mL, and the concentration of glycerol is 0.1-1 mL / mL.

10. The application according to claim 9, characterized in that, The concentration of α-cyclodextrin in the dye solution is 12.5~40 mg / mL.

11. The application according to claim 10, characterized in that, The concentration of α-cyclodextrin in the dye solution is 40 mg / mL.

12. The application according to any one of claims 9 to 11, characterized in that, The dye solution is used for sample detection based on flow cytometry.

13. A reactive oxygen species detection kit, characterized in that, Includes the dye solution and buffer reagent for detecting reactive oxygen species as described in any one of claims 1 to 8, which are packaged separately.

14. The reactive oxygen species detection kit according to claim 13, characterized in that, The buffer reagent includes phosphate buffer.

15. The use of the dye solution for detecting reactive oxygen species according to any one of claims 1 to 8 in any of the following: (a) Flow cytometry-based samples not intended for diagnostic or therapeutic purposes; (b) Used to prepare in vitro diagnostic reagents; said in vitro diagnostic reagents are used to detect diseases caused by abnormal levels of reactive oxygen species.

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